Patent Application
20120282700
The present invention relates to microorganisms that have attached on their surface a TNFα binding polypeptide. Peptides attached to the surface of the microorganism are more resistant to chemical and enzymatic degradation in the gastrointestinal tract than are secreted proteins. Microorganisms of the invention are capable of binding TNFα in the gastrointestinal tract and therefore reduce the content of free TNFα and alleviate its pro-inflammatory effects in the gut. Microorganisms of the invention can be used as medicament in the treatment of inflammatory bowel disease.
Inflammatory bowel disease (IBD) refers to a group of gastrointestinal disorders characterized by chronic, relapsing inflammatory disorders of the gastrointestinal tract. Crohn's disease and ulcerative colitis are the two main subtypes of IBD.
The etiology of IBD is unclear. IBD appears as multifactorial disease, with genetic and environmental factors probably cooperating in their development.
Patients typically suffer from frequent and chronically relapsing flares, resulting in diarrhea, abdominal pain, rectal bleeding and malnutrition. Crohn's disease can be distinguished from ulcerative colitis in that the inflammation associated with Crohn's disease is transmural and often discontinuous. By contrast, the inflammatory changes of ulcerative colitis typically involve only the superficial mucosal and submucosal layers of the intestinal wall. Crohn's disease most commonly involves the ileum and colon but can affect any region of the gut; ulcerative colitis always involves the rectum, and inflammation may extend as far as the caecum in a continuous pattern. Patients with IBD often have various extra-intestinal symptoms such as arthalgias, and are more likely to have other chronic inflammatory diseases, particularly primary sclerosing cholangitis, ankylosing spondylitis and psoriasis. Etiology, genetics and pathogenesis of IBD is described by Hugot (1999) and Cho (2008).
Since the etiology of both diseases is undetermined the causal therapy does not exist. The most commonly used conventional therapies are anti-inflammatory medicaments such as corticosteroids, salicilates, and immunosuppressives such as cyclosporine, mercaptopurine and azathioprine. Recently, biologic therapies have received a great amount of attention.
The mucosal immune system is the central effector of intestinal inflammation and injury, with cytokines playing a central role in modulating inflammation. Cytokines may, therefore, be a logical target for IBD therapy using specific cytokine inhibitors. In this context compounds described by Schwartz M (2008) blocking the effect of tumor necrosis factor α (TNFα) or its receptor are of interest.
TNFα is a cytokine produced by numerous cell types, including monocytes and macrophages, and was originally identified on the base its ability to induce the necrosis of certain mouse tumors as described by Old (1985). TNFα has been implicated in the pathophysiology of a variety of other human diseases and disorders, including sepsis, infections, autoimmune diseases, transplant rejection and graft-versus-host disease. TNFα promotes the inflammatory response, which, in turn, causes many of the clinical problems associated with autoimmune disorders such as rheumatoid arthritis, ankylosing spondylitis, Crohn's disease, psoriasis and refractory asthma. These disorders are sometimes treated by using a TNFα inhibitor or by counteracting TNFα activity.
In particular, monoclonal antibodies against TNFα are routinely used in parenteral therapy of IBD as described by Sandborn (1999). Commercially available are infliximab, adalimumab and certolizumab pegol. Since antibodies are administered mainly by subcutaneous injection, serious unwanted effects emerge such as headache, abscess, upper respiratory tract infection and fatigue.
De Silva (1992) reported on abundant presence of TNFα in the stool of IBD patients. Local delivery on the place of inflammation could solve unwanted effects associated with systemic delivery. This was achieved by Worledge (2000), with successful treatment of experimental colitis in rats by orally administering avian IgY anti TNFα antibodies with the capability of deactivating/removing TNFα.
Antibodies and antibody related derivatives against TNFα are not always the optimal choice. The high cost of production of these immunoglobulin preparations prohibits their large-scale application. Antibodies, like other protein molecules, are not stable in the gastrointestinal tract.
The use of genetically engineered bacteria that produce and deliver compounds that block the activity of TNFα could provide a solution. In this respect, lactic acid bacteria, which are normal commensals of the gut and other mucosal surfaces of human and animals and generally regarded as safe, represent ideal candidates.
WO2007/025977 discloses delivery of secreted antibodies against TNFα, and of fragments of such antibodies (so-called nanobodies), to the intestine for the treatment of enterocolitis using genetically engineered microorganisms such as lactic acid bacteria. The secreted antibodies of WO2007/025977 do not include a surface attachment domain, and are thus not attached to the cell's surface and more prone to chemical and biological degradation in the gut.
WO97/14806 discloses a delivery system of biologically active compounds in the intestine, wherein non-invasive bacteria such as lactic acid bacteria are used to deliver biological active polypeptides to the gut. WO00/23471 discloses recombinant lactic acid bacteria that can be used to deliver IL-10 and soluble TNF receptor via oral route to the ileum to treat IBD. No attachment of the polypeptides to the surface of the microorganism is disclosed. Hence, the polypeptides are subject to rapid degradation. WO97/14806 uses the Usp45 secretion domain to ensure efficient secretion of the proteins.
EP1477802 discloses vaccines against infection with Streptococcus pneunoniae. The vaccine comprises the antigenic part of pneumococcal proteins (PpmA, SlrA), which are fused with a cell wall anchoring domain derived from the AcmA protein. The pneumococcal proteins of EP 1477802, however, do not comprise a TNFα binding domain, hence do not exhibit the therapeutic effects of the present microorganisms.
