WO2022165609A1 - Anticorps dérivés de plante recombinés et variants fc, et méthodes associées - Google Patents
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Classifications
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- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
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- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
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- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B13/00—Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff
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- A—HUMAN NECESSITIES
- A24—TOBACCO; CIGARS; CIGARETTES; SIMULATED SMOKING DEVICES; SMOKERS' REQUISITES
- A24B—MANUFACTURE OR PREPARATION OF TOBACCO FOR SMOKING OR CHEWING; TOBACCO; SNUFF
- A24B13/00—Tobacco for pipes, for cigars, e.g. cigar inserts, or for cigarettes; Chewing tobacco; Snuff
- A24B13/02—Flakes or shreds of tobacco
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- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1228—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K16/1232—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia from Escherichia (G)
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/62—DNA sequences coding for fusion proteins
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- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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- C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
- C07K2317/13—Immunoglobulins specific features characterized by their source of isolation or production isolated from plants
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/52—Constant or Fc region; Isotype
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present application relates generally to recombinant plant-derived proteins, and more specifically to recombinant antibodies, and Fc variants thereof, and methods of producing the same.
- the application also relates to methods of preventing or reducing colonization of Escherichia coli in a mammal.
- the application also relates to methods of detecting the presence of E. coli in a sample.
- E. coli colonizes at mucosal sites primarily in the gastrointestinal (Gl) tract in an animal. E. coli is ultimately transmitted to humans through consumption of contaminated foods, such as undercooked or raw meat, milk, or vegetables, for example, and may cause severe gastrointestinal illness with lifethreatening consequences in some cases.
- E. coli colonization in the gastrointestinal tract in animals reduces the risk of contamination from fecal shedding or at slaughter and would ultimately reduce contamination of food sources for human consumption.
- the adhesion protein intimin expressed on the outer membrane in E. coli cells, mediates interaction between the bacteria and, for example, the epithelial cells lining the inner surface of the animal host’s gastrointestinal tract. Colonization is initiated when intimin binds to the translocated intimin receptor (Tir) located on epithelial cells. Interfering with this binding would reduce such colonization and facilitate subsequent expulsion of E. coli from an animal’s gastrointestinal tract.
- Tir translocated intimin receptor
- the availability of effective therapeutics and diagnostics for treatment or prevention of E. coli contamination remains a problem, and new therapies which can prevent binding of intimin to gut epithelial cells are desirable.
- slgA secretory immunoglobulin A
- administration of slgA can impart immediate, if transient, protection from a pathogen, it may be of value to beef producers and processors as a pre-harvest intervention for E. coli.
- slgA primarily functions to clear pathogens by immune exclusion: after binding to its target, glycans on the secretory component facilitate binding to the mucus lining of the Gl tract, enabling clearance of slgA-pathogen complexes by peristalsis.
- a slgA directed against intimin would thus be expected to prevent luminal E. coli cells from interacting with the host epithelium, clearing them by entrapment in the mucus layer and subsequent fecal shedding.
- Chimeric slgA antibodies which specifically recognize E. coli intimin and which comprise a single domain camelid antibody (V H or VHH) fused to a bovine IgA Fc chain have been reported by Saberianfar et al ⁇ Frontiers in Plant Science (2019), 10: 270) to show efficacy against several strains of enterohemorrhagic E. coli.
- one aspect of the invention provides an Fc variant polypeptide which is a variant of a native Fc polypeptide such that the variant sequence of the Fc variant polypeptide comprises one or more mutations of the native sequence of the native Fc polypeptide.
- the one or more mutations result in one or more of an increase in a net surface negative charge of the Fc variant polypeptide compared to a net surface negative charge of the native Fc polypeptide and introduction of cysteine residues adapted to form a disulfide bridge.
- the Fc variant polypeptide exhibits enhanced accumulation when expressed in a plant cell compared to accumulation of the native Fc polypeptide when expressed in the plant cell.
- the native sequence of the native Fc polypeptide is SEQ ID NO:47 and the one or more mutations are selected from N9D, N84D, N131 D, Q175E, Q195E, G196C/R219C and combinations thereof.
- the Fc variant polypeptide has a sequence selected from SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.
- Another aspect of the invention provides a method of producing an Fc variant polypeptide, the method comprising:
- selecting the solvent-exposed amino acid residue for mutation to a negatively charged amino acid residue comprises selecting an asparagine or glutamine residue for mutation to an aspartic acid or glutamic acid residue, respectively.
- the invention provides a method of producing a Fc variant polypeptide, the method comprising:
- a further aspect of the invention provides an Fc variant fusion polypeptide including an Fc variant polypeptide as described herein fused to a bioactive moiety.
- the bioactive moiety is a variable domain of an antibody.
- the bioactive moiety is a single domain antibody.
- the bioactive moiety is a VHH polypeptide as described herein.
- the variable domain of an antibody, the single domain antibody or the V H polypeptide specifically binds to intimin on an Escherichia coli cell.
- the present invention provides an antibody or antigen binding fragment thereof comprising an Fc variant fusion polypeptide as described herein comprising a variable domain of an antibody fused to an Fc variant polypeptide as described herein.
- the variable domain of the antibody is a single domain antibody.
- the variable domain of the antibody is a V H polypeptide as described herein.
- the antibody or antigen binding fragment thereof specifically binds to intimin on an Escherichia coli cell.
- the present invention provides a V H polypeptide comprising a first complementarity determining region (CDR1), a second complementarity determining region (CDR2) and a third complementarity determining region (CDR3), wherein:
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:4, the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:5 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:6;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:8
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:9
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NQ:10;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:12
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:13
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:14;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:16
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:17
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:18;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:20
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:21
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:22;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:24
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:25
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:26;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:28
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:29
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NQ:30;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:32
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:33
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:34;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:36
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:37
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:38;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NQ:40
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:41
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:42; or
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:44
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:45
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:46.
- the invention provides an antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises a V H polypeptide as described herein.
- the antibody or antigen binding fragment thereof comprises a VHH-FC fusion polypeptide comprising a VHH polypeptide as described herein fused to an Fc polypeptide.
- the Fc polypeptide is a native Fc polypeptide.
- the Fc polypeptide is an Fc variant polypeptide as described herein.
- the antibody or antigen binding fragment thereof comprises an Fc variant fusion polypeptide as described herein comprising a V H polypeptide as described herein fused to an Fc variant polypeptide as described herein.
- the antibody is an IgA antibody.
- nucleic acid encoding a V H polypeptide as described herein, an Fc variant polypeptide as described herein, a Fc variant fusion polypeptide as described herein, or a VHH-FC fusion polypeptide as described herein.
- the nucleic acid further comprises a chloroplast targeting signal sequence or an endoplasmic reticulum targeting signal sequence.
- the chloroplast targeting signal sequence is a stroma targeting signal sequence or a thylakoid targeting signal sequence.
- the thylakoid targeting signal sequence is a Sec signaling sequence.
- the thylakoid targeting signal sequence is a Tat signaling sequence.
- the present invention provides an expression vector comprising a nucleic acid as described herein.
- a further aspect of the invention provides a host cell comprising an expression vector as described herein.
- Yet another aspect of the invention provides a non-viable harvested plant material comprising a host cell as described herein.
- the invention provides a non-viable edible product comprising a host cell as described herein.
- a further aspect of the invention provides a tobacco product comprising a host cell as described herein.
- the invention provides an animal feed comprising a host cell as described herein.
- a further aspect of the invention provides a method of producing a V H polypeptide as described herein, an Fc variant polypeptide as described herein, an Fc variant fusion polypeptide as described herein, or a V H-FC fusion polypeptide as described herein, the method comprising transforming a host cell with an expression vector including a nucleic acid as described herein.
- An additional aspect of the invention provides a method of enhancing accumulation of a recombinant protein in a plant cell, the method comprising transforming the plant cell with a recombinant expression vector comprising a nucleic acid encoding a Fc variant fusion polypeptide as described herein comprising a bioactive moiety as described herein.
- the recombinant protein is a recombinant antibody and the bioactive moiety is a variable domain of the antibody.
- the nucleic acid further encodes a chloroplast targeting signal sequence.
- the chloroplast targeting signal sequence is a Sec signaling sequence.
