WO2019170899A1 - Vecteur pour l'expression de chaînes d'anticorps de cétuximab - Google Patents

Vecteur pour l'expression de chaînes d'anticorps de cétuximab Download PDF

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WO2019170899A1
WO2019170899A1 PCT/EP2019/055928 EP2019055928W WO2019170899A1 WO 2019170899 A1 WO2019170899 A1 WO 2019170899A1 EP 2019055928 W EP2019055928 W EP 2019055928W WO 2019170899 A1 WO2019170899 A1 WO 2019170899A1
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cetuximab
nucleic acid
chain
antibody
vector
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PCT/EP2019/055928
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English (en)
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Alexander Levitzki
Lital FRIEDMAN
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Targimmune Therapeutics Ag
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Publication of WO2019170899A1 publication Critical patent/WO2019170899A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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

Definitions

  • the present invention relates to the field of expression vectors, especially for expressing antibodies and antibody chains.
  • the present invention relates to a vector for expression of Cetuximab antibody chains comprising (i) at least one nucleic acid sequence encoding a chain of a Cetuximab antibody, and (ii) at least one regulatory element for expression in plant cells. Further the invention refers to a plant cell from the genus Nicotiana comprising the vector of the invention and a method for manufacturing a Cetuximab antibody and to the Cetuximab antibody or the chain of a Cetuximab antibody obtained by the method of the invention.
  • Cancer such as colorectal or lung cancers is still a devastating disease that has baffled researchers over the years. Elevated levels of the EGFR (erbB-l) and its cognate ligands have been identified as a common component of numerous cancer types.
  • a study by Nicholson et al. showed that several cancer types including head and neck, ovarian, cervical, bladder, oesophageal, gastric, breast, endometrial, colorectal cancer, and non- small cell lung cancer (NSCLC) express elevated levels of EGFR relative to normal tissues and have been studied in sufficient depth to allow sound judgements to be made concerning the association between EGFR and patient outlook (Nicholson et al,“EGFR and cancer prognosis,” Em: J. Cancer, vol. 37, pp. 9-15, 2001).
  • NSCLC non- small cell lung cancer
  • Cetuximab is approved for use in the treatment of EGFR- expressing, metastatic or recurrent colorectal carcinoma, locally or regionally advanced squamous cell carcinoma of the head and neck (SCCHN) and for use in combination with FOLFIRI (irinotecan, 5-fluorouracil, and leucovorin) for first-line treatment of patients with K-ras mutation-negative (wild-type), EGFR-expressing metastatic colorectal cancer (mCRC)(NIH, National Cancer Institute, FDA Approval for Cetuximab, July 2, 2013).
  • FOLFIRI irinotecan, 5-fluorouracil, and leucovorin
  • Cetuximab (IMC-C225, Erbitux®) is a recombinant, human/mouse chimeric IgGl monoclonal antibody that binds specifically to the epidermal growth factor receptor (EGFR, HER1, c-ErbB-l) on both normal and tumor cells and competitively inhibits the binding of epidermal growth factor (EGF) and other ligands, such as transforming growth factor-alpha.
  • EGFR epidermal growth factor receptor
  • HER1, c-ErbB-l epidermal growth factor receptor
  • Cetuximab Binding of Cetuximab to the EGFR blocks phosphorylation and activation of receptor- associated kinases, resulting in inhibition of cell growth, induction of apoptosis, and decreased matrix metalloproteinase and vascular endothelial growth factor production (NIH, National Cancer Institute, FDA Approval for Cetuximab, July 2, 2013).
  • Monoclonal antibodies are widely used in the diagnosis, management and treatment of infectious diseases and cancer, and represent an important component in the current pipeline of new biological compounds.
  • Recombinant mAbs have been expressed mainly in myeloma cell lines and in Chinese hamster ovary (CHO) cells.
  • Other expression systems have been explored, including Escherichia coli, yeast, transgenic animals and transgenic plants, and each has advantages and disadvantages (Hassan et al. , Considerations for extraction of monoclonal antibodies targeted to different subcellular compartments in transgenic tobacco plants, Plant Biotechnology Journal, 2008, vol. 6, pp. 733-748).
  • Plant genetic engineering can be applied to express recombinant biopharmaceutical proteins on an industrial scale (Ko, Expression of Recombinant Vaccines and Antibodies in Plants, Monoclonal antibodies in immunodiagnosis and immunotherapy 2014, vol. 33(3), pp. 192-198).
  • Transgenic tobacco plants are capable of expressing many recombinant proteins including full-length, secretory immunoglobulins at very high levels.
  • wet plant tissue has to be processed and the presence of toxic phenolics and alkaloids in the leaves complicates downstream processing (Hassan et al., 2008, op. cit.).
  • Cetuximab is produced by expressing clone C225 in the mouse myeloma cell line SP2/0, and a variant of Cetuximab, CHO-C225 is produced in Chinese hamster ovary (CHO) cell lines (Chung et al, Cetuximab-Induced Anaphylaxis and IgE Specific for Galactose-a-l,3- Galactose, N Engl J Med. 2008, vol. 358(11), pp. 1109-1117).
  • CHO Chinese hamster ovary
  • Adenovirus expressing full-length Cetuximab was used in gene therapy for cancer treatment.
  • Full-length Cetuximab antibody was cloned into two serotypes of adenoviral vectors.
  • In vivo studies showed that a single dose of AdC68-CTB or Hu5-CTB induced sustained Cetuximab expression (Xing et al, Gene therapy for colorectal cancer using adenovirus-mediated full-length antibody, Cetuximab, Oncotarget 2016, vol. 7(19), pp. 28262-28272).
  • the invention relates to a vector for expression of Cetuximab antibody chains, said vector comprises (i) at least one nucleic acid sequence encoding a chain of a Cetuximab antibody, and (ii) at least one regulatory element for expression in plant cells, wherein said at least one regulatory element is operably linked to said at least one nucleic acid sequence encoding the Cetuximab antibody.
  • the invention relates to a plant cell comprising the vector of the invention, wherein said plant cell is from the genus Nicotiana, preferably of Nicotiana tabacum or Nicotiana benthamiana, most preferably of Nicotiana tabacum.
  • the invention relates to a method for manufacturing a Cetuximab antibody comprising the steps of:
  • the invention relates to a Cetuximab antibody or a chain of a Cetuximab antibody obtained by the method of the invention.
  • the inventors developed an expression system for production of a Cetuximab antibody in plants of the genus Nicotiana.
  • This system is advantageous, because it is allows an efficient expression of a Cetuximab antibody in plants of the genus Nicotiana, it is scalable, has the capacity of large- scale production, and is cost-efficient. Moreover, the inventive expression system allows correct folding of the Cetuximab antibody and is at very low risk of being contaminated with mammalian disease agents like viruses or bacterial toxins. Moreover, it allows glycosylation of the Cetuximab antibody chains and assembly of the light and heavy chains of a Cetuximab antibody in order to form a full Cetuximab antibody.
  • FIGURE 1 Schematic design of (A) Cetuximab antibody (CTX), (B) Cetuximab antibody with a dsRNA binding domain (dsRBD) attached to the N terminus of the light chain of Cetuximab (Cetuximab-LC-dsRNA), (C) Cetuximab antibody with a dsRNA binding domain (dsRBD) attached to the C terminus of the heavy chain of Cetuximab (Cetuximab-HC- dsRBD)
  • FIGURE 2 Design of Cetuximab-DsRed pUC57 vector comprising genes encoding the heavy chain and light chain of the Cetuximab antibody and a gene expressing DsRed - a red fluorescent protein used as a screenable marker. These genes were codon-optimized for tobacco.
  • the heavy and light chains are each flanked by the rubisco small subunit promoter and terminator and each contain a signal peptide directing them to the apoplast, the space outside of the plant cell plasma membrane.
  • the DsRed gene is flanked by the CaMV 35 S promoter and terminator.
  • Surrounding each expression unit are matrix attachment regions (MARs) - labeled CHN S/M II, TM6 and Rb7 in the figure - which have been shown to enhance gene expression.
  • MARs matrix attachment regions
  • FIGURE 3 Intermediate vector pUC57 encoding Cetuximab heavy and light chains and screenable marker DsRed after successful Golden Gate assembly.
  • FIGURE 4 Cetuximab-heavy chain-dsRBD-DsRed in pUC57 (A); Cetuximab-light chain- dsRBD-DsRed in pUC57 (B).
  • FIGURE 5 Map of the binary agrobacterium expression vector pBINPLUS for plant transformation.
  • FIGURE 6 Western blot of selected Cetuximab expressing tobacco plants, using anti-human IgG HRP antibody. Bands at ⁇ 50 and ⁇ 25 kDa represent the heavy and light chains, respectively. 100 ng of commercial Cetuximab was run for comparison. A sample from wild type (WT) plant was used as control.
