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US-20260124286-A1 - E. COLI FIMH MUTANTS AND USES THEREOF

US20260124286A1US 20260124286 A1US20260124286 A1US 20260124286A1US-20260124286-A1

Abstract

This disclosure relates to the design of E. coli mutated FimH polypeptides that result in improved biochemical properties and immunogenicity, compositions comprising such polypeptides, and uses thereof.

Inventors

  • Ye CHE
  • Laurent Oliver Chorro
  • Robert George Konrad Donald
  • Matthew Curtis Griffor
  • Natalie Clare Silmon de Monerri

Assignees

  • PFIZER INC.

Dates

Publication Date
20260507
Application Date
20250618

Claims (20)

  1. 1 - 10 . (canceled)
  2. 11 . A nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence of a mutated FimH polypeptide, wherein the mutated FimH polypeptide comprises an amino acid substitution relative to the amino acid sequence of the wild-type FimH polypeptide, wherein the substitution is selected from the group consisting of: F1I; F1V; F1M; F1Y; F1W; P12C; G14C; G15A; G15P; G16A; G16P; A18C; P26C; V28C; Q32C; N33C; L34N; L34S; L34T; L34D; L34E; L34K; L34R; V35C; S62C; Y64C; G65A; L68C; F71C; T86C; L107C; Y108C; L109C; V112C; S113C; A115V; G116C; V118C; A119C; A119N; A119S; A119D; A119E; A119K; A119R; A127C; L129C; F144C; V154C; V155C; V156C; T158C; and V185I, and wherein the amino acid positions are numbered according to SEQ ID NO: 59.
  3. 12 - 23 . (canceled)
  4. 24 . The nucleic acid of claim 11 , wherein the mutated FimH polypeptide comprises the amino acid substitution V27A.
  5. 25 . The nucleic acid of claim 11 , wherein the nucleic acid is RNA.
  6. 26 . The nucleic acid of claim 25 , wherein the RNA is nucleoside modified mRNA (modRNA).
  7. 27 . A nanoparticle comprising the nucleic acid of claim 25 .
  8. 28 . An immunogenic composition comprising the nanoparticle of claim 27 .
  9. 29 . The immunogenic composition of claim 28 , comprising an E. coli saccharide antigen, wherein the saccharide antigen comprises a structure selected from the group consisting of Formula O25b, Formula O1A, Formula O2, and Formula O6.
  10. 30 . A nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence of a mutated FimH polypeptide, wherein the mutated FimH polypeptide comprises an amino acid substitution relative to the amino acid sequence of the wild-type FimH polypeptide, wherein the substitution is selected from the group consisting of: a) G15A and G16A; b) P12C and A18C; c) G14C and F144C; d) P26C and V35C; e) P26C and V154C; f) P26C and V156C; g) V27A and Q133K; h) V28C and N33C; i) V28C and P157C; j) Q32C and Y108C; k) N33C and L109C; I) N33C and P157C; m) V35C and L107C; n) V35C and L109C; o) S62C and T86C; p) S62C and L129C; q) Y64C and L68C; r) Y64C and A127C; s) L68C and F71C; t) V112C and T158C; u) S113C and G116C; v) S113C and T158C; w) V118C and V156C; x) A119C and V155C; y) L34N and V27A; z) L34S and V27A; aa) L34T and V27A; ab) L34D and V27A; ac) L34E and V27A; ad) L34K and V27A; ae) L34R and V27A; af) A119N and V27A; ag) A119S and V27A; ah) A119T and V27A; ai) A119D and V27A; aj) A119E and V27A; ak) A119K and V27A; al) A119R and V27A; am) G15A and V27A; an) G16A and V27A; ao) G15P and V27A; ap) G16P and V27A; aq) G15A, G16A, and V27A; ar) G65A and V27A; and as) G15A, G16A, V27A, and Q133K, and wherein the amino acid positions are numbered according to SEQ ID NO: 59.
  11. 31 . The nucleic acid of claim 30 , wherein the nucleic acid is RNA.
  12. 32 . The nucleic acid of claim 31 , wherein the RNA is nucleoside modified mRNA (modRNA).
  13. 33 . A nanoparticle comprising the nucleic acid of claim 31 .
  14. 34 . An immunogenic composition comprising the nanoparticle of claim 33 .
  15. 35 . The immunogenic composition of claim 34 , comprising an E. coli saccharide antigen, wherein the saccharide antigen comprises a structure selected from the group consisting of Formula O25b, Formula O1A, Formula O2, and Formula O6.
  16. 36 . A nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence of a mutated FimH polypeptide, wherein the mutated FimH polypeptide comprises the following amino acid substitutions relative to the amino acid sequence of the wild-type FimH polypeptide: G15A, G16A, and V27A, and wherein the amino acid positions are numbered according to SEQ ID NO: 59.
  17. 37 . The nucleic acid of claim 36 , wherein the nucleic acid is RNA.
  18. 38 . The nucleic acid of claim 37 , wherein the RNA is nucleoside modified mRNA (modRNA).
  19. 39 . A nanoparticle comprising the nucleic acid of claim 37 .
  20. 40 . An immunogenic composition comprising the nanoparticle of claim 39 .

