FimH MUTANT, COMPOSITION CONTAINING THE SAME, AND USE OF THE SAME

Abstract

To provide a vaccine that can induce highly inhibitory antibody to bacterial infection caused by Escherichia coil.SOLUTION: There is provided a polypeptide including an FimH lectin domain including at least one amino acid mutation which makes the FimH lectin domain become a three-dimensional structure having low affinity to mannose. Further there are provided a pharmaceutical composition containing such a polypeptide, and a method of performing stimulation of immune response by administration of polypeptide in a subject who needs it.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the fields of medical microbiology and vaccines. In particular, the present invention relates to Fim At least one of the H lectin domains is placed in a conformation that has low affinity for mannose. and upon administration to a subject, the FimH lectin domain contains two amino acid mutations, Polypeptides that induce high levels of antibody-mediated inhibition of E. coli adhesion to epithelial cells The present invention further relates to compositions comprising such polypeptides, and methods for enhancing immune responses. The stimulation is achieved by administration of an immunogenic polypeptide in a subject in need thereof. Regarding. [Background technology]

[0002] The E. coli strains responsible for extraintestinal infections are called extraintestinal pathogenic E. coli. ExPEC is an infectious disease caused by pathogenic E. coli (ExPEC), which is a type of infection that is often treated as an outpatient or long-term treatment. It is the most common enteric gram-negative organism causing extraintestinal infections in medical and hospital settings. Typical extraintestinal infections caused by E. coli include urinary tract infections (UTIs), bacteremia, and sepsis. E. coli is the leading cause of severe sepsis, with high morbidity and mortality. This is the cause.

[0003] ExPEC, like other members of the Enterobacteriaceae family, are endemic to mucosal epithelia. They produce type I pili that aid in attachment to surfaces. These type I pili are found in members of the Enterobacteriaceae family. Type I pili are hair-like structures that arise from the surface of the host. The main components of type I pili are arranged in a right-handed helix. The FimA subunits are repeated to form a 1 μm long molecule with a central pore. The main subunit, FimA, formed a filament with a diameter of 7 nm. Both fimbriae contain the accessory protein subunits FimF, FimG, and The accessory protein subunit FimH is involved in the synthesis of mannose on the surface of eukaryotic cells. is a mannan-binding adhesin that promotes the attachment of type I fimbriated bacteria to serotype-containing glycoproteins, Represents a family of proteins that bind to a variety of targets, including mannan and fibronectin. Immunoelectron microscopy studies have demonstrated that FimH is strategically located at the distal end of type I pili and that It is believed that it forms a complex with FimG to form a flexible pilus structure. It was found that the ribs were also spaced longitudinally at various intervals along the length of the tube.

[0004] The FimH adhesin protein was shown to be effective in preventing UTI in various preclinical models. It has been shown to induce protection when used as a stimulant (Langermann S,et al.,1997,Science,276:607-611,Lange rmann S, et al.,2000,J Infect Dis,181:774 -778, O'Brien VP et al.,2016, Nat Microbio l,2:16196).

[0005] Lectin domain of the adhesin FimH binds to mannosylated receptors during Escherichia coli infection. has a low affinity for mannose (tension) and a high affinity for mannose (stretch / relaxation) ) can adopt two different conformations (Kalas et al. ,2017,Sci Adv 10;3(2)). The low affinity conformation suppresses bacterial motility. and promotes colonization of new tissues. The high affinity conformation is Furthermore, antibodies against the low affinity variants are able to bind to the urinary tract. It has been shown to block bacterial binding to epithelial cells and reduce CFU numbers in the bladder (Tch esnokoca,2011 Infect Immun.79(10):3895-9 04;Kisiela,2013 Proc Natl Acad Sci,19;11 0(47):19089-94).

[0006] WO 02102974 describes a number of FimH mutants, which All of these contain amino acid modifications in the canyon region of the molecule. Publication No. 02102974 discloses a method for the preparation of a mannose-interacting peptide having mutated mannose-interacting residues in the binding pocket. The location of this mutation shifts the FimH mutant to a more open position. maintains a conformational structure, thereby exposing epitopes that are inaccessible in the wild-type protein However, to the best of the inventors' knowledge, None of these variants are being pursued further as vaccine candidates in clinical trials. , only wild-type FimH is used. Summary of the Invention

[0007] Therefore, it is possible to produce highly inhibitory antibodies against bacterial infections caused by E. coli. There remains a need in the art for inducible vaccines.

[0008] In a first aspect, the present invention provides a method for the preparation of a medicament for the treatment of atopic dermatitis comprising administering to a patient a medicament for the treatment of atopic dermatitis, ... A polypeptide containing the FimH lectin domain that contains an amino acid other than phenylalanine (F). Offer petite.

[0009] In a second aspect, the present invention relates to a method for the preparation of a medicament for the treatment of a pulmonary artery disease comprising administering to a patient a therapeutically effective amount ... The FimH receptor according to the first aspect, further comprising an amino acid other than phenylalanine (F). The present invention provides a polypeptide comprising a tin domain.

[0010] In a third aspect, the present invention provides a polynucleotide encoding a polypeptide according to the present invention. Provide punchlines.

[0011] In a fourth aspect, the present invention provides a vector comprising a polynucleotide according to the present invention. Provide.

[0012] In a fifth aspect, the present invention provides a method for the preparation of a nucleic acid sequence comprising a polynucleotide or vector according to the present invention, A host cell is provided.

[0013] In a sixth aspect, the present invention relates to a polypeptide, polynucleotide or A pharmaceutical composition comprising the vector is provided.

[0014] In a seventh aspect, the present invention relates to an immunogen against bacteria of the family Enterobacteriaceae. A polypeptide according to the invention, a polynucleotide according to the invention for use in inducing an immune response The present invention further provides a method for the preparation of a medicament for the treatment of a cancer, a vector according to the present invention, or a pharmaceutical composition according to the present invention. Method for treating or preventing a condition associated with enterobacteria in a subject in need thereof The method comprises the steps of: The present invention also relates to a method comprising administering a vector according to the present invention or a pharmaceutical composition according to the present invention.

[0015] In an eighth aspect, the present invention provides a method for producing a polypeptide comprising the steps of: The polypeptide is expressed from a recombinant cell containing the polynucleotide and / or vector of the present invention. and optionally recovering the polypeptide, which may include optionally Optionally, the method further comprises formulating the polypeptide into a pharmaceutical composition. do. [Brief description of the drawings]

[0016] [Figure 1A] Functionality of antibodies induced by different FimH variants. Wistar rats received four intramuscular immunizations on days 0, 7, 10, and 18 with 60ug / dose of different FimH variants combined with non-Freund's adjuvant (Speedy-rat model, Eurogentec). Serum samples were obtained on days 0 (pre-immunization) and 28 (post-immunization). A) Initial experiments with different FimH variants (indicated below the graph). Inhibitory antibody titers (IC50) were calculated based on a four-parameter logistic regression model fitted to a 12-step dilution curve. Data represent the average of duplicate serum samples from two animals / group. [Figure 1B]Functionality of antibodies induced by different FimH variants. Wistar rats received four intramuscular immunizations on days 0, 7, 10 and 18 with 60ug / dose of different FimH variants combined with non-Freund's adjuvant (Speedy-rat model, Eurogentec). Serum samples were obtained on days 0 (pre-immunization) and 28 (post-immunization). B) Separate experiments with FimH mutants F144V, F71Y and F144V / F71Y double mutant. Inhibitory antibody titers (IC50) were calculated based on a four-parameter logistic regression model fitted to a six-step dilution curve. The graph shows the individual IC50 titers pre- and post-immunization of serum samples measured in duplicate, as well as the GMT (geometric mean titer) ± 95% CI (confidence interval). LOD: limit of detection. [Figure 2A] Conformational states of different FimH lectin domain variants in the presence and absence of mannosidic ligands as determined by NMR spectroscopy. The left panel shows 15N HSQC NMR spectra of uniformly 15N-labeled FimH L D variants in the low affinity state (L) when no mannosidic ligand is bound (i.e., apo state), and the right panel shows the spectrum in the high affinity state (H) when the mannosidic ligand is bound (i.e., liganded state). The key amino acid residues that undergo chemical shifts upon binding of the mannosidic ligand are boxed. Residues were identified from the publicly available NMR spectrum from E. coli K12 (Rabbani S et al, J. Biol. Chem., 2018, 293(5):1835-1849), except for residues 1 and 2, which were specific to E. coli 23-10, indicating that the wild-type FimHLD 23-10 protein has a slightly different conformation in the apo state compared to E. coli K12 FimHLD. [Figure 2B]Conformational states of different FimH lectin domain variants in the presence and absence of mannosidic ligands as determined by NMR spectroscopy. The left panel shows 15N HSQC NMR spectra of uniformly 15N-labeled FimH L D variants in the low affinity state (L) when no mannosidic ligand is bound (i.e., apo state), and the right panel shows the spectrum in the high affinity state (H) when the mannosidic ligand is bound (i.e., liganded state). The key amino acid residues that undergo chemical shifts upon binding of the mannosidic ligand are boxed. Residues were identified from the publicly available NMR spectrum from E. coli K12 (Rabbani S et al, J. Biol. Chem., 2018, 293(5):1835-1849), except for residues 1 and 2, which were specific to E. coli 23-10, indicating that the wild-type FimHLD 23-10 protein has a slightly different conformation in the apo state compared to E. coli K12 FimHLD. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention relates to a novel polypeptide comprising a FimH lectin domain, The cutin domain has a conformation that has a low affinity for mannose (referred to herein as " The present invention provides a polypeptide that is "locked" in a "low affinity conformation" (also referred to as a "low affinity conformation"). The invention relates to a method for the production of FimH antigen, which is in a three-dimensional structure with low affinity for mannose, and which binds to mannosides via the mannosides. This is based, in part, on the observation that it is possible to induce antibodies capable of inhibiting adhesion to the host. These antibodies are highly inhibitory and have enhanced efficacy in the prevention or treatment of bacterial infections. As used herein, the FimH lectin domain having the F71Y mutation is, for example, For example, in a vaccine against UTI, for example to prevent or reduce recurrent UTI. It has a surprisingly good combination of desirable properties that make it very suitable for use. It was found that.

