WO2023215813A1 - Engineered probiotics for colorectal cancer screening, prevention, and treatment - Google Patents
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Definitions
- This disclosure generally relates to the fields of medicine and immunology. More specifically, the disclosure relates to orally-deliverable programmable bacteria cells (e.g., E.coli Nissle 1917 bacteria) that colonize colorectal tumors and produce diagnostic and therapeutic molecules, as well as related compositions and methods.
- orally-deliverable programmable bacteria cells e.g., E.coli Nissle 1917 bacteria
- CRC Colorectal cancer
- Non-invasive stool-based tests that exist presently have variable sensitivity levels (-5-40%) for pre-cancerous polyps, which are the primary precursor lesions of CRC and the target for CRC prevention. Beyond these limitations, patient adherence remains low for stool-based tests and is the main challenge to screening implementation. In contrast, serumbased tests are patient-preferred, but have lower sensitivity and specificity than the FIT tests.
- the ideal CRC prevention strategy would address current issues of cost, compliance, complication, and provide direct and effective chemo-, immune-, or bio-prevention to prevent progression from benign to malignant disease.
- the present disclosure relates programmable bacterial cells for diagnosing and/or treating colorectal tumors.
- Programmable bacterial cells described herein comprise a synchronized lysis circuit comprising a nucleic acid encoding a quorum-sensing gene, a nucleic acid encoding a lysis gene, a promoter, and a terminator contained on a single operon and one or more nucleic acids encoding a diagnostic agent for diagnosing colorectal tumors and/or one or more nucleic acids encoding a therapeutic agent for treating colorectal tumors.
- the diagnostic agent is luciferase, salicylate, or a combination of both.
- the therapeutic agent is an antibody that specifically binds to PD- Ll, an antibody that specifically binds to CTLA-4, or a cytokine such as GM-CSF.
- the programmable bacterial cells comprise one or more nucleic acids encoding a plurality of therapeutic agents.
- the programmable bacterial cells belong to at least one genus selected from the group consisting of Salmonella, Escherichia, Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacteria. In some embodiments, the programmable bacterial cells belong to the genus Escherichia. In particular embodiments, the programmable bacterial cells are Escherichia coli Nissle (EcN) cells. In one embodiment, the EcN cells comprise a knockout of the clbA gene (EcNZl clbA).
- the present disclosure also relates to methods of detecting the presence of a colorectal tumor in a subject comprising administering a programmable bacterial cell described herein to the subject and detecting the presence of a colorectal tumor in the subject.
- the present disclosure also relates to methods of monitoring the treatment of a colorectal tumor in a subject comprising: administering a programmable bacterial cell described herein to the subject, wherein the programmable bacterial cell comprises a nucleic acid encoding a diagnostic agent described herein, is detectable in a biological sample obtained from the subject; obtaining a first biological sample from the subject at a first time point; measuring the level of the diagnostic agent in the first biological sample; obtaining a second biological sample from the subject at a second time point; and measuring the level of the diagnostic agent in the second biological sample.
- the present disclosure also relates to methods of treating a colorectal tumor in a subject comprising administering a therapeutically effective amount of programmable bacterial cells described herein to the subject, wherein the programmable bacterial cells comprise a nucleic acid encoding a therapeutic agent described herein, which capable of treating the colorectal tumor.
- the present disclosure also relates to methods of reducing the rate of proliferation of a colorectal tumor cell comprising delivering a programmable bacterial cell described herein to the colorectal tumor cell.
- the present disclosure also relates to methods of killing a colorectal tumor cell comprising delivering a programmable bacterial cell described herein to the colorectal tumor cell.
- the programmable bacterial cells described herein may be administered to a subject or delivered to a tumor in the form of a pharmaceutical composition, which may comprise one or more pharmaceutically acceptable carriers, diluents, or excipients.
- the present disclosure also relates to articles of manufacture useful for treating a colorectal tumor.
- the articles of manufacture comprise a container comprising programmable bacterial cells described herein, or pharmaceutical compositions comprising the same, as well as instructional materials for using the same to treat a colorectal tumor.
- the articles of manufacture are part of a kit that comprises a bacterial culture vessel and/or bacterial cell growth media.
- FIG. 1 shows the tumor colonization of E. coli Nissle 1917 in mouse models and human CRC patients.
- A Schematic of spontaneous intestinal adenomas in APC min/+ model. 12- week-old APC miI1/+ mice were gavaged twice, 3-4 days apart with 10 9 CFU EcN-lux.
- CFU colony-forming- units
- J Histopathology of orthotopic tumor (left) and higher power image of boxed region (middle) showing tumor mucin lakes similar to (right) human CRC with overt mucous phenotype.
- K Schematic of human clinical trial.
- FIG. 2 shows how orally-delivered EcN colonizes intestinal adenomas. 12-week-old APC mm/+ mice were gavaged twice, 3-4 days apart with 10 9 CFU bioluminescent EcN (EcN-lux). After 7 weeks, mice were sacrificed, intestinal tissue was excised and ex vivo imaged for bioluminescence. Red circles (top images) indicate areas of macroadenomas on sections of intestinal tissue isolated from the duodenum, proximal jejunum, distal jejunum, and ileum. Bottom images show EcN-lux on the intestinal tissue sections. Figure 2 corresponds to data shown in Fig. 1 B-C.
- FIG. 3 shows that colibactin is not a requisite for EcN colonization of intestinal adenomas.
- A Schematic of colibactin-encoding operon in EcN whereby clbA is knocked out and colibactin production is disrupted.
- B-C 12-week-old APC mm/+ mice were gavaged twice, 3-4 days apart with 109 CFU bioluminescent EcNAc/M.
- FIG. 4 shows that EcN can colonize a wide range of adenoma sizes. 15-17-week-old APC mm/+ mice were gavaged twice, 3-4 days apart with 10 9 CFU EcN producing an HA-tagged reporter protein to enable protein detection in intestinal tissue by anti-HA immunohistochemistry after sacrifice at 4 weeks post-dosing.
- A distribution of polyp sizes where polyps were considered HA + if there was any dark staining in the outlined polyp area.
- Figure 5 shows a colonoscopic image of mouse CRC tumor.
- Figure 6 shows selective colonization of orthotopic CRC model following oral administration of EcN.
- A Schematic depicting experimental timeline.
- B In vivo luciferase activity from EcN-lux.
- C Ex vivo imaging of excised tumor (T), normal colon tissue adjacent to tumor tissue (NC), spleen (S), kidneys (K), liver (L), and stool (St).
- Figure 7 shows the strain specific EcN PCR detection assay: Primer design and culture enrichment for microbial sequences. Redesigning a strain-specific EcN PCR assay to avoid potential false positive detection of other gut microbes in human tissue samples.
- DNA region boxed in purple contains 283bp DNA specific to EcN pMUT2 (3372-2654bp) and not other closely related sequences. PCR primers and probe were designed to this region as indicated.
- FIG. 8 shows the application of EcN platform for CRC screening and reduction in tumor burden.
- A Schematic of utilization of orally-delivered EcN probiotic to produce a urine- detectable molecule if CRC tumors are present and therapeutic proteins to manipulate tumor size in situ.
- B Orthotopic CRC -bearing mice were orally dosed with EcN-lux, multiple stool pellets were collected per mouse and mice were sacrificed, intestinal tissue was excised, imaged on IVIS, homogenized, and plated for CFU on LB agar.
- NTC tumor control mice
- D 15-week-old APC mm/+ were dosed with 10 9 EcN-producing salicylate strains and urine was collected 24 hours after dosing.
- (E) Receiving operator curve (ROC) of urine collected from WT and APC min/+ dosed at 24 hours and feces collected at 24 hours and 48 hours.
- (F-M) 15-week-old APC 111111/+ mice were dosed with PBS (Unt), EcN genomically encoding a lysis circuit (SLIC) or SLIC producing granulocyte-macrophage colony-stimulating factor (GM-CSF) and blocking nanobodies against PD-L1 and CTLA-4 targets (SLIC-3).
- PBS Unt
- SLIC EcN genomically encoding a lysis circuit
- GM-CSF granulocyte-macrophage colony-stimulating factor
- SLIC-3 blocking nanobodies against PD-L1 and CTLA-4 targets
- FIG. 9 shows that EcN can produce salicylate molecules to be detected by liquid chromatography mass spectrometry.
- EcN was engineered to produce salicylate molecules (EcN-SA).
- A Overnight cultures of EcN and EcN-SA were optical density-matched and liquid chromatography mass spectrometry (LC-MS) was used to detect salicylate in both the cell pellet and supernatant of EcN and EcN-SA cultures. All samples were normalized to an internal isotope-labelled D4-salicylate standard.
- B Extracted ion chromatogram showing the characteristic retention time and detected salicylate peak from EcN (negative control) and EcN- SA in cell pellets and media.
- FIG 10 shows that orally-delivered EcN producing PD-L1 and CTLA-4 blocking nanobodies and GM-CSF reduces tumor burden in APC mul/+ mice.
- 15- 17- week-old APC mm/+ mice were dosed with PBS (Unt), EcN genomically encoding a lysis circuit (SLIC) or SLIC producing granulocyte macrophage colony-stimulating factor (GM-CSF) and blocking nanobodies against PD-L1 and CTLA-4 targets (SLIC-3).
- PBS Unt
- SLIC EcN genomically encoding a lysis circuit
- GM-CSF granulocyte macrophage colony-stimulating factor
- mice were sacrificed, intestines were bisected, Swiss-rolled, paraffin embedded, sectioned, stained with hemotoxin and eosin and quantified for (A) total tumor count and (B) percent of tumors ⁇ 1 mm 2 , 1-3 mm 2 , or ⁇ 3 mm 2 .
- A total tumor count
- B percent of tumors ⁇ 1 mm 2 , 1-3 mm 2 , or ⁇ 3 mm 2 .
- FIG 11 shows the immunophenotyping of SLIC-3 treated mice. 15-17-week-old APC mm/+ mice were dosed with PBS (Unt), EcN genomically encoding a lysis circuit (SLIC) or SLIC producing granulocyte-macrophage colony-stimulating factor (GM-CSF), and blocking nanobodies against PD-L1 and CTLA-4 targets (SLIC-3). 1 month after dosing, mice were sacrificed, intestines were bisected, Swiss-rolled, paraffin embedded, sectioned, stained using immunohistochemical (IHC) techniques.
