Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the current invention relates to a new form of a recombinant secretory component (rSC), obtainable from a recombinant Chinese Hamster Ovary (CHO) cell line, that can be crystallized and, hence, is susceptible to systematic studies of its 3-dimensional structure.
• the crystallisable form may be used directly (e.g. to stabilize immunoglobulins) or indirectly (e.g. via the derived structure) for lead finding, screening and binding studies.
• the secretory component is synthesized as the extracellular part of an integral membrane glycoprotein of specific cells which are responsible for the transport of polymeric immunoglobu' i n (Ig) to external mucosal surfaces.
• Such cells include epithelial cells found in several tissues including those in the lining of the respiratory, gastrointestinal, biliary and urogenital tracts and in the salivary, lacrimal, and mammalian gland and also hepatocytes.
• This membrane protein is termed polymeric immunogobulin receptor (plgR) and specifically binds polymeric IgA (poly-lgA) and IgM (poly-lgM) on the basal side of the cells.
• the plgR During transport of the plgR/poly-lg complex from the basal to the apical (extracorporal) side of the cell, the plgR is cleaved thus forming the SC. Most of the SC is released from the cell as part of the SC/poly-lg complex, but also free SC is found in external secretions. In the SC/poly-lg complex, SC is thought to stabilize the quaternary structure of poly-lg and to increase resistance of the complex to various proteolytic enzymes. This resistance, for example to digestive proteases, is an important if not essential prerequisite for the protective function of secretory immunoglobulins.
• plgR mediates the transport of IgA and IgM into mucosal secretions.
• mucosal antibodies are of enormous importance in the immune response. They are capable of inactivating pathogens that have not yet entered the body and those that do not enter the body but which cause disease by secreting toxins which are taken up by the body. Accordingly, antagonists and, preferable, agonists of SC are of great pharmaceutical value for the modulation of mucosal immune response.
• glycosylation pattern of a recombinant glycoprotein is determined by certain ill-understood determinants in the amino acid sequence of the protein, and by the availability of competing processing enzyme activities which determine the final structure of the glycans.
• different glycosylation variants, termed glycoforms, of the same polypeptide can be produced by different cell lines, depending on the distribution of competing oligosaccharide processing enzyme activities in these cell lines.
• the type of glycosylation of the glycoprotein influences functional and structural parameters of the protein. For example, glycosylation often enhances stability of the polypeptide in the presence of proteolytic enzymes. On the other hand, substantial glycosylation, as especially in the case of SC, often interferes with attempts to crystallize the protein.
• Recombinant glycoproteins expressed in CHO cells are known to contain carbohydrate chains terminally substituted with sialic acids.
• the most prominent sialic acids ⁇ 2-3-linked N,O-acetylneuraminic acids (NeuAc) and ⁇ 2-3-linked N-glycolylneuraminic acid (NeuGc), are present in a ratio of about of about 97:3 (Hokke et al., FEBS Lett. (1990), 275, 9-14).
• N-acetylneuraminate mono-oxygenase (EC 1.14.99.18) activity, responsible for conversion of CMP-Neu5Ac into CMP-Neu5GC acting as precursor for incorporation of Neu ⁇ Gc in the glycoprotein, has not been demonstrated to come to expression in normal adult human tissue. Extensive studies have shown that when normal adult humans are exposed to sera of animal species, immunogenic responses may occur. The so-called Hanganutziu-Deicher (serum sickness) antibodies are directed towards glycoconjugates containing terminal Neu5Gc, ⁇ 2-3-linked to ⁇ -Gal.
• CHO SSF 3 cells incorporates NeuGc in the carbohydrate chains of rSC in amounts much lower than thus far observed for a typical glycoprotein produced by any other CHO cells.
• the ratio of NeuGc for rSC produced in CHO SFF3 cells was below 0.25% of total sialic acid whereas a typical recombinant glycoprotein produced in CHO cells contains about 3%.
• the reduced content in NeuGc usually not present in human glycoproteins, makes rSC produced by CHO SSF 3 cells more human-like and thus less antigenic and safer for human applications.
• a further surprising fact is, that it is possible to produce glycoforms of recombinant SC (rSC), which are both active in binding polymeric immunoglobulin and can be crystallized.
• rSC recombinant SC
• inventive glycosylated rSC can be crystallized, e.g., by the 'hanging drop method' and provides an ideal starting point for the evaluation of the 3-dimensional structure of SC and facilitate the search for SC antagonists and, preferably, SC agonists including muteins of the SC polypeptide itself.
• the current invention relates to a recombinant secretory component (rSC) or a functional fragment thereof, obtainable by a process comprising culturing a CHO SSF 3 cell transfected with a vector comprising a DNA coding for said secretory component or a functional fragment thereof, and isolating the expressed protein from the culture medium.
• rSC recombinant secretory component
• the rSC as defined above has, for example, a content of N-glycolylneuraminic acid (NeuGc) that is below 0.5% in respect to total sialic acid.
• the inventive rSC exhibit, for example, decreased antigenicity in human.
