Patent Document

CROSS-REFERENCE TO RELATED APPLICATION 
     This application is a continuation of PCT International Patent Application No. PCT/EP2003/050424, filed on Jun. 19, 2003, designating the United States of America, and published, in English, as PCT International Publication No. WO 2004/01020 A2 on Dec. 31, 2003, the contents of the entirety of which is incorporated by this reference. 
    
    
     TECHNICAL FIELD 
     The present invention relates generally to biotechnology, and, more particularly, to epidermal growth factor (EGF) producing lactic acid bacteria and their use to increase intestinal villi height and to promote gut absorption. In particular, the invention relates to EGF producing  Lactococcus lactis  and  Lactobacillus casei . These organisms may be especially useful to treat Short Bowel Syndrome. 
     BACKGROUND 
     The efficiency of gut absorption is essential for good food conversion. Gut adsorption is largely determined by the gut surface, which is a function, amongst others, of the length of the gut and the height of the villi. In cases where an operative removal of a part of the gut is necessary, as in the case of cancer or Crohn&#39;s disease, this may result in decreased gut adsorption, resulting in insufficient food conversion and a shortage of nutrients, dehydration and even potentially lethal metabolic changes. These syndromes caused by the extensive resection of the small intestine are known as “Short Bowel Syndrome.” 
     Several methods have been proposed to improve the post-operational adaptation of, and to enhance, the gut absorption in patients with Short Bowel Syndrome. U.S. Pat. No. 5,288,703 discloses that both growth hormone and insulin-like growth factor have a positive effect on gut absorption in mammals. This positive effect can be enhanced by the administration of glutamine or a glutamine equivalent. Administration of glutamine and growth hormone results in an increase of the villi length (Gu et al., 2001; Zhou et al., 2001). U.S. Pat. No. 5,972,887 demonstrated a reversal of the reduced intestinal mucosal mass and absorptive function in patients by the administration of low doses of exogenous Hepatocyte Growth Factor. In addition, the glucagon-like peptides GLP-1 and GLP-2 have been used with positive results. Studies on laboratory animals (Scott et al., 1998), as well as on humans (Jeppesen et al., 2001), showed a positive correlation between an increase in concentration of GLP-2 and an improvement of the intestinal adaptation. Short Bowel patients, from whom the ileum has been removed, show a decrease in food-induced secretion of GLP-2 (Jeppesen et al., 1999). Those patients, especially, can be treated successfully with GLP-2. It has been shown that leptin also has a positive effect on intestinal adaptation in a rat model (Pearson et al., 2001). 
     A lot of interest has been paid to the effect of Epidermal Growth Factor (EGF, urogastron). EGF is a relatively acid stable hormone that is produced in the salivary and the Brunner&#39;s glands. It is found in a wide variety of external secretions, as well as in blood and amniotic fluid (Marti et al., 1989). The molecular weight of mature human EGF is 6.2 kDa (Carpenter et al., 1991). EGF is phylogenetically strongly conserved and is strongly cross-reactive between different species. 
     It is known that EGF increases the absorption of H 2 O, Na + , Cl −  and glucose in a rabbit model (Opleta-Madsen et al., 1991). Moreover, EGF is stimulating the elongation of the villi. This results in an increase of the apical surface and a general increase in absorption of nutrients (Hardin et al., 1999). Absorption of carbohydrates is further facilitated by the EGF-stimulated secretion of pancreatic amylase (Piiper et al., 1994). 
     Several studies have shown a positive effect of the application of EGF in experimental animal models for Short Bowel Syndrome (Helmrath et al., 1988; Chaet et al., 1994; O&#39;Loughlin et al, 1994; Swaniker et al., 1996; Lukish et al., 1997; Dunn et al., 1997). 
