CA3204634A1 - Recombinant plant-derived antibodies and fc variants and related methods - Google Patents

Recombinant plant-derived antibodies and fc variants and related methods Download PDF

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CA3204634A1
CA3204634A1 CA3204634A CA3204634A CA3204634A1 CA 3204634 A1 CA3204634 A1 CA 3204634A1 CA 3204634 A CA3204634 A CA 3204634A CA 3204634 A CA3204634 A CA 3204634A CA 3204634 A1 CA3204634 A1 CA 3204634A1
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seq
amino acid
polypeptide
acid sequence
sequence identity
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Rima Menassa
Adam Chin-Fatt
Reza Saberianfar
Kevin Henry
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Canada, Represented By Minis AS
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/743—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Agrobacterium; Rhizobium; Bradyrhizobium
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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
    • C12N15/8221—Transit peptides
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • 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)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—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
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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
    • C—CHEMISTRY; METALLURGY
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/522—CH1 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • 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®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/185—Escherichia
    • C12R2001/19—Escherichia coli
    • 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

Abstract

The present application describes polypeptides comprising a variant Fc chain that exhibits enhanced accumulation in an organism and methods of producing the same. A method of enhancing accumulation of a protein is also described. The present application also describes polypeptides which specifically bind to intimin on an Escherichia coli cell, including antibodies and antigen-binding fragments thereof, and methods of producing the same. Also described is a method of preventing or reducing colonization of E. coli in the gastrointestinal tract of a mammal. A method of detecting the presence of E. coli in a sample is also described.

Description

RECOMBINANT PLANT-DERIVED ANTIBODIES AND FC VARIANTS
AND RELATED METHODS
Field [0001] 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 co//in a mammal. The application also relates to methods of detecting the presence of E. coil in a sample.
Background
[0002] Food borne pathogens such as E. coil have consistently been one of the foremost foodborne pathogen threats worldwide. While there are many strategic interventions meant to prevent E. co//transmission to humans, conservative estimates indicate that the pathogen still causes 2.8 million acute illnesses annually.
[0003] Evading a host organism's defenses, E. coli colonizes at mucosal sites primarily in the gastrointestinal (GI) 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 life-threatening consequences in some cases.
[0004] Preventing or minimizing 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.
co/ifrom an animal's gastrointestinal tract. However, the availability of effective therapeutics and diagnostics for treatment or prevention of E. co//contamination remains a problem, and new therapies which can prevent binding of intimin to gut epithelial cells are desirable.
[0005] The use and efficacy of recombinant secretory immunoglobulin A (sIgA) in passive mucosal immunotherapy in livestock is documented to control pathogens in the GI tract. Because administration of sIgA 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. coll. In the GI tract, sIgA 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 GI tract, enabling clearance of sIgA-pathogen complexes by peristalsis. A sIgA directed against intimin would thus be expected to prevent lumina! E. coli cells from interacting with the host epithelium, clearing them by entrapment in the mucus layer and subsequent fecal shedding. Chimeric sIgA
antibodies which specifically recognize E. coli intim in and which comprise a single domain camelid antibody (VHH 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. coll.
[0006] One of the challenges in delivering therapeutics such as sIgA is the lack of cost-effective production strategies. Over the past twenty years, plants have become a preferred platform of choice for complex immunoglobulin proteins and related synthetics, including those that require glycosylation and disulfide bond formation for proper folding and assembly. However, low yield and the resulting high cost of production is arguably the greatest barrier for pushing these products to market. There are many strategies for improving the recombinant yield of plant-based biologics that include, for example, affecting the amount and stability of the transcript, affecting translation rates and susceptibility to gene silencing, the choice of host system/tissue, and affecting the physiological state of the plant to slow degradation of the accumulated recombinant protein through exogenous application of hormones or chemicals or changing environmental conditions. However, the improvements in yield provided by these strategies have been too modest to overcome the yield barrier to advancing these products to market and many require tailored optimization on a case-by-case basis.
[0007] While the use of a plant platform for folding and assembly of recombinant proteins and other synthetics in the endoplasmic reticulum (ER) is well established, some ER-targeted recombinant proteins have been associated with issues such as unfolded proteins, ER-associated degradation, and misfolding, potentially limiting the proper folding of certain antibodies, thus reducing antibody yield.
[0008] Accordingly, in addition to a need for therapies effective in curtailing E coil contamination of food and water supply, there is an associated need for strategies for improved recombinant protein yields to deliver plant produced therapeutics and diagnostics in a cost-effective manner.
Summary
[0009] The present applicant has found that using rational design methods to introduce mutations into the native Fc chain of an antibody can improve the accumulation of the antibody when recombinantly expressed in plant tissue. Therefore, one aspect of the invention provides an Fc variant polypeptide which is a variant of a native Fe 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.
[0010] In at least one embodiment, the native sequence of the native Fc polypeptide is SEQ ID NO:47 and the one or more mutations are selected from N9D, N84D, N131D, 0175E, 0195E, G196C/R219C and combinations thereof. In at least one embodiment, 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.
[0011] Another aspect of the invention provides a method of producing an Fc variant polypeptide, the method comprising:
(a) determining solvent accessibility of one or more amino acid residues in a native Fc polypeptide;
(b) selecting at least one solvent-exposed amino acid residue; and (c) mutating the at least one selected solvent-exposed amino acid residue to a negatively charged amino acid residue.
[0012] In at least one embodiment, 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.
[0013] In a further aspect, the invention provides a method of producing a Fc variant polypeptide, the method comprising:
(a) selecting a first amino acid and a second amino acid in a native Fe polypeptide, the second amino acid being within a predetermined distance from the first amino acid, wherein the first amino acid and the second amino acid are free from involvement in native disulfide bonding; and (b) mutating the first amino acid and the second amino acid respectively to a first cysteine residue and a second cysteine residue, such that a disulfide bond is formable between the first cysteine residue and the second cysteine residue.
[0014] 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. In at least one embodiment, 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 VHH polypeptide as described herein. In at least one embodiment, the variable domain of an antibody, the single domain antibody or the VHH polypeptide specifically binds to intimin on an Escherichia coli cell.
[0015] In an additional aspect, 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. In at least one embodiment, the variable domain of the antibody is a single domain antibody. In at least one embodiment, the variable domain of the antibody is a VHH polypeptide as described herein. In at least one embodiment, the antibody or antigen binding fragment thereof specifically binds to intimin on an Escherichia co//cell.
[0016] In another aspect, the present invention provides a VHH polypeptide comprising a first complementarity determining region (CDR1), a second complementarity determining region (CDR2) and a third complementarity determining region (CDR3), wherein:
(i) 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;
(ii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:10;
(iii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:14;
(iv) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:18;
(v) 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%, 5 99% or 100% amino acid sequence identity to SEQ ID NO:21 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:22;
(vi) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:26;
(vii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:30;
(viii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:34;
(ix) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:38;
(x) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:40, the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:41 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:42; or (xi) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:46.
[0017] In another aspect, the invention provides an antibody or antigen binding fragment thereof, wherein the antibody or antigen binding fragment thereof comprises a VHH
polypeptide as described herein. In at least one embodiment, 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. In at least one embodiment, 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 comprises an Fc variant fusion polypeptide as described herein comprising a VHH polypeptide as described herein fused to an Fc variant polypeptide as described herein. In at least one embodiment, the antibody is an IgA antibody.
[0018] Another aspect of the invention provides a nucleic acid encoding a VHH
polypeptide as described herein, an Fc variant polypeptide as described herein, a Fc variant fusion polypeptide as described herein, or a Viild-Fc fusion polypeptide as described herein. In at least one embodiment, the nucleic acid further comprises a chloroplast targeting signal sequence or an endoplasmic reticulum targeting signal sequence. In at least one embodiment, the chloroplast targeting signal sequence is a stroma targeting signal sequence or a thylakoid targeting signal sequence. In at least one embodiment, the thylakoid targeting signal sequence is a Sec signaling sequence. In at least one embodiment, the thylakoid targeting signal sequence is a Tat signaling sequence.
[0019] In another aspect, 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. In another aspect, 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. In another aspect, the invention provides an animal feed comprising a host cell as described herein.
[0020] 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, or a VHH-Fc fusion polypeptide as described herein, the method comprising transforming a host cell with an expression vector including a nucleic acid as described herein.
[0021] 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. In at least one embodiment, the recombinant protein is a recombinant antibody and the bioactive moiety is a variable domain of the antibody. In at least one embodiment, the nucleic acid further encodes a chloroplast targeting signal sequence. In at least one embodiment, the chloroplast targeting signal sequence is a Sec signaling sequence.
[0022] 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. In at least one embodiment, the recombinant protein is a recombinant antibody and the bioactive moiety is a variable domain of the antibody. In at least one embodiment, the nucleic acid further encodes a chloroplast targeting signal sequence. In at least one embodiment, the chloroplast targeting signal sequence is a Sec signaling sequence.
[0023] In another aspect, 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.
In at least one embodiment, the chloroplast targeting signal sequence is a Sec signaling sequence.
In at least one embodiment, the variable domain of the antibody is a single domain antibody. In at least one embodiment, the variable domain of the antibody is a VHH
polypeptide as described herein. In at least one embodiment, the recombinant antibody specifically binds to intimin on an Escherichia coli cell.
[0024] In another aspect, 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. In at least one embodiment, the chloroplast targeting signal sequence is a Sec signaling sequence. In at least one embodiment, the variable domain of the antibody is a single domain antibody. In at least one embodiment, the variable domain of the antibody is a VHH polypeptide as described herein. In at least one embodiment, the recombinant antibody specifically binds to intirnin on an Escherichia co//cell.
[0025] Another aspect of the invention provides a pharmaceutical composition comprising an antibody or an antigen binding fragment thereof as described herein, and a pharmaceutically acceptable carrier.
[0026] In another aspect, 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. co//in the gastrointestinal tract of a mammal, comprising administering to the mammal an antibody, or antigen binding fragment thereof as described herein.
[0027] In another aspect, 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. coil 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.
[0028] Another aspect of the invention provides a method of detecting the presence of E. coli in a sample, comprising:
(a) contacting the sample with a VHH polypeptide as described herein or with a VHH-Fc fusion polypeptide as described herein, and (b) detecting binding between intimin and the VHH polypeptide or the VHH-Fc fusion polypeptide.
In still another aspect, the invention provides use of a VHH polypeptide as described herein or a VHH-Fc fusion polypeptide as described herein for detecting the presence of E. co//in a sample. In another aspect, the invention provides a diagnostic kit for detecting the presence of E. co//in a sample, wherein the kit comprises a VHH
polypeptide as described herein or a VH H- Fc fusion polypeptide as described herein.