• the inventive process preferably comprises the following steps: a) constructing a vector capable of expressing the secretory component or a functional fragment thereof; b) transfecting a CHO SSF 3 cell with said vector; c) culturing the transfected cells; and d) isolating the secretory component or a fragment thereof from the culture medium.
• the plgR is a receptor capable to bind to poly-lg, especially IgA and IgM.
• Human plgR usually has nucleotide sequence as depicted in SEQ ID NO 1 , and an amino acid sequence basically as depicted in SEQ ID NO 2.
• the plgR is cleaved during transport of the plgR/poly-lg complex from the basal to the apical (extracorporal) side of the cell thus forming the SC.
• a functional fragment of rSC is a derivative of plgR that has one or more amino acid deletions or modifications, that is capable of binding to poly-lg, especially poly- IgA, and/or that reacts in the presence of antagonists and, preferable, agonists comparable to natural SC.
• a preferred fragment is, for example, a soluble fragment that lacks a membrane anchoring domain that resides, e.g., between amino acids 639-661 of the plgR, and/or the intracellular C-terminal domain between amino acids 662-764.
• a preferred fragment is, for example, a soluble fragment that lacks a membrane anchoring domain that resides, e.g., between amino acids 639-661 of the plgR, and/or the intracellular C-terminal domain between amino acids 662-764.
• the DNA fragments coding for said rSC or the fragment thereof may be modified, e.g., in so far as to adapt the codons to the preferred codon usage of the host, or the DNA sequence immediately in front or behind of the coding sequence may be modified in order to enhance the transcription, stabilize the mRNA produced, or to ease the genetic modifications, e.g., by an insertion of restriction sites.
• the DNA coding for the secretory component or a functional fragment thereof, as described above, usually is comprised in a polypeptide expression cassette capable of expressing said DNA.
• a promoter is operably linked to said DNA that is transcribed under the control of this promoter, and to a terminator.
• the promoter can be of almost any origin. It is for example possible to use a tightly regulated promoter or the promoter that is naturally adjacent to said DNA. Preferred are promoter that are active in CHO cells like viral promoters such as the 'early' promoter of SV40, the immediate early promoter of a cytomegalovirus (mouse, simian or human), or cellular promoters such the promoter of the ⁇ -actin gene, the metallothionein gene, or the heat shock genes. Especially preferred is the SV40 early promoter for the NEO gene, the mouse or human cytomegalovirus immediately early promoter for the SC and the human adenovirus type 2 promoter for the DHFR gene.
• viral promoters such as the 'early' promoter of SV40, the immediate early promoter of a cytomegalovirus (mouse, simian or human), or cellular promoters such the promoter of the ⁇ -actin gene, the metallothionein
• a DNA sequence containing the transcription termination signals is preferably the 3' flanking sequence of a gene which contains proper signals for transcription termination and polyadenylation for the desired host.
• Suitable signals are, for example, the polyadenylation signal of cellular genes such as the human growth hormone or the rabbit ⁇ -globin gene, or of viral genes the as those of the SV40 'early' and 'late' genes.
• the plasmids may also contain fragments of DNA that increase the stability of the plasmid in the desired host or that ease the integration of the plasmid DNA or the essential part thereof into the chromosome of the desired host.
• DNA fragments are the long terminal repeats of retroviruses, in case the recombinant genes are to be transferred as retrovirus particles, viral origins of replication, such as SV40, EBV, AAV, vaccinia, papillomavirus, Semliki forest virus etc., or DNA segments containing recognition sites for site-specific recombinases such as CRE and FLP.
• retrovirus particles viral origins of replication, such as SV40, EBV, AAV, vaccinia, papillomavirus, Semliki forest virus etc.
• viral origins of replication such as SV40, EBV, AAV, vaccinia, papillomavirus, Semliki forest virus etc.
• DNA segments containing recognition sites for site-specific recombinases such as CRE and FLP.
• the promoter, the DNA sequence coding for the secretory component or a functional fragment thereof and the DNA sequence containing transcription termination signals are operably linked to each other, i.e. they are juxtaposed in such a manner that their normal functions are maintained.
• the array is such that the promoter effects proper expression of the SC gene and the transcription termination signals effect proper termination of transcription and polyadenylation.
• the junction of these sequences may, for example, be effected by means of synthetic oligodeoxynucleotide linkers carrying the recognition sequence of a specific endonuclease.
• the expression cassettes according to the invention may be maintained in the desired host in form of a stable episome or plasmid or as part of the chromosome, wherein the latter case is preferred.
• the expression plasmids according to the invention include one or more, especially one or two, selective genetic markers for the host used for the construction, amplification and test of the plasmid, such a marker and an origin of replication for a bacterial host, especially Escherichia coli.
• any marker gene can be used which facilitates the selection for transformants due to the phenotypic expression of the marker gene.
• Suitable markers are, for example, those expressing resistance to an antibiotic or another antimetabolite or, in the case of auxotrophic host mutants, genes which complement host lesions.