  • WT wild type
  • FIGURE 7 Western blot of selected Cetuximab-LC-dsRBD expressing tobacco plants, using anti- human IgG HRP antibody (A) and mouse anti- human PKR followed by anti-mouse IgG antibody (B). Bands at ⁇ 50 and ⁇ 45 kDa represent the heavy chain and light chain-dsRBD, respectively.
  • FIGURE 8 Western blot of selected Cetuximab-HCdsRBD expressing tobacco plants, using anti- human IgG HRP antibody (A) and mouse anti- human PKR followed by anti-mouse IgG antibody (B). Bands at ⁇ 75 and ⁇ 25 kDa represent the heavy chain-dsRBD and light chain, respectively.
  • FIGURE 9 Western blots of Protein A bead purification results.
  • samples were run from the lysate before purification, the unbound proteins, the bound proteins (beads before elution) and the eluted protein. Blots were deteted using an anti-human IgG-HRP antibody.
  • mouse anti-PKR followed by anti- mouse-HRP antibody was also used for detection (right pane of B and C).
  • FIGURE 10 Protein A purification chromatography of plant derived Cetuximab. The elution peak was observed between 87 and 90 ml (x-axis) (A). Coommassie staining of selected fraction samples; the protein is found in fractions 6-11 (B). Western blot analysis using anti human IgG-HRP antibody shows Cetuximab heavy and light chains in lysate (before purification) as well as in eluted fractions, but not in unbound or wash fractions (C).
  • FIGURE 11 Protein A purification chromatography of plant derived Cetuximab-LC-dsRBD. The elution peak was observed between 82 and 90 ml (x-axis) (A). Western blot analysis using anti-human IgG-HRP antibody shows elution of Cetuximab heavy chain, and a ⁇ 25 kDa band that could be the original light chain, but not LC-dsRBD (B).
  • FIGURE 12 Specific binding of commercial Cetuximab, tobacco-expressed Cetuximab and Cetuximab-LC-dsRBD to EGFR.
  • Figure 13 TREATMENT OF HN5 AND A431 CELLS WITH THE COMMERCIAL
  • the invention relates to vector for expression of Cetuximab antibody chains, said vector comprises
  • At least one regulatory element for expression in plant cells wherein said at least one regulatory element is operably linked to said at least one nucleic acid sequence encoding the chain of the Cetuximab antibody.
  • vector refers to a nucleic acid molecule. It is used as a vehicle to artificially transfer nucleic acids and genetic material.
  • the vector can be a closed or open nucleic acid molecule and includes an expression cassette, plasmid, viral vector, phagemid, phage, cosmid, and artificial chromosomes (YAC, BAC, PAC etc.), expression vector and cloning vector.
  • a“regulatory element is operably linked to ... (an) encoding nucleic acid sequence” or vice versus means that the promoter and the encoding nucleic acid sequence are a functional combination, i.e. the promoter is suitably positioned in order to regulate and preferably initiate transcription of the encoding nucleic acid sequence.
  • Said at least one regulatory element for expression in plant cells is suitable for initiating transcription of an encoding nucleic acid sequence in plants.
  • said plant cells are from Nicotiana.
  • said Nicotiana is Nicotiana benthamiana or Nicotiana tabacum. More preferably, said Nicotiana is Nicotiana tabacum. Again more preferably, said Nicotiana is Nicotiana tabacum cv. Samsun.
  • Said nucleic acid sequence encoding the Cetuximab antibody is preferably codon optimized for expression in cells of Nicotiana, preferably Nicotiana tabacum.
  • Codon optimization means switching the codons used in the encoding nucleic acid sequence without changing the amino acid sequence, to remove codons that are rare in Nicotiana and replace them by codons abundantly used in Nicotiana.
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is (i) at least one nucleic acid sequence encoding a heavy chain of a Cetuximab antibody (CTX-HC) and at least one nucleic acid sequence encoding a light chain of a Cetuximab antibody (CTX-LC); or (ii) at least one nucleic acid sequence encoding a heavy chain of a Cetuximab antibody; or (iii) at least one nucleic acid sequence encoding a light chain of a Cetuximab antibody.
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is at least one nucleic acid sequence encoding a heavy chain of a Cetuximab antibody. In a preferred embodiment, said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is at least one nucleic acid sequence encoding a light chain of a Cetuximab antibody.
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is at least one nucleic acid sequence encoding a heavy chain of a Cetuximab antibody and at least one nucleic acid sequence encoding a light chain of a Cetuximab antibody.
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is one nucleic acid sequence encoding a heavy chain of a Cetuximab antibody
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is one nucleic acid sequence encoding a light chain of a Cetuximab antibody
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is one nucleic acid sequence encoding a heavy chain of a Cetuximab antibody and one nucleic acid sequence encoding a light chain of a Cetuximab antibody
  • said vector of the invention comprises (i) at least one nucleic acid sequence encoding a heavy chain of a Cetuximab antibody and at least one nucleic acid sequence encoding a light chain of a Cetuximab antibody, and (ii) said at least one regulatory element for expression in plant cells.
  • chain of a Cetuximab antibody refers to heavy and light chains of a Cetuximab antibody and functional variants and functional fragments thereof.
  • a chain of a Cetuximab antibody is a heavy or a light chain of a Cetuximab antibody.
  • Cetuximab (CTX, Erbitux®) has an epidermal growth factor receptor binding Fab (fragment, antigen-binding) region. Cetuximab is composed of the Fv (variable; antigen binding) regions of the 225 murine EGFR monoclonal antibody specific for the N-terminal portion of human EGFR with human IgGl heavy and kappa light chain constant (framework) regions.
  • Cetuximab full antibody includes two heavy chains and two light chains. Cetuximab heavy chain and light chain sequences are known in the art. For instance, a Cetuximab heavy chain or light chain can have a Cetuximab heavy chain sequence or Cetuximab light chain sequence as disclosed in any of (1) Fi et al, Structural basis for inhibition of the epidermal growth factor receptor by cetuximab, Cancer Cell 2005, vol. 7, pp. 301-311; (2) Dubois et al, Immunopurification and Mass Spectrometric Quantification of the Active Form of a Chimeric Therapeutic Antibody in Human Serum, Anal. Chem 2008; vol. 80: pp.
  • said Cetuximab heavy or light chain are Cetuximab full heavy or light chains of a Cetuximab antibody of fragments thereof, such as a chain of a Fab, Fab”, F(ab’) 2 , Fd, Fv or a single chain Fv (scFv). Chains forming a Cetuximab full antibody are more preferred, since it leads to a high level of Fc-mediated effects.
  • said Cetuximab antibody is a monospecific or multispecific antibody such as a bispecific antibody. More preferably, said Cetuximab antibody is a monospecific antibody.
  • Cetuximab heavy chain has the following sequence of SEQ ID NO:
  • Cetuximab light chain has the following sequence of SEQ ID NO: 13 :
  • said Cetuximab antibody chain is a Cetuximab chain as defined in the prior art.
  • said Cetuximab antibody chain is a Cetuximab chain as defined in (1) Fi et ai, 2005, op. cit.; (2) Dubois et ai, 2008, op. cit.; (3) HVIGT database, op. cit., ⁇ (4) Ayoub et ai, 2013, op. cit., ⁇ or (5) DrugBank, op. cit..
  • Cetuximab antibody encompasses Cetuximab and any antibody or antibody fragment that recognizes and specifically binds EGFR and has at least a heavy and light chain having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a sequence of SEQ ID NO: 12 and SEQ ID NO: 13, respectively.
  • Cetuximab antibody or Cetuximab also encompasses any antibody or antibody fragment that recognizes and specifically binds EGFR and has at least a heavy chain domain or light chain domain having at least 80% identity (e.g., at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to a sequence of Cetuximab, preferably to SEQ ID NO: 12 or SEQ ID NO: 13.
  • said heavy chain of a Cetuximab antibody has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 12.
  • said light chain of a Cetuximab antibody has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 13.
  • said nucleic acid encoding a heavy chain of a Cetuximab antibody (herein interchangeably used with the term“Cetuximab heavy chain”) has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 1.
  • a nucleic acid encoding a heavy chain of a Cetuximab antibody has the following sequence of SEQ ID NO: 1 :
  • said nucleic acid encoding a light chain of a Cetuximab antibody (herein interchangeably used with the term“Cetuximab light chain”) has at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 2
  • a nucleic acid encoding a light chain of a Cetuximab antibody has the following sequence of SEQ ID NO: 2:
  • said nucleic acid encoding a heavy and a light chain of a Cetuximab antibody have both at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • said nucleic acid encoding a chain of a Cetuximab antibody encodes a heavy or light chain of a Cetuximab antibody, wherein said heavy or light chain of a Cetuximab antibody has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the sequence of SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is (i) at least one nucleic acid sequence of SEQ ID NO: 1 and at least one nucleic acid sequence of SEQ ID NO: 2 or (ii) at least one nucleic acid sequence of SEQ ID NO: 1 or (iii) at least one nucleic acid sequence of SEQ ID NO: 2.
