CN1723283A - Method for manufacturing recombinant polyclonal proteins - Google Patents

Abstract

The invention relates to a method for manufacturing a recombinant polyclonal protein composition, in particular a recombinant polyclonal antibody composition. The method comprises obtaining a collection of cells transfected with a library of variant nucleic acid sequences, wherein each cell in the collection is transfected with and capable of expressing one member of the library, which encodes a distinct member of a polyclonal protein that binds a particular antigen and which is located at the same single site in the genome of individual cells in said collection, wherein said nucleic acid sequence is not naturally associated with said cell in the collection. The cells are cultured under suitable conditions for expression of the polyclonal protein, which is obtained from the cells or culture supernatant. The nucleic acid sequence id introduced into the cells by transfection with a library of vectors for site-specific integration. The present method is suitable for manufacturing recombinant polyclonal antibodies, thereby making available a superior replacement of plasma-derived therapeutic immunoglobulin products.

Description

Be used to produce the recombinant polyclonal method of protein

Invention field

The present invention has formed the basis that is used to produce the proteinic technology platform of recombinant polyclonal, described recombinant polyclonal protein can be used as treatment, improves or prevents various infection, inflammatory diseases, transplant rejection, cancer and anaphylactoid new therapeutic agent, described polyclone protein has the albumen that for example derives from immunoglobulin superfamily, for example the solubility of B or TXi Baoshouti and film combining form albumen.

Background of invention

Some infectious diseases and cancer lack effective treatment means.Monoclonal antibody can not successfully be resisted these targets usually, and partly cause is that the mutability of described complicated target and the adaptive mutation of target proteins cause immunity to escape the identification of monoclonal antibody.Can target most dynamic targets of polyclonal antibody on the other hand are for example to virus or cancer cells.In addition, if antigenic mutation, polyclonal antibody has the active maximum probability of maintenance.

The polyclonal antibody therapeutical agent that different commerce are buied includes: 1) isolating normal people's immunoglobulin (Ig) from the blood of normal people's donor; 2) from people's hyperimmunization immunoglobulin (Ig) in the blood of one people's donor, described donor carries for example antibody of virus of anti-specific virus target, and described disease targets is to run into by infection or vaccination before it; With 3) from the animal hyperimmunization immunoglobulin (Ig) of immune animal.

The immunoglobulin (Ig) of purifying has proved infection such as can resisting hepatitis b virus, respiratory syncytial virus, cytomegalovirus and other simplexvirus, rabies virus, Toxins, botulin effectively and has been effective in newborn rhesus monkey D prevention from human blood.The immunoglobulin (Ig) of purifying in the blood of the rabbit of personnel selection T cellular immunization is provided treatment or prevents that (for example, T cellular immunization Thymoglobulin) suppresses transplant rejection.Use normal people's immunoglobulin (Ig) to improve immune deficiency patient's immunity system and treated various autoimmune disorders.

Yet, the application of immunoglobulin (Ig) widely owing to the limited supply of donor blood raw material, batch with batch between different problem and the security of mutability be restricted.Particularly the immunoglobulin (Ig) of animal-origin be faced with the 1980's and nineteen ninety generation to the observed identical immunogenicity problem of the monoclonal antibody of animal-origin.

At last, for other blood products, propagate infectious for example the risk of HIV, bleb or hepatitis virus or Protein virus still exist.Therefore, be preferred therapeutical agent in some cases although the clinician admits polyclonal antibody, its use is still very limited.

