EP4073256A1 - Production de protéines recombinantes à l'aide de la fah en tant que marqueur de sélection - Google Patents

Production de protéines recombinantes à l'aide de la fah en tant que marqueur de sélection

Info

Publication number
EP4073256A1
EP4073256A1 EP20838382.8A EP20838382A EP4073256A1 EP 4073256 A1 EP4073256 A1 EP 4073256A1 EP 20838382 A EP20838382 A EP 20838382A EP 4073256 A1 EP4073256 A1 EP 4073256A1
Authority
EP
European Patent Office
Prior art keywords
fah
gene construct
gene
production
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.)
Pending
Application number
EP20838382.8A
Other languages
German (de)
English (en)
Inventor
Jonas Füner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Preclinics Gesellschaft fur Praklinische Forschung Mbh
Original Assignee
Preclinics Gesellschaft fur Praklinische Forschung Mbh
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Preclinics Gesellschaft fur Praklinische Forschung Mbh filed Critical Preclinics Gesellschaft fur Praklinische Forschung Mbh
Publication of EP4073256A1 publication Critical patent/EP4073256A1/fr
Pending legal-status Critical Current

Links

Classifications

    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/067—Hepatocytes
    • 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
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • C12N2015/8518—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic expressing industrially exogenous proteins, e.g. for pharmaceutical use, human insulin, blood factors, immunoglobulins, pseudoparticles