Brief Description of the Drawings
[0029] Exemplary embodiments are illustrated in referenced figures of the drawings. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive. Further features of the present invention will become apparent from the following written description and the accompanying figures, in which:
[0030] Figure 1 is a series of confocal images showing E. coli strain 0145 (C483) and E. co//strain 0145 (C625) incubated with HEp-2 cells, in the presence of PBS
(phosphate buffered saline) alone as a control, with VHH9-Fc or with VHH10-sIgA. Cells that are immunolabelled (white) are intimately adherent on HEp-2 cells (red) after repeated washes. The absence of immunolabeling suggests neutralization. Size bar = 20 urn.
[0031] Figure 2A is a graph showing accumulation levels of native Fc polypeptide compared to embodiments of the present Fc variant polypeptides at 4, 6, and 8 days post infiltration (dpi). Letters denote significantly different treatments as determined by one way ANOVA (analysis of variance) and post-hoc Tukey HSD (honest significant difference) test. P<0.05, n=3-5 biological replicates. Error bars shown are standard error of the mean.
[0032] 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.
[0033] Figure 2C is a graph showing accumulation levels of native Fc polypeptide compared to various embodiments of the present Fe variant polypeptides at 4, 6, and 8 dpi. Letters denote significantly different treatments as determined by one way ANOVA
and post-hoc Tukey HSD test. P<0.05, n=3-5 biological replicates. Error bars shown are standard error of the mean.
[0034] Figure 3A is a graph showing accumulation levels of an embodiment of the present VHH-Fc fusion polypeptide including a VHH polypeptide fused to a native Fc polypeptide compared to embodiments of the present VHH-Fc fusion polypeptide including a VHH polypeptide fused to embodiments of the present Fc variant polypeptides at 4, 6, and 8 dpi. Letters denote significantly different treatments as determined by one way ANOVA and post-hoc Tukey HSD test. P<0.05, n=3-5 biological replicates.
Error bars shown are standard error of the mean.
[0035] Figure 3B is a graph showing accumulation levels of an embodiment of the present VHH-Fc fusion polypeptide including a VHH polypeptide fused to a native Fc polypeptide compared to various embodiments of the present VHH-Fc fusion polypeptide including a VHH polypeptide fused to embodiments of the present Fc variant polypeptides at 8 dpi. Letters denote significantly different treatments as determined by one way ANOVA and post-hoc Tukey HSD test. P<0.05, n=3-5 biological replicates. Error bars 5 shown are standard error of the mean.
[0036] 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 VHH-Fc fusion polypeptide including a VHH
polypeptide fused to a native Fc polypeptide (VHH-native Fe) or of the present VHH-Fc fusion 10 polypeptide including a VHH polypeptide fused to an embodiment of the present Fc variant polypeptide (VHH-(5+1)-Fc) and JC subunits respectively. Leaf tissue was transformed with constructs of each subunit individually and also with combinations of VHH-Native-Fc/SC/JC and VHH-(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).
[0037] Figure 5 is a collection of confocal images showing the seven most prevalent E.
coli strains incubated with either an embodiment of the present VH H - Fc fusion polypeptide including a VHH polypeptide fused to a native Fc polypeptide (VHH-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). DAPI (4',6-diamidine-2'-phenylindole) has been used to visualize E. coil cells and a FITC (fluorescein 5(6)-isothiocyanate)-conjugated antibody has been used to immunolabel the Fc specifically. Size bar=10 pm.
[0038] Figure 6 is a collection of confocal images of the seven most prevalent E. coil 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 VHH polypeptide fused to a native Fc polypeptide (VHH-Native-Fc) or 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). As a control against nonspecific Fc binding, E. coil strain 0157:H7 was incubated with a Fc polypeptide only to confirm that neutralization was mediated through the VHH. Cells that are immunolabelled (white) are intimately adherent on HEp-2 cells (red) after repeated washes. The absence of immunolabeling suggests neutralization.
Size bar = 2011m.
[0039] Figure 7 is a graph showing the relative fluorescence of the seven most prevalent E. coil 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 VHH polypeptide fused to a native Fc polypeptide (VHH9-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 (VHH9-(5+1)-Fc; VHH9-Engineered Fc) and quantified by fluorometry.
As a negative control, HEp-2 cells were incubated with PBS instead of a bacterial strain or antibody. Letters indicate a significant difference of the amount of immunolabelled adherent bacteria as determined by a one-way ANOVA with a post-hoc Tukey HSD
test (p<0.05, N=3 biological replicates). Error bars indicate standard error.
[0040] Figure 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; VHH-Fc: an embodiment of the present VHH-Fc fusion polypeptide; nosT: nopaline synthase transcription terminator;
Xpress/C-Myc: detection/purification tags.
[0041] 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.
[0042] Figure 9B is an image of a Western blot showing relative accumulation of the embodiment of the present VHH-Fc fusion polypeptide of Figure 9A across the compartments of Figure 9A in reducing (left) and non-reducing conditions (right).
[0043] Figure 10 is a collection of confocal images showing an embodiment of the present VHH-Fc fusion polypeptide tagged with green fluorescent protein (VHH-Fc-GFP) and targeted to chloroplasts with either Sec, Tat or stromal targeting signals. Chlorophyll indicates the locations of the thylakoid grana. Fluorescence was sequentially captured, and the merged images show co-localization of GFP and chlorophyll. Size bar =
10 pm.
[0044] Figure 11 is a graph showing accumulation of an embodiment of the present VHH-Fc fusion polypeptide including a VHH polypeptide fused to a native Fc polypeptide (Native) targeted to the thylakoid lumen with a Sec targeting signal and an embodiment of the present VHH-Fc fusion polypeptide including a VHH 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.
[0045] Figure 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 (VHH-Fc) targeted to the chloroplast with either Sec, Tat or stromal signals incubated with E. coli 0157:H7. DAPI (orange) has been used to visualize E.
coh cells and a FITC-conjugated antibody (green) has been used to immunolabel the Fc specifically. Merged images appear yellow. Size bar=10 m.
[0046] Figure 13 is a collection of confocal images showing E. co//strain 0157:H7 that has been incubated with HEp-2 cells in the presence of either an embodiment of the present VHH-Fc fusion polypeptide including a VHH polypeptide fused to a native Fc polypeptide (VHH-Fc) targeted to Sec, Tat and stromal compartments or a Fc polypeptide (Fc) as a negative control targeted to the same compartments. Cells that are immunolabelled (white) are intimately adherent on HEp-2 cells (red) after repeated washes. The absence of immunolabeling suggests neutralization. Size bar = 20 m.
Detailed Description
[0047] One aspect of the invention provides an 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. In at least one embodiment, the native Fc polypeptide is an IgA Fe polypeptide.
In at least one embodiment, the native Fc polypeptide is a bovine IgA Fc.
[0048] 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:
1) enhancing the net surface negative charge of the Fe polypeptide chain through side chain alterations, also known as "supercharging". The resultant small charge-charge repulsive forces on the protein surface aid in reducing non-specific protein aggregation during recombinant protein production, and 2) introducing a de novo disulfide bridge in the Fc polypeptide chain to tether portions of the chain together, thereby providing further stability by preventing unfolding and exposure of reactive hydrophobic regions of the protein.
[0049] In at least one embodiment, the sequence of the native Fc polypeptide is SEQ ID
NO:47 and the one or more mutations are selected from N9D, N84D, N131D, 0175E, Q195E, G196C/R219C and combinations thereof. In at least one embodiment, the one or more mutations comprise N9D, N84D and N131D. In at least one embodiment, the one or more mutations comprise N9D, N84D, N131D, Q175E and Q195E. In at least one embodiment, the one or more mutations comprise N9D, N84D, N131D, 0175E, 0195E
and G196C/R219C. In at least one embodiment, 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.
[0050] Another aspect of the invention provides a method of producing an Fc variant polypeptide, the method comprising:
(a) determining solvent accessibility of one or more amino acid residues in a native Fc polypeptide;
(b) selecting at least one solvent-exposed amino acid residue; and (c) mutating the at least one selected solvent-exposed amino acid residue to a negatively charged amino acid residue.
[0051] In at least one embodiment, 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.
[0052] In at least one embodiment, the native Fc polypeptide is a native IgA
Fc polypeptide. In at least one embodiment, the native IgA Fc polypeptide has the sequence of SEQ ID NO:47. In at least one embodiment, selecting the solvent-exposed amino acid residue for mutation to a negatively charged amino acid residue comprises selecting an asparagine (Asn, N) or glutamine (Gln, Q) residue for mutation to an aspartic acid (Asp, D) or glutamic acid (Glu, E) residue, respectively. In at least one embodiment, at least one of asparagine-9, asparagine-84 or asparagine-131 of SEQ ID NO:47 is selected for mutation to aspartic acid. In at least one embodiment, at least one of glutamine-175 or glutamine-195 of SEQ ID NO:47 is selected for mutation to glutamic acid.
[0053] In a further aspect, the invention provides a method of producing a Fc variant polypeptide, the method comprising:
(a) selecting a first amino acid and a second amino acid in a native Fc polypeptide, the second amino acid being within a predetermined distance from the first amino acid, wherein the first amino acid and the second amino acid are free from involvement in native disulfide bonding; and (b) mutating the first amino acid and the second amino acid respectively to a first cysteine residue and a second cysteine residue, such that a disulfide bond is formable between the first cysteine residue and the second cysteine residue.
[0054] 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.
[0055] In at least one embodiment, the native Fc polypeptide is a native IgA
Fc polypeptide. In at least one embodiment, the native IgA Fc polypeptide has the sequence SEQ ID NO:47. In at least one embodiment, the first amino acid is glycine-196 (Gly, G) and the second amino acid is arginine-219 (Arg, R). In at least one embodiment, glycine-196 and arginine-219 are each mutated to cysteine (Cys, C) residues.
In at least one embodiment, a disulfide bond forms between cysteine-196 and cysteine-219.
In at least one embodiment, the predetermined distance is less than 5 A.
[0056] Without being bound by theory, it is contemplated that mutating the first and second amino acids to cysteine residues to form the disulfide bond stabilizes the tertiary structure of the Fc polypeptide. In at least one embodiment, such stabilization can be brought about by connecting at least two beta strands within the Fc polypeptide together.
[0057] In at least one embodiment, 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. In at least one embodiment, the plant cell is a cell of a Nicotiana plant or a Lactuca plant. In at least one embodiment, the plant cell is a cell of a Nicotiana benthamiana plant or a Nicotiana tabacum plant. In at least one embodiment, 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.
[0058] It is also contemplated that 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. Thus, 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. In at least one embodiment, 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. 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 5 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 10 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.
[0059] In at least one embodiment, 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 VHH polypeptide as described 15 herein. In at least one embodiment, accumulation of a Fc variant fusion polypeptide containing a Fc variant polypeptide fused to a VHH polypeptide as described herein, when expressed in a plant cell, is enhanced up to about 16-fold compared to accumulation of a VHH-Fc fusion polypeptide containing a native Fe polypeptide fused to a VHH polypeptide as described herein.
[0060] In another aspect, the present invention provides a VHH polypeptide comprising a first complementarity determining region (CDR1), a second complementarity determining region (CDR2) and a third complementarity determining region (CDR3), wherein:
(i) 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;
(ii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:10;
(iii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:14;
(iv) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:18;
(v) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:22;
(vi) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:26;
(vii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO :30;
(viii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:34;
(ix) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO :38;
(x) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:40, the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:41 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:42; or (xi) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:46.
[0061] The skilled person will appreciate that CDR and variable domain sequences may be highly homologous to the CDR and variable sequences specified herein and still retain antigen binding functionality.
In at least one embodiment of the VHH polypeptide:
(i) 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;
(iii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:14;
(ix) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:38;
(x) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:40, the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:41 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:42; or (xi) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID
NO:46.
[0062] In at least one embodiment, the VHH 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. In at least one embodiment, the VHH 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.
[0063] In at least one embodiment, the VHH polypeptide specifically binds to intimin on an Escherichia co//cell. In at least one embodiment, the VHH polypeptide binds to an epitope comprised in the C-terminal 277 residues of intimin. In at least one embodiment, the VHH 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. In at least one embodiment, the VHH polypeptide prevents binding of intimin on the E. coil cell to an epithelial cell. In at least one embodiment, the epithelial cell is from the gastrointestinal tract of a mammal. In at least one embodiment, the mammal is bovine.
[0064] In another aspect, the present invention provides an antibody or antigen binding fragment thereof. In at least one embodiment, 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. In at least one embodiment, the variable domain of the antibody is a VHH
polypeptide as described herein. In at least one embodiment, the antibody or antigen binding fragment thereof comprises a VHH polypeptide as described herein. In at least one embodiment, 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.
In at least one embodiment, 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 co//cell.
[0065] In at least one embodiment, the antibody is a chimeric IgA antibody comprising a VHH-Fc fusion polypeptide in which a VHH polypeptide as described herein is fused to an IgA Fc polypeptide. In at least one embodiment, the IgA Fc polypeptide is a bovine IgA
Fc polypeptide. In at least one embodiment, the Fc polypeptide is a native bovine IgA Fc polypeptide. In at least one embodiment, the Fc polypeptide is a bovine IgA Fc variant polypeptide as described herein. In at least one embodiment, 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. In at least one embodiment, the IgA Fc variant polypeptide is a bovine IgA Fc variant polypeptide as described herein. In at least one embodiment, the variable domain of the antibody is a VHH 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 VHH polypeptide may be fused to an Fc polypeptide or Fc variant polypeptide suitable for use in other animals.
[0066] Structurally, native sIgA antibodies include IgA subunits comprising two heavy chains, each including three constant domains (CH1, CH2 and CH3) and a variable domain (VH), and two light chains, each containing a constant domain (CL) and a variable domain (VL). 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 antigen-binding Fab regions, including the variable domains (VH and VL) of the heavy and light chains and the CL and CH1 constant domains. 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. A 70-kDa secretory component (SC) coils around the Fc regions of both IgA subunits.