Produced the novel method of producing human normal immunoglobulin by the transgenic animal technology.The transgenic mice at carrier's immunoglobulin (Ig) seat is established (U.S.Patent No.6,111,166).These mouse produce human normal immunoglobulin completely and can produce the antibody of anti-particular target by immunological technique commonly used.Yet, since the relatively little individual limit of mouse bigger antibody production.Large animal cow, sheep, hare and chicken (kuroiwa, people such as Y., Nature Biotechnology for example more; 2002; 20:889-893) once be used to carry out human immunoglobulin gene's transgenosis.Yet producing the polyclonal antibody that is used for the treatment of from the blood of these animals is not a simple thing.At first, the immunophysiology of animal and human's class may show suitable difference, and the immune storehouse (repertoire), the function that cause producing are reset multifarious different with antibody response.The second, the mitotic division unstable at the immunoglobulin (Ig) seat of described importing may influence the long-term production of antibody.The 3rd, the immunoglobulin (Ig) seat of deletion animal oneself so that for example the output of described animal's antibody can also not be challenging technically above the output of human antibodies.The 4th, the propagation infectious factor for example risk of virus, Protein virus or other virulence factor is accompanied by resulting from using of the intravital human antibodies of animal.

Just produced thus being used to produce for example needs of the production technology of antibody of recombinant polyclonal protein, described production technology can produce enough a large amount of and have the most small quantities of with batch between difference with so that the recombinant polyclonal protein of clinical safety use.Developed the effective ways that are used for producing homologous recombination protein to produce various albumen, comprised monoclonal antibody, interleukin-, Interferon, rabbit, tumour necrosis factor, proconvertin, VIII and IX by eucaryon (particularly Mammals) express cell system.Many these technology are based on: described goal gene is transformed and random integration in the genome of express cell system, then select, amplification and identify high yield person cloning by expression, be the breeding of increasing with this clone as main express cell then.

The expression of the alien gene that inserts may be subjected to the influence from " position effect " of genomic dna around it.Under many circumstances, described gene is inserted into position effect by force to the product synthetic site that is enough to suppress institute's quiding gene.In addition, since around reticent mechanism (promptly methylating) reason of applying of host chromosome DNA often cause unstable expression.

Developed permission special genes group position in mammalian cell and integrated and expressed the system of goal gene to express homologous recombination protein composition (U.S.Patent Nos.4,959,317 and 5,654,182; WO98/41645; WO01/07572).WO98/41645 has described in order to produce can secrete for example site-specific integration of the mammal cell line of antibody.Yet this expression is monoclonal and does not show that transfection can carry out with vector library.Also without any about how to keep by V specific in the library _H-V _LThe original multifarious prompting of combination results.

Summary of the invention

The invention provides and be used to produce the solution that is used to express and produce the proteinic production clone of recombinant polyclonal, avoided at the notable difference of forming between the proteinic single member of described polyclone.

Further, the present invention does not use animal in the proteinic production of described polyclone, has therefore avoided an ethics relevant with this method and a difficult problem clinically.

The invention summary

The invention provides the method that is used to produce recombinant polyclonal type of production clone, described recombinant polyclonal type of production clone is used to produce recombinant polyclonal protein, and described protein is selected from immunoglobulin superfamily usually.Polyclonal antibody, polyclone TXi Baoshouti or the segmental production of its polyclone are interested especially.The present invention allows to be used for the proteinic commercial production of recombinant polyclonal of pharmaceutical composition.A key character of the present invention is: the differential expression of forming the proteinic individual molecular of polyclone in process of production remains on inapparent level, minimized undesirable batch and batch between variation.

One aspect of the present invention relates to production purpose recombinant polyclonal method of protein, wherein said polyclone protein comprises different constituting in conjunction with the member of specific antigen or by it, described method comprises: the cell aggregation that comprises different IPs acid sequence library a) is provided, and wherein the proteinic different members of the described polyclone of each described nucleic acid sequence encoding and each described nucleotide sequence are incorporated on the genomic same single site of each single cell of described cell aggregation; B) under the condition that helps described polyclone protein expression, cultivate described cell aggregation; And c) the polyclone protein of the described expression of recovery from described cell culture, cell fraction or cell culture medium.