Definitions

  • Fumarylacetoacetate hydrolyase (FAH) deficiency is a rare condition called tyrosinemia type 1.
  • the deficiency leads to the fact that the tyrosine metabolites fumarylacetoacetate and maleyl acetoacetate are no longer broken down and thus accumulate and are then converted into the toxic substances succinylacetoacetate and succinylacetone. Toxic concentrations are reached quickly, especially in the hepatocytes.
  • NTBC nitisinone (2- (2-nitro-4-trifluoromethyl-benzoyl) -1, 3-cyclohexanedione
  • NTBC nitisinone
  • Tyrosinemia is a disease in which cell or gene therapy could provide a cure. Therefore, transgenic FAH-deficient animal models were developed to test such treatment methods. These studies have shown that it is possible, by introducing healthy liver cells into the liver of the FAH-knock-out (FAH-ko) animals, to cause the FAH-deficient hepatocytes to die and through the healthy cells be completely replaced. It was also possible, instead of normal, healthy liver cells, to use those that were successfully transferred with the FAH gene. The NTBC treatment was then discontinued and the FAH-deficient hepatocytes died and were completely replaced by the treated cells.
  • FAH-ko FAH-knock-out
  • a further field of application has developed from this.
  • This is a humanized animal model.
  • an animal which has an immunodeficiency in addition to an FAH deficiency it is also possible to implant human hepatocytes and thus to generate an animal model with human liver metabolism. This is a valuable model for some pharmacological testing.
  • this model is also used for the primary cell culture of human hepatocytes.
  • the hepatocytes from the liver of the FAH-k.o. treated with human hepatocytes are then removed. Taken from animal.
  • Antibodies have been obtained from animals for over a hundred years. There are therefore numerous technologies and protocols for the extraction and purification of antibodies from animals. The production of recombinant antibodies in animals is particularly promising because they only lead to toxic effects in the case of reactivity with a protein in the production host. This can be examined in advance and excluded.
  • the prior art also includes EP256082 B, in which a method for expanding human hepatocytes in vivo is described.
  • human hepatocytes are transplanted into FAH-deficient pigs and their expansion is regulated by controlling the administration of NTBC.
  • the animals are used to select agents for the treatment of human liver diseases.
  • the use as a production host for recombinant proteins is not envisaged.
  • the invention relates to a gene construct comprising at least two nucleic acid sequences, one of the nucleic acid sequences coding for FAH and a second nucleic acid sequence coding for a protein to be produced.
  • the special feature of the invention is that the gene constructs for recombinant production of proteins use the FAH gene as a selection marker in addition to the gene of the protein to be produced.
  • amino acid sequence for FAH (SEQ ID NO 1) is preferably as follows:
  • the gene construct of the invention therefore preferably comprises a nucleic acid sequence which codes for SEQ ID NO 1. It is also preferred that the gene construct comprises a nucleic acid sequence which codes for an amino acid sequence which is homologous or functionally analogous to SEQ ID NO 1. Preference is given to at least 80% homology, particularly preferably at least 90% homology, very particularly preferably 95% homology.
  • the nucleic acid sequence coding for SEQ ID N01 can also be present in a codon-optimized manner, depending on the selected production host. The person skilled in the art is able to carry out this codon optimization independently without becoming inventive.
  • a gene for the recombinant protein to be produced is contained in the gene construct.
  • Different promoters can be used.
  • the FAH nucleic acid sequence can be provided with the ubiquitous CMV promoter and the nucleic acid sequence for the recombinant protein with an albumin promoter.
  • the person skilled in the art is able to select a suitable promoter without becoming inventive.
  • the protein to be produced, for which the second nucleic acid sequence codes is preferably a human protein, in particular a human antibody.
  • a human protein in particular a human antibody.
  • these proteins or antibodies can either be found in humans or used in humans.
  • non-human proteins and antibodies can also be of particular interest for certain applications, so that the invention is not restricted thereto.
  • a “protein to be produced” is a protein specifically selected or desired by the user or experimenter.
  • a protein to be produced can be a naturally occurring or an artificial protein, for example a fusion protein.
  • non-limiting examples of such proteins to be produced can be: antibodies, preferably human antibodies, for passive immunization, such as IgG which binds to the RBD of the SARS-CoV-2 spike protein and neutralizes the virus, or IgG which binds to the Fragment C of the tetanus toxin binds and thus prevents the cell entry of the toxin.
  • the protein to be produced can also be antibodies for clinical diagnostics, which have to be obtained on a large scale, such as rabbit antibodies against human immunoglobulins (IgG, IgA, IgM, IgE), or goat antibodies against alpha antitrypsin and further antibodies from different species against, for example, transferrin, ferritin, C-reactive Protein, albumin, various complement factors (C3, C4 C1 inhibitor, etc.).
  • a protein to be produced can also be one of therapeutic, vaccine, agricultural, or veterinary interest.
  • Proteins of therapeutic interest can include, in particular, enzymes, blood derivatives, hormones, lymphokines (interleukins, interferons, TNF, etc.), growth factors, neurotransmitters or their precursors or synthetic enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5 etc.), apolipoproteins (ApoAl, ApoAIV, ApoE etc.), dystrophin or a minidystrophin, tumor suppressor genes (p53, Rb, Rap1A, DCC, k-rev, etc.), coagulation factors VII, VIII, IX, etc., or also all or part of a natural or artificial immunoglobulin (Fab, ScFv, etc.).
  • trophic factors BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5 etc.
  • apolipoproteins Apo
  • the protein to be produced can also be an antigen or immunogen capable of eliciting an immune response in humans or animals, for the production of vaccines.
  • they can be antigen peptides which are specific for bacteria, viruses or tumors.