- IHC immunohistochemical
- ranges provided in the specification and appended claims include both end points and all points between the end points. Therefore, a range of 1.0 to 2.0 includes 1.0, 2.0, and all points between 1.0 and 2.0.
- the inventions described herein relate to orally-deliverable programmable bacteria cells (e.g., E. coli Nissle 1917 bacteria) that that produce diagnostic and therapeutic agent, resulting in sensiti ve detection of early CRC lesions and reduction of tumor burden through local induction of robust anti-tumor immunity as described hereinbelow.
- orally-deliverable programmable bacteria cells e.g., E. coli Nissle 1917 bacteria
- diagnostic agents and/or therapeutic agents are produced by one or more programmable bacterial cells.
- the programmable bacterial cells comprise heterologous nucleic acid sequences, which include one or more sequences that encode the diagnostic agents and/or therapeutic agents and sequences that encode a synchronized lysis circuit (i.e., a quorum-sensing gene, a nucleic acid encoding a lysis gene, a promoter, and a terminator contained on a single operon).
- a synchronized lysis circuit i.e., a quorum-sensing gene, a nucleic acid encoding a lysis gene, a promoter, and a terminator contained on a single operon.
- the programmable bacterial cells are capable of lysing in response to one or more internal or external stimuli, such as achieving a certain concentration or cell density in a tumor microenvironment, thereby releasing the diagnostic agents and/or therapeutic agents and other cellular components into the surrounding environment (e.g., tumor microenvironment).
- the diagnostic agent is luciferase, salicylate, or a combination of both.
- the therapeutic agent is an antibody that specifically binds to PD- Ll, an antibody that specifically binds to CTLA-4, or a cytokine such as GM-CSF.
- the programmable bacterial cells comprise one or more nucleic acids encoding a plurality of therapeutic agents.
- heterologous nucleic acid sequence refers to a nucleic acid derived from a different organism that encodes for a protein and which has been recombinantly introduced into a cell
- the heterologous nucleic acid sequence is introduced by transformation in order to produce a recombinant bacterial cell.
- Methods for creating recombinant bacterial cells are well known to those of skill in the art. Such methods include, but are not limited to, different chemical, electrochemical and biological approaches, for example, heat shock transformation, electroporation, liposome-mediated transfection, DEAE-Dextran-mediated transfection, or calcium phosphate transfection. Multiple copies of the heterologous nucleic acid sequence (e.g., between 2 and 10,000 copies) may be introduced into the cell.
- the heterologous nucleic acid sequences are in a plasmid. In some embodiments, the heterologous nucleic acid sequences are in a single operon and are integrated into the genome of the programmable bacterial cells. In some embodiments, the programmable bacterial cells comprise at least one inducible promoter or non-constitutive promoter that is in operable linkage with one or more of the heterologous nucleic acid sequences.
- promoter means at least a first nucleic acid sequence that regulates or mediates transcription of a second nucleic acid sequence. A promoter may comprise nucleic acid sequences near the start site of transcription that are required for proper function of the promoter.
- a TATA element for a promoter of polymerase II type can include distal enhancer or repressor elements that may lie in positions from about 1 to about 500 base pairs, from about 1 to about 1,000 base pairs, from 1 to about 5,000 base pairs, or from about 1 to about 10,000 base pairs or more from the initiation site.
- inducible promoter refers to an operable linkage between a promoter and a nucleic acid sequence, whereby the promoter mediates the nucleic acid transcription in the presence or absence of at least one specific stimulus. In some embodiments, the inducible promoter mediates transcription of a nucleic acid sequence in the presence or absence of at least one, two, three, four, or five or more stimuli. In some embodiments, the one or more stimuli are produced in whole or in part by the programmable bacterial cells. In some embodiments, the only stimulus of the promoter is the presence of a certain concentration or density of programmable bacterial cell found in the subject of a patient (e.g., in a tumor).
- operable linkage refers to an operative connection between nucleic acid sequences, such as for example between a control sequence (e.g., a promoter) and another nucleic acid sequence that codes for a protein i.e., a coding sequence. If a promoter can regulate transcription of an exogenous nucleic acid sequence, then it is in operable linkage with the gene.
- a control sequence e.g., a promoter
- a promoter can regulate transcription of an exogenous nucleic acid sequence, then it is in operable linkage with the gene.
- the programmable bacterial cells are preferably non-pathogenic and colonize tumors.
- One of ordinary skill in the art would know how to attenuate pathogenic bacteria to create non-pathogenic bacteria.
- the bacteria are attenuated by removing, knocking out, or mutating a virulence gene such as altering genetic components of the bacterial secretion system.
- the programmable bacterial cells belong to at least one genus selected from the group consisting of Salmonella, Escherichia, Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacteria. In some embodiments, the bacterial cells belong to more than one genus selected from the group consisting of Salmonella, Escherichia, Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacteria.
- the programmable bacterial cells belong to the genus Escherichia.
- the programmable bacterial cells are Escherichia coli Nissle 1917 (EcN) cells.
- the EcN cells comprise a knockout of the clbA gene (EcNzlc/M).
- a culture comprises the programmable bacterial cells and a medium, for example, a liquid medium, which may also comprise: a carbon source, for example, a carbohydrate source, or an organic acid or salt thereof; a buffer establishing conditions of salinity, osmolarity, and pH, that are amenable to survival and growth; additives such as amino acids, albumin, growth factors, enzyme inhibitors (for example protease inhibitors), fatty acids, lipids, hormones (e.g., dexamethasone and gibberellic acid), trace elements, inorganic compounds (e.g., reducing agents, such as manganese), redox-regulators (e.g., antioxidants), stabilizing agents (e.g., dimethyl sulfoxide), polyethylene glycol, polyvinylpyrrolidone (PVP), gelatin, antibiotics (e.g.
- a carbon source for example, a carbohydrate source, or an organic acid or salt thereof
- a buffer establishing conditions of
- the culture may comprise an agent that induces or inhibits transcription of one or more genes in operable linkage with an inducible promoter, for example doxicycline, tetracycline, tamoxifen, IPTG, hormones, or metal ions. While the specific culture conditions depend upon the particular programmable bacterial cells, general methods and culture conditions for the generation of microbial cultures are well known to those of skill in the art.
- the inventions described herein also encompass methods of treating a colorectal tumor in a subject comprising administering a therapeutically effective amount of programmable bacterial cells described herein to the subject, wherein the programmable bacterial cells comprise a nucleic acid encoding a therapeutic agent described herein, which capable of treating the colorectal tumor.
- the present disclosure also relates to methods of reducing the rate of proliferation of a colorectal tumor cell comprising delivering a programmable bacterial cell described herein to the colorectal tumor cell.
- the present disclosure also relates to methods of killing a colorectal tumor cell comprising delivering a programmable bacterial cell described herein to the colorectal tumor cell.
- treatment refers to all processes wherein there may be a slowing, interrupting, arresting, controlling, stopping, alleviating, or ameliorating symptoms or complications, or reversing of the progression of colorectal cancer, but does not necessarily indicate a total elimination of all disease or all symptoms.
- Non-limiting examples of treatment include reducing the rate of growth of a colorectal tumor or colorectal cancer cell disease, reducing the size of a tumor, or preventing the metastases of a tumor.
- a therapeutically effective dose means the number of cells per dose administered to a subject in need thereof that is sufficient to treat the hyperproliferative disorder.
- a therapeutically effective dose can be at least about l x10 4 cells, at least about IxlO 5 cells, at least about I xlO 6 cells, at least about I xlO 7 cells, at least about IxlO 8 cells, at least about IxlO 9 cells, or at least about IxlO 10 cells.
- programmable bacterial cells may be delivered to a subject in the form of a pharmaceutical composition, which may comprise one or more pharmaceutically acceptable carriers, diluents, or excipients.
- Pharmaceutical compositions may be formulated as desired using art recognized techniques.
- Various pharmaceutically acceptable carriers which include vehicles, adjuvants, and diluents, are readily available from numerous commercial sources.
- an assortment of pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are also available.
- Certain non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
- compositions may be frozen and thawed prior to administration or may be reconstituted in WFI with or without additional additives (e.g., albumin, dimethyl sulfoxide).
- additional additives e.g., albumin, dimethyl sulfoxide.
- Programmable bacterial cells described herein are preferably formulated for oral administration, but other routes of administration known in the art may be utilized.
- Particular dosage regimens i.e., dose, timing, and repetition, will depend on the particular subject being treated and that subject’s medical history. Empirical considerations such as pharmacokinetics will contribute to the determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy and is based on reducing the number of tumor cells or tumor mass, maintaining the reduction of such tumor cells or tumor mass, reducing the proliferation of tumor cells or an increase in tumor mass, or delaying the development of metastasis. A therapeutically effective dose may depend on the mass of the subject being treated, his or her physical condition, the extensiveness of the condition to be treated, and the age of the subject being treated. [0056] Articles of Manufacture
- the inventions disclosed herein also encompass articles of manufacture useful for treating a colorectal tumor comprising a container comprising programmable bacterial cells described herein, or a pharmaceutical composition comprising the same, as well as instructional materials for using the same to treat the colorectal tumor.
- the articles of manufacture are part of a kit that comprises a bacterial culture vessel and/or bacterial cell growth media.
- All bacterial strains used were luminescent (integrated luxCDABE cassette) so they could be visualized with the In Vivo Imaging System (IVIS).
- the EcNdc/M strain was engineered using the lambda-red recombineering method.
- the salicylate-encoding plasmid was constructed using Gibson assembly methods or restriction enzyme-mediated cloning methods whereby pchA and pchB genes were cloned onto a high-copy origin plasmid and driven by the lac promoter.
- the SLIC and SLIC-3 strains were constructed as previously described.
- Culture medium was Advanced Dulbecco’s modified Eagle medium/F12 (Life Technologies) supplemented with lx gentamicin/antimycotic/antibiotic (Life Technologies), 10 mM HEPES, 2mM GlutaMAX, lxB27 (Life Technologies), lxN2 (Life Technologies), 50 ng/mL mouse recombinant EGF (Peprotech), 100 ng/mL mouse recombinant noggin (Peprotech), 10 ng/mL human recombinant TGF-
- organoids were cultured in 10 pM Y-27632 (In Vitro Technologies), 3 pM iPSC (Calbiochem Cat #420220), 3 pM GSK-3 inhibitor (XVI, Calbiochem, # 361559) for the first 3 days.