Hence it is known to produce peptides by lactic acid bacteria and secrete them into the gastrointestinal tract. Such peptides then generally undergo fast degradation.
The present invention addresses these problems by producing genetically modified microorganisms that have attached to their surface, or express on their surface, a TNFα binding polypeptide. Surface expression of TNFα binding polypeptides, or attachment of TNFα binding polypeptides, on lactic acid bacteria provides protection from chemical and enzymatic factors in the gastrointestinal tract. Such peptides are markedly more acid resistant. TNFα binding polypeptide expressed on the surface of mentioned microorganism may be TNFα binding Z domain (affibody). The peptide has stable and robust structure due to Z domain and is able to bind TNFα in the sample, for example body fluid.
The present invention thus relates to a gene construct coding for a polypeptide comprising a secretion signal peptide, a TNFα binding domain and surface attachment domain, said TNFα binding domain and said surface attachment domain being preferably separated by a spacer region, said gene construct preferably being under the control of a suitable promoter.
In a preferred embodiment of the invention, said secretion signal peptide is a signal peptide of Usp45 or its homologue.
In another preferred embodiment of the invention, said secretion signal peptide is adjacent a peptide sequence selected from LEISSYCDA, LEISSTCDA, LQVDDIPSA or LGISSTCNA, or homologues thereof.
In another preferred embodiment of the invention, said surface attachment domain comprises at least 10, or at least 20 consecutive amino acid residues of the C terminal part of AcmA, or of homologues of the AcmA protein. In one embodiment, the at least 10, or at least 20 consecutive amino acid residues include the last (C-terminal) amino acid residue of the C terminal part of the AcmA protein, or of homologues of the AcmA protein. In another embodiment, the consecutive amino acid residues are in the C-terminal half, preferably in the C-terminal third, of the AcmA protein, or of homologues of the AcmA protein.
In another preferred embodiment of the invention, said surface attachment domain comprises 1 to 12, preferably 1 to 6, more preferably 2 to 4, or most preferred 3 LysM repeats.
In another preferred embodiment of the invention, said spacer region comprises at least 3, 5, 10, 20, or 40 consecutive amino acids of the AcmA protein, or of homologues of the AcmA protein.
In another preferred embodiment of the invention, the length of spacer region is 5 to 50 amino acids, preferably 10 to 30, more preferably 15-25, most preferably 20 amino acids.
In another preferred embodiment of the invention, said construct is coding for a protein sequence
(a) of any one of SEQ ID NO:6 to SEQ ID NO:10, or
(b) for a protein sequence which is at least 50, 80, 90, 95, 98, or 99% identical to any one of said sequences under (a), and conferring the same biological function as the said sequence under (a).
The invention further relates to a protein construct encoded by a gene construct of any one of the above claims.
The invention also relates to a microorganism, preferably a GRAS microorganism, having attached to its surface a protein comprising a TNFα binding domain. In a preferred embodiment, said attachment to said surface is a non-covalent attachment. The attachment, however, can also be covalent attachment.
In a preferred embodiment, said attachment to said surface is by at least one protein domain of said protein, said protein domain being selected from the group consisting of LysM repeat, choline binding domain, lipobox and LPxTG motif. The LysM repeat domain is preferred.
In a preferred embodiment, said protein comprising a TNFα binding domain is a protein construct as defined above. Hence, said protein comprising a TNFα binding domain may have a sequence
(a) of any one of SEQ ID NO:6 to SEQ ID NO:10, or
(b) a sequence which are at least 80% identical to any one of said sequences under (a), and preferably conferring the same biological function as said sequence under (a).
In a preferred embodiment, said microorganism is genetically modified to express said protein comprising a TNFα binding domain on its surface. This shall include microorganisms that express the protein, secrete the protein into the medium, wherein said protein is thereafter attached to the surface of said microorganism.
The invention further relates to a genetically modified microorganism preferably produced from a GRAS microorganism, said genetically modified microorganism expressing on its surface a protein comprising a TNFα binding domain.
In a preferred embodiment of the invention, microorganisms of the invention are lactic acid bacterium, Lactococcus lactis, or Lactococcus spec.
In another preferred embodiment of the invention, the microorganism of the invention comprises a gene construct as defined above.
In another preferred embodiment of the invention, the microorganism of the invention comprises a protein construct as defined above.
The invention also relates to a method for producing a genetically modified microorganism as described above, said method comprising introducing of a the above gene construct into a GRAS microorganism.
In a preferred embodiment of the invention, said gene construct is in a plasmid vector capable of replicating in said microorganism, or said gene construct is integrated into the genome of said microorganism.
The invention further relates to a genetically modified microorganism as described above for use as a medicament.
The invention further relates to the above microorganism for the treatment of inflammatory bowel disease, chronic colitis, Crohn's disease or ulcerative colitis.
The invention further relates to the above microorganism for reducing the content of free TNFα in the gastrointestinal tract of a subject.
The invention further relates to a pharmaceutical composition comprising a modified microorganism as described above. The pharmaceutical composition is preferably for oral administration, for example a tablet, or a capsule, preferably an enteric-coated tablet or capsule. The pharmaceutical preparation may also be in form of a suppository. The microorganism in the pharmaceutical composition can be viable or non-viable. In one embodiment the microorganism is non-viable.
In a preferred embodiment said pharmaceutical composition is adapted for oral administration.
The invention also relates to a food composition comprising the microorganism above.