• Corresponding genes confer, for example, resistance to the antibiotics tetracyclin, ampicillin, G418, hygromycin, puromycin or bleomycin or provide for prototrophy in an (conditionally) auxotrophic mutant, for example the thymidine kinase (TK) gene, dihydrofolate reductase (DHFR) gene and the E. coli gpt, HisD or Trp genes.
• TK thymidine kinase
• DHFR dihydrofolate reductase
• Trp genes can also be provided by cotransfection of the expression gene with a physically unlinked selection gene. After such a cotransfection the enzymatic machinery will with
• a prokaryote such as E. coli.
• a prokaryote e.g. E. coli, genetic marker and a prokaryote, e.g. E. coli, replication origin are included advantageously.
• corresponding prokaryotic plasmids for example E. coli plasmids, such as pBR322, pTZ18R, or a pUC plasmid, for example pUC18 or pUC19, which contain both prokaryotic, e.g. £ coli, replication origin and genetic marker conferring resistance to antibiotics, such as ampicillin and tetracyclin.
• the expression plasmids according to the invention can contain optionally additional expression cassettes, such as 1 to 3 additional polypeptide expression cassettes, which may be the same or different.
• suitable vectors are mammalian cell expression vectors based, for example, on pEUK-C1 (Clontech Inc., Palo Alto, California, USA ), pcDNAlneo (Invitrogen Corp. San Diego California, USA) pCGA28 (Asselbergs et al. Fibrinolysis (1993), 7, 1 -14) or PCGA93D-PPREN (Asselbergs etal., Biotech. (1994), 32, 191-202).
• the ligation mixture is then transformed into a suitable prokaryotic or eukaryotic host depending on the nature of the regulatory elements used, and a transformant containing the desired vector is selected according to conventional procedures.
• the plasmids can be multiplicated by means of the transformed hosts and can be isolated in conventional manner. The choice of the host depends on the regulatory sequences located on the vector. For the construction and multiplication of the vector a prokaryotic host, e.g., E. coli, is preferred. Hosts, transfection and culturing
• a suitable host for the production of rSC is a CHO SSF 3 cell (Gandor, C.R. (1993) Establishment and characterization of growth-factor-prototrophic Chinese hamster ovary (CHO) cell lines for the production of recombinant proteins, Zurich: Dissertation Nr 10087, Swiss Federal Institute of Technology.) or a cell that is derived therefrom and produces the same glycoforms of rSC.
• the suitable host can be transfected by the standard methods in genetic engineering, as for example with the aid of cationic lipid vesicles, electroporation or particle gun.
• a high copy plasmid or the plasmid DNA is integrated into the genome in several copies.
• the latter can be achieved, for example, via an amplification with methotrexate as described for example in (Asselbergs et al. J. Biotechnol. (1994), 32, 191-202,; Asselbergs et al. J. Biotechnol. (1992), 23, 143-151 ; Asselbergs etal. J. Mol. Biol. (1986), 189, 401-411 and Kaufman et al. Mol. Cell Biol. (1985.), 5, 1750-1759,).
• the modified CHO SSF 3 cell can be cultured by standard methods in cell culture.
• the cells are cultured in a serum-free medium and more preferred in a serum- and protein-free medium.
• the amount of Pluronic , especially Pluronic F-68 , added to the culture medium is preferably about 0.005 to 0.5% (w/v) and more preferred 0.01 to 0.1 % (w/v).
• the rSC produced by the inventive method is secreted predominantly in to the culture medium. It can be isolated therefrom by conventional means. During the isolation conventional additives like protein stabilizers, inhibitors of proteinases and the like may be added.
• the first step consists usually in separating the cells from the culture fluid by means of centrifugation or filtration. In the presence of additional proteins and impurities, the resulting supernatant can be enriched for rSC.
• Representative purification schemes include, e.g., treatment with polyethyleneimine as to remove most of the non- proteinaceous material, and precipitation of proteins by saturating the solution with ammonium sulfate or the like, ultrafiltration, diafiltration, gel electrophoresis, carrier-free electrophoresis, chromatographic processes such as ion exchange chromatography, size exclusion chromatography, partition chromatography, affinity chromatography, HPLC, reverse phase HPLC, treatment with Sephadex , dialysis, or by other processes, especially those known from the literature.
• chromatographic processes such as ion exchange chromatography, size exclusion chromatography, partition chromatography, affinity chromatography, HPLC, reverse phase HPLC, treatment with Sephadex , dialysis, or by other processes, especially those known from the literature.
• chromatographic processes such as ion exchange chromatography, size exclusion chromatography, partition chromatography, affinity chromatography, HPLC, reverse phase HPLC, treatment with Sephadex , di
• a further embodiment of the invention is a method for the crystallization of the secretory component (SC) according to the invention comprising placing a solution of said secretory component in a vessel containing a precipitating agent buffer, wherein the solution and the buffer are separated.
• SC secretory component
• the solution of the secretory component or the functional fragment thereof can contain a precipitating agent buffer. It is, for example, preferred to mix the solution comprising the inventive rSC and the precipitating agent buffer in an amount of 1:2 to 2:1 or, preferred, in about equal amounts.