  • the therapeutic protein to be produced has no or at least no harmful effect in the production host. The prerequisite for this is the lowest possible homology of the protein to be produced to the host proteins.
  • This gene construct is preferably used to be introduced into the hepatocytes of FAH-deficient animals (production host) via gene transfer, e.g. transfection or transduction. This then causes the cells in the liver that received the gene construct to produce the recombinant protein and, when a secretory signal is used, secrete it into the blood. In addition, the cells produce FAH, which can then be used as a selection marker. Cells that produce no or no functioning FAH accumulate the toxic substances succinylacetoacetate and succinylacetone and die. This can only be prevented with balancing substances (e.g. NTBC), which ensure that succinylacetoacetate and succinylacetone are not accumulated.
  • balancing substances e.g. NTBC
  • FAH can be used to select which cells have undergone a gene transfer.
  • Gene transfer refers to the transfer or passing on of one or more genes or genetic material within the gene construct to a eukaryotic cell.
  • the one or more genes are only passed on to a specific cell population (target cell population) or to a tissue, here preferably liver tissue.
  • the selective gene transfer can be promoted by introducing a targeting domain, for example on the virus surface.
  • the nucleic acid sequence coding for the protein to be produced additionally comprises a signal sequence for secretion.
  • the recombinant protein can be secreted into the blood by a secretory signal. The choice of secretory signal depends on the recombinant protein chosen.
  • the gene construct preferably comprises the respective nucleic acid sequences that code for these amino acid sequences:
  • Sequences which are homologous or functionally analogous to the sequences mentioned are also suitable. Preference is given to at least 80% homology, particularly preferably at least 90% homology, very particularly preferably 95% homology. It is accordingly preferred that the gene construct comprises a nucleic acid sequence which codes for such a homologous signal sequence.
  • the gene which codes for the protein to be produced with a secretory signal and the gene which codes for FAH are preferably cloned or inserted into a common vector. Liver cells of the FAH deficient animal are then transfected and / or transduced with this vector.
  • the invention relates to a vector comprising a gene construct according to the invention. It is preferred that the vector is a viral vector, particularly preferably a lentiviral vector.
  • vector and “viral vector”, as used herein, thus preferably relate to virus particles.
  • the invention relates to a plasmid comprising a gene construct according to the invention.
  • the invention relates to a liver cell into which the gene construct according to the invention has been introduced.
  • the liver cell was preferably deficient in FAH before the gene transfer, so that it either lacked the FAH gene or it was mutated and ultimately no FAH could be produced.
  • the invention relates to a transgenic non-human mammal which has received the gene construct according to the invention through a gene transfer and which was an FAH (- / -) animal before the gene transfer.
  • production hosts all non-human mammals are conceivable as production hosts.
  • the animal is preferably a pig.
  • pigs are used as the production host, an advantage arises from the affinity of porcine IgG for protein G and a lack of affinity for protein A, while most immunoglobulin G groups from other species have an affinity for protein A or for proteins A and G.
  • the recombinant antibodies can be purified separately from the porcine IgG by protein A binding.
  • double transgenic production hosts which do not form immunoglobulins can also be used.
  • the animal is a sheep. Sheep are particularly well suited for the production of serum, since plasmapheresis is easier and therefore less stressful for the animals due to the easily accessible vessels.
  • non-human mammals for example mouse, rat, rabbit, rabbit, cattle, horse, camel or others.
  • the use of larger animals has the advantage that the amount of antibodies to be produced is larger. It is preferred that the animal has an immunodeficiency. This has the advantage that an immune reaction against the protein to be produced is less likely.
  • the invention relates to a method for the production of recombinant proteins, preferably antibodies, comprising the following steps: a) provision of a non-human FAH (- / -) mammal as a production host, which is kept under administration of NTBC, b) provision of one according to the invention Gene construct, plasmid, vector and / or a liver cell, c) gene transfer, wherein the nucleic acid of the gene construct, plasmid, vector and / or liver cell from step b is introduced into hepatocytes of the production host, d) reduction or discontinuation of NTBC administration for expansion the FAH positive liver cells, e) Isolation of the recombinant proteins.
  • NTBC another substance that interferes with the breakdown of tyrosine can also be used.
  • shRNAs such as CEHPOBA or other substances that prevent the accumulation of succinylacetoacetate and succinylacetone are possible.
  • NTBC is mentioned in connection with the invention, such an alternative substance can also be used.
  • the gene transfer is preferably a transfection or transduction.
  • the gene transfer it is preferably possible here for the gene transfer to take place by in vivo transfection of the hepatocytes.
  • in vivo transfection of the hepatocytes There are various methods known to the person skilled in the art which are suitable for the invention.
  • hydrodynamic transfection can be used, in which the blood in the liver is displaced by infusion technology and the infusion solution with the vectors is perfused through the liver under increased pressure.
  • specific lipid nanoparticles in which the vectors are packaged and which are thus taken up directly by the hepatocytes.
  • a rather unspecific transfection can also be carried out with systemically or intravenously administered viral vectors, preferably lentiviruses, since it is not disadvantageous if other cell types are also transfected. Due to the selection in the hepatocytes, the majority of the transgenic expression will ultimately take place in the liver in any process.
  • the vectors can also be injected directly into the liver tissue in introduced into the liver. This can also be done percutaneously. In the case of direct injection into the liver tissue, the transfection efficiency can be increased by in vivo electroporation.