- a further aspect of the invention provides a method of producing a recombinant protein in a plant or portion thereof, the method comprising transforming the plant or portion thereof with a recombinant expression vector comprising a nucleic acid encoding a Fc variant fusion polypeptide as described herein comprising a bioactive moiety as described herein.
- the recombinant protein is a recombinant antibody and the bioactive moiety is a variable domain of the antibody.
- the nucleic acid further encodes a chloroplast targeting signal sequence.
- the chloroplast targeting signal sequence is a Sec signaling sequence.
- the invention provides a method of enhancing expression of a recombinant antibody in a plant cell, the method comprising transforming the plant cell with a recombinant expression vector comprising a nucleic acid encoding a variable domain of the antibody fused to an Fc polypeptide or to an Fc variant polypeptide as described herein and further encoding a chloroplast targeting signal sequence.
- the chloroplast targeting signal sequence is a Sec signaling sequence.
- the variable domain of the antibody is a single domain antibody.
- the variable domain of the antibody is a VHH polypeptide as described herein.
- the recombinant antibody specifically binds to intimin on an Escherichia coli cell.
- the invention provides a method of producing a recombinant antibody in a plant or a portion thereof, the method comprising transforming the plant or portion thereof with a recombinant expression vector comprising a nucleic acid encoding a variable domain of the antibody fused to a Fc polypeptide or to an Fc variant polypeptide as described herein and further encoding a chloroplast targeting signal sequence.
- the chloroplast targeting signal sequence is a Sec signaling sequence.
- the variable domain of the antibody is a single domain antibody.
- the variable domain of the antibody is a V H polypeptide as described herein.
- the recombinant antibody specifically binds to intimin on an Escherichia coli cell.
- Another aspect of the invention provides a pharmaceutical composition
- a pharmaceutical composition comprising an antibody or an antigen binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.
- the invention provides use of an antibody or antigen binding fragment thereof as described herein, for preventing or reducing E. coli cell colonization of the gastrointestinal tract of a mammal.
- Yet another aspect of the invention provides use of an antibody, or antigen binding fragment thereof as described herein, in preparation of a medicament for preventing or reducing E. coli cell colonization of the gastrointestinal tract of a mammal.
- Another aspect of the invention provides a method of preventing or reducing colonization of E. coli in the gastrointestinal tract of a mammal, comprising administering to the mammal an antibody, or antigen binding fragment thereof as described herein.
- the invention provides use of an antibody or an antigen binding fragment thereof as described herein for neutralizing the ability of an Escherichia coli cell to bind to a mammalian gastrointestinal epithelial cell.
- Another aspect of the invention provides use of an antibody, or antigen binding fragment thereof as described herein, in preparation of a medicament for neutralizing the ability of an E. coli cell to bind to a mammalian gastrointestinal epithelial cell.
- Another aspect of the invention provides a method of neutralizing the ability of an E. coli cell to bind to a mammalian gastrointestinal epithelial cell, comprising exposing the E. coli cell to an antibody, or antigen binding fragment thereof as described herein.
- Another aspect of the invention provides a method of detecting the presence of E. coli m a sample, comprising:
- the invention provides use of a V H polypeptide as described herein or a V H-FC fusion polypeptide as described herein for detecting the presence of E. coli in a sample.
- the invention provides a diagnostic kit for detecting the presence of E. coli in a sample, wherein the kit comprises a V H polypeptide as described herein or a V H-FC fusion polypeptide as described herein.
- Figure 2B is a graph showing accumulation levels of native Fc polypeptide compared to another embodiment of the present Fc variant polypeptide at 4, 6, and 8 dpi. * represents statistically significant difference from native Fc as determined by a T-test.
- Figure 3A is a graph showing accumulation levels of an embodiment of the present V H-FC fusion polypeptide including a V H polypeptide fused to a native Fc polypeptide compared to embodiments of the present V H-FC fusion polypeptide including a V H polypeptide fused to embodiments of the present Fc variant polypeptides at 4, 6, and 8 dpi.
- Figure 3B is a graph showing accumulation levels of an embodiment of the present V H H-FC fusion polypeptide including a V H polypeptide fused to a native Fc polypeptide compared to various embodiments of the present V H-FC fusion polypeptide including a V H polypeptide fused to embodiments of the present Fc variant polypeptides at 8 dpi.
- Figure 4 is a collection of images of Western blots probed with either anti-c-Myc (panels A and B) or anti-HA (panels C and D) which correspond to differently tagged embodiments of the present V H-FC fusion polypeptide including a V H polypeptide fused to a native Fc polypeptide (VnH-native Fc) or of the present VHH-FC fusion polypeptide including a V H polypeptide fused to an embodiment of the present Fc variant polypeptide (V H H-(5+1 )-FC) and JC subunits respectively.
- VnH-native Fc native Fc polypeptide
- VHH-FC fusion polypeptide including a V H polypeptide fused to an embodiment of the present Fc variant polypeptide (V H H-(5+1 )-FC) and JC subunits respectively.
- Leaf tissue was transformed with constructs of each subunit individually and also with combinations of V H-Native-Fc/SC/JC and V H-(5+1 )-FC/SC/JC for intended co-expression and assembly. Detection was done for both crude leaf extract (panels A and C) and for the eluent after the extract had been co-immunoprecipitated using an anti-FLAG column (panels B and D).
- FIG. 5 is a collection of confocal images showing the seven most prevalent E. coli strains incubated with either an embodiment of the present V H H-FC fusion polypeptide including a V H polypeptide fused to a native Fc polypeptide (V H-Native Fc) or with an embodiment of the present VHH-FC fusion polypeptide including a VHH polypeptide fused to an embodiment of the present Fc variant polypeptide (VHH-(5+1 )-FC).
- Figure 6 is a collection of confocal images of the seven most prevalent E. coli strains that have been immunolabelled and incubated with HEp-2 cells in the presence of PBS as a control, or in the presence of an embodiment of the present VHH-FC fusion polypeptide including a V H H polypeptide fused to a native Fc polypeptide (V H-Native- Fc) or an embodiment of the present V H-FC fusion polypeptide including a V H polypeptide fused to an embodiment of the present Fc variant polypeptide (V H H-(5+1)-FC).
- V H-Native- Fc native Fc polypeptide
- V H-FC fusion polypeptide including a V H polypeptide fused to an embodiment of the present Fc variant polypeptide
- FIG. 7 is a graph showing the relative fluorescence of the seven most prevalent E.
- coli strains that have been immunolabelled are adherent on HEp-2 cells and either incubated on HEp-2 cells in PBS alone, with an embodiment of the present VHH-FC fusion polypeptide including a V H polypeptide fused to a native Fc polypeptide (VHH9-Native-Fc) or with an embodiment of the present VHH-FC fusion polypeptide including a V H polypeptide fused to an embodiment of the present Fc variant polypeptide (V H9-(5+1 )-FC; V H9-Engineered Fc) and quantified by fluorometry.
- VHH9-Native-Fc native Fc polypeptide
- VHH-FC fusion polypeptide including a V H polypeptide fused to an embodiment of the present Fc variant polypeptide (V H9-(5+1 )-FC; V H9-Engineered Fc) and quantified by fluorometry.
- VHH9-Native-Fc native Fc polypeptide
- FIG. 8 is a schematic diagram representing the thylakoid expression vector.
- 2x35S double-enhanced promoter from Cauliflower Mosaic Virus 35S gene
- tCUP translational enhancer from a tobacco cryptic upstream promoter
- transit peptide a signaling sequence targeting the expressed protein to a subcellular compartment
- attB1/attB2 cloning sites used for GatewayTM cloning
- V H-FC an embodiment of the present V H-FC fusion polypeptide
- nosT nopaline synthase transcription terminator
- Xpress/C-Myc detection/purification tags.
- Figure 9A is a graph showing accumulation levels of an embodiment of the present VHH-FC fusion polypeptide including a VHH9 polypeptide fused to a native Fc polypeptide across the subcellular compartments cytoplasm, thylakoid lumen (Sec pathway), thylakoid lumen (Tat pathway), stroma and endoplasmic reticulum (ER) extracted in reducing (left) or non-reducing conditions (right). Error bars indicate standard error.
- Figure 9B is an image of a Western blot showing relative accumulation of the embodiment of the present V H-FC fusion polypeptide of Figure 9A across the compartments of Figure 9A in reducing (left) and non-reducing conditions (right).