[0018] Thus, in a first aspect, the present invention provides a method for the preparation of a medicament comprising administering to a subject the medicament a medicament comprising the amino acid sequence of position 71 of the reference amino acid sequence of SEQ ID NO:1. The FimH lectin domain contains an amino acid other than phenylalanine (F) at the position corresponding to The present invention provides a polypeptide, preferably an immunogenic polypeptide, comprising the polypeptide.

[0019] In a second aspect, the present invention further comprises a method for the preparation of a medicament comprising administering to a subject a medicament the steps of: and Fi containing an amino acid other than phenylalanine (F) at both positions corresponding to position 144. A polypeptide, preferably an immunogenic polypeptide, comprising an mH lectin domain is provided. This "double mutant" remains locked in a low affinity conformation and therefore Similarly, antibodies can be induced that can inhibit mannoside-mediated adhesion. In addition to inducing anti-inflammatory antibodies, the F71Y and F144V variants in the FimH lectin domain The double mutant is unable to switch back to the high affinity conformation, thereby It has now been found that the double mutant confers surprisingly high stability. The inability of the double mutant to switch back to the high affinity conformation of For example, in a vaccine against UTI, for example to prevent or reduce recurrent UTI. These are very desirable properties that make them very suitable for use in applications where Test whether the conformation is stable during storage or under other storage or use conditions. This is because it is certain that no additional quality or stability controls will be required to

[0020] As used herein, amino acid positions 71 and 144 are respectively represented by SEQ ID NOs: 1. In the amino acid sequences of the invention other than number 1, preferably the amino acid positions 71 and 144 respectively, sequence alignment, preferably using Cluster with default settings In the lW(1.83) sequence alignment, the 7th amino acid of SEQ ID NO:1 of other amino acid sequences It is present at positions corresponding to positions 1 and 144. Using a sequence alignment algorithm, the FimH lectin domain other than SEQ ID NO:1 One would know how to identify corresponding amino acid positions in an amino acid sequence.

[0021] Throughout this application, a phenylalanine atom is shown at a position corresponding to position 71 of the amino acid sequence of SEQ ID NO:1. Polypeptides of the invention containing the FimH lectin domain containing amino acids other than lanin (F) The sequence represented by SEQ ID NO: phenylalanine at both positions, corresponding to positions 71 and 144, respectively, of the amino acid sequence of Polypeptides of the invention containing FimH lectin domains containing amino acids other than nin (F) is referred to herein as "FimH(F71mut / F144mut)."

[0022] In certain embodiments, FimH(F71mut) corresponds to position 71 of SEQ ID NO:1. and an amino acid selected from the group consisting of tyrosine (Y) and tryptophan (W) at the corresponding position. nothing.

[0023] In certain embodiments, FimH(F71mut) corresponds to position 71 of SEQ ID NO:1. It contains a tyrosine (Y) at the corresponding position.

[0024] In certain embodiments, FimH(F71mut / F144mut) is Valine (V), isoleucine (I), and leucine (L) at the position corresponding to position 144 of No. 1; An amino acid selected from the group consisting of glycine (G), methionine (M), and alanine (A). Contains acetic acid.

[0025] In certain embodiments, FimH(F71mut / F144mut) is It contains a valine (V) at the position corresponding to position 144 of no. 1.

[0026] In certain embodiments, FimH(F71mut) and FimH(F71mut) / F144mut) At least one of the lectin domains has at least one identical sequence to SEQ ID NO: 1 Approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or have an amino acid sequence with 99% sequence identity.

[0027] In one embodiment, FimH(F71mut) or FimH(F71mut / F14 4mut) confers low affinity to the FimH lectin domain for mannose conformations In one embodiment, FimH(F71mut) comprises the sequence In one embodiment, FimH is mutated at a position corresponding to position 71 of FimH. (F71mut / F144mut) is the position corresponding to positions 71 and 144 of SEQ ID NO: 1 It has been mutated in

[0028] In this context, the FimH lectin domain has low affinity for mannose. In the present specification, for example, under the conditions specified in Example 2, surface plasmon resonance The dissociation constant (K) is at least 1000 nM when measured using a D ) at Mannoshi It is defined as the FimH lectin domain that binds to the mannose ligand. A FimH lectin domain with high affinity is defined herein as a domain that binds to 1 K below 00nM D The FimH lectin domain is defined as the domain that binds mannoside ligands in Typically, wild-type FimH falls into this category. K from 0nM to 1000nM D The FimH lectin domain having the following structure is defined herein as: It is said to have a moderate affinity for mannose.

[0029] In this context, "mutant," "mutated," "mutated," or "substitution" or "substituted" The term "has" refers to the corresponding parent molecule (here, Fim having F at positions 71 and 144). A polypeptide containing an H lectin domain This means that there are additional amino acids present. Such parent molecules are physically It may be present as an amino acid or in the form of a nucleic acid encoding such a polypeptide, The corresponding nucleic acid sequence that encodes the amino acid sequence may be used simply in silico or theoretically. Thus, mutations or substitutions in this context also include, for example, When a protein is expressed from a nucleic acid that has been synthesized to encode the mutation or substitution, the corresponding even if the nucleic acid encoding the parent molecule was not actually prepared first during the process. For example, even when a nucleic acid molecule is prepared entirely by chemical synthesis, It is thought that.

[0030] Preferably, the mutation is a substitution of a single amino acid residue. and at least one of amino acid residues corresponding to positions 71 and 144 of SEQ ID NO:1. Preferably, the mutation is a substitution of the phenyl at the position corresponding to position 71 of SEQ ID NO:1. Alanine (F) is substituted with another amino acid. Preferably, the polypeptide of the invention has phenylalanine (F) at a position corresponding to position 144 of SEQ ID NO:1; In the case of FimH(F71mut), the nucleotide sequence corresponding to position 71 of SEQ ID NO:1 is further replaced by The corresponding position is preferably selected from the group consisting of tyrosine (Y) and tryptophan (W). In one preferred embodiment, FimH (F71m ut) contains a phenylalanine (F) to tyrosine (Y) substitution at position 71. In certain embodiments, FimH(F71mut) is a non-mutated nucleotide that contains a tyrosine at position 71. It is a naturally occurring polypeptide. In certain embodiments, FimH(F71mut) is , which has a tyrosine instead of the naturally occurring phenylalanine at position 71 ("FimH( In certain embodiments, FimH(F71mut) is , having a tyrosine at position 71 in place of a naturally occurring amino acid other than phenylalanine ("FimH(x71Y)" where x is phenylalanine or tyrosine in the parent molecule. (amino acids other than cyclic amines).

[0031] In one embodiment, FimH(F71mut / F144mut) is selected from the group consisting of amino acid sequence 7 of SEQ ID NO:1. The mutation at position 71 is preferably The mutation at position 144 is preferably 144 of SEQ ID NO:1. Preferably, the mutation is a substitution of an amino acid residue corresponding to position 144 of SEQ ID NO:1. A substitution of a phenylalanine (F) amino acid residue at the corresponding position. The amino acid at position 144 is valine (V), isoleucine (I), leucine (L), and glycine (G). An amino acid selected from the group consisting of lysine (G), methionine (M), and alanine (A). In one preferred embodiment, FimH (F71mut / F 144mut) is a substitution of phenylalanine (F) to valine (V) at position 144 Includes.

[0032] In certain embodiments, FimH(F71mut / F144mut) is and valine at position 144. In the form FimH(F71mut / F144mut) is naturally occurring at position 71 It has tyrosine instead of phenylalanine and the naturally occurring phenylalanine at position 144. In certain embodiments, FimH (F71mut / F144mut) is a nucleotide that replaces a naturally occurring amino acid other than phenylalanine at position 71. and tyrosine at position 143, and phenylalanine at position 144 instead of a naturally occurring amino acid other than phenylalanine. Contains valine.

[0033] Full length FimH FL The two domains are separated by a short tetrapeptide loop linker: Terminal pyrin domain (FimH PD ) connected to the N-terminal lectin domain (FimH L DIn a particular embodiment of the invention, the FimH receptor according to the invention is A polypeptide that contains a pilin domain does not contain a FimH pilin domain. In an embodiment, the polypeptide comprising the FimH lectin domain according to the present invention is In one embodiment, FimH(F71mut) or F imH(F71mut / F144mut) has the FimH lectin domain, About FimH(F71mut) and FimH(F71mut / F144mut) The present invention relates to a full-length FimH polypeptide comprising the amino acid sequence defined hereinabove. In one particular embodiment of the invention, a polyclonal antibody comprising the FimH lectin domain according to the invention is The peptide is a fusion polypeptide of the FimH lectin domain fused to another polypeptide. The other polypeptide may be any polypeptide of interest, which may be However, it is not necessary that the FimH protein is related to FimH or that it is naturally associated with FimH. In one embodiment, the polypeptide comprising the FimH lectin domain of the present invention is A fusion polypeptide further comprising an H pilin domain and further comprising another polypeptide. The other polypeptide may be any polypeptide of interest, including, for example, Fim The fusion polypeptides of the present invention do not necessarily have to be related to FimH or naturally associated with FimH. The peptides can also be used as immunogens, for example, for vaccination purposes.

[0034] In one embodiment, FimH FL The polypeptide has at least about 80% identity with SEQ ID NO:2. %, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% %, or 99% sequence identity, thereby preferably Positions 71 and 144 are FimH(F71mut) and FimH( F71mut / F144mut) as defined herein above In certain embodiments, FimH FL The polypeptide may be any of the polypeptides described herein. It may comprise a sequence having SEQ ID NO:2, except for the F71Y and optionally the F144V substitutions. In another embodiment, the polypeptide has a sequence similar to, or preferably at least similar to, SEQ ID NO:4. At least about 80%, 85%, 90%, 91%, or 92% of the amino acids 22 to 300 , 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity FimH FL whereby positions 71 or 71 and 144 are Fi mH(F71mut) and FimH(F71mut / F144mut) are described herein. In certain embodiments, FimH comprises the amino acid residues defined above. F L The polypeptide comprises, except for the F71Y and / or F144V substitutions described herein, It may comprise SEQ ID NO: 4 or a sequence having at least amino acids 22-300 thereof.