     EGF-mediated effects after intestinal resection are strongly dose dependent; up to a certain limit, the adaptation increases with increasing doses. In intestinal studies, the normal dose is situated between 30 and 300 μg/kg body weight/day. Systemic, as well as enteral, applications seem effective. However, systemic application may be unwanted for possible side effects; several neoplasmas do have EGF receptors and a general increase in EGF concentration in the blood might stimulate the formation of tumors. Enteral application of EGF, however, is less efficient as pepsin can process mature EGF into a truncated form that has only 25% of the initial biological activity (Playford et al., 1995). 
     DISCLOSURE OF THE INVENTION 
     Surprisingly, demonstrated is that EGF can be delivered in situ by recombinant lactic acid bacteria producing EGF. Efficient production and secretion of EGF by lactic acid bacteria is not evident, and needs optimization of the coding sequence. Moreover, it cannot be predicted that the lactic acid bacteria sufficiently survive the passage through the stomach to produce the appropriate amount of EGF to stimulate growth of the villi, to promote nutrient absorption and to treat the Short Bowel Syndrome. 
     It is a first aspect of the invention to provide an EGF producing lactic acid bacterium. Preferably, the lactic acid bacterium is secreting the EGF produced in the growth environment. Preferably, the lactic acid bacterium is a  Lactococcus lactis  or a  Lactobacillus casei . Even more preferably, the lactic acid bacterium comprises SEQ ID NO:1 and/or SEQ ID NO:3 of the accompanying and incorporated herein SEQUENCE LISTING. A preferred embodiment is an EGF producing  Lactococcus lactis  comprising SEQ ID NO:3. Another preferred embodiment is an EGF producing  Lactobacillus casei comprising SEQ ID NO:3. 
     Another aspect of the invention is the use of an EGF producing lactic acid bacterium according to the invention to promote gut absorption. Methods to measure gut absorption are known to the person skilled in the art. Still another aspect of the invention is the use of an EGF producing lactic acid bacterium according to the invention to treat the Short Bowel Syndrome. Preferably, the lactic acid bacterium according to the invention is applied orally; it may be treated by any treatment known to the person skilled in the art to improve its survival during the passage of the intestinal system. As a non-limiting example, it may be freeze-dried or spray dried and/or encapsulated in a suitable recipient so that the bacteria are only released in the small intestine. Encapsulation and treatments for delivery in the small intestine have been described, amongst others in U.S. Pat. No. 5,972,685, International Publication Nos. WO0018377 and WO0022909. 
     The lactic acid bacterium, according to the invention, may be combined with other compounds, having a positive effect on gut absorption and/or enhancing the positive effect of EGF. As a non-limited example, glutamine can be used in combination of the lactic acid bacterium according to the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         FIG. 1 : Outline of the construction of pT1hEGF. The construction of pT1mEGF is carried out in a similar way. 
         FIG. 2 : Expression of mEGF (A) and hEGF (B) in  L. lactis  and  L. casei . Supernatant of the cultures as indicated is separated on a 20% polyacrylamide gel and the proteins are detected using a Western blot. 
         FIG. 3 : Average villus length of the mice treated with either  Lactococcus lactis  or  Lactobacillus casei , transformed with the empty vector pT1NX (pT1NX), with the vector pT1mEGF, expressing murine EGF (mEGF) or with the vector pT1hEGF expressing human EGF (hEGF). Medium BM9 treated mice are used as additional negative control (BM9). 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     EXAMPLES 
     Media and strains 
     M17:
         5 g Bacto Tryptone   5 g Bacto Soytone   5 g Meat Digest   2.5 g Yeast Digest   0.5 g ascorbic acid   0.25 g MgSO 4      19 g disodium-β-glycerolphosphate   in 1 l deionized H 2 0       