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is at least one nucleic acid sequence of SEQ ID NO: 1.
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is at least one nucleic acid sequence 2.
  • said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is at least one nucleic acid sequence of SEQ ID NO: 1 and at least one nucleic acid sequence of SEQ ID NO: 2.
  • said nucleic acid sequence encoding a heavy chain of a Cetuximab antibody is SEQ ID NO: 1
  • said nucleic acid sequence encoding a light chain of a Cetuximab antibody is SEQ ID NO: 2.
  • said vector of the invention comprises (i) at least one nucleic acid sequence encoding a heavy chain of a Cetuximab antibody of SEQ ID NO: 1 and one nucleic acid sequence encoding a light chain of a Cetuximab antibody, and (ii) at least at least one regulatory element for expression in plant cells.
  • said at least one nucleic acid sequence encoding said Cetuximab antibody chain comprises (i) at least one sequence of SEQ ID NO: 1 and (ii) at least one sequence of SEQ ID NO: 2. In another preferred embodiment, said at least one nucleic acid sequence encoding said Cetuximab antibody chain comprises (i) at least one sequence of SEQ ID NO: 1 or (ii) at least one sequence of SEQ ID NO: 2. In another preferred embodiment, said at least one nucleic acid sequence encoding said Cetuximab antibody chain comprises at least one sequence of SEQ ID NO: 1. In another preferred embodiment, said at least one nucleic acid sequence encoding said Cetuximab antibody chain comprises at least one sequence of SEQ ID NO: 2.
  • said at least one nucleic acid sequence encoding said Cetuximab antibody chain is (i) at least one sequence of SEQ ID NO: 1 and (ii) at least one sequence of SEQ ID NO: 2. In another preferred embodiment, said at least one nucleic acid sequence encoding said Cetuximab antibody chain is (i) at least one sequence of SEQ ID NO: 1 or (ii) at least one sequence of SEQ ID NO: 2. In another preferred embodiment, said at least one nucleic acid sequence encoding said Cetuximab antibody chain is at least one sequence of SEQ ID NO: 1. In another preferred embodiment, said at least one nucleic acid sequence encoding said Cetuximab antibody chain is at least one sequence of SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of the Cetuximab antibody has at least 80% identity with SEQ ID NO: 1, and said nucleic acid encoding the light chain of the Cetuximab antibody has at least 80% identity with SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of the Cetuximab antibody has at least 80% identity with SEQ ID NO: 1.
  • said nucleic acid encoding the light chain of the Cetuximab antibody has at least 80% identity with SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of the Cetuximab antibody has at least 90% identity with SEQ ID NO: 1
  • said nucleic acid encoding the light chain of the Cetuximab antibody has at least 90% identity with SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of the Cetuximab antibody has at least 90% identity with SEQ ID NO: 1.
  • said nucleic acid encoding the light chain of the Cetuximab antibody has at least 90% identity with SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of the Cetuximab antibody has at least 95% identity with SEQ ID NO: 1
  • said nucleic acid encoding the light chain of the Cetuximab antibody has at least 95% identity with SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of the Cetuximab antibody has at least 95% identity with SEQ ID NO: 1.
  • said nucleic acid encoding the light chain of the Cetuximab antibody has at least 95% identity with SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of a Cetuximab antibody has at least 99% identity with SEQ ID NO: 1, and said nucleic acid encoding the light chain of the Cetuximab antibody has at least 99% identity with SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of a Cetuximab antibody has at least 99% identity with SEQ ID NO: 1.
  • said nucleic acid encoding the light chain of the Cetuximab antibody has at least 99% identity with SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of the Cetuximab antibody has 100% identity with SEQ ID NO: 1
  • said nucleic acid encoding the light chain of the Cetuximab antibody has 100% identity with SEQ ID NO: 2.
  • said nucleic acid encoding the heavy chain of the Cetuximab antibody is SEQ ID NO: 1.
  • said nucleic acid encoding the light chain of the Cetuximab antibody is SEQ ID NO: 2.
  • said Nicotiana is Nicotiana benthamiana or Nicotiana tabacum. More preferably, said Nicotiana is Nicotiana tabacum. Again more preferably, said Nicotiana is Nicotiana tabacum cv. Samsun.
  • Said at least one regulatory element is preferably selected from the group consisting of one or more promoters, enhancers, repressors, silencers, terminators, binding sites for regulatory proteins that promote or inhibit transcription, and matrix attachment region (MAR) or a combination thereof.
  • the regulatory sequence does not have to be one continuous nucleotide sequence, but can composed of more than one nucleotide sequence, optionally present as separate or discontinuous nucleotide sequences in the vector.
  • the regulatory element is selected from a group consisting of a promoter, terminator and matrix attachment region (MAR).
  • each of said nucleic acid sequences encoding a Cetuximab antibody chain is operably linked to at least one regulatory element.
  • each of said nucleic acid sequences encoding a Cetuximab antibody chain is operably linked to at least one regulatory element including a promoter, a terminator and at least one MAR.
  • said vector includes as regulatory element a promoter and a terminator, wherein said promoter and said terminator are from a cauliflower mosaic virus (CaMV) 35S or a ribulose-l,5-bisphosphate carboxylase (rubisco) small subunit.
  • CaMV cauliflower mosaic virus
  • rubisco ribulose-l,5-bisphosphate carboxylase
  • each of said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is operably connected with at least two of said regulatory elements, wherein said at least two regulatory elements comprises a promoter and a terminator, both from a CaMV 35 S or a rubisco small subunit.
  • said vector comprises least one sequence of SEQ ID NO: 1 and/or at least one sequence of SEQ ID NO: 2, wherein each sequence of SEQ ID NO: 1 and each sequence of SEQ ID NO: 2 is operably linked to at least two of said regulatory elements, wherein said at least two regulatory elements are a promoter and a terminator, both from a CaMV 35 S or a rubisco small subunit.
  • said vector includes as regulatory element a plant promoter of a ribulose-l,5-bisphosphate carboxylase (rubisco) small subunit and a plant terminator from a ribulose-l,5-bisphosphate carboxylase (rubisco) small subunit.
  • a plant promoter of a ribulose-l,5-bisphosphate carboxylase (rubisco) small subunit and a plant terminator from a ribulose-l,5-bisphosphate carboxylase (rubisco) small subunit.
  • said vector includes as regulatory element a plant promoter of a ribulose-l,5-bisphosphate carboxylase (rubisco) small subunit.
  • said vector includes as regulatory element a plant terminator from a ribulose- l,5-bisphosphate carboxylase (rubisco) small subunit.
  • rubisco ribulose-l,5-bisphosphate carboxylase
  • each of said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is operably connected with at least two of said regulatory elements, wherein said at least two regulatory elements comprises a promoter and a terminator, both from a rubisco small subunit.
  • said vector comprises least one sequence of SEQ ID NO: 1 and/or at least one sequence of SEQ ID NO: 2, wherein each sequence of SEQ ID NO: 1 and each sequence of SEQ ID NO: 2 is operably linked to at least two of said regulatory elements, wherein said at least two regulatory elements are a promoter and a terminator, both from a rubisco small subunit.
  • said vector includes as regulatory element a plant promoter and a plant terminator, wherein said plant promoter and said plant terminator are both from a rubisco small subunit of Chrysanthemum morifolium Ramat..
  • said vector includes as regulatory element a plant promoter from a rubisco small subunit of Chrysanthemum morifolium Ramat..
  • said vector includes as regulatory element a plant terminator from a rubisco small subunit of Chrysanthemum morifolium Ramat..
  • each of said at least one nucleic acid sequence encoding chain of a Cetuximab antibody is operably connected with at least two of said regulatory elements, wherein said at least two regulatory elements comprises a promoter and a terminator, both from a rubisco small subunit of Chrysanthemum morifolium Ramat..
  • said vector comprises least one sequence of SEQ ID NO: 1 and/or at least one sequence of SEQ ID NO: 2, wherein each sequence of SEQ ID NO: 1 and each sequence of SEQ ID NO: 2 is operably linked with a promoter and a terminator, both from a rubisco small subunit of Chrysanthemum morifolium Ramat. as regulatory elements.
  • said vector includes as regulatory element a plant promoter from a rubisco small subunit of the nucleic acid sequence of SEQ ID NO: 3.
