WO2008151405A1 - Expression de protéines de fusion contenant un fragment d'anticorps simple chaîne lié à des unités répétitives d'élastine dans des plantes transgéniques - Google Patents

Expression de protéines de fusion contenant un fragment d'anticorps simple chaîne lié à des unités répétitives d'élastine dans des plantes transgéniques Download PDF

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WO2008151405A1
WO2008151405A1 PCT/CA2007/001065 CA2007001065W WO2008151405A1 WO 2008151405 A1 WO2008151405 A1 WO 2008151405A1 CA 2007001065 W CA2007001065 W CA 2007001065W WO 2008151405 A1 WO2008151405 A1 WO 2008151405A1
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nucleic acid
acid sequence
dna construct
apa
repeats
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PCT/CA2007/001065
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English (en)
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Jim Brandle
Hong Zhu
Anthony Michael Jevnikar
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Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food
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Priority to PCT/CA2007/001065 priority Critical patent/WO2008151405A1/fr
Publication of WO2008151405A1 publication Critical patent/WO2008151405A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • C12N15/8258—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 for the production of oral vaccines (antigens) or immunoglobulins
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to a transgenic plant. More particularly, the invention relates to expression of foreign proteins in a transgenic plant.
  • transgenic plants may be used as general expression systems for economically competitive production of antibodies.
  • Transgenic plants like transgenic animals, have the advantage to fold and assemble the antibodies correctly, but not the disadvantageous possibility of contamination with pathogenic organisms or oncogenic
  • transgenic plants further provide cost advantages over the fermentation based methods and transgenic animals.
  • Tobacco plants when used in large scale transgenic production can advantageously achieve large quantities of biomass, as tobacco plants may be harvested three to four times per growing season with over 100 tons of biomass per acre, or about 10 to 20 tons of dry solid biomass. Proteins in tobacco plants are substantially allergen-free. In addition, tobacco plants are easily genetically engineered. Tobacco plants with low levels of, or no nicotine production are attractive as transgenic plants for expressing commercially valuable products such as antibodies.
  • Single chain antibody fragments is an engineered antibody derivative, made from the Fv portions of the heavy and light chain of antibodies held together by a polypeptide linker. Single chain antibody fragments can have the same binding properties as their full length two chain counterparts. The reduced size of 27-30 kDa may facilitate the expression in transgenic plants.
  • HAI Hospital acquired infections
  • Nosocomial Infections include urinary tract infections, respiratory system infections, dermatitis, soft tissue infections, bacteremia, bone and joint infections, gastrointestinal infections and a variety of systemic infections. These infections typically result from the use of urinary catheters, central venous catheters and ventilators in intensive care units (ICU) and high-risk nurseries. In addition surgical site infections also occur following a wide variety of operative procedures, patients with severe burns, cancer and AIDS patients who are immuno-suppressed are also prone to HAI.
  • ICU intensive care units
  • Pseudomonas aeruginosa is Gram-negative, aerobic, and common inhabitant of soil and water. Pseudomonas aeruginosa occur regularly on the surfaces of plants and occasionally on the surfaces of animals. Pseudomonas aeruginosa, an opportunistic pathogen of humans, is responsible for about 4% of all HAI. Pseudomonas aeruginosa exploits some break in the host defenses to initiate an infection. Pseudomonas aeruginosa infection causes serious problems in patients hospitalized with cancer, cystic fibrosis, and burns, resulting in a 50 percent fatality rate.
  • HAI HAI are needed.
  • Preventative topical application of antibodies is one of the available solutions, which would prevent the infection and the spread of bacteria to the blood.
  • Antibodies can be administered to patients to clear the infection, but the high cost and limited availability of antibodies prevents topical application from being widely adopted.
  • Elastin-like polypeptides are synthetic proteins made from penta-peptide repeats of the amino acids "Val-Pro-Gly-Xaa-Gly", where Xaa is any amino acid but proline that occur in all mammalian elastin proteins.
  • elastin co-occurs with collagen and is the major matrix protein of large arteries, lung tissue, intestines and skin where it imparts extensibility and elastic recoil on the tissue.
  • ELP can be used as thermally responsive tags for the temperature based nonchromatographic separation of recombinant proteins. Schaller et al.
  • the embodiments of the present invention provide for recombinant single chain antibodies expressed in transgenic plants.
  • a method for expressing a single chain fragment in transgenic plants comprising the steps of (a) introducing into a plant cell a DNA construct comprising: (1 ) a first nucleic acid sequence encoding a variable heavy chain; (2) a second nucleic acid sequence operably linked to the first nucleic acid sequence, encoding a variable light chain; the variable light chain and the variable heavy chain being part of the single chain fragment and forming a binding site for an antigen; and (3) a third nucleic acid sequence encoding an elastin-like polypeptide (ELP), the third nucleic acid sequence operably linked to the first nucleic acid sequence and the second nucleic acid sequence, the ELP comprising a number of repeats of Val-Pro-Gly-Xaa-Gly, wherein Xaa is any amino acid and the number of repeats is between 2 and 80; and (b) growing said plant cell to produce the single chain fragment.