Further aspect of the present invention relates to the method that produces the cell aggregation that is suitable as recombinant polyclonal type of production clone, described method comprises: the vector library that comprises different IPs acid sequence colony a) is provided, wherein each described carrier comprises 1) the different IPs acid sequence of the coding polyclone protein different members of a single copy, described polyclone protein comprise in conjunction with the different members of specific antigen or by its forms and 2) one or more recombinase recognition sequences; B) introduce described vector library to host cell system, the genome of each cell of wherein said host cell system comprises the recombinase recognition sequence, described recognition sequence on its genomic single specific site and with the recombinase recognition sequence coupling of carrier; C) guarantee the existence of one or more recombinases in the described cell, thereby the different IPs acid sequence in the step of making (a) can carry out site-specific integration in the cell of host cell system, wherein said one or more recombinases can be I) import the cell expressing of described nucleotide sequence; II) by the carrier efficient coding of step a; III) provide by expression from second carrier; Or IV) provides to described cell as protein; And d) screening comprises the cell from the copy through integrating in described different IPs acid sequence library.

In two methods of the present invention, should be appreciated that all described polyclone protein generally is the sort of natural incoherent protein of cell of expressing with realization therein.

The present invention describes several methods that different IPs acid sequence library importing host cell system are suitable as the cell aggregation of polyclone type of production clone with generation.These methods comprise the whole transfection of using the cell aggregation that the library carries out, semi-monolithic transfection or the individual transfection of using the equal portions cell in part library, in described individual transfection, use individual member's transfection host cell in library, then compile the clone who obtains by selecting.Preferably the present invention utilizes mammalian cell (clone or cell type) as host cell to be.

In one aspect of the invention, the proteinic individual member of polyclone is by paired separate gene fragment coding.When described individual member was made up of two polypeptide chains, polyclone protein comprised antibody and TXi Baoshouti solubility or film combining form.In the further embodiment of the present invention, the heavy chain of a pair of gene fragment encoding antibody and variable region of light chain, or the γ chain of the α chain of TXi Baoshouti and β chain variable region or TXi Baoshouti and δ chain variable region.

The present invention further provides recombinant polyclonal type of production clone, described clone comprises with the cells transfected set of different IPs acid sequence library, each cell in wherein gathering is with member's transfection in the described library and can express this member, described member encodes in conjunction with the proteinic different members of the polyclone of specific antigen and is arranged on the genomic identical single site of described set individual cells, does not have natural getting in touch between the described cell in wherein said nucleotide sequence and the set.Preferably for example Chinese hamster ovary (CHO) cell, COS cell, bhk cell, myeloma cell be (for example from mammal cell line for clone, the Sp2/0 cell, NS0), the human cell of YB2/0, NIH3T3, inoblast or immortalization for example Hela cell, HEK293 cell or PER.C6 cell.But also can use nonmammalian eucaryon or prokaryotic cell prokaryocyte, for example vegetable cell, insect cell, yeast cell, bacterium, fungi etc.

The present invention also comprises the vector library that is used for site-specific integration, described library comprises the colony of the various nucleotide sequences of natural generation, and wherein each described carrier comprises 1) coding of a copy is in conjunction with the different IPs acid sequence and 2 of the proteinic different members of polyclone of specific antigen) one or more recombinase recognition sequences.

In yet another aspect, the invention provides pharmaceutical composition, recombinant polyclonal antibody (or its fragment) or polyclone TXi Baoshouti (or its fragment) that it comprises as activeconstituents preferably obtain by the inventive method.The recombinant polyclonal protein of described composition has specificity or reactivity to predetermined disease targets.This pharmaceutical composition is used in Mammals for example treatment in people, domestic animal or the pet, improvement or preventing disease, for example cancer, infection, inflammatory diseases, anaphylaxis, asthma and other respiratory disease, autoimmune disease, immunodeficiency disease, cardiovascular disorder, central nervous system disease, metabolism and endocrinopathy, transplant rejection or the pregnancy do not expected.