- Figure 10 is a collection of confocal images showing an embodiment of the present V H H-FC fusion polypeptide tagged with green fluorescent protein (VHH-FC-GFP) and targeted to chloroplasts with either Sec, Tat or stromal targeting signals.
- Figure 11 is a graph showing accumulation of an embodiment of the present VHH-FC fusion polypeptide including a V H polypeptide fused to a native Fc polypeptide (Native) targeted to the thylakoid lumen with a Sec targeting signal and an embodiment of the present V H H-FC fusion polypeptide including a V H polypeptide fused to an embodiment of the present Fc variant polypeptide with an added disulfide bridge (+ Disulfide) targeted to the thylakoid lumen with a Sec targeting signal.
- *indicates statistical significance as determined by a T-test with p ⁇ 0.05, n 3 biological replicates. Error bars shown are standard error of the mean.
- FIG 12 is a collection of confocal images showing an embodiment of the present VHH-FC fusion polypeptide including a VHH polypeptide fused to a native Fc polypeptide (V H-FC) targeted to the chloroplast with either Sec, Tat or stromal signals incubated with E. coli O157:H7.
- DAPI red
- FITC-conjugated antibody green
- FIG. 13 is a collection of confocal images showing E. coli strain O157:H7 that has been incubated with HEp-2 cells in the presence of either an embodiment of the present V H-FC fusion polypeptide including a V H polypeptide fused to a native Fc polypeptide (V H-FC) targeted to Sec, Tat and stromal compartments or a Fc polypeptide (Fc) as a negative control targeted to the same compartments.
- V H-FC native Fc polypeptide
- Fc Fc polypeptide
- Fc variant polypeptide which is a variant of a native Fc polypeptide wherein the variant sequence of the Fc variant polypeptide comprises one or more mutations of the native sequence of the native Fc polypeptide.
- the native Fc polypeptide is an IgA Fc polypeptide.
- the native Fc polypeptide is a bovine IgA Fc.
- the present applicant used rational design strategies to introduce mutations which stabilize the structure of the Fc chain, thereby improving the accumulation of the Fc variant polypeptide when expressed in plant tissue.
- the strategies include:
- the sequence of the native Fc polypeptide is SEQ ID NO:47 and the one or more mutations are selected from N9D, N84D, N131 D, Q175E, Q195E, G196C/R219C and combinations thereof.
- the one or more mutations comprise N9D, N84D and N131 D.
- the one or more mutations comprise N9D, N84D, N131 D, Q175E and Q195E.
- the one or more mutations comprise N9D, N84D, N131 D, Q175E, Q195E and G196C/R219C.
- the Fc variant polypeptide has a sequence selected from SEQ ID NO:51 , SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:59.
- Another aspect of the invention provides a method of producing an Fc variant polypeptide, the method comprising:
- solvent accessibility of a candidate amino acid is determined by determining the average number of neighbouring atoms (within 10 A) per side-chain atom (AvNAPSA). Other methods of measuring solvent accessibility are known in the art and may be used as well.
- the native Fc polypeptide is a native IgA Fc polypeptide.
- the native IgA Fc polypeptide has the sequence of SEQ ID NO:47.
- selecting the solvent-exposed amino acid residue for mutation to a negatively charged amino acid residue comprises selecting an asparagine (Asn, N) or glutamine (Gin, Q) residue for mutation to an aspartic acid (Asp, D) or glutamic acid (Glu, E) residue, respectively.
- at least one of asparagine-9, asparagine-84 or asparagine- 131 of SEQ ID NO:47 is selected for mutation to aspartic acid.
- at least one of glutamine-175 or glutamine-195 of SEQ ID NO:47 is selected for mutation to glutamic acid.
- the invention provides a method of producing a Fc variant polypeptide, the method comprising:
- Selection of de novo intrachain disulfide bonds can be carried out by manual inspection of a model of the molecule for disulfide bonds that are expected to stabilize the tertiary structure of the protein, for example, by tethering beta strands in the Fc polypeptide together, or by other means known in the art.
- the native Fc polypeptide is a native IgA Fc polypeptide.
- the native IgA Fc polypeptide has the sequence SEQ ID NO:47.
- the first amino acid is glycine-196 (Gly, G) and the second amino acid is arginine-219 (Arg, R).
- glycine-196 and arginine-219 are each mutated to cysteine (Cys, C) residues.
- a disulfide bond forms between cysteine-196 and cysteine-219.
- the predetermined distance is less than 5 A.
- the Fc variant polypeptide exhibits enhanced accumulation when expressed in a plant cell compared to accumulation of the native Fc polypeptide when expressed in the plant cell.
- the plant cell is a cell of a Nicotiana plant or a Lactuca plant.
- the plant cell is a cell of a Nicotiana benthamiana plant or a Nicotiana tabacum plant.
- the Fc variant polypeptide exhibits at least a 3-fold increase in accumulation compared to accumulation of the native Fc polypeptide in the plant cell. In at least one embodiment, the Fc variant polypeptide exhibits up to about a 22-fold increase in accumulation compared to accumulation of the native Fc polypeptide in the plant cell. In at least one embodiment, the Fc variant polypeptide exhibits about a 22-fold increase in accumulation compared to accumulation of the native Fc polypeptide in the plant cell.
- the Fc variant polypeptide can act as a stabilization partner when fused to a variable domain of an antibody or to another bioactive moiety, so as to enhance accumulation and recombinant production of the fusion protein.
- a further aspect of the invention provides an Fc variant fusion polypeptide including an Fc variant polypeptide as described herein fused to a bioactive moiety.
- the bioactive moiety is a protein, including but not limited to an enzyme, a cytokine, an antigen, an antibody, an antibody fragment, a polypeptide, a signalling molecule, a receptor, or a ligand.
- Fc-fusion molecules Due to the wide variety of bioactive moieties that may be used as fusion partners with the Fc chain, the skilled person will recognize that these Fc-fusion molecules have numerous biological and pharmaceutical applications. In addition to their use in vaccines, intravenous immunoglobulin therapy, and drug therapies, in vitro applications may include, for example, protein binding assays, microarray applications, flow cytometry, and immunohistochemistry. Fusion with the Fc chain may also provide the bioactive moiety with a number of beneficial biological and pharmacological properties. For example, fusion with an Fc chain may significantly increase bioactive moiety’s plasma half-life, facilitate interaction with immune cell Fc receptors, and improve solubility and stability both in vivo and in vitro.
- the bioactive moiety is a variable domain of an antibody. In at least one embodiment, the bioactive moiety is a single domain antibody. In at least one embodiment, the bioactive moiety is a V H polypeptide as described herein. In at least one embodiment, accumulation of a Fc variant fusion polypeptide containing a Fc variant polypeptide fused to a V H polypeptide as described herein, when expressed in a plant cell, is enhanced up to about 16-fold compared to accumulation of a V H-FC fusion polypeptide containing a native Fc polypeptide fused to a VHH polypeptide as described herein.
- the present invention provides a V H H polypeptide comprising a first complementarity determining region (CDR1), a second complementarity determining region (CDR2) and a third complementarity determining region (CDR3), wherein:
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:4, the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:5 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:6;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:8
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:9
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NQ:10;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:12
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:13
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:14;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:16
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:17
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:18;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NQ:20
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:21
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:22;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:24
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:25
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:26;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:28
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:29
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NQ:30;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:32
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:33
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:34;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:36
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:37
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:38;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NQ:40
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:41
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:42; or
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:44
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:45
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:46.
- CDR and variable domain sequences may be highly homologous to the CDR and variable sequences specified herein and still retain antigen binding functionality.
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:4, the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:5 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:6;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:12
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:13
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:14;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:36
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:37
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:38;
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NQ:40
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:41
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:42; or
- the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:44
- the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:45
- the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:46.
- the V H polypeptide has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11 , SEQ ID NO:15, SEQ ID NO:19, SEQ ID NO:23, SEQ ID NO:27, SEQ ID NO:31 , SEQ ID NO:35, SEQ ID NO:39 or SEQ ID NO:43.
- the V H polypeptide has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:3, SEQ ID NO:11 , SEQ ID NO:35, SEQ ID NO:39 or SEQ ID NO:43.
- the VHH polypeptide specifically binds to intimin on an Escherichia coli cell.