[0067] In contrast, in at least one embodiment, the present chimeric IgA
antibody includes two VHH-Fc fusion polypeptides in place of the two heavy chains and two light chains. Although the VHH-Fc fusion polypeptide lacks the light chains and CH1 domains found in native mammalian sIgA, 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 sIgA complex with the JC and SC subunits. Thus, in at least one embodiment, the chimeric sIgA antibody comprises four VHH-Fc fusion polypeptides, one SC
subunit, and one JC subunit.
[0068] Another aspect of the invention provides a nucleic acid encoding a VHH
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. In at least one embodiment, the nucleic acid further comprises a chloroplast targeting signal sequence or an endoplasmic reticulum targeting signal sequence. In at least one embodiment, the chloroplast targeting signal sequence is a stroma targeting signal sequence or a thylakoid targeting signal sequence. In at least one embodiment, the thylakoid targeting signal sequence is a Sec signaling sequence. In at least one embodiment, the thylakoid targeting signal sequence is a Tat signaling sequence.
[0069] In another aspect, the present invention provides an expression vector 5 comprising a nucleic acid as described herein. A further aspect of the invention provides a host cell comprising an expression vector as described herein. In at least one embodiment, the host cell is a bacterial cell. In at least one embodiment, the bacterial cell is Agrobacterium tumefaciens. In at least one embodiment, the host cell is a plant cell. In at least one embodiment, the plant cell is a Nicotiana plant cell. In at least one 10 embodiment, the plant cell is a Nicotiana benthamiana plant cell or a Nicotiana tabacum plant cell. In at least one embodiment, the plant cell is a Lactuca plant cell.
[0070] Yet another aspect of the invention provides a non-viable harvested plant material comprising a host cell as described herein. In at least one embodiment, the non-viable plant harvested material comprises a leaf or a stem. In another aspect, the 15 invention provides a non-viable edible product comprising a host cell as described herein. In at least one embodiment, 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. In at least one embodiment, the tobacco product is cut, shredded, powdered, loose, ground, granulated, or extruded. In another aspect, the invention 20 provides an animal feed comprising a host cell as described herein.
[0071] 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 sIgA antibody comprising an Fe variant polypeptide as described herein or a VH 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. In at least one embodiment, the host cell is a plant cell. In at least one embodiment, the plant is a Nicotiana plant or a Lactuca plant. In one embodiment, 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.
[0072] In at least one embodiment, the expression vector is delivered through Agrobacterium-mediated plant transformation. In such embodiments, Agrobacterium strains are transformed with a plant-optimized expression vector comprising a nucleic acid as described herein. In at least one embodiment of a method of producing an assembled chimeric sIgA antibody comprising an Fc variant polypeptide as described herein or a VHH-Fc fusion polypeptide 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.
[0073] Plant leaves are then co-infiltrated with transformed strains. In at least one embodiment, the plant is harvested after infiltration. In at least one embodiment, the plant is harvested more than 3 days post infiltration (dpi). In at least one embodiment, the plant is harvested from between about 4 dpi to about 12 dpi. In at least one embodiment, the plant is harvested at about 12 dpi. In at least one embodiment, the plant is harvested at a stage of harvest in which accumulation of the VHH
polypeptide, Fc variant polypeptide, VHH-Fc fusion polypeptide, Fc variant fusion polypeptide or assembled chimeric sIgA antibody in the plant is maximal.
[0074] 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. In at least one embodiment, 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. In at least one embodiment, 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. In at least one embodiment, 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.
[0075] In another aspect, 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 Fe 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. In at least one embodiment, the plant is transiently transformed. In at least one embodiment, the plant is stably transformed. In at least one embodiment, the chloroplast targeting signal sequence is a stroma targeting signal sequence, a Sec signaling sequence or a Tat signaling sequence. In at least one embodiment, the chloroplast targeting signal sequence is a Sec signaling sequence. In at least one embodiment, 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 coil cell.
[0076] Without being bound by theory, it is contemplated that including 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. Because folding of both a VHH polypeptide and an Fc polypeptide requires the formation of stabilizing intra-chain 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 VHH 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 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.
[0077] Another aspect of the invention provides a pharmaceutical composition comprising an antibody, or antigen binding fragment thereof as described herein, and a pharmaceutically acceptable carrier. As used herein, the term "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. As used herein, the term "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. Preferably, as used herein, the term "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.
[0078] In another aspect, the invention provides use of an antibody or antigen binding fragment thereof as described herein, for preventing or reducing Escherichia co//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. In at least one embodiment, administering the polypeptide to the mammal comprises causing the mammal to ingest plant material from a plant as described herein that produces the polypeptide. In at least one embodiment, the polypeptide or the plant material is for oral administration. In at least one embodiment, the polypeptide or plant material is for rectal administration.
[0079] In another aspect, the invention provides use of an antibody or an antigen binding fragment thereof as described herein for neutralizing the ability of an Escherichia coil 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.
[0080] In at least one embodiment, the E. co//cell is a Shiga toxin-producing E. coli (STEC) cell or a cell of an enterohemorrhagic E. coli (EHEC) strain. In at least one embodiment, the E. coli cell is a cell of strain 026:H11, strain 0111:Hnm, strain 0145:Hnm, or strain 0157:H7. In at least one embodiment, the antibody or an antigen binding fragment thereof as described herein thus neutralizes the capacity of different E
coil strains to bind to a host's cells thereby conferring cross-serotype inhibition of bacterial adhesion crucial to pathogenicity in a host system.
[0081] Another aspect of the invention provides a method of detecting the presence of E. co//in a sample, comprising:
(a) contacting the sample with a VHH polypeptide as described herein or a VHH-Fc fusion polypeptide as described herein, and (b) detecting binding between intimin and the VHH polypeptide or a VHH-Fc fusion polypeptide.
[0082] In still another aspect, the invention provides use of a VHH
polypeptide as described herein or a VHH-Fc fusion polypeptide as described herein for detecting the presence of E. co//in a sample. In another aspect, 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 VHH-Fc fusion polypeptide as described herein. The presence of E.
coil may be confirmed by Western blotting analysis, ELISA, or any of the various antigen-antibody detection methods known in the art. In at least one embodiment, the sample is a food sample, environmental sample, or a sample from an animal or microorganism. In at least one embodiment, 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.
Definitions
[0083] Terms defined herein are provided solely to aid in the understanding of the present disclosure and should not be construed to have a scope less than understood by a person of ordinary skill in the art.
[0084] As used herein, 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. For example, 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 ( /0), 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).
Alternatively, and particularly in biological systems, 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.
[0085] As used herein, 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. For example, an object that is "substantially" aligned would mean that the object is either completely aligned or nearly completely aligned. For an additional example, 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 5 same as if it completely lacked that material. In other words, a composition that is "substantially free or 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 10 so as to have the same overall result as if absolute and total completion were obtained.
[0086] As used herein, unless otherwise required by context, the terms "a" and "an" are intended to mean "at least one" and include both singular and plural. Any examples following the term "for example" or "e.g." are not meant to be limiting or exhaustive.
[0087] As used herein, the terms "comprises", "comprising", "include", "includes", 15 "including", "contain", "contains" and "containing" are meant to imply inclusion of the stated element or step but not to the exclusion of other elements or steps.
[0088] 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, 20 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.
25 [0089] 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).
[0090] As used herein, 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. In certain embodiments, 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.
[0091] As used herein, the term "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. In certain embodiments, 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. In certain embodiments, 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.
[0092] As used herein, the term "percent identity" or " /c, 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.
[0093] In at least one embodiment, a variant polynucleotide sequence can hybridize to a polyribonucleotide or polydeoxyribonucleotide as described herein under at least moderately stringent conditions. By "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. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrid, is determined by the melting temperature (Tm), which in sodium-containing buffers is a function of the sodium ion concentration ([Na.-1) and temperature (Tm = 81.5 C - 16.6 (Logic [Nal) +
0.41(%(G+C) - 600/1), where %G+C is the percentage of cytosine and guanine nucleotides in the nucleic acid andl is the length of the nucleic acid in base pairs, or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule, a 1%
mismatch may be assumed to result in about a 1 C decrease in Tm. For example, if nucleic acid molecules are sought that have a >95% identity, 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.
[0094] In some embodiments, stringent hybridization conditions are selected.
By way of example 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 Tm - 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.
Additional guidance regarding hybridization conditions may be found in:
Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in:
Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.
[0095] As used herein, the term "single domain antibody" is intended to mean an immunoglobulin molecule consisting of only a single variable domain which includes the antigen binding site. In conventional immunoglobulins (i.e. four chain antibodies), a heavy chain variable domain (VH) and a light chain variable domain (VL) 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, in contrast, 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. Thus, the VH and VL regions interact to form an antigen binding site defined by a total of six CDRs. In contrast, single domain antibodies are capable of binding to an epitope without an additional variable domain, with the antigen-binding site formed by a single VH/VHH, VNAR or VL domain. As such, the antigen binding site of a single variable domain is generally formed by not more than three CDRs in conjunction with its associated FRs. Thus, 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.
[0096] The terms "VHH domains", "VH1-I domains", "VHH", or "VHH" 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 "VH" domains) and the light chain variable domains (referred to "as "VL" or "VL" domains) present in conventional four chain antibodies. The term "VNAR" refers to the variable domain of single domain antibodies (IgNAR) found in sharks.
[0097] The terms "specifically binds" or "binds specifically" are well understood in the art, and methods to determine such specific binding between an antibody and antigen are also well known in the art. An antibody "specifically binds" or "binds specifically" to a target if it binds with greater affinity, avidity, more readily, and/or with greater duration to the target than it binds to other present substances.
[0098] As used herein, the term "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.
[0099] As used herein, the term "Fc polypeptide" is intended to refer to the polypeptide found in the constant region of an antibody. The term "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.
[0100] 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.
[0101] The term "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). Among other features of vectors known to the skilled person, 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).
[0102] As used herein, the term "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. As used herein, the term "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.
[0103] The term "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.
[0104] As used herein, the term "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.
[0105] As used herein, the terms "transforming" or "transformation" refer 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). 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.
[0106] The term "solvent accessibility", as used herein, 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.
[0107] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure have the meanings that are commonly 5 understood by a person of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.
EXAMPLES
10 [0108] Other features of the present invention will become apparent from the following non-limiting examples which illustrate, by way of example, the principles of the invention.
The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present 15 specification unless otherwise indicated. See, e.g., Sambrook J. &
Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998).
[0109] In the specific experiments discussed herein, for the ease of reference and 20 understanding, the resulting data are reported as an example of the disclosure according to an embodiment. Exemplary methods and materials are also described herein, although methods and materials similar or equivalent to those described therein can also be used in the practice or testing of aspects of the disclosure. It is to be understood that these examples, materials, and methods are for illustrative purposes only and should not 25 be used to limit the scope of the present invention in any manner.
Example 1: Chimeric VHH-secretory IgA antibodies against E. coil intimin Preparation and characterization of variable domains of came/id heavy chain only antibodies (VHHs) [0110] Camelid variable domains of heavy chain only antibodies (VHHs) recognizing the 30 Escherichia coil intimin protein were prepared and characterized as described in detail in Saberianfar et al, Frontiers in Plant Science (2019), 10: 270, herein incorporated by reference. Briefly, a fusion protein (MBP-Int277, SEQ ID NO:1) including the C-terminal 277 residues (SEQ ID NO:2) of E. coli 0157:H7 strain EDL933 intimin y (intimin-277) fused C-terminally to maltose-binding protein (MBP) was expressed in E. coli (DE3) cells and used to immunize a male llama (Lama glama). The resulting expressed VHH genes were incorporated into a phage-displayed library and panned to identify VHH
domains which specifically recognize intimin. The amino acid sequences of identified VHH domains (VHH-1 to VHH-10) are listed in Table 1, along with the amino acid sequence of an additional VHH domain (VHH-fl) 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 VHH 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.
Table 1: Amino acid sequences of VHH domains and complementarity determining regions Sequence VHH Sequence Identifier QVQLVESGGGLVQAGGSLRLTCTASGRIFNLTDMSWY