Definition

" protein " or " polypeptide " is meant any amino acid chain, not length of tube or posttranslational modification.Protein can monomer or the polymer form exist, polymer comprises polypeptide chain, protein fragments, polypeptide, oligopeptides or the peptide of two or more assemblings.

As used herein, term " polyclone protein " or " polyclone (polyclonality) " are meant and comprise the different still protein composition of homologous protein molecule, preferably be selected from immunoglobulin superfamily.So other molecule homology in each protein molecule and the described composition, but also contain one or more variable peptide sequence fragments, it is characterized in that the difference of sequence between the single member of polyclone protein.Known this class polyclone protein example comprises antibody or immunoglobulin molecules, TXi Baoshouti and B-cell receptor.Polyclone protein may be made of the protein molecule subclass of determining, described protein molecule subclass for example defines in conjunction with activity the common of wanting of purpose target by common trait, for example under the situation at the antigenic polyclonal antibody of wanting of purpose.

Term " purpose polyclone albumen " comprises definite polyclone protein subclass of enjoying common trait, for example to the binding ability of the target wanted, for example according under the situation to the antigenic polyclonal antibody of describing in conjunction with activity or specificity of purpose, described antigen is any other structure, molecule or the material in conjunction with target that one or more protein that for example separate, microorganism, parasite, cell type, allergen or glycan molecule maybe may become specific antibodies, or described antigenic mixture.

Term " member of recombinant polyclonal protein composition " or " the proteinic member of recombinant polyclonal " are meant a protein molecule in the protein composition, described protein composition comprises different but the homologous protein molecule, other molecule homology in each protein molecule and the composition but also contain one or more variable peptide sequence fragments wherein, described variable peptide sequence fragment is a feature with the aminoacid sequence difference between the proteinic single member of described polyclone.

Term " variable peptide sequence " and " variable region " but mutual alternative use.

Term " the proteinic different members of recombinant polyclonal " is meant a protein molecule in the protein composition, described protein composition comprises different but the homologous protein molecule, other molecule homology in each protein molecule and the composition wherein, but also contain one or more variable peptide sequence fragments, described variable peptide sequence fragment is a feature with the aminoacid sequence difference between the proteinic single member of described polyclone.

Term " antibody " is described the functional component of serum and is often referred to molecule (antibody or immunoglobulin (Ig)) set or individual molecule (antibody molecule or immunoglobulin molecules).Antibody molecule can be attached to or react with specific antigen determinant (antigen or epitope), and this can cause inducing of immunoeffectors function conversely.Single antibody molecule is considered to monospecific usually, and the antibody molecule composition can be monoclonal (promptly, constitute by identical antibody molecule) or polyclonal (promptly be made of different antibody molecules, the identical or different epi-position that exists on described antibody and same antigen or the antigen not even together reacts).Each antibody molecule has makes its specificity be attached to the unique texture of its corresponding antigen, and all natural antibody molecules have identical overall basic structure, promptly is made up of with two consistent heavy chains the light chain of two unanimities.Antibody also is generically and collectively referred to as immunoglobulin (Ig).That term antibody as used herein also comprises mosaic type and single-chain antibody, and the binding fragment of antibody (as Fab, Fv fragment or scFV fragment), and the polymer form IgM of dimerization IgA molecule or pentavalent for example.

The composition of different antibodies molecule described in term " polyclonal antibody ", described different antibodies molecular energy and several different specific antigens determinants combinations or reactions on identical or different antigen.Normally, the mutability of polyclonal antibody is considered to be positioned at so-called polyclonal antibody variable region.Yet, in the context of the invention, be appreciated that polyclone also describes the difference that is present between the monospecific antibody molecule on the so-called constant region, for example comprising for example human homogeneous type IgG1 of two or more antibody isotypes, IgG2, IgG3, IgG4, IgA1 and IgA2, or under the situation of the mixtures of antibodies of mouse IgG1, IgG2a, IgG2b, IgG3 and IgA.