[0035] In certain embodiments, FimH FL The polypeptides are herein incorporated in their entirety. SEQ ID NOs: 23 to 45 and 5 described in U.S. Patent No. 6,737,063. 5 with at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of By this, positions 71 or 71 and 144 are FimH(F71mut) and FimH(F71mut / F144mut) is defined herein above as It contains amino acid residues.

[0036] The FimH polypeptide is highly conserved among various strains of E. coli. It is also highly conserved among a wide range of Gram-negative bacteria. The high degree of conservation of FimH among E. coli strains As a result, FimH polypeptides derived from one strain are not recognized by the FimH lectin in other strains. inhibit or prevent the E. coli strain from binding to cells and / or from being inhibited by other E. coli strains Inducing an antibody response that provides protection against and / or treatment of infections caused by Thus, in one embodiment, FimH FL The polypeptide has the sequence At least about 80% with number 5, or preferably with at least amino acids 22 to 300 thereof %, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity and whereby positions 71 or 71 and 144 are each independently imH (F71mut) and FimH (F71mut / F144mut) In certain embodiments, FimH comprises the amino acid residues defined above in the literature. FL The polypeptide comprises a F71Y substitution as described herein, and optionally a F144V substitution. It may comprise a sequence having SEQ ID NO:5 or at least amino acids 22 to 300 thereof, excluding substitutions. In another embodiment, FimH FL The polypeptide has a sequence similar to that of SEQ ID NO:6. 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity and whereby positions 71 or 71 and 144 are, respectively , FimH(F71mut) and FimH(F71mut / F144mut) In certain embodiments, the amino acid residues are as defined hereinabove. mH FL The polypeptide may comprise a F71Y substitution as described herein, and optionally a F144 It may comprise a sequence having SEQ ID NO:6, except for the V substitution.

[0037] In certain embodiments, FimH is preferably E. coli FimH.

[0038] As used herein, the term "periplasmic chaperone" refers to a common binding enzyme. Form complexes with various chaperone-binding proteins through recognition of a pitope(s) Chaperones are defined as proteins located in the bacterial periplasm that can mediate the function of the reported that proteins exported from bacterial cells to the periplasm fold into their native conformation. The association of chaperone-binding proteins with chaperones acts as a template for the folding of the protein. It also protects the binding protein from degradation by proteases located in the periplasm. role, increasing their solubility in aqueous solutions and their association with the assembling pili. Chaperone proteins mediate such assembly, resulting in the proper order of incorporation. In Gram-negative bacteria, it is involved in the assembly of pili by binding to them but is not incorporated into the structure. FimC is a class of proteins that act as periplasmic chaperones for FimH. The FimC polypeptide for use in the present invention has at least one sequence similar to SEQ ID NO:3. At least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 9 9%, or about 100% sequence identity. In the present invention, the FimC polypeptide is described in U.S. Pat. A sequence similar to SEQ ID NO:29 in US Pat. No. 737,063, which is at least 70%, 75%, 80%, 8 5%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100% sequence identity The non-covalent complex of FimC and FimH has the amino acid sequence It is named H.

[0039] Thus, in a further aspect, the present invention relates to a method for the treatment of osteoclasts comprising administering to the patient a therapeutically effective amount of FimH (F71 mut) or FimH(F71mut / F144mut), as defined herein. A polypeptide further comprising the FimH pyrin domain or full-length FimH, as defined herein. and a FimC polypeptide which is

[0040] In one embodiment of the present invention, FimH(F71mut) or FimH(F71mut The lectin domain is a polypeptide further comprising the FimH pilin domain. This polypeptide forms a complex with FimC to form the FimCH complex. Form.

[0041] The inventors of the present application have identified several FimH lectin domains with different amino acid changes. They are generating variants and testing them for efficacy in various assays. (See examples).

[0042] FimH(F71mut) and FimH(F71mut / F14 4mut) were all able to form FimCH complexes.

[0043] In one embodiment, FimH(F71mut) or FimH(F71mut / F14 4mut) The lectin domain has at least about 80%, 85%, 90%, or FimH with 95%, 96%, 97%, 98%, or 99% sequence identity FL Polype a portion of a peptide, which optionally forms a complex with a FimC polypeptide, Formation of the FimCH complex. Final form of FimH FL The polypeptide, i.e., the mature F imH FL The polypeptide typically comprises, for example, amino acids 1 to 21 of SEQ ID NOs: 4 and 5. i.e., the mature F of SEQ ID NO: 4 or SEQ ID NO: 5 does not contain the signal peptide shown in imH FL Polypeptides contain amino acids 22 to 300 of these sequences, but typically It is understood that amino acids 1 to 21 are missing. FL Polypeptide Set For recombinant production, mature FimH containing the signal peptide is produced in a recombinant host cell. FL P The general secretory pathway that encodes the polypeptide and leads to the periplasmic location of the polypeptide. transport across the inner (cytoplasmic) membrane via the Although some of the more useful are the final mature forms, which can be isolated and used, for example, in pharmaceutical compositions, FimH FL In a polypeptide, a signal peptide typically serves to direct the polypeptide It is no longer present as a result of processing by the expressing recombinant cell.

[0044] In one embodiment, the FimCH complex has at least 80%, 85% , 90%, 95%, 96%, 97%, 98%, or at least about 99%, or 100 % sequence identity with the FimC protein as defined herein above. H(F71mut) or FimH(F71mut / F144mut) lectin domain In certain embodiments, the method comprises the steps of: , FimCH complexes have at least 80%, 85%, 90%, 95%, 90% or 90% homology with SEQ ID NO:3. 6%, 97%, 98%, or at least about 99%, or 100% sequence identity and FimH(F71mut) or FimH(F71mut) as defined herein above. is the FimH protein containing the FimH(F71mut / F144mut) lectin domain. Quality or FimH FL and a protein, thereby H FL The protein is preferably a polypeptide having a sequence similar to that of the polypeptide of the present invention (e.g., SEQ ID NO: 1, still including the signal peptide). Optionally, the FimCH complex comprises a F92 substitution at SEQ ID NO: 3. At least 80%, 85%, 90%, 95%, 96%, 97%, 98% or less of A FimC protein having at least about 99% or 100% sequence identity with a lectin FimH protein containing the F71(Y) and F144(V) substitutions in the FimH domain, or For example, F92(Y) and F92(Y) in SEQ ID NO: 4 or 5, which still contain the signal peptide. and F165(V) substitutions.

[0045] In the full-length FimH, which still contains the signal peptide, amino acid position 92 corresponds to the Fim Corresponding to amino acid position 71 in the H lectin domain, position 165 corresponds to amino acid position 144. Those skilled in the art will know how to use the amino acid sequence alignment algorithms defined above to identify the corresponding amino acid positions in the full-length FimH amino acid sequence and the FimH lectin domain amino acid sequence.

[0046] In one embodiment, a complex comprising the E. coli chaperone FimC and a polypeptide comprising FimH (F71mut) or FimH (F71mut / F144mut) can be formed by co-expressing the polypeptide comprising FimH (F71mut) or FimH (F71mut / F144mut) with FimC from recombinant cells.

[0047] In one embodiment, the FimCH complex comprises FimC originating from one bacterial strain, while FimH originates from a different bacterial strain. In another embodiment, the FimCH complex comprises both FimC and FimH originating from the same bacterial strain. In certain embodiments, FimH, or FimC, or both FimH and FimC may be artificial sequences not derived from an actual bacterial isolate that occurs in nature; for example, they may also be based on the consensus sequence or combination of natural isolates.

[0048] In one embodiment, the FimCH complex comprises at least one polypeptide comprising a His tag. In one embodiment, the full-length FimH described herein comprises a His tag, or the FimC described herein comprises a His tag. Preferably, in the FimCH complex, FimC comprises a His tag. As used herein, the His tag ​​​​​​​​​​​​​is a stretch of histidine (His) residues, e.g., six His residues, which is , can be added internally or, preferably, to the N-terminus or C-terminus of the protein. Such tags are well known for their use to facilitate purification.

[0049] In a further aspect, the present invention relates to a method for the treatment of osteoarthritis of the ovarian tumor, comprising administering to the patient a therapeutically effective amount of FimH (F71m Polypeptides containing the FimH (F71mut / F144mut) or FimH (F71mut / F144mut) lectin domains The polynucleotide relates to a polynucleotide that encodes a nucleotide sequence operatively associated with the It may be preceded by a linked promoter. In certain embodiments, the promoter The promoter is endogenous to the FimH coding sequence. The motor drives the expression of FimH in bacteria of the Enterobacteriaceae family. In another embodiment, the promoter is an endogenous promoter that encodes the FimH gene. The sequences are heterologous to the gene sequences and are known to those skilled in the art for use in, for example, recombinant expression systems. A strong promoter is used, for example pET-D, which contains an inducible Lac promoter. The UET vector is a vector encoding FimH(F71mut) or FimH(F71mut) of the present invention. / F144mut) polypeptide and / or FimC polypeptide Inducible promoters such as the Lac promoter or the Tac promoter can be used. In the case of a polynucleotide vector, expression can be induced using IPTG. The leotide is isolated from its natural environment. The present invention provides an isolated polynucleotide according to the present invention. A polynucleotide may be any form of nucleic acid, e.g. For example, it may be DNA or RNA, preferably DNA. One or more nucleotides that are not present in the polynucleotide encoding FimH present Preferably, the polynucleotide may include a nucleotide sequence at its 5' end and / or 3' end. One or more nucleotides that are not present in the naturally occurring polynucleotide encoding FimH The sequences of the encoded mature FimC and / or FimH preferably have A signal peptide is contained in the polypeptide encoded by each polynucleotide. and the signal peptide may be preceded by the FimC and / or FimH polypeptides, respectively. endogenous signal peptides for the peptides (i.e., those proteins that are naturally They may be heterologous signal peptides, i.e., signal peptides present in the host. That is, even if it is a signal peptide derived from another protein or a synthetic signal peptide, Signal peptides are useful for periplasmic expression, but are typically cleaved off. It is not present in the FimC and / or FimH, respectively, that are ultimately produced and purified.