RQAPGEMRALVAAITRDGQTSYGDSVKGRFTISRDNA E ID NO
SQ
H :3 KNTVYLQMNSLKPEDTAVYLCNADHYTYGLGHTDYW
GQGTQVTVSS

NO:4 NO:5 NO:6 QVQLVESGGGLVQAGGSLRLTCTASGRIFNLTDMSWY
RQAPGEMRALVAAITRDGQTSYGDSVKGRFTISRDNA
VH H2 SEQ ID NO:7 RNTVYLQMNSLKPEDTAVYLCNADHYTYGLGHTEYW
GOGTOVTVSS

NO:8 NO:9 NO:10 QVKLEESGGGLVQPGGSLRLSCAASGSFFEIDEMGW
YRQAPGKMRELVAGFTSGGMTNYADSVKGRFTFSRD
VHH3 SEQ ID NO:11 NAKNTVYLQMNSLSPEDTATYLCNAEIRLSAGWGLTE
YWGQGTQVTVSS

NO:12 NO:13 NO:14 QVKLEESGGGLVQPGGSLRLSCAASGSFFEIDEMGW
YRQAPGKMRELVAGFTSGGMTKYADSVKGRFTFSRD
VH H4 SEQ ID NO:15 NAKNTVYLQMNNLSPEDTAVYLCNAEIRLSAGWGLTD
YWGQGTQVTVSS

NO:16 NO:17 NO:18 QVKLEESGGGLVQPGGSLRLSCAVSGTFFEIETMAWY
RQAPDKMRELVAIISDGDSTRYGDSVKGRFTISRDNAK

NO:19 NTAFLQMNSLKYEDTAVYLCNADIHLSAGWGLTDYWG
QGTQVTVSS

NO:20 Sequence VHH Sequence Identifier SEQ ID NO:21 SEQ ID NO:22 QVKLE ESGGGLVQPGGSLRLSCAVSGTFFE I ETMAWY
RQAPDKMRELVAIISDGDSTRYGDSVKGRFTISRDNAK
VHH6 SEQ ID NO:23 NTAFLQMNSLKYEDTAVYLCNADIHLSAGWGLTGYWG
QGTQVTVSS

SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 EVQLVESGGGLVQAGGSLTLTCTASG RI FNLTDMNWY
RC:MPG EM RALVAAITI NGHTSYG DSVKG RFTISRDNAK
VHH7 SEQ ID NO:27 NTVYLQMNSLKPEDTAVYLCNANHYTYGLGHTDYWG
RGTQVTVSS

SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:30 EVOLVESGGGLVQAGGSLTLTCTASGPVFNLTDMNW
YRQAPGEMHALVAAITINGHTSYGDSVKGRFTISRDNA
VHH8 SEQ ID NO:31 KNIVYLQMNSLKPEDTAVYLCNANHYTYGLGHTDYW
GRGTQVTVSS

SEQ ID NO:32 SEQ ID NO:33 SEQ ID NO:34 QVQLVESGGGLVQSGGSLRLSCAASGNFFEVDTMDW
YROAPGKMRELVAGSYSGGSTNYGDSVKGRFTISRD
VHH9 SEQ ID NO:35 NAKNTVYLQMNSLKPEDTAVYLCNAQIRLQRDFGLTD
YWGQGTQVTVSS

SEQ ID NO:36 SEQ ID NO:37 SEQ ID NO:38 QVQLVESGGGLVQAGGSLRLSCVLSRSTFTENDMGW

SQ

AKNTAYLQMNSLKPEDTAVYYCNAVVGWAGSIANPRR
:39 EYWGQGTQVTVSS

SEQ ID NO:40 SEQ ID NO:41 SEQ ID NO:42 QVQLVESRGGSVQAGGSLRLSCAAPEWTGRQFCVA
WFRQSPGKVHELVARTHIDGFDTTYTDSVKGRFTISPD
VHHn SEQ ID NO:43 KAKSTVHLQMNDLKGGDTGNYYCRAKFANYCSDNWG
RLDDFPYWGQGTQVTVSS

SEQ ID NO:44 SEQ ID NO:45 SEQ ID NO:46 [0111] Monovalent binding affinity data for intimin of selected VHH sequences based on surface plasmon resonance (SPR) are shown in Table 2. No binding to MBP alone was observed. Based on these results, the skilled person would understand that at least these isolated VHH sequences bind specifically to intimin.

Table 2: Monovalent affinities and kinetics of the interaction between VHHs and MBP-Int277 by SPR (pH 7.4, 25 C) VHH ka (1/MS) kd (us) KD (nM) VHH1 7.5x105 5.3x10-3 7.1 VHH3 7.1x105 3.2x10-4 0.5 VHH9 1.3x106 1.5x10-3 1.1 VHH10 6.8x105 1.4x10-4 0.2 VHHn 5.3x105 1.1x10-3 2.1 Preparation and characterization of chimeric VHH-secretory IgA antibodies [0112] Chimeric antibodies containing the VHH domains VHH1, VHH3, VHH9 and fused to the Fc region of bovine immunoglobulin A (IgA) were prepared and characterized as described in detail in Saberianfar et al, Frontiers in Plant Science (2019), 10: 270. Briefly, each VHH sequence was fused to a bovine IgA Fc sequence (SEQ ID NO:47, derived from GenBank accession no. ANN46383) and a construct containing each fused VHH-Fc sequence was cloned into a plant expression vector including a PR1b 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:
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK SAEGASFTWN
PIGGKIAVQG SPKRDSCGCY SVSSVLPGCA DPWNSGQTYS CSVIHPESKS
SLTATIKKDL GNTFRPQVHL LPPPSEELAL NELVTLTCLV RGESPKEVLV
RWLQGNQELP REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC Y
(SEQ ID NO:47) and the corresponding encoding DNA sequence is:
gatagttcca gttgctgtgt cccaaattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgcttctggg ttctaacgct tctctaactt gtacactgag tggactgaag agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tggatgttac agcgtctcat cagtcttacc aggttgtgct gatccatgga actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca tcactcacag caacaatcaa gaaggacctg ggaaatacgt tccgtcctca agtgcattta ctccctccac cttcagagga actcgcattg aatgagctcg taacacttac ctgcttggta agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc agggaaaaat atttgacctg ggggcccctt ccggaagctg gccaatctgt tactactttt gctgttactt ctgttcttcg agttgatgct gaagtttgga aacagggcga tacttttagc tgcatggttg ggcacgaagu ucttuugutt guutttactc agaaaaccat agatcggtta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg (SEQ ID NO:48) [0113] As an example, the DNA sequence (SEQ ID NO:49, below) encoding the VHH9-Fc protein was prepared by fusing a DNA sequence encoding the VHH9 protein (SEQ ID
NO:35) to the DNA sequence (SEQ ID NO:48) encoding the Fc protein.
caagtacagc tagtagagtc cggtggtgga ttagttcagt ctggtggatc tcttagactt tcctgtgcag ctagtggaaa tttcttcgag gttgatacga tggattggta ccgtcaggca ccaggaaaga tgagagaact tgttgctggt tcatacagtg gaggttctac aaactacggg gattcagtta agggcaggtt tacaatctca agggataacg ctaaaaatac cgtctatctg cagatgaata gcctcaaacc tgaagacacc gccgtgtatt tgtgcaatgc ccaaattcgg ttgcaacgag actttgggtt gactgattat tgggggcaag gcactcaagt gactgtctct agcgagatag ttccagttgc tgtgtcccaa attgtgagcc atctctaagt gtccagcctc cagcactaga agatttgctt ctgggttcta acgcttctct aacttgtaca ctgagtggac tgaagagtgc agagggtgca tcatttacat ggaaccctac aggaggtaag acagctgtac aaggaagtcc aaagagagat tcctgtggat gttacagcgt ctcatcagtc ttaccaggtt gtgctgatcc atggaactcc ggacaaacgt tctcctgctc tgtaactcat cctgagtcta agtcatcact cacagcaaca atcaagaagg acctgggaaa tacgttccgt cctcaagtgc atttactccc tccaccttca gaggaactcg cattgaatga gctcgtaaca cttacctgct tggtaagagg tttcagccct aaggaggttt tggttaggtg gcttcaaggt aatcaggagc ttcccaggga aaaatatttg acctgggggc cccttccgga agctggccaa tctgttacta cttttgctgt tacttctgtt cttcgagttg atgctgaagt ttggaaacag ggcgatactt ttagctgcat ggttgggcac gaagcccttc cgcttgcctt tactcagaaa accatagatc ggttagccgg gaaaccgacc cacgttaatg tgtctgtggt gatgtctgaa gtggatggcg tgtgctatgg (SEQ ID NO:49) [0114] The amino acid sequence of the VHH9-Fc protein is:
QVQLVESGGG LVQSGGSLRL SCAASGNFFE VDTMDWYRQA PGKMRELVAG
SYSGGSTNYG DSVKGRFTIS RDNAKNTVYL QMNSLKPEDT AVYLCNAQIR
LQRDFGLTDY WGQGTQVTVS SDSSSCCVPN CEPSLSVQPP ALEDLLLGSN
ASLTCTLSGL KSAEGASFTW NTTGGKTAVQ GSPKRDSCGC YSVSSVLPGC
ADPWNSGQTF SCSVTHPESK SSLTATIKKD LGNTFRPQVH LLPPPSEELA
LNELVTLTCL VRGFSPKEVL VRWLQGNQEL PREKYLTWGP LPEAGQSVTT
FAVTSVLRVD AEVWKQGDTF SCMVGHEALP LAFTQKTIDR LAGKPTHVNV
SVVMSEVDGV CY
(SEQ ID NO:50) [0115] Nicotiana benthamiana leaves were infiltrated with an Agrobacterium tumefaciens strain transformed with an expression vector including DNA encoding either VHH1-Fc, VHH3-Fc, VHH9-Fc (SEQ ID NO:50) or VHH1O-Fc, in addition to A. tumefaciens strains each transformed with a vector encoding the bovine joining chain sequence (JC;
NCB!
accession no. NP 786967), the bovine secretory component sequence (SC; NCB!
accession no. NP 776568) or p19, a suppressor of gene silencing from Cymbidium ringspot virus. At six days post-infiltration, expression of each of the VHH1, VH1-13-Fc, VHH9-Fc, VHH1O-Fc, JO and SC subunits was detected in the leaves.
[0116] For correct assembly of secretory IgA (sIgA) subunits into a hetero-multimeric protein complex and optimal accumulation, the nascent subunit polypeptides should be temporally and spatially coordinated in a 4:1:1 stoichiometric ratio of VHH-Fc:SC:JC.
Optimization of the infiltration conditions required to obtain accumulation of an assembled VHH-sIgA complex and purification and characterization of the assembled VHH-sIgA 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 VHH-Fc subunits, 5 SC, JC, and p19 at optical densities (OD at A600) 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.
[0117] Furthermore, it was confirmed, using procedures described in detail in Saberianfar et al, Frontiers in Plant Science (2019), 10: 270, that the VHH-Fc, SC and JC
10 subunits associate in vivo after expression in the plant leaves to form a chimeric VHH-sIgA complex which can bind intimin, including intimin on the surface of cells of Escherichia coli strains 026:H11, 0145:Hnm, 0111:Hnm and 0157:H7. It was also confirmed, using procedures described in detail in Saberianfar et al, Frontiers in Plant Science (2019), 10: 270, that binding of VH H10-sIgA to E. co//strains 026:H11, 15 0111:Hnm and 0157:H7 could neutralize the ability of the bacteria to adhere to epithelial cells. A further experiment carried out using the conditions described in Saberianfar et al, Frontiers in Plant Science (2019), 10: 270 confirmed that both VHH10-sIgA and the VHH9-Fc subunit alone abrogated the adhesion of an alternative 0145 strain obtained from a different supplier (ATCC, C625) to HEp-2 cells and reduced the relative 20 fluorescence caused by adherent bacteria for to background levels. Thus, both VHH10-sIgA and VH H9- Fc were able to completely neutralize the alternative 0145 strain (Figure 1). As described in detail in Saberianfar et al, Frontiers in Plant Science (2019), 10: 270, E. coli strains 0157, 0111, 0145 and 026 group together based on sequence similarity of the C-terminal 277 residues of intimin.
25 Example 2: Bovine IgA Fc variants Selection of mutation sites in bovine IgA Fc [0118] The structure of bovine IgA Fc was predicted using the I-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 30 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.