  • a DNA construct comprising: (1 ) a first nucleic acid sequence en
  • a DNA construct for expressing a single chain fragment in transgenic plants comprising: (1 ) a first nucleic acid sequence encoding a variable heavy chain; (2) a second nucleic acid sequence operably linked to the first nucleic acid sequence, encoding a variable light chain; the variable light chain and the variable heavy chain being part of the single chain fragment and forming a binding site for an antigen; and (3) a third nucleic acid sequence encoding an elastin-like polypeptide
  • ELP the third nucleic acid sequence operably linked to the first nucleic acid sequence and the second nucleic acid sequence, the ELP comprising a number of repeats of Val-Pro-Gly-Xaa-Gly, wherein Xaa is any amino acid and the number of repeats is between 2 and 80.
  • a transgenic plant comprising: plant cells containing a DNA construct for expressing a single chain fragment in transgenic plants, the DNA construct comprising: (1 ) a first nucleic acid sequence encoding a variable heavy chain; (2) a second nucleic acid sequence operably linked to the first nucleic acid sequence, encoding a variable light chain; the variable light chain and the variable heavy chain being part of the single chain fragment and forming a binding site for an antigen; and (3) a third nucleic acid sequence encoding an elastin-like polypeptide (ELP), the third nucleic acid sequence operably linked to the first nucleic acid sequence and the second nucleic acid sequence, the ELP comprising a number of repeats of Val-Pro-Gly-Xaa-Gly, wherein
  • Xaa is any amino acid and the number of repeats is between 2 and 80.
  • a plant seed containing a DNA construct for expressing a single chain fragment in transgenic plants, the DNA construct comprising: (1 ) a first nucleic acid sequence encoding a variable heavy chain; (2) a second nucleic acid sequence operably linked to the first nucleic acid sequence, encoding a variable light chain; the variable light chain and the variable heavy chain being part of the single chain fragment and forming a binding site for an antigen; and (3) a third nucleic acid sequence encoding an elastin-like polypeptide (ELP), the third nucleic acid sequence operably linked to the first nucleic acid sequence and the second nucleic acid sequence, the ELP comprising a number of repeats of Val-Pro-Gly-Xaa-Gly, wherein Xaa is any amino acid and the number of repeats is between 2 and 80.
  • ELP elastin-like polypeptide
  • a polypeptide comprising: (1 ) a variable heavy chain; (2) a variable light chain; the variable light chain and the variable heavy chain being part of a single chain fragment and forming a binding site for an antigen; and (3) an elastin-like polypeptide, comprising a number of repeats of Val-Pro-Gly-Xaa-Gly, wherein Xaa is any amino acid and the number of repeats is between 2 and 80.
  • the DNA construct further comprises a fourth nucleic acid sequence encoding an effector.
  • At least one of the first and second nucleic acid sequences is optimized for expression in the transgenic plants.
  • the DNA construct further comprises a linker linking the first nucleic acid sequence and the second nucleic acid sequence.
  • the number of repeats is between 10 and 65.
  • the number of repeats is between 15 and 50.
  • the number of repeats is between 20 and 40.
  • the DNA construct further comprises a fourth nucleic acid sequence encoding constant heavy chain region.
  • the DNA construct further comprises a fourth nucleic acid sequence encoding human constant heavy chain region.
  • the DNA construct further comprises a polyhistidine-tag.
  • the plant is a tobacco plant
  • the antigen is a bacterium.
  • the antigen is a pathogen.
  • the antigen is Pseudomonas aeruginosa.
  • Figure 1 (a) describes component parts of an antibody
  • Figure 1 (b) describes structure of an scFv fragment
  • Figure 1 (c) illustrates a single chain variable fragment with an effector
  • Figure 2 illustrated DNA construct Apa-4 comprising a linker, a polyhistidine-tag (HIS), a Lys-Asp-Glu-Leu (KDEL) retention signal and sequences encoding the signal peptide from the tobacco pathogenesis related protein 1b (PM b), the variable light chain and the variable heavy chain;
  • HIS polyhistidine-tag
  • KDEL Lys-Asp-Glu-Leu
  • Figure 2 illustrated DNA construct Apa-6 comprising a linker, HIS, KDEL, a cleavage site that allows the removal of the remaining protein using a tobacco etch virus protease (TEV) and sequences encoding Pr1 b, the variable light chain and the variable heavy chain, and elastin-like polypeptide (ELP);
  • TEV tobacco etch virus protease
  • ELP elastin-like polypeptide
  • Figure 2 (c) illustrated DNA construct Apa-8 comprising a linker, HIS, KDEL, and sequences encoding PrI b, the variable light chain and the variable heavy chain, and human IgGI heavy chain constant region (huFc);
  • Figure 2 (d) illustrated DNA construct Apa-10 comprising a linker, HIS, KDEL and sequences encoding PrIb, the variable light chain and the variable heavy chain, huFc, and ELP;
  • Figure 3 shows Western blot analysis of protein extracts from the two highest accumulating selections from the populations of Apa-4 and Apa-6 transgenic plants; lanes 1 , 2 and 3 contain 1 , 5 and 10 ng of pure scFv, lane 4 is a non-transgenic control protein from the cultivar 81 V9, lanes 5-9 contains Apa-4 products, lanes 10-14 contains Apa-6 products;
  • Figure 4(a) illustrates examples of Western blot analysis results with the transgenic plants 4T1-1 , 4T1-2, 4T1-3, 4T1-4, 4T1-7, 4T1-10, and 4T1-19 from lanes 4 to 10, respectively, Controls with Apa-4 are shown from lanes 1 to 3, in the amount of 1 ng, 5 ng and 10 ng, respectively;
  • Figure 4(b) illustrates examples of Western blot analysis results with the transgenic plants 6T1 -4, 6T1 -13, 6T1 -21 , 6T1 -24, 6T1 -25, 6T1 -26, 6T1 -27 and 6T1 -30 from lanes 3 to 11 , respectively, Controls with Apa-6 are shown from lanes 1 to 2, in the amount of 1 ng and 5ng, respectively;