  • SEQ ID NO: 3 Nucleic acid sequence of a plant promoter from a rubisco small subunit: AATT C G ATAT C AC GCTTAG AC AAAC ACC CCTT GTTATAC A AAG AATTT C GCTTTAC A AAAT C AA ATT C G AG A AAATAATATAT GC ACTAAATAAG AT C ATTCC G ATCC AATCTA ACC AATTACGATACGCTTT GGGTAC ACTT GATTTTTGTTT C AGTAGTTAC ATATATCT T GTTTTATAT GCTATCTTTAAGGAT CTT C ACTC AAAGACTATTT GTT GAT GTT CTT GA T GGGGCTCGG AAGATTT G ATAT G ATAC ACT CTAAT CTTTAGG AGATACC AGCC AGGA TTATATTC AGTAAGAC AAT C AAATTATCTTTTC ATTTAA T GG AT G AGC C AG AAT CTCTATAG AAT GATT GC AATCG, AAT GATT GC AATCGAA
  • said at least one regulatory element includes a plant terminator from a rubisco small subunit of the nucleic acid sequence of SEQ ID NO: 4.
  • SEQ ID NO: 4 (Nucleic acid sequence of a plant terminator from a rubisco small subunit):
  • said vector includes as regulatory element a plant promoter of the nucleic acid sequence of SEQ ID NO: 3 and a plant terminator of the nucleic acid sequence of SEQ ID NO: 4.
  • each of said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is operably connected with at least one of said regulatory element, wherein said one regulatory element has the nucleic acid sequence of SEQ ID NO: 3.
  • each of said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is operably connected with at least one of said regulatory elements, wherein said one regulatory element has the nucleic acid sequence of SEQ ID NO: 4.
  • each of said at least one nucleic acid sequence encoding a heavy or light chain of a Cetuximab antibody is operably connected with at least two of said regulatory elements, wherein said two regulatory element have the nucleic acid sequence of SEQ ID NO: 3 and 4.
  • said vector includes as regulatory elements a plant promoter and terminator, both from a ribulose-l ,5-bisphosphate carboxylase (rubisco) small subunit, preferably of Chrysanthemum morifolium Ramat., wherein said promoter is located at the 5’ end of each of said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody and said terminator is located at the 3’ end of each of said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody.
  • a plant promoter and terminator both from a ribulose-l ,5-bisphosphate carboxylase (rubisco) small subunit, preferably of Chrysanthemum morifolium Ramat.
  • said vector comprises least one sequence of SEQ ID NO: 1 and/or at least one sequence of SEQ ID NO: 2, and said vector comprises as regulatory element a plant promoter from a ribulose-l ,5- bisphosphate carboxylase (rubisco) small subunit, preferably of Chrysanthemum morifolium Ramat. located at the 5’ end of each sequence of SEQ ID NO: 1 and each sequence of SEQ ID NO: 2, and said vector comprises as a further regulatory element a plant terminator from a ribulose-l ,5-bisphosphate carboxylase (rubisco) small subunit, preferably of Chrysanthemum morifolium Ramat. located at the 3’ end of each sequence of SEQ ID NO: 1 and each sequence of SEQ ID NO: 2.
  • said vector comprises as regulatory element a plant promoter from a ribulose-l ,5- bisphosphate carboxylase (rubisco) small subunit, preferably of Chrysanthemum morifolium Ramat. located at the
  • the vector of the invention further comprises at least one nucleotide sequence encoding a signal peptide (signal sequence), wherein said signal peptide targets the expressed Cetuximab (light and/or heavy) chain to a cell compartment, provided that said cell compartment is not the endoplasmic reticulum (ER).
  • signal sequence signal sequence
  • the vector of the invention further comprises at least one nucleotide sequence encoding a signal peptide (signal nucleotide sequence), wherein said signal nucleotide sequence is located directly at the 5’ end of each of said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody, wherein said signal peptide targets the expressed Cetuximab antibody sequences to a cell compartment, provided that said cell compartment is not the endoplasmic reticulum (ER).
  • signal nucleotide sequence signal nucleotide sequence
  • one of said at least one signal nucleotide sequence is located directly at the 5’ end of SEQ ID NO: 1 and another or the same signal nucleotide sequence is located directly at the 5’ end of SEQ ID NO: 2, wherein said signal peptide(s) targets the expressed Cetuximab antibody sequences to a cell compartment, provided that said cell compartment is not the endoplasmic reticulum (ER).
  • ER endoplasmic reticulum
  • said signal peptide targets the expressed Cetuximab antibody sequences to a cell compartment selected from the group consisting of cytoplasm, apoplast and vacuole.
  • said signal peptide targets the expressed Cetuximab antibody sequences to the apoplast, i.e. said signal peptide is an apoplast signal peptide.
  • the apoplast is preferably defined as the space outside of the plasma membrane of the plant cell.
  • each of said at least one signal peptide is an apoplast signal peptide.
  • said at least one signal peptide is at least two different apoplast endoglucanases.
  • said apoplast signal peptide is a human or plant apoplast signal peptide. In another preferred embodiment, said apoplast signal peptide is a plant apoplast peptide. In another preferred embodiment, said apoplast signal peptide is selected from the group consisting of an apoplast signal peptide from human, Gossypium and Nicotiana. Preferably said apoplast signal peptide is from Gossypium or Nicotiana, more preferably from Nicotiana tobacco.
  • said vector of the invention further comprises at least two nucleotide sequences encoding a signal peptide (signal sequence), wherein said signal peptide targets the expressed Cetuximab (light and/or heavy) chain to a cell compartment, provided that said cell compartment is not the endoplasmic reticulum (ER), and wherein said at least two signal peptides are different from each other.
  • said vector of the invention further comprises at least a first and a second nucleotide sequence encoding a first and second signal peptide (first and second signal sequence), wherein said first signal sequence encodes a tobacco apoplast signal peptide and said second signal sequence encodes a cotton apoplast signal peptide.
  • said vector of the invention further comprises at least a first and a second nucleotide sequence encoding a first and second signal peptide (first and second signal sequence), wherein said first signal sequence is located 5’ to the nucleic acid sequence encoding a light chain of a Cetuximab antibody and is a tobacco apoplast signal peptide; and said second signal sequence is located 5’ to the nucleic acid sequence encoding a heavy chain of a Cetuximab antibody and is a cotton apoplast signal peptide.
  • said first and second apoplast peptide are different endoglucanases.
  • said first and second apoplast peptide are different endoglucanases from different species.
  • first and second apoplast peptide are different endoglucanases, wherein the first is from cotton and the second is from tobacco.
  • first and second apoplast peptide are different endoglucanases, wherein the first is of SEQ ID NO: 5 and the second is of SEQ ID NO: 6.
  • first and second apoplast peptide are different endoglucanases, wherein the first is of SEQ ID NO: 5 attached to the heavy chain and the second is of SEQ ID NO: 6 attached to the light chain
  • said at least one nucleotide sequence encoding a signal peptide has the nucleotide sequence of SEQ ID NO: 5 or 7. In another preferred embodiment, said at least one nucleotide sequence encoding a signal peptide has the nucleotide sequence of SEQ ID NO: 5. In another preferred embodiment, said at least one nucleotide sequence encoding a signal peptide has the nucleotide sequence of SEQ ID NO: 7. In a further preferred embodiment, said vector comprises one nucleotide sequence encoded by SEQ ID NO: 5 and one nucleotide sequence encoded by SEQ ID NO: 7.
  • SEQ ID NO: 5 Nucleic acid sequence encoding a cotton apoplast signal peptide: ATGGCTAGGAAGTCCCTTATTTTCCCAGTGATCCTTCTCGCCGTGCTCCTTTTTTCT CC ACC AAT CTACT CT GCT GGCC ACGATTAC AGGGAT GCTCTCCGTAAAT CTTCC AT G GCT
  • SEQ ID NO: 6 (Amino acid sequence encoding a cotton apoplast signal peptide): MARKSLIFPVILLAVLLFSPPIYSAGHDYRDALRKSSMA
  • SEQ ID NO: 7 (Nucleic acid sequence of a tobacco apoplast signal peptide):
  • SEQ ID NO: 8 (Amino acid sequence of the tobacco apoplast signal peptide):
  • said vector comprises a signal nucleotide sequence encoded by SEQ ID NO: 5 located directly at the 5’ end of SEQ ID NO: 1 or 2, and a nucleotide sequence encoded by SEQ ID NO: 7, located directly at the 5’ end of SEQ ID NO: 1 or 2.
  • said signal nucleotide sequence encodes an apoplast signal peptide
  • each of said at least one sequence of SEQ ID NO: 1 and each of said at least one sequence of SEQ ID NO: 2 is operably linked to a promoter and a terminator, each from a rubisco small subunit, preferably of Chrysanthemum morifolium Ramat. as regulatory element.
  • said signal nucleotide sequence encoding said apoplast signal peptide is located directly at the 5’ end of each SEQ ID NO: 1 and 2.
