BRPI0813963B1 - NUCLEIC ACID, PLASMID, AND METHODS FOR PRODUCING AND IMPROVING EXPRESSION OF AN IMMUNOGLOBULIN OF THE IgG CLASS IN A MAMMALIAN CELL - Google Patents

Abstract

HEAVY CHAIN MUTANT LEADING TO BETTER IMMUNOGLOBULIN PRODUCTION. The present invention relates to a nucleic acid encoding the amino acid sequence of the C-terminal part of the CH3 domain of an immunoglobulin of the IgA or IgG class or of the C-terminal part of the CH4 domain of an immunoglobulin of the IgE or IgM class, wherein the dipeptide-glycine-lysine comprised in the amino acid sequence of the C-terminal part of the CH3 or CH4 domain is encoded by the ggaaaa nucleic acid, or the ggcaaa nucleic acid, or the gggaaa nucleic acid, or the gggaag acid, or the ggcaag nucleic acid, or the ggaaag nucleic acid.

Description

[0001] The present invention relates to methods and nucleic acids used in the production of immunoglobulins in mammalian cells.

Background of the Invention

[0002] Expression systems for the production of recombinant polypeptides are well known and are reported in the prior art literature. For the production of polypeptides used in pharmaceutical applications, mammalian cells such as CHO cells, BHK cells, NS0 cells, Sp2/0 cells, COS cells, HEK cells, PER.C6® cells and others are used. The nucleic acid encoding the polypeptide is introduced into the cell, for example, on a plasmid, such as an expression plasmid. The essential elements of an expression plasmid are a prokaryotic plasmid propagation unit, for example for Escherichia coli, comprising an origin of replication and a selection marker, a eukaryotic selection marker and one or more expression cassettes for the expression of the nucleic acid(s) of interest, each comprising a promoter, a structural gene and a transcription terminator including a polyadenylation signal. For transient expression in mammalian cells, one can include a mammalian origin of replication, such as the SV40 Ori or OriP. As a promoter, one can select a constitutive or inducible promoter. For optimized transcription, a Kozak sequence can be included in the 5' untranslated region. For mRNA processing, a polyadenylation signal can also be included.

[0003] Proteins and particularly immunoglobulins play an important role in the current medical arsenal. For human application, every pharmaceutical substance must meet different criteria. To ensure the safety of biopharmaceutical agents for humans, substances that would cause serious harm must be particularly removed.

[0004] mRNA splicing is regulated by the occurrence of a splice donor site in combination with a splice acceptor site, which are located at the 5' end and 3' end of an intron, respectively. According to Watson et al. (Watson et al. (Eds), Recombinant DNA: A Short course, Scientific American Books, distributed by W.H. Freeman and Company, New York, New York, USA (1983)) are the 5' splice donor site consensus sequence ag|gtragt (exon|intron) and the 3' splice acceptor site (y)nNcag|g (intron|exon) (r=purine base; y=pyrimidine base; n=integer; N=any natural base).

[0005] In 1980, the first articles were published dealing with the origin of secreted and membrane-bound forms of immunoglobulins. The formation of secreted (sIg) and membrane-bound (mIg) isoforms results from alternative splicing of heavy chain pre-mRNA. In the mIg isoform, a splice donor site in the exon encoding the C-terminal domain of the secreted form (i.e., the CH3 or CH4 domain, respectively) and a splice acceptor site located a distance downstream of it are used to bind the region constant to exons downstream that encodes the transmembrane domain.

[0006] A method for preparing synthetic nucleic acid molecules that are less inappropriate or have undesirable transcriptional characteristics in a particular host cell is reported in WO 2002/016944. In WO 2006/042158, modified nucleic acid molecules are reported to increase the expression of the recombinant protein and/or reduce or eliminate poorly spliced and/or read by-products through the intron.

[0007] Consequently, there is a need for a recombinant production method for immunoglobulins with reduced by-products.

Summary of the Invention

[0008] The present invention comprises, in a first aspect, a nucleic acid that encodes the amino acid sequence of the C-terminal part of the CH3 domain of an immunoglobulin of the IgA or IgG class, or of the C-terminal part of the CH4 domain of an immunoglobulin of the IgE or IgM class, in which the glycine-lysine dipeptide comprised in the primary amino acid sequence of the C-terminal part of the CH3 or CH4 domain is encoded by the nucleic acid ggaaaa, or the nucleic acid ggcaaa, or the nucleic acid gggaaa, or the ggaaag nucleic acid, or the ggcaag nucleic acid, or the gggaag nucleic acid.

[0009] In one embodiment, the nucleic acid according to the invention encodes an amino acid sequence selected from the amino acid sequences SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8. In another embodiment, it is the nucleic acid encoding the glycine-lysine dipeptide preceded by the nucleotide g or a. In another embodiment, the nucleic acid encoding the glycine-lysine dipeptide is the gga-aaa nucleic acid, or the ggcaaa nucleic acid, or the gggaaa nucleic acid.

[00010] The second aspect of the present invention is a plasmid comprising the nucleic acid according to the invention, and the third aspect of the invention is a cell comprising the nucleic acid according to the invention.

[00011] An additional aspect of the invention is a nucleic acid with the nucleotide sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 30, or 31.

[00012] The fifth aspect of the invention is a method for producing an immunoglobulin in a mammalian cell comprising the following steps:

[00013] transfecting a mammalian cell with a nucleic acid encoding an immunoglobulin heavy chain comprising a nucleic acid of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 30, or 31, encoding the C-terminal part of the immunoglobulin heavy chain, a) culturing the transfected mammalian cell under conditions suitable for expression of the immunoglobulin, b) recovering the immunoglobulin from the culture or cell.

[00014] In one embodiment, the mammalian cell is a CHO cell, a BHK cell, an NS0 cell, an Sp2/0 cell, a COS cell, a HEK cell, or a PER.C6® cell. Preferably, the mammalian cell is a CHO cell, or a BHK cell, or a PER.C6® cell. In another embodiment, the mammalian cell is transfected with two nucleic acids, wherein the first nucleic acid comprises an expression cassette encoding an immunoglobulin light chain, and wherein the second nucleic acid comprises an expression cassette encoding an immunoglobulin light chain. an immunoglobulin heavy chain comprising a nucleic acid of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 30, or 31 that encodes the C-terminal part of the immunoglobulin heavy chain.

[00015] The final aspect of the invention is a method for improving the expression of an immunoglobulin in a mammalian cell, wherein the nucleic acid encoding the immunoglobulin heavy chain comprises the ggaaaa nucleic acid, or the ggcaaa nucleic acid, or the gggaaa nucleic acid, or ggaaag nucleic acid, or ggcaag nucleic acid, or gggaag nucleic acid encoding the dipeptide-glycine-lysine contained in the CH3 or CH4 domain.

Detailed Description of the Invention

[00016] The present invention relates to a nucleic acid that encodes the amino acid sequence of the C-terminal part of the CH3 domain of an immunoglobulin of the IgA or IgG class, or of the C-terminal part of the CH4 domain of an immunoglobulin of the class IgE or IgM, in which the dipeptide-glycine-lysine comprised in the amino acid sequence of the C-terminal part of the CH3 or CH4 domain is encoded by the nucleic acid ggaaaa, or the nucleic acid ggcaaa, or the nucleic acid ggga-aa, or the ggaaag nucleic acid, or the ggcaag nucleic acid, or the gggaag nucleic acid, and wherein the nucleic acid encoding the dipeptide-glycine-lysine is optionally preceded by the nucleotide g or the nucleotide a.

[00017] Methods and techniques usable for carrying out the present invention are known to those skilled in the art and are described, for example, in Ausubel, F.M., ed., Current Protocols in Molecular Biology, Volumes I to III (1997), and Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). As known by those skilled in the art, it is possible to use recombinant DNA technology for the production of numerous derivatives of a nucleic acid and/or polypeptide. These derivatives can be, for example, modified at an individual position or at several by substitution, alteration, exchange, deletion or insertion. Modification or derivatization can be, for example, carried out by means of site-directed mutagenesis. These modifications can be easily carried out by those skilled in the art (see, for example, Sambrook, J., et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA). The use of recombinant technology allows those skilled in the art to transform various host cells with heterologous nucleic acid(s). Although the transcription and translation machinery, i.e. expression, of different cells use the same elements, cells belonging to different species may have, among other things, a different so-called codon usage. In this way, identical polypeptides (with respect to amino acid sequence) can be encoded by different nucleic acids. Likewise, due to degeneration of the genetic code, different nucleic acids can encode the same polypeptide.

