EP1226178A1 - Recombinant fusion molecules - Google Patents

Recombinant fusion molecules

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Publication number
EP1226178A1
EP1226178A1 EP00973028A EP00973028A EP1226178A1 EP 1226178 A1 EP1226178 A1 EP 1226178A1 EP 00973028 A EP00973028 A EP 00973028A EP 00973028 A EP00973028 A EP 00973028A EP 1226178 A1 EP1226178 A1 EP 1226178A1
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EP
European Patent Office
Prior art keywords
antibody
antigen
constant region
recombinant
fusion protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00973028A
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German (de)
English (en)
French (fr)
Inventor
Julian Ma
Kristian Dalsgaard
Palle Hoy Jakobsen
Fabrizio Advanced Biotechnology Center MANCA
Daniel Maurice King's College London CHARGELEGUE
Pascal Mark Wayne Drake
Thomas Lehner
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Kings College London
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Kings College London
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Publication date
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Publication of EP1226178A1 publication Critical patent/EP1226178A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • This invention relates to a method for increasing the production of a protein in a cell and more particular the production in a plant cell.
  • This invention also relates to recombinant immune fusion complexes and more particularly to their expression in plants.
  • Immune complexes comprise antibodies and antigens, and give rise to the classical immune response. Immune complexes are conventionally produced by mixing antigen with antibody and allowing the molecules to associate via the binding site of the antibody and the specific epitope of the antigen. The disadvantage of this process is that immune complexes in vitro which require careful mixing of antigen with antibody at optimal concentrations.
  • the present invention provides a method of increasing the production of a protein in a cell comprising expressing recombinant DNA encoding a recombinant fusion protein comprising a desired protein fused to the constant region of a heavy chain of an antibody or to part of said constant region.
  • the term 'heavy chain constant region will be used throughout the following description to refer either to the full length immunoglobulin heavy chain constant region or to a part (fragment) of this region, as the context permits.
  • the constant region comprises a number of regions (domains) depending of the class of the antibody.
  • the constant region comprises three regions (domains) known as CHI, CH2, and CH3; the CH3 domain being the C-terminal domain.
  • truncated versions of this may be used comprising the conjoined CH1/CH2, CH2/CH3 or CH1/CH3 domains.
  • the desired protein can be any protein including reporter molecules such as ⁇ -galactosidase, luciferase and GFP.
  • the desired protein may also be a selectable marker such as an antibiotic.
  • the desired protein is a food protein such as a storage protein, i.e. phaseolin or wheat gluten.
  • a storage protein i.e. phaseolin or wheat gluten.
  • the desired protein is a therapeutic protein.
  • Therapeutically useful proteins include receptors, e.g. the cystic fibrosis receptor (CFTR), enzymes including prodrug activating enzymes, e.g. nitroreductases, ligands, regulatory factors, hormones, and structural proteins. Therapeutic proteins also include sequences encoding nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma-associated proteins and serum proteins.
  • CFTR cystic fibrosis receptor
  • Therapeutic proteins also include sequences encoding nuclear proteins, cytoplasmic proteins, mitochondrial proteins, secreted proteins, plasmalemma-associated proteins and serum proteins.
  • the desired protein may also be selected from lipoproteins, glycoproteins and phosphoproteins, hormones, growth factors, enzymes, clotting factors such as Factor XHI and Factor TX etc., apolipoproteins, receptors, erythropoietin, drugs, oncogenes and tumor suppressors.
  • these compounds include proinsulin, growth hormone, androgen receptors, insulin-like growth factor I, insulin-like growth factor II, insulin-like growth factor binding proteins, epidermal growth factors, angiogenesis factors (acidic fibroblast growth factor, basic fibroblast growth factor, vascular endothelial growth factor and angiogenin), matrix proteins (Type IV collagen, Type N ⁇ collagen, laminin), phenylalanine hydroxylase, tyrosine hydroxylase, oncogenes (ras, fos, myc, erb, src, sis, jun), E6 or E7 transforming sequence, p53 protein, Rb gene product, cytokine receptor, II- 1, IL-6, IL-8, viral capsid protein, and other proteins of useful significance in the body.