  • Figure 4(c) illustrates examples of Western blot analysis results with the transgenic plants 8T1 -2, 8T1 -4, 8T1 -5, 8T1 -7, 8T1 -8, 8T1 -9, 8T1 -10, 8T1 -11 , 8T1 -12, 8T1 -17, 8T1-18, and 8T1-19 from lanes 3 to 14, respectively, controls with purified Apa-4 standard and cultivar 81 V9 are shown from lanes 1 to 2, in the amount of10 ng and 20 ⁇ g, respectively;
  • Figure 4(d) shows the transgenic plants 10T1-1 , 10T1-6, 10T1-9, 10T1-11 , 10T1-12, 10T1-13, 10T1-14, 10T1-16, 10T1-17, 10T1-18, 10T1-19 and 10T 1-20 from lanes 3 to 14, respectively, controls with unpurified Apa-10 and cultivar 81 V9 are shown from lanes 1 to 2, in the amount of 20 ⁇ g total soluble protein of each;
  • Figure 5(a) and (b) depict ELISA test results to determine the binding of the recombinant scFv produced in transgenic plants to Pseudomonas aeruginosa;
  • Figure 5 (c) shows the binding of huFc in Apa-8 to Protein G, compared with 81 V9, Apa-4 and human IgG as control;
  • Figure 5 (d) shows binding of huFc in Apa-8 to anti-human IgG, compared with 81 V9, Apa-4 and human IgG as control;
  • Figure 6 shows the result of a human complement CIC-CIq binging assay
  • Figure 7 shows a Western blot of purification products at different steps of the purification process
  • lane 1 is a control with 2 ng of Apa-4
  • lanes 2-4 are the 100K TFF retentate, flow through and flow through stored overnight at 4 0 C, respectively, indicating that Apa-4 can be safely stored at 4 0 C overnight without being degraded in 100K flow through;
  • Figure 8 shows a Western blot of the ion exchange purification process
  • Figure 9 shows a Western blot of IMAC purified Apa-8.
  • nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
  • Numeric ranges recited within the specification are inclusive of the numbers defining the range and include each integer within the defined range.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may be referred to by their commonly accepted single-letter codes.
  • transgenic plant is intended to refer to a plant which comprises within its genome a heterologous polynucleotide.
  • the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations.
  • the heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • transgenic is intended to describe any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
  • tobacco or "tobacco plants” are intended to include any plant, species, or hybrids of the genus Nicotiana, including but not limited to N. rustica, N. tabacum and N. glutinosa.
  • low nicotine is intended to describe a transgenic tobacco plant that contains less than half, preferably less than 25%, and more preferably less than 20% of the nicotine content of the non-transgenic parent plant from which the transgenic plant is produced.
  • antibody is intended to include any of several classes of structurally related proteins that function as part of the immune response of an animal, which proteins include IgG, IgD, IgE, IgA, and IgM and related proteins. Antibodies are found in plasma and other body fluids and in the membrane of certain cells. Under normal physiological conditions (e.g. absent immunological dysfunction or human intervention) antibodies are produced by B cells (or the functional equivalent) of an animal in reaction to the entry of proteins or other chemical substances which that animal is not immunologically tolerant of the tissue or body fluids of that animal.
  • heterologous in reference to a nucleic acid is a nucleic acid that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and/or genomic locus by deliberate human intervention.
  • a heterologous protein may originate from a foreign species or, if from the same species, is substantially modified from its original form by deliberate human intervention.
  • vector is intended to include a nucleic acid used in introduction of a polynucleotide of the present invention into a host cell.
  • DNA construct is intended to include a deoxyribonucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to include segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.
  • promoter is intended to include a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription.
  • operably linked is intended to include a functional linkage between a first sequence and a second sequence, wherein the products encoded by the first sequence and the second sequence are functionally linked.
  • operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame.
  • fragment and "antibody fragment” are intended to include the component parts of an antibody. These component parts are described in Figure 1 by way of example with reference to an IgG molecule 100, but it will be understood by those skilled in the art that these component parts may be derived from any of the other antibody classes. All antibodies are bivalent molecules with two antigen binding regions to antigen 101. IgG, IgD or IgE molecule has two antigen binding sites. Secreted IgA is a dimer and secreted IgM is a pentamer.
  • the IgG molecule 100 may be described as a "Y" shaped protein made up of four polypeptide chains 102, 104, 106, 108 linked together by disulfide bonds 110.
  • the tops of the "Y 1 are the N-terminals of the protein chains which include the antigen binding site.
  • Each polypeptide has both constant regions (C regions) 112 and variable regions (V regions) 114.
  • the V regions are located in the N-terminal domains of the H and L-chains.
  • the V region 114 are three areas of greatest sequence variability known as the complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • the H-and L-chain CDRs together form the antigen binding site 116.
  • Sequence variability in chain CDRs underlies the range of antibody specificities that the immune system produces. All antibodies of a given type have the same constant regions 1 12, but the variable regions differ from one clone of B cells to another.
