Improved asparaginase administration, production and use thereof

Abstract

A pharmaceutical preparation including asparaginase characterised in that the asparaginase is encapsulated or contained within bacterial extracellular vesicles (BEVs).

Description

[0001]Improved Asparaginase Administration, Production and Use thereof The present invention relates to bacterial extracellular vesicles (BEVs) including asparaginase and the methods of production and the uses of said BEVs including enzymes. Although the following description refers to BEVs from the genus Bacteroides the skilled person will appreciate that other genus of bacteria can be used, in particular other commensal bacteria. For over 50 years the anti-tumour activity of bacteria-derived L- asparaginase enzyme has been utilised as an essential front-line treatment for acute lymphocytic leukaemia (ALL) and more recently for acute myeloid leukaemia (AML). However, frequent and high dose treatment regimens (6000 IU / m2three times a week) are required to overcome the short half-life of soluble non- encapsulated forms of L-asparaginase. This can cause early relapse and serious adverse effects such as hypersensitivity,pancreatitis, l iver toxicity and thrombosis.AML is the most common form of acute leukaemia, with ~3100 new diagnosis in the UK each year, with 42% aged over 75, and only 13.6% surviving after 5 years. ALL is less frequent, with ~790 people diagnosed in the UK each year, however ~60% of cases are diagnosed in children aged 0-4 years, with around 400 children developing ALL in the UK each year. It is an aim of the present invention to provide an asparaginase containing composition that addresses the abovementioned problems. It is a further aim of the present invention to provide an improved method of delivery of asparaginase that addresses the abovementioned problems. It is a yet further aim of the present invention to provide a method of producing asparaginase containing compositions that addresses the abovementioned problems. In a first aspect of the invention there is provided bacterial extracellular vesicles (BEVs) characterised in that said BEVs encapsulate L-asparaginase. Typically the BEVs are generated by human commensal gut bacteria. Further typically the BEVs naturally encapsulate L- asparaginase. In one embodiment the asparaginase containing BEVs are derivedfrom Bacteroides.In a preferred embodiment the BEV-encapsulated asparaginase formulations are substantially free from endotoxins. In a second aspect of the invention there is provided a L- asparaginase encapsulated in BEVs for the treatment of cancer. Typically the cancer is leukaemia. Further typically the leukaemia is acute lymphocytic leukaemia (ALL) and / or acute myeloid leukaemia (AML). In a third aspect of the invention there is provided a pharmaceutical preparation including encapsulated L- asparaginase characterised in that the L-asparaginase is encapsulated or contained within BEVs. In one embodiment the pharmaceutical preparation is in an oral dosage form. Typically the preparation includes at least one pharmaceutically acceptable carrier. In one embodiment the preparation is in a form suitable for intravenous administration. Typically the preparation is in liquid form. In a further aspect of the invention there is provided a method of producing encapsulated L-asparaginase, said method including the step of introducing the asparaginase gene to a bacterial genome such that the expressed asparaginase is contained or encapsulated within BEVs. In a yet further aspect of the invention there is provided a method of treating cancer, said method including the administration of BEV encapsulated asparaginase. Specific embodiment of the invention are now described with reference to the following figures wherein; Figure 1 shows the biogenesis and composition of BEVs. LPS: lipopolysaccharide, OM: outer membrane, PG: peptidoglycan, IM: Inner membrane; Figure 2 shows a confocal microscopy image of THP-1 showing the uptake and internalisation of fluorescently labelled gut commensal BEVs. BEVs (DiO; green), cell membrane (phalloidin; red), nucleus (Hoechst; blue). Scale bar 20µm; Figure 3 shows the mechanism of action of asparaginase; Figure 4 shows a schematic of BEV isolation and characterisation; Figure 5 shows a graph demonstrating cytotoxicity of two gut commensal BEV-encapsulated asparaginase formulations on THP-1 and HL-60. Asparaginase (ASNase), wild-type (WT), and overexpressing strain (O / E); and Figure 6 shows a graph of asparaginase activity of BEV formulations. Asparaginase activity was quantified in 2.5x1010 BEVs isolated from Bt (Bacteroides thetaiotaomicron) and Bs (Bacteroides stercoris) wild-type (WT), overexpressing (O / E) and knockout (- / -) strains or those produced in the presence of 0.4% Asparagine (Asp) using the Asparaginase assay