Description

RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63/130,153 filed Dec. 23, 2020, U.S. Provisional Application No. 63/185,425 filed May 7, 2021 and U.S. Provisional Application No. 63/282,244 filed Nov. 23, 2021. The entire content of each of the foregoing applications is herein incorporated by reference in its entirety. REFERENCE TO SEQUENCE LISTING This application is being filed electronically via EFS-Web and includes an electronically submitted sequence listing in .xml format. The .xml file contains a sequence listing entitled “PC072713A Sequence Listing.xml” created on Mar. 4, 2024 and having a size of 220,025 bytes. The sequence listing contained in this .xml file is part of the specification and is incorporated herein by reference in its entirety. TECHNICAL FIELD OF THE INVENTION The present disclosure relates to mutated Escherichia coli FimH polypeptides and methods of their use. BACKGROUND OF THE INVENTION Urinary tract infections (UTI) affect 1 in 5 women at least once during their lifetime and are responsible for significant morbidity and mortality, resulting in a substantial burden on healthcare systems. While several different bacteria can cause UTI, the most common cause (90-95% of cases) is the Gram-negative bacteria Escherichia coli (E. coli). Most E. coli UTI are caused by uropathogenic E. coli (UPEC) that colonise the gastrointestinal tract and migrate from the faecal flora to the urogenital tract, where they adhere to host uroepithelial cells, thus establishing a reservoir for ascending infections of the urinary tract. Adhesion is facilitated by fimbrial adhesins including type 1 fimbriae, which bind to mannosylated glycoproteins in the epithelial layer or secreted into the urine. Type 1 fimbriae are highly conserved among clinical UPEC isolates and are encoded by a cluster of genes called fim, which encode accessory proteins (FimC, FimD), various structural subunits (FimE, FimF, FimG) and an adhesin called FimH. FimH is essential for all characteristics of UTI infection in mouse models that mimic aspects of human bladder infection (Hannan et al. PLoS Pathog. 2010 Aug. 12; 6(8):e1001042; doi: 10.1371/journal.ppat.1001042; Schwartz et al. Infect Immun. 2011 October; 79(10):4250-9. doi: 10.1128/IAI.05339-11). Small molecule inhibitors that target FimH by mimicking mannosylated receptors further validate the role of FimH in UTI, and are showing promise as therapeutics in animal models (Cusurnano C K, et al. Sci Transl Med. 2011; 3(109):109ra115. doi:10.1126/scitranslmed.3003021). In addition, FimH is under positive selection in E. coli human cystitis isolates (Chen S L, et al. Proc Natl Acad Sci USA. 2009 Dec. 29; 106(52):22439-44. doi: 10.1073/pnas.0902179106) and positively selected residues may influence virulence in mouse models of cystitis (Schwartz, D. J. et al. Proc Natl Acad Sci USA 110, 15530-15537, doi:10.1073/pnas.1315203110 (2013)). FimH is composed of two domains, the lectin binding domain (FimHLD) responsible for binding to mannosylated glycoproteins, and the pilin domain. The pilin domain serves to link FimH to other structural subunits of the pilus such as FimG, via a mechanism called donor strand exchange (Le Trong, I et al., J. Struct Biol. 2010 December; 172(3):380-8. doi: 10.1016/j.jsb.2010.06.002). The FimH pilin domain forms an incomplete immunoglobulin fold, resulting in a groove that provides a binding site for the N-terminal β-strand of FimG, forming a strong intermolecular linkage between FimH and FimG. While FimHLD can be expressed in a soluble, stable form, full length FimH is unstable alone (Vetsch, M., et al. J. Mol. Biol. 322:827-840 (2002); Barnhart M M, et al., Proc Natl Acad Sci USA. (2000) July 5; 97(14):7709-14) unless in a complex with the chaperone FimC or complemented with the donor strand peptide of FimG in peptide form or as a fusion protein (Barnhart M M, et al., Proc Natl Acad Sci USA. (2000) July 5; 97(14):7709-14; Sauer M M, et al. Nat Commun. (2016) March 7; 7:10738; Barnhart M M, et al. J Bacteriol. 2003 May; 185(9):2723-30). The design and expression of a full length FimH molecule by linking the FimG donor peptide to full length FimH via a glycine-serine linker has been previously described (PCT Intl. Publication No. WO2021/084429, published May 6, 2021), and is designated FimH-DSG. FimHLD is thought to be a poor immunogen in terms of its ability to stimulate functional immunogenicity. Some studies suggest that although binding antibody titers can be elicited with FimHLD with and without adjuvant, functional neutralizing titers were only observed in the presence of adjuvant (PCT Intl. Publication No. WO2021/084429, published May 6, 2021). Studies suggest that locking FimH in an open conformation, with reduced affinity for mannoside ligands, improves functional immunogenicity (Kisiela, D. I. et al., Proc Natl Acad Sci USA 110, 19089-19094 (2013). Accordingly, there is a need in the art for novel FimH mutan