  • the L- and H-chain variable regions fold to create an antigen binding site comprising the CDRs as described above.
  • the H-chains are about 50 kD in size, and the L-chains are about 25 kD in size.
  • the point at which the H-chains separate to form the top of the "Y” is known to the art as the hinge region 118.
  • the IgG may be cleaved by papain enzyme above the hinge region 118 into two Fab fragments and one Fc fragment.
  • the top of the "Y” is known to include the variable region, which performs the specific binding function of the antibody.
  • the Fc fragment represents the bottom of the "Y” and serves complement fixation and other non-binding functions.
  • the Fab fragment is a heterodimer cleaved from each side of the top of the "Y", and is formed of intact L-chains and of an approximately 25 kD partial H-chain known to the art as an Fd fragment.
  • the Fv fragment is a 25 kD heterodimer 118 similar in structure to the Fab fragment described above, but including shorter segments of the N-terminal sequences of the H- and L-chains 102, 104 respectively from the top of the "Y" 100.
  • the Fv includes the variable regions 114 of the constituent H- and L-chain fragments.
  • the Fv fragment may be separated into two approximately 12.5 kD single chain fragments or components referred to as a variable fragment of an H-chain (VH) 120 and a variable fragment of an L-chain (V L ) 122.
  • VH H-chain
  • V L variable fragment of an L-chain
  • V H - and V L -chain may be produced de novo as a single chain fragment (scFv) linked by a linker 124 by one of the recombinant genetic techniques known to the art from a gene construct, which after folding includes an antigen binding site 126.
  • scFv single chain fragment
  • effector functions as provided by the Fc region of the secreted antibody are needed to neutralize many antigens, but are missing in scFvs. Addition of the Fc portion 130 of IgG confers those effector functions to the scFvs for initiating the complement cascade; thus activating immunological responses.
  • Example 1 describes the construction of synthetic scFv gene.
  • a DNA construct Apa-4 the native scFv gene directed against Pseudomonas aeruginosa strain 06, was constructed by using the gene cloned by reverse transcripting total RNA isolated from hybridoma cells developed from XenoMouse producting human MAb against Pseudomonas aeruginoas into cDNA as described in Hemachandra, S. et al. "Human monoclonal antibodies against Pseudomonas aeruginosa lipopolysaccharide derived from transgenic mice containing megabase human immunoglobulin loci are opsonic and protective against fatal
  • the high expression dataset was used to create a codon usage table for entry into the DNABuilder backtranslation software tool. Codons used at a frequency of less than 10% in tobacco or which inherently contain potential inhibitory motifs such as CG dinucleotides or CXG triplets were set to 0% of synonymous codon usage for the particular amino acid and the frequency of the remaining codons adjusted accordingly to account for 100% codon usage for the given amino acid.
  • the coding sequences for the antibody genes were optimized for expression in Nicotiana tabacum by reverse translating the amino acid sequence, including the PR1 b secretory signal sequence (GenBank accession #X12486) and a carboxy terminal 6xHis/KDEL tag using the Nicotiana tabacum high expression dataset codon usage table in DNABuilder to incorporate optimal and avoid inhibitory Nicotiana tabacum codons.
  • the back-translated sequence was then analyzed for the presence of potential inhibitory elements which are replaced manually by conservative codon.
  • the optimized nucleotide sequence for the variable light chain is represented by SEQ ID NO:1 ; and the optimized nucleotide sequence for the variable heavy chain is represented by SEQ ID NO:2.
  • the corresponding polypeptide sequence for the variable light chain is represented by SEQ ID NO:3; and the corresponding polypeptide sequence for the variable heavy chain is represented by SEQ ID NO:4.
  • the synthetic gene Apa-4 was then constructed using oligonucleotides between 20 or 40 bases long and the ligation chain reaction (LCR) followed by polymerase chain reaction (PCR) with 5' and 3' outside primers.
  • LCR ligation chain reaction
  • PCR polymerase chain reaction
  • the LCR and the PCR reactions are described in Chalmers, F. M. and Curnow, K.M.: "Scaling up the ligase chain reaction based approach to gene synthesis", Biotechniques 30, (2001 ) 249-252, the entirety of which is incorporated herein by reference.
  • the procedure allows for rapid synthetic gene construction and elements within the gene can be easily altered by changing oligonucletide sequences and resynthesizing the gene.
  • PM b is the signal peptide from the tobacco pathogenesis related protein 1b, which targets the protein to the plant secretory system and is cleaved.
  • the Lys-Asp-Glu-Leu (KDEL) results in the retention of the recombinant antibody in the endoplasmic reticulum and enhances accumulation.
  • a polyhistidine-tag (HIS) is included between the V L and the KDEL for purification by affinity chromatography.
  • SEQ ID NO:5 The nucleotide sequence for Apa-4 is represented by SEQ ID NO:5; and the corresponding polypeptide sequence is represented by SEQ ID NO:6.
  • SEQ ID NO:6 SEQ ID NO:6 includes a sequence of "catggatccaaa” fo BamHI restriction site and RNA polymerase initiation at the 5' end, and a sequence of "tgaattccc" for Eco Rl restriction site at the 3' end.
  • ELP elastin-like polypeptide tag
  • An ELP is a polypeptide with the repeating pentapeptide sequence Val-Pro-Gly-Xaa-Gly, where the residue Xaa can be any amino acid, except Pro.