     GM17: M17 with 0.5% glucose 
     Recuperation medium:
         1 ml 2×M1   0.5 ml 2 M sucrose   50 μl 20% glucose   40 μl 1 M MgCl 2      4 μl 1 M CaCl 2      406 μl H 2 O       

     Agar medium is obtained by adding 1.2% agar 
     BM9 expression medium
         60 g Na 2 HPO 4      30 g KH 2 PO 4,      10 g NH 4 Cl   5 g NaCl.   50 Mm CO 3 -buffer   2 mM MgSO 4      0.1 mM CaCl 2      0.5% casiton (Difco)   0.5% glucose   in 1 liter H 2 O       

       L. lactis  MG1363 is a plasmid and prophage free derivative of the  L. lactis  strain NCDO 712 (Gasson, 1983). 
     Example 1 
     Optimizing the EGF Coding Sequence for Expression in  Lactococcus    
     Both the murine as well as the human EGF (accession number X04571 for hEGF and NM 010113 for mEGF) are available in the public databases at the National Center for Biotechnology Information (accession number X04571 for hEGF and NM — 010113 for mEGF). The coding sequences were adapted to optimize the expression in  Lactococcus . On the base of these sequences, primer sets were designed to assemble the optimized coding sequences of both hEGF and mEGF. At the 3′ end of the coding sequence, a SpeI restriction site was introduced. The primers are shown in Table 1 (hEGF) and Table 2 (m EGF). 
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 1 
               
               
                   
               
               
                 oligos used for assembly of hEGF, and the amount available 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Sense 
                   
                   
               
               
                 HEGF01 
                 AACTCAGATTCAGAATGTCCACTTTCACACGATGGTTACT 
                 33.3 nmol 
               
               
                   
                 (SEQ ID NO:5) 
               
               
                   
               
               
                 HEGF02 
                 GTTTGCACGATGGTGTTTGTATGTACATCGAAGCTCTTGA 
                 34.8 nmol 
               
               
                   
                 (SEQ ID NO:6) 
               
               
                   
               
               
                 HEGF03 
                 TAAATACGCTTGTAACTGTGTTGTTGGTTACATCGGTGAA 
                 26.9 nmol 
               
               
                   
                 (SEQ ID NO:7) 
               
               
                   
               
               
                 HEGF04 
                 CGTTGTCAATACCGTGATTTGAAATGGTGGGAACTTCGTT 
                 28.8 nmol 
               
               
                   
                 (SEQ ID NO:8) 
               
               
                   
               
               
                 HEGF05 
                 AACTAGTCTGCAGAATCTAG 
                 29.7 nmol 
               
               
                   
                 (SEQ ID NO:9) 
               
               
                   
               
               
                 Antisense 
               
               
                 HEGF06 
                 CTAGATTCTGCAGACTAGTTAACGAAGTTCCCACCATTTC 
                 31.1 nmol 
               
               
                   
                 (SEQ ID NO:10) 
               
               
                   
               
               
                 HEGF07 
                 AAATCACGGTATTGACAACGTTCACCGATGTAACCAACAA 
                 22.5 nmol 
               
               
                   
                 (SEQ ID NO:11) 
               
               
                   
               
               
                 HEGF08 
                 CACAGTTACAAGCGTATTTATCAAGAGCTTCGATGTACAT 
                 23.6 nmol 
               
               
                   
                 (SEQ ID NO:12) 
               
               
                   
               
               
                 HEGF09 
                 ACAAACACCATCGTGCAAACAGTAACCATCGTGTGAAAGT 
                 28.4 nmol 
               
               
                   
                 (SEQ ID NO:13) 
               
               
                   
               
               
                 HEGF10 
                 GGACATTCTGAATCTGAGTT 
                 37.8 nmol 
               
               
                   
                 (SEQ ID NO:14) 
               
               
                   
               
             
          
         
       
     
     
       
         
               
             
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                 oligos used for assembly of mEGF, and the amount available 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 Sense 
                   
                   
               
               
                 MEGF01 
                 AACTCATACCCAGGTTGTCCATCATCATACGATGGTTACT 
                 29.7 nmol 
               
               
                   
                 (SEQ ID NO:15) 
               
               
                   
               
               
                 MEGF02 
                 GTTTGAACGGTGGTGTTTGTATGCACATCGAATCACTTGA 
                 28.0 nmol 
               
               
                   
                 (SEQ ID NO:16) 
               
               
                   
               
               
                 MEGF03 
                 TTCATACACTTGTAACTGTGTTATCGGTTACTCAGGTGAT 
                 20.0 nmol 
               
               
                   
                 (SEQ ID NO:17) 
               
               
                   
               
               
                 MEGF04 
                 CGTTGTCAAACTCGTGATTTGCGTTGGTGGGAACTTCGTT 
                 25.5 nmol 
               
               
                   
                 (SEQ ID NO:18) 
               
               
                   
               
               
                 MEGF05 
                 AACTAGTCTGCAGAATCTAG 
                 29.7 nmol 
               
               
                   
                 (SEQ ID NO:19) 
               
               
                   
               
               