• the precipitating agent buffer usually contains in addition to the compounds used to establish a certain pH, one or more hygroscopic compounds and preservatives.
• suitable ingredients are NaN 3 , Na-citrate, HEPES, ammonium phosphate, and/or Li 2 SO 4 .
• Preferred buffers comprise for example a mixture of Na-citrate, ammonium phosphate and NaN 3 ; or a mixture of HEPES, Li 2 SO 4 and NaN 3 .
• the solution comprising rSC is placed in a hanging manner over the precipitating agent buffer (hanging drop method).
• the crystallization is carried out preferred at temperatures from 3°C to 30°C, more preferred from 5°C to 25°C, and especially preferred at room temperature.
• the isolated and crystallized rSC as described above can be used to identify the 3- dimensional structure of the whole protein or at least of the areas responsible for binding and secretion of poly-lg.
• Conventional methods for the identification of the 3-dimensional structure are, for example, X-ray studies or NMR studies.
• the data received with these or comparable methods may be used directly or indirectly for the identification of antagonists or, preferably, agonists of the rSC mediated IgA transport.
• a commonly used method in this respect is, for example, computer aided drug design or molecular modeling.
• a further embodiment of the invention concerns the antagonist or, preferably, agonist identified with the inventive rSC, or with the aid of the 3-dimensional structure derived therefrom, for use in a method of treatment.
• the inventive rSC has valuable pharmaceutical properties because of its lower immunogenicity in respect to previously known SC.
• This lower immunogenicity is based, e.g., on a low content of NeuGc (0.25% or less of the total sialic acid content).
• a further embodiment of the invention concerns the use of the inventive rSC as defined above in a method of treatment, e.g., in the stabilization of poly Ig, especially poly IgA.
• the cDNA sequence of the human polymeric immunoglobuline receptor (plgR, SEQ ID NO:1) is known (Krajci et al., Biochem. Biophys. Res. Commun. (1989), 158, 783-789; Krajci et al., Hum. Genet. (1991), 87, 642-648; Piskurich et al., Mol. Immunol. (1993), 30, 413-421 ; SEQ ID NO 1).
• Such cDNA can be generated using standard methods in genetic engineering, e.g., by reverse transcription of mRNA from samples of tissue expressing plgR (Krajci et al., 1989, alphabet cit.) or from a publicly available cell line such as HT29 (ATCC HTC-38, Piskurich et al., Mol. Immunol. (1993) 30, 413 -421).
• the cloned cDNA can be identified by hybridization with PCR fragments generated from the cDNA mixture with primers designed using the cDNA sequences in the public domain.
• the cDNA is cloned in a plasmid vector, which can be multiplied in E. coli.
• Plasmid DNA is prepared according conventional procedures (Sambrook et al., Molecular Cloning: A laboratory manual, 2 nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1989) and the nucleotide sequence of the cDNA sequence is determined.
• the natural coding sequence of the precursor of the plgR protein (SEQ ID NO 2) starts with an ATG methionine codon and ends with a TAG stop codon.
• the leader peptide is cleaved off from the precursor polypeptide within the cell prior to presentation of the receptor protein on the cell membrane.
• An artificial DNA molecule encoding secretory component is generated by creation of a stop codon immediately before the transmembrane segment using PCR mutagenesis.
• the information contained in DNA sequence encoding pre-plgR is sufficient, but optionally, vector DNA sequences lying upstream of the ATG initiation codon can be used in the creation of a DNA molecule coding for secretory component.
• the forward PCR primer is chosen such that the functional properties of the leader peptide are preserved. The simplest way to achieve this is, is not altering the natural leader peptide sequence.
• the primer sequence upstream of the ATG codon is further chosen such that a convenient restriction site, which is compatible with a restriction site in a suitable expression vector, is created.
• One preferred restriction site is that of the restriction endonuclease Hindlll, AAGCTT.
• a preferred DNA molecule for PCR modification is plgR cDNA cloned in vector pCB6. In this vector the plgR cDNA is cloned downstream of the major immediate early promoter of the human cytomegalovirus.
• a primer hybridizing to the cytomegalovirus promoter segment can be used, preserving restriction sites already present in the vector between the priming site and the position of the ATG codon.
• a preferred oligonucleotide primer is 5' PCR primer 1 (SEQ ID NO 5).
• a second criterion used in the design of the oligonucleotide is that the sequence around the ATG codon is such that the initiation of protein synthesis at the ATG is efficient.
• the DNA sequence found upstream of the ATG codon in natural mRNAs is not always optimal as under natural conditions only limited amounts of a protein are needed.
• the criteria for optimal initiation efficiency are known (Kozak et al., Nucleic Acids Res. (1987), 15, 8125- 8148; Peabody D.S. in Setlow J.K. ed. Genetic Engineering Vol. 12, pp. 99-76). It is preferred that the nucleotide immediately following the ATG is G.
• Ncol site is compatible with Ncol sites is many efficient expression vectors.
• Another preferred DNA sequence immediately upstream of the ATGG is GCCACC.