kit (Abcam). We show here the promising cytotoxic effects of gut commensal BEV-encapsulated asparaginase formulations on the prototypicalleukaemia cell l ines HL-60 and THP-1 highlighting their potentialfor clinical applications. This novel BEV-based therapeutic offers the possibility to provide a safer and more effective treatment for AML and ALL by reducing the frequency and severity of side effects associated with current therapies. Acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) are aggressive blood cancers with limited treatment options that often lead to severe side effects. Traditional therapies, including E.coli-derived L-asparaginase, are essential but can cause hypersensitivity and other adverse reactions due to their instability and high dosing regimens. Our invention provides a novel approach using bacterial extracellular vesicles (BEVs) generated by human commensal gut bacteria, which naturally encapsulate L-asparaginase. Our data demonstrate the efficacy of BEV-encapsulated asparaginase produced by different gut bacteria and our current research focuses on benchmarking against clinical formulations of E. coli L-asparaginase . Results to date demonstrate promising cytotoxiceffects of Bacteroides derived BEVs on the prototypical leukaemiacell l ines HL-60 and THP-1 with potential for clinical application.This BEV based therapeutic has the potential to provide a safer and more effective treatment for AML and ALL by reducing the frequency and severity of side effects associated with current therapies. Our current research focuses on evaluating the efficacy of BEV- encapsulated asparaginase formulations produced by commensal gut bacteria and benchmarking against clinical formulations of L- asparaginase. Using BEVs as a therapeutic has the potential for delivering a more safe and effective treatment for AML and ALL. Through intimate host-microbe interactions, BEVs can be internalised and transfer bioactive molecules to cells including theAMoL cell l ine, THP-1 (Figure 2). They are therefore excellentnanocarriers for therapeutic drug delivery. Lymphoblastic leukaemia cells typically lack the enzyme asparagine synthetase (ASNS) and are susceptible to asparagine depletion. Clinical asparaginase treatments deplete serum asparagine, eventually leading to leukemic cell death. The present invention the nano vesicles carrying the asparaginase could be internalised by the cancer cell and deplete internal, (cytosolic)asparagine, beside the serum pool. This represents anotherimprovement over the current therapies.L-asparaginase's primary activity - hydrolyses L-asparagine intoL-aspartic acid and ammonia. Secondary activity - hydrolyses L- glutamine into L-glutamic acid and ammonia (glutaminaseactivity).Turning to Figure 5 which shows the cytotoxicity of BEV- encapsulated asparaginase formulations on THP-1 and HL-60leukaemia cell l ines. 2.5x1010 BEVs isolated from Bt (Bacteroides thetaiotaomicron) and Bs (Bacteroides stercoris) wild-type (WT), overexpressing (O / E) and knockout (- / -) strains or those produced in the presence of 0.4% Asparagine (Asp) were incubated with 2x104 THP-1 or HL-60 cells for 72 hours at 37°Cwith 5% CO2. Cytotoxicity (%) was quantified using the CellTiterGlo 2.0 assay (Promega) and BEV formulations compared to PBS control and L-asparaginase (ASNase) standard. Figure 6 shows asparaginase activity of BEV formulations. Asparaginase activity was quantified in 2.5x1010 BEVs isolated from Bt (Bacteroides thetaiotaomicron) and Bs (Bacteroides stercoris) wild-type (WT), overexpressing (O / E) and knockout (- / -) strains or those produced in the presence of 0.4% Asparagine (Asp) using the Asparaginase assay kit (Abcam). ASNase gene deletion and overexpression in Bt The Bt ∆ASNase deletion mutant is described in Stentz et al. 2022 (https: / / doi.org / 10.1128 / aem.00533-22). For ASNase overexpression, the construct was made based on thework we published in 2016 (Horn et al . , 2016,https: / / doi.org / 10.3389 / fmicb.2016.01080). Bs ATCC 43183 asparaginase type II gene (B0NLF6_BACSE) was cloned downstream from the mannan promoter in the pGH117 vector [described in WO2017187190 “Engineering gut commensal bacteria to express heterologous proteins…”) and transferred into the Bt∆ASNase deletion mutant.BEV preparationsAll Bacteroides strains were grown in 500 ml of BDM+ (Stentz etal. , 2022 https: / / link.springer.com / protocol / 10.1007 / 978-1-0716-1900-1_11 ) for 16 hours. Bacteroides defined medium Plus (BDM+): To prepare 500 ml, dissolve 2.61 g of KH2PO4and 7.03 g of K2HPO4* 3 H2O into 481 mL of deionized water, add the following solutions to a final concentration of 15 mM NaCl and 8.5 mM (NH4)2SO4, adjust the pH to 7.4 using 5 M NaOH, autoclave and place in the anaerobic cabinet to equilibrate for a minimum of 24 h. Add the rest of the solutions to