In a preferred embodiment the food composition is selected from the group consisting of milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice cream, fermented cereal products, and milk based powders.
a and 2b show the results of flow cytometry of lactococcal cells harboring different plasmids.
The present invention relates to genetically modified GRAS (e.g., food grade) microorganisms that express, or have attached, a TNFα binding domain on their surface. Peptides expressed on the surface of microorganisms are more resistant to degradation in the gastrointestinal tract. Such microorganisms are capable of binding TNFα and therefore reducing the content of free (non-bound) TNFα and alleviating its pro-inflammatory effects in the gut. Such microorganisms can be used as medicament, e.g., in the treatment of IBD.
A TNFα binding domain may comprise antibodies or antibody fragments with ability to bind TNFα. The term “antibodies” refers to monoclonal antibodies, chimeric antibodies, humanized and fully human antibodies. “Antibody fragments” means a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab)′ 1 and Fv fragments, diabodies, linear antibodies, single chain antibody molecules and multispecific antibodies formed from antibody fragments, and nanobodies.
A TNFα binding domain may also be an affibody, as disclosed in WO2006/092338. Affibody molecules are small and robust high affinity protein molecules that can be engineered to bind specifically to a large number of target proteins. The scaffold used for construction of affibodies is Z domain, an engineered version of B domain, which is one of the five stable three-alpha-helix bundle domains from the immunoglobulin binding region of staphylococcal protein A. Affibody molecules with unique binding properties are usually acquired by randomization of 13 amino acids located in two alpha-helices involved in the binding activity of the parent protein domain. Affibodies with affinity towards specific target protein are obtained by library construction and affinity selection.
A “TNFα binding domain”, within the meaning of the present invention, shall thus be understood to be any peptide or domain to which TNFα binds, or which binds to TNFα. A TNFα binding domain can be a part of a larger protein, which then binds to TNFα, or to which TNFα binds. Preferred TNFα binding domains of the invention have a dissociation constant (Kd) with TNFα of less than 0.01, 0.1, 1, 10, 100, 1000 nM. With regard to the formation of a ligand-protein complex (C) from a protein (P) and a ligand (L) according to CP+L the corresponding dissociation constant is defined as
where [P], [L] and [C] represent the concentrations of the protein, ligand and complex, respectively.
Kd, within the context of the present invention, is determined by the use of surface plasmon resonance technology, e.g. using the Biacore® instrument. Kd is determined in an experiment wherein the TNFα binding domain is immobilized on a sensor chip of the instrument, and samples containing TNFα prepared by serial dilution are passed over the chip. The skilled person may than interpret the obtained sensograms to establish the Kd value (the apparent Kd value) for the interaction of TNFα binding domain and TNFα. Apparent Kd are calculated from the results, using the 1:1 Langmuir binding model of the BlAevaluation 3.2 software provided by the instrument manufacturer, according to the manufacturer's instructions.
Kd, according to the invention is preferably measured for the free protein construct, i.e., the protein of the invention in a state not bound to the surface of the producing microorganism. In another embodiment Kd is measured for the protein bound to the surface of the microorganism.
A “surface attachment domain” of a protein, according to the present invention, shall be understood as being any domain of said protein which is capable of attaching the said protein to the surface of a microorganism, e.g., the microorganism producing the protein. In preferred embodiments, the surface attachment domain comprises a lipobox and preferably a corresponding signal peptide (anchoring to membrane via a lipid group), or at least one LysM repeat (sometimes also referred to as a peptidoglycan binding domain, PBD), at least one choline binding domain (CBD) or at least one LPxTG motif (cell wall binding domain). A surface attachment domain of the invention preferably comprises at least one of the following consensus sequences:
LysM repeat consensus sequence: [YV]-X(0-4)-G-D-[ST]-[VLIA]-X(0-2)-[VLIA]
CBD consensus sequence: G-X(0-5)-G-X-[WYI]-[WYT]-[YVL]-[FV]
Lipobox consensus sequence (+signal peptide): M-X(1-10)-[RK]-{DERK}(7-17)-[LVTIMG]-[ASTIVGMLCPFL]-[AGLISVTFP]-C
LPxTG consensus sequence: L-P-X-T-G,
in which X denotes any naturally occurring amino acid, and in which square brackets indicate one position which can be filled with any of the amino acids indicated between the square brackets. Rounded brackets (“{...}”) indicate a position which can be filled with any amino acid except the ones mentioned between the rounded brackets. The numbers in subscript “(X-Y)” indicate that the corresponding amino acid is repeated the indicated number of times, i.e., from X to Y. Preferred surface attachment domains comprise LysM repeats.