• the backward primer is designed with two main criteria in mind. Firstly, it serves to generate a stop codon at position 1906 just upstream of the hydrophobic transmembrane segment of the plgR protein. The primary translation product will thus terminate with Ser- Glu-Glu-Gln-Gly-Gly-COOH. Secondly, it serves to create downstream of the new stop codon a restriction endonuclease site compatible with a restriction site in a suitable expression vector.
• One preferred stop codon is TGA and a preferred restriction site is that of Xbal, TCTAGA as created by preferred 3' PCR primer 1 (SEQ ID NO 7).
• a DNA fragment is transcribed by a heat-stable DNA polymerase using a specific forward and 3' PCR primer as described in example 2 and with plgR cDNA cloned in a plasmid vector as template.
• the PCR reaction is done (according to Sambrook et al., Molecular Cloning: A laboratory manual, 2nd Edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY, 1989) in a buffered solution containing the four deoxyribonucleotide triphosphates and the temperature is of the incubation controlled such that multiple cycles of primer-driven transcription of the SC-coding fragment occur. This produces a DNA fragment terminating with DNA sequences of the two oligonucleotide primers.
• a Hindlll- Xbal fragment encoding SC is generated from this DNA fragment by first separating the SC DNA fragment from the primers and other components of the PCR reaction and then digesting this DNA to completion with Hindlll and Xbal. Subsequently, the 1.9 kb Hindlll- Xbal fragment purified from the restriction enzyme reaction and is ready for ligation to the restricted expression vector DNA.
• the PCR product generated in this way with 5' PCR primer 1 (SEQ ID NO 5) and 3' PCR primer 1 (SEQ ID NO 7) is called SC DNA fragment 1 (SEQ ID NO 3).
• the PCR product generated in this way with 5' PCR primer 2 (SEQ ID NO 6) and 3' PCR primer 1 (SEQ ID NO 7) is called SC DNA fragment 2 (SEQ ID NO 4).
• the artificial DNA segment encoding secretory component is inserted between restriction endonuclease sites in a suitable mammalian cell expression vector.
• a suitable mammalian cell expression vector is pCB6, of which the main features are listed in Table 1 and that can be synthesized easily with standard methods in genetic engineering:
• the vector pCB6 or pCB6 containing the plgR cDNA (pCB6plgR) is cut to completion with Hindlll and Xbal and the 6.1 kb fragment is isolated.
• This DNA fragment is ligated using DNA ligase (from bacteriophage T4 or another enzyme with ligation activity) to SC DNA fragment 1.
• This ligation product is used to transform E. coli DH5 ⁇ and ampicillin resistant strains are isolated.
• a plasmid DNA is isolated from such an ampicillin resistant strain is termed pCB6-SC and is structured as pCB6, but with the 1.9 kb SC DNA fragment 1 (Example 3) replacing the small Hindlll-Xbal fragment of pCB6.
• An alternative expression vector is constructed from pCB6-SC, by replacing the major immediate early promoter from the human cytomegalovirus, by the more powerful major immediate early promoter from the mouse cytomegalovirus (mCMV). This promoter is obtained together with the beginning of the ampicillin resistance gene as a 1.1 kb Pvul- Hindlll fragment from plasmid pCGA93D-PPREN (Asselbergs et al. J. Biotechnol. (1994), 32, 191-202).
• pCB6 is cut with Pvul and Hindlll and the largest fragment (6.1 kb), which contains all the structural elements of the expression vector (see table 1 ) except of the human cytomegalovirus promoter and the beginning of the pTZ18R-derived ampicillin resistance gene, is purified.
• the 1.1 kb and the 6.1 kb fragment are ligated and after transformation of E. coli with the ligation product ampicillin resistant colonies containing plasmid pMC-SC are obtained.
• pMC-SC is functionally equivalent to pCB-6 (see table 1) except that the human cytomegalovirus promoter is replaced by the murine one.
• Example 5 Construction of SC expression plasmid pCGA93D-SC
• the artificial DNA segment encoding secretory component is inserted between restriction endonuclease sites in a suitable mammalian cell expression vector.
• a suitable mammalian cell expression vector is pCGA93D-PPREN which is constructed according to Asselbergs et al., J. Biotech. (1994), 32, 191-202, and of which the main features are listed in Table 2:
• This DNA fragment is ligated using DNA ligase (from bacteriophage T4 or another enzyme with ligation activity) to SC DNA fragment 2 (Example 3).
• This ligation product is used to transform E. co// DH5 ⁇ and ampicillin resistant strains are isolated.
• a plasmid DNA is isolated from such an ampicillin resistant strain is termed plNTERMED2 (6.8 kb).
• plNTERMED2 is cut to completion with Sail and Xbal and the large fragment (6.5 kb) is isolated.
• pCGA93D-PPREN is cut to completion with Sail and Xbal and the 3.3 kb fragment is isolated.
• the two DNA fragments are ligated and the product is used to transform E. coli DH5 ⁇ .
• pCGA93D-SC From an ampicillin and tetracyclin resistant strain thus obtained pCGA93D-SC is isolated.
• This plasmid is structured like pCGA93D-PPREN, but with the SC coding DNA replacing the preprorenin coding DNA.