                 Antisense 
               
               
                 MEGF06 
                 CTAGATTCTGCAGACTAGTTAACGAAGTTCCCACCAACGC 
                 33.4 nmol 
               
               
                   
                 (SEQ ID NO:20) 
               
               
                   
               
               
                 MEGF07 
                 AAATCACGAGTTTGACAACGATCACCTGAGTAACCGATAA 
                 30.2 nmol 
               
               
                   
                 (SEQ ID NO:21) 
               
               
                   
               
               
                 MEGF08 
                 CACAGTTACAAGTGTATGAATCAAGTGATTCGATGTGCAT 
                 27.3 nmol 
               
               
                   
                 (SEQ ID NO:22) 
               
               
                   
               
               
                 MEGF09 
                 ACAAACACCACCGTTCAAACAGTAACCATCGTATGATGAT 
                 26.2 nmol 
               
               
                   
                 (SEQ ID NO:23) 
               
               
                   
               
               
                 MEGF10 
                 GGACAACCTGGGTATGAGTT 
                 40.3 nmol 
               
               
                   
                 (SEQ ID NO:24) 
               
               
                   
               
             
          
         
       
     
     The oligonucleotides were dissolved in water at a concentration of 100 μM, and used in a 10 times diluted concentration. 
     1 μl of each oligonucleotide is added to 10 μl Taq buffer, 8 μl 2 mM Mg 2+ , 2 μl 0.5 mM XTP, 5 u Taq DNA polymerase (Boehringer, Mannheim, Germany) and 1 u Pfu DNA polymerase (Promega, Madison, USA). The reaction mixture is added up to 100 μl with water. The PCR reaction is carried out for 300 seconds at 94° C., followed by 30 times the cycle of 45 seconds at 94° C., 30 seconds at 48° C. and 30 seconds at 72° C., with a final step of 10 seconds at 15° C. After the assembly, hEGF and mEGF are amplified in a PCR mixture containing 1 μl Vent DNA-polymerase (New England Biolabs, Beverly, USA), 10 μl Taq buffer, 4 μl 0.5 mM XTP, 5 μl 0.5 μM of each primer, 1μl template DNA, 1 μl mM Mg 2 SO 4  and 74 μl H 2 O. 
     In the case of hEGF, HEGF01 and HEGF06 were used as primer; for mEGF, MEGF01 and MEGF06 were used. For hEGF, the same temperature schedule was used as for the first step. In the case of mEGF, the hybridization step was carried out at 52° C. in stead of 48° C. 
     After the assembly, the size of the optimized gene fragments was confirmed on a 2% agarose gel. 
     Example 2 
     Construction of pT1hEGF and pT1mEGF and transformation into  Lactococcus lactis    
     SpeI cut assembled EGF (both for hEGF and mEGF) is ligated into a NaeI and SpeI digested pT1NX (Steidler et al., 1995), resulting in pT1hEGF and pT1mEGF. A schematic overview of the construction of pT1hEGF is shown in  FIG. 1 . Plasmids are transformed into competent cells of  L. lactis  by electroporation. 50 μl of cells are electroporated in a precooled cuvet of 2 mm, at 25 μF, 2.5 kV and 400 Ω (Bio-Rad electroporator).  L. lactis  is made competent by growing a 1/100 dilution of a saturated culture, in 200 ml GM17 with 2.5% glycine, until an OD 600  of 0.5 (Wells et al., 1993). After electroporation, 1 ml of recuperation medium is added, and the cells are incubated for 1.5 hour at 28° C. Cells are plated on GM17 solid medium, comprising 5 μg/ml erythromycin. 