  • T t inverse transition temperature
  • ELPs are structurally disordered and soluble in aqueous solutions, but when the temperature is raised above their T t , they undergo a sharp (2-3 0 C range) disorder-to-order transition, leading to desolvation and aggregation of the polypeptide.
  • the TEV site is a cleavage site that allows the removal of the remaining protein using a tobacco etch virus protease.
  • a Kpn I site was added onto the 5' of ELP gene by PCR to generate a Kpn l-ELP-KDEL-EcoR I fragment.
  • Both Apa-4 and Kpn l-ELP-KDEL-EcoR I fragments were digested with Kpn I and gel purified. The resulted Apa-4-Kpn I and Kpn 1-ELP-KDEL-EcoR I fragment were ligated to form Apa-6 construct.
  • SEQ ID NO:7 The nucleotide sequence for Apa-6 is represented by SEQ ID NO:7; and the corresponding polypeptide sequence is represented by SEQ ID NO:8.
  • SEQ ID NO:8 SEQ ID NO:7 includes a sequence of "catggatccaaa” fo BamHI restriction site and RNA polymerase initiation at the 5' end, and a sequence of "taggaattccgg” for Eco Rl restriction site at the 3' end.
  • SEQ ID NO:9 The nucleotide sequence for Apa-8 is represented by SEQ ID NO:9; and the corresponding polypeptide sequence is represented by SEQ ID NO: 10.
  • SEQ ID NO:9 includes a sequence of "catggatccaaa” fo BamHI restriction site and RNA polymerase initiation at the 5' end, and a sequence of "tgaattccc" for Eco Rl restriction site at the 3' end.
  • SEQ ID NO:11 The nucleotide sequence for Apa-10 is represented by SEQ ID NO:11 ; and the corresponding polypeptide sequence is represented by SEQ ID NO: 12.
  • SEQ ID NO.11 includes a sequence of "catggatccaaa” fo BamHI restriction site and RNA polymerase initiation at the 5' end, and a sequence of "taggaattccgg” for Eco Rl restriction site at the 3' end.
  • Example 2 describes scFv expression in E. coli and anti-scFv antibody production.
  • the BamHI-EcoRI fragment was then excised from pBS KS+ and cloned into pET30a (Novagen, Mississauga, Ontario).
  • the resulted plasmid was then transformed by electroporation into E. coli strain BL21 (DE3, Cod+, Novagen).
  • Expression of recombinant protein was conducted according to manufacturer's instructions (pET System Manual, Novagen, the entirety of which is incorporated herein by reference).
  • the inclusion bodies were collected and refolded with Protein Refolding Kit (Novagen) according to the manufacturer's instructions, the entirety of which is incorporated herein by reference.
  • the refolded soluble Apa-14 protein was used to generate of polyclonal antibodies using the standard protocols (Invitrogen, Camarillo, CA, USA).
  • Figure 3 shows Western analysis of protein extracts from the two highest accumulating selections from the populations of Apa-4 and Apa-6 transgenic plants.
  • Lanes 1 , 2 and 3 contain 1 , 5 and 10 ng of pure scFv
  • lane 4 is a non-transgenic control protein from the cultivar 81 V9.
  • the Apa-4 lanes (lanes 5-9) show a distinct band at 36 kDa and the Apa-6 lanes (lanes 10-14) show a distinct band at 48 kDa, the shift in size represents the additional amino acids resulting from the ELP tag and TEV cleavage site.
  • Example 3 describes Agrobacterium-mediated transient expression assays.
  • the tobacco plants were maintained in a controlled environment chamber at 20 0 C, with 16 hr day length for 4 days and the individual infiltrated panels were collected and analysed separately. The average of the 3 or 4 leaf panels was then used to represent the concentration of a given recombinant protein.
  • Example 4 describes the plant protein extraction.
  • Leaf tissue was weighed and homogenized in 4 volumes of extraction buffer (phosphate buffered saline, pH7.4, 0.1% Tween 20 (v/v), 2% (w/v) polyvinylpolypyrrolidone, 100 mM ascorbic acid, 1 mM phenylmethylsulfonyl fluoride, and 1 ⁇ g/ml leupeptin).
  • extraction buffer phosphate buffered saline, pH7.4, 0.1% Tween 20 (v/v), 2% (w/v) polyvinylpolypyrrolidone, 100 mM ascorbic acid, 1 mM phenylmethylsulfonyl fluoride, and 1 ⁇ g/ml leupeptin.
  • the extract was clarified by centrifugation at 13,000 x g for 15 min at 4°C. Total soluble protein concentrations were determined by dye binding.
  • Example 5 describes immunoblot analysis.
  • Proteins from one-dimensional PAGE gels were transferred to a Sequi-BlotTM PVDF (Bio-Rad) membrane in a semi-dry electroblotting apparatus (Bio-Rad) using transfer buffer (48 mM Tris/39 mM glycine) containing 20% methanol (v/v).
  • Membranes were blocked with 1 % Western Blocking Reagent (Roche Applied Science, Laval, Quebec) in TBS overnight at 4 0 C and were probed with one of the primary antibodies: anti-His antibody (Amersham Biosciences, Baie d'Urfe, Quebec, Canada), anti-ELP antibody (Patel et al, supra) or anti-Apa antibody for one hour at room temperature and washed with TBS-Tween 20 and 0.5% blocking reagent.