- the V H H polypeptide binds to an epitope comprised in the C-terminal 277 residues of intimin.
- the V H polypeptide specifically binds to an epitope having at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to an epitope sequence which is a sub-sequence of SEQ ID NO:2.
- the V H polypeptide prevents binding of intimin on the E. coli cell to an epithelial cell.
- the epithelial cell is from the gastrointestinal tract of a mammal. In at least one embodiment, the mammal is bovine.
- the present invention provides an antibody or antigen binding fragment thereof.
- the antibody or antigen binding fragment thereof comprises an Fc variant fusion polypeptide as described herein comprising a variable domain of an antibody fused to an Fc variant polypeptide as described herein.
- the variable domain of the antibody is a VHH polypeptide as described herein.
- the antibody or antigen binding fragment thereof comprises a V H polypeptide as described herein.
- the antibody or antigen binding fragment thereof comprises a V H-FC fusion polypeptide comprising a VHH polypeptide as described herein fused to an Fc polypeptide.
- the Fc polypeptide is a native Fc polypeptide. In at least one embodiment, the Fc polypeptide is an Fc variant polypeptide as described herein. In at least one embodiment, the antibody or antigen binding fragment thereof specifically binds to intimin on an Escherichia coli cell.
- the antibody is a chimeric IgA antibody comprising a VHH-FC fusion polypeptide in which a V H polypeptide as described herein is fused to an IgA Fc polypeptide.
- the IgA Fc polypeptide is a bovine IgA Fc polypeptide.
- the Fc polypeptide is a native bovine IgA Fc polypeptide.
- the Fc polypeptide is a bovine IgA Fc variant polypeptide as described herein.
- the antibody is a chimeric IgA antibody comprising an Fc variant fusion polypeptide as described herein comprising a variable domain of an antibody fused to an IgA Fc variant polypeptide as described herein.
- the IgA Fc variant polypeptide is a bovine IgA Fc variant polypeptide as described herein.
- the variable domain of the antibody is a V H polypeptide as described herein. The skilled person will recognize that the present antibody or antigen binding fragment thereof could be adapted for use in other animals and thus appreciate that the variable domain of an antibody or V H polypeptide may be fused to an Fc polypeptide or Fc variant polypeptide suitable for use in other animals.
- native slgA antibodies include IgA subunits comprising two heavy chains, each including three constant domains (CH1 , CH2 and CH3) and a variable domain (V H ), and two light chains, each containing a constant domain (CL) and a variable domain (V L ).
- the CL domains of the light chains are each bound to the CH1 domains of the heavy chains by disulfide bonds.
- Each IgA subunit thus includes a Fc region, including the CH2 and CH3 constant domains of the heavy chains, and two antigenbinding Fab regions, including the variable domains (V H and V L ) of the heavy and light chains and the CL and CH1 constant domains.
- IgA units Two such IgA units are linked at the ends of their respective Fc regions by a 15-kDa joining chain (JC) to form an IgA dimer.
- JC 15-kDa joining chain
- SC secretory component
- the present chimeric IgA antibody includes two VHH-FC fusion polypeptides in place of the two heavy chains and two light chains.
- the V H-FC fusion polypeptide lacks the light chains and CH1 domains found in native mammalian slgA, assembly with the joining chain (JC) and secretory component (SC) subunits is directed specifically via disulfide bond formation with the IgA Fc region. Therefore, it is contemplated that the present VHH-FC fusion polypeptide forms an slgA complex with the JC and SC subunits.
- the chimeric slgA antibody comprises four V H-FC fusion polypeptides, one SC subunit, and one JC subunit.
- nucleic acid encoding a V H polypeptide as described herein, an Fc variant polypeptide as described herein, a Fc variant fusion polypeptide as described herein, or a V H-FC fusion polypeptide as described herein.
- the nucleic acid further comprises a chloroplast targeting signal sequence or an endoplasmic reticulum targeting signal sequence.
- the chloroplast targeting signal sequence is a stroma targeting signal sequence or a thylakoid targeting signal sequence.
- the thylakoid targeting signal sequence is a Sec signaling sequence.
- the thylakoid targeting signal sequence is a Tat signaling sequence.
- the present invention provides an expression vector comprising a nucleic acid as described herein.
- a further aspect of the invention provides a host cell comprising an expression vector as described herein.
- the host cell is a bacterial cell.
- the bacterial cell is Agrobacterium tumefaciens.
- the host cell is a plant cell.
- the plant cell is a Nicotiana plant cell.
- the plant cell is a Nicotiana benthamiana plant cell ora Nicotiana tabacum plant cell.
- the plant cell is a Lactuca plant cell.
- non-viable harvested plant material comprising a host cell as described herein.
- the non- viable plant harvested material comprises a leaf or a stem.
- the invention provides a non-viable edible product comprising a host cell as described herein.
- the non-viable edible product comprises a leaf or a stem.
- a further aspect of the invention provides a tobacco product comprising a host cell as described herein.
- the tobacco product is cut, shredded, powdered, loose, ground, granulated, or extruded.
- the invention provides an animal feed comprising a host cell as described herein.
- a further aspect of the invention provides a method of producing a VHH polypeptide as described herein, an Fc variant polypeptide as described herein, an Fc variant fusion polypeptide as described herein, a VHH-FC fusion polypeptide as described herein, or an assembled chimeric slgA antibody comprising an Fc variant polypeptide as described herein or a V H-FC fusion polypeptide as described herein, the method comprising transforming a host cell as described herein with an expression vector as described herein including a nucleic acid as described herein.
- the host cell is a plant cell.
- the plant is a Nicotiana plant or a Lactuca plant.
- the plant is a Nicotiana benthamiana plant or a Nicotiana tabacum plant.
- Other plant systems may be selected, as will be understood by the skilled person.
- the expression vector is delivered through Agrobacterium-mediated plant transformation.
- Agrobacterium strains are transformed with a plant-optimized expression vector comprising a nucleic acid as described herein.
- transforming the host cell with the nucleic acid molecule comprises preparing Agrobacterium strain cultures comprising a VHH-FC fusion polypeptide as described herein or a Fc variant fusion polypeptide as described herein, an SC subunit, and a JC subunit at optical densities (OD) of about 0.57, 0.14, and 0.14, respectively, for infiltration in the plant.
- Plant leaves are then co-infiltrated with transformed strains.
- the plant is harvested after infiltration.
- the plant is harvested more than 3 days post infiltration (dpi).
- the plant is harvested from between about 4 dpi to about 12 dpi.
- the plant is harvested at about 12 dpi.
- the plant is harvested at a stage of harvest in which accumulation of the V H polypeptide, Fc variant polypeptide, V H-FC fusion polypeptide, Fc variant fusion polypeptide or assembled chimeric slgA antibody in the plant is maximal.
- An additional aspect of the invention provides a method of enhancing accumulation of a recombinant protein in a plant cell as described herein, the method comprising transforming the plant cell with a recombinant expression vector comprising a nucleic acid encoding a Fc variant fusion polypeptide as described herein comprising a bioactive moiety as described herein.
- a further aspect of the invention provides a method of producing a recombinant protein in a plant or portion thereof, the method comprising transforming the plant or portion thereof with a recombinant expression vector comprising a nucleic acid encoding a Fc variant fusion polypeptide as described herein comprising a bioactive moiety as described herein.
- the plant is transiently transformed. In at least one embodiment, the plant is stably transformed. In at least one embodiment, the recombinant protein is a recombinant antibody and the bioactive moiety is a variable domain of the antibody.
- the nucleic acid further comprises an endoplasmic reticulum targeting signal sequence. In at least one embodiment, the nucleic acid further comprises a chloroplast targeting signal sequence. In at least one embodiment, the nucleic acid further comprises a Sec signaling sequence.
- the variable domain of the antibody is a VHH polypeptide as described herein. In at least one embodiment, the antibody is an IgA antibody. In at least one embodiment, the antibody is a chimeric IgA antibody. In at least one embodiment, the antibody specifically binds to intimin on an Escherichia coli cell.
- the invention provides a method of enhancing expression of a recombinant antibody in a plant cell, the method comprising transforming the plant cell with a recombinant expression vector comprising a nucleic acid encoding a variable domain of the antibody fused to a Fc polypeptide or to an Fc variant polypeptide as described herein and further encoding a chloroplast targeting signal sequence.
