Developing a new class of engineered live bacterial therapeutics to treat human diseases

In the paper

“Indeed, several recombinant bacterial therapeutics are currently being explored clinically (Table 1), and patients are actively enrolling in these trials and accepting the associated risks.”

In the paper

“Several engineered live bacterial therapeutics are currently entering early or mid-stage clinical development and are poised to deliver the proof of concept needed to unlock the potential for this new class of therapeutics.”

In the paper

“Escherichia coli Nissle 1917 (EcN) has been used as a probiotic since its isolation over 100 years ago21.”

What the sources say

• Not checked yetcites [21] Beimfohr, C. A review of research conducted with probiotic E. coli marketed as symbioflor. Int. J. Bacteriol. 2016, 3535621 (2016). source

In the paper

“Similar to EcN, these genera do not colonize the human gut, thus allowing for predictive pharmacokinetic profiling of therapeutic strains41,42.”

What the sources say

• Not checked yetcites [41] Song, A. A., In, L. L. A., Lim, S. H. E. & Rahim, R. A. A review on Lactococcus lactis: from food to factory. Microb. Cell Factories 16, 55 (2017). source
• Not checked yetcites [42] Vesa, Pochart & Marteau. Pharmacokinetics of Lactobacillus plantarum NCIMB 8826, Lactobacillus fermentum KLD, and Lactococcus lactis MG 1363 in the human gastrointestinal tract. Aliment. Pharmacol. Ther. 14, 823–828 (2000). source

In the paper

“Acto Bio Therapeutics currently has three engineered candidate strains in clinical development using the chassis organism Lactococcus lactis (Table 1).”

In the paper

“They must also be studied in well-controlled clinical trials in the intended patient population to establish safety and efficacy10–12.”

What the sources say

• Not checked yetcites [10] U.S. Department of Health and Human Services, Food and Drug Administration Guidance for Industry. Early clinical trials with live biotherapeutic products: Chemistry, manufacturing, and control information. www.fda.gov/downloads/Biologi%E2%80%A6/UCM292704.pdf (2016).
• Not checked yetcites [11] Deal, C. Science and Regulation of Live Microbiome-Based Products Used to Prevent, Treat, or Cure Diseases in Humans - 09/17/2018 - 09/17/2018. (U.S. Food and Drug Administration, 2019). http://www.fda.gov/vaccines-blood-biologics/workshops-meetings-conferences-biologics/science-and-regulation-live-microbiome-based-products-used-prevent-treat-or-cure-diseases-humans.
• Not checked yetcites [12] Ross, J. J. et al. Considerations in the development of live biotherapeutic products for clinical use. Curr. Issues Mol. Biol. 10, 13–16 (2008).

In the paper

“However, the genetic stability of an engineered live bacterial therapeutic is a concern for organisms that are intended to replicate within and/or colonize the patient’s microbiota, and it may be possible for the strain to transfer engineered genetic material to other members of the endogenous microbiota.”

In the paper

“However, the need for strict anaerobiosis may complicate manufacturing of these organisms.”

In the paper

“However, if there is a risk that the organism may reach other tissues, additional studies could be required to support the safety of the LBP.”

In the paper

“Additionally, EcN may interact with the intestinal epithelium to stimulate anti-inflammatory activities24, as well as to restore and maintain intestinal barrier function25.”

What the sources say

• Not checked yetcites [24] Fábrega, M. J. et al. Intestinal anti-inflammatory effects of outer membrane vesicles from Escherichia coli Nissle 1917 in DSS-experimental colitis in mice. Front. Microbiol. 8, 1274 (2017). source
• Not checked yetcites [25] Guo, S. et al. Escherichia coli Nissle 1917 protects intestinal barrier function by inhibiting NF-κB-mediated activation of the MLCK-P-MLC signaling pathway. Mediators Inflamm. 2019, 5796491 (2019). source

In the paper

“Recently, Shepherd et al. engineered Bacteroides ovatus to metabolize porphyran, a marine polysaccharide that is rarely encountered in a Western diet44. The resulting strain, B. ovatus NB001, was shown to stably engraft in the colonic microbiota of mice supplemented with porphyran in the diet, and the fecal abundance of this strain was titratable by modulating dietary porphyran44.”