- IVIS imaging Background (stage alone) subtracted total flux (photons/second) was used to capture the light signal emitted by EcN in identically sized areas for each live mouse in vivo. Following necropsy, individual tissues were collected into individual wells of a 6-well plate, weighed and average radiance (photons/s/cm 2 /sr) used for ex vivo tissue imaging to correct for the area being measured which differed for each tissue analyzed.
- CFU Excised tissues were placed aseptically into 5 mL 20% glycerol in PBS and homogenized in MACS Gentle cell dissociator C tubes, one tissue per tube using program C. lOOul of each tissue homogenate glycerol stock was serially diluted 1:100 six times. 10 pL of each dilution was spotted onto an LB agar plate with erythromycin selection at 50 pg/mL with 5 technical replicates. Plates were incubated at 37°C overnight (16 hours). Colony forming units (CFU) were calculated for each sample normalized to weight of tissue input to generate CFU/g tissue.
- CFU/g stool To generate CFU/g stool, one pellet of stool was placed into an Eppendorf and manually homogenized in PBS with a pipette tip and rigorous pipetting. Serial dilutions were spotted onto an LB agar place with 50 pg/mL erythromycin and incubated at 37°C overnight. CFU was normalized to weight of the stool.
- This study was an interventional, double-blind, dual-center, prospective clinical trial (WHO Universal Trial Number U1111-1225-7729, ANZCTR number ACTRN12619000210178).
- the study was approved by the Human Research Ethics Committee of the Central Sydney Local Health Network (HREC/18/CALHN/751) to meet the requirements of the National Statement on Ethical Conduct in Human Research in accordance with the Declaration of Helsinki for medical research involving human subjects.
- the study objective was to evaluate the colonization of matched normal and neoplastic bowel tissue by the probiotic E.coli Nissle.
- Tissue samples were weighed and collected in sterile 20% glycerol in PBS. Tissue was homogenized in GENTLEMACSTM C Tubes (Miltenyi Biotec, 130-093-237), with a GENTLEMACSTM Dissociator (Miltenyi Biotec, 130-093-235), program E. Aliquoted, homogenized tissue was stored at -80°C until further use. For culture enrichment, the equivalent of 10 mg of human tissue in homogenate was added to 1.2 mL of LB broth/sample and incubated with shaking at 37 °C for 24 hours.
- E. coli Nissle pMUT2 primers ECN7/8 and 9/10 were tested to detect E. coli Nissle in mouse fecal samples but they generated unacceptable false positives using gDNA isolated from human tissue samples from untreated patients. Alignment of PCR primer sets ECN7/8 or 9/10 against DNA sequences using Primer-BLAST suggested that Edwardsiella and Plesiomonas contain highly related sequences potentially also found in the human gut that may cause false positive calls via PCR assay using these primers.
- the external 283 bp amplicon spans the unique pMUT2 DNA region: ext-F 5’ TCGCGAACGTTAAATAATCATC (SEQ ID NO: 1); ext-R 5’ TCTGTTTTAGATAAGGCCATGTCTTC (SEQ ID NO: 2), and was amplified from 50 ng DNA input using KAPA Probe qPCR Master Mix (Roche, KK4716) with PCR conditions: denaturation 95°C for 20 seconds; 10 cycles of 95°C for 1 second, 60°C for 20 seconds, and 72°C for 25 seconds. Then 1 pL of this reaction was used as the template for the second 114 bp nested primer/probe-based assay. Nested primer and probe sequences were: int-F 5’ ACCCATCGATACCAAATGTATGT (SEQ ID NO: 3); int-R 5’
- FAM/CCCGCAGAT/ZEN/CACTGACCTCAATACA (SEQ ID NO: 5)/31ABkFQ/ using KAPA Probe qPCR Master Mix with PCR conditions as follows: 95°C for 20 seconds, 40 cycles of 9 °C for 1 second, 60°C for 20 seconds, and 72°C for 25 seconds.
- standard KAPA SYBR (non-nested) qPCR Master Mix (Roche, KK4602) with primers reported to amplify a 466 bp amplicon covering 331-797 of the E. coli 16S rRNA gene 16S-F 5’
- E. coli Nissle 1917 PCR standards were generated from serially diluted DNA isolated from exponentially growing cultures from crushed MUTAFLOR® capsule in LB at 37°C, with CFU determined by plating of matched samples on LB agar plates.
- an orally deliverable probiotic bacterium was engineered for both non-invasive diagnosis and tumor burden reduction of early-stage CRC.
- CRC precursor lesions, adenomas were modeled using APC min/+ mice, which develop spontaneous intestinal polyps and are representative of initiating genetic mutations seen in human familial adenomatous polyposis (Fig. 1 A).
- Neoplasia colonization was established by orally-delivering E.coli Nissle 1917 (EcN) encoding a genomically-integrated luxCDABE cassette (EcN-lux).
- a clbA knockout strain (EcN c/M) was generated in order to disrupt colibactin production and subsequently delivered to tumor-bearing APC m,n/+ (Fig. 3A). Similar to the EcN-lux strain, bioluminescent EcN c/M co-localized with visible macroadenomas as observed by ex vivo intestinal imaging and EcNzlc/M was detectable in APC mm feces for multiple days after oral dosing, suggesting that colonization does not rely on the presence of clbA gene or an intact colibactin-encoding operon (Fig. 3B-C).
- EcN-lux was engineered to produce a human influenza hemagglutinin (HA)-tagged reporter protein and orally delivered to APC""" /+ mice.
- Immunohistochemical staining against HA in Swiss-rolled intestinal tissue showed EcN-lux localization across early and late adenoma stages (Fig. IE) and sizes ranging from -0.16 mm 2 to 3 mm 2 (Fig. 4).
- Murine CRC organoids were injected into the distal murine colon and tumor grade tracked via weekly colonoscopy (Fig. 5, Fig. IF).
- mice were pre-treated with broad-spectrum antibiotics, which induces gut dysbiosis, a common phenomenon in gastrointestinal diseases including CRC.
- EcN-lux was orally delivered, and in vivo imaging five days post dosing revealed colocalization of bioluminescent EcN-lux with colon tumors (Fig. 1G, 1H, Fig. 6).
- Subsequent homogenization and plating of excised organs on antibiotic- selective LB plates confirmed EcN-lux was significantly enriched in tumors compared to adjacent healthy tissue and peripheral organs (Fig. II).
- the median diameter of EcN-lux colonized tumors was 2 mm (+/- 1.2 mm), suggesting the size of neoplastic lesions detected using this EcN-lux platform was similar to colonoscopic reporting of diminutive (0 to 5 mm) polyps in humans. Additional histological interrogation of these tumors also indicated the presence of tumor mucin lakes, demonstrating this model phenocopies mucinous human adenocarcinomas (Fig. 1J), and does not inhibit EcN-lux colonization.
- Example 16 [00118] Non-Invasive Methods of Treating CRC
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Abstract
Orally-deliverable programmable bacteria cells that colonize colorectal tumors and produce diagnostic and therapeutic molecules, as well as related compositions and methods.
Description
TITLE OF THE INVENTION
[0001] Engineered Probiotics for Colorectal Cancer Screening, Prevention, and Treatment
CROSS-REFERENCE TO RELATED APPLICATIONS
[0002] The instant application claims priority to U.S. Provisional Patent Application No. 63/338,132, filed 4 May 2022, which is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy, created on 3 May 2023, is named 39700051W001SEQL.xml and is 7,245 bytes in size.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0004] This invention was made with government support under grants U01CA247573 and R01CA249160 awarded by the National Institutes of Health and 1644869 awarded by the National Science Foundation. The government has certain rights in the invention.
TECHNICAL FIELD OF THE INVENTION
[0005] This disclosure generally relates to the fields of medicine and immunology. More specifically, the disclosure relates to orally-deliverable programmable bacteria cells (e.g., E.coli Nissle 1917 bacteria) that colonize colorectal tumors and produce diagnostic and therapeutic molecules, as well as related compositions and methods.
BACKGROUND OF THE INVENTION
[0006] Colorectal cancer (CRC) is the second leading cause of morbidity and mortality worldwide, with incidence rates significantly rising in younger populations. Colonoscopies are effective at reducing CRC incidence and mortality, allowing for simultaneous detection and treatment with removal of precancerous and cancerous lesions, however they are (1) inconvenient, as they require bowel preparation and a day off work; (2) costly due to hospital and personnel expenses; and (3) carry some risk, both procedural and related to anesthesia. Given this, CRC screening often occurs as a two-step approach, firstly a non-invasive step such as the
immunochemical fecal occult blood test (iFOBT), which if positive is then investigated by a diagnostic colonoscopy. Non-invasive stool-based tests that exist presently have variable sensitivity levels (-5-40%) for pre-cancerous polyps, which are the primary precursor lesions of CRC and the target for CRC prevention. Beyond these limitations, patient adherence remains low for stool-based tests and is the main challenge to screening implementation. In contrast, serumbased tests are patient-preferred, but have lower sensitivity and specificity than the FIT tests.
[0007] None of the foregoing alternatives offer the opportunity to manipulate lesions in situ, resulting in the eventual need for therapeutic intervention. Colonoscopies and colectomies are therapeutic options to remove local disease. If the cancer become metastatic, chemotherapy is often administered and, in some cases, the lesion is able to be surgically resected. More recently, targeted immunotherapies have become available to manage disease, but patient response is variable, and micro satellite stable CRC (MSS-CRC) is not responsive to immunotherapy in most cases due to the lack of immune cell infiltration.
[0008] The ideal CRC prevention strategy would address current issues of cost, compliance, complication, and provide direct and effective chemo-, immune-, or bio-prevention to prevent progression from benign to malignant disease.
BRIEF SUMMARY OF THE INVENTION
[0009] The present disclosure relates programmable bacterial cells for diagnosing and/or treating colorectal tumors. Programmable bacterial cells described herein comprise a synchronized lysis circuit comprising a nucleic acid encoding a quorum-sensing gene, a nucleic acid encoding a lysis gene, a promoter, and a terminator contained on a single operon and one or more nucleic acids encoding a diagnostic agent for diagnosing colorectal tumors and/or one or more nucleic acids encoding a therapeutic agent for treating colorectal tumors.