- a further aspect provides a method of producing a recombinant antibody in a plant or a portion thereof, the method comprising transforming the plant or portion thereof with a recombinant expression vector comprising a nucleic acid encoding a variable domain of the antibody fused to an Fc polypeptide or to an Fc variant polypeptide as described herein and further encoding a chloroplast targeting signal sequence.
- the plant is transiently transformed.
- the plant is stably transformed.
- the chloroplast targeting signal sequence is a stroma targeting signal sequence, a Sec signaling sequence or a Tat signaling sequence.
- the chloroplast targeting signal sequence is a Sec signaling sequence.
- variable domain of the antibody is a VHH polypeptide as described herein.
- the antibody is an IgA antibody.
- the antibody is a chimeric IgA antibody.
- the antibody specifically binds to intimin on an Escherichia coli cell.
- a chloroplast targeting signal and specifically a thylakoid targeting signal, such as a Sec signalling sequence or a Tat signalling sequence, in the expression vector will target the expressed protein to the thylakoid compartment of the chloroplast for folding.
- V H polypeptides Because folding of both a V H polypeptide and an Fc polypeptide requires the formation of stabilizing intrachain disulfide bonds, and because the thylakoid compartment provides an environment in which such oxidative folding is facilitated, it was contemplated that the expression and accumulation of a V H polypeptide as described herein, an Fc variant polypeptide as described herein, a Fc variant fusion polypeptide as described herein, or a V H-FC fusion polypeptide as described herein may be enhanced by targeting the expressed polypeptides to the thylakoid lumen in transplastomic plants, compared to targeting to the endoplasmic reticulum in transgenic plants.
- compositions comprising an antibody, or antigen binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.
- carrier is intended to refer to a diluent, adjuvant, excipient, or vehicle with which an antibody, or antigen binding fragment thereof as described herein can be administered to an animal in need thereof.
- pharmaceutically acceptable is intended to refer to carriers and compositions containing such carriers that are tolerable and do not typically produce untoward reactions to a subject being treated with or exposed to such carriers and compositions.
- pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government for use in pharmaceutical applications. Such pharmaceutically acceptable carriers are well known in the art and would be readily identified and used by the skilled person.
- the invention provides use of an antibody or antigen binding fragment thereof as described herein, for preventing or reducing Escherichia coli cell colonization of the gastrointestinal tract of a mammal. Yet another aspect of the invention provides use of an antibody or antigen binding fragment thereof as described herein, in preparation of a medicament for preventing or reducing E. coli cell colonization of the gastrointestinal tract of a mammal. Another aspect of the invention provides a method of preventing or reducing colonization of E. coli in the gastrointestinal tract of a mammal, comprising administering to the mammal an antibody or antigen binding fragment thereof as described herein.
- administering the polypeptide to the mammal comprises causing the mammal to ingest plant material from a plant as described herein that produces the polypeptide.
- the polypeptide or the plant material is for oral administration.
- the polypeptide or plant material is for rectal administration.
- the invention provides use of an antibody or an antigen binding fragment thereof as described herein for neutralizing the ability of an Escherichia coli cell to bind to a mammalian gastrointestinal epithelial cell.
- Another aspect of the invention provides use of an antibody or antigen binding fragment thereof as described herein, in preparation of a medicament for neutralizing the ability of an E. coli cell to bind to a mammalian gastrointestinal epithelial cell.
- Another aspect of the invention provides a method of neutralizing the ability of an E. coli cell to bind to a mammalian gastrointestinal epithelial cell, comprising exposing the E. coli cell to an antibody or antigen binding fragment thereof as described herein.
- the E. coli cell is a Shiga toxin-producing E. coli (STEC) cell or a cell of an enterohemorrhagic E. coli (EHEC) strain.
- the E. coli cell is a cell of strain O26:H11 , strain 0111 :Hnm, strain O145:Hnm, or strain O157:H7.
- the antibody or an antigen binding fragment thereof as described herein thus neutralizes the capacity of different E. coli strains to bind to a host’s cells thereby conferring cross-serotype inhibition of bacterial adhesion crucial to pathogenicity in a host system.
- Another aspect of the invention provides a method of detecting the presence of E. coli m a sample, comprising:
- the invention provides use of a V H polypeptide as described herein or a V H-FC fusion polypeptide as described herein for detecting the presence of E. coli in a sample.
- the invention provides a diagnostic kit for detecting the presence of E. coli in a sample comprising a VHH polypeptide as described herein or a V H-FC fusion polypeptide as described herein. The presence of E. coli may be confirmed by Western blotting analysis, ELISA, or any of the various antigenantibody detection methods known in the art.
- the sample is a food sample, environmental sample, or a sample from an animal or microorganism.
- the sample is a fecal sample, a carcass swab sample, a water sample, a sample from a packaged meat, a sample from a vegetable, a soil sample, or a sample from a food-contacting surface.
- the terms “about” or “approximately” as applied to a numerical value or range of values, including but not limited to a measurable value such as an amount, a temporal duration, and the like, are intended to mean that the recited values can vary within an acceptable degree of error for the quantity measured given the nature or precision of the measurements, such that the variation is considered in the art as equivalent to the recited values and provides the same function or result.
- the degree of error can be indicated by the number of significant figures provided for the measurement, as is understood in the art, and includes but is not limited to a variation of ⁇ 1 in the most precise significant figure reported for the measurement.
- Typical exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values, or within the experimental error of the indicated value (e.g. within the 95% confidence interval for the mean).
- the terms “about” and “approximately” can mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.
- the term “substantially” refers to the complete or nearly complete extent or degree of an action, characteristic, property, state, structure, item, or result, or of a lack thereof.
- an object that is “substantially” aligned would mean that the object is either completely aligned or nearly completely aligned.
- a composition that is “substantially free of” a material would either completely lack that material, or so nearly completely lack that material that the effect would be the same as if it completely lacked that material.
- a composition that is “substantially free of” an ingredient or element may still actually contain such an ingredient or element as long as there is no measurable effect thereof.
- the exact allowable degree of deviation from absolute completeness may in some cases depend on the specific context. However, generally speaking, the nearness of completion will be so as to have the same overall result as if absolute and total completion were obtained.
- polypeptide As used herein, the term “polypeptide”, “peptide”, and “protein” may be used interchangeably to refer to chains of amino acids of any length and may comprise amino acids modified naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.
- nucleic acid As used herein, the terms “nucleic acid”, “nucleic acid molecule”, “oligonucleotide”, or “polynucleotide” may be used interchangeably to refer to a polymer of nucleic acid residues in single or double stranded form, including but not limited to deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- the term “variant” when used in reference to a polynucleotide is intended to refer to a polynucleotide which differs in its nucleotide sequence from the sequence of a reference polynucleotide to which the variant is being compared by one or more nucleotide residues.
- the differences between the sequence of the variant and the sequence of the reference polynucleotide also referred to herein as variations or mutations, can include substitution of one or more nucleotide residues with different nucleotide residues, insertion of additional nucleotide residues or deletion of nucleotide residues.
- a variant can differ from a reference polynucleotide by substitution of one or more nucleotide residues with replacement nucleotide residues which do not alter the open reading frame(s) of the polynucleotide or the amino acid sequence of any protein(s) encoded by the polynucleotide.
- variant when used in reference to a polypeptide is intended to refer to a polypeptide which differs in its amino acid sequence from the sequence of a reference polypeptide to which the variant is being compared by one or more amino acid residues.
- the differences between the sequence of the variant and the sequence of the reference polypeptide can include substitution of one or more amino acid residues with different amino acid residues, insertion of additional amino acid residues or deletion of amino acid residues.
- a variant can differ from a reference polypeptide by conservative substitution of one or more amino acid residues with replacement amino acid residues which may have similar properties, including but not limited to charge, size and hydrophilicity, to the amino acid residues which the new residues replace.
- variants may completely or partially retain one or more biological functions of the reference polypeptide. In certain embodiments, variants may not retain one or more biological functions of the reference polypeptide.
- percent identity or “% identity” when used in reference to the sequence of a polypeptide or a polynucleotide is intended to mean the percentage of the total number of amino acid or nucleotide residues, respectively, in the sequence which are identical to those at the corresponding position of a reference polypeptide or polynucleotide sequence. In at least one embodiment, when the length of the variant sequence and the length of the reference sequence are not identical, percent identity can be calculated based on the total number of residues in the variant sequence or based on the total number or residues in the reference sequence.