[00018] A "nucleic acid", as used herein, refers to a polymeric molecule consisting of the individual nucleotides (also called bases) a, c, g, and t (or u in RNA), e.g., for DNA , RNA or modifications thereof. Such a polynucleotide molecule may be a naturally occurring polynucleotide molecule or a synthetic polynucleotide molecule or a combination of one or more naturally occurring polynucleotide molecules with one or more synthetic polynucleotide molecules. Also covered by this definition are naturally occurring polynucleotide molecules in which one or more nucleotides are altered (for example, by mutagenesis), deleted or added. A nucleic acid may be isolated or integrated into another nucleic acid, for example, in an expression cassette, a plasmid, or in the chromosome of a host cell. A nucleic acid is also characterized by its nucleic acid sequence consisting of individual nucleotides.

[00019] For those skilled in the art, procedures and methods for converting an amino acid sequence, for example, from a polypeptide, into the corresponding nucleic acid sequence encoding that amino acid sequence are well known. Consequently, a nucleic acid is characterized by its nucleic acid sequence consisting of individual nucleotides and also by the amino acid sequence of a polypeptide encoded by it.

[00020] The term "plasmid" includes, for example, launcher and expression plasmids, as well as transfection plasmids. The term "vector" is used synonymously for "plasmids" in this application. Typically, a "plasmid" will also comprise an origin of replication (e.g., the ColE1 or oriP origin of replication) and a selection marker (e.g., an ampicillin, kanamycin, tetracycline, or chloramphenicol selection marker). , for replication and selection, respectively, of the plasmid in bacteria.

[00021] An "expression cassette" refers to a construct that contains the necessary regulatory elements, such as a promoter and polyadenylation site, for expression of at least the nucleic acid contained in a cell.

[00022] A "selection marker" is a nucleic acid that allows cells carrying the selection marker to be specifically selected for or against, in the presence of a corresponding selection agent. Typically, a selection marker will confer resistance to a drug or compensate for a metabolic or catabolic defect in the host cell. A selection marker can be positive, negative, or bifunctional. A usable positive selection marker is an antibiotic resistance gene. This selection marker allows the host cell transformed with it to be positively selected in the presence of the corresponding selection agent, for example, the antibiotic. An untransformed host cell is not capable of growing or surviving under the conditions of selective culture, that is, in the presence of the selection agent in the culture. Positive selection markers allow the selection of cells carrying the marker, whereas negative selection markers allow cells carrying the marker to be selectively eliminated. Selection markers used with eukaryotic cells include, for example, the genes for aminoglycoside phosphotransferase (APH), such as the selection markers for hygromycin (hyg), neomycin (neo), and G418, dihydrofolate reductase (DHFR). , thymidine kinase (tk), glutamine synthetase (GS), asparagine synthetase, tryptophan synthetase (indole selection agent), histidinol dehydrogenase (histidinol D selection agent), and nucleic acids that confer resistance to puromycin, bleomycin, phleomycin, chloramphenicol, Zeocin and mycophenolic acid. Additional marker genes are reported, for example, in WO 92/08796 and WO 94/28143.

[00023] The term "expression", as used herein, refers to transcription and/or translation processes that occur within a cell. The level of transcription of a nucleic acid sequence of interest in a cell can be determined based on the amount of corresponding mRNA that is present in the cell. For example, mRNA transcribed from a sequence of interest can be quantified by RT-PCR or by Northern hybridization (see Sambrook et al., 1989, supra). Polypeptides encoded by a nucleic acid of interest can be quantified by various methods, for example, by ELISA, by assaying the biological activity of the polypeptide, or by using assays that are independent of this activity, such as Western blotting or radioimmunoassay, using immunoglobulins. that recognize and bind to the polypeptide (see Sambrook et al., 1989, supra).

[00024] The term "cell" or "host cell" refers to a cell into which a nucleic acid, for example, encoding a heterologous polypeptide, can be or is introduced/transfected. The term "cell" includes both prokaryotic cells, which are used for propagation of plasmids, and eukaryotic cells, which are used for expression of a nucleic acid. Preferably, the eukaryotic cells are mammalian cells. Preferably, the mammalian cell is selected from the group of mammalian cells comprising CHO cells (e.g. CHO K1, CHO DG44), BHK cells, NS0 cells, Sp2/0 cells, HEK 293 cells, HEK 293 EBNA cells, PER cells .C6®, and COS cells. As used herein, the term "cell" includes the present cell and its progeny. Thus, the words "transformant" and "transformed cell" include the present primary cell and cells in cultures derived therefrom, without regard to the number of transfers. It must also be understood that not all offspring may be precisely identical in DNA content, due to deliberate or inadvertent mutations. A variant progeny is included that has the same functional or biological activity screened for the originally transformed cell.

[00025] A "polypeptide" is a polymer consisting of amino acids joined by peptide bonds, produced naturally or synthetically. Polypeptides of less than about 20 amino acid residues may be called "peptides," whereas molecules consisting of two or more polypeptides or comprising a polypeptide of more than 100 amino acid residues may be called "proteins." . A polypeptide may also comprise non-amino acid components, such as carbohydrate groups, metal ions or carboxylic acid esters. Non-amino acid components may be added by the cell in which the polypeptide is expressed and may vary with cell type. Polypeptides are defined here in terms of their main amino acid structure or the nucleic acid that encodes it. Additions, such as carbohydrate groups, are generally not specified, but may nevertheless be present.

[00026] The term "amino acid", as used in this application, designates a group of carboxy-amino acids, which directly or in precursor form can be encoded by a nucleic acid. Individual amino acids are encoded by nucleic acids each consisting of three nucleotides, called codons or base triplets. Each amino acid is encoded by at least one codon. For example, the amino acid glycine can be encoded by each of the four nucleic acids (codons) gga, ggc, ggg, and ggt, whereas the amino acid lysine can only be encoded by the two nucleic acids aaa, and aag. This phenomenon is known as "degeneration of the genetic code." The group of amino acids comprises alanine (three-letter code: ala, one-letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y ), and valine (val, V).

[00027] As used herein, the term "immunoglobulin" designates a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. This definition includes variants such as mutated forms, that is, forms with substitutions, deletions and insertions of one or more amino acids, truncated forms at the N terminus, fused forms, chimeric forms, as well as humanized forms. The recognized immunoglobulin genes include the different constant region genes, as well as the myriad immunoglobulin variable region genes of, for example, primates and rodents. Monoclonal immunoglobulins are preferred. Each of the heavy and light polypeptide chains of an immunoglobulin can comprise a constant region (generally, the carboxy terminal part).

[00028] The term "monoclonal immunoglobulin", as used herein, refers to an immunoglobulin obtained from a population of substantially homogeneous immunoglobulins, that is, the individual immunoglobulins that make up the population are identical, except for possible mutations naturally occurring substances that may be present in small quantities. Monoclonal immunoglobulins are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal immunoglobulin preparations, which include different immunoglobulins directed against different antigenic sites (determinants or epitopes), each monoclonal immunoglobulin is directed against a single antigenic site on the antigen. In addition to their specificity, monoclonal immunoglobulins are advantageous because they can be synthesized without being contaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the immunoglobulin as being obtained from a substantially homogeneous population of immunoglobulins and should not be taken to require production of the immunoglobulin by any particular method.

[00029] "Humanized" forms of non-human immunoglobulins (e.g., from rodents) are chimeric immunoglobulins that contain partial sequences derived from non-human immunoglobulin and human immunoglobulin. In most cases, humanized immunoglobulins are derived from a human immunoglobulin (recipient immunoglobulin), in which residues from a hypervariable region are replaced by residues from a hypervariable region from a non-human species (donor immunoglobulin), such as mouse, rat , rabbit or non-human primate, with the desired specificity and affinity (see, e.g., Morrison, S.L., et al., Proc. Natal. Acad. Sci. USA 81 (1984) 6851-6855; U.S. patent No. 5,202,238; US Patent No. 5,204,244). In some cases, residues from the framework region (FR) of human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized immunoglobulins may comprise additional modifications, for example, amino acid residues that are not found in the recipient immunoglobulin or donor immunoglobulin. These modifications result in variants of that recipient or donor immunoglobulin that are homologous, but not identical, to the corresponding sequence of origin. These modifications are made to further refine the performance of the immunoglobulin. In general, the humanized immunoglobulin will comprise substantially all of at least one and typically two variable domains, wherein all or substantially all of the hypervariable loops correspond to those of a non-human donor immunoglobulin, and all or substantially all of the FRs are of a human receptor immunoglobulin. The humanized immunoglobulin will optionally also comprise at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. Methods for humanizing non-human immunoglobulin have been described in the art. Preferably, a humanized immunoglobulin has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often called "imported" residues, which are typically taken from an "imported" variable domain. Humanization can essentially be carried out according to the method of Winter and collaborators, by replacing the corresponding sequences of a non-human immunoglobulin with hypervariable region sequences. Therefore, these "humanized" immunoglobulins are chimeric immunoglobulins, in which substantially less than one intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized immunoglobulins are typically human immunoglobulins in which some residues of the hypervariable region and possibly some residues of the scaffold region are replaced by residues from analogous sites in immunoglobulins from rodents or non-human primates.