  • said vector of the invention does not contain untranslated regions at the 3’ end or 5’ end of the nucleic acid sequences encoding a Cetuximab heavy or light chain.
  • said vector of the invention does not contain any genes involved in glycosylation.
  • said vector of the invention further comprises at least one matrix attachment region (MAR).
  • said at least one MAR is selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7. These MAR have been shown to enhance gene expression in the vector of the invention.
  • said vector of the invention comprises more than one matrix attachment region (MAR) from which at least two, preferably at least three are different from each other.
  • said vector of the invention comprises CHN 50 S/M II, TM6 and Rb7.
  • said vector of the invention comprises two MARs of CHN 50 S/M II, one TM6 and one Rb7.
  • said vector of the invention comprises CHN 50 S/M II of SEQ ID NO: 9, TM6 of SEQ ID NO: 10 and Rb7 of SEQ ID NO: 11.
  • said MAR is selected from the group consisting of a sequence of SEQ ID NO: 9, 10 and 11.
  • SEQ ID NO: 9 (Nucleic acid sequence of CHN 50 S/M II):
  • said at least one regulatory element includes a promoter flanked upstream by a MAR, wherein said MAR is preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7.
  • said vector includes as regulatory element a terminator flanked downstream by a MAR, wherein said MAR is preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7.
  • said vector includes as regulatory element a promoter flanked upstream by a MAR and a terminator flanked downstream by a MAR, wherein said MAR is preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7.
  • each of said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is operably connected with at least two of said regulatory elements; said at least two regulatory elements include (i) a promoter flanked upstream by a MAR, and (ii) a terminator flanked downstream by a MAR.
  • each of said at least one sequence of SEQ ID NO: 1 and (ii) each of said at least one sequence of SEQ ID NO: 2 is operably linked to promoter and a terminator as regulatory elements, said promoter is flanked upstream by a MAR, and said terminator is flanked downstream by a MAR.
  • Said MAR are preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7.
  • each of said at least one nucleic acid sequence encoding a chain of a Cetuximab antibody is operably connected with a promoter and a terminator, both from a rubisco small subunit as regulatory elements; said promoter is flanked upstream by a MAR, and said terminator is flanked downstream by a MAR; said MARs are preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7.
  • each of said at least one sequence of SEQ ID NO: 1 and (ii) each of said at least one sequence of SEQ ID NO: 2 is operably linked to a promoter and a terminator as regulatory elements, both from a rubisco small subunit; said a promoter is flanked upstream by a MAR, and said terminator is flanked downstream by a MAR; said MARs are preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7.
  • the vector of the invention comprises from 5’ to 3’ direction (i.e. in downstream direction): (1) a first MAR; (2) a first regulatory elements defined as a promoter; (3) a signal sequence encoding an apoplast signal peptide, preferably of Gossypium or Nicotiana (4) a sequence encoding a heavy or light chain of a Cetuximab antibody, preferably of SEQ ID NO: 1 or 2; (5) a second regulatory element defined as a terminator; and (6) a second MAR. Said elements of (1) to (6) form an expression cassette.
  • Such an arrangement has the advantage that the expression of the sequence encoding a heavy or light chain of a Cetuximab antibody is at a high level and that the heavy or light chain of the Cetuximab antibody are fully expressed.
  • the vector of the invention comprises from 5’ to 3’: (1) a first of said MAR selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7; (2) a first of said regulatory elements, which is a promoter, wherein said promoter is a rubisco small subunit promoter; (3) said signal sequence encoding an apoplast signal peptide, preferably of Gossypium or Nicotiana (4) said nucleic acid sequence encoding a chain of a Cetuximab antibody, wherein said chain is a heavy or light chain of a Cetuximab antibody, preferably said nucleic acid sequence is SEQ ID NO: 1 or 2; (5) a second of said regulatory elements, which is a terminator, wherein said terminator is a rubisco small subunit terminator; and (6) a second of said MAR selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and R
  • the vector of the invention comprises from 5’ to 3’ (i.e. in downstream direction): (1) a first MAR, wherein said first MAR is preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7; (2) a regulatory elements defined as a first promoter, wherein said first promoter is preferably a rubisco small subunit promoter; (3) a signal sequence encoding an apoplast signal peptide, preferably of Gossypium or Nicotiana (4) one of a sequence encoding a heavy chain or a light chain of a Cetuximab antibody, preferably one of SEQ ID NO: 1 or 2; (5) a further regulatory element defined as a first terminator, wherein said first terminator is preferably a rubisco small subunit terminator; and (6) a second MAR, wherein said second MAR is preferably TM6 or Rb7; (7) a further regulatory elements defined as a second promoter
  • said vector further comprises at least one marker cassette comprising a marker gene and at least one regulatory element for expression in plant cells, wherein said at least one regulatory element is operably linked to said marker gene.
  • said marker gene is a fluorescent protein, excluding green fluorescent proteins, or an antibiotic resistance gene.
  • said at least one regulatory element for expression in plant cells is a plant promoter and a plant terminator.
  • said plant promoter of the marker cassette is a CaMV 35S promoter and a NOS terminator.
  • a preferred marker cassette comprises a red fluorescent marker gene, such as DsRed gene, a CaMV 35S promoter preferably flanked by a MAR and NOS terminator preferably flanked by a MAR, wherein said MAR are preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7. Said marker gene expressed from the marker cassette is useful as selective marker for screening transformed cells.
  • MAR preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7.
  • said vector further comprises a first marker cassette comprising a fluorescent protein, such as DSRed, and a second marker cassette comprising an antibiotic resistance gene, such as a Kanamycin resistance gene; each marker cassette comprises at least one regulatory element for expression in plant cells, wherein each of said at least one regulatory element is operably linked to the marker gene of its marker cassette.
  • said at least one regulatory element for expression in plant cells is a plant promoter flanked upstream by a MAR and plant terminator flanked downstream by a MAR, wherein said MAR is preferably selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7.
  • said marker cassette comprises a sequence of the following SEQ ID NO: 14:
  • SEQ ID NO: 14 is flanked downstream and upstream by a MAR selected from the group consisting of CHN 50 S/M I, CHN 50 S/M II, TM6 and Rb7.
  • the invention relates to a plant cell comprising the vector of the invention, wherein said plant cell is from the genus Nicotiana.
  • said plant cell is from Nicotiana tabacum or Nicotiana benthamiana. Most preferably of said plant cell is from Nicotiana tabacum. In an again more preferred embodiment, said plant cell is from Nicotiana tabacum cv. Samsun.
  • the invention relates to a method for manufacturing a Cetuximab antibody comprising the steps of:
  • said plant cell is from the genus Nicotiana. In another preferred embodiment, said plant cell is from Nicotiana tabacum or Nicotiana benthamiana. More preferably, said plant cell is from Nicotiana tabacum. In an again more preferred embodiment, said plant cell is from Nicotiana tabacum cv. Samsun.
  • Cetuximab antibodies assemble in the presence of the Cetuximab heavy and light chain. Plant cells from the genus Nicotiana are preferred for assembly, especially from Nicotiana tabacum ox Nicotiana benthamiana.
  • said plant cells are transformed with the vector of the invention in advance of the expression step.
  • said vector is transformed into said plant cells, e.g., via electroporation or a calcium chloride based method.
  • said vector is transformed into said plant cells, preferably Nicotiana tabacum, via incubation with bacteria, preferably with agrobacteria, more preferably with Agrobacterium tumefaciens, again more preferably with Agrobacterium tumefaciens strain GV3101.
  • the plant cells are screened for the expressed Cetuximab antibody chains.
  • cell samples such as leaf samples were analyzed with Western blot.
  • the step of purifying the expressed Cetuximab antibody comprises using a protein A column protein purification, followed by column gel filtration; or using a Protein- A- Cellulose-Binding-Domain.
  • the eluted protein can be concentrated and loaded on a gel filtration column in order to remove any protein aggregates that may have formed.
  • the invention relates to a Cetuximab antibody or a chain of a Cetuximab antibody obtainable by the method of the invention.
  • Constructs were designed for expression of the anti-EGFR antibody Cetuximab, as well as two Cetuximab-dsRBD chimeras; one with the dsRBD bound to the light chain of the antibody at the N terminus (Cetuximab-LC-dsRBD), and one with the dsRBD bound to the heavy chain at the C terminus (Cetuximab-HC-dsRBD)
  • the dsRBD was bound to the antibody via a short (Gly 4 Ser) 3 peptide.
  • Cetuximab-DsRed The Cetuximab-DsRed pUC57 vector contains genes encoding the heavy chain and light chain of the Cetuximab antibody, and a gene expressing DsRed - a red fluorescent protein used as a marker for screening. These genes were codon-optimized for tobacco.