[0050] In a further aspect, the present invention relates to a FimH(F71mut) or FimH(F A vector comprising a polynucleotide encoding a polypeptide of In certain embodiments, the vector is a plasmid or a viral vector. The vector is preferably a DNA, e.g., a DNA In certain embodiments, the vector is in the form of a promoter- operably linked to the polynucleotide of the present invention, which is The promoter means that the polypeptide of the present invention is under the control of the promoter. The gene may be located upstream of the loading polynucleotide, for example in an expression cassette of a plasmid.

[0051] In a still further aspect, the present invention relates to a FimH(F71mut) or F A polynucleotide encoding the imH(F71mut / F144mut) polypeptide or The present invention relates to a host cell comprising the vector described herein. A polynucleotide encoding the FimH (F71mut / F144mut) or FimH (F71mut / F144mut) polypeptide The leutidine can be introduced into cells by standard molecular biology methods. The nucleic acid may be extrachromosomal, for example on a plasmid or other vector, or It may be integrated into the genome of the host cell. Preferably, the nucleic acid encodes a protein. is operable with a sequence (e.g., a promoter) that drives expression of the nucleic acid in a host cell. The promoter may be a constitutive promoter or its activity may be can be regulated, for example by changes in temperature or by the regulation of certain chemicals or proteins within the cell. It may be a promoter that can be repressed or induced under certain conditions, such as the presence of all of which are well known in the art.

[0052] The host cell may be an isolated cell. The host cell may be in culture medium, e.g., in a culture medium containing The cells can be cultured in a culture vessel such as a bioreactor. 71mut) or FimH (F71mut / F144mut) polypeptide The cell may be any microbial, prokaryotic, or eukaryotic cell suitable for expressing polynucleotides. Preferably, the host cell is a bacterial host cell. Preferably, the bacterial host cell is a Gram-positive cell. Preferably, the host cell is a microbial strain selected from the group consisting of Escherichia coli (E. coli) and Klebsiella (K. Preferably, the host cell is selected from Escherichia coli.

[0053] If necessary and / or desired, the FimH(F71mut) or or the FimH(F71mut / F144mut) polypeptide of the present invention. FimH (F71mut) or FimH (F71mut / F144mut) polypeptide. The polynucleotide to be administered can be made pharma- ceutical by adding a pharma- ceutical acceptable carrier. It can be incorporated into the active mixture.

[0054] Therefore, in a further aspect, the present invention also provides the FimH(F71mut) of the present invention. or FimH(F71mut / F144mut) polypeptide, or the Fim of the present invention. H(F71mut) or FimH(F71mut / F144mut) polypeptide Compositions, preferably pharmaceutical compositions, containing the encoding polynucleotide are provided.

[0055] The (pharmaceutical) composition of the present invention may comprise a carrier, a filler, a preservative, a solubilizer, and / or a diluent. The pharmaceutical composition may contain any pharma- ceutically acceptable excipient, including saline and aqueous dextrose. and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. These include starch, glucose, lactose, sucrose, gelatin, malt, rice, and small grains. Wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate , talc, sodium chloride, dried skim milk, glycerol, propylene, glycol Examples of suitable pharmaceutical carriers are known and are described, for example, in It is described in books and manuals.

[0056] In certain embodiments, the compositions of the invention contain one or more buffers, such as Tris Buffered saline, phosphate buffer, HEPES, or sucrose phosphate glutamate buffer The liquid further comprises:

[0057] In certain embodiments, the compositions of the invention contain one or more salts, such as tris-salts. Acid salts, sodium chloride, calcium chloride, potassium chloride, sodium phosphate, glutamine monosodium phosphate, and aluminum salts (e.g., aluminum hydroxide, aluminum phosphate, The aluminum salts may further comprise potassium aluminum sulfate, potassium aluminum sulfate, or a mixture of such aluminum salts.

[0058] The compositions of the present invention can be used to induce an immune response in a host to which the compositions are administered. Therefore, the composition of the present invention is capable of inhibiting enterobacteria (Enterobacteria). against infections caused by bacteria of the family Bacteriaceae, preferably Klebsiella Klebsiella or E. coli, more preferably E. coli. and thus can be used as a vaccine against infectious diseases caused by and may include any additional components suitable for use in a vaccine. An additional optional component of the composition is an adjuvant, as described herein.

[0059] In certain embodiments, the compositions of the present invention contain preservatives, such as phenols, chlorides, etc. Further comprising benzethonium, 2-phenoxyethanol, or thimerosal. In some embodiments, the (pharmaceutical) composition of the present invention contains 0.001% to 0.01% of a preservative. In another embodiment, the (pharmaceutical) composition of the invention is free of preservatives.

[0060] In certain embodiments, the compositions of the invention are suitable for the intended route of administration to a subject. For example, the compositions of the present invention can be administered subcutaneously, parenterally, orally, intradermally, or intravenously. Can be formulated to be suitable for dermal, colorectal, intraperitoneal, vaginal, or rectal administration In certain embodiments, the pharmaceutical composition is administered intravenously, orally, bucally, intraperitoneally, intranasally, intratracheally, or intravenously. The formulation may be for intravenous, subcutaneous, intramuscular, topical, intradermal, transdermal, or pulmonary administration, preferably intramuscular administration. It can be made into

[0061] The compositions of the invention can be included in a container, pack, or dispenser together with instructions for administration. This can be done.

[0062] In certain embodiments, the compositions of the present invention can be stored prior to use, e.g., the compositions can be The product may be stored frozen (e.g., at about -20°C or at about -70°C) or under refrigerated conditions (e.g., It may be stored at about 2-8°C, for example, about 4°C, or it may be stored at room temperature.

[0063] In certain embodiments, the pharmaceutical compositions of the invention herein further comprise an adjuvant. As used herein, the term "adjuvant" refers to a compound that is used in combination with the composition of the present invention. augment, enhance, or increase the immune response to FimH when administered to, or as part of, and / or boost, but when the adjuvant compound is administered alone, the conjugate An adjuvant refers to a compound that does not generate an immune response against Gate and / or FimH. For example, lymphocyte recruitment, stimulation of B cells and / or T cells, and stimulation of antigen-presenting cells. The immune response can be enhanced by several mechanisms, including:

[0064] In certain embodiments, the pharmaceutical composition of the invention comprises an adjuvant or The composition of the present invention is administered in combination with an adjuvant. The adjuvant may be administered prior to, simultaneously with, or after administration of the immunogenic composition. In certain embodiments, FimH(F71mut) or FimH(F 71mut / F144mut) and the adjuvant are administered in the form of a single composition.

[0065] In another embodiment, the pharmaceutical composition of the present invention does not include an adjuvant. Not administered in combination with

[0066] Specific examples of adjuvants include aluminum salts (alum) (aluminum hydroxide) Aluminum phosphate, aluminum sulfate, and aluminum oxide. nanoparticles containing alum or nanoalum formulations), calcium phosphate (e.g. Masson JD et al,2017,Expert Rev Vaccin es 16:289-299), monophosphoryl lipid A (MPL), or 3-de-O- Acylated monophosphoryl lipid A (3D-MPL) (see, for example, UK Patent No. 2220211 No. 0971739, No. 1194166, U.S. Pat. No. 64919 19), AS01, AS02, AS03, and AS04 (all GlaxoSmithKline SmithKline, e.g., European Patent No. 1126876 for AS04, US AS02 is European Patent No. 0671948 and AS03 is European Patent No. 0671948. 761231, U.S. Pat. No. 5,750,110), imidazopyridine compounds (see WO 2007 / 109812), imidazoquinoxaline compounds (see WO 2007 / 109813), delta-inulin (e.g. , Petrovsky N and PD Cooper, 2015, Vaccine 33:5920-5926), STING-activating synthetic cyclic dinucleotides (e.g., U.S. Patent Application Publication No. 20150056224), lecithin and carbomer homopolymers combinations with polymers (e.g., U.S. Pat. No. 6,676,958), as well as saponins, e.g. For example, Quil A and QS21 (e.g., Zhu D and W Tuo, 2016 ,Nat Prod Chem Res 3:e113(doi:10.4172 / 23 29-6836.1000e113), but are not limited to: and optionally combined with QS7 (Kensil et al., in Vac cine Design:The Subunit and Adjuvant App roach(eds. Powell & Newman, Plenum Press, N Y, 1995), U.S. Patent No. 5,057,540. In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). In certain embodiments, the adjuvant is Quil-A, e.g., Brenntag ( These include those commercially available from QuilA (now Croda) or Invivogen. is a saponin derived from the Quillaja saponaria Molina tree. These saponins share a common triterpenoid structure. Saponins belong to the group of triterpenoid saponins, which have the ability to inhibit T-dependent antigens as well as T-non-dependent antigens. Potent adjuvant responses to dependent antigens and potent cytotoxic CD8+ lymphocytes These are known to induce responses and enhance responses to mucosal antigens. In combination with sterols and phospholipids, they can form immunostimulating complexes (ISCOMs). The QuilA adjuvant enhances antibody-mediated immunity against a wide range of antigens from different sources. In certain embodiments, the agonist may activate both the inflammatory and cell-mediated immune responses. The adjuvant is AS01, preferably AS01B. S01 is MPL (3-O-deoxyglucose). Suacyl-4'-monophosphoryl lipid A), QS21 (Quillaja Saponaria Molina, Fraction 21), and liposomes. AS01 is commercially available (GSK) or is available from the US Pat. No. 6,313,663, which is incorporated herein by reference. Certain adjuvants may be prepared as described in WO 96 / 33739. Examples include emulsions, which are mixtures of two immiscible fluids, e.g., oil and water. one of them is suspended as droplets inside the other and stabilized by a surfactant Oil-in-water emulsions have water forming a continuous phase that surrounds droplets of oil. However, water-in-oil emulsions have oil forming the continuous phase. Certain emulsions are , squalene (a metabolisable oil). Certain adjuvants include block copolymers It contains two monomers that cluster together to form a block of repeating units. Block copolymers, squalene, and microparticles are copolymers formed when An example of a water-in-oil emulsion that contains a polymer stabilizer is TiterMax®. It is commercially available from Sigma-Aldrich. The emulsion may be combined with additional immune stimulating components, such as TLR4 agonists. One particular adjuvant is MF59 (see, e.g., European Patent No. 0 See U.S. Patent No. 399843, U.S. Patent No. 6,299,884, and U.S. Patent No. 6,451,325. (e.g., squalene or water-in-water emulsions, which are also used in AS03) peanut oil, and optionally an immunostimulant, e.g., monoamine ester in AS02. Combined with phosphoryl lipid A and / or QS21 (Stoute et al. (See, e.g., 1997, N. Engl. J. Med. 336, 86-91). Further examples of bunts include immunostimulants, such as M in AS01E and AS01B. A liposome containing PL and QS21 (see, e.g., U.S. Patent Application Publication No. 2011 / 0133625). No. 0206758. Other examples of adjuvants include CpG, and imidazoquinolines (e.g. For example, imiquimod and R848. For example, Reed G, et al., 2013 , Nature Med, 19:1597-1608.