“LysM repeats” (or “Lysin motifs”) are well known in the art. More than 4000 proteins of both prokaryotes and eukaryotes have been found to contain one or more LysM repeats (see http://www.genome.jp/dbget-bin/www_bget?pfam:PF01476). Notably, this collection contains not only truly secreted proteins, but also (outer-)membrane proteins, lipoproteins or proteins bound to the cell wall in a (non-) covalent manner. LysM repeats typically range in length from 40 to 65 amino acid residues and binds to various types of peptidoglycan and chitin, recognizing the N-acetylglucosamine moiety. Most bacterial LysM-containing proteins are peptidoglycan hydrolases with various cleavage specificities. LysM repeats occur frequently in bacterial lysins, in bacteriophage proteins and in certain proteins of eukaryotes (Pfam PF01476 and Prodom PD407905). They are also present in bacterial peptidoglycan hydrolases and in peptidases, chitinases, esterases, reductases or nucleotidases. Multiple LysM repeats within one surface attachment domain are separated by spacing sequences mostly consisting of Ser, Thr and Asp or Pro residues, which may form a flexible region between the LysM repeats. The intervening sequences vary in length, composition and do not share significant homology. LysM repeats are present in the N-terminal as well as the C-terminal domains of proteins; they are also present in the central part of proteins, possibly connecting two (catalytic) domains. Prokaryotic LysM repeats, in contrast to their eukaryotic counterparts, do not possess possible disulphide bridges. Their domains contain extensive secondary structures and hydrogen bond networks, and consequently, disulphide bridges are non-essential for their structure and functions. The isoelectric points (pIs) of the LysM proteins range from 4 to 12, with most having a pI of 5 or 10. The best-characterized LysM-repeat containing protein is the N-acetylglucosaminidase AcmA of L. lactis. AcmA binds in a non-covalent manner to the cell wall and is responsible for cell lysis of producing cells.
In a preferred embodiment, the LysM repeat comprises the consensus sequence [YV]-X(0-4)-G-D-[ST]-[VLIA]-X(0.2)-[VLIA].
The expression “to express on its surface”, in relation to microorganisms expressing proteins, shall be understood to include microorganisms which express the protein, secrete the protein into the surrounding medium, wherein the protein is subsequently attached to the surface of the microorganism by means of covalent or non-covalent attachment.
“GRAS”, in respect to a microorganism within the context of the present invention, shall be understood to be a microorganism which is generally regarded as safe in accordance with the US Food And Drug Administration guidelines (http://www.fda.gov/Food/FoodIngredientsPackaging/GenerallyRecognizedasS afeGRAS/default.htm). The expression “GRAS microorganism” and “food grade organism” can be used interchangeably. A GRAS microorganism is a microorganism that has a long history of safe usage and is a constituent of fermented foods. Such microorganisms have “generally recognized as safe” status. Preferably genetically modified microorganism of food grade strain relates to lactic acid bacteria. Lactic acid bacteria are a group of phylogenetically related microorganisms. Preferred GRAS organisms are: Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pedicococcus, Streptococcus, Tetragenococcus, Vagococcus and Weisela. According to their physiological characteristics, genus Bifidobacterium spp. is usually classified as lactic acid bacterium because it occupies the same environmental niches, even though it is phylogenetically more distant. Preferably genetically modified microorganism of food grade strain is Lactococcus species. In one preferred embodiment said genetically modified organism is Lactococcus lactis strain.
Genetically modified GRAS microorganisms such as lactic acid bacteria with desirable new traits can be obtained by application of molecular biology techniques and production recombinant microorganism.
The “surface” of a microorganism, according to the invention, shall be understood to mean the outer surface of the cell wall of the microorganism.
A “homologue” of a reference protein, according to the invention, is a protein which has a similar, but not a fully identical amino acid sequence as the reference protein, and which has the same biological function or activity as the reference protein. The homologue has preferably an amino acid sequence which is 50, 70, 80, 90, 95, or 99% identical to the amino acid sequence of the original protein.
Within the context of the present invention, the “% identity” of a protein relative to a reference protein of a defined length shall be understood as follows: A peptide that is 50 percent identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide, which is 50 percent identical to the reference polypeptide over its entire length. Of course, other polypeptides will meet the same criteria. The term “sequence identity” as used herein means that the sequences are compared as follows. The sequences are aligned using Version 9 of the Genetic Computing Group's GAP (global alignment program), using the default (BLOSUM62) matrix (values −4 to +11) with a gap open penalty of −12 (for the first null of a gap) and a gap extension penalty of −4 (per each additional consecutive null in the gap). After alignment, percentage identity is calculated by expressing the number of matches as a percentage of the number of amino acids in the reference polypeptide.
To achieve anchoring in the extracellular site of cell membrane, TNFα binding domain is expressed as fusion protein containing signal peptide for extracellular secretion, TNFα binding domain and surface attachment domain. Signal peptide leads the fusion protein into the extracellular space, where surface attachment domain anchors it on the membrane by binding to peptidoglycan. Signal peptide is processed and detached from fusion protein when reaching extracellular space.
Quantitative measure of affinity (Kd) may be determined also for mentioned isolated fusion protein with signal peptide detached by using surface plasmon resonance. Said fusion protein may be isolated from the growth medium using methods known to the person skilled in the art and immobilized on a sensor chip e.g. of the Biacore® instrument. Samples containing TNFα prepared by serial dilution are passed over the chip. The skilled person may than interpret the obtained sensograms to establish a quantitative measure, apparent Kd value for the interaction of fusion protein and TNFα. Apparent Kd values may then be calculated from the results, using e.g. the 1:1 Langmuir binding model of the BIAevaluation 3.2 software provided by the instrument manufacturer.
A genetically modified microorganism expressing TNFα binding domain on its surface, may be prepared by introduction of gene construct to the mentioned microorganism. The gene construct comprises secretion signal peptide coding sequence, TNFα binding domain coding sequence and surface attachment domain coding sequence, wherein the TNFα binding domain coding sequence and the surface attachment domain coding sequence are separated with a spacer region coding sequence. Said gene construct is under the control of suitable promoter. Gene construct may be a part of plasmid vector capable of replicating in said microorganism or may be introduced into the genome of said microorganism. Introduction of gene construct may be performed by electroporation.