[0010] In some embodiments, the diagnostic agent is luciferase, salicylate, or a combination of both. In some embodiments, the therapeutic agent is an antibody that specifically binds to PD- Ll, an antibody that specifically binds to CTLA-4, or a cytokine such as GM-CSF. In some embodiments, the programmable bacterial cells comprise one or more nucleic acids encoding a plurality of therapeutic agents.
[0011] In some embodiments, the programmable bacterial cells belong to at least one genus selected from the group consisting of Salmonella, Escherichia, Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacteria. In some embodiments, the programmable bacterial cells belong
to the genus Escherichia. In particular embodiments, the programmable bacterial cells are Escherichia coli Nissle (EcN) cells. In one embodiment, the EcN cells comprise a knockout of the clbA gene (EcNZl clbA).
[0012] The present disclosure also relates to methods of detecting the presence of a colorectal tumor in a subject comprising administering a programmable bacterial cell described herein to the subject and detecting the presence of a colorectal tumor in the subject.
[0013] The present disclosure also relates to methods of monitoring the treatment of a colorectal tumor in a subject comprising: administering a programmable bacterial cell described herein to the subject, wherein the programmable bacterial cell comprises a nucleic acid encoding a diagnostic agent described herein, is detectable in a biological sample obtained from the subject; obtaining a first biological sample from the subject at a first time point; measuring the level of the diagnostic agent in the first biological sample; obtaining a second biological sample from the subject at a second time point; and measuring the level of the diagnostic agent in the second biological sample.
[0014] The present disclosure also relates to methods of treating a colorectal tumor in a subject comprising administering a therapeutically effective amount of programmable bacterial cells described herein to the subject, wherein the programmable bacterial cells comprise a nucleic acid encoding a therapeutic agent described herein, which capable of treating the colorectal tumor.
[0015] The present disclosure also relates to methods of reducing the rate of proliferation of a colorectal tumor cell comprising delivering a programmable bacterial cell described herein to the colorectal tumor cell. The present disclosure also relates to methods of killing a colorectal tumor cell comprising delivering a programmable bacterial cell described herein to the colorectal tumor cell.
[0016] In some embodiments, the programmable bacterial cells described herein may be administered to a subject or delivered to a tumor in the form of a pharmaceutical composition, which may comprise one or more pharmaceutically acceptable carriers, diluents, or excipients.
[0017] The present disclosure also relates to articles of manufacture useful for treating a colorectal tumor. In some embodiments, the articles of manufacture comprise a container comprising programmable bacterial cells described herein, or pharmaceutical compositions comprising the same, as well as instructional materials for using the same to treat a colorectal tumor. In some embodiments, the articles of manufacture are part of a kit that comprises a bacterial culture vessel and/or bacterial cell growth media.
[0018] The foregoing is a summary and thus contains, by necessity, simplifications, generalizations, and omissions of detail; consequently, those skilled in the art will appreciate that the summary is illustrative only and is not intended to be in any way limiting. Other aspects, features, and advantages of the methods, compositions and/or devices and/or other subject matter described herein will become apparent in the teachings set forth herein. The summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description of the Invention. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0019] Figure 1 shows the tumor colonization of E. coli Nissle 1917 in mouse models and human CRC patients. (A) Schematic of spontaneous intestinal adenomas in APCmin/+ model. 12- week-old APCmiI1/+ mice were gavaged twice, 3-4 days apart with 109 CFU EcN-lux. (B) EcN-lux was visualized using an IVIS for bioluminescence in vivo 96 hours post dosing. After 7 weeks, mice were sacrificed, intestinal tissue was excised, and ex vivo imaged for bioluminescence. Red arrows point to macroadenomas on distal intestinal tissue (n = 20 WT, n=25 APCmin/+). (C) Plot of adenoma area per H&E-stained intestine and bioluminescence signal (r = 0.75, Spearman correlation coefficient). In separate cohorts, mice were sacrificed at 1 week post dosing and homogenized small intestines plated on antibiotic- selective plates to (D) quantify colony-forming- units (CFU) per gram of tissue (n=5 WT, n=5 APCmin/+) or (E) gavaged twice, 3-4 days apart, with EcN producing an HA-tagged reporter protein to enable protein detection in intestinal tissue by anti-HA immunohistochemistry after sacrifice at 4 weeks post-dosing (n=3 mice, scale bars depict 200 pm). Dark brown stain depicts HA-tagged protein in early and late- stage adenomas. (F) Schematic of a murine model showing orthotopic tumor growth in the distal colon following mucosal injection of colorectal organoids. Tumor growth was monitored by colonoscopy, with tumor and non-tumor bearing control animals orally-dosed twice, 2 days apart, with EcN-lux or PBS before (G) imaging 5 days after last dose for bioluminescence, with (H) luminescence quantified in organs ex vivo (L, liver n = 20, S, spleen n = 20, NC, normal colon n = 20, Tumor grade 1/2 n = 15, Tumor grade 3/4 n = 12, PBS tumors n = 6, ** p < 0.01, **** p < 0.0001, oneway ANOVA with Tukey’s multiple comparisons). Organs and tumors (grade 1-4) were homogenized, plated on antibiotic-selective plates, and quantified for CFU per gram of tissue (liver
n = 22, spleen n = 22, normal colon n = 22, grade 1/2: n = 9 and grade 3/4: n = 6, PBS tumors n = 4). **** p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons.) (J) Histopathology of orthotopic tumor (left) and higher power image of boxed region (middle) showing tumor mucin lakes similar to (right) human CRC with overt mucous phenotype. (K) Schematic of human clinical trial. (L) Matched normal and tumor tissue homogenates from CRC patients administered placebo (n=3) or MUTAFLOR® (n=6) for 2 weeks prior to tissue sampling were used to inoculate overnight aerobic liquid culture. DNA isolated from culture was subjected to qPCR with the EcN specific assay or pan-microbial 16S. Mean value of 4 technical replicates shown per sample, box plot depicts 25th-75th percentile and median per group. Red dashed line depicts the limit of detection of each assay based on standard curve dilution series, dot points above the line have detectable PCR amplicon signal. No EcN S2 PCR amplicon signal was detected in no template or buffer only DNA prep controls. *p<0.05, Wilcoxon test. (M) Representative images of patient tissue pathology, right depicts boxed necrotic tumor region from middle image at higher power. Scale bars 100 pm (left, right) or 200 pm (middle).
[0020] Figure 2 shows how orally-delivered EcN colonizes intestinal adenomas. 12-week-old APCmm/+ mice were gavaged twice, 3-4 days apart with 109 CFU bioluminescent EcN (EcN-lux). After 7 weeks, mice were sacrificed, intestinal tissue was excised and ex vivo imaged for bioluminescence. Red circles (top images) indicate areas of macroadenomas on sections of intestinal tissue isolated from the duodenum, proximal jejunum, distal jejunum, and ileum. Bottom images show EcN-lux on the intestinal tissue sections. Figure 2 corresponds to data shown in Fig. 1 B-C.
[0021] Figure 3 shows that colibactin is not a requisite for EcN colonization of intestinal adenomas. (A) Schematic of colibactin-encoding operon in EcN whereby clbA is knocked out and colibactin production is disrupted. (B-C) 12-week-old APCmm/+ mice were gavaged twice, 3-4 days apart with 109 CFU bioluminescent EcNAc/M. (B) After 1 week, mice were sacrificed, intestinal tissue was excised and ex vivo imaged for bioluminescence. Red arrows point to macroadenomas on distal intestinal tissue (representative image from sample size of n=5 mice). (C) one stool pellet was collected 24, 48, and 72h after last dose, homogenized, plated on antibiotic- selective plates and quantified for CFU (n=3, n=l stool per mouse)
[0022] Figure 4 shows that EcN can colonize a wide range of adenoma sizes. 15-17-week-old APCmm/+ mice were gavaged twice, 3-4 days apart with 109 CFU EcN producing an HA-tagged reporter protein to enable protein detection in intestinal tissue by anti-HA immunohistochemistry
after sacrifice at 4 weeks post-dosing. (A) distribution of polyp sizes where polyps were considered HA+ if there was any dark staining in the outlined polyp area. (B) Representative images of polyps considered HA’ and HA+ (n=3 mice, scale bars depict 200 qm).
[0023] Figure 5 shows a colonoscopic image of mouse CRC tumor.
[0024] Figure 6 shows selective colonization of orthotopic CRC model following oral administration of EcN. (A) Schematic depicting experimental timeline. (B) In vivo luciferase activity from EcN-lux. (C) Ex vivo imaging of excised tumor (T), normal colon tissue adjacent to tumor tissue (NC), spleen (S), kidneys (K), liver (L), and stool (St).
[0025] Figure 7 shows the strain specific EcN PCR detection assay: Primer design and culture enrichment for microbial sequences. Redesigning a strain-specific EcN PCR assay to avoid potential false positive detection of other gut microbes in human tissue samples. (A) Full length endogenous pMUT2 plasmid sequence from EcN was used to BLAST against human gut microbiota DNA sequences to identify regions specific to EcN. Red bars indicate regions with a BLAST alignment score of >200, magenta indicates a score of 80-200, green indicates a score of 50-80, grey line indicates non-homologous sections. DNA region boxed in purple contains 283bp DNA specific to EcN pMUT2 (3372-2654bp) and not other closely related sequences. PCR primers and probe were designed to this region as indicated. (B) Microbial growth dynamics as measured by ODeoo over 24 hours in batch liquid culture from n=15 individual patient-tissue homogenization in normoxia at 37°C.