[0067] In certain embodiments, the adjuvant is a saponin, preferably Quillaja saponin. In certain embodiments, the water-extractable fraction of saponins obtained from Naria The adjuvant includes QS-21.

[0068] In certain embodiments, the adjuvant is a toll-like receptor 4 (TLR4) antagonist. TLR4 agonists are well known in the art and include, for example: Ireton GC and SG Reed,2013,Expert Rev Va See, e.g., 12:793-807. The adjuvant is a TLR4 agonist, including lipid A, or an analog or derivative thereof. It is.

[0069] For example, adjuvants containing TLR4 agonists can be formulated in a variety of ways, e.g., water-in-oil formulations. (w / o) emulsion or oil-in-water (o / w) emulsion (e.g., MF59, AS0 3) Emulsions, stable (nano)emulsions (SEs), lipid suspensions, lipo virosomes, (polymeric) nanoparticles, alum adsorbed, aqueous The immunomodulatory molecule and / or immunosuppressant in the adjuvant can be formulated in a folate formulation (AF) or the like. Various delivery systems for antigens are presented (e.g., Reed et al., 2013 (supra) , Alving CR et al, 2012, Curr Opin Immunol 24:310-315).

[0070] The immunostimulatory TLR4 agonist may optionally contain other immunomodulatory components, such as saponins. (e.g., QuilA, QS7, QS21, Matrix M, Iscoms, Isco matrix, aluminum salts, and activators for other TLRs (e.g., imidazolium salts). zoquinoline, flagellin, CpG, dsRNA analogs, etc.) (See, e.g., Reed et al., 2013, supra).

[0071] As used herein, the term "Lipid A" refers to the hydrophobic lipid portion of the LPS molecule. This part contains glucosamine and is linked by a ketosidic bond to the inner core of the LPS molecule. In gram-negative bacteria, LPS molecules are linked to keto-deoxyoctulosonate. The synthesis of LPS and lipid A structures is outlined in, for example, Raetz, 1993, J. Bacteriology 175:5745-575 3,Raetz CR and C Whitfield,2002,Annu Rev Biochem 71:635-700, U.S. Pat. No. 5,593,969 and U.S. Pat. See, e.g., 191,072. Lipid A, as used herein, refers to naturally occurring lipids. The term includes lipid A, mixtures, analogs, derivatives, and precursors thereof, which are present in the Classes such as lipid A precursors called lipid X, disaccharide lipid A, heptaacyl lipids A, hexaacyl lipid A, pentaacyl lipid A, tetraacyl lipid A, e.g., lipid The tetraacyl precursor of lipid A, called lipid IVA, dephosphorylated lipid A, monophospho Phosphoryl lipid A, diphosphoryl lipid A, e.g., Escherichia coli and Rhodobacter Lipid A from Rhodobacter sphaeroides. The immunostimulatory lipid A structure of IgG contains six acyl chains, each of which is directly linked to a glucosamine sugar. The four primary acyl chains are usually 3-hydroxyacyl chains of 10-16 carbons in length. Two additional acyl chains are often attached to the 3-hydroxy group of the primary acyl chain. E. coli lipid A, for example, typically contains four C14 3-hydroxyl groups bound to the sugar. the 3-hydroxyl group of the primary acyl chain at the 2'- and 3'-positions, respectively; It has one C12 and one C14 bonded to an oxy group.

[0072] As used herein, the term "Lipid A analog or derivative" refers to a lipid A Molecules that are similar in structure and immunological activity to, but do not necessarily occur in nature. A lipid A analog or derivative may be, for example, shortened or condensed, and / or or alternatively, having a glucosamine residue substituted with a galactosamine residue. To achieve this, 2-deoxy-2-amino glucosamine-1-phosphate was added to the reducing end. It is designed to have a galacturonic acid moiety instead of a phosphate at the 4' position so as to contain gluconic acid. Lipid A analogs or derivatives are lipid A analogs isolated from bacteria. It may be prepared, for example, by chemical derivatization from dope A, or it may be prepared, for example, by first To determine the structure of lipid A and to synthesize its analogs or derivatives Lipid A analogs or derivatives may also be used as TLR4 agonist adjuvants. (e.g., Gregg KA et al, 2017, MBio 8 , eDD492-17, doi:10.1128 / mBio.00492-17 For example, lipid A analogs or derivatives can be converted to wild-type lipid A by, for example, alkaline treatment. It can be obtained by deacylation of the native lipid A molecule. Lipid A analogs or derivatives The bodies can be prepared, for example, from lipid A isolated from bacteria. It can also be chemically synthesized. Another example of a lipid A analog or derivative is lipid A. Mutations, deletions or insertions of enzymes involved in lipid A biosynthesis and / or lipid A modification MPL and 3D-MPL are lipid A molecules isolated from bacterial cells that have Lipid A analogs or derivatives that have been modified to reduce the toxicity of A. Lipid A, MP L and 3D-MPL have a sugar backbone with long chain fatty acids attached, and this backbone is a glycoside. It contains two linked 6-carbon sugars and a phosphoryl moiety at position 4. It typically has 5-8 long chains. Fatty acids (usually 12-14 carbon atoms) are attached to a sugar backbone. Originates from natural sources. In addition, MPL and 3D-MPL have several fatty acid substitution patterns with various fatty acid lengths. As complexes or mixtures of heptaacyls, hexaacyls, pentaacyls, etc. This may also be the case for some of the other lipid A analogs or derivatives described herein. Although this is true for a few species, synthetic lipid A variants can also be defined and are homogeneous. MPL and its preparation are described, for example, in U.S. Pat. No. 4,436,727. 3D-MPL is described, for example, in U.S. Pat. No. 4,912,094 (B1). 3-hydroxymyristyl ester bonded to the reducing end glucosamine at the 3-position It differs from MPL by the selective removal of acid acyl residues (see, e.g., U.S. Pat. No. 4,912, (See also the structure of the MPL in column 1 of No. 094(B1) with the 3D-MPL in column 6.) In the technical field, 3D-MPL is often used, but it is sometimes called MPL. (See, for example, Table 1 of Ireton GC and SG Reed, 2013 (above)) The first structure is called MPL (registered trademark), but it is actually 3D-MPL. (The structure of the lipid A analogs and derivatives according to the present invention is shown.) L, 3D-MPL, RC529 (e.g., European Patent No. 1385541), PET-lipid Glycopyranosyl lipid adjuvant, synthetic disaccharide lipid, e.g., U.S. Pat. No. 5,399,433; Application Publication No. 20100310602, U.S. Patent No. 8,722,064), SLA (e.g., Carter D et al,2016,Clin Transl Immunolo gy 5:e108(doi:10.1038 / cti.2016.63)), PHAD (phosphorylated hexaacyl disaccharide, the structure of which is the same as that of GLA), 3D-PHA D, 3D-(6-acyl)-PHAD (3D(6A)-PHAD) (PHAD, 3D-P HAD and 3D(6A)PHAD are synthetic lipid A variants, e.g., ava Please see ntilipids.com / divisions / adjuvants , which also provides the structures of these molecules), E6020 (CAS number 287180-6 3-6), ONO4007, OM-174, etc. 3D-MPL, RC529 , PET-Lipid A, GLA / PHAD, E6020, ONO4007, and OM-17 Exemplary chemical structures of 4 are described, for example, in Ireton GC and SG Ree For the structure of the SLA, see, for example, Ree dSG et al,2016,Curr Opin Immunol 41:85- See Figure 1 at 90. In certain preferred embodiments, a TLR4 agonist The adjuvant is a lipid A analogue selected from 3D-MPL, GLA, or SLA. includes derivatives.

[0073] Exemplary adjuvants that include lipid A analogs or derivatives include GLA-LSQ ( Synthetic MPL [GLA], QS21, lipids formulated as liposomes), SLA-LS Q (synthetic MPL [SLA], QS21, lipids formulated as liposomes), GLA- SE (synthetic MPL [GLA], squalene oil / water emulsion), SLA-SE (synthetic M PL[SLA], squalene oil / water emulsion), SLA-nanoalum (synthetic MP L[SLA], aluminum salt), GLA-nanoalum (synthetic MPL[GLA], aluminium salt) Aluminum salt), SLA-AF (synthetic MPL [SLA], aqueous suspension), GLA-AF ( Synthetic MPL [GLA], aqueous suspension), SLA-alum (synthetic MPL [SLA], a MPL [GLA], aluminum salt), GLA-alum (synthetic MPL [GLA], aluminum salt), and AS01 (MPL, QS21, liposome), AS02 (MPL, QS21, oil / water extract) emulsion), AS25 (MPL, oil / water emulsion), AS04 (MPL, aluminum GSK AS15 (MPL, QS21, CpG, liposomal) Some of the adjuvants in the xx series are mentioned. For example, see International Publication No. 201 No. 3 / 119856, No. 2006 / 116423, U.S. Pat. No. 4,987,237 , No. 4,436,727, No. 4,877,611, No. 4,866,034, Same No. 4,912,094, Same No. 4,987,237, Same No. 5191072, Same No. 5 No. 593969, No. 6,759,241, No. 9,017,698, No. 9,14 No. 9,521, No. 9,149,522, No. 9,415,097, No. 9,415 ,101, ... ), Johnson et al., 1999, J Med Chem, 42:4640 -4649, and Ulrich and Myers, 1995, Vaccine De sign:The Subunit and Adjuvant Approach;P owell and Newman,Eds.;Plenum:New York,49 Please see 5-524.