  • Membranes were then incubated with horseradish peroxidase conjugated goat anti-mouse IgG (for anti-His antibody) (Bio-Rad) or goat anti-rabbit IgG (for anti-ELP antibody and anti-Apa antibody) (Bio-Rad) for one hour at room temperature and washed with TBS-Tween 20. The blot was detected using ECL Western Blotting Detection Reagent (Amersham Biosciences, Baie d'Urfe, Quebec) and exposed to X-ray film for 1 to 5 min.
  • Example 6 describes stable transformation of tobacco with the four expression constructs.
  • Stable transgenic plants have been regenerated carrying all four of the synthetic genes.
  • a total of 241 transgenic plants, 55 with Apa-4, 58 with Apa-6, 63 with Apa-8 and 65 with Apa-10 were regenerated.
  • the Apa-4 and Apa-6 plants were tested by both PCR and Western blot and the Apa-8 and Apa-10 plants were tested by Western alone.
  • the two transgenic lines that gave the highest relative levels of scFv accumulation as primary transformants and have single gene copy insertion showing as 3:1 kanamycin resistance segregation were chosen for seed production and scale up.
  • the selected transgenic lines were: Apa-4 T1-22, Apa-4 T2-3, Apa-6 T1-10, Apa-6 T2-39, Apa-8 T1-9, Apa-8 T1-11 , Apa-10 T1-39, Apa-10 T1 -46.
  • Figure 4 (a) illustrates examples of Western blot analysis results using anti His tag monoclonal antibodies.
  • Figure 4 (a) shows the transgenic plants 4T1-1 , 4T1-2, 4T1-3, 4T1-4, 4T1-7, 4T1-10, and 4T1-19 from lanes 4 to 10, respectively.
  • Controls with purified Apa-4 are shown from lanes 1 to 3, in the amount of 1 ng, 5 ng and 10 ng, respectively.
  • Figure 4 shows Western blot analysis for the transgenic plants 6T1-4, 6T1 -13, 6T1 -21 , 6T1 -24, 6T1 -25, 6T1 -26, 6T1 -27 and 6T1 -30 from lanes 3 to 11 , using anti ELP antibodies, respectively. Controls with purified Apa-6 are shown from lanes 1 to 2, in the amount of 1 ng and 5 ng, respectively.
  • Figure 4 (c) shows Western blot analysis for the transgenic plants 8T1-2, 8T1-4, 8T1-5, 8T1-7, 8T1-8, 8T1-9, 8T1-10, 8T1 -11 , 8T1 -12, 8T1 -17, 8T1 -18, and 8T1 -19 from lanes 3 to 14, using anti His tag monoclonal antibodies, respectively.
  • Controls with Apa-4 and cultivar 81 V9 are shown from lanes 1 to 2, in the amount of 10 ng and 20 ⁇ g, respectively.
  • Figure 4(d) shows the transgenic plants 10T1-1 , 10T1-6, 10T1-9, 10T1-11 , 10T1-12, 10T1-13, 10T1-14, 10T1-16, 10T1-17, 10T1-18, 10T1-19 and 10T1-20 from lanes 3 to 14, using anti ELP antibodies, respectively.
  • Controls with Apa-10 and cultivar 81 V9 total soluble protein are shown from lanes 1 to 2, in the amount of 20 ⁇ g each respectively.
  • Example 6 describes enzyme linked immunosorbent assay (ELISA) for quantification of antibody binding.
  • ELISA enzyme linked immunosorbent assay
  • Pseudomonas aeruginosa 06 strain ATCC 27312 and non-06 strain ATCC 27853 and ATCC 19660 were cultured on LB agar plate at 37°C for overnight.
  • the bacteria were suspended in 0.02 M phosphate buffer, pH 7.4.
  • the cell density was measured with a spectrophotometer and was adjusted to 5 x 10 7 cell/ml.
  • a 96-well, flat-bottom immuno MaxiSorp plate (Nalge Nunc International, Rochester, New York,
  • the E.coli strain DH5 ⁇ or Pseudomonas syringe strain DC3000 was used as a negative control.
  • the plate was washed with PBS and blocked with 200 ⁇ l/well 1 % bovine serum albumin in PBS for two hours at room temperature. After washing the plate with PBS, antibodies against Pseudomonas aeruginosa extracted from transgenic tobacco were added into each well and incubated for one hour at room temperature.
  • the plate was washed again with PBS and incubated with either anti-His antibody (Amersham Biosciences, Baie d'Urfe, Quebec) or anti-ELP antibody (Patel et al., supra) for one hour at room temperature and washed with PBS.
  • the plate was incubated with horseradish peroxidase conjugated goat anti-mouse (for anti-His) or goat anti-rabbit
  • Figure 5 (a) illustrates an ELISA test conducted to determine if the recombinant scFv produced in transgenic plants was capable of binding Pseudomonas aeruginosa, because antigen recognition and antibody binding is critical to their ability to facilitate removal of pathogens.
  • ELISA experiment comparing binding of protein extract from non-transgenic (81 V9) and transgenic plants expressing one of four genes (Apa-4, Apa-6, Apa-8, Apa-10) to P. aeruginosa strain 06, P. aeruginosa strain non-06 and to P. syringae strain DC3000.
  • scFv from Apa-4 and Apa-8 transgenics was purified with Immobilized Metal Affinity Chromatography (IMAC).