[0026] Figure 8 shows the application of EcN platform for CRC screening and reduction in tumor burden. (A) Schematic of utilization of orally-delivered EcN probiotic to produce a urine- detectable molecule if CRC tumors are present and therapeutic proteins to manipulate tumor size in situ. (B) Orthotopic CRC -bearing mice were orally dosed with EcN-lux, multiple stool pellets were collected per mouse and mice were sacrificed, intestinal tissue was excised, imaged on IVIS, homogenized, and plated for CFU on LB agar. Plot shows detection of CRC in tumor-bearing mice and no tumor control (NTC) mice by percentage of tumors colonized with EcN-lux above background, as determined by ex vivo IVIS of tissue (n=12 tumor-bearing mice, n=3 NTC mice), CFU assays (n=6 tumor-bearing mice, n=5 NTC mice) or by clinical guaiac fecal occult blood test (gFOBT) in stool (n=8 mice Grade 3/4 tumors, n=21 stools; n=4-5 mice no tumor control, NTC, n=12 stools), for animals bearing orthotopic grade 3/4 tumors. (C) 15-week-old APCmin/+ mice were dosed orally 3-4 days apart with 109 CFU EcN and one stool pellet was collected at 3, 7, 24, 48, and 72h after last dose, homogenized, plated on antibiotic- selective plates, and quantified for
CFU (n=5 mice per group). (D) 15-week-old APCmm/+ were dosed with 109 EcN-producing salicylate strains and urine was collected 24 hours after dosing. Graph is of LC-MS of salicylate molecules in urine of wild-type (WT) and APCmiI1/+ mice normalized to pre-treated urine values (p<0.05, unpaired T test, n=3-5 mice per group). (E) Receiving operator curve (ROC) of urine collected from WT and APCmin/+ dosed at 24 hours and feces collected at 24 hours and 48 hours. (F-M) 15-week-old APC111111/+ mice were dosed with PBS (Unt), EcN genomically encoding a lysis circuit (SLIC) or SLIC producing granulocyte-macrophage colony-stimulating factor (GM-CSF) and blocking nanobodies against PD-L1 and CTLA-4 targets (SLIC-3). One month after dosing, mice were sacrificed, intestines were bisected, Swiss-rolled, paraffin embedded, sectioned, stained with hemotoxin and eosin and quantified for (F) overall tumor area and (G) tumor area at different gut locations (* p<0.05, ** p < 0.01, *** P , 0.001, ns, not significant, ordinary one-way ANOVA test with Holm-Sidak multiple comparisons test, n=5-10 mice per group). (H) Representative H&E-stained histology images of SLIC and SLIC-3 treated mice. (I-M) Using immunohistochemical (IHC) techniques, intestinal tissue sections from all mouse groups were stained and quantified for (I) CD3+, (J) CD8+, and (K) granzyme B+ cells (n=3-5 mice per group, each marker represents a polyp, n=50-100 polyps per group, **** p < 0.0001 ordinary one-way ANOVA with Tukey post-test). (L) Representative IHC images of granzyme B+ staining depicted as brown puncta in SLIC and SLIC-3 -treated mice. Scale bars represent 500 pm.
[0027] Figure 9 shows that EcN can produce salicylate molecules to be detected by liquid chromatography mass spectrometry. EcN was engineered to produce salicylate molecules (EcN-SA). (A) Overnight cultures of EcN and EcN-SA were optical density-matched and liquid chromatography mass spectrometry (LC-MS) was used to detect salicylate in both the cell pellet and supernatant of EcN and EcN-SA cultures. All samples were normalized to an internal isotope-labelled D4-salicylate standard. (B) Extracted ion chromatogram showing the characteristic retention time and detected salicylate peak from EcN (negative control) and EcN- SA in cell pellets and media.
[0028] Figure 10 shows that orally-delivered EcN producing PD-L1 and CTLA-4 blocking nanobodies and GM-CSF reduces tumor burden in APCmul/+ mice. 15- 17- week-old APCmm/+ mice were dosed with PBS (Unt), EcN genomically encoding a lysis circuit (SLIC) or SLIC producing granulocyte macrophage colony-stimulating factor (GM-CSF) and blocking nanobodies against PD-L1 and CTLA-4 targets (SLIC-3). 1 month after dosing, mice were sacrificed, intestines were bisected, Swiss-rolled, paraffin embedded, sectioned, stained with hemotoxin and eosin and
quantified for (A) total tumor count and (B) percent of tumors <1 mm2, 1-3 mm2, or <3 mm2. Corresponds to data shown in Figure 8F- H.
[0029] Figure 11 shows the immunophenotyping of SLIC-3 treated mice. 15-17-week-old APCmm/+ mice were dosed with PBS (Unt), EcN genomically encoding a lysis circuit (SLIC) or SLIC producing granulocyte-macrophage colony-stimulating factor (GM-CSF), and blocking nanobodies against PD-L1 and CTLA-4 targets (SLIC-3). 1 month after dosing, mice were sacrificed, intestines were bisected, Swiss-rolled, paraffin embedded, sectioned, stained using immunohistochemical (IHC) techniques. Representative IHC images of CD3+ and CD8+ staining are shown where positive staining is depicted as brown puncta in SLIC and SLTC-3-treated mice (n=3-5 mice per group, each marker represents a polyp, n=50-100 polyps per group, **** p < 0.0001 ordinary one-way ANOVA with Tukey post-test). Scale bars represent 500 pm. Corresponds to data shown in Figure 8 I- J.
DETAILED DESCRIPTION OF THE INVENTION
[0030] While the present invention may be embodied in many different forms, disclosed herein are specific illustrative embodiments thereof that exemplify the principles of the invention. It should be emphasized that the present invention is not limited to the specific embodiments illustrated. Moreover, any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.
[0031] Unless otherwise defined herein, scientific, and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a protein" includes a plurality of proteins; reference to "a cell" includes mixtures of cells, and the like.
[0032] In addition, ranges provided in the specification and appended claims include both end points and all points between the end points. Therefore, a range of 1.0 to 2.0 includes 1.0, 2.0, and all points between 1.0 and 2.0.
[0033] The term "about" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of .+-.20%, .+-.10%,
.+-.5%, .+-.1%, or .+-.0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0034] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one of a number or lists of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of "consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.
[0035] In the claims, as well as in the specification above, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases "consisting of’ and "consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively.
[0036] Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclature used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art.
[0037] The inventions described herein relate to orally-deliverable programmable bacteria cells (e.g., E. coli Nissle 1917 bacteria) that that produce diagnostic and therapeutic agent, resulting in sensiti ve detection of early CRC lesions and reduction of tumor burden through local induction of robust anti-tumor immunity as described hereinbelow.
[0038] Programmable Bacteria Cells
[0039] In some embodiments of the inventions described herein, diagnostic agents and/or therapeutic agents are produced by one or more programmable bacterial cells. The programmable bacterial cells comprise heterologous nucleic acid sequences, which include one or more sequences that encode the diagnostic agents and/or therapeutic agents and sequences that encode a synchronized lysis circuit (i.e., a quorum-sensing gene, a nucleic acid encoding a lysis gene, a promoter, and a terminator contained on a single operon). By including a synchronized lysis circuit, the programmable bacterial cells are capable of lysing in response to one or more internal or external stimuli, such as achieving a certain concentration or cell density in a tumor microenvironment, thereby releasing the diagnostic agents and/or therapeutic agents and other cellular components into the surrounding environment (e.g., tumor microenvironment).
[0040] In some embodiments, the diagnostic agent is luciferase, salicylate, or a combination of both. In some embodiments, the therapeutic agent is an antibody that specifically binds to PD- Ll, an antibody that specifically binds to CTLA-4, or a cytokine such as GM-CSF. In some embodiments, the programmable bacterial cells comprise one or more nucleic acids encoding a plurality of therapeutic agents.
[0041] The term "heterologous nucleic acid sequence" refers to a nucleic acid derived from a different organism that encodes for a protein and which has been recombinantly introduced into a cell, In some embodiments, the heterologous nucleic acid sequence is introduced by transformation in order to produce a recombinant bacterial cell. Methods for creating recombinant bacterial cells are well known to those of skill in the art. Such methods include, but are not limited to, different chemical, electrochemical and biological approaches, for example, heat shock transformation, electroporation, liposome-mediated transfection, DEAE-Dextran-mediated transfection, or calcium phosphate transfection. Multiple copies of the heterologous nucleic acid sequence (e.g., between 2 and 10,000 copies) may be introduced into the cell.
[0042] In some embodiments, the heterologous nucleic acid sequences are in a plasmid. In some embodiments, the heterologous nucleic acid sequences are in a single operon and are integrated into the genome of the programmable bacterial cells. In some embodiments, the programmable bacterial cells comprise at least one inducible promoter or non-constitutive promoter that is in operable linkage with one or more of the heterologous nucleic acid sequences.
[0043] As used herein, the term "promoter" means at least a first nucleic acid sequence that regulates or mediates transcription of a second nucleic acid sequence. A promoter may comprise nucleic acid sequences near the start site of transcription that are required for proper function of the promoter. As an example, a TATA element for a promoter of polymerase II type. Promoters of the present invention can include distal enhancer or repressor elements that may lie in positions from about 1 to about 500 base pairs, from about 1 to about 1,000 base pairs, from 1 to about 5,000 base pairs, or from about 1 to about 10,000 base pairs or more from the initiation site.
[0044] The term "inducible promoter" refers to an operable linkage between a promoter and a nucleic acid sequence, whereby the promoter mediates the nucleic acid transcription in the presence or absence of at least one specific stimulus. In some embodiments, the inducible promoter mediates transcription of a nucleic acid sequence in the presence or absence of at least one, two, three, four, or five or more stimuli. In some embodiments, the one or more stimuli are produced in whole or in part by the programmable bacterial cells. In some embodiments, the only stimulus of the promoter is the presence of a certain concentration or density of programmable bacterial cell found in the subject of a patient (e.g., in a tumor).
[0045] An "operable linkage" refers to an operative connection between nucleic acid sequences, such as for example between a control sequence (e.g., a promoter) and another nucleic acid sequence that codes for a protein i.e., a coding sequence. If a promoter can regulate transcription of an exogenous nucleic acid sequence, then it is in operable linkage with the gene.
[0046] In accordance with the purposes of the inventions described herein, the programmable bacterial cells are preferably non-pathogenic and colonize tumors. One of ordinary skill in the art would know how to attenuate pathogenic bacteria to create non-pathogenic bacteria. In some embodiments, the bacteria are attenuated by removing, knocking out, or mutating a virulence gene such as altering genetic components of the bacterial secretion system.
[0047] In some embodiments, the programmable bacterial cells belong to at least one genus selected from the group consisting of Salmonella, Escherichia, Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacteria. In some embodiments, the bacterial cells belong to more than one genus selected from the group consisting of Salmonella, Escherichia, Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacteria.
[0048] In some embodiments, the programmable bacterial cells belong to the genus Escherichia. In particular embodiments, the programmable bacterial cells are Escherichia coli
Nissle 1917 (EcN) cells. In one embodiment, the EcN cells comprise a knockout of the clbA gene (EcNzlc/M).