[0074] Non-glycolipid molecules, e.g., synthetic molecules such as neoseptin-3 or natural molecules such as LeIF can also be used as a TLR4 agonist adjuvant, e.g., Reed See SG et al., 2016 (supra).

[0075] In another aspect, the present invention relates to a method for the preparation of a FimH (F71 mut) or FimH(F71mut / F144mut) polypeptide of the present invention, imH(F71mut) or FimH(F71mut / F144mut) polypeptide The present invention relates to a polynucleotide encoding the polypeptide of the present invention, or a pharmaceutical composition of the present invention.

[0076] In a further aspect, the present invention relates to a gram-negative bacterium of the family Enterobacteriaceae. The present invention provides a method for the induction of an immune response against FimH(F71mut) or The FimH(F71mut / F144mut) polypeptide of the present invention is 1mut) or FimH (F71mut / F144mut) polypeptide. The present invention relates to the use of a polynucleotide comprising the polypeptide or a pharmaceutical composition as described herein.

[0077] As used herein, the term "immunogen" or "immunogenic" or "antigen" means Either alone, in combination with an adjuvant, or presented on a display vehicle or induces an immunological response against itself upon administration to the recipient. The terms "antibody" and "antibody-specific" refer to molecules that can be used interchangeably.

[0078] As used herein, an "immunological response" or "immune response" to an antigen or composition. " refers to the humoral and / or cellular response in a subject to an antigen or to an antigen present in a composition. Refers to the development of an immune response.

[0079] In a further aspect, the present invention relates to a gram-negative bacterium of the family Enterobacteriaceae. The present invention relates to a method for the induction of an immune response against a bacterial infection caused by imH(F71mut) or FimH(F71mut / F144mut) polypeptide The FimH(F71mut) or FimH(F71mut / F144mu) of the present invention t) a polynucleotide encoding a polypeptide or a pharmaceutical composition as described herein In certain embodiments, the bacterial infection is caused by Klebsiella spp., is caused by E. coli. In a preferred embodiment, the bacterial infection is caused by E. coli. Thus, in one embodiment, The invention is directed to E. coli or Klebsiella, preferably E. coli. The present invention also provides a method for the induction of an immune response to a medicament comprising administering to a patient a therapeutically effective amount of FimH(F71mut) or FimH(F71mut / F144mut) polypeptide of the present invention ut) or FimH(F71mut / F144mut) polypeptide, or The present invention relates to the use of a pharmaceutical composition according to the present invention.

[0080] In a preferred embodiment, the infection is caused by a gram-negative bacterium of the family Enterobacteriaceae. Bacterial infections caused by E. coli, e.g., extraintestinal pathogenic E. coli (E xPEC), for example, the infection can be a urinary tract infection (UTI). In an embodiment, the present invention provides a method for treating symptoms and / or sequelae associated with a UTI in a subject. and a method for treating, preventing or suppressing a disease comprising administering to a subject a therapeutically effective amount of a lectin domain comprising the FimH lectin domain described herein. a polypeptide comprising the polypeptide described herein, a polynucleotide described herein, or a pharmaceutical composition described herein In certain embodiments, the UTI is a rUTI. The main causes of UTIs and rUTIs are important health care problems in older adults and women Thus, in a preferred embodiment, the bacterial infection is caused by E. coli. These are UTIs or rUTIs caused by

[0081] In one embodiment, the present invention provides a method for the treatment of a gut microbe-associated condition in a subject in need thereof. The present invention relates to a method for treating, preventing, or inhibiting symptoms and / or sequelae of a compound comprising administering to said subject an effective amount of The FimH(F71mut) or FimH(F71mut / F144mut) of the present invention ) polypeptide of the present invention, FimH(F71mut) or FimH(F71mut / A polynucleotide encoding a F144mut) polypeptide, or a pharmaceutical composition as described herein. and administering the pharmaceutical composition to a subject. Preferably, the administration is for treating an intestinal bacteria-associated condition. Preferably, the intestinal bacteria-associated condition is a urinary intestinal bacteria-associated condition. A tract infection, more specifically a UTI or rUTI.

[0082] The present invention also relates to an antibacterial agent caused by gram-negative bacteria of the family Enterobacteriaceae. and preferably a bacterial infection caused by E. coli. The present invention relates to a method for the manufacture of a medicament for the treatment, prevention, or suppression of FimH(F71mut ) or FimH(F71mut / F144mut) polypeptide, FimH of the present invention (F71mut) or FimH(F71mut / F144mut) polypeptide. The present invention relates to the use of a polynucleotide encoding a medicament for the treatment of a disease, or a pharmaceutical composition as described herein. Alternatively, the bacterial infection may be UTI or recurrent UTI (rUTI) caused by Escherichia coli. I).

[0083] The present invention further relates to a method for the production of a polypeptide of the invention, comprising the steps of: (F71mut) or FimH(F71mut / F144mut) polypeptide. Recombinant cells comprising the polynucleotides and / or vectors described herein. wherein the culturing is carried out under conditions conducive to the production of the polypeptide. do.

[0084] In certain embodiments, the method further comprises recovering the polypeptide, optionally Optionally, this is followed by formulation into a pharmaceutical composition.

[0085] Preferably, the E. coli cell, e.g., an E. coli BL21 derivative cell, expresses the FimH( FimH (F71mut) or FimH (F71mut / F144mut) polypeptides. The present invention is used in a method for

[0086] Recovery of the polypeptide is preferably carried out by conventional protein purification methods well known in the art. Such methods include purification and / or isolation steps that may be performed using methods such as For example, ammonium sulfate or ethanol precipitation, acid extraction, anion and / or cation extraction ion exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography Chromatography, affinity chromatography, hydroxylapatite chromatography - and / or lectin chromatography.

[0087] Typical examples of such purification and / or isolation include antibodies against the protein, or His-tag or cleavable leader or tag expressed as part of a protein structure In certain embodiments, antibodies against the polypeptides described herein may be utilized. The peptides contain His tags and can be purified by methods such as IMAC affinity purification. In certain embodiments, the polypeptides described herein contain a His tag. In such cases, purification may be by chromatography, e.g., ion exchange chromatography. Chromatography (IEX), Hydrophobic Interaction Chromatography (HIC), and / or Size This can be done by exclusion chromatography.

[0088] definition Various publications, articles and patents are cited in the Background section and throughout this specification. or described herein, each of which is incorporated by reference in its entirety. Any discussion of documents, operations, materials, devices, articles or the like which has been included in the specification is in accordance with the context of the present invention. Such discussion is intended to provide an overview of the present invention. or admitted to constitute part of the prior art to any claimed invention. It is not.

[0089] Unless otherwise defined, all technical and scientific terms used herein are understood to be within the meaning of the present invention. It has the same meaning as commonly understood by a person skilled in the art to which the invention pertains. When used herein, certain terms are intended to have the meanings set forth herein. All patents, published patent applications and publications cited herein are hereby incorporated by reference. The entire contents of this specification and the accompanying patents are incorporated herein as if set forth in their entirety. As used in the claims, the singular forms "a," "an," and "the" shall be used unless the context clearly indicates otherwise. It should be noted that unless otherwise specified, a single word includes multiple referents.

[0090] Throughout this specification and the following claims, unless the context otherwise requires, the words "comprises" and "includes" are used interchangeably. The term "rise" and variations such as "comprises" and "comprising" The form includes the specified integer or step or group of integers or steps, but may also include any other integer is understood to mean not excluding any whole number or step or group of steps. As used herein, the term "comprising" means "containing "containing" or "including" may be substituted; may sometimes be replaced with the term "having" when used herein. It is also possible.

[0091] As used herein, "consisting of" means The term "compound" as used herein excludes any element, step, or ingredient not specified in the description. When used herein, "consisting essentially of" is used in the claims. Materials or steps that do not materially affect the basic and novel characteristics of the present invention are not excluded. When used herein in connection with an aspect or embodiment, to vary the scope of the disclosure "comprising," "containing," "including," and "having" may be replaced with the term "consisting of" or "consisting essentially of" can be substituted.

[0092] As used herein, the conjunction "and / or" between multiple listed elements is intended to be construed as meaning "and / or" in any manner whatsoever. The term is to be understood as embracing both individual and combined options. For example, when two elements are connected by "and / or," the first option is the second option. The second option refers to the applicability of the second element without the first element. The third option indicates that the first and second elements are applicable together. Any one of these alternatives is encompassed within the meaning and therefore the present specification When used in the present specification, it is understood that the requirements of the term "and / or" are met. The simultaneous applicability of two or more of these is also included in the meaning and therefore the element of the term "and / or". It is understood that the conditions are met.

[0093] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutical "refers to a non-toxic material that does not interfere with the efficacy of or the biological activity of the composition according to the present invention." A "pharmaceutically acceptable carrier" includes any excipient, diluent, filler, salt, buffer, stabilizer, soluble Solubilizers, oils, lipids, lipid-containing vesicles, microspheres, liposomal encapsulates, or pharmaceutical formulations The composition may include other materials well known in the art for use in pharmaceutical compositions. It will be understood that the characteristics of the acceptable carrier will depend on the route of administration for a particular application. According to embodiments, in view of the present disclosure, any pharma- ceutical acceptable salt suitable for use in a vaccine may be used. Any acceptable carrier can be used in the present invention. Suitable excipients include sterile water, saline, etc. Saline, dextrose, glycerol, ethanol, etc., and combinations thereof; a stabilizer, such as human serum albumin (HSA) or other suitable protein and a reducing sugar These include, but are not limited to:

[0094] As used herein, the term "effective amount" refers to a desired biological effect in a subject. An effective amount refers to the amount of active ingredient or component that elicits a therapeutic or medicinal response for the described purpose. For example, the amount of the in vivo Toro assays can be used to help identify optimal dosage ranges.