What the sources say

• Not checked yetcites [44] Shepherd, E. S., DeLoache, W. C., Pruss, K. M., Whitaker, W. R. & Sonnenburg, J. L. An exclusive metabolic niche enables strain engraftment in the gut microbiota. Nature 557, 434–438 (2018). source

In the paper

“Many bacterial species have been evolutionarily selected for metabolic function within the mammalian gastrointestinal tract, and some probiotic organisms have a long history of safe use in humans18,19.”

What the sources say

• Not checked yetcites [18] Sonnenborn, U. Escherichia coli strain Nissle 1917-from bench to bedside and back: history of a special Escherichia coli strain with probiotic properties. FEMS Microbiol. Lett. 363, fnw212 (2016). source
• Not checked yetcites [19] Schultz, M. Clinical use of E. coli Nissle 1917 in inflammatory bowel disease. Inflamm. Bowel Dis. 14, 1012–1018 (2008). source

In the paper

“While this genetic transfer is unlikely to be directly harmful to the patient, it may eliminate the competitive advantage of the engineered strain and undermine the efficacy of treatment.”

In the paper

“LBPs are distinguished from probiotic supplements on the basis of their labeling claims, as most probiotics are regulated as dietary supplements and cannot make claims to treat or prevent disease10–12.”

What the sources say

• Not checked yetcites [10] U.S. Department of Health and Human Services, Food and Drug Administration Guidance for Industry. Early clinical trials with live biotherapeutic products: Chemistry, manufacturing, and control information. www.fda.gov/downloads/Biologi%E2%80%A6/UCM292704.pdf (2016).
• Not checked yetcites [11] Deal, C. Science and Regulation of Live Microbiome-Based Products Used to Prevent, Treat, or Cure Diseases in Humans - 09/17/2018 - 09/17/2018. (U.S. Food and Drug Administration, 2019). http://www.fda.gov/vaccines-blood-biologics/workshops-meetings-conferences-biologics/science-and-regulation-live-microbiome-based-products-used-prevent-treat-or-cure-diseases-humans.
• Not checked yetcites [12] Ross, J. J. et al. Considerations in the development of live biotherapeutic products for clinical use. Curr. Issues Mol. Biol. 10, 13–16 (2008).

In the paper

“Engineered LBPs can be designed to perform natural biological processes, such as the assimilation of ammonia into amino acids, at significantly increased rates14 and to produce effectors that are not native to bacteria, including human proteins16.”

What the sources say

• Not checked yetcites [14] Kurtz, C. B. et al. An engineered E. coli Nissle improves hyperammonemia and survival in mice and shows dose-dependent exposure in healthy humans. Sci. Transl. Med. 11, eaau7975 (2019). source
• Not checked yetcites [16] Braat, H. et al. A phase I trial with transgenic bacteria expressing interleukin-10 in Crohn’s disease. Clin. Gastroenterol. Hepatol. 4, 754–759 (2006). source

In the paper

“Gene cassettes conferring the ability to utilize porphyran, for example, could be horizontally transferred to other members of the gut microbiota.”

In the paper

“Moreover, while the colon harbors a diverse microbiota, these organisms are predominantly localized to a loosely adherent layer of mucus49.”

What the sources say

• Not checked yetcites [49] Welch, J. L. M., Hasegawa, Y., McNulty, N. P., Gordon, J. I. & Borisy, G. G. Spatial organization of a model 15-member human gut microbiota established in gnotobiotic mice. Proc. Natl Acad. Sci. USA 114, E9105–E9114 (2017). source

In the paper

“Studies of radiolabeled E. coli in gnotobiotic mice have demonstrated that mucus-adherent E. coli display significantly higher rates of replication than those in the colonic lumen50. Interestingly, no differences in replication rates were observed for Bacteroides thetaiotaomicron50.”

What the sources say

• Not checked yetcites [50] Li, H. et al. The outer mucus layer hosts a distinct intestinal microbial niche. Nat. Commun. 6, 8292 (2015). source

In the paper

“Strict anaerobes, including Bacteroides spp., may be useful chassis organisms due to their abundance in the colonic microbiota of humans, as well as their capacity to consume complex dietary and host-derived glycans51.”