  • liver gene transfer be performed on newborn production hosts. This has the advantage, especially with larger animals, that the use of NTBC can be reduced and thus considerable cost savings can be achieved. In all species, however, it is advantageous that this makes it more likely that the production host will develop an immunological tolerance to the recombinant protein to be produced.
  • NTBC discontinuation not occur immediately after gene transfer.
  • NTBC administration is discontinued or reduced depends on both the animal and the gene transfer method. Weaning can take place, for example, one day after the gene transfer or several days later. Sufficient time must have passed for the nucleic acids of the gene construct to integrate into the genome of the liver cells so that FAH can actually be used as a selection marker.
  • NBTC blocks the enzyme hydroxyphenylpöyruvate dioxygenase upstream of FAH and thus prevents the accumulation of hepatotoxic metabolites.
  • the non-transfected cells die when NTBC is withdrawn, while all transfected cells survive due to the co-expression of FAH and colonize the liver tissue.
  • the liver only has hepatocytes, which also produce the recombinant protein. Depending on the species, this process takes different lengths of time, but is usually completed after a few weeks.
  • the method uses animals in which the gene for fumarylacetoacetate hydrolyase (FAH) is missing or defective.
  • FH fumarylacetoacetate hydrolyase
  • the proteins are taken from the blood. Proteins, in particular antibodies, can thus be produced in a very simple and stress-free manner for the animal and then isolated.
  • An advantage of the production of recombinant proteins, in particular recombinant antibodies, in this system is the extensive experience in obtaining proteins and antibodies from the blood. In this case, plasmapheresis can be used in order to obtain as many and long recombinant proteins as possible from each production host.
  • the purification protocols and the systems for immunoglobulin precipitation be used. Purification and further processing on an industrial scale are thus easily possible without creating a high cost aspect.
  • the recombinant proteins are preferably obtained by plasmapheresis. This method is particularly suitable for antibodies, as these are plasma proteins and are therefore in their natural matrix in the plasma.
  • the non-human FAH (- / -) mammal has an immunodeficiency.
  • immunosuppressive treatment such as cyclosporine can be used.
  • this is only preferable in certain cases, as this method leads to significantly higher keeping costs and also to a higher burden on the animal.
  • proteins in particular antibodies
  • proteins can be produced at significantly lower costs and, above all, quickly in large quantities than was previously possible.
  • Such quantities are not possible in cell culture, so that the invention is primarily used for the production of therapeutic antibodies, e.g. in the event of a pandemic - i.e. when particularly large quantities are required quickly.
  • the invention relates to the use of FAH (- / -) non-human mammals for the production of recombinant proteins, in particular antibodies.
  • the FAH (- / -) non-human mammals are immunodeficient at the same time.
  • a gene construct according to the invention is introduced into the FAH (- / -) non-human mammal.
  • the use of the animals according to the invention, in combination with the gene construct, is very suitable for the rapid and economical production of medium-sized to very large amounts of recombinant proteins, in particular antibodies.
  • the invention also relates to a kit comprising a gene construct according to the invention and / or a vector according to the invention and / or a plasmid according to the invention and auxiliaries for gene transfer, preferably for transfection and / or transduction.
  • the advantageous embodiments of the invention have at least one or more of the advantages mentioned.
  • a lentiviral vector For the planned production of a therapeutic antibody, which is directed against the nucleoprotein of influenza virus A, for example, a lentiviral vector is first produced.
  • the plasmid pSMP-anti-NP-FAH (FIG. 1) is a lentiviral expression vector with two promoters EF-1a and PGK.
  • the sequences of the heavy and light chain of the human NP-specific antibody are separated with the sequence for a P2A peptide and cloned under the control of an EF-1a promoter.
  • the FAH sequence is cloned downstream of the PGK promoter.
  • a third generation lentiviral packaging system (with VSV-G) is used together with the expression plasmid pSMP-Anti-NP-FAH for the production of anti-NP-FAH lentiviral vectors.
  • the expression cassettes of anti-NP and FAH are integrated into the genome of the transduced cells.
  • the hepatocytes are selected through the expression of FAH.
  • the anti-NP antibody can now be produced by the selected hepatocytes.
  • the lentiviral vectors are transcutaneously injected into the large lobes of the liver under anesthesia in 3 to 4 week old FAH (- / -) mice (alternatively, the lentiviral vectors can also be injected intravenously).
  • the administration of NTBC (4 mg / ml in the drinking water) is discontinued one day after the transfection, thus starting the selection process.
  • the antibody can be isolated from the serum or liver tissue.
  • the plasmid contains the following sequences:
  • SEQ ID NO 8 codes for the following amino acid sequence:
  • SEQ ID NO 10 codes for the following amino acid sequence:
  • a gene segment of the SEQ ID NO 1 or a functionally analogous homologous sequence preferably 80% homology, particularly preferably 90% homology, very particularly preferably 95% homology.
  • FIG. 1 shows the expression plasmid pSMP-Anti-NP-FAH. It is a lentiviral expression vector with two promoters EF-1a and PGK. The sequences of the heavy and light chain of the human NP-specific antibody (nucleoprotein of influenza virus A) are separated with the sequence for a P2A peptide and cloned under the control of an EF-1a promoter. The FAH sequence is cloned downstream of the PGK promoter.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
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  • Wood Science & Technology (AREA)
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  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
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Abstract