     For the transformation of  L. casei , plasmid is isolated from  L. lactis  on a Qiagen-tip 100, according to the instructions of the manufacturer. The DNA is transformed into competent  L. casei  cells.  L. casei  cells are made competent by growing a 1/50 dilution of an overnight culture in 50 ml MRS (Oxoid LTD., Basingstoke, Hampshire, England) with 1% glycine at 37° C., untill an OD 600  of 0.6. The cells are harvested and washed twice with 10 ml 5 mM Na 3 PO 4  pH 7.4, 1 mM MgCl 2 , and resuspended in 500 μl electroporation buffer (0.3 M sucrose, 5 mM Na 3 PO 4  pH 7.4, 1 mM MgCl 2 ). 10 μl of DNA is added to 50 μl of competent cells and the electroporation is carried out in a BioRad electroporator. After electroporation, 450 μl MRS is added and the cells are incubated for two hours at 37° C. Cells are plated on MRS agar with 5 μg/ml erythromycin. The presence of the plasmid is confirmed using PCR. 
     Example 3 
     Expression of EGF in  L. lactis  and  L. casei    
     The transformed  L. lactis  strains MG1363 [pT1NX], MG1363 [pT1mEGF] and MG1363 [pT1hEGF] are pitched in 5 ml GM17 comprising 5 μg/ml erythromycin, and grown overnight at 30° C. This preculture is diluted 1/100 in 5 ml GM17 with erythromycin, and incubated for three hours at 28° C. The culture is centrifuged and resuspended in BM9 expression medium, and incubated overnight at 28° C. The transformed  L. casei  strains are grown under similar conditions, but using MRS as preculture, and BM9 as expression medium. 
     To the culture supernatant, 1/10 volume sodium desoxycholate is added, and the mixture is kept on ice for 10 minutes. 1/10 of volume 100% TCA is added and the mixture is incubated on ice for 15 minutes. After centrifugation, the pellet is dissolved in 50 μl H 2 O and 50 μl 1 M Tris-HCl pH 9.5. The proteins are analyzed on a 20% Laemmli protein gel. Detection is carried out using a Western blot, with mouse polyclonal anti hEGF and rabbit anti mEGF as primary antibodies. Alkaline phosphatase labeled anti-mouse and anti-rabbit secondary antibodies were from Southern Biotechnology (Birmingham, USA). The results are summarized in  FIG. 2 . 
     Example 4 
     In Vivo Testing of Mice, Using the Transformed Lactic Acid Bacteria Strains 
     In order to assess the effect of the transformed lactic acid bacteria and the growth of the villi and the gut adsorption, seven groups of Balb/c mice (IFFA CREDO CR Broekman/Sulzfield) were treated either with a mEGF or hEGF expressing lactic acid bacterium strain.  L. lactis  and  L. casei  transformed with an empty vector pT1NX, or with BM9 medium was given to mice as a negative control. 
     600 μl of  L. casei  is pitched in 15 ml MRS with 10 μg/ml erythromycin. In the case of  L. lactis , GM17 is used instead of MRS, and only 5 μg/ml erythromycin is used for selection.  L. casei  is incubated overnight at 37° C., for  L. lactis , 30° C. is used. The overnight culture is harvested by centrifugation, and the pellet is resuspended in 1.5 ml BM9 expression medium. 100 μl of this solution is supplied daily, for a period of four weeks. At the end of the experiment, the mice are sacrificed and the intestine is isolated. The tissue is fixated in buffered formaldehyde and thin sections are colored using hematoxylin and eosin G, for microscopic analysis of the villi. The length of the villi is measured at several points to obtain a representative average. All sections were taken from the terminal ileum. 
     The results are summarized in  FIG. 3 .  L. casei  [pT1hEGF], especially, has a positive effect on villus growth and should promote gut absorption. 
     REFERENCES 
     