What the sources say

• Not checked yetcites [51] Grondin, J. M., Tamura, K., Déjean, G., Abbott, D. W. & Brumer, H. Polysaccharide Utilization Loci: Fueling Microbial Communities. J. Bacteriol. https://doi.org/10.1128/JB.00860-16 (2017). source

In the paper

“Chen et al. demonstrated that an N-acylphosphatidylethanolamine (NAPE)-producing strain of EcN could significantly ameliorate symptoms associated with high-fat diet feeding in mice. Mice treated with this recombinant EcN displayed reduced adiposity, insulin resistance, and hepatosteatosis compared to animals treated with an unengineered EcN control37.”

What the sources say

• Not checked yetcites [37] Chen, Z. et al. Incorporation of therapeutically modified bacteria into gut microbiota inhibits obesity. J. Clin. Invest. 124, 3391–3406 (2014). source

In the paper

“In 2016, the FDA issued a guidance document describing the regulatory considerations for conducting clinical trials with LBPs10.”

What the sources say

• Not checked yetcites [10] U.S. Department of Health and Human Services, Food and Drug Administration Guidance for Industry. Early clinical trials with live biotherapeutic products: Chemistry, manufacturing, and control information. www.fda.gov/downloads/Biologi%E2%80%A6/UCM292704.pdf (2016).

In the paper

“The European Pharmacopeia published a monograph setting the quality standards for LBPs for human use, in European Pharmacopoeia, Supplement 9.7; effective in April 201913.”

What the sources say

• Not checked yetcites [13] The European Pharmacopoeia Commission. Live biotherapeutic products for human use 9.7 (2019).

In the paper

“However, chemically inducible promoters are less amenable to intratumoral applications, since achieving an effective concentration of the inducer molecule in situ may be challenging, and some such compounds are not Generally Regarded As Safe (GRAS) for human use.”

In the paper

“In recent years, automated parallel bioreactor systems have accelerated fermentation process development by combining smaller volumes and higher throughput to enable greater iteration than traditional benchtop bioreactors84–86.”

What the sources say

• Not checked yetcites [84] Xu, P. et al. Characterization of TAP Ambr 250 disposable bioreactors, as a reliable scale-down model for biologics process development. Biotechnol. Prog. 33, 478–489 (2017). source
• Not checked yetcites [85] Sandner, V., Pybus, L. P., McCreath, G. & Glassey, J. Scale-down model development in ambr systems: an industrial perspective. Biotechnol. J. 14, e1700766 (2019). source
• Not checked yetcites [86] Manahan, M. et al. Scale-down model qualification of ambr® 250 high-throughput mini-bioreactor system for two commercial-scale mAb processes. Biotechnol. Prog. https://doi.org/10.1002/btpr.2870 (2019). source

In the paper

“The selection of an appropriate animal model depends on the question being addressed, as the translational value of animal models varies by species and genotype.”

In the paper

“Human gastrointestinal anatomy and physiology is more closely approximated by pigs and non-human primates than by rodent models80, but models of disease states may be unavailable in these large animal species.”

What the sources say

• Not checked yetcites [80] Hatton, G. B., Yadav, V., Basit, A. W. & Merchant, H. A. Animal farm: considerations in animal gastrointestinal physiology and relevance to drug delivery in humans. J. Pharm. Sci. 104, 2747–2776 (2015). source

In the paper

“Moreover, these in vitro simulations can elucidate the kinetics of engineered circuit regulation and function over timescales that are relevant to human biology.”

In the paper

“In a similar study, Wei et al. engineered Bifidobacterium longum to express the proapoptotic compound, tumstatin68. This strain was shown to inhibit tumor growth in a mouse model by various routes of administration68.”

What the sources say

• Not checked yetcites [68] Wei, C. et al. Bifidobacteria expressing tumstatin protein for antitumor therapy in tumor-bearing mice. Technol. Cancer Res. Treat. 15, 498–508 (2016). source

In the paper

“While several examples of effectors have shown promise preclinically, rigorous clinical trials will be required to determine whether these results translate to heterogeneous human cancers that have been recalcitrant to current modes of therapy.”

In the paper

“To help address these concerns, nutritional auxotrophies or kill switches can be utilized in engineered strains to prevent replication inside the host organism as well as to control the duration of therapeutic activity and limit the potential toxicity of an engineered strain to patients.”

In the paper

“However, the requirements for clinical development of engineered biotherapeutics, including diligent toxicology studies and adherence to regulatory guidelines, do not scale similarly and represent significant cost and effort for each candidate strain.”