  • angiogenesis factors acidic fibroblast growth factor, basic fibroblast growth factor, vascular endothelial growth factor and angiogenin
  • matrix proteins Type IV collagen, Type N ⁇ collagen, laminin
  • the desired protein which can be fused, is only limited by the availability of the nucleic acid sequence encoding the protein or polypeptide to be incorporated.
  • One skilled in the art will readily recognise that as more proteins and polypeptides become identified they can be integrated into the recombinant fusion protein of the present invention.
  • the desired protein is an antigen.
  • antigen refers to any protein that gives rise to an immune response in an animal such as a mammal, preferably a human.
  • the term "antigen” as used herein refers to a protein that stimulates a series of reaction in an animal that are mediated by white blood cells including lymphocytes, neutrophils and monocytes.
  • Preferred antigens include viral antigens, bacterial antigens, protozoal antigens, parasitic antigens, tumor antigens, and proteins from viral, bacterial and parasitic organisms which can be used to induce an immune response. It is also preferred that the antigen is not a toxin.
  • a toxin is defined herein as a protein which has a direct toxic effect on a cell and causes cell death. It is most preferred that the antigen is human immunodeficiency virus gpl20, the non-toxic C-tetanus toxin fragment or bovine respiratory syncytial virus (BRSV) F-protein.
  • gpl20 the non-toxic C-tetanus toxin fragment
  • BRSV bovine respiratory syncytial virus
  • the desired protein may be linked to the constant region or part thereof directly or indirectly through an intermediate peptide linker which may include a peptide cleavage site. This will allow separation of the desired protein from the constant region of part thereof (if required) for purification purposes. Attachment of the desired protein to the constant region or part thereof may be made via the C-terminus of the heavy chain or part thereof. Alternatively, the antigen may be fused to the antibody at a site within the constant region or part thereof.
  • the method of the present invention is applicable to the production of the desired protein in any eukaryotic cell.
  • the desired protein is produced in a eukaryotic cell which is capable of producing immunoglobulins. More preferably the desired protein is produced in a mammalian cell and most preferably in a plant cell.
  • the method of the present invention has the benefit of allowing the production of a desired protein in a cell at a higher level than when the desired protein is not produced as part of a fusion protein comprising a constant region of a heavy chain of an antibody. Desired proteins are generally expressed at low levels in transgenic plants, whereas antibody molecules usually accumulate to much higher levels. By genetically fusing the desired protein with a component of the antibody molecule, the antibody component acts as a carrier and stabilising molecule and allows much higher levels of production and accumulation of the desired protein.
  • the present invention also provides a first recombinant fusion protein comprising an antigen fused to the constant region of the heavy chain of an antibody or to a part of said constant region.
  • the recombinant fusion protein is as defined above in connection with the method of the present invention, except that the fusion protein comprises an antigen as defined above.
  • the present invention also provides a second recombinant fusion protein comprising a desired protein fused to the constant region of a heavy chain of an antibody or to part of said constant region, wherein the fusion protein does not comprise a functional antigen binding domain.
  • the recombinant fusion protein is as defined above in connection with the method of the present invention, except that the fusion protein does not comprise a functional antigen binding domain.
  • a functional antigen binding domain is defined herein as the antibody domain which contacts and binds an antigen.
  • the domain comprises the complementarity determining regions (CDRs) of the antibody, which are well known to those skilled in the art and can be easily identified.
  • the domain is preferably the variable domain of the heavy and/or light chain of an antibody.
  • the second recombinant fusion protein of the present invention can comprise an antibody which has had one or more of its variable domains removed.
  • the second recombinant fusion protein of the present invention can comprise an antibody which has one or more non-functional variable domains.
  • the variable domains can be made non-functional by deleting the CDRs. Other methods for producing an antibody with a non- functional antigen binding domain are well known to those skilled in the art.
  • the present invention also provides a recombinant antibody molecule having an antigen fused to an antibody molecule, wherein the antibody molecule has affinity for the antigen.