[00030] Depending on the amino acid sequence of the constant region of their heavy chains, immunoglobulins are divided into classes: IgA, IgD, IgE, IgG and IgM. Some of these can be further divided into subclasses (isotypes), for example, IgG1, IgG2, IgG3, and IgG4, IgA1 and IgA2. According to the heavy chain constant regions, the different classes of immunoglobulin heavy chains are designated as a-, δ-, ε-, γ- and μ- chain respectively. The constant region of a heavy chain of a full-length human immunoglobulin of the IgA, IgD, and IgG class consists of a constant domain 1 (herein designated as CH1), a hinge region, a constant domain 2 (CH2) , and a constant domain 3 (CH3). Human immunoglobulins of the IgE and IgM class comprise an additional fourth constant domain (CH4). Furthermore, immunoglobulins of the IgM class are polymers comprising multiple immunoglobulins, for example, five or six, covalently linked by disulfide bonds. The amino acid sequences of the C-terminal constant domain of these different classes of human immunoglobulins are listed in table 1. The first amino acid of SEQ ID NO: 01 to 08 may or may not be present, because this amino acid is encoded by two exons, the that encodes the C-terminal constant domain and the exon that encodes the preceding domain. Table 1: C-terminal amino acid sequence of different classes of immunoglobulins. Glycine-lysine dipeptides are underlined.

[00031] The term "C-terminal part of the CH3 domain of an immunoglobulin of the IgA or IgG class, or C-terminal part of the CH4 domain of an immunoglobulin of the IgE or IgM class" designates the amino acid sequences of a heavy chain of immunoglobulin, which is located at the C-terminal end of a full-length or naturally occurring immunoglobulin heavy chain, whereby the C-terminus of said C-terminal part is identical to the C-terminus of the primary amino acid sequence of the immunoglobulin heavy chain. The term "primary amino acid sequence" designates the amino acid sequence of an immunoglobulin heavy chain after translation of the corresponding mRNA. This primary amino acid sequence can be further modified in the expressing cell after translation of the mRNA, for example, by peptidases that cleave one or more C-terminal amino acids from the primary amino acid sequence. Consequently, the primary amino acid sequence and the secreted amino acid sequence may not be identical, but may differ by a few amino acids at the C terminus. In one embodiment, the C-terminal part comprises at least the C-terminal 100 amino acids of a primary immunoglobulin heavy chain amino acid sequence or, preferably, at least the C-terminal 50 amino acids of a primary amino acid sequence. of immunoglobulin heavy chain or, preferably, at least the C-terminal 20 amino acids of a primary immunoglobulin heavy chain amino acid sequence. In one embodiment, the nucleic acid according to the invention encodes the amino acid sequence of the C-terminal part of the CH3 domain of an immunoglobulin of the IgG class, or of the C-terminal part of the CH4 domain of an immunoglobulin of the IgG class. IgE. In a further embodiment, the nucleic acid according to the invention encodes the amino acid sequence of the C-terminal part of the CH3 domain of an immunoglobulin of the IgG class.

[00032] The amino acid sequences of the C-terminal constant domain of different human immunoglobulins are encoded by corresponding DNA sequences. In the genome, these DNA sequences contain coding (exonic) and non-coding (intronic) sequences. After transcription of DNA into pre-mRNA, pre-mRNA also contains these intronic and exonic sequences. Before translation, non-coding intronic sequences are removed during mRNA processing by splicing the primary mRNA transcript to generate mature mRNA. Splicing of primary mRNA is controlled by a splice donor site in combination with an appropriately spaced splice acceptor site. The splice donor site is located at the 5' end, and the splice acceptor site is located at the 3' end of an intronic sequence, and both are partially removed during the pre-mRNA splicing process.

[00033] The term "appropriately spaced" designates that a splice donor site and a splice acceptor site in a nucleic acid are arranged so that all elements required for the splicing process are available and are in an appropriate position to allow for the splicing process to occur.

[00034] The present invention comprises a nucleic acid that encodes the amino acid sequence of the C-terminal part of the CH3 domain of an immunoglobulin of the IgA or IgG class, or of the C-terminal part of the CH4 domain of an immunoglobulin of the IgE or IgM class. , wherein the dipeptide-glycine-lysine comprised in said amino acid sequence of the C-terminal part of the CH3 or CH4 domain is encoded by the nucleic acid ggaaaa, or the nucleic acid ggcaaa, or the nucleic acid gggaaa.

[00035] It was surprisingly discovered that, with the nucleic acid according to the invention, the formation of undesirable by-products by undesirable splicing events can be reduced.

[00036] The term "dipeptide-glycine-lysine", as used with the present application, designates the peptide comprising in the N-terminal to C-terminal direction the two amino acids glycine and lysine linked by a peptide bond. The term "dipeptide-glycine-lysine", as used in this application, designates a dipeptide moiety of a larger polypeptide or protein, which can be found at the beginning, within or at the end of the larger polypeptide. The amino acid lysine can be encoded by the nucleic acids aaa and aag. Accordingly, it is another embodiment of the present invention that the dipeptide-glycine-lysine comprised in the amino acid sequence of the C-terminal part of the CH3 or CH4 domain of an immunoglobulin heavy chain is encoded by the nucleic acid ggaaag, or the nucleic acid ggcaag, or the gggaag nucleic acid. In another embodiment, the nucleic acid encoding the dipeptide-glycine-lysine is preceded by the nucleotide a or g. ]

[00037] Nucleic acid sequences encoding the C-terminal constant domain of different classes and subclasses of human immunoglobulins are listed in table 2. For the CH3 domain of human IgG1 and IgG2, two variant forms are known. In one embodiment, the nucleic acid encodes a portion of the C-terminal constant domain of an immunoglobulin heavy chain. Table 2: Nucleic acid sequences encoding the C-terminal part of the heavy chain of different classes of human immunoglobulins.

[00038] In one embodiment, the nucleic acid according to the invention encodes an amino acid sequence selected from the amino acid sequences of SEQ ID NO: 1, 3, 4, 5, 6, 7, or 8.

[00039] In one embodiment, the nucleic acid encodes a part of the C-terminal constant domain of an immunoglobulin heavy chain of the IgA, IgE, IgM, or IgG class and is selected from the nucleic acids of SEQ ID NO: 17, 18, 19, 20, 21, 22, or 23, or 30, or 31. In another embodiment, the nucleic acid encodes a part of the C-terminal constant domain of an immunoglobulin heavy chain of the IgA, IgE, IgM class , or IgG and is selected from the nucleic acids of SEQ ID NO: 17, 18, 19, 22, 23, 30, or 31. In a further embodiment, the nucleic acid encodes a portion of the C-terminal constant domain of a heavy chain of immunoglobulin of the IgA, IgE, IgM, or IgG class and is selected from the nucleic acids of SEQ ID NO: 17, 18, 19, 22, or 23.

[00040] Nucleic acids with the nucleotide sequence of SEQ ID NO: 17 to 23 and 30 to 31 are also an aspect of the present invention. In one embodiment, the nucleotide sequences of SEQ ID NO: 17, 18, 19, 22, 23, 30, or 31 are an aspect of the present invention. In another embodiment, the nucleotide sequences of SEQ ID NO: 17, 18, 19, 22, or 23 are an aspect of the present invention.

[00041] In one embodiment, the nucleic acid encodes the C-terminal constant domain of an immunoglobulin of the IgA, IgE, IgM, or IgG class, and comprises a nucleic acid selected from the nucleic acids of SEQ ID NO: 17, 18 , 19, 20, 21, 22, or 23, or 30, or 31, which encodes a part of the C-terminal domain of the immunoglobulin heavy chain. In another embodiment, the nucleic acid encodes the C-terminal constant domain of an immunoglobulin of the IgA, IgE, IgM, or IgG class, and comprises a nucleic acid selected from the nucleic acids of SEQ ID NO: 17, 18, 19, 22, 23, 30, or 31, which encodes a part of the C-terminal domain of the immunoglobulin heavy chain. In a further embodiment, the nucleic acid encodes the C-terminal constant domain of an immunoglobulin of the IgA, IgE, IgM, or IgG class, and comprises a nucleic acid selected from the nucleic acids of SEQ ID NO: 17, 18, 19 , 22, or 23, which encodes a portion of the C-terminal domain of the immunoglobulin heavy chain. Table 3: Nucleic acid sequences according to the invention encoding a part of the C-terminal constant domain amino acid sequence of a heavy chain of immunoglobulins of different classes.