In the paper

“Oral administration of SYNB1618 was shown to significantly lower blood Phe concentrations in a mouse model of PKU, as well as to result in dose-dependent production of the PAL-specific urinary biomarker, hippuric acid, in healthy non-human primates.”

What the sources say

• Not checked yetcites [54] Isabella, V. M. et al. Development of a synthetic live bacterial therapeutic for the human metabolic disease phenylketonuria. Nat. Biotechnol. 36, 857–864 (2018). source

In the paper

“For example, E. coli has been shown to colonize the region surrounding the necrotic core of tumors after intravenous injection9, and several reports have demonstrated the use of engineered E. coli strains to treat solid tumors in preclinical models15,62–65.”

What the sources say

• Not checked yetcites [9] Stritzker, J. et al. Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice. Int. J. Med. Microbiol. IJMM 297, 151–162 (2007). source
• Not checked yetcites [15] Chowdhury, S. et al. Programmable bacteria induce durable tumor regression and systemic antitumor immunity. Nat. Med. 25, 1057–1063 (2019). source
• Not checked yetcites [62] Yamada, T. et al. Apoptosis or growth arrest: modulation of tumor suppressor p53’s specificity by bacterial redox protein azurin. Proc. Natl Acad. Sci. USA 101, 4770–4775 (2004). source
• Not checked yetcites [63] Zhang, Y. et al. Escherichia coli Nissle 1917 targets and restrains mouse B16 Melanoma and 4T1 breast tumors through expression of Azurin protein. Appl. Environ. Microbiol. 78, 7603–7610 (2012). source
• Not checked yetcites [64] Li, R. et al. Expressing cytotoxic compounds in Escherichia coli Nissle 1917 for tumor-targeting therapy. Res. Microbiol. 170, 74–79 (2019). source
• Not checked yetcites [65] Ho, C. L. et al. Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nat. Biomed. Eng. 2, 27–37 (2018). source

In the paper

“However, the selection of engineered therapeutic effectors is likely to be critical for efficacy in patients.”

In the paper

“For example, while mice are readily available for preclinical studies, the oral bioavailability of small molecule drugs in humans and rodents have demonstrated poor correlation80.”

What the sources say

• Not checked yetcites [80] Hatton, G. B., Yadav, V., Basit, A. W. & Merchant, H. A. Animal farm: considerations in animal gastrointestinal physiology and relevance to drug delivery in humans. J. Pharm. Sci. 104, 2747–2776 (2015). source

In the paper

“In another report, Chowdhury et al. engineered a non-pathogenic E. coli strain to lyse specifically within the tumor microenvironment and release an anti-CD47 antagonist nanobody. The authors demonstrated the activation of tumor-infiltrating T cells, tumor regression, and long-term survival in a syngeneic tumor model in mice, as well as abscopal effects on untreated tumors15.”

What the sources say

• Not checked yetcites [15] Chowdhury, S. et al. Programmable bacteria induce durable tumor regression and systemic antitumor immunity. Nat. Med. 25, 1057–1063 (2019). source

In the paper

“Quantitative systems pharmacology approaches have been widely used to accelerate drug development for other modalities, including small molecules and recombinant proteins, by modeling the pharmacokinetic and pharmacodynamic properties of drug candidates across a wide assortment of therapeutic indications82.”

What the sources say

• Not checked yetcites [82] Milligan, P. A. et al. Model-based drug development: a rational approach to efficiently accelerate drug development. Clin. Pharmacol. Ther. 93, 502–514 (2013). source

In the paper

“Zhang et al. developed a strain of EcN to express azurin, a small bacterial protein that induces apoptosis in tumor cells62. This engineered strain suppressed the growth of tumors and prevented pulmonary metastasis in mice63.”

What the sources say

• Not checked yetcites [62] Yamada, T. et al. Apoptosis or growth arrest: modulation of tumor suppressor p53’s specificity by bacterial redox protein azurin. Proc. Natl Acad. Sci. USA 101, 4770–4775 (2004). source
• Not checked yetcites [63] Zhang, Y. et al. Escherichia coli Nissle 1917 targets and restrains mouse B16 Melanoma and 4T1 breast tumors through expression of Azurin protein. Appl. Environ. Microbiol. 78, 7603–7610 (2012). source

In the paper

“More recently, Ho et al. engineered EcN for the treatment of colorectal cancer by expressing HlpA, a protein that binds specifically to a heparan sulphate proteoglycan, enabling engineered EcN to specifically target polyps in a murine colorectal cancer model65.”