- Percent identity can be measured by various local or global sequence alignment algorithms well known in the art, including but not limited to the Smith-Waterman algorithm and the Needleman- Wunsch algorithm. Tools using these or other suitable algorithms include but are not limited to BLAST (Basic Local Alignment Search Tool) and other such tools well known in the art.
- BLAST Basic Local Alignment Search Tool
- a variant polynucleotide sequence can hybridize to a polyribonucleotide or polydeoxyribonucleotide as described herein under at least moderately stringent conditions.
- at least moderately stringent hybridization conditions it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length.
- T m melting temperature
- T m melting temperature
- T m 81 .5 °C - 16.6 (Logw [Na + ]) + 0.41 (%(G+C) - 600/I), where %G+C is the percentage of cytosine and guanine nucleotides in the nucleic acid and I is the length of the nucleic acid in base pairs, or similar equation).
- the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature.
- a 1% mismatch may be assumed to result in about a 1 °C decrease in T m .
- the final wash temperature may be reduced by about 5 °C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions.
- stringent hybridization conditions are selected.
- the following conditions may be employed to achieve stringent hybridization: hybridization at 5x sodium chloride/sodium citrate (SSC)/5x Denhardt's solution/1 .0% sodium dodecylsulfate (SDS) at T m - 5 °C based on the above equation, followed by a wash of 0.2x SSC/0.1% SDS at 60 °C.
- Moderately stringent hybridization conditions include a washing step in 3x SSC at 42 °C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures.
- single domain antibody is intended to mean an immunoglobulin molecule consisting of only a single variable domain which includes the antigen binding site.
- a heavy chain variable domain (V H ) and a light chain variable domain (V L ) each contain three complementarity determining regions (CDRs) interconnected by framework regions (FRs).
- the hypervariable CDRs vary widely in sequence and are in direct contact with the antigen, providing the specificity of binding between a particular antibody and its antigen.
- the FRs are less variable in sequence and aid in maintaining the structure of the variable domains so that the CDRs are positioned for binding to the antigen.
- the V and V L regions interact to form an antigen binding site defined by a total of six CDRs.
- single domain antibodies are capable of binding to an epitope without an additional variable domain, with the antigen-binding site formed by a single V H /VHH, VNAR or VL domain.
- the antigen binding site of a single variable domain is generally formed by not more than three CDRs in conjunction with its associated FRs.
- the single variable domain may be a light chain variable domain (e.g. VL sequence), or a heavy chain variable domain (e.g. VH, VHH or VNAR sequence), or a fragment thereof capable of forming the single antigen binding unit, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit.
- VL sequence e.g. VL sequence
- VH, VHH or VNAR sequence e.g. VH, VHH or VNAR sequence
- VHH domains refer to the variable domain of camelid “heavy chain antibodies” (that is, antibodies which do not include a light chain), and are used to distinguish these variable domains from the heavy chain variable domains (referred to as “VH” or “V ” domains) and the light chain variable domains (referred to “as “VL” or “V L ” domains) present in conventional four chain antibodies.
- VNAR refers to the variable domain of single domain antibodies (IgNAR) found in sharks.
- neutralizes or “neutralizing antibody” means an antibody that reduces or abolishes the biological activity (for example, binding and/or infectivity) of the target to which it binds.
- Fc polypeptide is intended to refer to the polypeptide found in the constant region of an antibody.
- native Fc polypeptide is intended to mean an Fc polypeptide having a sequence substantially identical to the sequence of an Fc polypeptide found in nature and lacking any artificially induced mutations.
- the term “competes”, as used herein with regard to an antibody, means that a first antibody, antigen binding fragment thereof, ligand/receptor, or other protein binds to an epitope in a manner sufficiently similar to the binding of a second antibody, antigen binding fragment thereof, ligand/receptor, or other protein such that the result of binding of the first antibody antigen binding fragment thereof, ligand/receptor, or other protein to its cognate epitope is detectably decreased in the presence of the second antibody, antigen binding fragment thereof, ligand/receptor, or other protein compared to binding in the absence of the second antibody, antigen binding fragment thereof, ligand/receptor, or other protein.
- expression vector includes plasmid vectors, cosmid vectors, phage vectors, viral vectors, or any other vectors known to the skilled person.
- Expression vectors contain a desired coding sequence and promoter sequences for the expression of the operably linked coding sequence in a particular host organism (e.g., higher eukaryotes, lower eukaryotes, prokaryotes).
- the vector may also contain features relating to expression control (e.g., inducible and constitutive promoters) and identification (e.g., markers suitable for identifying vector-transformed cells such as tetracycline resistance or ampicillin resistance).
- chloroplast targeting signal sequence is intended to mean a nucleotide sequence encoding a chloroplast targeting transit peptide, such that any expressed protein fused to the chloroplast targeting transit peptide would be targeted to the chloroplast.
- endoplasmic reticulum targeting signal sequence is intended to mean a nucleotide sequence encoding an endoplasmic reticulum targeting signal peptide, such that any expressed protein fused to the endoplasmic reticulum targeting signal peptide would be targeted to the endoplasmic reticulum.
- animal feed is used herein to refer to food suitable for consumption by an animal, in solid or liquid form, that comprise nutrients for the sustenance and/or health of the recipient animal and may comprise additional components and/or supplements.
- sample includes biological samples such as cell samples, bacterial samples, virus samples, samples of other microorganisms, samples obtained from a mammalian subject, such as tissue samples, cell culture samples, stool or fecal samples, carcass swab samples, and biological fluid samples (e.g., blood, plasma, serum, saliva, urine, cerebral or spinal fluid, and lymph liquid), environmental samples, such as samples from food-contacting surfaces, air samples, water samples, dust samples and soil samples, and food samples, such as from raw or undercooked meat, packaged meat, milk, or vegetables.
- biological fluid samples e.g., blood, plasma, serum, saliva, urine, cerebral or spinal fluid, and lymph liquid
- environmental samples such as samples from food-contacting surfaces, air samples, water samples, dust samples and soil samples
- food samples such as from raw or undercooked meat, packaged meat, milk, or vegetables.
- transforming refers to a process whereby exogenous or heterologous DNA (i.e. , a nucleic acid construct) is introduced into a recipient host cell (e.g., prokaryotic cells, plant cells).
- a recipient host cell e.g., prokaryotic cells, plant cells.
- the transfer of genetic information to a host may be heritable (i.e. integrated within the host genome) and stable, or the transfer may be non-heritable and transient.
- solvent accessibility refers to the degree of exposure of a given amino acid residue of a protein to the surrounding solvent. For example, an amino acid residue located near the surface of the protein would be more accessible to solvent than would an amino acid residue located within interior folds of the protein. There are a variety of methods known in the art used to measure such exposure, including determining the average number of neighbouring atoms per side chain atom (AvNAPSA) for a given amino acid residue.
- AvNAPSA average number of neighbouring atoms per side chain atom
- Example 1 Chimeric V H H-secretory IgA antibodies against E. coli intimin
- V H HS variable domains of camelid heavy chain only antibodies
- V HS heavy chain only antibodies
- V H H-1 to V H-10 The amino acid sequences of identified V H domains (V H H-1 to V H-10) are listed in Table 1 , along with the amino acid sequence of an additional VHH domain (VnH-n) isolated from pooled peripheral blood lymphocytes of unimmunized alpacas, camels and llamas, which also binds to intimin.
- Complementarity determining regions CDR1 , CDR2 and CDR3 of each V H H domain were identified based on comparative analysis using the international ImMunoGeneTics (IMGT) database, as described, for example, in Lefranc et al, Developmental & Comparative Immunology (2003), 27(1): 55-77, and are listed in Table 1 .
- IMGT international ImMunoGeneTics
- ANN46383 a construct containing each fused V H-FC sequence was cloned into a plant expression vector including a PR1 b signal peptide and a KDEL retrieval signal peptide to enable targeting of the fused peptide to the endoplasmic reticulum (ER), as described by Pereira et al (BMC Biotechnol (2014), 14: 59).