  • the heavy and light chains were each flanked by the rubisco small subunit promoter and terminator and fused to an apoplast signal peptide (from either tobacco or cotton) directing them to the apoplast, the space outside of the plant cell plasma membrane.
  • DsRed a red fluorescent protein from reef coral ( Discosoma spp .) was used as a marker gene and flanked by the CaMV 35 S promoter and terminator. Surrounding each expression unit are four matrix attachment regions (MARs), CHN S/M II, TM6 and Rb7 (Fig. 2), which have been shown to enhance gene expression.
  • the recombinant anti-EGFR antibody Cetuximab and the chimeric antibody Cetuximab-dsRBD were designed using Genome Compiler and ordered from General Biosystems, Inc., North Carolina, USA, who also provided codon optimization of the sequence for tobacco.
  • the sequences encoding Cetuximab heavy and light chains were obtained from DrugBank (https://www.drugbank.ca/drugs/DB00002).
  • the Cetuximab-DsRed pUC57 vector was assembled using the Golden Gate assembly method, which enables assembly of multiple inserts into a vector backbone in a one pot reaction using a single type IIS restriction enzyme and T4 DNA ligase.
  • Type IIS restriction enzymes cut outside of their recognition site, leaving a 4 nucleotide overhang. Inserts can be designed with overhangs that allow assembly only in the desired order.
  • the sequence was ordered in seven parts ranging from 1-3 Kbp, and was supplied in pUC57 plasmids (Fig. 3). Each part was flanked with recognition sites for the type IIS restriction enzyme Bsal and 4 nucleotide overhangs allowing them to be assembled in the correct order using the NEB Golden Gate Assembly Tool (https://goldengate.neb.com/editor).
  • Figure 3 depicts the vector map of Cetuximab-DsRed in pUC57 after successful Golden Gate assembly, which was validated with DNA sequencing.
  • Each of the seven sequence parts contained restriction sites, allowing for traditional ligation of the seven parts in case Golden Gate assembly was not successful.
  • the destination vector for Golden Gate assembly was prepared by inserting a short sequence containing two Bsal restriction sites in to a pUC57 vector.
  • the four nucleotide base pair sequences on the outside of the Bsal restriction sites were complimentary to the first and last of the seven sequence parts.
  • the Golden Gate assembly reaction was comprised of 100 ng of the destination vector, each of the seven parts at a 2:1 insertvector molar ratio, 1.5 m ⁇ lOx NEB T4 buffer, 1.5 m ⁇ lOx BSA, 1 m ⁇ NEB T4 ligase (2,000,000 units/ml), 1 m ⁇ Bsal (10,000 units/ml) in a final volume of 15 m ⁇ .
  • the assembly reaction was performed in a thermocycler as follows: (3 min 37°, 4 min 16°)c24, 5 min 50°, 5 min 80°, hold 16°. 5 m ⁇ of the reaction was transformed into competent E. coli, strain DH5a.
  • Cetuximab-dsRBD chimeras The obtained Golden Gate assembly product, Cetuximab-DsRed pUC57 vector, was further modified to create the Cetuximab-dsRBD chimeras.
  • the vector was cut (with Apal and Xhol) to remove the heavy chain stop codon, and then ligated with a fragment encoding a flexible protein linker (Gly 4 Ser)3 and the dsRBD, thus creating Cetuximab-Heavy Chain-dsRBD-DsRed (Fig. 4B).
  • the vector was cut (with Agel and Spel) to remove the light chain, and then ligated with a fragment encoding the dsRBD, linker and light chain, thus creating Cetuximab-dsRBD-Light Chain-DsRed (Fig. 4A).
  • the inserts (Cetuximab-DsRed, Cetuximab-Heavy Chain-dsRBD-DsRed and Cetuximab-dsRBD-Light Chain-DsRed) were then each cut out of the pUC57 with EcoRI and Hindlll and transferred to the multiple cloning site (MCS) of pBINPLUS binary expression vectors (Fig. 5).
  • the T-DNA area of pBINPLUS also contained Kanamycin resistance near the left border, which was used as a selective marker.
  • the MCS was located on the plasmid within the T-DNA (between the left and right T-DNA borders) - the area of DNA that was transferred via agrobacterium mediated transformation to the tobacco.
  • EXAMPLE 2 Agrobacterium Electroporation
  • the pBINPLUS vectors encoding Cetuximab, Cetuximab-LC-dsRBD and Cetuximab- HC-dsRBD were then transformed into Agrobacterium tumefaciens strain GV3101 using electroporation. Due to poor efficiency of DNA isolation in agrobacterium, in order to validate successful transformation, DNA was isolated from the agrobacterium and then re- transformed into E. coli. DNA was then isolated and sequenced from the re-transformed E. coli. Agrobacterium clones with validated plasmid sequences were then used for tobacco transformation.
  • Nicotiana tabacum cv Samsun plants were transformed via agrobacterium mediated tobacco transformation. Leaf pieces from sterile grown tobacco were incubated with the recombinant agrobacterium, and then grown on MS medium plates containing Kanamycin for about 3-5 weeks. Shoots that developed were grown on rooting medium, and plantlets were moved to the greenhouse once roots developed. Leaves were sampled soon after being moved to greenhouse, approximately 8-10 weeks post transformation.
  • sterile Nicotiana tabacum cv. Samsun plants were grown for 6-8 weeks.
  • Each of the three engineered agrobacterium strains was grown in 50 ml LB with 50 pg/ml Kanamycin for approximately 48 hours, on a shaker, at 28°.
  • the bacterial cultures were centrifuged for 10 minutes, 5000 RPM, at room temperature.
  • Tobacco leaves were cut into 1 cm 2 pieces and incubated for 5 minutes with the agrobacterium.
  • Leaf pieces were then plated on Petri dishes with solid MS medium (liquid MS medium with 7 g/l plant agar (Duchefa (cat# P 1001.1000)), containing 0.8 ml/L indole-3 -acetic acid (LAA) and 2 ml/L kinetin. Plates were incubated in the dark at 25° for 48 hours, after which the explants were transferred to new Petri dishes containing selective MS medium (0.8 ml/L IAA, 2 ml/L kinetin, 400 mg/L carbenicillin and 100 mg/L kanamycin).
  • selective MS medium 0.8 ml/L IAA, 2 ml/L kinetin, 400 mg/L carbenicillin and 100 mg/L kanamycin.
  • the light chain was 20 kDa larger due to the addition of the PKR dsRNA binding domain.
  • Western blots using anti-IgG antibody showed a band at ⁇ 50 kDa, for the heavy chain, and a faint band at ⁇ 45 kDa, the molecular weight for the light chain-dsRBD chimera.
  • the band at ⁇ 45 kDa was more prominent.
  • some smaller bands were detected when using the anti-IgG antibody, including a band at ⁇ 25 kDa, the molecular weight of the light chain without the dsRBD.
  • the heavy chain was 20 kDa larger.
  • bands were detected at ⁇ 50 and ⁇ 25 kDa, representing the original heavy and lights chains, but not the chimeric heavy chain-dsRBD (Fig. 8). Because of the addition of the dsRBD, the anti-IgG antibody is obscured from binding the chimeric heavy chain-dsRBD.
  • bands were detected at the expected molecular weight of ⁇ 75 kDa, in addition to a smaller band of ⁇ 65 kDa.
  • the dsRBD of PKR is comprised of two double-stranded RNA binding domains, so the smaller band in Fig. 8B represents a chimeric heavy chain-dsRBD with only one dsRNA binding domain. Thus, the desired chimera was also present.
  • Plant sample preparation for SDS-PAGE and Western Blot analysis In detail, pre-weighed micro fuge tubes were prepared with 100 m ⁇ grinding buffer (100 mM Tris-HCl pH 8, 25 mM NaCl, 10 mM EDTA, 1 mM potassium metabisulfite (PMBS), lx Complete Protease Inhibitor Tablets). Approximately 10 weeks post transformation; four leaf discs ( ⁇ 80 mg) from each plant were clipped with the microfuge tubes lid into the tube, which was then placed on ice and weighed. The samples were ground for 30 seconds with a plastic micro- pestle attached to an overhead stirrer. Samples were centrifuged for 30 minutes at a speed of 14000 RPM at 4° and supernatant was transferred to new tubes for analysis.
  • PMBS potassium metabisulfite
  • a mouse derived anti-PKR primary antibody was incubated with the membrane overnight at 4°, the membrane was washed 3 times for 5 minutes with TBST and then incubated for one hour with a secondary anti-mouse HRP conjugated antibody.
  • a sandwich ELISA was performed using an EGFR coated plate. A positive reaction was observed with the expressed Cetuximab as well as the Cetuximab-LC-dsRBD. A positive reaction was also observed with the Cetuximab HC-dsRBD at a lower intensity. This lower reactivity is most likely due to the difficulty of the detection reagent, an anti-Cetuximab antibody, to bind the chimeric HC-dsRBD.