[0049] Some aspects of this invention implicitly relate to culturing the programmable bacterial cells described herein. In some embodiments, a culture comprises the programmable bacterial cells and a medium, for example, a liquid medium, which may also comprise: a carbon source, for example, a carbohydrate source, or an organic acid or salt thereof; a buffer establishing conditions of salinity, osmolarity, and pH, that are amenable to survival and growth; additives such as amino acids, albumin, growth factors, enzyme inhibitors (for example protease inhibitors), fatty acids, lipids, hormones (e.g., dexamethasone and gibberellic acid), trace elements, inorganic compounds (e.g., reducing agents, such as manganese), redox-regulators (e.g., antioxidants), stabilizing agents (e.g., dimethyl sulfoxide), polyethylene glycol, polyvinylpyrrolidone (PVP), gelatin, antibiotics (e.g., Brefeldin A), salts (e.g., NaCl), chelating agents (e.g., EDTA, EGTA), and enzymes (e.g., cellulase, dispase, hyaluronidase, or DNase). In some embodiments, the culture may comprise an agent that induces or inhibits transcription of one or more genes in operable linkage with an inducible promoter, for example doxicycline, tetracycline, tamoxifen, IPTG, hormones, or metal ions. While the specific culture conditions depend upon the particular programmable bacterial cells, general methods and culture conditions for the generation of microbial cultures are well known to those of skill in the art.
[0050] Therapeutic Methods and Compositions
[0051] The inventions described herein also encompass methods of treating a colorectal tumor in a subject comprising administering a therapeutically effective amount of programmable bacterial cells described herein to the subject, wherein the programmable bacterial cells comprise a nucleic acid encoding a therapeutic agent described herein, which capable of treating the colorectal tumor. The present disclosure also relates to methods of reducing the rate of proliferation of a colorectal tumor cell comprising delivering a programmable bacterial cell described herein to the colorectal tumor cell. The present disclosure also relates to methods of killing a colorectal tumor cell comprising delivering a programmable bacterial cell described herein to the colorectal tumor cell.
[0052] As used interchangeably herein, “treatment” or “treating” or “treat” refers to all processes wherein there may be a slowing, interrupting, arresting, controlling, stopping, alleviating, or ameliorating symptoms or complications, or reversing of the progression of
colorectal cancer, but does not necessarily indicate a total elimination of all disease or all symptoms. Non-limiting examples of treatment include reducing the rate of growth of a colorectal tumor or colorectal cancer cell disease, reducing the size of a tumor, or preventing the metastases of a tumor.
[0053] Programmable bacterial cells described herein are preferably administered in one or more therapeutically effective doses. As used herein the terms "therapeutically effective dose" means the number of cells per dose administered to a subject in need thereof that is sufficient to treat the hyperproliferative disorder. In some embodiments, a therapeutically effective dose can be at least about l x104 cells, at least about IxlO5 cells, at least about I xlO6 cells, at least about I xlO7 cells, at least about IxlO8 cells, at least about IxlO9 cells, or at least about IxlO10 cells.
[0054] In some embodiments, programmable bacterial cells may be delivered to a subject in the form of a pharmaceutical composition, which may comprise one or more pharmaceutically acceptable carriers, diluents, or excipients. Pharmaceutical compositions may be formulated as desired using art recognized techniques. Various pharmaceutically acceptable carriers, which include vehicles, adjuvants, and diluents, are readily available from numerous commercial sources. Moreover, an assortment of pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents, and the like, are also available. Certain non-limiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. Pharmaceutical compositions may be frozen and thawed prior to administration or may be reconstituted in WFI with or without additional additives (e.g., albumin, dimethyl sulfoxide). Programmable bacterial cells described herein are preferably formulated for oral administration, but other routes of administration known in the art may be utilized.
[0055] Particular dosage regimens, i.e., dose, timing, and repetition, will depend on the particular subject being treated and that subject’s medical history. Empirical considerations such as pharmacokinetics will contribute to the determination of the dosage. Frequency of administration may be determined and adjusted over the course of therapy and is based on reducing the number of tumor cells or tumor mass, maintaining the reduction of such tumor cells or tumor mass, reducing the proliferation of tumor cells or an increase in tumor mass, or delaying the development of metastasis. A therapeutically effective dose may depend on the mass of the subject being treated, his or her physical condition, the extensiveness of the condition to be treated, and the age of the subject being treated.
[0056] Articles of Manufacture
[0057] The inventions disclosed herein also encompass articles of manufacture useful for treating a colorectal tumor comprising a container comprising programmable bacterial cells described herein, or a pharmaceutical composition comprising the same, as well as instructional materials for using the same to treat the colorectal tumor. In some embodiments, the articles of manufacture are part of a kit that comprises a bacterial culture vessel and/or bacterial cell growth media.
EXAMPLES
[0058] The following examples have been included to illustrate aspects of the inventions disclosed herein. In light of the present disclosure and the general level of skill in the art, those of skill appreciate that the following examples are intended to be exemplary only and that numerous changes, modifications, and alterations may be employed without departing from the scope of the disclosure.
[0059] Example 1
[0060] Strains and plasmids
[0061] All bacterial strains used were luminescent (integrated luxCDABE cassette) so they could be visualized with the In Vivo Imaging System (IVIS). The EcNdc/M strain was engineered using the lambda-red recombineering method. The salicylate-encoding plasmid was constructed using Gibson assembly methods or restriction enzyme-mediated cloning methods whereby pchA and pchB genes were cloned onto a high-copy origin plasmid and driven by the lac promoter. The SLIC and SLIC-3 strains were constructed as previously described.
[0062] Example 2
[0063] Bacterial preparation for oral administration
[0064] Overnight cultures of EcN-lux were diluted 1 : 100 into LB with 50 ng/mL erythromycin and cultured to an ODeoo of -0.5 on a shaker at 37°C. Bacteria were collected by centrifugation at 3000 r.c.f., washed three times with sterile PBS, resuspended in sterile ice-cold PBS with a total of 100-200 pL dosed at orally at a concentration of -1010 CFU/mL. SLIC strains were prepared
as previously described. Briefly, growth media for SLIC and SLIC-3 strains also contained 0.2% glucose to suppress premature lysis in culture. Additionally, SLIC-3 strains were grown with 50 mg/mL kanamycin.
[0065] Example 3
[0066] Organoid culture
[0067] Mouse CRC Braf600E;Tgfbr2^;Rnf43 ^/Znrf3 ^;pl6 Ink4a^ (Braf600E TRZI) organoids were generated using CRISPR/Cas9 genome engineering and expanded for injection into mice in matrigel culture as described. Culture medium was Advanced Dulbecco’s modified Eagle medium/F12 (Life Technologies) supplemented with lx gentamicin/antimycotic/antibiotic (Life Technologies), 10 mM HEPES, 2mM GlutaMAX, lxB27 (Life Technologies), lxN2 (Life Technologies), 50 ng/mL mouse recombinant EGF (Peprotech), 100 ng/mL mouse recombinant noggin (Peprotech), 10 ng/mL human recombinant TGF-|31 (Peprotech). Immediately after each split, organoids were cultured in 10 pM Y-27632 (In Vitro Technologies), 3 pM iPSC (Calbiochem Cat #420220), 3 pM GSK-3 inhibitor (XVI, Calbiochem, # 361559) for the first 3 days.
[0068] Example 4
[0069] Orthotopic mouse model of CRC
[0070] All animal experimentation related to the orthotopic CRC model was approved by the institutional animal ethics committee (SAM-319, SAM-20-031). Orthotopic injections to generate distal colon tumors were undertaken as previously described. In brief, NOD.Cg- Prkd cldH2rgtmlw^l/SzJ (NSG) mice (male and female, 6-12 weeks old) were obtained from the SAHMRI Bioresources facility and housed under SPF conditions. Digested Braf600EATRZI organoid clusters (equivalent to -150 organoids) were resuspended in 20 pL 10% GFR matrigel 1:1000 India ink, 10 pM Y-27632 in PBS and injected into the mucosa of the distal colon of anesthetized NSG mice using colonoscopy-guided orthotopic injection (2 injection sites/mouse). Injection sites were monitored by weekly colonoscopy. EcN administration began once the tumors were clearly established, at 3 weeks post organoid injection. Broad-spectrum antibiotic treatment to generate gut dysbiosis in specific groups involved administration of 0.5 g/L neomycin and 1 g/L ampicillin in ad libitum drinking water for 5 days. This was halted 6h prior to EcN admini tration.
[0071] Example 5
[0072] APCn""/+ mouse model of CRC
[0073] All animal experimentation related to the APCmm/+ mouse model of CRC was approved by the Institutional Animal Care and Use Committee (Columbia University, protocols AC- AAAN8002 and AC-AAAZ4470). All mice were regularly monitored and euthanized based on veterinarian recommendation or when they reached -20 weeks of age. In all therapeutic studies wild-type (WT) littermates of APCMm/+ on the C57BL/6 background were used and both males and females were treated and evenly distribute among groups. For diagnostic studies, APCM1I1/+ mice were purchased from Jackson Laboratories and C57BL/6 mice were used as WT.
[0074] Example 6
[0075] E. coli Nissle (EcN) 1917 biodistribution
[0076] IVIS imaging: Background (stage alone) subtracted total flux (photons/second) was used to capture the light signal emitted by EcN in identically sized areas for each live mouse in vivo. Following necropsy, individual tissues were collected into individual wells of a 6-well plate, weighed and average radiance (photons/s/cm2/sr) used for ex vivo tissue imaging to correct for the area being measured which differed for each tissue analyzed.
[0077] CFU: Excised tissues were placed aseptically into 5 mL 20% glycerol in PBS and homogenized in MACS Gentle cell dissociator C tubes, one tissue per tube using program C. lOOul of each tissue homogenate glycerol stock was serially diluted 1:100 six times. 10 pL of each dilution was spotted onto an LB agar plate with erythromycin selection at 50 pg/mL with 5 technical replicates. Plates were incubated at 37°C overnight (16 hours). Colony forming units (CFU) were calculated for each sample normalized to weight of tissue input to generate CFU/g tissue. To generate CFU/g stool, one pellet of stool was placed into an Eppendorf and manually homogenized in PBS with a pipette tip and rigorous pipetting. Serial dilutions were spotted onto an LB agar place with 50 pg/mL erythromycin and incubated at 37°C overnight. CFU was normalized to weight of the stool.