a final concentration of 30 mM of glucose, 0.2 mM L-histidine, 100 nM vitamin B12, 6 µM vitamin K3 (menadione),0.1 mM MgCl2, 50 µM CaCl2 , 4.1 mM L-cysteine and 1.4 µMFeSO4* 7 H2O. Leave the medium for a minimum of 24 h in the anaerobic cabinet to fully deoxygenate. Add 2 µM of Protoporphyrin IX freshly made before using the media. After 2 hours of growth, 25 mg / ml of mannan inducer was added to the medium for Bt (∆ASNase) overexpressing the Bs asparaginase enzyme.). Bs (ATCC 43183) was grown either with or without asparagine (0.4 %). Asparagine naturally induces the expression of asparaginase. The bacterial cultures were centrifuged at 5500 g, 4°C for 45 min and the supernatant filtered through polyethersulfone (PES) membranes (0.22 µm pore-size) (Sartorius) to remove debris andremaining cells. Vesicle suspensions were concentrated bycrossflow ultrafiltration (100 kDa MWCO, Vivaflow 200, Sartorius) to 5 mL, rinsed by addition of 500 mL of PBS, pH 7.4,concentrated again by crossflow fi ltration to 5 mL and theretentates were concentrated to 1 mL with a Vivaspin 20 centrifugal concentrator (100 kDa molecular weight cut-off,Sartorius). Further purification of BEVs was performed by size-exclusion chromatography (SEC) using a qEV / 35 nm series SEC column according to the manufacturer’s instructions (IZON Science, Lyon, France) with the pooled collected fractions filtered through PES membranes (0.22 µm pore-size) and stored at 4°C. Nanoparticle analysis The size and concentration of the isolated BEVs was determined using a ZetaView PMX-220 TWIN instrument according to manufacturer’s instructions (Particle Metrix GmbH, Germany). Aliquots of BEV suspensions were diluted 1,000- to 20,000-fold in particle-free PBS or water for analysis. Size distribution video data were acquired using the following settings: temperature: 25°C; frames: 60; duration: 2 s; cycles: 2; positions: 11; camera sensitivity: 80 and shutter value: 100. Data were analyzed using the ZetaView NTA software (version 8.05.12) with the following post acquisition settings: minimum brightness: 20; max area: 2000; min area: 5 and tracelength: 30. Cytotoxicity assay 2.5x1010BEVs isolated from Bt (Bacteroides thetaiotaomicron) and Bs (Bacteroides stercoris) were incubated with 2x104THP-1 or HL-60 cells in a total volume of 500µL complete medium in a 24-wellplate for 72 hours at 37°C with 5% CO2. An L-asparaginase(ASNase) standard was diluted in PBS and cells treated as abovewith 0.1 or 0.0001 IU / well. Cell viability was quantified using theCellTiter Glo 2.0 assay (Promega) according to manufacturer’s instructions. Briefly, 100µL of CellTiter-Glo 2.0 reagent was added to 100µL of the BEV treated cells in a white 96 well plate. The plate was mixed for 2 minutes on an orbital shaker to induce cell lysis and incubated at room temperature for 10 minutes. Luminescence was recorded using a ClarioStar microplate reader (BMG Labtech). Cytotoxicity (%) was quantified by comparison of cell viability between BEV-formulations and PBS control Asparaginase activity assay Asparaginase activity was quantified in BEVs isolated from Bt (Bacteroides thetaiotaomicron) and Bs (Bacteroides stercoris) using the Asparaginase Activity Assay Kit (Abcam) according to manufacturer’s instructions. Briefly, 50µL of 2.5x1010BEVs or aspartate standards were incubated with 50µL asparaginase reaction mix in a 96 well plate and mixed on an orbital shaker for 10 minutes. OD570 nm was recorded every 2 minutes for 120 minutes total at 25°C protected from light using a ClarioStar microplate reader (BMG Labtech). Asparaginase activity was calculated as mU / mL using the aspartate standard curve. 1 Unit Asparaginase is equivalent to the amount of asparaginase which generates 1.0 µmol of aspartate per min at 25°C. In yellow is the degradation of glutamine by the E. coli recombinant ASNase, while for the BEVs in orange, glutamine is not decreased (possibly increased following cell death and glutamine release in the medium). Most asparaginases used to treat leukemia break down glutamine due to their secondary glutaminase activity. This unintended glutaminase activity can cause significant side effects, including liver toxicity, problems with blood clotting, pancreatitis, immunosuppression, neurological issues, and severe depletion of glutamine, an amino acid essential for healthy cells. As shown in the results, asparagine was fully consumed (down to 0) in the 4-day BEV / asparaginase-treated samples, indicating complete enzymatic activity over this period. Note: Concentrations are in mM. VPI + glu: BEVs from commercial B. stercoris grown in presence of glutamate. FMH05 (BEVs): QIB strain (aand b are 2 separate preparations). 2.5 1010 BEV added to 0.5 ml cel lculture. These are the original concentrations in the RPMI 1640 medium used to maintain and grow THP-1 cells.