[0095] As used herein, a "subject" or "patient" refers to a subject of a method according to an embodiment of the present invention. Any animal that is to be vaccinated or has been vaccinated with the composition, preferably Preferably, the subject is a mammal, and most preferably, a human. As used herein, the term " "Mammal" includes any mammal. Examples of mammals include cows, horses, sheep, etc. The most popular are: cats, pigs, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans. Preferably, the subject is a human, but is not limited to such a human. In certain embodiments, The subject is a human adult. As used herein, the term "human adult" refers to a subject aged 18 In certain embodiments, the subject is under 18 years of age, e.g., 0 to 18 years of age. 18 years of age, for example, 9 to 18 years of age, or 12 to 18 years of age. The subject is a human subject between about 18 and about 50 years of age. In certain embodiments, the subject is 50 to about 100 years old, e.g., 50 to 85 years old, 60 to 80 years old, 50 years old or older, 55 years old or older, 6 Humans aged 0 years or older, 65 years or older, 70 years or older, 75 years or older, 80 years or older, or 85 years or older In some embodiments herein, the subject is 85 years of age or younger, 80 years of age or younger, 75 years of age or older, In certain embodiments, the human subject is male. In the present invention, the human subject is a female.

[0096] As used herein, "UTI" means an infection of the kidneys, bladder, ureters, or urethra. Symptoms of a UTI include a burning sensation when urinating, a frequent or urgent need to urinate, incomplete urination, and abnormal urine flow. urine with a bad appearance and / or odor, elevated white blood cells in the urine, fatigue or tremors, disorientation, One of the following: fever or chills, fatigue, pain or pressure in the back, lower abdomen, pelvis, or bladder However, in some patients, symptoms are absent or nonexistent. Sequelae of UTIs may include invasive disease or systemic complications such as sepsis. In certain embodiments, the UTI may be clinically and / or microbiologically documented. and confirmed, for example, by bacterial culture of urine and / or by molecular or other methods. In an embodiment, the subject is a human subject who has previously had or currently has a UTI. In certain embodiments, the subject has had schizophrenia for the past two years, the past year, or the past six months. In certain embodiments, the subject has had a UTI in the past. As used herein, "rUTI" refers to a condition that occurs during or after a course of This means at least two infections in a six-month period or at least three UTIs in a year. In a particular embodiment, FimH(F71mut) or FimH(F71mut / F144mut), the FimCH complex of the present invention, the fusion polypeptide of the present invention, or the The subject to whom the composition is administered is a person who has had at least one In certain embodiments, the subject has had at least two UTIs. As used herein, a "complication of UTI" refers to a structural disorder of the genitourinary tract. It also refers to UTIs that are associated with a condition such as a pulmonary or functional abnormality or the presence of an underlying disease. In certain embodiments, a UTI results in elevated white blood cell counts in the urine or other urinary abnormalities. In certain embodiments, a subject with a UTI has a large number of bacteria in the urine, i.e. , the urine is not sterile, e.g., at least about 10 cells / mL, at least about 100 cells / mL, at least approximately 10 3 cells / mL, e.g., at least about 10 4 Pieces cells / mL, e.g., at least about 10 5 With a bacterial cell count of 10 cells / mL.

[0097] As used herein, an "immunological response" or "immune response" to an antigen or composition. " refers to the humoral and / or cellular response in a subject to an antigen or to an antigen present in a composition. Refers to the development of an immune response.

[0098] Unless otherwise stated, the term "at least" preceding a series of elements refers to every element in the series. should be understood to refer to.

[0099] The words "about" or "approximately" when used in connection with a numerical value (e.g., about 10) , preferably the value is around 10% of the given value (10), preferably 5% before the value This means it could be later.

[0100] The terms "homology," "sequence identity," and the like are used interchangeably herein. Sequence identity, as used herein, refers to the degree of similarity between two or more sequences, as determined by comparing the sequences. Between amino acid (polypeptide or protein) sequences or between two or more nucleic acids (polynucleotides In the art, "identity" is also used in In some cases, the amino acid or nucleoside sequences may be identical, as determined by the correspondence between such linear sequences. The degree of sequence relatedness between two amino acid sequences is called "similarity." The amino acid sequence of a polypeptide and its conservative amino acid substitutes are compared to the sequence of a second polypeptide. "Identity" and "similarity" may be determined by comparing sequences of the same gene with those of the same sequence. This can be easily calculated.

[0101] "Sequence identity" and "sequence similarity" can be global or local, depending on the length of the two sequences. A local alignment algorithm is used to align two peptides or two nucleotide sequences. Similar length sequences can be determined by alignment of the entire sequence. Global alignment algorithms that optimally align sequences over a long period of time (e.g. For example, alignment is performed using the Needleman Wunsch method, while Sequences of different lengths are preferably aligned using a local alignment algorithm (e.g., Sm The sequences are then aligned using the Waterman algorithm. (For example, by the programs GAP or BESTFIT using default parameters At least a certain minimum percentage of the sequences (when optimally aligned by "Substantially identical" or "essentially similar" if they share identity (defined below) GAP is an abbreviation for the Needleman and Wunsch global alignment algorithm. Align two sequences over their entire length using an algorithm The global alignment is done by maximizing the number of matches and minimizing the number of gaps. It is preferably used to determine sequence identity when the sequences have similar lengths. The default parameters for GAP are: gap creation penalty = 50 (nucleotides ) / 8(protein) and gap extension penalty=3(nucleotides) / 2(protein For nucleotides, the default scoring matrix used is The scoring matrix is ​​nwsgapdna, and for proteins, the default scoring matrix is Ricks is Blosum62 (Henikoff & Henikoff, 1999). 92, PNAS 89, 915-919). Sequence alignment and percent sequence identity Accelrys Inc., 9685 Scranton Road GCG W is available from Computer programs such as the isconsin Package, version 10.3 or by using the program "ne edle" (using the global Needleman Wunsch algorithm) "water" (using a local Smith Waterman algorithm) Using open source software, with the same parameters as GAP above, or default settings (both "needle" and "water", and protein and For both nucleotide and DNA alignments, the default gap opening penalty is 10.0. the default gap extension penalty is 0.5; the default scoring matrix The enzymes used were Blosum62 for proteins and DNAFu If the sequences have substantially different overall lengths, the locus can be determined using Cal alignment, e.g. using the Smith Waterman algorithm But it is preferred.

[0102] Alternatively, the similarity or identity percentage may be calculated using algorithms such as FASTA, BLAST, etc. The rhythm may be used to search public databases. Thus, the nucleic acid and protein sequences of the present invention may further be used to identify, for example, other family members. or a "query" to conduct a search against public databases to identify related sequences. Such searches can be performed using the method described in Altschul, et al. ,(1990,J.Mol.Biol,215:403-10) This can be done using the ASTx program (version 2.0). BLAST nucleotide searches were performed using the T program, score = 100, word length = 12. This can be carried out to obtain nucleotide sequences that are homologous to the nucleic acid molecules of the present invention. BLAST protein searches were performed using the ASTx program, score = 50, word length = 3. This can be carried out to obtain amino acid sequences that are homologous to the protein molecules of the present invention. To obtain gapped alignments for comparison purposes, use Gapped BLAST with Alts chul et al.,(1997) Nucleic Acids Res.25(1 7):3389-3402. When using the Gapped BLAST program, the respective programs (e.g., Default parameters for BLASTX and BLASTn may be used. onal Center for Biotechnology Information See the NBI homepage at http: / / www.ncbi.nlm.nih.gov / I want to be.

[0103] Array description:

[0104] [Table 1]

[0105] Other examples of sequences of FimH polypeptides are described in U.S. Pat. No. 6,737,063, e.g., It is described in any one of SEQ ID NOs: 23 to 45 or 55, all of which are It is incorporated herein by reference. EXAMPLES

[0106] The following examples of the present invention are presented to further illustrate the principles of the present invention. The examples are not intended to limit the invention, the scope of which is defined by the appended claims. I want you to understand this.

[0107] To understand the impact of FimH conformational changes on vaccine efficacy, we performed bacterial inoculation of bladder Improved FimH induces functional antibodies capable of reducing adhesion and colonization Potentially locking a protein into a low affinity state with the goal of identifying variants We designed several variants of FimH that contain different mutations that can To optimize the chances of finding mH lectin domain variants, we used different predicted functional groups. We selected a variant with the same expression pattern as previously described mutant FimH_Q133. K and FimH_R60P, as well as wild-type (WT) FimH, were used as controls.

[0108] Example 1: Ability to induce inhibitory antibodies Antibodies raised against the low affinity conformation of FimH block bacterial cells and It has been shown that the colony formation of . As a result, we have demonstrated that the low affinity To evaluate the functionality of antibodies induced by different conformationally locked FimH variants It was worth it.

[0109] Materials and Methods Design and expression of FimH FimC and FimH are expressed with a heterologous signal sequence for expression in the periplasm, Fim for affinity purification using immobilized metal affinity chromatography (IMAC) The C-terminal His tag on C was used to express the ribozyme in the pET-DUET or pET-22b vectors. Expression was induced using IPTG, and the protein was extracted and IMAC purified. (Talon).

[0110] Immunization Wistar rats were immunized with non-Freund's adjuvant (Speedy rat 28-day model, Eurogentec) in combination with different FimH variants (60 μg each variant) g / dose) on days 0, 7, 10, and 18, Serum antibody functionality was evaluated on days 0 (pre-immunization) and 28 (post-immunization). was examined by the adhesion inhibition assay (AIA) described below.

[0111] Adhesion inhibition assay (AIA) Bacteria (Escherichia coli strain J96) were labeled with fluorescein isothiocyanate (FITC). The labeled bacteria were incubated with bladder urothelial cells (cell line 5637) for 1 h at 37°C. The percentage of adherent bacteria was determined by flow cytometry. Evaluation of serum inhibition For titration, bacteria were preincubated with serum samples at 37°C for 30 min and then incubated at 56°C for 30 min. 37 cells were mixed.