[0119] Engineering of negatively supercharged Fc was performed computationally by first ranking residues for solvent accessibility by their average number of neighboring atoms (within 10 A) per side-chain atom (AvNAPSA) and then identifying highly polar solvent-exposed asparagine (Asn, N) and glutamine (Gln, Q) residues for mutation to their negatively charged counterparts, aspartic acid (Asp, D) and glutamic acid (Glu, E) respectively. Residues involved in the interaction of sIgA molecules with the Fc a receptor (FcaR), which may be involved in activation of the immune response, were excluded, as were residues expected to be involved in native glycosylation.
Three asparagine residues (at positions 9, 84 and 131 of SEQ ID NO:47) and two glutamine residues (at positions 175 and 195 of SEQ ID NO:47) on the surface of the Fc chain were selected for mutation to aspartic acid and glutamic acid, respectively.
Modelling the substitutions to the five selected residues predicted an increase in net negative charge from -5.30 to -10.29 at pH 7 with a corresponding change in pl from 5.39 to 4.85.
[0120] For the selection of de novo intrachain disulfide bonds, based on modelling of the predicted Fc structure, disulfide candidates were chosen by manual inspection of the molecule in PyMol (Schrodinger LLC (2010). The PyMOL molecular graphics system.
Version, 1(5), 0). To retain the functionality of the native Fc, native disulfide sites were avoided. The IgA Fc secondary structure includes a characteristic beta sandwich of seven anti-parallel beta strands for both its CH2 and CH3 domains. Both domains exhibit Greek key connectivity (ABED CFG) forming two distinct beta sheets that fold over each other. For both domains, an intra-chain disulfide in the centre of the beta sheet stabilizes the tertiary structure. 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.
Expression of Fe mutants in Nicotiana benthamiana leaf tissue [0121] 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, N131D, 0175E, 0195E 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 Fe sequences prepared are listed in Table 3.
Table 3: Mutant Fc amino acid sequences Sequence Mutant Sequence Identifier DSSSCCVPDC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL

NO:51 REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWDSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
N84D LPPPSEELAL NELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:52 REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
N131D LPPPSEELAL DELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:53 REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
0175E LPPPSEELAL NELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:54 REKYLTWGPL PEAGESVTTF AVTSVLRVDA EVWKQGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL
0195E LPPPSEELAL NELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:55 REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKEGDTFS
CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPNC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA
DPWNSGQTFS CSVTHPESKS SLTATIKKDL GNTFRPQVHL

LPPPSEELAL NELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:56 REKYLTWGPL PEAGQSVTTF AVTSVLRVDA EVWKQCDTFS
CMVGHEALPL AFTQKTIDCL AGKPTHVNVS VVMSEVDGVC

Sequence Mutant Sequence Identifier DSSSCCVPDC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK
SAEGASFTWN PTGGKTAVQG SPKRDSCGCY SVSSVLPGCA

N84D/ LPPPSEELAL DELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:57 CMVGHEALPL AFTQKTIDRL AGKPTHVNVS VVMSEVDGVC
DSSSCCVPDC EPSLSVQPPA LEDLLLGSNA SLTCTLSGLK