  • the ELISA was used to assess expression levels of the four highest expressing plants of each construct, based on the Western blot results.
  • a Cetuximab ELISA kit was purchased from SomruBioScience. Prepared plant samples were diluted 1 :100 in assay buffer (for a final dilution of 1 :1000) and 100 m ⁇ of each sample was added to EGFR coated wells. A standard curve was prepared with concentrations of Cetuximab ranging from 0.156 - 10 ng/well. The plate was incubated on a plate shaker at 23° for one hour at 300 RMP. Plate content was discarded and the plate was washed 3 times 3 minutes with 250 m ⁇ wash buffer. 100 m ⁇ detection reagent was added, and the plate was incubated on a plate shaker at 23° for one hour at 300 RMP.
  • Cetuximab-LC- dsRBD, and Cetuximab-HC-dsRBD, maxisorp 96-well plates were coated overnight at 4 °C with 1 pg/ml R-Hum-hEGFR (Extracellular domain active EGFR AA 1-645, product no. ABIN2001843 from Antibodies Online) in 0.1 M Carbonate buffer pH 9.6. The plate was then washed four times with PBS pH 7.4 0.1% v/v Tween-20, and blocked with the same wash buffer including 2% skim milk for 2 h at 37 °C. Following washing, dsRBD-LC was added, with serial 1 :1 dilutions.
  • a calibration curve with Cetuximab ranging from 0.1 pg/ml to 0.002 pg/ml was included.
  • the plate was incubated for 45 min at 37 °C.
  • the plate was washed as above, and incubated with peroxidase-labeled anti-human IgG, diluted 1/200000 in PBS 0.1% tween-20 2% skim milk, for 30 min at 37 °C.
  • the plate was washed as above, and 100 pl of TMB solution was added to each well.
  • the plate was incubated in the dark for 2 min at room temperature, and the reaction was stopped by adding 50 pl of 1M sulfuric acid to each well. Absorbance was recorded at 450 nm, with background correction at 620 nm.
  • Cetuximab-HC-dsRBD did not seem to be able to bind to the protein A beads, since the addition of the dsRBD to the heavy chain obscured protein A-Fc binding. Thus, protein A column purification was carried out only with Cetuximab and Cetuximab-LC- dsRBD.
  • elution buffer (lOOmM citric acid) was incubated with the beads for 5 minutes, followed by centrifugation for 2 minutes at 2500 g and supernatant was collected. The elution step was repeated and supernatants were combined. 37 m ⁇ 1M Tris pH 8.5 was added to elution to restore physiological pH.
  • Cetuximab and Cetuximab-LC-dsRBD plant lysates from high expressing plants were purified on a protein A column using an AKTA design chromatography system. Elution was carried out under acidic conditions, and fractions were collected. Fractions from elution peak were run on SDS-PAGE for Coomassie staining and Western blot analysis. For Cetuximab purification, heavy and light chains were detected in the eluted fractions at ⁇ 50 and ⁇ 25 kDa, respectively (Fig. 10).
  • the eluted protein was concentrated and loaded on a gel filtration column in order to remove any protein aggregates that may have formed. After collecting the protein fractions in the desired buffer, the protein was again concentrated and frozen at -80°C. Protein concentration was measured with a NanoDrop. The concentration of purified Cetuximab was ⁇ l0 mg/kg and the concentration of purified Cetuximab-LC-dsRBD was ⁇ lmg/kg.
  • prepared plant extracts were filtered through a 0.2 pm filter and passed through a HiTrap Protein A High Performance 1 ml column, using an AKTA chromatography system.
  • Sodium phosphate buffer (10 mM sodium phosphate pH 7.4, 150 mM NaCl) was used to wash the column. Elution was performed with 100 mM citric acid, pH 3.
  • Fractions were collected in tubes containing neutralization buffer (Tris pH 9.5) at the volume required to achieve a final pH of 7.4 and analyzed by SDS PAGE. Fractions that contained protein were combined, concentrated, and purified using a gel filtration column. Fractions containing protein were again combined, concentrated, and frozen at -80°C. Protein concentration was measured using a NanoDrop.
  • Protein-A-Cellulose-Binding- Domain the chimeric protein Protein-A-Cellulose-Binding- Domain
  • ProtA-CBD chimeric protein Protein-A-Cellulose-Binding- Domain
  • Protein A which binds specifically to the Fc of IgG molecules, is fused to a cellulose binding domain.
  • Cellulose is commercially available in many forms at a very low price and is thus an attractive matrix for affinity purification.
  • Tobacco plants are cross-bred with high yield of recombinant Cetuximab or Cetuximab-dsRBD with tobacco plants expressing ProtA-CBD. Therefore, in these crossbred plants, the expressed recombinant antibody is bound via the Fc to ProtA-CBD.
  • nitrocellulose is added to the protein extract and filtered through a 0.2 micron ho lo fiber.
  • the ProtA-CBD- Antibody compound binds to the nitrocellulose and is not able to pass through the ho lo fiber, while the other unwanted proteins pass through and are discarded.
  • a series of pH changes and washes cause the recombinant protein to be released from the ProtA-CBD and eluted, ready for use in further experiments.
  • the assembly of the heavy and light chain of the chimeric protein took place in the cells of the expression system used, e.g. in the cells of the tobacco plants.
  • the light chain and heavy chain are expressed separately, then form S-S bonds (both within each heavy and light chain and between the heavy and light chains), and are folded in to the final structure, which is made up of 2 heavy and 2 light chains.
  • This is a process that happens inside the cells, using the cell machinery (Feige and Buchner, Principles and engineering of antibody folding and assembly, Biochimica et Biophysica Acta (BBA) Proteins and Proteomics, 2014, vol. 1844 (11), pp. 2024-2031; Ma et ai, Assembly of monoclonal antibodies with IgGl and IgA heavy chain domains in transgenic tobacco plants, Eur. J. Immunol. 1994. 24: 131-138).
  • an electrophoretic mobility shift assay (EMSA) test is performed. Different amounts of the purified chimera are incubated with dsRNA (i.e. Cy3-labeled polylC or dsRNA of a defined length) and electrophoresed on agarose gel. After visualization of the gel, migration of the dsRNA incubated with the chimera is compared with dsRNA without the chimera. Retarded migration shows the ability of the chimera to bind dsRNA.
  • dsRNA i.e. Cy3-labeled polylC or dsRNA of a defined length
  • Cetuximab chimera with dsRBD attached to the N-terminus of the light chain is able to bind to EGFR.
  • the ability of the chimera to bind EGFR was shown by ELISA, which is performed on plates coated with EGFR.
  • EGFR attached to the plate was followed by Cetuximab or dsRBD-Cetuximab chimera, followed by peroxidase conjugated anti human IgG. Binding of the Cetuximab-dsRBD chimera with polylC and without polylC to EGFR was comparable to binding of commercially available Cetuximab.
  • EGF peroxidase conjugated anti-IgG
  • EGFR over-expressing cells are plated in 96-well plates (5000 cells per well). Medium is changed the next day, and the cells are treated with polylC, Cetuximab, Cetuximab-dsRBD chimera (“CTX-dsRBD”), or polylC which has been pre-incubated with Cetuximab-dsRBD chimera (“polylC/CTX-dsRBD”) at a predetermined ratio. Cell survival is measured with the methylene blue colorimetric assay 72 hours after treatment.
  • CTX-dsRBD in the absence of polylC, will be as effective as Cetuximab (leading to 20-80% survival, depending on the cell line), but polylC/CTX-dsRBD will strongly decrease the survival of cells that are barely affected by Cetuximab, e.g. MDA-MB-468.
  • EGFR over-expressing cells are plated in 6-well plates (500,000 cells per well). Medium is changed the next day, and cells are treated with polylC, Cetuximab, CTX-dsRBD, or polylC/CTX-dsRBD at different concentrations for 48 hours. Cells are dissociated with trypsin and centrifuged for 10 min at 500 g. Cell pellets are washed twice with PBS, and cell membranes are damaged by repeated freeze-thaw cycles in liquid nitrogen. Alternatively, cells can be fixed and permeabilized with ethanol.
  • Cells are incubated with 0.2 ml of ribonuclease A (1 mg/ml) and stained with 0.2 ml of propidium iodide solution (100 pg/ml). Fluorescence of cells is analyzed using flow cytometry, BD FACS ARIAIII (BD Biosciences, USA). Cell cycle distributions are calculated using appropriate software. Various kits are available to simplify the procedure, e.g. Bio Vision’s EZ Cell Cycle Analysis kit.
  • CTX-dsRBD in the absence of polylC, will arrest cells in Gl, as does Cetuximab.
  • polylC/CTX-dsRBD will drive most of the cells into apoptosis, and that the remaining cells may be arrested in Gl .