  • the recombinant antibody molecule of the present invention can form an immune complex by associating with other recombinant antibody molecules of the present invention through the antigen binding sites of the antibody.
  • Such an immune complex resembles, but is not identical to, a classical immune complex.
  • a significant advantage of the immune complex formed from the recombinant antibody molecules of the present invention is that it is easy to prepare in contrast to conventional preparation of immune complexes in vitro which require careful mixing of antigen with antibody at optimal concentrations. In our case, the expression ratio of antibody to antigen molecules is generally fixed at 1 : 1 , which optimises the potential for forming immune complexes.
  • the recombinant antibody molecule of the present invention is highly immunogenic, especially when it is forms an immune complex, and is suitable for both systemic and mucosal immunisation without the need for an adjuvant.
  • Adjuvants are generally incorporated into vaccine compositions to improve the immune response.
  • the recombinant antibody molecules of the present invention are highly immunogenic, it is possible to obtain immunisation without the need of an adjuvant.
  • the immune complex formed using recombinant antibody molecules of the present invention will possess substantially the same enhanced immunogenic properties as an endogenously produced immune complex or a conventionally produced immune complex.
  • the recombinant antibody molecule of the present invention can comprise any antibody molecule provided it is has affinity for the antigen to which it is fused.
  • the antibody molecule can be a complete monoclonal antibody or an antigen binding fragment thereof, such as a Fv, Fab or F(ab')2 fragment.
  • Preferably the antibody molecule comprises a constant region of a heavy chain of an antibody.
  • antigen-antibody fusion proteins there is no restriction as to the species of either of these components.
  • the invention is of especial interest in relation to human medicine but other primate and other animal antigen/antibody combinations may be used depending on the intended biological application of the product.
  • the antigens and the antibody heavy and light chains may be of human, rodent, rabbit, bovine, ovine, caprine, fowl, canine, camel, feline or primate origin.
  • human immunodeficiency virus gpl20 the non-toxic C-tetanus toxin fragment and bovine respiratory syncytial virus (BRSV) F-protein, with their respective specific monoclonal antibodies.
  • BRSV bovine respiratory syncytial virus
  • the antigen is fused to either the C-terminus of a monoclonal antibody (Mab) full length heavy chain, or to truncated heavy chain domains consisting of 1, 2 or 3 constant region domains.
  • the antigen is fused to the constant region at a point within the constant region.
  • the present invention also provides a transgenic plant expressing a recombinant fusion protein comprising a desired protein fused to a constant region of a heavy chain of an antibody.
  • the present invention also provides a transgenic plant expressing the first or second recombinant protein of the present invention, or the recombinant antibody molecule of the present invention.
  • the transgenic plants of the present invention may express a number of molecular forms of fusion protein for each antigen, permitting the selection of the correctly formed molecular form of fusion protein and its expression at the highest levels and to extract and purify the fusion protein. It is also possible to characterise the plant derived fusion proteins by analysis of assembled molecular forms, recognition by and affinity for a panel of antibodies.
  • the advances deriving from this work are the development of a new type of engineered molecule suitable for vaccination in a range of infectious diseases.
  • the recombinant antibody molecule resulting from the fusion of an antigen with an antibody molecule has all the components required for immune complex mediated stimulation of an immune response and our approach offers a convenient method for ensuring antigen/antibody complexing.
  • the recombinant antibody molecules of the present invention can also form larger immune complexes planta (see Figure 1) which can be used for immunisation.
  • antibody molecules are essential, such as the sites for FcR binding and complement activation that reside in the constant domains of IgG antibodies.
  • truncated antibody molecules which lack either the C ⁇ 3 domain (that is not involved in antigen recognition), or the C ⁇ l domain.
  • first recombinant fusion protein of the present invention or the second recombinant fusion protein of the present invention for immunisation.
  • a fusion protein comprising an antibody molecule which does not comprise a functional antigen binding domain and an antigen can be used to deliver the antigen to phagocytes and also to initiate complement activation.