The secretion signal peptide coding sequence codes for a secretion signal peptide. The secretion signal peptide enables the secretion of protein construct from the cell to the growth medium. Such secretion signal peptides include, but are not limited to signal peptide of Usp45 protein (SPUsp45), SP310 signal peptide and its improved mutants. Other secretion signal peptides are known in the art. Secretion may be increased by the use of synthetic pro-peptides. Such pro-peptides are homologues which contain acidic or neutral amino acids at position +2 and +8 or acidic amino acids at position +4 and +5 and have neutral or positive global net charge. Such pro-peptides include but are not limited to LEISSTCDA, LEISSYCDA, LQVDDIPSA and LGISSTCNA. Preferably the secretion signal peptide is the signal peptide of Usp45, where the second amino acid K is changed to A and is straightened by the synthetic pro-peptide LEISSYCDA.
The usp45 gene encodes the major extracellular protein from Lactococcus lactis. The deduced sequence of the 27 residue leader peptide reveals the tripartite characteristics of a signal peptide. This leader peptide enables efficient secretion of various proteins. The Usp45 protein, according to the invention, is the one accessible in GenBank under the following GenBank record:
L. lactis secreted protein (usp45) gene, complete cds.
Lactococcus lactis subsp. cremoris MG1363
Lactococcus lactis subsp. cremoris MG1363
Lactococcus.
The Usp45 protein is thus understood to be the protein having the amino acid sequence of SEQ ID NO:12. First 27 amino acids represent a preferred signal peptide SPUsp45.
The protein product of mentioned gene construct is secreted to the medium and from there attached to the surface of said microorganism by binding to peptidoglycan. The attachment to the peptidoglycan on cell surface may be non-covalent, where protein construct comprises different number of peptidoglycan binding domains derived from C terminal part of AcmA protein. The attachment to the peptidoglycan on cell surface may be covalent. This may be achieved by addition of LPXTG signal peptide to the protein construct to be covalently attached to the cell surface peptidoglycan. The attachment of such assembly to surface peptidoglycan is catalysed by enzyme sortase. Surface attachment domain coding sequence preferably codes for different number of peptidoglycan binding domains derived from C terminal part of AcmA. Preferably surface attachment domain consists of one to six LysM repeats. More preferably it consists of two to four LysM repeats, more preferably of three LysM repeats.
Spacer region coding sequence between TNFα binding domain coding sequence and surface attachment domain coding sequence may be derived from AcmA protein. The length of spacer region may be 5-50 amino acids, preferably 10-30, more preferably 20 amino acids.
The AcmA protein, according to the invention, is the one accessible in GenBank under the following GenBank record:
Lactococcus lactis N-acetylmuramidase (acmA)
Lactococcus lactis
Lactococcus lactis
Lactococcus.
The AcmA protein is thus understood to be the protein having the amino acid sequence of SEQ ID NO:11.
Examples of described gene constructs of the invention are shown in sequence listing as SEQ ID NO:6 to SEQ ID NO:10 and in
The promoter that controls the expression of mentioned gene construct may be constitutive or inducible. Constitutive promoters enable continuous protein production, but are usually prone to lower level of expression. Inducible promoters are also referred to as controlled promoters. Inducible promoters usually provide higher expression level. Inducible promoters include but are not limited to bacteriocin controlled promoters, carbohydrate controlled promoters, pH controlled promoters, phage promoter Φ31 and temperature controlled promoters.
Bacteriocin controlled promoters are controlled by addition of bacteriocins. Such bacteriocins include, but are not limited to nisin and pediocin. pH controlled promoters may be P170, induced by accumulation of lactic acid at pH 6.0-6.5, Pgad, induced by chloride ions and F1F0-ATPase promoter, induced by acidification. Temperature controlled promoter may be P2, where inactivation of repression is achieved at temperatures above 40° C. Carbohydrate promoters may be lacA and xylA operons that are induced by changing the carbon source in the growth medium.
Anti-TNFα binding domain displaying microorganism binding to TNFα refers to a property which may be tested by the use of flow cytometry or confocal microscopy. The testing of binding of surface expressed peptide to TNFα in accordance with invention is described in examples.
According to another embodiment the present invention relates to the use of anti-TNFα peptide displaying microorganism for the purpose of reducing the content of free TNFα in gastrointestinal tract.
A genetically modified microorganism of the invention can be used as medicament to treat IBD. Said disease or disorder is characterized by imbalance in TNFα production, and can be treated by TNFα inactivating compounds, such as TNFα binding polypeptide expressed on the surface of genetically modified microorganisms. More preferably, said disease is an irritable bowel disease, including but not limited to chronic colitis, ulcerative colitis and Crohn's disease.
Another embodiment of the present invention is the pharmaceutical composition for treating IBD. Preferably pharmaceutical composition, comprising anti-TNFα peptide displaying microorganisms is for oral administration.
The pharmaceutical composition may be liquid, comprising microorganism expressing anti TNFα peptide on surface, or it may be solid, comprising dried microorganism that can be reactivated when put in a suitable environment. Microorganisms may be dried by any system, including freeze drying and spray drying. “Anti-TNFα peptide displaying microorganism” as used here, means that the microorganism is viable and can express anti-TNFα peptide when placed in a suitable environment, not necessarily expressing the anti-TNFα peptide in the pharmaceutical composition. The pharmaceutical composition may further comprise agents known to the person skilled in the art to improve viability of the microorganisms and stability of surface expressed anti-TNFα peptide, such as but not limited to skimmed powdered milk.