N131D/ LPPPSEELAL DELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:58 LPPPSEELAL DELVTLTCLV RGFSPKEVLV RWLQGNQELP SEQID NO:59 (5+1) Y
[0122] The DNA sequences encoding the mutant Fc chains are identified in Table 4.
Table 4: Mutant Fc DNA sequences Sequence Mutant Sequence Identifier gatagttcca gttgctgtgt cccagattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgcttctggg ttctaacgct tctctaactt gtacactgag tggactgaag agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tggatgttac agcgtctcat cagtcttacc aggttgtgct gatccatgga actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca tcactcacag caacaatcaa gaaggacctg ggaaatacgt tccgtcctca agtgcattta N9D ctccctccac cttcagagga actcgcattg aatgagctcg SEQID
NO:60 taacacttac ctgcttggta agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc agggaaaaat atttgacctg ggggcccott ccggaagctg gccaatctgt tactactttt gctgttactt ctgttcttcg agttgatgct gaagtttgga aacagggcga tacttttagc tgcatggttg ggcacgaagc ccttccgctt gcctttactc agaaaaccat agatcggtta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg Sequence Mutant Sequence Wentifier gatagttcca gttgctgtgt cccaaattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgcttctggg ttctaacgct tctctaactt gtacactgag tggactgaag agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tqqatqttac agcqtctcat cagtottacc aqqttqtqct gatccatggg actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca tcactcacag caacaatcaa gaaggacctg ggaaatacgt tccgtcctca agtgcattta N840 ctccctccac cttcagagga act cgcattg aatgagctcg SEQID
NO:61 taacacttac ctgottggta agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc agggaaaaat atttgacctg ggggcccctt ccggaagctg gccaatctgt tactactttt gctgttactt ctgttottcg agttgatgct gaagtttgga aacagggcga tacttttagc tgcatggttg ggcacgaagc ccttccgctt gcctttactc agaaaaccat agatcggtta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg gatagttcca gttgctgtgt cccaaattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgottctggg ttctaacgct tctctaactt qtacactgag tqqactqaaq agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tggatgttac agcgtctcat cagtcttacc aggttgtgct gatccatgga actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca tcactcacag caacaatcaa gaaggacctg ggaaatacgt tccgtcctca agtgcattta N131D ctocctccac cttcagagga actcgcattg gatgagctcg SEQID NO:62 taacacttac ctgcttggta agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc agggaaaaat atttgacctg ggggcccctt ccggaagctg gccaatctgt tactactttt gctgttactt ctgttcttcg agttgatgct gaagtttgga aacagggcga tacttttagc tgcatggttg ggcacgaagc ccttccgctt gcctttactc agaaaaccat agatcggtta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg Sequence Mutant Sequence Wentifier gatagttcca gttgctgtgt cccaaattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgcttctggg ttctaacgct tctctaactt gtacactgag tggactgaag agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tqqatqttac agcqtctcat cagtottacc aqqttqtqct gatccatgga actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca tcactcacag caacaatcaa gaaggacctg ggaaatacgt tccgtcctca agtgcattta 0175E ctccctccac cttcagagga actcgcattg aatgagctcg SEOID NO:63 taacacttac ctgottggta agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc agggaaaaat atttgacctg ggggcccctt ccggaagctg gcgaatctgt tactactttt gctgttactt ctgttottcg agttgatgct gaagtttgga aacagggcga tacttttagc tgcatggttg ggcacgaagc ccttccgctt gcctttactc agaaaaccat agatcggtta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg gatagttcca gttgctgtgt cccaaattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgottctggg ttctaacgct tctctaactt qtacactgag tqqactqaaq agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tggatgttac agcgtctcat cagtcttacc aggttgtgct gatccatgga actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca tcactcacag caacaatcaa gaaggacctg ggaaatacgt tccgtcctca agtgcattta 0195E ctocctccac cttcagagga actcgcattg aatgagctcg SEQID NO:64 taacacttac ctgcttggta agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc agggaaaaat atttgacctg ggggcccctt ccggaagctg gccaatctgt tactactttt gctgttactt ctgttcttcg agttgatgct gaagtttgga aagagggcga tacttttagc tgcatggttg ggcacgaagc ccttccgctt gcctttactc agaaaaccat agatcggtta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg Sequence Mutant Sequence Identifier gatagttcca gttgctgtgt cccaaattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgcttctggg ttctaacgct tctctaactt gtacactgag tggactgaag agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tqqatqttac aqcqtctcat caqtcttacc aqqttqtqct gatccatgga actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca tcactcacag caacaatcaa gaaggacctg ggaaatacgt tccgtcctca agtgcattta G196C/ R219C ctccctccac cttcagagga actcgcattg aatgagctcg SEQID NO:65 taacacttac ctgottggta agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc agggaaaaat atttgacctg ggggcccctt ccggaagctg gccaatctgt tactactttt gctgttactt ctgttottcg agttgatgct gaagtttgga aacagtgtga tacttttagc tgcatggttg ggcacgaagc ccttccgctt gcctttactc agaaaaccat agattgttta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg gatagttcca gttgctgtgt cccagattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgottctggg ttctaacqct tctctaactt qtacactqaq tqqactqaaq agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tggatgttac agcgtctcat cagtcttacc aggttgtgct gatccatggg actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca tcactcacag caacaatcaa N9D/N840/ gaaggacctg ggaaatacgt tccgtcctca agtgcattta N131 ctocctccac cttcagagga actcgcattg gatgagctcg SEQID
NO:66 D
taacacttac ctgcttggta agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc agggaaaaat atttgacctg ggggcccctt ccggaagctg gccaatctgt tactactttt gctgttactt ctgttcttcg agttgatgct gaagtttgga aacagggcga tacttttagc tgcatggttg ggcacgaagc ccttccgctt gcctttactc agaaaaccat agatcggtta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg Sequence Mutant Sequence Identifier gatagttcca gttgctgtgt cccagattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgcttctggg ttctaacgct tctctaactt gtacactgag tggactgaag agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tqqatqttac agcqtctcat cagtottacc aqqttqtqct gatccatggg actccggaca aacgttctcc tgctctgtaa ctcatcctga gtctaagtca tcactcacag caacaatcaa N9D/N84D/ gaaggacctg ggaaatacgt tccgtcctca agtgcattta N131 0/01 ctccctccac cttcagagga act cgcattg gatgagctcg SEQID NO:67 75E/0195E taacacttac ctgottggta agaggtttca gccctaagga ggttttggtt aggtggcttc aaggtaatca ggagcttccc agggaaaaat atttgacctg ggggcccctt ccggaagctg gcgaatctgt tactactttt gctgttactt ctgttottcg agttgatgct gaagtttgga aagagggcga tacttttagc tgcatggttg ggcacgaagc cottccgott gcctttactc agaaaaccat agatcggtta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg gatagttcca gttgctgtgt cccagattgt gagccatctc taagtgtcca gcctccagca ctagaagatt tgottctggg ttctaacgct tctctaactt qtacactgag tqqactqaaq agtgcagagg gtgcatcatt tacatggaac cctacaggag gtaagacagc tgtacaagga agtccaaaga gagattcctg tggatgttac agcgtctcat cagtcttacc aggttgtgct N9D/N84D/ gatccatggg actccggaca aacgttctcc tgctctgtaa N13-ID/ ctcatcctga gtctaagtca tcactcacag caacaatcaa 0175E/ gaaggacctg ggaaatacgt tccgtcctca agtgcattta 0195E/ ctocctccac cttcagagga actcgcattg gatgagctcg SEQID NO:68 G1960/ taacacttac ctgcttggta agaggtttca gccctaagga R2190 ggttttggtt aggtggcttc aaggtaatca ggagcttccc (5+1) agggaaaaat atttgacctg ggggcccctt ccggaagctg gcgaatctgt tactactttt gctgttactt ctgttcttcg agttgatgct gaagtttgga aagagtgtga tacttttagc tgcatggttg ggcacgaagc ccttccgctt gcctttactc agaaaaccat agattgttta gccgggaaac cgacccacgt taatgtgtct gtggtgatgt ctgaagtgga tggcgtgtgc tatgg [0123] To enable expression in leaf tissue, each construct was cloned into a pCaMGate plant expression vector including an N-terminal PR1b 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). Without being bound by theory, it was contemplated that 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 VHH-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 m2 s1 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.
[0124] As seen from the results shown in Figure 2A, each of the supercharged Fe mutants, N9D (SEQ ID NO:51), N84D (SEQ ID NO:52), N131D (SEQ ID NO:53), Q175E
(SEQ ID NO:54) and 0195E (SEQ ID NO:55), showed a three- to four-fold improvement in accumulation across the time course, compared to the accumulation of native Fc (SEQ ID NO:47). Similarly, the de novo disulfide mutant, G196C/R219C (SEQ ID
NO:56), showed a six to seven-fold improvement in accumulation compared to the native Fc after six dpi (Figure 2B).
[0125] To test if these mutations could be combined to further improve accumulation, the mutations were combined in a step-wise manner and accumulation was measured in transformed leaf extract by Western blot. As seen from the results shown in Figure 2C, the Fc mutant containing the three N D supercharging mutations, N9D/N84D/N131D
(SEQ ID NO:57) gave a progressive increase in accumulation after transient expression.
Further, the Fc mutant containing all five supercharging mutations, N9D/N84D/N131D/0175E/0195E (SEQ ID NO:58), showed a ten-fold improvement in accumulation compared to native Fc. In addition, adding the mutations required for de novo disulfide formation to these five supercharging mutations to give the Fc mutant N9D/N84D/N131D/Q175E/0195E/G196C/R219C (5+1) (SEQ ID NO:59), further improved accumulation by approximately twenty-two-fold_ Expression of VHH9-Fc mutants [0126] To test if these Fc mutants could also enhance accumulation as an Fc scaffold protein, each Fc mutant was fused to VHH9. Genetic fusions of Fc mutant sequences to anti-E. coil VHH9 (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 VHH9-Fc mutant constructs were expressed in Nicotiana benthamiana leaf tissue as described above. As seen from the results shown in Figure 3A, each of the individual mutant VHH9-Fc fusions, either with a supercharged residue or with a de novo disulfide, showed a three- to four-fold improvement in accumulation when compared to the native VHH9-Fc fusion, as seen in the comparison of the unfused Fc mutants. When fused to VHH9, 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 VH H-Fc fusions.
Assembly of mutant VHH9-Fc sIgA
[0127] To determine if the presence of the Fc mutations in the VHH9-Fc affected the ability of the Fc subunit to assemble with the SC and JC subunits to form IgA, all three subunits (VHH9-Fc, SC and JC) were co-expressed and immunoprecipitation experiments were conducted. Each subunit was labelled with a different tag (VHH9-Fc-c-myc; SC-Flag; JC-HA). Crude extracts were immunoprecipitated with the anti-FLAG antibody specific to the SC subunit, then separated and detected on a Western blot probing for either anti-c-myc (VHH9-Fc subunit) or anti-HA (JC
subunit).
Bands matching the predicted 44 kDa size of VHH9-Fc were detected with anti-c-myc antibody in crude extract transformed with Vi1H9-Fc, VH H9-N9D/N84D/N131D/Q175E/Q195E/G196C/R219C-Fc (VHH9-(5+1)-Fc), co-expressed VHH9-Fc/SC/JC and co-expressed VHH9-(5+1)-Fc/SC/JC, but no bands were detected in crude extract expressing only JC or SC (Figure 4, panel A).
After co-imnnunoprecipitation, -44 kDa bands were seen only in extracts co-expressing VHH9-Fc/SC/JC and VHH9-(5+1)-Fc/SC/JC (Figure 4, panel B). This indicated that both SC and VHH9-Fc or SC and VHH9-(5+1)-Fc interact, and that the mutations in Fc did not hinder this interaction. Similarly, detection with anti-HA indicated bands of -20 kDa, matching the predicted size of JC, in crude extract transformed with JC, VHH-Fc/SC/JC
and VHH9-(5+1)-Fc/SC/JC (Figure 4, panel C). After co-innmunoprecipitation, -20 kDa bands were seen only for the co-expressed VHH9-Fc/SC/JC and VHH9-(5+1)-Fc/SC/JC, indicating that SC and JC are present in the same complex (Figure 4, panel D).
Binding of mutant VHH9-Fc to Escherichia coli strains [0128] To determine if the rationally designed mutations impact the cross-serotype pattern of VHH-Fc binding against E. coli, either VHH9-Fc or VHH9-(5+1)-Fc was incubated with E. co/1026:H11, 045:H2, 0103:H2, 0145:Hnm, 0121:H19, 0111:Hnm or 0157:H7. E. col/ 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 VHH9-Fc binding visualized using a secondary fluorescent antibody (rabbit anti-bovine-FITC) that binds Fc. As seen from the results presented in Figure 5, consistent co-localization of FITC signal with strains 026:H11, 0145:Hnm, 0111:Hnm and 0157:H7 cells for both VHH9-Fc and VHH9-(5+1)-Fc was observed, indicating multi-serotype detection of E.coli . As a negative control, E. coli cells were also treated with PBS containing 0.1% Tween-20 (PBS-T) instead of antibodies and similarly stained but did not show fluorescence under FITC-related imaging 5 conditions (480 nm excitation and 520-540 nm detection).
Neutralization of adherence of Escherichia coil strains to epithelial cells by mutant V/41-19-Fc [0129] To determine the ability of mutant VHH9-Fc to prevent E. coli from adhering to epithelial cells by blocking intimin, HEp-2 adherence inhibition assays were performed as 10 described above. HEp-2 cells were incubated with a culture of one of seven E. coli strains (026:H11, 045:H2, 0103:H2, 0145:Hnm, 0121:H19, 0111:Hnm and 0157:H7) in the presence or absence of either VHH9-Fc or VHH9-(5+1)-Fc, washed to remove any non-adherent bacteria and then visualized by immunofluorescence microscopy. As seen from the results presented in Figure 6, the addition of either VHH9-Fc or VHH9-(5+1)-Fc 15 abrogated the adhesion of E. coli strains 026:H11, 0111:Hnm, 0145:Hnm and 0157:H7 to HEp-2 cells to HEp-2 cells compared to the respective positive controls of no VI IH-Fc (+ PBS treatment).
[0130] To quantify the relative neutralization capacity of the VHH9-Fc compared to the VHH9-(5+1)-Fc, the adhesion assay for fluorometry was adapted as described above and 20 the relative fluorescence of HEp-2 cells incubated with a culture of each of the seven E.
coli strains with and without either VHH9-Fc or VHH9-(5+1)-Fc was measured.
The addition of either VHH9-Fc or VHH9-(5+1)-Fc showed the same pattern of reducing the relative fluorescence caused by adherent bacteria for strains 026:H11, 0111:Hnm, 0145 and 0157:H7 to background levels, as seen in Figure 7. Thus, VHH9-(5+1)-Fc 25 retains the ability to neutralize adherence of E. coil strains 0157, 026, 0111, and 0145 to HEp-2 cells.
Example 3: Targeting of VHH-Fc fusion proteins to plant cell sub-compartments Cloning and expression of targeting vectors [0131] Plant expression vectors (schematically illustrated in Figure 8) were adapted from 30 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 VHH9-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 35 sequencing.