  • antigen recognition it is possible to help prevent the phenomenon of modulation of antigen processing that results from antibody masking of T cell epitopes, as the antigen will be a fusion protein with the constant region of an antibody.
  • plants are ideally suited for the production of the recombinant fusion proteins of the present invention not only because of the potential requirement for large quantities for vaccination purposes, but also because those complexes that involve full length antibody, or assembly with light chain are not readily produced in most other expression systems.
  • DNA encoding an antigen or antigenic fragment may be amplified by PCR.
  • the genes encoding the light and heavy chains of murine, human or other mammalian monoclonal antibodies specific for these antigens are also cloned.
  • the gene constructs can be made encoding the desired recombinant fusion protein.
  • the following gene constructs for each of the antigens can be prepared for plant transformation:
  • the linker peptide as shown in Figure 2 may conveniently be (GGGGS) 3 .
  • a conventional ⁇ xHistidine tag and a protease cleavage site e.g. enterokinase, Factor Xa or thrombin
  • a further different protease cleavage site e.g. enterokinase, Factor Xa or thrombin
  • Additional or alternative peptide tags and protease cleavage sites may be incorporated.
  • the gene constructs are sequenced for confirmatory purposes and inserted into plant expression vectors for plant transformation.
  • Nicotiana tabacum is a convenient plant host using Agrobacterium mediated transformation.
  • constructs I and ⁇ in order to generate the recombinant fusion proteins , the immunoglobulin light chain genes and the heavy chain constructs may be introduced into separate plant lines as shown in Figure 1. Following regeneration of these first generation transformants, the light chain and heavy chain transformed plants are cross-fertilised and the second generation plants screened for production of the final product. For constructs IH and IN, there is no requirement for the light chain and the final product can be produced in the first generation transformed plants.
  • transgenic plants expressing correctly assembled products are used to generate homozygous plant stocks.
  • the plants are grown to maturity, self fertilised, and the resulting seeds screened by back crossing with non-transformed plants to determine those that are homozygous. Further stocks can be generated by self- fertilisation and stored as seeds.
  • the primary plant transformants are screened to determine which types of products are expressed and assembled optimally. This investigation can be performed by Western blot analysis and ELISA of crude plant extracts, using a range of antisera and monoclonal antibodies that are commercially available. Extraction and purification of the selected recombinant products may be achieved by ammonium sulphate precipitation, followed by filtration and affinity chromatography using either the immunoglobulin regions or specific peptides as affinity tags. Further characterisation is performed to determine molecular structure, recognition of the antigen moiety by a panel of antibodies and binding affinity.
  • Figure 1 shows schematically immune complexes in plants and potential assembly arrangements.
  • Figure 2 shows schematically the basic molecular design.
  • Figure 3 shows schematically the pEXG13 principle cloning sites.
  • Figure 4 shows RT-PCR analysis of transgenic plants.
  • Figure 5 shows results of a capture ELISA for CH2-CH3-gpl20.
  • Figure 6 shows results of a capture ELISA for recombinant gpl20 expressed in plants
  • Figure 7 shows a 10% SDS gel under reducing and non-reducing conditions.
  • the source of DNA for the HIV gpl20 antigen was an infectious cloned isolate of HIV DIB.
  • DNA can be obtained from the Medical Research Council AIDS Reagents Project (NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, UK).
  • the genes encoding the heavy and light chains of an HIV gpl20 specific monoclonal antibody e.g. Gorny et al., 1991, Proceedings of the National Academy of Sciences, USA 88:3238-3242
  • were cloned as described (Ma, J. K-C, T. Lehner, P. Stumble, CL Fux and A. Hiatt.
  • the ohgonucleotide primers 1 and 2 corresponded to the 5' and 3' ends of the antibody heavy chain and the ohgonucleotide primers 3 and 4 corresponded to the 5' and 3' ends of the antigen.
  • the 5' primer for the antigen also includes sequences encoding a linker peptide and a protease cleavage site.