[00042] The nucleic acid according to the invention encodes at least a part of the C-terminal constant domain of an immunoglobulin of the IgA, IgE, IgM or IgG class. The term "C-terminal constant domain" designates the CH3 domain of an immunoglobulin heavy chain of the IgA or IgG class, or the CH4 domain of an immunoglobulin heavy chain of the IgE or IgM class. The term "a part of" means a C-terminal moiety of the C-terminal constant domain of an immunoglobulin heavy chain of the IgA, IgE, IgM, or IgG class, of at least 20 consecutive amino acids, or of at least 50 consecutive amino acids. , or at least 100 consecutive amino acids of the primary amino acid sequence counted from the C terminus toward the N terminus of the immunoglobulin heavy chain.

[00043] The recombinant production of immunoglobulins is well known in the art and is described, for example, in the review articles by Makrides, S.C., Protein Expr. Purif. 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purif. 8 (1996) 271-282; Kaufman, R.J., Mol. Biotechnology. 16 (2000) 151-160 Werner, R.G., Arzneimittelforschung - Drug Research 48 (1998) 870-880.

[00044] For the production of an immunoglobulin comprising an amino acid sequence encoded by a nucleic acid according to the invention, the invention also comprises a method for producing an immunoglobulin in a mammalian cell comprising the following steps: a) transfection of a mammalian cell having a nucleic acid encoding an immunoglobulin heavy chain, wherein a nucleic acid selected from the nucleic acids of SEQ ID NO: 17, 18, 19, 20, 21, 22, or 23, or 30, or 31 encodes a part of the C-terminal domain of the immunoglobulin heavy chain, b) culturing the transfected mammalian cell under conditions suitable for expression of the immunoglobulin, c) recovering the immunoglobulin from the culture or cell.

[00045] The term "under conditions suitable for expression of the immunoglobulin" designates conditions that are used for culturing a mammalian cell that expresses an immunoglobulin and that are known or can be easily determined by those skilled in the art. Those skilled in the art also know that these conditions can vary depending on the type of mammalian cell cultured and the type of immunoglobulin expressed. In general, the mammalian cell is cultured at a temperature, for example, between 20°C and 40°C, and for a period of time sufficient to permit effective production of immunoglobulin protein, for example, for 4 to 28 days. .

[00046] In one embodiment, transfection of a mammalian cell is with a nucleic acid encoding an immunoglobulin heavy chain, wherein a nucleic acid selected from the nucleic acids of SEQ ID NO: 17, 18, 19 , 22, 23, 30, or 31 encodes a part of the C-terminal domain of the immunoglobulin heavy chain. In a further embodiment, transfection of a mammalian cell is with a nucleic acid encoding an immunoglobulin heavy chain, wherein a nucleic acid selected from the nucleic acids of SEQ ID NO: 17, 18, 19, 22, or 23 encodes a part of the C-terminal domain of the immunoglobulin heavy chain. In one embodiment, the mammalian cell is transfected with a single, i.e., nucleic acid comprising an expression cassette encoding an immunoglobulin heavy chain and an expression cassette encoding an immunoglobulin light chain. In another embodiment, the mammalian cell is transfected with two nucleic acids, one comprising an expression cassette that encodes an immunoglobulin light chain, and one comprising an expression cassette that encodes an immunoglobulin heavy chain.

[00047] The immunoglobulin produced with the method according to the invention is, preferably, a heterologous immunoglobulin. The term "heterologous immunoglobulin" designates an immunoglobulin that is not naturally produced by said mammalian cell. The immunoglobulin produced according to the method of the invention is produced by recombinant means. These methods are widely known in the art and comprise the expression of proteins in prokaryotic and eukaryotic cells with subsequent recovery and isolation of the heterologous immunoglobulin, and usually purification to a pharmaceutically acceptable purity. For the production, i.e. expression, of an immunoglobulin, a nucleic acid encoding the light chain and a nucleic acid encoding the heavy chain, comprising the nucleic acid according to the invention, are each inserted into an expression cassette. by standardized methods. Nucleic acids encoding immunoglobulins are readily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of these nucleic acids. The expression cassette can be inserted into an expression plasmid(s), which is then transfected into host cells, which otherwise do not produce immunoglobulins. Expression is carried out in appropriate prokaryotic or eukaryotic host cells, and the immunoglobulin is recovered from the cells after lysis or from the culture supernatant.

[00048] Different methods are well established and widely used for protein recovery and purification, such as affinity chromatography with microbial proteins (e.g., protein A or protein G affinity chromatography), ion exchange chromatography ( e.g., cation exchange resins (carboxymethyl resins), anion exchange resins (amino ethyl resins), and mixed mode exchange resins), thiophilic adsorption chromatography (e.g., with beta-mercaptoethanol and other SH), hydrophobic interaction or aromatic adsorption (e.g., with phenyl-Sepharose, azarenophilic resins, or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., with Ni(II) affinity material and Cu(II)), size exclusion chromatography and electrophoretic methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, M.A. Appl. Biochem. Biotech. 75 (1998) 93-102).

[00049] The present invention also comprises a nucleic acid that encodes an immunoglobulin heavy chain comprising the nucleic acid according to the invention. Furthermore, the invention comprises a plasmid comprising the nucleic acid according to the invention and a cell comprising that plasmid.

[00050] Another aspect of the present invention is a method for improving the expression of an immunoglobulin in a mammalian cell, wherein the nucleic acid encoding the immunoglobulin heavy chain comprises the nucleic acid ggaaaa, or the nucleic acid ggcaaa, or the nucleic acid gggaaa, or the nucleic acid ggaaag, or the nucleic acid ggcaag, or the nucleic acid gggaag, which encodes the di-peptide-glycine-lysine contained in the CH3 or CH4 domain. With this nucleic acid, undesirable splicing events can be reduced or suppressed.

[00051] In one embodiment of the invention, the immunoglobulin heavy chain is an immunoglobulin heavy chain of a human antibody of the IgG4 subclass or an immunoglobulin heavy chain of a human antibody of the IgG1, IgG2 or IgG3 subclass. In one embodiment, the immunoglobulin heavy chain is a human immunoglobulin heavy chain and preferably of the human IgG4 subclass or a mutated immunoglobulin heavy chain of the human IgG1 subclass. In another embodiment, the immunoglobulin heavy chain is of the human IgG1 subclass with the L234A and L235A mutations. In a further embodiment, the immunoglobulin heavy chain is a human IgG4 immunoglobulin heavy chain with the S228P mutation. In one embodiment, the immunoglobulin heavy chain is of the IgG4 subclass or of the IgG1 or IgG2 subclass, with a mutation at L234, L235 and/or D265, and/or contains the PVA236 mutation. In other embodiments, the mutations are S228P, L234A, L235A, L235E and/or PVA236 (PVA236 means the ELLG amino acid sequence (given in the one-letter amino acid code) from amino acid position 233 to 236 of IgG1 or EFLG of IgG4 is replaced by PVA). The mutations S228P of IgG4, and L234A and L235A of IgG1 are preferred (numbering according to Kabat's EU index).

[00052] "Regulatory elements", as used herein, refer to nucleotide sequences present in cis, necessary for transcription and/or translation of the nucleic acid sequence encoding a polypeptide of interest. Transcriptional regulatory elements typically comprise a promoter upstream of the nucleic acid sequence to be expressed, transcription start and stop sites, and a polyadenylation signal sequence. The term "transcription start site" refers to the nucleic acid base in the nucleic acid corresponding to the first nucleic acid incorporated into the primary transcript, i.e., the mRNA precursor; the transcription start site may overlap with the promoter sequence. The term "transcription stop site" refers to a nucleotide sequence typically represented at the 3' end of a gene of interest to be transcribed, which causes RNA polymerase to terminate transcription. The polyadenylation signal sequence, or poly-A addition signal, provides the signal for cleavage at a specific site at the 3' end of eukaryotic mRNA and the post-transcriptional addition into the nucleus of a sequence of about 100-200 adenine nucleotides (polyA tail) to the cleaved 3' end. The polyadenylation signal sequence may include the consensus sequence AATAAA located about 10-30 nucleotides upstream of the cleavage site.

[00053] To produce a secreted polypeptide, the nucleic acid of interest includes a segment of DNA that encodes a leader signal/peptide sequence. The signal sequence directs the newly synthesized polypeptide to and across the ER membrane, where the polypeptide can be routed for secretion. The signal sequence is cleaved by signal peptidases as the protein crosses the ER membrane. As for the function of the signal sequence, recognition by the host cell's secretion machinery is essential. Consequently, the signal sequence used must be recognized by the proteins and enzymes of the host cell's secretion machinery.