         
         Carpenter C. D., Ingraham H. A., Cochet C., Walton G. M., Lazar C. S., Sodawski J. M., Rosenfeld M. G. and Gill G. N. (1991) Structural analysis of the transmembrane domain of the epidermal growth factor receptor.  J. Biol. Chem.  266, 5750-5755. 
         Chaet M. S., Arya G., Ziegler M. M. and Warner B. W. (1994) Epidermal growth factor enhances intestinal adaptation after massive small bowel resection.  J. Pediatr. Surg.  29, 1035-1039. 
         Chaet M. S., Arya G., Ziegler M. M. and Warner B. W. (1994) Epidermal growth factor enhances intestinal adaptation after massive small bowel resection.  J. Pediatr. Surg.  29, 1035-1039. 
         Dunn J. C., Parungo C. P., Fonkalsrund E. W., McFadden D. W. and Ashley S. W. (1997) Epidermal growth factor selectively enhances functional enterocyte adaptation after massive small bowel resection.  J. Surg. Res.  67, 90-93. 
         Gasson M. J. (1983) Plasmid complements of  Streptococcus lactis  NCDO 712 and other  lactic streptococci  after protoplast-induced curing. J. Bacteriol. 154, 1-9. 
         Gu Y., Wu Z. H., Xie J. X., Jin D. Y. and Zhuo H. C. (2001) Effects of growth hormone (rhGH) and glutamine supplemented parenteral nutrition on intestinal adaptation in short bowel rats.  Clin. Nutr.  20, 159-166. 
         Hardin J. A., Chung B., O&#39;Loughlin E. V. and Gal, D. G. (1999) The effect of epidermal growth factor on brush border surface area and function in the distal remnant following resection in the rabbit.  Gut  44, 26-32. 
         Helmrath M. A., Shin C. E., Fox J. W., Erwin C. R. and Warner B. W. (1988) Adaptation after small bowel resection is attenuated by sialoadenectomy: the role for endogenous epidermal growth factor.  Surgery  124, 848-854. 
         Jeppesen P. B., Hartmann B., Hansen B. S., Thulesen J., Holst J. J., Mortensen P. B. (1999) Impaired meal stimulated glucagon-like peptide 2 response in ileal resected short bowel patients with intestinal failure.  Gut  45, 559-563. 
         Jeppesen P. B., Hartmann B., Thulesen J., Graff J., Lohmann J., Hansen B. S., Tofteng F., Poulsen S. S., Madsen J. L., Holst J. J. and Mortensen P. B. (2001) Glucagon-like peptide 2 improves nutrient absorption and nutritional status in short-bowel patients with no colon.  Gastroenterology  120, 806-815. 
         Lukish J., Schwartz M. Z., Rushin J. M. and Riordan G. P. (1997) A comparison of the effect of growth factors on intestinal function and structure in short bowel syndrome.  J. Pediatr. Surg.  32, 1652-1655. 
         Marti U., Burwen S. J. and Jones A. L. (1989) Biological effects of epidermal growth factor, with emphasis on the gastrointestinal tract and liver: an update.  Hepatology  9, 126-138. 
         O&#39;Loughlin E., Winter M., Shun A., Hardin J. A. and Gall D. G. (1994) Structural and functional adaptation following jejunal resection in rabbits: effect of epidermal growth factor.  Gastroenterology  107, 87-93. 
         Opleta-Madsen K., Hardin J. and Gall D. G. (1991) Epidermal growth factor upregulates intestinal electrolyte and nutrient transport.  Am. J. Physiol.  260, G807-814. 
         Pearson P. Y., O&#39;Connor D. M. and Schwartz M. Z. (2001) Novel effect of leptin on small intestine adaptation.  J. Surg. Res.  97,192-195. 
         Piiper A., Stryjek-Kaminska D., Stein J., Caspary W. F. and Zeuzem S. (1994) Tyrphostins inhibit secretagogue-induced 1,4,5-IP3 production and amylase release in pancreatic acini.  Am. J. Physiol.  266 G363-371. 
         Playford R. J., Marchbank T., Calnan D. P., Calam J., Royston P., Batten J. J. and Hansen H. F. (1995) Epidermal growth factor is digested to smaller, less active forms in acidic gastric juice.  Gastroenterology  108, 92-101. 
         Scott R. B., Kirk D., MacNaughton W. K. and Meddings J. B. (1998) GLP-2 augments the adaptive response to massive intestinal resection in rat.  Am. J Physiol.  275, G911-921. 
         Swaniker F., Guo W., Diamond J. and Fonkalsrud E. W. (1996) Delayed effects of epidermal growth factor after extensive small bowel resection.  J. Pediatr. Surg.  31, 56-60. 
         Wells J. M., Wilson P. W. and Le Page R. W. (1993) Improved cloning vectors and transformation procedure for  Lactococcus lactis. J. Appl. Bacteriol.  74, 629-636. 
         Zhou X., Li Y. X., Li N. and Li J. S. (2001) Effect of bowel rehabilitative therapy on structural adaptation of remnant small intestine: animal experiment.  World J. Gastroenterol.  7, 66-73.

Technology Category: 1