  • the primers included appropriate restriction enzyme sites (such as 5' BamHI and 3' Xmal for gpl20; 5' Xhol and 3' BamHI for Ig heavy chain) and extra sequences encoding linker peptides (GGGSGGGSGGGS) or protease cleavage sites (e.g. Factor Xa IEGR).
  • the gene can be cloned according to the methodology described by Drake et al., Antibody production in plants. P. Shepherd and Dean (eds). Monoclonal Antibodies - A practical approach. Oxford University Press. 2000)
  • Positive transformants were identified by the release of a DNA fragment of the correct size following digestion with the appropriate restriction enzymes, or PCR using the appropriate 5' and 3' ohgonucleotide primers.
  • Four chimeric immunoglobulin heavy chains constructs were engineered in which the antigen was expressed in fusion with varying portions of the immunoglobulin heavy chain as shown in Figure 1.
  • the specific example of cloning the Ig heavy chain CH2-CH3 domain fusion with an HIV gpl20 fragment (construct HI in Figure 1) is shown in Figure 2.
  • Two PCR products were amplified using ohgonucleotide pairs 1-2 and 3-4.
  • the HIV gpl20 DNA sequence encodes a truncated peptide starting at the N-terminus, up to and including the sequence KEYAL (aal47) with a stop codon immediately after. These were cloned into a vector based on pET32 which had previously been engineered with the key features of the cloning site within this vector shown in Figure 3.
  • the gpl20 gene fragment was ligated into the BamHI - Xmal site of the vector, then the Ig heavy chain gene was cloned upstream in the Ncol - BamHI site.
  • the entire construct was then re-amplified by PCR to include the downstream enterokinase cleavage site and His tag, and to include 5' Xhol and 3' EcoRI terminal restriction sites. This fragment was cloned into pBluescript for confirmatory sequencing.
  • Plasmids of this type are widely available, such as the pGreen system (www.pgreen.ac.uk).
  • the plant expression cassette in this case contains the 5' untranslated region from tobacco etch virus which stabilises mRNA in plant cells, as well as an upstream murine IgG leader sequence ( Figure 3), which directs secretion of the recombinant protein out of the plant cells.
  • This recombinant vector was used to transform E. coli (DH5- ⁇ ). Screening of transformed clones was by Southern blotting, using radiolabelled DNA probes derived from the original PCR products. Plasmid DNA was purified from positive transformants (PromegaQiaprep kit) and used for transformation of Agrobacterium tumefaciens (strain LBA4404 - Gibco/BRL, UK) using a freeze/thaw procedure as follows:
  • Plant transformation and regeneration All gene constructs were introduced into Nicotiana tabacum, var. xanthii. Tobacco transformation with A. tumefaciens was by standard procedures. Leaf discs are cut from surface sterilised tobacco leaves and incubated with a culture of the recombinant A. tumefaciens, containing cDNA inserts. The infected discs are transferred to culture plates containing a medium that induces regeneration of shoots, supplemented with kanamycin and carbenicillin (Sigma, UK). Shoots developing after this stage are excised and transplanted onto a root inducing medium, supplemented with kanamycin. Rooted plantlets are transplanted into soil after the appearance of roots. The detailed methodology was as follows:
  • Developing shoots are removed when they reach a convenient size (approx. 0.5 cm in length) and transferred to rooting medium (3-4 shoots/40 ml medium/175 ml glass jar). Incubate for 14 d at 25°C with a 16 h photoperiod.
  • Rooted shoots are transferred to compost in plant pots, watered and supplied with nutrients. Plants are kept in seed trays and covered with a lid for 24 h after transfer to compost, to minimise initial water loss.
  • Regenerated plants were screened for expression of immunoglobulin chains and each of the antigens by Western blot and ELISA of crude leaf extracts using available antisera and monoclonal antibodies (e.g. from the MRC AIDS Directed reagents programme, address given above).
  • Transgenic plants were self- fertilised to establish homozygous plant lines and cross fertilised to generate antibody producing plants.