  • IMAC Immobilized Metal Affinity Chromatography
  • For Apa-6 and Apa-10 crude extract was used. Results are normalized for total protein.
  • An anti-HIS antibody was used for detection of Apa-4 and Apa-8 protein in IMAC purified extracts.
  • An anti-ELP antibody was used for detection in Apa-6 and Apa-10 in crude extracts. The results from the ELISA indicated that all four of the scFv variations were capable of binding to Pseudomonas aeruginosa 06 strain and to the non-06 strain.
  • Apa-8 scFvs did not bind Pseudomonas syringae indicating that the binding is species specific.
  • Apa-6 and Apa-10 partially bound to Pseudomonas syringae. This may be caused by the impurity in the crude plant extract cross reacting with either anti-ELP or anti-rabbit, the secondary antibody. Since the scFvs bind both the 06 and non-06 strain, the scFv may be useful for controlling infections caused by a broader range of
  • Figure 5 (b) shows the results of another ELISA experiment where the scFv (Apa-4) and ScFv + huFc (Apa-8) bind three different Pseudomonas aeruginosa strain
  • ATCC 19660 is a strain with violence to mouse.
  • Figure 5 (c) shows the binding of huFc in Apa-8 to Protein G, compared with 81V9, Apa-4 and human IgG as control.
  • Figure 5 (d) shows binding of huFc in Apa-8 to anti-human IgG, compared with
  • Example 7 describes human complement C1q binding assay.
  • Antibodies can neutralize a pathogen by opsonization, which involves recognition of the bound antibody by Fc receptors of the surface of phagocytic cells and subsequent uptake by those cells. In addition those same bound antibodies can activate the proteins of the complement system, which then opsonize it by binding to complement receptors on phagocytes. Other complement components recruit phagocytes and the terminal components can lyse certain pathogens directly. scFv's do not have the constant regions associated with the parent IgG molecule and therefore lack these effector functions.
  • C1q is the first component in the classical complement activation pathway and binding by IgG triggers the pathway.
  • Figure 6 illustrates the result of the complement activation, it indicates that the
  • Apa-8 with ScFv and huGc is positive in CIC-CIq binding assay.
  • the known amount of heat aggregated human gamma globulin (HAGG) standard A, B, C were included as control.
  • Standard A is 0 ⁇ g Eq/ml
  • B is 16 ⁇ g Eq/ml
  • C is 1.05 ⁇ g Eq/ml.
  • the untransformed plant 81 V9 and scFc without huFc (Apa-4) are negative.
  • Example 8 describes the purification of the single chain antibody fragment, for example, Apa-4 from the transgenic plant.
  • Frozen leaf tissue frozen at -80 0 C was homogenized in 4 0 C extraction buffer (0.02M potassium phosphate buffer, pH 7.4, 0.5M KCI, 0.1% tween 20, 2% PVPP (poly vinylpolypyrrolidone), 1 mM PMSF (phenylmenthanesulfonyl fluoride), 1 ug/ml leupeptin, and 10OmM ascorbate) with a blender, and filtered through nylon bags to remove fiber. After centrifuge at 12,000 rpm for 15 min, the supernatant was filtered through MiraclothTM to remove debris remaining in the solution.
  • 4 0 C extraction buffer 0.02M potassium phosphate buffer, pH 7.4, 0.5M KCI, 0.1% tween 20, 2% PVPP (poly vinylpolypyrrolidone), 1 mM PMSF (phenylmenthanesulfonyl fluoride), 1 ug/ml leupeptin, and 10OmM ascorbat
  • the filtrate is then passed through a 100K Tangential Flow Filters (TFF) cartridge (Millipore, Mississauga, Ontario) to remove particles larger than 100K.
  • TFF Tangential Flow Filters
  • the extract was then concentrated with a 10K TFF cartridge, and applied through a polyhistidine affinity column.
  • the column is washed with start buffer (0.02M potassium phosphate buffer, pH 7.4, 0.5M KCI) followed by start buffer plus 30 mM imidazole and eluted with start buffer plus 200 mM imidazole. Elution was collected as 10 ml fraction and tested by Western blot.
  • the fraction with Apa-4 was dialyzed against PBS to remove imidazole and reduce the concentration of salt.
  • the purified protein was concentrated with JumbosepTM centrifugal devices (Pall Life Science, Ann Arbor, Ml USA) or CentriprepTM column (Millipore, Mississauga, Ontario ) according to the volume of the extract.
  • the purified Apa-4 is stored at -80 0 C in 15% glycerol.
  • Figure 7 shows a Western blot of the different steps during the purification process.
  • Lane 1 is a control with 2 ng of Apa-4.
  • Lanes 2-4 are the 100K TFF retentant, flow through and flow through overnight at 4 0 C, indicating Apa-4 is stable in the extract at 4 0 C for overnight without degrading, which enable finishing the purification process in multiple days, respectively.
  • Lane 5 and 6 is 1 OK TFF flow through and retentant, respectively.
  • Lane 7-14 are samples from IMAC purification procedures which indicated that Apa-4 was bound to a chelating column (Bio Rad) charged with nickel ion (Ni 2+ ) and eluted in fraction number 4.
  • Example 10 describes the purification of the single chain antibody fragment with ELP, for example, Apa-6 from the transgenic plant.
  • Adding ELP tag can increase the accumulation of recombinant protein in tobacco leaves, but the tag interferes the access of polyhistidine (HIS) tag to Ni +2 ion on an affinity column. Therefore, an ion exchange technique was used to purify Apa-6, instead of using IMAC to purify the recombinant protein Apa-6.