[00054] Translation regulatory elements include a translation start (AUG) and a stop (TAA, TAG or TGA) codon. An internal ribosome entry site (IRES) can be included in some constructs.

[00055] "Promoter" refers to a polynucleotide sequence that controls the transcription of a gene/structural gene or nucleic acid sequence to which it is operably linked. A promoter includes signals for binding RNA polymerase and initiating transcription. The promoters used will be functional in the cell type of the host cell in which expression of the selected sequence is considered. A large number of promoters, including constitutive, inducible, and repressible promoters from a variety of different sources, are well known in the art (and identified in databases such as GenBank) and are available as or within cloned polynucleotides (from , for example, depositories such as ATCC, as well as other commercial or individual sources). A "promoter" comprises the nucleotide sequence that directs the transcription of a structural gene. Typically, a promoter is located in the 5' non-coding or non-translated region of a gene, proximal to the transcription start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee, R.E., et al., Mol. Endocrinol. 7 (1993) 551-560), cyclic AMP response (CREs), serum response elements (SREs; Treisman, R., Seminars in Cancer Biol. 1 (1990) 47-58), glucocorticoid response elements (GREs), and binding sites for other factors of transcription, such as CRE/ATF (Treisman, R., Seminars in Cancer Biol. 1 (1990) 47-58; O'Reilly, M.A., et al., J. Biol. Chem. 267 (1992) 19938), AP2 (Ye, J., et al., J. Biol. Chem. 269 (1994) 25728), SP1, cAMP response element binding protein (CREB; Loeken, M.R., Gene Expr. 3 (1993) 253) and octameric factors (see, generally, Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre, F.P. and Rousseau, G.G., Biochem. J .303 (1994) 1-14). If the promoter is an inducible promoter, then the rate of transcription increases in response to an inducing agent. In contrast, the rate of transcription is not regulated by an inducing agent if the promoter is a constitutive promoter. Repressible promoters are also known. For example, the c-fos promoter is specifically activated by the binding of growth hormone to its receptor on the cell surface. Tetracycline (tet)-regulated expression can be achieved by artificial hybrid promoters consisting, for example, of a CMV promoter followed by two Tet operator sites. The Tet repressor binds to the two Tet operator sites and blocks transcription. Upon addition of the inducer tetracycline, the Tet repressor is released from the Tet operator sites, and transcription proceeds (Gossen, M. and Bujard, H. PNAS 89 (1992) 5547-5551). For other inducible promoters, including metallothionein and heat shock promoters, see, for example, Sambrook et al. (supra) and Gossen, M., Curr. Opinion. Biotech. 5 (1994) 516-520. Among the eukaryotic promoters that have been identified as strong promoters for high-level expression are the SV40 early promoter, adenovirus major late promoter, mouse metallothionein-I promoter, Rous sarcoma virus long terminal repeat, Chinese hamster (CHEF-1, see, for example, US patent no. 5,888,809), human EF-1 alpha, ubiquitin, and human cytomegalovirus immediate early promoter (CMV IE).

[00056] The "promoter" can be constitutive or inducible. An enhancer (i.e., a cis-acting DNA element that acts on a promoter to increase transcription) may be required to work in conjunction with the promoter to increase the level of expression obtained from the promoter alone and may be included as a transcriptional regulatory element. Often, the polynucleotide segment containing the promoter will also include enhancer sequences (e.g., CMV or SV40).

[00057] An "enhancer", as used herein, refers to a polynucleotide sequence that enhances the transcription of a gene or coding sequence to which it is operably linked. Unlike promoters, enhancers are relatively independent of orientation and position and have been found to be 5' or 3' (Lusky, M., et al., Mol. Cell Bio., 3 (1983) 1108-1122) with respect to transcription unit, within an intron (Banerji, J., et al., Cell, 33 (1983) 729-740), as well as within the coding sequence itself (Osborne, T.F., et al., Mol. Cell Bio. , 4 (1984) 1293-1305). Consequently, enhancers may be upstream or downstream of the transcription start site or at considerable distances from the promoter, although, in practice, enhancers may be physically and functionally superimposed on promoters. A large number of enhancers, from many different sources, are well known in the art (and identified in databases such as GenBank) and are available as or within cloned polynucleotide sequences (from, for example, depositories such as ATCC , as well as other commercial or individual sources). A number of polynucleotides comprising promoter sequences (such as the commonly used CMV promoter) also comprise enhancer sequences. For example, all of the strong promoters listed above may also contain strong enhancers (see, for example, Bendig, M.M., Genetic Engineering, 7 (Academic Press, 1988) 91-127).

[00058] An "internal ribosome entry site" or "IRES" describes a sequence that functionally promotes translation initiation independently of the 5' gene of the IRES and allows two cistrons (open reading frames) to be translated to from a single transcript in an animal cell. The IRES provides an independent ribosome entry site for translation of the open reading frame immediately downstream (downstream is used here interchangeably with 3') of it. Unlike bacterial mRNA, which can be polycistronic, that is, encoding several different polypeptides that are sequentially translated from mRNAs, most animal cell mRNAs are monocistronic and encode the synthesis of just one protein. With a monocistronic transcript in a eukaryotic cell, translation would begin at the 5' most translation start site, end at the first stop codon, and the transcript would be released from the ribosome, resulting in translation of only the first polypeptide encoded on the mRNA . In a eukaryotic cell, a polycistronic transcript with an IRES operably linked to the second or subsequent open reading frame in the transcript allows sequential translation of this downstream open reading frame to produce the two or more polypeptides encoded by the same transcript. The use of IRES elements in the construction of a vector has been previously described, see, for example, Pelletier, J., et al., Nature 334 (1988) 320-325; Jang, S. K., et al., J. Virol. 63 (1989) 1651- 1660; Davies, M. V., et al., J. Virol. 66 (1992) 1924-1932; Adam, M.A., et al. J. Virol. 65 (1991) 49854990; Morgan, R. A., et al. Nucl. Acids Res. 20 (1992) 1293-1299; Sugimoto, Y, et al. Biotechnology 12 (1994) 694-698; Ramesh, N., et al. Nucl. Acids Res. 24 (1996) 2697-2700; and Mosser, D.D. et al, BioTech-niques 22 (1997) 150-161).

[00059] "Operationally linked" refers to a juxtaposition of two or more components, wherein the components so described are in a relationship that allows them to function in their desired manner. For example, a promoter and/or enhancer are operably linked to a coding sequence if they act in cis to control or modulate transcription of the linked sequence. Generally, but not necessarily, DNA sequences that are "operationally linked" are contiguous and, when necessary to join two protein-coding regions, such as a secretory leader and a polypeptide, contiguous and in phase (reading). However, although an operably linked promoter is generally located upstream of the coding sequence, it is not necessarily contiguous with it. Enhancers do not have to be contiguous. An enhancer is operably linked to a coding sequence if the enhancer increases transcription of the coding sequence. Operably linked enhancers can be located upstream, within or downstream of the coding sequences and at a considerable distance from the promoter. A polyadenylation site is operably linked to a coding sequence if it is located at the downstream end of the coding sequence such that transcription proceeds from the coding sequence to the polyadenylation sequence. A translation stop codon is operably linked to an exonic nucleic acid sequence if it is located at the downstream end (3' end) of the coding sequence such that translation proceeds from the coding sequence to the stop codon and is then terminated . Ligation is carried out by recombinant methods known in the art, for example, using PCR methodology and/or by ligation at convenient restriction sites. If suitable restriction sites do not exist, then synthetic oligonucleotide adapters or linkers are used in accordance with conventional practice.

[00060] "Heterologous DNA" or "heterologous polypeptide" refers to a DNA molecule or a polypeptide or a population of DNA molecules or a population of polypeptides that do not naturally exist within a given host cell. DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA), as long as the host DNA is combined with non-host DNA (i.e., exogenous DNA). For example, a DNA molecule containing a non-host DNA segment that encodes a polypeptide operably linked to a host DNA segment comprising a promoter is considered a heterologous DNA molecule. Conversely, a heterologous DNA molecule may comprise an endogenous structural gene operably linked to an exogenous promoter.

[00061] A peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.

[00062] An "expression plasmid" is a nucleic acid molecule that encodes a protein to be expressed in a host cell. Typically, an expression plasmid comprises a prokaryotic plasmid propagation unit, for example for E. coli, comprising an origin of replication and a selection marker, a eukaryotic selection marker and one or more expression cassettes for expression of the structural gene(s) of interest, each comprising a promoter, a structural gene and a transcription terminator including a polyadenylation signal. Gene expression is normally placed under the control of a promoter, and this structural gene is said to be "operationally linked to" the promoter. Likewise, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

[00063] An "isolated polypeptide" is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrates, lipids or other proteinaceous impurities associated with the polypeptide in nature. Typically, an isolated polypeptide preparation contains the polypeptide in a highly purified form, that is, at least about 80% pure, at least about 90% pure, at least about 95% pure, more than 95% pure , or more than 99% pure. One way to show that a particular protein preparation contains an isolated polypeptide is by the appearance of a single band after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the protein preparation and Coomassie Brilliant Blue staining of the gel. However, the term "isolated" does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.