  • the upper (aqueous) phase was collected, mixed with pre-cooled ethanol (0.7 volumes) and 1M acetic acid (0.2 volumes), and incubated at -200°C for 16 hours. After centrifugation, the precipitate was washed three times with 3M sodium acetate, pH5.2, and once with 70% ethanol. The pellet was dissolved in sterile RNAse free water (Sigma, UK) containing RNAse free DNAse (Promega, UK), and incubated at 37°C for 1 hour, then at 70°C for 5 minutes.
  • RNA concentration and purity were assessed using the GeneQuant II RNA/DNA Calculator (Pharmacia BioTech, UK). RNA was stored in sterile RNAse free water at -20°C.
  • RT-PCR was performed using appropriate ohgonucleotide primers to determine that the correct RNA transcript was being made by putative transformed plants.
  • leaf extracts were examined by ELISA and Western blot analysis.
  • Samples were extracted in 150mM NaCl and 20mM tris, pH8 (TBS) with leupeptin (lOmg ml) (Calbiochem).
  • Capture ELISA analyses were performed by incubating the plant extracts on microtiter plates that had been pre-coated with a monoclonal antibody specific for HIV gpl20 (ADP401 from the MRC Aids Directed Program) and blocked with 5% non-fat dry milk in TBS.
  • the plates were washed in TBS with 0.05% Tween 20, then incubated with either a horseradish peroxidase labelled Sheep anti-Mouse IgG antiserum (The Binding Site, UK), or one of a panel of gpl20 specific monoclonal antibodies (MRC Aids Directed Program), followed by the relevant horseradish peroxidase antiserum (The Binding Site, UK).
  • the assay was developed using the TMB substrate (Sigma, UK) and absorbance was read at 450nm.
  • Glycosylation is determined by Western blot, examining binding to lectins such as concanavalin A, or by using glycans specific antisera (kindly provided by Dr. Loic Faye, University of Rouen). Functional studies of antigen binding affinity to available monoclonal antibodies may be performed using surface plasmon resonance techniques.
  • Purification was performed using a procedure which we have previously determined for IgG extraction from transgenic plants. Following initial precipitation from crude plant extract with ammonium sulphate, the recombinant antibody was concentrated by stirred cell filtration using a YM30 molecular weight cut-off filter (Amicon, UK). Purification was by affinity chromatography using agarose coupled to anti-mouse or human IgG antibodies (Sigma, UK) as the ligand. Elution was in 0.1M glycine-HCl pH 2.5.
  • the recombinant heavy chains may also be designed to allow a further affinity purification step using the 6xHis fusion peptide tag with immobilised metal chelate affinity chromatography, which can subsequently be cleaved by thrombin.
  • fusion proteins are not limited to plants, and could be carried out in any other eukaryotic cells, especially cells that can produce immunoglobulins such as mammalian cells.
  • the immunoglobulin chain sequences can be exchanged for sequences from other classes of immunoglobulins.
  • sequences can be incorporated into the construct in addition to the epitope tags and protease cleavage sites.
  • these could code for other functional regions, such as markers, enzyme activity, or sites for chemical coupling to other active molecules.
  • Antigen and whole antibody are genetically linked to form a type of immune complex.
  • antigen and antibody fragments that may or may not include the antigen binding site
  • fusion proteins are linked to form fusion proteins.
  • the recombinant antibody molecule and the antigen are automatically expressed at 1 :1 ratio. In the case of constructs involving whole IgG, larger complexes may form spontaneously.
  • Antigen-antibody fusion molecules are easy to produce.
  • the use of plants to make these molecules significantly reduces the cost, particularly if production is increased to agricultural scale.

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US8029803B2 (en) 2002-06-20 2011-10-04 Paladin Labs, Inc. Chimeric antigens for eliciting an immune response
US8007805B2 (en) 2003-08-08 2011-08-30 Paladin Labs, Inc. Chimeric antigens for breaking host tolerance to foreign antigens
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ZA200804078B (en) * 2005-10-13 2009-09-30 Virexx Medical Corp Chimeric hepatitis C virus antigens for eliciting an immune response
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US8444976B2 (en) 2008-07-02 2013-05-21 Argen-X B.V. Antigen binding polypeptides
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