  • Apoptosis assay EGFR over-expressing cells are plated in 24-well plates (100,000 cells per well). Medium is changed the next day and the cells are treated with polylC, Cetuximab, CTX-dsRBD, or polylC/CTX-dsRBD at a predetermined ratio for 8 hours. Annexin V/Propidium iodide (PI) staining is performed using the MBF MEBCYTO apoptosis kit according to the manufacturer's guidelines and analyzed using flow cytometry, BD FACS ARIAIII (BD Biosciences, USA).
  • PI Propidium iodide
  • CTX-dsRBD in the absence of polylC, will cause low levels of apoptosis ( ⁇ 20 %), but polylC/CTX-dsRBD will drive most of the cells into apoptosis.
  • EGFR over-expressing cells are plated in 6-well plates (500,000 cells per well). Medium is changed the next day and the cells are treated with polylC, Cetuximab-dsRBD, or polylC which has been pre-incubated with Cetuximab-dsRBD at different concentrations. At different time points, the cells are lysed with boiling Faemmli sample buffer (10% glycerol, 50 mmol/F Tris-HCl, pH 6.8, 3% SDS, and 5% 2- mercaptoethanol).
  • Faemmli sample buffer (10% glycerol, 50 mmol/F Tris-HCl, pH 6.8, 3% SDS, and 5% 2- mercaptoethanol.
  • the lysates are subjected to western blot analysis with antibodies against the following: EGFR, phospho-EGFR, ERK, phospho-ERK, CDK2, phospho-pl30, phospho- Rb, p27, BCL2 and Bax, caspase 3, caspase 9 and PARP.
  • EGFR over-expressing cells are plated in 96-well plates (5000 cells per well). Medium is changed the next day and the cells are treated with polylC, Cetuximab-dsRBD, or polylC which has been pre-incubated with Cetuximab-dsRBD at a predetermined ratio. Medium is collected after 24 hours, and Interferon gamma- induced protein 10 (IP 10), chemokine (C-C motif) ligand 5 (CCL5) and tumor necrosis factor alpha (TNFa) proteins are quantified using ABTS ELISA Development Kits (PeproTech) according to the manufacturer’s protocol. Interferon beta (IFN-b) protein is quantified using a bio luminescent ELISA kit (LumiKine) according to the manufacturer’s protocol.
  • IP 10 Interferon gamma- induced protein 10
  • C-C motif chemokine
  • TNFa tumor necrosis factor alpha
  • EGFR over-expressing cells are plated in 6-well plates (500,000 cells per well). Medium is changed the next day and the cells are treated with polylC which has been pre-incubated with Cetuximab-dsRBD at a predetermined ratio. 48 hrs after treatment, 0.5 ml of medium from the transfected cells (“conditioned medium”) is added to 500,000 PBMCs which have been seeded 24 hrs earlier into 24 well plates and grown in 0.5 ml medium. 0.1 ml of medium from the conditioned PBMCs is then exchanged for 0.1 ml medium from additional non-treated EGFR over-expressing cells (“indicator cells”) seeded on 96 well plates 24 hrs earlier. Survival of these cells is determined by methylene blue assay 48 hours after addition of medium from the PBMCs.
  • Direct bystander effects In parallel to previous assay, to show the direct bystander effect, 0.1 ml of conditioned medium is used to replace 0.1 ml medium from non-transfected indicator cells seeded 24 hrs earlier onto 96 well plates and grown in 0.2 ml medium. Survival of these cells is determined 48 hours after addition of the conditioned medium using methylene blue.
  • PBMCs In vitro cancer cell killing by activated PBMCs: 20,000 EGFR-over expressing cells are seeded onto 24 well plates and grown overnight in 1 ml RPMI medium supplemented with 10% FCS and antibiotics. Cells are then treated with polylC which has been pre-incubated with Cetuximab-dsRBD at a predetermined concentration. 24 hrs later 500,000 PBMCs/well are added to the cancer cells and co-incubated for another 24 hrs. Apoptotic cells are visualized using an Annexin-V-Biotin kit (Biosource, Inc.). To distinguish tumor cells from PBMCs, tumor cells are labeled with FITC-conjugated EGFR antibody (Biosource, Inc., green fluorescence).
  • apoptosis can be analyzed by FACS, with gating to ignore the PBMCs.
  • Results When treating EGFR-over-expressing cell lines with Cetuximab-dsRBD- polylC, cell growth will be inhibited, and apoptosis, Gl population, p27 and Bax will be increased, whereas CDK2, phosphor-pl30, phospho-Rb, phospho-EGFR, phosphor-ERK, and BCL2 will be decreased.
  • cytokine and chemokine levels When treating with Cetuximab-dsRBD-polylC, cytokine and chemokine levels will be elevated, and direct as well as PBMC-mediated bystander effects will occur. Due to the synergistic effect of polylC combined with Cetuximab, the effects induced by treatment with Cetuximab-dsRBD-polylC will exceed the effects evoked by Cetuximab monotherapy.
  • EXAMPLE 13 Treatment of HN5 and A431 cells with tobacco expressed Cetuximab 5000 cells/per well are plated in a 96-well plate (100 ul per well). The next day, the medium was replaced with fresh medium, and triplicate wells were treated. Serial dilutions of commercial and plant-expressed Cetuximab (CTX) were prepared xlO, and final concentrations in each well range from 1-8 nM. Viability was assayed using methylene blue after 72h of treatment. Cells were fixed with 25 m ⁇ of 2.5 % glutaraldehyde (diluted from 25% in DDW) per well (final concentration 0.5%).
  • CX commercial and plant-expressed Cetuximab
  • Plates were incubated for 10 min at room temperature in the fume cabinet and then washed 3 times with DDW by dipping the plates face up in a water bath. Plates were blotted well on paper and dried at 50° for a little under an hour. 100 m ⁇ of 1% Methylene Blue (dissolved in 0.1 M Boric Acid pH 8.5 and filtered) was added per well. Plates were incubated 1 h at room temperature and then washed ⁇ 6 times in DDW. Plates were dried for 1 h at 50° and 200 m ⁇ of 0.1 M HC1 were added per well, followed by incubation for 1 h at 37°. Optical density was read with a plate reader at 630 nm.
  • HN5 and A431 cells Viability of HN5 and A431 cells could be shown, quantified by methylene blue assay, 3 days after treatment with commercial Cetuximab or protein-A purified tobacco expressed CTX ( ⁇ 95% pure). Shown are 3 independent experiments for each HN5 and A431 cell line.

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Abstract

La présente invention concerne un vecteur d'expression de chaînes d'anticorps de cétuximab comprenant (i) au moins une séquence d'acide nucléique codant pour une chaîne d'un anticorps de cétuximab, et (ii) au moins un élément régulateur pour l'expression dans des cellules végétales. L'invention concerne en outre une cellule végétale du genre Nicotiana comprenant le vecteur de l'invention ainsi qu'un procédé de fabrication d'un anticorps de cétuximab et l'anticorps cétuximab ou la chaîne d'un anticorps de cétuximab obtenu par le procédé de l'invention.
PCT/EP2019/055928 2018-03-08 2019-03-08 Vecteur pour l'expression de chaînes d'anticorps de cétuximab WO2019170899A1 (fr)

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10894812B1 (en) 2020-09-30 2021-01-19 Alpine Roads, Inc. Recombinant milk proteins
US10947552B1 (en) 2020-09-30 2021-03-16 Alpine Roads, Inc. Recombinant fusion proteins for producing milk proteins in plants
US10988521B1 (en) 2020-09-30 2021-04-27 Alpine Roads, Inc. Recombinant milk proteins
US11034743B1 (en) 2020-09-30 2021-06-15 Alpine Roads, Inc. Recombinant milk proteins
US11072797B1 (en) 2020-09-30 2021-07-27 Alpine Roads, Inc. Recombinant fusion proteins for producing milk proteins in plants
US11142555B1 (en) 2020-09-30 2021-10-12 Nobell Foods, Inc. Recombinant milk proteins
US11401526B2 (en) 2020-09-30 2022-08-02 Nobell Foods, Inc. Recombinant fusion proteins for producing milk proteins in plants
US11685928B2 (en) 2020-09-30 2023-06-27 Nobell Foods, Inc. Recombinant fusion proteins for producing milk proteins in plants
US11840717B2 (en) 2020-09-30 2023-12-12 Nobell Foods, Inc. Host cells comprising a recombinant casein protein and a recombinant kinase protein
US11952606B2 (en) 2020-09-30 2024-04-09 Nobell Foods, Inc. Food compositions comprising recombinant milk proteins
WO2022103797A1 (fr) * 2020-11-10 2022-05-19 Massachusetts Institute Of Technology Protéine modifiée pour capture rapide et efficace d'anticorps spécifiques d'un agent pathogène

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