Lactic acid bacteria thrive in nutrient rich environments such as milk. They are integral part of mammalian GI flora and are considered beneficial to human health. As such they are added to the diet in the form of probiotic food. The present invention also relates to a food containing such recombinant microorganisms. Food composition may be milk, yogurt, curd, cheese, fermented milks, milk based fermented products, ice cream, fermented cereal based products and milk based powders.
The present invention further relates to a system for immobilization of a heterologous protein construct containing a TNFα binding polypeptide coupled to a surface attachment domain on the cell surface of microorganisms, preferably a GRAS microorganism, in cases for which the use of genetically modified bacteria is less desirable. For example, in applications that involve uncontrolled release into the environment, and foods products. This system is based on non-genetically modified GRAS microorganisms, which are used as carrier (support) to bind an externally added heterologous protein construct of the invention. The binding of the protein construct is preferably by a surface attachment domain as described above. Thus the expressed protein construct is not anchored or attached to the producing cells. The cells for production of the protein construct of the invention and the carrier cells may be different, thereby allowing the use of a non-genetically modified (non-GMO) cells as carrier cells.
Preferred GRAS non-GMO carrier cells are: Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc, Oenococcus, Pedicococcus, Streptococcus, Tetragenococcus, Vagococcus, Weisela and Bifidobacterium spp. Preferably non-GMO carrier microorganism strain is Lactococcus species. In one preferred embodiment said non-GMO carrier microorganism is Lactococcus lactis strain.
Non-GMO carrier microorganisms may be viable or non-viable (i.e., they cannot proliferate). Such non-viable cells can be generated various methods known in the art, such as UV, chemical inactivation, heat and gamma-irradiation. Preferably non-viable non-GMO carrier microorganisms are prepared by boiling in various acids, for example trichloroacetic acid.
Protein constructs of the invention, composed of signal peptide for extracellular secretion, TNFα binding domain and surface attachment domain, are expressed in a recombinant expression host. Protein constructs are then secreted in the growth medium. The protein construct may be purified from growth medium or cell free growth medium may be added to non-GMO carrier microorganism. Protein constructs of the invention composed of the TNFα binding domain and anchoring domain (i.e., lacking the secretion signal peptide) may be expressed intracellularly in a recombinant expression host. The protein construct is then purified from host cell lysate and added to the non-GMO carrier microorganism.
Recombinant expression host is a system suitable for the production of a protein construct of the invention, such as bacterial expression systems, yeast expression systems, baculovirus/insect expression systems, and/or mammalian cells.
The expression system preferably includes an expression vector, including the DNA encoding the protein construct of the invention, and the host cell. The expression system preferably allows foreign gene expression at a high level. Preferred recombinant GRAS microorganisms are lactic acid bacteria (e.g., L. lactis) for use as an expression host. In this case the cell free culture medium is directly suitable for addition to non-GMO carrier microorganism and further purification steps are not necessary.
The use of a non-GMO carrier microorganism is demonstrated in Example 6.
Design of Gene Constructs for Lactococcal Surface Display
Several gene constructs were prepared to obtain fusion protein with optimal surface binding capabilities and are shown on
Peptidoglycan binding domains were derived from AcmA protein. They consisted of one peptidoglycan binding LysM repeat, as in the case of pSDBA1 or pSDLBA1, or three peptidoglycan binding LysM repeats in remaining constructs. pSDLBA3a, pSDLBA3b and pSDLBA3c contained three peptidoglycan binding LysM repeats with variable length of spacer region between binding domain and first LysM repeat.
Bacterial Strains, Media and Culture Conditions
Bacterial strains used in this study are shown in Table 1. E. coli DH5α was grown at 37° C. with aeration in LB medium supplemented with 100 μg/ml ampicillin.
L. lactis NZ9000 and L. lactis NZ9000ΔHtrA were grown in M-17 medium supplemented with 0.5% glucose at 30° C. without aeration. 10 μg/ml of chloramphenicol or erythromycin or both was added when appropriate.
DNA Manipulation and Plasmid Construction
General cloning procedures were performed using conventional molecular biology methods. Electroporation of L. lactis was performed, using Gene Pulser II apparatus. Primers and plasmids are listed in Table 1.