• the SC coding DNA starts with an Ncol site and the DNA sequence around the ATG initiation codon of SC allows efficient initiation of translation of the recombinant SC mRNA.
• Example 6 Expression of human secretory component in CHO SSF3 cell transfected with pCB6-SC or pMC-SC
• CHO SSF 3 cells are known (Gandor C.R., Establishment and characterization of growth- factor-prototrophic Chinese hamster ovary (CHO) cell lines for the production of recombinant proteins, Zurich: Dissertation #10087, Swiss Federal Institute of Technology, 1993).
• a cell stock is maintained in FMX-8 medium (Dr. F. Messi Cell Culture Technologies, Rohrstrasse 29, CH-8152 Glattbrugg/Z ⁇ rich) without further additives. If transfection is going to be performed without serum, one to ten million cells are pelleted at low speed in a centrifuge and the cells are resuspended at 200O00 cells/ml in fresh FMX-8 medium of 37°C.
• CHO SSF 3 cells In the alternative procedure, a dense culture of CHO SSF 3 cells is diluted 10-fold in FMX-8 medium with 4% fetal calf serum and plated in 3 cm diameter tissue culture Petri dishes. Cultured in this way the CHO SSF 3 cells form a cell monolayer, which adheres to the plastic of the Petri dish. When this monolayer is 30-50% confluent, they can be used for transfection.
• the solutions for transfection are prepared in a polystyrene vessels to prevent adsorption of the cationic lipid used and the complex formed of the cationic lipid and DNA to the vessel wall.
• the pCB6-SC (example 3) DNA is prepared for transfection as follows: 4 ⁇ g plasmid DNA is dissolved in 0.1 ml FMX-8 medium. Separately 14 ⁇ l cationic lipid solution (lipofectin, GIBCO) is diluted in 0.1 ml FMX-8 medium. Subsequently, the DNA solution and the lipofectin solution are carefully mixed and incubated at room temperature for 15 min to allow formation of a lipofectin-DNA complex.
• One cell line CHO SSF 3 producing human SC is designated SSF3-HSC-1 and is used for production of human SC in stirred tank bioreactor.
• the cell population produces more than 10 ⁇ g SC per million cells day.
• CHO-SSF3/pMC-SC3 DSM ACC2203
• DSM ACC2203 DSM ACC2203
• CHO SSF 3 cells and lipofectin-DNA complex are prepared as described above. (Example 6), except that instead of plasmid pCB6-SC plasmid pCGA993D-SC (Example 5) is used.
• the serum-containing medium is suctioned off, the cells are rinsed with FMX-8-medium without serum and 0.3 ml FMX-8-medium without serum is added.
• the lipofectin-DNA complex solution is added and the cells are incubated for 5 h in a CO 2 incubator (5% CO 2 ) at 37°C, after which 1 ml of FMX-8-minus with 4% dialyzed serum is added. 24h later the cells are trypsinized, diluted 20-fold in FMX- 8-minus with 4% dialyzed serum and 5 nM methotrexate and plated in Petri dishes.
• methotrexate-resistant cells After 2- 3 weeks colonies of methotrexate-resistant cells have developed, which are individually scraped off and transferred to a 24-well microtiter plate. FMX-8-minus without serum with 5 nM methotrexate is added to the cells. After a week a dense culture of mostly non- adherent cells has developed. The concentration of SC in the conditioned medium from each well is measured and cells from the wells in which a high amount SC is detected are transferred to larger culture vessels and expanded until sufficient cells are obtained for selection of cell lines with increased methotrexate resistance (Example 8) or to inoculate a stirred tank bioreactor.
• One CHO SSF 3 cell line producing human SC is designated SSF3-HSC-M1 and is used for production of human SC in stirred tank bioreactor. The cell population produces more than 10 ⁇ g SC per million cells day.
• Example 8 Methotrexate selection of cell lines with increased number of copies of DCGA93D-SC
• the cells cultured in FMX-8-minus (FMX-8 lacking glycine, hypoxanthine and thymidine) plus 5 nM methotrexate are diluted to a cell density of approximately 500 cells/ml in medium with the new methotrexate concentration and divided over several 96-well microtiter culture plates. It is known that the frequency of amplification is approximately one in 10000 and that small increments of the methotrexate concentration favor the development of resistance due to gene amplification rather than other gene alterations (Kaufman R.J., Methods in Enzymology (1990), 185, 537-566). Therefore, the selection is initiated at about twice the initial concentration of 5 nM methotrexate.
• Cells with higher specific SC production are obtained with a frequency of 20-40% of the more resistant subcultures. This selection procedure is repeated several times, each time raising the methotrexate concentration 1.5-2.5-fold. When a concentration of 50-150 nM methotrexate is reached a cell population producing more than 10 ⁇ g SC per million cells day is obtained. The cell line thus obtained is readapted to growth in protein- free FMX-8 minus by gradually over a period of 1 -2 weeks lowering the serum concentration. These cells are then transferred to an appropriate bioreactor for large scale SC production.