  • HIS polyhistidine
  • Plant tissue was homogenized, cleared, and pre-purified and concentrated with 100K and 10K TFF cartridge, as described above for the purification procedures of Apa-4 except different extraction buffer (0.02M phosphate buffer, pH6.2, 0.1 % tween 20, 2% PVPP (poly vinylpolypyrrolidone), 1 mM PMSF (phenylmenthanesulfonyl fluoride), 1 ug/ml leupeptin, and 10OmM ascorbate) was used . After concentration with 10K TFF cartridge, the extract was applied through HiTrap SP Sepharose ion exchange column (Bio Rad).
  • extraction buffer 0.02M phosphate buffer, pH6.2, 0.1 % tween 20, 2% PVPP (poly vinylpolypyrrolidone), 1 mM PMSF (phenylmenthanesulfonyl fluoride), 1 ug/ml leupeptin, and 10OmM ascorbate
  • Figure 8 shows a Western blot of the ion exchange purification process.
  • Lane 1 is a control with 2 ng of Apa-6.
  • Lanes 2 is the extract concentrated with 10K TFF cartridge before applied into ion exchange column.
  • Lanes 3 and 4 are flow through and wash, respectively.
  • Lane 5-14 are elution fractions 1 to 10. The majority of Apa-6 fusion protein was eluted in fractions 3 and 4.
  • Example 11 describes the purification of the single chain antibody fragment with huFc, for example, Apa-8 from the transgenic plant.
  • the huFc adds effector functions to scFv which enable the recognition of scFv by human immune system.
  • the recombinant protein reduces stability in the process of purification. Apa-8 broke into small fragments when the extract was filtered through 10K and 100K TFF cartridge. Therefore, the TFF cartridges were replaced with an OptcanTM (Millipore) filter with 0.5 ⁇ m pore size to remove large particles, and the extract was applied into IMAC column without concentration.
  • Figure 9 shows a western blot of IMAC purified Apa-8. Although some Apa-8 shown as reduced size, full size (70 Kd) Apa-8 was purified successfully.

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Abstract

La présente invention concerne un procédé permettant l'expression d'un fragment simple chaîne dans une plante transgénique en introduisant un produit recombinant d'ADN dans une cellule de plante. Le produit recombinant d'ADN présente des séquences d'acide nucléique codant une chaîne variable lourde, une chaîne variable légère et un polypeptide d'élastine. La chaîne variable légère et la chaîne variable lourde font partie du fragment simple chaîne et forment un site de liaison pour un antigène. Le polypeptide d'élastine présente plusieurs répétitions Val-Pro-Gly-Xaa-Gly, où Xaa est un acide aminé quelconque, le nombre de répétitions étant compris entre 2 et 80.
PCT/CA2007/001065 2007-06-15 2007-06-15 Expression de protéines de fusion contenant un fragment d'anticorps simple chaîne lié à des unités répétitives d'élastine dans des plantes transgéniques WO2008151405A1 (fr)

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WO2012109624A3 (fr) * 2011-02-11 2012-12-06 Zyngenia, Inc. Complexes plurispécifiques monovalents et multivalents et leurs utilisations
WO2014028776A1 (fr) * 2012-08-15 2014-02-20 Zyngenia, Inc. Complexes multispécifiques monovalents et multivalents et leurs utilisations
WO2017203426A1 (fr) * 2016-05-25 2017-11-30 University Of Cape Town Production d'une protéine de fusion d'igg-peroxydase de raifort
CN116694625A (zh) * 2023-06-26 2023-09-05 中国人民解放军空军军医大学 一种dna四面体及其复合物和用途

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US20060248615A1 (en) * 2000-06-09 2006-11-02 Jurgen Scheller Synthetic spider silk proteins and expression thereof in transgenic plants
WO2003041493A1 (fr) * 2001-11-14 2003-05-22 Ipk - Institut Für Pflanzengenetik Und Kulturpflanzen Forschung Production d'anticorps recombines par fusion avec des peptides similaires a l'elastine

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MEYER D.E. ET AL.: "Protein purification by fusion with an environmentally responsive elastin-like polypeptide: effect of polypeptide length on the purification of thioredoxin", BIOTECHNOL. PROG., vol. 17, no. 4, July 2001 (2001-07-01) - August 2001 (2001-08-01), pages 720 - 728, XP002955911 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012109624A3 (fr) * 2011-02-11 2012-12-06 Zyngenia, Inc. Complexes plurispécifiques monovalents et multivalents et leurs utilisations
WO2014028776A1 (fr) * 2012-08-15 2014-02-20 Zyngenia, Inc. Complexes multispécifiques monovalents et multivalents et leurs utilisations
WO2017203426A1 (fr) * 2016-05-25 2017-11-30 University Of Cape Town Production d'une protéine de fusion d'igg-peroxydase de raifort
US11788067B2 (en) 2016-05-25 2023-10-17 University Of Cape Town Production of a horseradish peroxidase-igg fusion protein
CN116694625A (zh) * 2023-06-26 2023-09-05 中国人民解放军空军军医大学 一种dna四面体及其复合物和用途
CN116694625B (zh) * 2023-06-26 2024-01-02 中国人民解放军空军军医大学 一种dna四面体及其复合物和用途

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