[0112] ELISA A 96-well plate was coated overnight with 1ug / mL FimH. After washing, The coated wells were then filled with blocking buffer [phosphate-buffered saline (PBS) + 2% The cells were incubated with PBS + 0.0 mM sodium bicarbonate (BSA) for 1 hour at room temperature. After washing with 5% Tween 20, serum was added to the plates, which were then incubated at room temperature. After 1 hour of incubation, the cells were washed and then resuspended in horseradish percolator diluted with 2% BSA in PBS. Add goat anti-rat antibody conjugated to oxidase to each well for 1 hour at room temperature. After a final wash, the reaction was developed with tetramethylbenzidine substrate. It is stopped with phosphoric acid and the absorbance is measured at 450 nm.

[0113] result Serum antibody inhibitory titers were estimated based on a four-parameter logistic (4PL) regression model. The half-maximal inhibitory concentration (IC50) was calculated based on the concentration of FimH. The levels of serum antibodies (total IgG) induced by the antibody were assessed by ELISA. IC50 potency, defined as the effective concentration, was analyzed using the 4PL nonlinear regression model. The calculation was based on replicate six-step titration curves.

[0114] In preliminary experiments, several different FimH variants were identified as those that induce inhibitory antibodies. As can be seen in FIG. 1A, the variant F mH(F71Y) and FimH(F144V) induced the highest levels of functional antibodies. The FimH(F144V) variant was identified on January 16, 2020 by Janssen Ph.D. European Patent Application No. 2015221 filed in the name of Armaceuticals, Inc. 7, which is incorporated herein by reference. It is surprising that mH(F71Y) also induced high levels of inhibitory antibodies. , which was completely unpredictable prior to the present invention.

[0115] To confirm the preliminary results, the AIA assay was repeated in a larger number of animals. Both FimH(F71Y) and FimH(F144V) ensured high levels of inhibitory antibodies It was confirmed that the IL-11 expression could be induced in the IL-11 expression vector (Figure 1B).

[0116] The FimH(F71Y) and FimH(F144V) variants have a steric structure in the binding pocket. It appears that the low affinity conformation is locked by a different mechanism that induces a conformational change. The substitution in the mH(F71Y) variant allows the residue to fold in a hydrophobic pocket (relaxed state). It prevents the belt from folding and makes the tension more favorable, but FimH(F144V) In the mutant, the substitutions are relatively small in the mannose-binding pocket that stabilize a low-affinity conformation. The conformational change to the low affinity state occurs via these two distinct mechanisms. Therefore, the present inventors have developed a lectin domain containing both the F71Y and F144V substitutions. The main domain may be more stably locked into a low affinity conformation, thereby reducing the number of Fs of that type. We hypothesized that this would provide an additional advantage to imH variants. To achieve this, we introduced both F71Y and F144V substitutions into the lectin domain. We generated a FimH double mutant (FimH(F71Y / F144V)) containing the FimH double mutant (FimH(F71Y / F144V)). As can be seen in Fig. 1, FimH(F71Y / F144V) induced high levels of inhibition. It was also able to induce specific antibodies.

[0117] Based on these results, FimH(F71Y) and FimH(F71Y / F144V ) were selected as lead candidates. Their properties were further analyzed as described below. .

[0118] Example 2: Design of new variants - SPR data SPR FimH lectin domain variants and n-heptyl-α-D-mannopyranoside drug To gain detailed insight into the binding affinity and kinetics of the interaction with the surface plasmon Briefly, we performed spin resonance (SPR) measurements on FimH variants. The antibody was titrated onto the surface of Gand 5 (Rabbani S et al, J Biol. Chem., 2018,293(5):1835-1849), where HBS-N (0.01MH EPES, pH7.4, 0.15M NaCl, 3mM EDTA, 0.05% surfactant The highest concentration of 3 or 10 μM in the preparation P20 was used. was ranged from 0.036 to 3.0 μM and injected using single cycle kinetic injections.

[0119] result The previously described mutants FimH_Q133K and FimH_R60P have These mutations affect the binding interaction with mannose. These mutants were used as positive controls. The mutant R60P_Q133K was also used to investigate its potentially enhanced effect. The wild-type (WT) FimH lectin domain was used as a negative control.

[0120] The affinity of the variants for binding to mannose was assessed using SPR. As expected, the leukocytes carrying mutations in the mannoside-binding pocket of FimH were The chin domain variants (Q133K and R60P_Q133K) were completely insensitive to mannosides. The R60P mutant, as expected, showed a lower affinity for mannosides. did.

[0121] Although capable of inducing high levels of inhibitory antibodies, The riants still showed some affinity for mannosides, suggesting that this mutation FimH containing the F144V substitution did not completely abolish binding to the cid. In the lectin domain variant, binding to mannosides was completely abolished (see reference Janssen Pharmaceuticals, Inc., on January 16, 2020, which is incorporated herein by reference. In European Patent Application No. 20152217 filed in the name of Artificials, Inc. As previously described for the single F144V mutant. In mutants containing both V, binding to mannosides was also completely abolished.

[0122] [Table 2] * low affinity K D ≥ 1000 nM, moderate affinity K D 100~1000nM, high affinity K D <=100nM

[0123] Example 3: Ability to bind to characterized inhibitory mAbs 475 and 926 Mutations in the lectin domain of FimH are important for eliciting strong and functional immune responses This leads to the loss of important epitopes, such as those in the binding pocket of FimH. It is to be ensured that the integrity of the binding pocket is not compromised by the mutations described herein. To achieve this, we developed mutant F of monoclonal antibodies (mAbs) mAb475 and mAb926. Binding to the imH lectin domain was evaluated. mAb475 and mAb926 bind to the FimH lectin domain. Overlapping but distinct epitopes on the FimH lectin domain within the mannose-binding pocket of H Recognize the group (Kisiela et al. 2013&2015).

[0124] result The mutant FimH lectin domain was previously described in WO 02102974. WO 02102974 identifies possible mutations, most of which are unidentified. The authors report an extensive list of mutations (65 possible mutation sites in a gene approximately 159 amino acids long). However, the lectin domain of FimH, which is a marker for mitochondrial endothelial cell death, has not been suggested. No. 02102974 is a Fi having an amino acid substitution at position 54, 133, or 135. The mH lectin domain was shown to be the most promising candidate, FimH_Q133K is explicitly mentioned as the highly preferred option. It is quite surprising that 10_Q133K was not recognized by the functional mAb475. This indicates a problem with the integrity of the binding pocket (Table 3). In contrast, FimH (F Both FimH(F144V) and FimH(F71Y) were associated with both mAb475 and mAb926. The binding pocket was fully intact (Table 3). The FimH(F71Y / F144V) double mutant was still recognized by both antibodies. This indicates that the binding pocket also remained intact in this double mutant ( Table 3).

[0125] [Table 3]

[0126] Example 4: Conformational state analysis of FimH lectin domain variants by NMR 15 was produced as described in the Methods section by adding N to the growth medium. 15 N Uniformly labeled FimH LD Variant 1 H, 15 N heteronuclear single quantum coherence nuclear magnetism The resonance (HSQC NMR) spectrum of n-heptyl-α-D-mannopyranoside was The structural differences were assessed at a residue-by-residue level in the absence and presence of the band. Proteins were concentrated to 150 μM and dissolved in 20 mM phosphate buffer containing 7% to 10% DO. The n-heptyl-α-D-mannopyranoside ligand was measured in D2O The samples were dissolved in 100 mM KOH and added in a 10-fold molar excess to the protein. The torr were compared with publicly available reference spectra (Rabbani S et al. , J Biol. Chem., 2018, 293(5):1835-1849).

[0127] result FimH variants FimH(F71Y), FimH(F144V), and FimH( F71Y / F144V) remained in a low affinity conformation in the presence of mannoside ligands. In the absence of ligand, all three variants were highly Comparing the chemical shifts of residues known to represent affinity states, we see that the low affinity conformation (Rabbani S et al, J Biol. Chem., 2018, 29 3(5):1835-1849). In contrast, FimH LD23-10 wild type, Man In the absence of a ligand, it is locked into a high affinity conformation. In the presence of β-actin, FimH(F71Y) and FimH(F144V) are regulated by the high affinity conformation. FimH(F71Y / F144V) switches back to the low-affinity conformation. For example, chemical shifts were observed after addition of ligand to FimH(F71Y / F144V). No changes were observed (Figure 2 and Table 4).

[0128] [Table 4]

[0129] Apart from two unknown residues that differ from the K12 wild type, FimH LD 23-10 wild The single mutant variant is thought to be already in the high affinity (H) conformation without any ligand. Ants F71Y and F144V are in a low affinity (L) conformation without ligand, In the presence of ligand, it switches to the H conformation, whereas the double mutant variant F71Y / F144V remained in the L conformation even in the presence of ligand. The inability to switch back to the three-dimensional conformation leads to FimH(F71Y / F1 44V) appears to be particularly promising for use in pharmaceutical vaccine compositions, as For example, to test whether the conformation is stable during storage and under other storage conditions. This is because it is certain that no additional quality or stability controls will be required for high The inability of FimH(F71Y) to switch back to the affinity conformation / F144V) as a research tool to study the mechanism of action or to clarify the three-dimensional structure. This will allow us to obtain knowledge about FimH (F71Y / F144V). to generate specific antibodies that have useful research, diagnostic, and possibly pharmaceutical applications. This makes it possible.

[0130] Therefore, in conclusion, of all the FimH variants tested, FimH(F7 1Y), FimH(F144V), and FimH(F71Y / F144V) are FimH It is possible to induce the highest levels of functional inhibitory antibodies while keeping the binding pocket of the antibody intact. FimH(F71Y / F144V) completely abolished the binding to mannosides and It has the added advantage of remaining locked in a low affinity conformation even in the presence of ligand. Yes.

Claims

[Claim 1] The invention described in the specification.

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