[0078] Example 7
[0079] Clinical trial design
[0080] This study was an interventional, double-blind, dual-center, prospective clinical trial (WHO Universal Trial Number U1111-1225-7729, ANZCTR number ACTRN12619000210178).
The study was approved by the Human Research Ethics Committee of the Central Adelaide Local Health Network (HREC/18/CALHN/751) to meet the requirements of the National Statement on Ethical Conduct in Human Research in accordance with the Declaration of Helsinki for medical research involving human subjects. The study objective was to evaluate the colonization of matched normal and neoplastic bowel tissue by the probiotic E.coli Nissle.
[0081] Adult participants undergoing routine colonoscopy or surgical resection for primary colorectal cancer were recruited from St. Andrew’s Hospital and Royal Adelaide Hospital, Adelaide (n=36). Written, informed consent was provided before participants were assigned to take either 2 tablets (109 CFU) per day of non-genetically modified EcN (MUTAFLOR®) or placebo for 14 days, prior to their procedure. Patients and treating physicians were blind to active or placebo status. Mucosal biopsies (colonoscopy) or surgical resection samples from normal and neoplastic tissue were collected from each participant at the time of their procedure. Participants were excluded if they took probiotics or antibiotics during the trial period.
[0082] Example 8
[0083] Human tissue sample analysis
[0084] Tissue samples were weighed and collected in sterile 20% glycerol in PBS. Tissue was homogenized in GENTLEMACS™ C Tubes (Miltenyi Biotec, 130-093-237), with a GENTLEMACS™ Dissociator (Miltenyi Biotec, 130-093-235), program E. Aliquoted, homogenized tissue was stored at -80°C until further use. For culture enrichment, the equivalent of 10 mg of human tissue in homogenate was added to 1.2 mL of LB broth/sample and incubated with shaking at 37 °C for 24 hours. Culture OD was monitored hourly for the first 14 hours to ensure exponential growth, samples from three patients were excluded due to inability to attain log phase cultures from tissue homogenates. 1 mL of saturated culture at 24 hours was centrifuged at 10,000xg to collect cells and DNA extracted from cell pellet using DNEASY® POWERSOIL® Pro kit (Qiagen, 47016).
[0085] Example 9
[0086] Development of E. coli Nissle 1917 strain specific PCR assay for human samples [0087] E. coli Nissle pMUT2 primers ECN7/8 and 9/10 were tested to detect E. coli Nissle in mouse fecal samples but they generated unacceptable false positives using gDNA isolated from human tissue samples from untreated patients. Alignment of PCR primer sets ECN7/8 or 9/10
against DNA sequences using Primer-BLAST suggested that Edwardsiella and Plesiomonas contain highly related sequences potentially also found in the human gut that may cause false positive calls via PCR assay using these primers. To avoid this confounding amplification during E coli Nissle 1917 detection, a different nested PCR strategy was devised to boost specificity and sensitivity for use with human samples using DNA sequence from pMUT2 unique to E. coli Nissle 1917 in comparison with human gut microbiota sequences. The external 283 bp amplicon spans the unique pMUT2 DNA region: ext-F 5’ TCGCGAACGTTAAATAATCATC (SEQ ID NO: 1); ext-R 5’ TCTGTTTTAGATAAGGCCATGTCTTC (SEQ ID NO: 2), and was amplified from 50 ng DNA input using KAPA Probe qPCR Master Mix (Roche, KK4716) with PCR conditions: denaturation 95°C for 20 seconds; 10 cycles of 95°C for 1 second, 60°C for 20 seconds, and 72°C for 25 seconds. Then 1 pL of this reaction was used as the template for the second 114 bp nested primer/probe-based assay. Nested primer and probe sequences were: int-F 5’ ACCCATCGATACCAAATGTATGT (SEQ ID NO: 3); int-R 5’
TCAATGCGTACTCGACTATTCAAA (SEQ ID NO: 4); probe 5’ 156-
FAM/CCCGCAGAT/ZEN/CACTGACCTCAATACA (SEQ ID NO: 5)/31ABkFQ/ using KAPA Probe qPCR Master Mix with PCR conditions as follows: 95°C for 20 seconds, 40 cycles of 9 °C for 1 second, 60°C for 20 seconds, and 72°C for 25 seconds. For 16S PCR, standard KAPA SYBR (non-nested) qPCR Master Mix (Roche, KK4602) with primers reported to amplify a 466 bp amplicon covering 331-797 of the E. coli 16S rRNA gene 16S-F 5’
TCCTACGGGAGGCAGCAGT (SEQ ID NO: 6) and 16S-R 5’
GGACTACCAGGGTATCTAATCCTGTT (SEQ ID NO: 7). E. coli Nissle 1917 PCR standards were generated from serially diluted DNA isolated from exponentially growing cultures from crushed MUTAFLOR® capsule in LB at 37°C, with CFU determined by plating of matched samples on LB agar plates.
[0088] Example 10
[0089] Mass spectrometry
[0090] For in vitro samples, overnight cultures of EcN-SA were ODeoo matched to be 1. Cultures were then centrifugated at 3000 r.c.f. and 1 mL of the supernatant was collected and stored at -80"C, the rest was decanted, and the pellet was stored at -80"C as well until analysis. To 1 mL of supernatants, 1000 pL of extraction solvent (MeOH/McCN/FEO containing 0.1 mg/mL of D4-salicylate) was added. Similarly, to cell pellets 800 u L of the same extraction solvent with
the internal D4- salicylate standard was added. All samples were then dried and resuspended in 200 pL of MeCN/thO before LC-MS analysis. To 30 pL of urine, 120 pL MeOH extraction solvent was added. Samples were then centrifugated at maximum speed and then 50 pL of the supernatant was taken before LC-MS analysis.
[0091] Example 11
[0092] Histology
[0093] All intestinal tissue was excised with the caecum removed and tissue was bisected such that there were 5 total sections: duodenum, proximal jejunum, distal jejunum, ileum, and colon. Intestines were flushed with PBS, splayed open, Swiss-rolled, and fixed overnight in 4% paraformaldehyde. After 24 hours the Swiss rolls were switched to 70% ethanol and sent for histology services at Histowiz, where they were paraffin-embedded, sectioned, and stained with either H&E or specific immunohistochemistry markers (HA-Tag C29F4 #3724 from Cell Signaling Technology; Granzyme B, CD3 Abeam 16669; CD8 catalog #CST98941 clone D4W2Z). Tumor sizes and IHC quantification were determined using FIJI software image analysis tools.
[0094] Example 12
[0095] EcN-lux
[0096] To address the challenges pertaining to CRC screening and therapy described hereinabove, an orally deliverable probiotic bacterium was engineered for both non-invasive diagnosis and tumor burden reduction of early-stage CRC. CRC precursor lesions, adenomas, were modeled using APCmin/+ mice, which develop spontaneous intestinal polyps and are representative of initiating genetic mutations seen in human familial adenomatous polyposis (Fig. 1 A). Neoplasia colonization was established by orally-delivering E.coli Nissle 1917 (EcN) encoding a genomically-integrated luxCDABE cassette (EcN-lux). In vivo imaging of mice dosed with EcN- lux showed elimination of bioluminescent bacteria in healthy mice and retention in the APCIlun/+ mouse gut for weeks after oral administration (Fig. IB). Enrichment of EcN-lux in neoplasia was further demonstrated with ex vivo imaging of intestinal tissue, where bioluminescence co-localized with visible macroadenomas and generally, more bioluminescence was observed in the distal gut where polyp burden was the greatest (Fig. 1C, Fig. 2).
[0097] Example 13
[0098] EcN c/M
[0099] Due to concerns of colibactin-producing bacteria like EcN being pro -carcinogenic, a clbA knockout strain (EcN c/M) was generated in order to disrupt colibactin production and subsequently delivered to tumor-bearing APCm,n/+ (Fig. 3A). Similar to the EcN-lux strain, bioluminescent EcN c/M co-localized with visible macroadenomas as observed by ex vivo intestinal imaging and EcNzlc/M was detectable in APCmm feces for multiple days after oral dosing, suggesting that colonization does not rely on the presence of clbA gene or an intact colibactin-encoding operon (Fig. 3B-C). Subsequent plating of homogenized intestinal tissue on antibiotic-selective Luria broth (LB) plates specific for EcN-lux, indicated that no detectable EcN- lux could be recovered from wild-type mouse tissue, suggesting that a long-term niche is not formed in the gut unless neoplastic tissue is present (Fig. ID). This result was unexpected because colibactin has been thought to play a critical role in EcN colonization.
[00100] Example 14
[00101] Bacterial Localization
[00102] To further investigate bacteria localization, EcN-lux was engineered to produce a human influenza hemagglutinin (HA)-tagged reporter protein and orally delivered to APC"""/+ mice. Immunohistochemical staining against HA in Swiss-rolled intestinal tissue showed EcN-lux localization across early and late adenoma stages (Fig. IE) and sizes ranging from -0.16 mm2 to 3 mm2 (Fig. 4).
[00103] Example 14
[00104] Orthotopic Model of CRC
[00105] Murine CRC organoids were injected into the distal murine colon and tumor grade tracked via weekly colonoscopy (Fig. 5, Fig. IF). To enhance colonization, mice were pre-treated with broad-spectrum antibiotics, which induces gut dysbiosis, a common phenomenon in gastrointestinal diseases including CRC. EcN-lux was orally delivered, and in vivo imaging five days post dosing revealed colocalization of bioluminescent EcN-lux with colon tumors (Fig. 1G, 1H, Fig. 6). Subsequent homogenization and plating of excised organs on antibiotic- selective LB plates confirmed EcN-lux was significantly enriched in tumors compared to adjacent healthy tissue and peripheral organs (Fig. II). The median diameter of EcN-lux colonized tumors was 2 mm (+/-
1.2 mm), suggesting the size of neoplastic lesions detected using this EcN-lux platform was similar to colonoscopic reporting of diminutive (0 to 5 mm) polyps in humans. Additional histological interrogation of these tumors also indicated the presence of tumor mucin lakes, demonstrating this model phenocopies mucinous human adenocarcinomas (Fig. 1J), and does not inhibit EcN-lux colonization.