[00064] "Transcription terminator", as designated in this application, is a DNA sequence 50-750 base pairs in length that gives RNA polymerase the signal to terminate mRNA synthesis. Very efficient (strong) terminators at the 3' end of an expression cassette are advisable to prevent RNA polymerase from reading beyond, particularly when using strong promoters. Inefficient transcription terminators can lead to the formation of an operon-like mRNA, which can be the reason for undesirable gene expression, e.g. plasmid-encoded.

[00065] The following examples, sequence listing and figures are presented to assist the understanding of the present invention, the true scope of which is set out in the attached claims. It must be understood that modifications can be made to the exposed procedures, without departing from the spirit of the invention. Description of Sequences SEQ ID NO: 01 C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgA class immunoglobulin SEQ ID NO: 02 C-terminal part of the amino acid sequence noacids of the heavy chain constant domain (CH3) of an immunoglobulin of the human IgD class SEQ ID NO: 03 C-terminal part of the amino acid sequence of the constant heavy chain domain (CH4) of an immunoglobulin of the class Human IgE SEQ ID NO: 04 C-terminal part of the amino acid sequence of the heavy chain constant domain (CH4) of an immunoglobulin of the human IgM class SEQ ID NO: 05 C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgG1 class immunoglobulin SEQ ID NO: 06 C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of an IgG2 class immunoglobulin human SEQ ID NO: 07 C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgG3 class immunoglobulin SEQ ID NO: 08 C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgG4 class immunoglobulin SEQ ID NO: 09 nucleic acid sequence encoding the C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgG4 immunoglobulin human IgA class SEQ ID NO: 10 nucleic acid sequence encoding the C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgD class immunoglobulin SEQ ID NO: 11 nucleic acid sequence encoding the C-terminal part of the amino acid sequence of the heavy chain constant domain (CH4) of a human IgE class immunoglobulin SEQ ID NO: 12 nucleic acid sequence encoding the C-terminal part of the amino acid sequence of the constant chain domain heavy chain (CH4) of a human IgM class immunoglobulin SEQ ID NO: 13, 28 nucleic acid sequence encoding the C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgG1 class immunoglobulin ( variants 1 and 2) SEQ ID NO: 14, 29 nucleic acid sequence encoding the C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgG2 class immunoglobulin (variants 1 and 2) SEQ ID NO: 15 nucleic acid sequence encoding the C-terminal part of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgG3 class immunoglobulin SEQ ID NO: 16 nucleic acid sequence encoding the C- part terminal of the amino acid sequence of the heavy chain constant domain (CH3) of a human IgG4 class immunoglobulin SEQ ID NO: 17 nucleic acid sequence according to the invention encoding a part of the amino acid sequence of the C-terminal constant domain ( CH3) of an immunoglobulin of the IgA class SEQ ID NO: 18 nucleic acid sequence according to the invention encoding a part of the amino acid sequence of the C-terminal constant domain (CH4) of an immunoglobulin of the IgE class SEQ ID NO: 19 nucleic acid sequence according to the invention that encodes a part of the amino acid sequence of the C-terminal constant domain (CH4) of an immunoglobulin of the IgM class SEQ ID NO: 20, 30 nucleic acid sequence according to the invention that encodes a part of the amino acid sequence of the C-terminal constant domain (CH3) of an immunoglobulin of the IgG1 class (variants 1 and 2) SEQ ID NO: 21, 31 nucleic acid sequence according to the invention encoding a part of the sequence of amino acids of the C-terminal constant domain (CH3) of an IgG2 class immunoglobulin (variants 1 and 2) SEQ ID NO: 22 nucleic acid sequence according to the invention encoding a part of the amino acid sequence of the C-terminal constant domain (CH3) of an immunoglobulin of the IgG3 class SEQ ID NO: 23 nucleic acid sequence according to the invention encoding a part of the amino acid sequence of the C-terminal constant domain (CH3) of an immunoglobulin of the IgG4 class SEQ ID NO: 24 to 27 first nucleic acids used in the examples.

Description of Figures

[00066] Figure 1 Annotated plasmid map of p4831.

[00067] Figure 2 SDS-PAGE and Western blot analysis of antibodies secreted by clone no. 23; a) Coomassie staining, b) Western blot analysis with anti-human immunoglobulin gamma chain antibody coupled to peroxidase, c) Western blot analysis with anti-human immunoglobulin kappa light chain antibody coupled to peroxidase. Lane 1: reference human anti-IFG-1R antibody; lane 2: culture supernatant from CHO-DG44 clone no. 23 comprising the antibody from clone no. 23.

[00068] Figure 3 Annotated plasmid map of p4817.

[00069] Figure 4 Western blot analyzes of immunoglobulins secreted by CHO-DXB11 transfected with p4831 or 4855. Lane 1: CHO cells transfected with p4831, lane 2: CHO cells transfected with p4855.

[00070] Figure 5 Annotated plasmid map of p5031.

[00071] Figure 6 SDS-PAGE analysis of antibodies secreted by HEK-293-EBNA cells after transfection with plasmid p5031 or p5032; Lanes 1 + 2: p5031, lanes 3 + 4: p5032, lanes 5 - 8: anti-human IGF-IR antibody as reference antibody; 5: 0.2 μg, 6: 0.7 μg, 7: 2 μg, 8: 6 μg.

[00072] Figure 7 CHO-DG44 cells transfected with p5032 and selected for stable integration of the plasmid with Methotrexate (MTX). Antibodies from six clones were purified and analyzed by SDS-PAGE and Coomassie staining.

Materials and methods Recombinant DNA techniques

[00073] Standard methods were used to manipulate DNA as described in Sambrook et al., 1989 (supra). All molecular biological reagents were commercially available (if not otherwise noted) and were used according to the manufacturer's instructions.

Determination of nucleic acid sequence

[00074] The DNA sequences were determined by double-strand sequencing carried out at MediGenomix GmbH (Martinsried, Germany).

Analysis of DNA and protein sequences and control of sequence data

[00075] GCG's software package (Genetics Computer Group, Madison, Wisconsin, USA) version 10.2 and Infomax's Vetor NTI Advance suite version 8.0 were used for sequence creation, mapping, analysis, annotation and illustration.

Cell culture techniques

[00076] CHO-DXB11 cells were cultured in MEM alpha medium (Invitrogen Corp., Gibco®, Cat. No.: 22571) with 10% FCS (fetal calf serum obtained from Hyclone, Thermo Fisher Scientific Inc., Cat. No.: SH3007.03). HEK-293-EBNA cells (ATCC no. CRL-10852) were cultured in DMEM, supplemented with 2 mM glutamine (Gibco®, Cat. No.: 25030), 1% (v/v) non-essential amino acids MEM (Gibco®, Cat. No.: 11140), 10% (v/v) ultra-low IgG FCS (Gibco®, Cat. No.: 16250), and 250 μg/mL G418 (Roche Applied Sciences, Roche Diagnostics GmbH, Germany, Cat. No.: 1464981). The medium for culturing CHO-DG44 cells was MEM alpha medium (Gibco®, Cat. No.: 22561) supplemented with 10% (v/v) dialyzed FCS (Gibco®, Cat. No.: 26400 ) and 2% (v/v) HT supplement (Gibco®, Cat. No.: 41065). For the selection of stably transfected CHO DG44 cell lines, the HT supplement was omitted, and 20 to 500 nM of Methotrexate (MTX) were added, alone or in combination with 400 μg/mL of Hygromycin B (Roche Diagnostics GmbH, Roche Applied Sciences, Germany, Cat. No.: 10843555001). All cell lines were maintained in humidified incubators at 37°C with 5% CO2. Cell transfection was performed by nucleofection (Amaxa GmbH, Germany) or by lipofection using FuGENE 6 (Roche Diagnostics GmbH, Roche Applied Sciences, Germany, Cat. No.: 1815075).

[00077] In addition, standardized cell culture techniques were applied as described, for example, in Bonifacino, J. S., et al. (Eds) (2000) Current Protocols in Cell Biology, John Wiley and Sons, Inc.

Precipitation with Protein A, SDS-PAGE and Western Blot

[00078] Immunoglobulins from cell culture supernatants were precipitated with Protein A-Sepharose beads and then analyzed by SDS/polyacrylamide gel electrophoresis (sodium dodecyl sulfate, SDS-PAGE) and Western-blotting.