What the sources say

• Not checked yetcites [65] Ho, C. L. et al. Engineered commensal microbes for diet-mediated colorectal-cancer chemoprevention. Nat. Biomed. Eng. 2, 27–37 (2018). source

In the paper

“Engineered LBPs have the potential for delivering a living medicine that can sense signals within the patient, respond at the site of disease, and alleviate concerns of systemic exposure and toxicity.”

In the paper

“In this case, assessment of viability using commercial live/dead stains to detect intact cell membranes may be more appropriate for enumerating cells in the drug product88.”

What the sources say

• Not checked yetcites [88] Boulos, L., Prévost, M., Barbeau, B., Coallier, J. & Desjardins, R. LIVE/DEAD® BacLightTM: application of a new rapid staining method for direct enumeration of viable and total bacteria in drinking water. J. Microbiol. Methods 37, 77–86 (1999). source

In the paper

“Since no recombinant LBPs have been approved for use in humans, the field will be greatly advanced by the first proof of concept demonstration of an engineered bacterial therapeutic delivering a clinically meaningful impact on disease in patients.”

In the paper

“However, it may be desirable to establish longer-term colonization by live bacterial therapeutics in the gastrointestinal tract44 to allow for continuous delivery of a therapeutic effector, thereby reducing the need for repeat dosing and enabling lower efficacious bacterial dose levels.”

What the sources say

• Not checked yetcites [44] Shepherd, E. S., DeLoache, W. C., Pruss, K. M., Whitaker, W. R. & Sonnenburg, J. L. An exclusive metabolic niche enables strain engraftment in the gut microbiota. Nature 557, 434–438 (2018). source

In the paper

“Finally, expansion of the tools for synthetic biology, to allow commensal organisms to be considered as chassis for engineered bacterial therapeutics, could enhance compatibility of the strain with the human host.”

In the paper

“Odor and color masking may also be needed for any LBP to ensure patient compliance93.”

What the sources say

• Not checked yetcites [93] Tan, X., Feldman, S. R., Chang, J. & Balkrishnan, R. Topical drug delivery systems in dermatology: a review of patient adherence issues. Expert Opin. Drug Deliv. 9, 1263–1271 (2012). source

In the paper

“Technological advances including microencapsulation and cryoprotectants could improve the stability of future LBP formulations90,91, and buffering may be considered to preserve cell activity and viability in the stomach.”

What the sources say

• Not checked yetcites [90] Yus, C. et al. Targeted release of probiotics from enteric microparticulated formulations. Polymers 11, 1668 (2019). source
• Not checked yetcites [91] Guowei, S. et al. Comprehensive optimization of composite cryoprotectant for Saccharomyces boulardii during freeze-drying and evaluation of its storage stability. Prep. Biochem. Biotechnol. 49, 846–857 (2019). source

In the paper

“In other words, engineered microbes could act as biological thermostats and synthesize therapeutic effectors only as needed.”

In the paper

“However, for outpatient studies, formulations that require frozen storage and at-home reconstitution present challenges for patients, potentially leading to compliance issues89 and risks of product instability.”

What the sources say

• Not checked yetcites [89] Shahiwala, A. Formulation approaches in enhancement of patient compliance to oral drug therapy. Expert Opin. Drug Deliv. 8, 1521–1529 (2011). source

In the paper

“LBP formulations must also be palatable to patients to ensure compliance with dosing89.”

What the sources say

• Not checked yetcites [89] Shahiwala, A. Formulation approaches in enhancement of patient compliance to oral drug therapy. Expert Opin. Drug Deliv. 8, 1521–1529 (2011). source

In the paper

“Predicting the translational potential of engineered bacterial therapeutics necessitates a move toward mathematical frameworks for integrating data from in vitro and in vivo model systems to predict the behavior of engineered strains in these dynamic conditions.”

In the paper

“Hydrogel formulations have also been used for delivery of intratumoral drugs and may improve the concentration of the LBP within the tumor92.”

What the sources say

• Not checked yetcites [92] Fakhari, A. & Anand Subramony, J. Engineered in-situ depot-forming hydrogels for intratumoral drug delivery. J. Control. Release 220, 465–475 (2015). source