[00106] Example 15
[00107] Clinical Trial
[00108] To explore the applicability of the obtained preclinical data to humans, a clinical trial was carried out in a small number of CRC patients see Table 1).
[00110] A commercially available, non-genetically modified form of EcN, MUTAFLOR®, or placebo, was orally administered to CRC patients for two weeks, prior to tissue resection as part of standard care treatment (WHO Universal Trial Number Ull l l-1225-7729, ANZCTR number ACTRN12619000210178). Homogenates from matched normal and tumor tissue (n=8 patients) were cultured to enrich for microbial content, DNA was then isolated and subjected to qPCR assays. EcN-specific PCR amplicons indicated significant enrichment of this bacteria in cultures from tumor tissue in patients administered MUTAFLOR®, but not placebo controls (Fig. 1K-L, Fig. 7). Subsequent histological analysis of healthy and diseased tissue from patients in this cohort highlighted areas of necrosis in tumors (Fig. IM), consistent with microenvironments that enable bacterial growth.
[00111] Example 15
[00112] Non-invasive Methods of Screening for CRC
[00113] The selective colonization potential of EcN suggested its utility as a platform for both CRC detection and in situ disease manipulation (Fig. 8A). Since stool-based tests are the most common non-invasive screening tool available for CRC, the sensitivity of EcN colonization as a screening modality was initially compared to FOBT. Using the orthotopic CRC model with advanced grade 3/4 tumors, mice were dosed with EcN-lux and detected 92% as having positive luminescence signal, 83% as having recoverable CFU after tissue homogenization and plating, and 37.5% as positive with a FOBT (Fig. 8B). These data suggest that EcN colonization is a more sensitive metric than blood found in fecal matter in our mouse model.
[00114] Shifting focus to early-stage intervention, APCmin/+ mice were orally dosed with EcN- lux and fecal matter from the mice was subsequently homogenized and plated on antibiotic- selective LB agar plates. During the first seven hours post-dosing, both healthy wild-type (WT) and APCmm/+ mice had comparable shedding of EcN-lux CFU in their stool, corresponding to material transit time through the gut. However, by twenty-four hours, levels of EcN-lux were undetectable in some mice and by forty-eight hours EcN-lux was unable to be recovered from the stool of WT mice (Fig. 8C).
[00115] While the stool test is a one example of a non-invasive screen, a more accessible urine screen was developed in an effort to improve upon already available tests and increase patient compliance. To this end, EcN-lux were engineered to produce salicylate (EcN-SA), a safe metabolite that is easily detected in urine using liquid chromatography-mass spectrometry (LC- MS) (Fig. 9). After establishing baselines, WT and APCI™11/+ mice were orally dosed with the EcN- SA strain. Urine was collected twenty-four hours later and probed for the presence of salicylate using LC-MS. While urine from both the WT and APCmm/+ mice had detectable levels of salicylate, the tumor-bearing mice had ~20 times more relative salicylate in the urine (Fig. 8D). Furthermore, the ROC analysis of the CRC screen yielded an area under the curve (AUC) of 1 at the twenty- four-hour time point, suggesting greater sensitivity and specificity of the urine readout when compared to the stool screening assay at the same time point (Fig. 8E).
[00116] This result was unexpected because there was no suggestion that detectable levels of salicylate would passively diffuse into the vasculature from adenomas, much less at the levels observed.
[00117] Example 16
[00118] Non-Invasive Methods of Treating CRC
[00119] With the ability to have a urinary readout of colorectal neoplasia, the screening methods described in the previous example were further modified in order to reduce polyp burden in APCmm/+ mice. An EcN-lux strain was initially genomically-encoded with a systemic lysis circuit (SLIC) optimized to maximize therapeutic delivery. SLIC was specifically used to deliver nanobodies blocking PD-L1 and CTLA-4 targets and GM-CSF (SL1C-3), which has been shown to work in combination to enhance efficacy of checkpoint blockade therapy. The APCmin/+ mice were either dosed twice with PBS or SLIC-3 and then sacrificed ~1 month later. Histological analysis of hematoxylin and eosin-stained tumors demonstrated an overall reduction of tumor area (Fig. 2F) and number (Fig. 10A) by -47% with SEIC-3 treatment. Notably, an increased percentage of smaller tumors was observed in SEIC-3 treated mice, whereas PBS-treated mice tended to have larger tumors (Fig. 10B). Moreover, this reduction was not specific to a location and was observed throughout the small intestine (Fig. 8G, Fig. 8H). Interrogation of immunophenotype on tissue sections suggested that reduction in tumor burden is associated with increased tumor infiltration of CD3+, CD8+ cells and increased production of intratumoral granzyme B (GrB), suggesting immune-mediated tumor cell killing in SEIC-3 treated mice (Fig. 8I-M, Fig. 11).
[00120] While this invention has been disclosed with reference to particular embodiments, it is apparent that other embodiments and variations of the inventions disclosed herein can be devised by others skilled in the art without departing from the true spirit and scope thereof. The appended claims include all such embodiments and equivalent variations.
Claims
1. A method of detecting the presence of a colorectal tumor in a subject comprising: administering a programmable bacterial cell to the subject, wherein the programmable bacterial cell comprises a nucleic acid encoding a diagnostic agent that is detectable in a biological sample obtained from the subject; and detecting the presence of a colorectal tumor in the subject.
2. The method of claim 1, wherein the programmable bacterial cell belongs to a genus selected from the group consisting of Salmonella, Escherichia, Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacteria.
3. The method of claim 2, wherein the programmable bacterial cell belongs to the genus Escherichia.
4. The method of claim 3, wherein the programmable bacterial cell is an Escherichia coli Nissle 1917 (EcN) cell.
5. The method of claim 4, wherein the programmable bacterial cell comprises a knockout of the clbA gene (EcN clbA).
6. The method of claim 1, wherein the diagnostic agent is selected from the group consisting of luciferase, salicylate, or a combination of both.
7. The method of claim 1, wherein the nucleic acid is a luxCDABE operon.
8. The method of claim 1, wherein the programmable bacterial cell further comprises a synchronized lysis circuit comprising a nucleic acid encoding a quorum-sensing gene, a nucleic acid encoding a lysis gene, a promoter, and a terminator contained on a single operon.
9. The method of claim 8, wherein the programmable bacteria cell further comprises one or more nucleic acids encoding a therapeutic agent selected from the group consisting of:
(a) an antibody that specifically binds to PD-L1;
(b) an antibody that specifically binds to CTLA-4;
(c) a cytokine;
(d) an antibody that specifically binds to PD-L1 and an antibody that specifically binds to CTLA-4;
(e) an antibody that specifically binds to PD-L1 and a cytokine;
(f) an antibody that specifically binds to CTLA-4 and a cytokine; and
(g) an antibody that specifically binds to PD-L1, an antibody that specifically binds to
CTLA-4, and a cytokine (e.g., GM-CSF).
10. The method of claim 1, wherein the biological sample is selected from the group consisting of urine and feces.
11. A method of treating a colorectal tumor in a subject comprising administering a therapeutically effective amount of programmable bacterial cells to the subject, wherein the programmable bacterial cells comprise a nucleic acid encoding a therapeutic agent capable of treating the colorectal tumor.
12. The method of claim 11, wherein the programmable bacterial cells belong to at least one genus selected from the group consisting of Salmonella, Escherichia, Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacteria.
13. The method of claim 12, wherein the programmable bacterial cells belong to the genus Escherichia.
14. The method of claim 13, wherein the programmable bacterial cells are Escherichia coli Nissle 1917 (EcN) cells.
15. The method of claim 14, wherein the programmable bacterial cells comprise a knockout of the clbA gene (EcNzl clbA).
16. The method of claim 11, wherein the programmable bacterial cells further comprise a synchronized lysis circuit comprising a nucleic acid encoding a quorum- sensing gene, a nucleic acid encoding a lysis gene, a promoter, and a terminator contained on a single operon.
17. The method of claim 11, wherein the therapeutic agent is selected from the group consisting of:
(a) an antibody that specifically binds to PD-L1 ;
(b) an antibody that specifically binds to CTLA-4;
(c) a cytokine;
(d) an antibody that specifically binds to PD-L1 and an antibody that specifically binds to CTLA-4;
(e) an antibody that specifically binds to PD-L1 and a cytokine;
(f) an antibody that specifically binds to CTLA-4 and a cytokine; and
(g) an antibody that specifically binds to PD-L1, an antibody that specifically binds to
CTLA-4, and a cytokine (e.g., GM-CSF).
18. The method of claim 11, wherein the biological sample is selected from the group consisting of urine and feces.
19. The method of claim 11, wherein the programmable bacterial cells further comprise at least one nucleic acid encoding a diagnostic agent selected from the group consisting of luciferase, salicylate, or a combination of both.
20. A method of monitoring the treatment of a colorectal tumor in a subject comprising: administering a programmable bacterial cell to the subject, wherein the programmable bacterial cell comprises a nucleic acid encoding a diagnostic agent that is detectable in a biological sample obtained from the subject; obtaining a first biological sample from the subject at a first time point; measuring the level of the diagnostic agent in the first biological sample;
obtaining a second biological sample from the subject at a second time point; and measuring the level of the diagnostic agent in the second biological sample.
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US20080193373A1 (en) * | 2006-07-11 | 2008-08-14 | Jochen Harald Stritzker | Methods and compositions for detection of microorganisms and cells and treatment of diseases and disorders |
US20160033485A1 (en) * | 2013-03-15 | 2016-02-04 | Massachusetts Institute Of Technology | Compositions and methods for cancer diagnosis |
US20190160115A1 (en) * | 2016-01-11 | 2019-05-30 | Synlogic, Inc. | Microorganisms programmed to produce immune modulators and anti-cancer therapeutics in tumor cells |
US20210308195A1 (en) * | 2018-07-20 | 2021-10-07 | The Trustees Of Columbia University In The City Of New York | Programmable bacteria for the treatment of cancer |
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US20080193373A1 (en) * | 2006-07-11 | 2008-08-14 | Jochen Harald Stritzker | Methods and compositions for detection of microorganisms and cells and treatment of diseases and disorders |
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US20190160115A1 (en) * | 2016-01-11 | 2019-05-30 | Synlogic, Inc. | Microorganisms programmed to produce immune modulators and anti-cancer therapeutics in tumor cells |
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