• Example 9 ELISA assay for human SC in conditioned medium of transfected CHO SSF 3 cells
• Polystyrene microtiter plates are coated overnight at 4°C with 100 ⁇ l/well of 10 ⁇ g/ml rabbit anti-human SC immunoglobulin (DAKO Code nr. A187) dissolved in PBS without MgCI 2 and CaCI 2 (GIBCO) containing 0.1 mg/ml of the bacteriostatic sodium ethyl mercurithiosalicylate. Subsequently, the plates are rinsed three times with in washing solution: PBS lacking MgCI 2 and CaCI 2 containing 0.05% Tween-20 (EIA-grade, BIORAD) and 0.1 mg/ml sodium ethyl mercurithiosalicylate.
• DAKO Code nr. A187 rabbit anti-human SC immunoglobulin
• Non-specific protein binding sites on the polystyrene are neutralized by incubation with blocking buffer: PBS containing 2% bovine serum albumin (BSA), 0.5% rabbit serum and 0.05% Tween-20. Subsequently, the plates are rinsed three times with washing solution. A 100 ⁇ l sample of an SC-containing solution (conditioned medium, column fraction from a purfication etc.) diluted in blocking solution is added to each well and incubated overnight at 4°C. Subsequently, the plates are rinsed three times with washing solution. Next, 50 ⁇ l of a solution containing 250 ng/ml biotinylated anti-human SC immunoglobulin in blocking buffer is added followed by an incubation of 1 h at room temperature.
• blocking buffer PBS containing 2% bovine serum albumin (BSA), 0.5% rabbit serum and 0.05% Tween-20.
• BSA bovine serum albumin
• This biotinylated antibody is prepared by treatment of rabbit anti-human SC immunoglobulin (DAKO Code nr. A187) with aminohexanoyl-biotin-n-hydroxysuccinimide ester (Zymed) according to the instructions of the manufacturer. Subsequently, the plates are rinsed three times with washing solution. Next, 50 ⁇ l of avidin crosslinked to horseradish peroxidase (Zymed) 1000-fold diluted in blocking buffer is added followed by an incubation of 1 h at room temperature.
• DAKO Code nr. A187 rabbit anti-human SC immunoglobulin
• aminohexanoyl-biotin-n-hydroxysuccinimide ester Zymed
• Example 10 ELISA assay for binding of SC to human IgA
• Polystyrene microtiter plates are coated overnight at 4°C with 100 ⁇ l/well of either 10 ⁇ g/ml human serum albumin (negative control, Sigma A-6003), human IgG (Sigma I-4506), IgM (Sigma I-8640), IgA (Sigma, I-0633) or rabbit anti-human SC immunoglobulin (DAKO Code nr. A187) dissolved in PBS without MgCI 2 and CaCI 2 (GIBCO) containing 0.1 mg/ml of the bacteriostatic sodium ethyl mercurithiosalicylate.
• human serum albumin negative control, Sigma A-6003
• human IgG Sigma I-4506
• IgM Sigma I-8640
• IgA Sigma, I-0633
• DAKO Code nr. A187 rabbit anti-human SC immunoglobulin
• the plates are rinsed three times with in washing solution : PBS lacking MgCI 2 and CaCI 2 containing 0.05% Tween-20 (EIA-grade, BIORAD) and 0.1 mg/ml sodium ethyl mercurithiosalicylate.
• Non-specific protein binding sites on the polystyrene are neutralized by incubation with blocking buffer: PBS containing 2% bovine serum albumin (BSA), 0.5% rabbit serum and 0.05% Tween-20.
• BSA bovine serum albumin
• the plates are rinsed three times with washing solution.
• a 100 ⁇ l sample of an SC-containing solution diluted in blocking solution is added to each well and incubated overnight at 4°C. Subsequently, the plates are rinsed three times with washing solution.
• Example 11 Production of SC of IgA in small pilot scale suspension culture in serum and protein free cultivated CHO SSF 3 cells.
• All cell cultivations are performed as suspended repeated step-wise-fed-batch cultivations in 10 L glass-bioreactors with marine type impellers. After having reached maximal cell density and working volume, indicated by beginning stationary growth phase, 90% of the cell suspension is harvested and the remaining 10% are diluted by a factor 1 :10 with fresh medium. Tne process is controlled by on-line control loops for temperature, pH and pO 2 . Cell concentration, cell viability and product concentration (end concentration) are off-line determined.
• the basal medium consists of FMX-8 (Dr. F. Messi AG, Zurich, Switzerland). The cells however are proved to be shear sensitive with respect to their production kinetics. The growth kinetics, however, are only slightly affected (see below). Medium is therefore supplied with Pluronic F-68 (P-1300, SIGMA) as productivity enhancer. The two different medium configurations are compared below.
• the cells After inoculation of the bioreactor with 1.2 x 10 5 cells/ml and 2.5 L working volume the cells are grown up to 9.8 x 10 5 cells/ml. During growth phase the working volume is increased twice by the addition of 3 L of fresh medium after 4 d and 2 L of fresh medium after 7 d respectively. This batch cultivation results in a final working volume of 8 L after 9 d of culture and with a final concentration of 109 mg/L of SC of IgA. The mean doubling time of the cells is 2.82 d and the daily yield of the SC of IgA is 12.1 mg/L d.