AcmA1, acmA3a, acmA3b and acmA3c genes were amplified from lactococcal genomic DNA using colony PCR with AcmA-EcoRI/AcmA-XbaI, AcmA-EcoRI/AcmA-R3-XbaI, AcmA-Fb-EcoRI/AcmA-R3-XbaI and AcmA-Fc-EcoRI/AcmA-R3-XbaI primer pairs, respectively. SpUsp45 was amplified from pGEM::Usp using Usp1-NcoUUspR-BamHI primer pair. B domain gene was amplified from pGEM::B using Bdom-F-BamHU Bdom-R-EcoRI primer pair. PCR amplicons were digested with restriction enzymes in the following manner: spUsp45 with BamHI, b-dom with BamHI and EcoRI and acmA1 with EcoRI. Digested fragments were ligated and fusion gene termed sdb was PCR amplified using Usp1-NcoI/AcmA-XbaI primer pair. Sdb was digested with NcoI and XbaI and cloned to equally prepared pNZ8148, yielding pSDBA1. LEISSYCDA synthetic propeptide gene was added to spUsp45 gene by PCR using Usp1-NcoI/LeisR-BamHI primer pair and pGEM::Usp as a template, yielding spUsp45-LEIS. SpUsp45-LEIS gene was used to substitute spUsp45 in pSDBA1 via NcoI/BamHI restriction sites, yielding pSDLBA1. AcmA3a, acmA3b and acmA3c genes were used to substitute acmA1 gene in pSDLBA1 via EcoRI/XbaI restriction sites, yielding pSDLBA3a, pSDLBA3b and pSDLBA3c, respectively. TNFα binding Z domain gene labeled z-tnf, with BamHI and EcoRI restriction sites was designed on the basis of Z00185 polypeptide described in WO2006/092338 by codon optimization and ordered from ATG:biosynthetics. Z-tnf was BamHI/EcoRI digested and substituted b-dom in equally prepared pSDLBA3a, yielding pSDZ-TNF. All the constructs are shown in
Expression of Fusion Proteins in L. lactis
Overnight cultures of L. lactis NZ9000 or NZ9000ΔHtrA harboring appropriate plasmid: pSDBA1, pSDLBA1, pSDLBA3a, pSDLBA3b, pSDLBA3c or pSDZ-TNF, were diluted in 10 ml of fresh GM-17 medium, grown to optical density A600=0.5-0.8 and induced with 25 ng/ml nisin. 3 hours after induction we split the culture in half. 5 ml were centrifuged at 5000 g for 10 min. Supernatant was decanted; cell pellet was resuspended in 0.1 M potassium phosphate buffer pH 7.00.
Surface Display of Model B Domain, Characterization of Secretion, Optimization of Protein Yield and its Functionality
SDS PAGE and Western Blot
SDS PAGE was performed using a mini-Protean II apparatus. Prestained standards were used for molecular weight comparison. Protein concentration was determined by Bio Rad protein assay and equal amounts were used to enable comparison between samples. Samples were denatured by heating at 100° C. in the presence of DTT before loading. Proteins were stained with Coomassie Brilliant Blue or transferred to polyvinylidene fluoride membrane. The membrane was blocked in 1% Western blocking solution and incubated overnight at 4° C. with FITC conjugated anti-protein A antibody. After washing with 50 mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, pH 7.5, the fluorescence was detected with Typhoon 9410 imager using blue excitation at 488 nm.
Expression of fusion proteins was confirmed by Western blot using FITC conjugated anti-protein A antibody, which bound the B domain of the fusion proteins. Cell lysate, containing cytoplasmic and surface bound fusion protein, and growth medium, containing secreted unbound fusion protein, were tested. All fusion proteins were detected in both, cell lysate and growth medium, and their observed molecular weights corresponded to the calculated. Molecular weight of fusion proteins was: Sdba1 18.4 kDa, Sdlba1 19.4 kDa, Sdlba3a 34.0 kDa, Sdlba3b 32.9 kDa and Sdlba3c 31.8 kDa.
Two bands of similar size were detected with all fusion proteins, which corresponded to fusion proteins with or without secretion signal with calculated molecular weight difference of 2.9 kDa.
N-Terminal Sequencing
The processing of secretion signals was evaluated with growth medium fraction of Sdba1 and Sdlba1 proteins. Their N-terminal sequence was determined to be GSADN and LEISS, respectively, which corresponds to the N-terminal sequences of fusion proteins without Usp45 signal.
Growth media of L. lactis NZ9000 expressing Sdba1 or Sdlba1 were concentrated on Amicon Ultra-4, separated on SDS PAGE and blotted to PVDF membrane. Membrane was stained briefly with Coomassie Brilliant Blue, dried and corresponding bands were excised. N-terminal amino acid sequences of Sdba1 and Sdlba1 were determined by automated Edman degradation using an Applied Biosystems 492 Protein sequencer.
Staining of Cells, Flow Cytometry and Fluorescent Microscopy
Surface display of B domain was characterized by flow cytometry and fluorescent microscopy. Control and Sdlba3b-expressing cells stained with unspecific antibodies were visualized using fluorescent microscopy. Stronger binding of antibodies and increased fluorescence was observed with Sdlba3b-expressing cells.
For flow cytometric analysis, cells were gated as shown in
For the flow cytometric analysis 10 μL of cell cultures, approximately 107 cells/ml, were added to 500 μL of 50 mM Tris-HCl, 150 mM NaCl, pH 7.5 and centrifuged 3 min at 5000 g at 4° C. Supernatants were decanted and cells were resuspended in 500 μL of the same buffer. Next, either 1 μg of specific, FITC conjugated anti-protein A antibody or 2 μg of unspecific, Alexa Fluor 488 conjugated rabbit anti-mouse antibody was added to the suspensions and incubated 2 hours at RT with constant shaking at 100 rpm. Cells were than washed three times with 200 μL 50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5 and resuspended in 500 μt of 50 mM Tris-HCl, 150 mM NaCl, pH 7.5. Stained sample and control cells were analyzed with FACS Calibur flow cytometer. At least 100000 bacterial cells were counted for each sample. Cells were gated using FSC vs. SSC to isolate the bacterial cells.
For the purpose of fluorescent microscopy the staining protocol was similar except the starting volume of cell cultures was 20 μL and final volume of TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.5) for re-suspension was 200 μL. Fluorescence microscopy was performed using a Carl Zeiss LSM 510 confocal microscope. Alexa Fluor 488 was excited with an argon laser, and emission was filtered using narrow-band 505-530 nm filter. Images were analyzed using Carl Zeiss LSM image software 3.0.