- the amino acid sequence of native bovine IgA Fc is: D SSSCCVPNC EP SLSVQPPA LEDLLLGSNA SLTCTLSGLK SAEGASFTWN
- the DNA sequence (SEQ ID NO:49, below) encoding the V H9- Fc protein was prepared by fusing a DNA sequence encoding the V H9 protein (SEQ ID NO:35) to the DNA sequence (SEQ ID NO:48) encoding the Fc protein.
- amino acid sequence of the VHH9-FC protein is:
- Nicotiana benthamiana leaves were infiltrated with an Agrobacterium tumefaciens strain transformed with an expression vector including DNA encoding either VHH1-FC, V H H3-FC, V H9-FC (SEQ ID NQ:50) or V H10-FC, in addition to A. tumefaciens strains each transformed with a vector encoding the bovine joining chain sequence (JC; NCBI accession no. NP 786967), the bovine secretory component sequence (SC; NCBI accession no. NP 776568) or p19, a suppressor of gene silencing from Cymbidium ringspot virus.
- JC bovine joining chain sequence
- SC bovine secretory component sequence
- p19 a suppressor of gene silencing from Cymbidium ringspot virus.
- the nascent subunit polypeptides should be temporally and spatially coordinated in a 4:1 :1 stoichiometric ratio of V H-FC:SC:JC. Optimization of the infiltration conditions required to obtain accumulation of an assembled V H-slgA complex and purification and characterization of the assembled V H-slgA complex were carried out as described in detail in Saberianfar et al, Frontiers in Plant Science (2019), 10: 270.
- the results show that, while a range of different optical densities may be used, the Agrobacterium strains expressing various V H-FC subunits, SC, JC, and p19 at optical densities (OD at A S oo) of about 0.57, 0.14, 0.14, and 0.14 respectively, provide increased accumulation levels of all three subunits up to 12 days post-infiltration.
- E. coli strains 0157, 0111 , 0145 and 026 group together based on sequence similarity of the C-terminal 277 residues of intimin.
- bovine IgA Fc was predicted using the l-TASSER (Iterative Threading Assembly Refinement) method (Wu et al. (2008). Proteins 72:547-556; Zhang Y (2008). BMC Bioinformatics 9:40), using the publicly available crystal structure of human IgA (Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) structure 11GA), with which the bovine IgA Fc shares 70% sequence similarity, as a threading template.
- the resulting predicted structure had a high confidence score of 1 .35 (given a range of -5 to 2) and was used to determine rational design candidates.
- De novo disulfide candidates at residue positions 196 and 219 of SEQ ID NO:47 were chosen based on neighboring proximity (under 5 A) for tethering the C-terminal end of a portion of the Fc structure referred to as “strand G” to the N terminal end of a portion of the Fc structure referred to as “strand F”. Without being bound by theory, it was contemplated that this additional disulfide bond could act to stabilize the Fc protein by tethering the end of strand G to adjacent strand F, thus preventing access by proteolytic enzymes to vulnerable hydrophobic regions of the unstructured tailpiece leading out from the end of strand G.
- the DNA sequence (SEQ ID NO:48) encoding the native bovine IgA Fc amino acid sequence (SEQ ID NO:47) was synthesized by Bio Basic Inc. (Markham, ON, Canada). Individual mutations N9D, N84D, N131 D, Q175E, Q195E and G196C/R219C were then made using an in vitro single primer site-directed mutagenesis method (Huang et al, (2017). Methods Mol. Biol. 1498: 375-383). A multi-site-directed mutagenesis method (Liang et al. (2012). Anal. Biochem. 427: 99-101) was used to combine mutations. The mutant Fc sequences prepared are listed in Table 3.
- each construct was cloned into a pCaMGate plant expression vector including an N-terminal PR1 b tobacco signal peptide sequence and a C-terminal KDEL tag for targeting the expressed protein to the endoplasmic reticulum (ER) (Pereira et al. (2014). BMC Biotechnol 14: 59).
- ER endoplasmic reticulum
- targeting the constructs to the ER was desirable because of the requirement for disulfide formation for correct folding assembly and because targeting to the ER using the vector described in Example 1 above gave robust accumulation of V H H-FC fusion proteins.
- the vectors were transformed into Agrobacterium tumefaciens (EHA105) strains.
- Transient expressions were performed by syringe infiltration into leaf tissue of N. benthamiana plants. Plants were grown in a growth chamber at 22 °C with a 16 h photoperiod at a light density of 110 pmol nr 2 s 1 for 7 weeks and fertilized with water soluble N:P:K (20:8:20) at 0.25 g/L (Plant products, Brampton, ON, Canada). Plant extracts were prepared under native conditions as described in Example 1 above. Purification was performed using an anti-c-myc purification kit (MBL International Corp., Woburn, MA, USA). Screening of ER-targeted wild type and mutant Fc was done by semi-quantitative Western blotting at four, six and eight days post-infiltration (dpi) as described in Example 1 above.
- each of the supercharged Fc mutants showed a three- to four-fold improvement in accumulation across the time course, compared to the accumulation of native Fc (SEQ ID NO:47).
- the de novo disulfide mutant, G196C/R219C showed a six to seven-fold improvement in accumulation compared to the native Fc after six dpi ( Figure 2B).
- each Fc mutant was fused to V H H9.
- Genetic fusions of Fc mutant sequences to anti-E. coli V H H9 (SEQ ID NO:35) were carried out using a sequence and ligation independent cloning (SLIC) method (Li et al. (2007). Nature Methods 4:251 -256), and the fused V H9-FC mutant constructs were expressed in Nicotiana benthamiana leaf tissue as described above.
- each of the individual mutant V H H9-FC fusions showed a three- to four-fold improvement in accumulation when compared to the native V H H9-FC fusion, as seen in the comparison of the unfused Fc mutants.
- the combined Fc mutants also showed progressively improved accumulation, with five mutations showing an approximately sixteen-fold improvement compared to the native VHH9-FC fusion, as seen from the results shown in Figure 3B. Based on these results, the skilled person would understand that the mutations introduced in the Fc chain led to multiple fold increases in accumulation for both Fc and VHH-FC fusions.
- V H H9-N9D/N84D/N131 D/Q175E/Q195E/G196C/R219C-Fc V H H9-(5+1 )-FC
- co-expressed V H9-FC/SC/JC co-expressed V H9-(5+1 )-FC/SC/JC
- no bands were detected in crude extract expressing only JC or SC
- ⁇ 44 kDa bands were seen only in extracts co-expressing V H9-FC/SC/JC and V H9-(5+1 )-FC/SC/JC ( Figure 4, panel B).
- V H9-FC or V H9-(5+1 )-FC was incubated with E. coli O26:H11 , O45:H2, O103:H2, O145:Hnm, 0121 :H19, 0111 :Hnm or O157:H7.
- E. coli binding assays were performed as described in Example 1 and/or according to methods known in the art. After washing and fixing with paraformaldehyde, bacteria was visualized with DAPI and V H H9-FC binding visualized using a secondary fluorescent antibody (rabbit anti-bovine-FITC) that binds Fc.
- HEp-2 adherence inhibition assays were performed as described above. HEp-2 cells were incubated with a culture of one of seven E. coli strains (O26:H11 , O45:H2, O103:H2, O145:Hnm, 0121 :H19, 0111 :Hnm and O157:H7) in the presence or absence of either V H9-FC or V H9-(5+1 )-FC, washed to remove any non-adherent bacteria and then visualized by immunofluorescence microscopy.
- E. coli strains O26:H11 , O45:H2, O103:H2, O145:Hnm, 0121 :H19, 0111 :Hnm and O157:H7
- V H9-FC or V H9-(5+1 )-FC abrogated the adhesion of E. coli strains O26:H11 , 0111 :Hnm, O145:Hnm and O157:H7 to HEp-2 cells to HEp-2 cells compared to the respective positive controls of no VHH-FC (+PBS treatment).
- V H9-FC retains the ability to neutralize adherence of E. coli strains 0157, 026, 0111 , and 0145 to HEp-2 cells.
- Plant expression vectors (schematically illustrated in Figure 8) were adapted from a cytosolic expression vector described by Pereira et al. ⁇ BMC Biotechnol (2014), 14: 59). Transit peptide sequences were synthesized and cloned using a sequence and ligation independent cloning method (Li et al, Nature Methods (2007), 4: 251 -256). The V H9-FC sequence described in Example 1 (SEQ ID NO:49) was then cloned into each vector by Gateway cloning and the reading frame for each vector was confirmed by sequencing.