• Example 11.2 Batch cultivation with FMX-8 medium, supplied with Pluronic F-68
• the FMX-8 medium is supplied with 0.05% (w/v) Pluronic F-68 as productivity enhancer.
• Pluronic F-68 as productivity enhancer.
• the cells After inoculation of the bioreactor with 2.3 x 10 5 cells/ml (from Example 8)and 2.5 L working volume the cells are grown up to 1.1 x 10 6 cells/ml.
• the working volume is increased 3 times by the addition of 2.5 L of fresh medium after 1 d, 3 L after 3 d and 1 L after 4 d respectively.
• This batch cultivation results in a final working volume of 9 L after 7 d of culture and with a final cell concentration of 1 .1 x 10 6 cells/ml and a final concentration of 229 mg/L of SC of IgA.
• Example 12 Human secretory component produced by Chinese hamster ovary cells in the absence and presence of Pluronic.
• the immobilized lectin is collected above a glassfilter, washed with 5 mM sodiumacetate pH 5.6, containing 0.5 M NaCI, 1 mM CaCI 2 , and 1 mM MnCI 2 (binding buffer), and packed into a 2.6 cm x 30 cm column-housing.
• Concanavalin A-bound protein is eluted with binding buffer, containing 0.5 M methyl- ⁇ -D-mannopyranoside.
• the hSC- containing fraction are concentrated and dialyzed against 50 mM sodiumacetate pH 5.5, containing 0.15 M NaCI and 0.02 (w/v) NaN 3 , by ultrafiltration using an YM10® membrane (AMICON) in an Amicon cell.
• Example 13 Crystallization of recombinant soluble polymeric Ig receptor (hSC)
• Example 14 Sialic acid analysis of recombinant soluble polymeric Ig receptor (hSC)
• sialic acids The analysis of sialic acids is carried out essentially as described in Harra et al., Anal. Biochem. (1989), 179, 162-166.
• An aliquot of 236 ⁇ g recombinant soluble polymeric Ig receptor (hSC) in 10 ⁇ l 0.05 M sodiumacetate pH 5.5, containing 0.15 M sodiumchloride is dried under reduced pressure and solved in 200 ⁇ l 2 M acetic acid, heated for 3 h at 80°C.
• Released sialic acids are converted into fluorescent derivatives by the addition of 200 ⁇ l 7.0 mM 1 ,2-diamino-4,5-methylenedioxybenzene (DMB, Sigma) in 1.4 M acetic acid, containing 0.75 M ⁇ -mercaptoethanol and 18 mM sodium dithionate at 50°C for 2.5 h.
• DMB diamino-4,5-methylenedioxybenzene
• HPLC analysis is carried out on a Waters Novapak C 8 4- ⁇ m (60 A) column (3.9 x 150 mm) fitted into a Waters 840 chromatography system equipped with two model 510 HPLC pumps, a WISP model 712 sample processor, a model 490 programmable multi-wavelength detector and a Kratos GM 970 fluorescence detector operating at an excitation wavelength of 373 nm, detecting emission at wavelength >418 nm using a cut-off filter. Simultaneously, the absorbance of the eluent is monitored at a wavelength of 373 nm.
• Elutions are performed isocratically using acetonitrile : methanol : water (6.4 : 4.9 : 88.7, v/v/v) as eluent at a flow rate of 0.7 ml/min.
• the HPLC profiles of the DMB sialic acids derived from hST and bGP show only Neu ⁇ Ac for hST (as described in Spik et al., FEBS Lett. (1975), 50, 296-299 and Hokke et al., FEBS Lett. (1990), 275, 9-14) and a mixture of Neu ⁇ Gc and Neu ⁇ Ac in a ratio of 1.0 : 1.0 for bGP.
• the HPLC pattern of the DMB sialic acids derived from hSC show peaks at the elution positions of Neu ⁇ Gc and Neu ⁇ Ac, respectively.
• the content of Neu5Ac in hSC is more than 99.9% and that of Neu ⁇ Gc less than 0.1%.
• microorganism strains were deposited at the Deutsche Sammlung von Mikroorganismen (DSM), Mascheroder Weg 1 b, D-38124 Braunschweig (accession numbers and deposition dates given):
• AAG TCC TTC TAC AAG CAG ATA GGC CTG TAC CCT GTG CTG GTC ATC GAC 644 Lys Ser Leu Tyr Lys Gin He Gly Leu Tyr Pro Val Leu Val He Asp 165 170 175
• ATCCCAACTA TACAGGAAGA ATACGCCTTG ATATTCAGGG TACTGGCCAG TTACTGTTCA 720
• TCACTGTCAT CCTCAACCAG CTCACCAGCC GGGACGCCGG CTTCTACTGG TGTCTGACCA 1440
• GAGACTCTCA AGGTCCCCTC TCACTTTCCA TGCAAATTCT CCTCGTACGA GAAATACTGG 1500

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