[00079] For immunoglobulin precipitation, cell culture supernatants containing up to 7 μg of immunoglobulin were diluted with TBS buffer (50 mM TRIS/HCl, pH 7.5, supplemented with 150 mM NaCl), 1% (v/ v) of Nonidet-P40 (Roche Diagnostics GmbH, Roche Applied Sciences, Cat. No.: 1754599) to a final volume of 1 mL, and thereafter incubated for one hour with 15 μL liquid volume of Protein A globules -Sepharose. The globules were recovered by centrifugation and washed with TBS with 1% (v/v) Nonidet P-40, thereafter with PBS (phosphate buffered saline) concentrated 2 times and finally with 100 mM sodium citrate buffer, pH 5. After the final washing step, the supernatant was completely removed from the globules. Bound immunoglobulins were eluted with 20 μL of 2-fold concentrated LDS (lithium dodecyl sulfate) sample buffer (Invitrogen Corp.) containing 50 mM DTT (dithiothreitol). After 5 minutes of incubation at 95°C, the suspension was centrifuged, and the supernatant was recovered for further analysis.

[00080] SDS-PAGE: SDS-PAGE was performed using the NuPAGE® gel system (Invitrogen Corp.) according to the manufacturer's recommendations. Samples were loaded onto 10% NuPAGE® Novex Bis/TRIS gels (Invitrogen Corp., Cat. No.: NP0301), and proteins were separated in reducing NuPAGE® MES SDS operating buffer (4-morpholinoethanesulfonic acid/ sodium dodecyl sulfate). Typically, 2 to 3 μg of immunoglobulin per lane was loaded for Coomassie staining with Simply Blue Safe Stain® (Invitrogen Corp.) and 0.4 to 0.6 μg for Western-blotting.

[00081] Western Blot: For the electrotransfer of SDS/polyacrylamide gel proteins, standardized PVDF (polyvinylidene difluoride) or nitrocellulose membranes were used. After electrotransfer, the membranes were washed in TBS (Tris-buffered saline, 50 mM TRIS/HCl, pH 7.5, 150 mM NaCl). Non-specific binding sites were blocked by incubation in TBS with 1% (w/v) Western Blocking Reagent (Roche Diagnostics GmbH, Roche Applied Sciences, Cat. No.: 11921673001). Human immunoglobulin gamma heavy chains and kappa light chains were detected with peroxidase-coupled polyclonal detection antibodies (see the following paragraph for details) diluted in TBS with 0.5% (w/v) Western Blocking Reagent. . After three TBS wash steps with 0.05% (v/v) Tween® 20 and one TBS wash step, bound peroxidase-coupled detection antibodies were detected by chemiluminescence using LumiLightPlus substrate solution (Roche Diagnostics GmbH , Roche Applied Sciences, Cat. No.: 12015196001) and the LUMI-Imager F1 analyzer (Roche Diagnostics GmbH, Roche Applied Sciences).

[00082] Gamma heavy chains (H) and kappa light chains (L) of human immunoglobulin were detected simultaneously or separately. For simultaneous detection, peroxidase-coupled goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch Laboratories Inc., Cat. No.: 109-035-088) was used at a dilution of 1:2500 (v /v). For consecutive detection, membranes were first probed with peroxidase-coupled rabbit anti-human Ig gamma antibody (DAKO GmbH, Germany, code no. P0214) at a dilution of 1:1000 (v/v) or anti-Fc antibody. human gamma goat F(ab')2 coupled to peroxidase (Jackson ImmunoResearch Laboratories, Cat. No.: 109-036-008) at a dilution of 1:7500 (v/v). After detection of the immunoglobulin gamma heavy chain, the membranes were distilled for 30 minutes in 62.5 mM TRIS/HCl, pH 6.7, supplemented with 2% (w/v) SDS and 100 mM β-mercaptoethanol, at 50°C. For the second detection, the membranes were again probed with peroxidase-coupled rabbit anti-human Ig kappa antibody (DA-KO GmbH, code no. P0129) at a dilution of 1:1000 (v/v).

Example 1 Preparation of an expression plasmid for an immunoglobulin of the IgG1 class

[00083] Plasmid 4831 (designated as p4831 below) is the expression plasmid for the expression of an anti-IGF-1R antibody (genomically organized expression cassette with retained exon-intron organization) in eukaryotic cells (for the sequences, see, for example, U.S. patent no. 2005/0008642, or European patent 1,646,720). It comprises the following functional elements: - an origin of replication derived from the pUC18 vector (pUC origin), - a β(beta)-lactamase gene that confers resistance to ampicillin in E. coli (Amp), - an expression cassette for the expression of a gamma 1 heavy chain comprising the following elements - the human cytomegalovirus major immediate-early promoter and enhancer (hCMV IE1 promoter), - a synthetic 5'UTR including a Kozak sequence, - a heavy chain signal sequence of murine immunoglobulin including the signal sequence intron (L1_Intron_L2), - the cDNA for a heavy chain variable region (VH) arranged with a splice donor site at the 3' end, - the mouse immunoglobulin μ enhancer region , - the human immunoglobulin gamma 1 heavy chain gene (IGHG1) including the CH1, Joint, CH2 and CH3 exons, intervening introns and the 3'UTR carrying the polyadenylation signal sequence and optionally containing mutations, - a cassette of expression for the expression of a kappa light chain comprising the following elements - the human cytomegalovirus major immediate-early promoter and enhancer (hCMV IE1 promoter), - a synthetic 5'UTR including a Kozak sequence, - a signal sequence of murine immunoglobulin heavy chain including the signal sequence intron (L1_Intron_L2), - the cDNA for a light chain variable region arranged with a splice donor site at the 3' end (VL), - the Ig enhancer region - intronic mouse kappa, - the human immunoglobulin kappa (IGK) gene including the IGKC exon and the IGK 3'UTR that carries the polyadenylation signal sequence. - a Hygromycin B phosphotransferase transcription unit suitable for selection in eukaryotic cells including - the SV40 early promoter and origin, - the hygromycin B phosphotransferase (HygB) coding sequence, - the SV40 early polyadenylation signal - a expression cassette for the expression of murine dihydrofolate reductase (DHFR) suitable for auxotrophic selection in eukaryotic cells including - a shortened version of the early promoter and the origin of SV40, - the coding sequence for murine DHFR, - the early polyadenylation signal of SV40. An annotated plasmid map of p4831 is shown in Figure 1.

[00084] P4831 was transfected into CHO-DG44 cells, and stable cell lines were isolated after selection with Hygromycin B and Methotrexate (MTX). The antibodies secreted by the selected clone no. 23 were precipitated with Protein A-Sepharose beads and analyzed by SDS-PAGE and Coomassie staining (figure 2 a)). In addition to the expected 50 kDa gamma-1 immunoglobulin heavy chain and 25 kDa kappa immunoglobulin light chain, considerable amounts of an 80 kDa protein byproduct were detected. This protein was recognized by antibodies anti-human immunoglobulin gamma chain (figure 2 b)), as well as by antibodies anti-human immunoglobulin kappa chain (figure 2 c)).

Example 2 Preparation of an expression plasmid for an immunoglobulin of the IgG1 class with a modified CH3 domain

[00085] To avoid the generation of by-products resulting from aberrantly spliced gamma 1 pre-mRNA, the internal splice site of the CH3 exon of p4831 was destroyed by mutating the T at position 4573 to C. At the same time, T4567 was replaced with C to remove a BsmA I restriction site.

[00086] P4855 was constructed as follows. P4817, an ancestral plasmid of p4831 with the same gamma 1 heavy chain transcription unit, is composed of the following elements: - an origin of replication of the pUC18 vector that allows replication of this plasmid in E. coli (pUC Ori) - a gene of beta-lactamase that confers resistance to ampicillin in E. coli (Amp) - an expression cassette for the expression of a gamma 1 heavy chain comprising the following elements - the major immediate-early promoter and enhancer of human cytomegalovirus (hCMV IE1 promoter), - a synthetic 5'UTR including a Kozak sequence, - a murine immunoglobulin heavy chain signal sequence including the signal sequence intron (L1_Intron_L2), - the cDNA for a chain variable region heavy (VH) arranged with a splice donor site at the 3' end, - the mouse immunoglobulin μ enhancer region, - the human immunoglobulin gamma 1 heavy chain (IGHG1) gene including exons CH1, Joint, CH2 and CH3 , intervening introns and the 3'UTR carrying the polyadenylation signal sequence and optionally containing mutations. The plasmid map of p4817 is shown in Figure 3. P4817 was manipulated by site-directed mutagenesis using the site-directed mutagenesis kit. QuickChange site (Stratagene, Cat. No.: 200518) and sequence-specific oligonucleotides 1 agcctctccc tgtccccggg caaatgagtg cgacggccg SEQ ID NO: 24 and 2 cggccgtcgc actcatttgc ccggggacag ggagaggct SEQ ID NO: 25.