L'invention concerne une construction génique comprenant au moins deux séquences d'acides nucléiques, une des séquences d'acides nucléiques codant pour la FAH et une seconde séquence d'acides nucléiques codant pour une protéine à produire. Il est ainsi possible d'utiliser la FAH comme marqueur de sélection pour la production de protéines recombinantes, en particulier d'anticorps. L'invention concerne en outre des plasmides, des vecteurs ou des hépatocytes, comprenant ladite construction génique. L'invention concerne en outre un procédé de production de protéines recombinantes dans des FAH (-/-) de mammifères non humains faisant appel à ladite la construction génique et à la FAH comme marqueur de sélection.
EP20838382.8A 2019-12-12 2020-12-14 Production de protéines recombinantes à l'aide de la fah en tant que marqueur de sélection Pending EP4073256A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE102019134122 2019-12-12
PCT/EP2020/086028 WO2021116495A1 (fr) 2019-12-12 2020-12-14 Production de protéines recombinantes à l'aide de la fah en tant que marqueur de sélection

Publications (1)

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EP4073256A1 true EP4073256A1 (fr) 2022-10-19

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Family Applications (1)

Application Number Title Priority Date Filing Date
EP20838382.8A Pending EP4073256A1 (fr) 2019-12-12 2020-12-14 Production de protéines recombinantes à l'aide de la fah en tant que marqueur de sélection

Country Status (3)

Country Link
US (1) US20230029363A1 (fr)
EP (1) EP4073256A1 (fr)
WO (1) WO2021116495A1 (fr)

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0237196A1 (fr) 1986-02-14 1987-09-16 Meri-Mate Limited Récipients en matière plastique

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WO2021116495A1 (fr) 2021-06-17
US20230029363A1 (en) 2023-01-26

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