- a vector designed to target the expressed construct to the chloroplast thylakoid by the secretory (Sec) pathway includes a bipartite transit peptide sequence corresponding to the N-terminal 75 amino acids of the Arabidopsis thaliana thylakoid luminal 15.0 kDa protein 2 (At5g52970; NCBI accession no. NP 001318791) and shown below.
- MAMLFRPPP S QCRSFSPFVF NYSSREVSSS SRLSLKTSGD EENWVSRFRS KSLSLVFSGA LALGLSLSGV GFADA
- This bipartite N-terminal transit peptide sequence consists of two signal regions in tandem.
- the first signal region targets the peptide to the outer double membrane of the chloroplast, where it is cleaved, releasing the remaining peptide into the stroma.
- the remaining second signal region targets the peptide to the thylakoid membrane, where it is cleaved, releasing the peptide into the thylakoid lumen.
- ChloroP and TargetP online tools Almagro Armenteros et al, Life Sci. Alliance (2019), 2: e201900429; Emanuelsson et al, Protein Sci. (1999), 8:978-984)
- the cleavage site between the two signals was predicted to be between serine-28 and serine-29 of SEQ ID NO:69 (indicated in underlined bold italics) .
- a vector designed to target the expressed construct to the chloroplast thylakoid by the twin-arginine translocation (Tat) pathway includes a transit peptide sequence corresponding to the N-terminal 71 amino acids of the Arabidopsis thaliana FKBP-type peptidyl-prolyl cis-trans isomerase (At1g20810; NCBI accession no. NP 001321139) and shown below.
- this bipartite N-terminal transit peptide sequence consists of two signal regions in tandem, the first targeting the peptide to the outer double membrane of the chloroplast, and the second targeting the peptide to the thylakoid membrane.
- the cleavage site between the two signals was predicted to be between alanine-38 and phenylalanine-39 of SEQ ID NQ:70 (indicated in underlined bold italics) .
- Vectors designed to target the expressed construct to the stroma, the endoplasmic reticulum or the cytoplasm were adapted from those described in Pereira et al., BMC Biotechnol (2014), 14: 59.
- the vector designed for targeting to the cytoplasm lacks a transit peptide sequence.
- Total accumulation is highest in the ER at 51 .1 mg/kg fresh weight (FW), followed by the thylakoid via Sectargeting at 30.5 mg/kg FW.
- Accumulation in the stroma and thylakoid via Tat-targeting are substantially lower at 6.6 mg/kg FW and 5.4 mg/kg FW respectively.
- an enriched band at 44 kDa is detected matching the predicted size of the V H H9-FC monomer for the ER, stromal, thylakoid via Sec and thylakoid via Tat compartments suggesting that the VHH9-FC dimer in these compartments is stabilized by an interchain disulfide bond.
- the V H9-FC was also encoded in the chloroplast by transforming the chloroplast genome through homologous recombination using vector pCEC5 as described in Kolotilin et al. (2013), Biotechnology for Biofuels 6:65.
- the V H9-FC was targeted to the thylakoid within the chloroplast using the Sec import pathway, using an N-terminally truncated Seq transit peptide having the sequence:
- the V H H9-FC fusion carrying the G196C/R2190 mutation was targeted to the thylakoid lumen via the Sec pathway and accumulation by Western blot after agroinfiltration was measured.
- the G196C/R219C mutant VHH9-FC showed a significant yield improvement over the native V H9-FC when targeted to the Sec pathway.
- introduction of a rationally designed de novo disulfide does not impede, but rather significantly enhances in vivo accumulation when introduced into the Sec-targeted Fc chain. Without being bound by theory, this indicates that the disulfide bridge is introduced in the lumen of the thylakoid after import of the unfolded polypeptide.
- VHH9-FC retained the ability of ER-targeted V H9-FC (Examples 1 and 2) to bind E. coli
- purified V H9-FC from each compartment was incubated with the pathogen then fixed in paraformaldehyde, washed and probed for immunofluorescence using a FITC labelled anti-c-myc secondary antibody.
- HEp-2 cells were incubated with E. coli O157:H7 in the presence or absence of purified VHH9-FC from each of the compartments. Cells were then washed to remove non-adherent bacteria, fixed in paraformaldehyde and incubated with immunofluorescent labels. HEp-2 cells were visualized by fluorescent actin staining using rhodamine phalloidin and O157:H7 cells visualized using a donkey anti-rabbit AlexaTM 350 secondary antibody. As seen in Figure 13, the addition of purified V H9-FC from any of the compartments abrogated adhesion of any labelled E.
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Abstract
La présente invention concerne des polypeptides comprenant une chaîne Fc variante qui présente une accumulation améliorée dans un organisme et leurs méthodes de production. L'invention concerne également une méthode d'amélioration de l'accumulation d'une protéine. La présente invention concerne en outre des polypeptides qui se lient spécifiquement à l'intimine sur une cellule d'Escherichia coli, comprenant des anticorps et des fragments de liaison à l'antigène de ceux-ci, et leurs méthodes de production. L'invention concerne aussi une méthode de prévention ou de réduction de la colonisation d'E. coli dans le tractus gastro-intestinal d'un mammifère. L'invention concerne enfin une méthode de détection de la présence d'E. coli dans un échantillon.
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PCT/CA2022/050177 WO2022165609A1 (fr) | 2021-02-08 | 2022-02-08 | Anticorps dérivés de plante recombinés et variants fc, et méthodes associées |
Country Status (2)
Country | Link |
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CA (2) | CA3108573A1 (fr) |
WO (1) | WO2022165609A1 (fr) |
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2021
- 2021-02-08 CA CA3108573A patent/CA3108573A1/fr active Pending
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2022
- 2022-02-08 CA CA3204634A patent/CA3204634A1/fr active Pending
- 2022-02-08 WO PCT/CA2022/050177 patent/WO2022165609A1/fr active Application Filing
Non-Patent Citations (5)
Title |
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ANONYMOUS: "Communicating Innovation in Plant Science", PLANT CANADA, 1 January 2019 (2019-01-01), XP055960805, Retrieved from the Internet <URL:https://web.archive.org/web/20220311190540if_/https://cspb-scbv.ca/resources/Documents/Meetings/NEW%20Program%20Booklet%20with%20Abstracts%20Plant%20Canada%202019%20July%205(2).pdf> [retrieved on 20220914] * |
CHIN-FATT, A., YIELD IMPROVEMENT OF A PLANT-MADE ANTIBODY AGAINST ENTEROHEMORRHAGIC E. COLI, 2020, Retrieved from the Internet <URL:https://ir.lib.uwo.ca/etd/7142> [retrieved on 20220311] * |
IMPROVING THE STABILITY OF A BOVINE SECRETORY IGA BY RATIONAL DESIGN OF THE FRAGMENT CRYSTALLISABLE CHAIN, 28 July 2017 (2017-07-28), Retrieved from the Internet <URL:https://web.archive.org/web/2O22O31O191124/https://twitter.com/MandanaMiri/status/89O993O93952118784?cxt=HHwWgIC95fHfuNOYAAAA> * |
PUBLICATION DATE OF THE FOLLOWING DOCUMENT, IN THE FORM OF A TWEETED PHOTOGRAPH OF A POSTER WHICH IS OF SUFFICIENT QUALITY TO PERMIT READING OF ITS TEXT, IS ESTABLISHED BY DATED TWITTER POST, Retrieved from the Internet <URL:https://twitter.com/MandanaMiri/status/89O993O93952118784?cxrHHwWgIC95fHfuNOYAAAA> * |
SABERIANFAR, R ET AL.: "lant-Produced Chimeric VHH-sIgA Against Enterohemorrhagic E. coli Intimin Shows Cross-Serotype Inhibition of Bacterial Adhesion to Epithelial Cells", FRONTIERS IN PLANT SCIENCE, vol. 10, no. 270, 12 March 2019 (2019-03-12), XP055960814, DOI: 10.3389/fpls.2019.00270 * |
Also Published As
Publication number | Publication date |
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CA3204634A1 (fr) | 2022-08-11 |
CA3108573A1 (fr) | 2022-08-08 |
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