[00087] The 839 bp SfiI/SgrAI fragment of the mutated p4817 was excised and ligated with the 13133 bp SgrAI/SfiI fragment of p4831 to form p4855.

Example 3 Expression of nucleic acids according to examples 1 and 2, isolation of the immunoglobulin produced, and analysis of the immunoglobulin produced

[00088] Plasmids p4831 and p4855 were transiently transfected into CHO-DXB11 cells. Cells were cultured under non-selective conditions. After three days of cultivation, cell culture supernatants were harvested, and secreted immunoglobulins were purified with Protein A-Sepharose beads. Western blot analysis of immunoglobulins with anti-human IgG antibodies (H+L) showed that the 80 kDa byproduct had been expressed by cells transfected with p4831, but not by cells transfected with p4855 (figure 4).

Example 4 Preparation of an expression plasmid for an immunoglobulin of the IgG4 class

[00089] Plasmid 5031 was designed for transient and stable expression of human anti-P-selectin antibody in eukaryotic cells in tissue culture. For exemplary anti-P-selectin antibodies, see, for example, European patent 1,737,891 or U.S. patent no. 2005/0226876. P5031 is composed of the following elements - an origin of replication of the pUC18 vector that allows the replication of this plasmid in E. coli (pUC origin), - a β-lactamase gene that confers resistance to ampicillin in E. coli (Amp) , - an expression cassette for the expression of a human gamma 4 heavy chain comprising the following elements: - the human cytomegalovirus major immediate-early promoter and enhancer (hCMV IE1 promoter), - a synthetic 5'UTR including a sequence Kozak, - a murine immunoglobulin heavy chain signal sequence including the signal sequence intron (L1_Intron_L2), - the cDNA for a heavy chain variable region (VH) arranged with a splice donor site at the 3' end , - the μ enhancer region of mouse Ig, - the heavy chain of human immunoglobulin gene gamma 4 (IGHG4) including the CH1, Joint, CH2 and CH3 exons, intervening introns and the 3'UTR carrying the polyade signal sequence - nilation and optionally containing mutations, - an expression cassette for expression of the human kappa light chain comprising the following elements - the human cytomegalovirus major immediate-early promoter and enhancer (hCMV IE1 promoter), - a synthetic 5'UTR including a Kozak sequence, - a murine immunoglobulin heavy chain signal sequence including the signal sequence intron (L1_Intron_L2), - the cDNA for a light chain variable region arranged with a splice donor site at the 3' end (VL), - the intronic mouse Ig-kappa enhancer region, - the human immunoglobulin kappa (IGK) gene including the IGKC exon and the IGK 3'UTR that carries the polyadenylation signal sequence, - a Hygromycin B phosphotransferase transcription unit suitable for selection in eukaryotic cells including - the SV40 early promoter and origin, - the hygromycin B phosphotransferase (HygB) coding sequence, - the SV40 early polyadenylation signal, - a expression cassette for the expression of murine dihydrofolate reductase (DHFR) suitable for auxotrophic selection in eukaryotic cells including - a shortened version of the early promoter and the origin of SV40, - the coding sequence for murine DHFR, - the early polyadenylation signal of SV40. A plasmid map of p5031 is shown in Figure 5.

[00090] When HEK-293-EBNA cells were transfected with the p5031 plasmid, the cells produced immunoglobulins comprising an 80 kDa byproduct (Figure 6, lanes 1 and 2). This protein was bound by an anti-human Ig gamma antibody, as well as by an anti-human Ig kappa antibody in Western-Blot analysis.

Example 5 Preparation of an expression plasmid for an immunoglobulin of the IgG4 class with a modified CH3 domain

[00091] The modification was introduced according to example 3. In summary, T4565 was mutated to C along with a second nucleotide, T4559, which was also changed to C to remove a BsmAI restriction site. Oligonucleotide 3 gcctctccct gtccctgggc aaatgagtgc cagg SEQ ID NO: 26 and oligonucleotide 4 cctggcactc atttgcccag ggacagggag aggc SEQ ID NO: 27 were used for site-directed mutagenesis. The plasmid obtained was called p5032.

Example 6 Expression of nucleic acids according to example 5, isolation of the produced immunoglobulin, and analysis of the produced immunoglobulin

[00092] Plasmids p5031 and p5032 were transiently transfected into HEK-293-EBNA cells. Cells were cultured under non-selective conditions. After three days of cultivation, cell culture supernatants were harvested, and secreted immunoglobulins were purified with Protein A-Sepharose beads. Western blot analysis of immunoglobulins with anti-human IgG antibodies (H+L) showed that cells transfected with p5031 expressed an 80 kDa byproduct (Figure 6, lanes 1+2), whereas no byproduct was expressed by cells transfected with p5032 (Figure 6, lanes 3+4).

[00093] The p5032 plasmid was transfected into HEK-293-EBNA cells, the 80 kDa protein was not expressed (figure 6, lanes 3 and 4). This clearly demonstrates that the 80 kDa fusion protein is the result of aberrant pre-mRNA splicing and that production of this undesirable protein during transient expression can be efficiently suppressed by mutation of the internal CH3 splice site of the immunoglobulin heavy chain gene. range 4 (IGHG4).

[00094] Mutation of the internal CH3 splice site of IGHG4 prevented the expression of 80 kDa also in stable cell lines. CHO-DG44 cells were transfected with p5032 and selected for stable plasmid integration with Methotrexate (MTX). Antibodies from the 6 clones were purified and analyzed by SDS-PAGE and Coomassie staining (figure 7). None of the antibodies contained the 80 kDa subunit.

Claims (9)

1. Nucleic acid encoding a heavy chain of a humanized immunoglobulin of an immunoglobulin of the IgG1 and IgG4 class comprising a nucleic acid encoding the amino acid sequence of the C-terminal part of the CH3 domain, characterized by the fact that the glycine-lysine dipeptide comprised in said amino acid sequence of the C-terminal part of the CH3 domain is encoded by the nucleic acid ggcaaa; wherein said nucleic acid encoding the amino acid sequence of the C-terminal part of the CH3 domain preserves the genomic architecture of the organization of exons and introns; and wherein said nucleic acid is selected from the nucleic acids of SEQ ID NO: 20, 23 or 30. 2. Nucleic acid according to claim 1, characterized by the fact that the nucleic acid encoding said glycine-lysine dipeptide is preceded by the nucleotide g or a. 3. Plasmid, characterized in that it comprises the nucleic acid as defined in claim 1 or 2, and a prokaryotic plasmid propagation unit comprising an origin of replication and a selection marker. 4. Method for producing an immunoglobulin of the IgG class in a mammalian cell, characterized in that it comprises the following steps: (a) transfecting said mammalian cell with the nucleic acid encoding an immunoglobulin heavy chain, as defined in claim 1 or 2, (b) culturing the transfected mammalian cell under conditions suitable for expression of the immunoglobulin, (c) recovering the immunoglobulin from the culture or cell. 5. Method according to claim 4, characterized by the fact that said mammalian cell is transfected with a nucleic acid comprising an expression cassette encoding an immunoglobulin heavy chain and an expression cassette encoding an immunoglobulin light chain. immunoglobulin. 6. Method according to claim 4, characterized by the fact that said mammalian cell is transfected with two nucleic acids, wherein the first nucleic acid comprises an expression cassette encoding an immunoglobulin light chain, and the second nucleic acid comprises an expression cassette encoding an immunoglobulin heavy chain. 7. Method according to any one of claims 4 to 6, characterized by the fact that said transfection of said mammalian cell is with a nucleic acid encoding an immunoglobulin heavy chain, wherein a nucleic acid of SEQ ID NO: 23 or 30, encodes the C-terminal part of the immunoglobulin heavy chain. 8. Method according to claim 7, characterized by the fact that said transfection of said mammalian cell is with a nucleic acid encoding an immunoglobulin heavy chain, wherein a nucleic acid of SEQ ID NO: 23 encodes the C-terminal part of the immunoglobulin heavy chain. 9. Method for improving the expression of an immunoglobulin of the IgG class in a mammalian cell, characterized by the fact that the nucleic acid encoding the immunoglobulin heavy chain comprises the nucleic acid ggcaaa, which encodes the glycol dipeptide kine-lysine contained in the CH3 domain of the immunoglobulin heavy chain; and wherein said nucleic acid is selected from the nucleic acids of SEQ ID NO: 20, 23 or 30.