[0132] 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 lumina! 15.0 kDa protein 2 (At5g52970; NCB! accession no. NP 001318791) and shown below.
MAMLFRPPPS QCRSFSPFVF NYSSREVSSS SRLSLKTSGD EENWVSRFRS
KSLSLVFSGA LALGLSLSGV GFADA
(SEQ ID NO:69) [0133] 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. Using the 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).
[0134] 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; NCB! accession no. NP
001321139) and shown below.
MASISSLHRW ASNQHSRLPR ITSISEADQS RPINQVVAFS VPISRRDASI
ILLSSIPLTS FFVLTPSSSE A
(SEQ ID NO:70) [0135] Again, 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 NO:70 (indicated in underlined bold italics).
[0136] Vectors designed to target the expressed construct to the stroma, the endoplasnnic 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.
[0137] After transiently transforming leaves of N. benthamiana, tissue was harvested and crude extract separated by SDS-PAGE in either a reducing buffer or a non-reducing buffer. Detection by Western blot using an anti-c-myc antibody showed accumulation of the VHH9-Fc in the thylakoid lumen via both pathways, in the stroma, and in the ER, but lacked sufficient signal for detection in the cytoplasm, as seen in Figures 9A
and 9B.
Under non-reducing extraction conditions, the VHH9-Fc is detected predominantly as an 88 kDa band matching the predicted size of the VHH9-Fc dimer. Total accumulation is highest in the ER at 51.1 mg/kg fresh weight (FW), followed by the thylakoid via Sec-targeting 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. Under reducing extraction conditions of the same samples, an enriched band at 44 kDa is detected matching the predicted size of the VHH9-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.
[0138] Based on these results, while ER-targeting produced the highest yields in the VHH9-Fc tested, the Sec-targeting accumulation remained significantly high, in contrast to stromal, cytoplasm, and Tat-import pathways.
Expression of VHH9-Fc in chloroplasts [0139] In addition to transient expression, the VHH9-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 VHH9-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:
MASSSRLSLKTSGDEENWVSRFRSKSLSLVFSGALALGLSLSGVGFADA
(SEO ID NO:71) [0140] The DNA sequence comprising the truncated Seq transit peptide, the VHH9 and the Fc chain was optimized for expression in the chloroplast genome.
Localization of VHH9-Fc in the thylakoid [0141] To verify that the Sec and Tat transit peptides indeed target VHH9-Fc to the thylakoid compartment, subcellular localization of the VHH9-Fc was tracked by fusing GFP to the Fc chain in each of the expression vectors. As seen in Figure 10, visualization by confocal microscopy showed the Sec and Tat-targeted GFP-tagged protein to consistently colocalize with chlorophyll, which accumulates in the thylakoid and autofluoresces at -735 nm. On the other hand, the construct targeting the recombinant protein to the stroma showed a very distinct pattern surrounding the thylakoid grana, and into stromules. Therefore, the Sec and Tat transit peptides identified indeed target the recombinant protein to the thylakoid.

Accumulation of VHH9-G196C/R219C-Fc in the thylakoid [0142] To determine if the oxidative folding of the thylakoid can recapitulate the yield-improving effects of an engineered disulfide bond, the VHH9-Fc fusion carrying the G196C/R219C mutation was targeted to the thylakoid lumen via the Sec pathway and accumulation by Western blot after agroinfiltration was measured. As seen from the results shown in Figure 11, the G196C/R219C mutant VHH9-Fc showed a significant yield improvement over the native VHH9-Fc when targeted to the Sec pathway.
Based on these results, 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.
Binding of targeted VHH9-Fc to Escherichia coli strains [0143] To determine if the thylakoid-targeted VHH9-Fc retained the ability of ER-targeted VHH9-Fc (Examples 1 and 2) to bind E. coli, purified VHH9-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. As seen in Figure 12, visualization by confocal microscopy showed consistent co-localization between DAPI-stained bacterial cells and the FITC-labelled VHH9-Fc for the thylakoid via Sec, thylakoid via Tat, and stromal compartments, indicating that the chloroplast-targeted VHH9-Fc retains the ability to bind intimin on E. co//surfaces. As a negative control, 0157:H7 cells were also treated with PBS containing 0.1% Tween-20 (PBS-T) instead of the VHH9-Fc and similarly stained but did not show fluorescence under FITC-related imaging conditions (480 nm excitation and 520-540 nm detection). Based on these results, the VHH9-Fc is folded correctly through Sec, Tat and stroma targeted pathways regardless of the status of the Fc chain.
Neutralization of adherence of Escherichia coil strains to epithelial cells by targeted VHH9-Fc [0144] HEp-2 cells were incubated with E. coli 0157: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 0157:H7 cells visualized using a donkey anti-rabbit AlexaTM 350 secondary antibody. As seen in Figure 13, the addition of purified VHH9-Fc from any of the compartments abrogated adhesion of any labelled E. coli 0157:H7 to the incubated HEp-2 cells as visualized using confocal microscopy, while the addition of Fc alone did not abrogate adhesion of E. coli to Hep-2 cells. Without being bound by theory, given that Tat and stromal imported antibodies retain functionality and show dimerized banding under non-reducing conditions as seen in Figure 9B, this suggests disulfide formation in the stroma despite its reducing environment.
[0145] These results indicate that the chloroplast-targeted VHH9-Fc retains the ability to neutralize E. co//from colonizing epithelial cells and that the inhibition of adhesion is mediated by the VHH and not by non-specific interactions of the Fc chain of the antibody.
[0146] The embodiments described herein are intended to be illustrative of the present compositions and methods and are not intended to limit the scope of the present invention. Various modifications and changes consistent with the description as a whole and which are readily apparent to the person of skill in the art are intended to be included. The appended claims should not be limited by the specific embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

Claims (20)

PCT/CA2022/050177
1. An Fc variant polypeptide, wherein the Fc variant polypeptide is a variant of a native Fc polypeptide, the native Fc polypeptide having a native sequence and 5 the Fc variant polypeptide having a variant sequence comprising one or more mutations of the native sequence, wherein 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 Fe polypeptide or 10 introduction of cysteine residues adapted to form a disulfide bridge; and wherein 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.
2. The Fc variant polypeptide according to claim 1 wherein the native sequence is 15 SEQ ID NO:47 and the one or more mutations are selected from N9D, N84D, N131D, 0175E, 0195E, G196C/R219C and combinations thereof.
3. An Fc variant fusion polypeptide comprising an Fc variant polypeptide according to claim 1 or 2 fused to a bioactive moiety.
4. The Fc variant fusion polypeptide according to claim 3 wherein the bioactive 20 moiety is a variable domain of an antibody
5. The Fc variant fusion polypeptide according to claim 4 wherein the variable domain of the antibody is a VHH polypeptide.
6. 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 25 expression vector comprising a nucleic acid encoding a Fc variant fusion polypeptide according to any one of claims 3 to 5.
7. A method of producing a recombinant antibody 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 variable domain of the 30 antibody fused to an Fc polypeptide or to an Fc variant polypeptide according to claim 1 or 2 and further encoding a chloroplast targeting signal sequence.
8. A VHH polypeptide comprising a first complementarity determining region (CDR1), a second complementarity determining region (CDR2) and a third complementarity determining region (CDR3), wherein:
(i) 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;
(ii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:10;
(iii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:14;
(iv) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:18;
(v) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:22;
(vi) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:26;

(vii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:30;
(viii) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:34;
(ix) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:38;
(x) the CDR1 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:40, the CDR2 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:41 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:42; or (xi) 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 and the CDR3 has at least 80%, 85%, 90%, 95%, 97%, 99% or 100% amino acid sequence identity to SEQ ID NO:46.
9. A VHH-Fc fusion polypeptide comprising a VHH polypeptide according to claim 8 fused to an Fc polypeptide.
10. A method of detecting the presence of E. coli in a sample, comprising:
(a) contacting the sample with a VHH polypeptide according to claim 8 or with a VHH-Fc fusion polypeptide according to claim 9, and (b) detecting binding between intimin and the VHH polypeptide or the VHH-Fc fusion polypeptide.
11. An antibody or antigen-binding fragment thereof comprising an Fc variant fusion polypeptide according to claim 4 or 5, a VHH polypeptide according to claim 8 or a VHH-Fc fusion polypeptide according to claim 9.
12. The antibody or antigen-binding fragment thereof according to claim 11, wherein the antibody or antigen-binding fragment thereof is an lgA antibody or antigen-binding fragment thereof.
13. The antibody or antigen-binding fragment thereof according to claim 12, wherein the antibody or antigen-binding fragment thereof specifically binds to Escherichia coli intimin.
14. Use of the antibody or antigen-binding fragment thereof according to claim 13 for preventing or reducing E. coli cell colonization of the gastrointestinal tract of a mammal.
15. Use of the antibody or antigen-binding fragment thereof according to claim 13 for neutralizing the ability of an E. co/icell to bind to a mammalian gastrointestinal epithelial cell.
16. A pharmaceutical composition comprising an antibody or an antigen binding fragment thereof according to claim 13, and a pharmaceutically acceptable carrier.
17. A nucleic acid encoding an Fc variant polypeptide according to claim 1 or 2, an Fc variant fusion polypeptide according to any one of claims 3 to 5, a VHH
polypeptide according to claim 8 or a VHH-Fc fusion polypeptide according to claim 9.
18. The nucleic acid according to claim 17 wherein the nucleic acid further comprises a chloroplast targeting signal sequence.
19. The nucleic acid according to claim 18 wherein the chloroplast targeting sequence is a Sec signaling sequence.
20. An expression vector comprising a nucleic acid according to any one of claims 17 to 19.
CA3204634A 2021-02-08 2022-02-08 Recombinant plant-derived antibodies and fc variants and related methods Pending CA3204634A1 (en)

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