Surface Display of TNFα-Binding Z Domain and its Functionality
Staining of Cells, Flow Cytometry and Fluorescent Microscopy
Surface display of TNFα-binding Z domain was characterized by the capability to bind Alexa Fluor 488-conjugated TNFα.
Stronger fluorescence of Sdz-tnf-expressing cells was also observed with the use of fluorescent microscopy.
For flow cytometric analysis and fluorescent microscopy samples were prepared as described in example 2, except 10 μL of Alexa 488-labelled TNFα was added to the suspensions.
Soluble TNFα was labelled using Alexa Fluor 488 Microscale Protein Labeling kit according to manufacturer's protocol. Final concentration of labeled protein was 0.5 mg/ml determined with Nanodrop 1000 Spectrophotometer.
Stability of Surface Displayed Model B Domain in Simulated Gastric Conditions
Stability of the surface displayed B domain was tested against simulated gastric juice. 500 μl of pNZ8148 as control or pSDLBA3b-containing cells were mixed with either 500 μl of TBS buffer, pH 7.0 or 500 μl of simulated gastric juice, which was prepared using 3 mg/ml pepsin and pH was adjusted to 2.0 or 4.0 with concentrated HCl. The mixture was than incubated at room temperature for 30 min. 50 μl of the mixture was washed with 500 μl of TBS buffer, stained with Alexa Fluor 488 rabbit anti-mouse antibody and analyzed with flow cytometer as described earlier.
Capability of surface displayed B domain to bind Fc region of antibody was tested after the incubation of cells containing pSDLBA3b in simulated gastric conditions and compared to incubation at pH 7.0; positive control or to cells containing pNZ8148 at pH 7.0; negative control.
The loss of fluorescence intensity in samples, exposed to simulated gastric juice was less than 14% even after 30 min at pH 2.0 as shown in
Determination of Maximum Unspecific Antibody Binding Capacity to B Domain-Displaying Cells
Control cell cultures containing pNZ8148 or cell cultures containing pSDLBA3b were washed with PBS buffer twice and resuspended in PBS to the cell density of 1×109 cells/mL. 100 μL of cells were centrifuged and resuspended in 100 μl PBS containing M13/HRP antibody with various concentrations (0.45, 0.9, 1.8, 4.6 and 9.0 μg/mL) and incubated for 2 h at room temperature with constant shaking in preblocked Eppendorf tubes. After incubation, cells were pelleted and 20 μL of supernatant was transferred to microtiter plate and incubated with 180 μL TMB substrate for 15 minutes at room temperature. Colour development was terminated by the addition of 50 μL 2 M H2SO4 and absorbance was read at 450 nm using Rainbow reader. All samples were measured in duplicate. Amount of adsorbed antibody; Γ was calculated as follows: Γ=CAb×V×(1−A450(pSDLBA3b)/A450(pNZ8148)). Langmuir adsorption isotherm was assumed for the adsorption of antibody to the surface of Sdlba3b-expressing L. lactis. Langmuir linear regression method (CAb/Γ=CAb/Γmax+1/(K×Γmax)) was used to fit the Langmuir equation to the data and maximal amount of adsorbed antibody; Γmax was calculated.
The amount of antibody adsorbed on 1×108 cells was calculated and plotted against the concentration of antibody, shown in
Langmuir linear regression method was applied and yielded the equation CAb/Γ=6,837×CAb+22,688 (r2=0.9897). From this equation, the maximum amount of adsorbed antibody was calculated to be 0.146 μg per 1×108 cells.
Immobilization of Heterologous Protein Constructs Containing TNFα-Binding Z Domain or Fc-Binding B Domain on Non-GMO Carrier Microorganism L. lactis
Overnight cultures of L. lactis NZ9000 harboring plasmid pSDLBA3a or pSDZ-TNF were diluted in 10 ml of fresh GM-17 medium, grown to optical density A600=0.5-0.8 and induced with 25 ng/ml nisin. Three hours after induction cultures were centrifuged at 5000 g for 10 min. Supernatant containing secreted protein construct was separated from the cells and filtered through 0.22 μm Minisart filter (Sartorius). Overnight culture of non-recombinant carrier microorganism L. lactis NZ9000 was grown in fresh GM-17 medium and centrifuged. Supernatant was separated from cells.
500 μL of cell-free supernatant from culture expressing either TNFα-binding fusion peptide or Fc-binding fusion peptide were added to approximately 107 of non-recombinant L. lactis cells and incubated for 2 h at room temperature. Next, cells were separated from supernatants by 3 min centrifugation at 5000 g and resuspended in 500 μL of TBS. Next, either 10 μL of Alexa 488-labelled TNFα or 2 μg of Alexa Fluor 488 conjugated rabbit anti-mouse antibody (Invitrogen; A-11059) was added to the suspensions and incubated 2 hours at room temperature with constant shaking at 100 rpm. Cells were than washed three times with 200 μL 0.1% TBST and resuspended in 500 μL of TBS. For control, non-recombinant L. lactis without previous incubation with cell-free supernatants from culture expressing either TNFα-binding fusion peptide or Fc-binding fusion peptide were stained as described. Stained sample and control cells were analyzed with FACS Calibur (Becton Dickinson Inc.) flow cytometer. At least 100,000 bacterial cells were counted for each sample. Cells were gated using FSC vs. SSC to isolate the bacterial cells.
The results are shown in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10000074.4 | Jan 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP11/00019 | 1/5/2011 | WO | 00 | 7/16/2012 |