Claims

16 1. A preparation including asparaginase characterised in that the asparaginase is encapsulated or contained within bacterial extracellular vesicles (BEVs).

2. A preparation according to claim 1 wherein the BEVs are generated by human commensal gut bacteria.

3. A preparation according to claim 1 or 2 wherein the preparation is a pharmaceutical preparation including a pharmaceutically acceptable carrier.

4. A pharmaceutical preparation according to claim 3 wherein the BEVs naturally encapsulate L-asparaginase.

5. A pharmaceutical preparation according to claim 4 wherein theasparaginase containing BEVs are derived from Bacteroides .

6. A preparation according to any preceding claim wherein the BEV-encapsulated asparaginase preparations are substantially free from endotoxins.

7. Asparaginase encapsulated or contained in BEVs for the treatment of cancer.

8. Asparaginase encapsulated in BEVs according to claim 7 wherein the asparaginase is L-asparaginase.

9. Asparaginase encapsulated in BEVs according to claim 7 or 8 wherein the cancer is leukaemia.

10. Asparaginase encapsulated in BEVs according to claim 9 wherein the leukaemia is acute lymphocytic leukaemia (ALL) or acute myeloid leukaemia (AML).17 11. A pharmaceutical preparation including encapsulated L- asparaginase according to claims 7-10 wherein preparation is in an oral dosage form or an injectable dosage form.

12. A pharmaceutical preparation according to claim 11 wherein preparation includes at least one pharmaceutically acceptable carrier for intramuscular or intravenous delivery.

13. A method of producing encapsulated asparaginase, said method including the step of introducing the asparaginase gene to a bacterial genome such that the expressed asparaginase is contained or encapsulated within BEVs.

14. A method of treating leukaemia using asparaginase encapsulated or contained within BEVs.

15. A method of delivering a therapeutic amount of asparaginase using BEVs to encapsulate the asparaginase.

Images