" reorganization purpose polyclonal antibody " describes the recombinant polyclonal antibody subclass of determining, described subclass is to be feature with itself and the target wanted or one group of target bonded ability wanting, described target can be for example isolating protein, microorganism, parasite, cell, allergen or glycan molecule, maybe can become other structure, molecule or the material in conjunction with target of specific antibody, or its mixture.

Term " immunoglobulin (Ig) " is used as collective's title of the mixtures of antibodies of finding usually in blood or serum, but the also available mixtures of antibodies that is used for calling from other source.

Term " immunoglobulin molecules " expression monospecific antibody molecule is for example as the part of immunoglobulin (Ig) or the part of any polyclone or monoclonal antibody combination.

When mentioning the proteinic member of polyclone and combine with antigen, be meant to have the combination that binding constant is lower than 1mM herein, be preferably lower than 100nM, even more preferably be lower than 10nM.

Term " various objectives nucleic acid molecule library " is used for describing the nucleic acid molecule set of common coding " reorganization purpose polyclone protein ".When being used for transfection, various objectives nucleic acid molecule library is contained in the expression vector library.This class library has at least 3,5,10,20,50,1000,10 usually ⁴, 10 ⁵Or 10 ⁶Individual different members.

Term " copy of various objectives nucleotide sequence " can not be interpreted as corresponding to the segmental single nucleic acid fragment of term single gene from literal as used herein, and be meant a copy of needed all gene fragments of all subunits that produce a target protein molecule, and be assembled into for example carrier of a nucleic acid molecule.Ask for something surpasses fragment that example that a gene fragment produces the complete molecule of target protein matter comprises B-cell receptor, antibody and antibody for example Fab ' s and variable domains, or TXi Baoshouti.When being imported into cell, the gene fragment of the target protein matter of the described completed assembled of encoding together is present on the same carrier, so be connected to a nucleotide sequence, may become the element of transcribing of separation under different promoters control.

Term as used herein " goal gene " is meant the nucleotide sequence of being made up of a member's of one or more coding target protein matter gene fragment (genomic or cDNA)." goal gene " of described plural form is meant coding purpose polyclone nucleic acid sequences to proteins library.Term " GOI " is as the abbreviation of goal gene.

Term " carrier " is meant nucleic acid molecule as used herein, and described nucleic acid molecule is inserted being used for by other nucleic acid molecule and transports and/or express in host cell at different genotypic environments.Can be called " site-specific integration carrier " herein by carrier predetermined in genome, that specific position is incorporated in the host genome.If described carrier carries the controlling element (suitable at least promotor) that the nucleotide sequence that is used to regulate and control to insert carrier is transcribed, described carrier is called " expression vector " herein.If expression vector can be incorporated on predetermined particular seat in the genome of host cell, described expression vector can be described as " site-specific integration expression vector ".If be inserted into the target protein matter that the nucleic acid sequence encoding in the carrier of above-mentioned evaluation defines herein, available following term " purpose carrier ", " the purpose carrier that is used for site-specific integration ", " purpose expression vector " and " the purpose expression vector that is used for site-specific integration ".Term " isotype code carrier " is meant the carrier of the nucleotide sequence that carries the coding isotype antibody.In this specification sheets, " phagemid carrier " and " phage vector " commutative use.Term " plasmid " and " carrier " commutative use.The present invention also comprises the carrier of this other form of class, and the carrier of described other form has same function, plasmid, phagemid and viral genome or any nucleic acid molecule that for example can instruct the albumen wanted to produce in appropriate host.

Term " each member of purpose vector library " is used to describe the single carrier molecule from the purpose vector library with different IPs acid sequence, the proteinic member of wherein said nucleic acid sequence encoding purpose recombinant polyclonal.

Term " global transfer " is used to describe purpose nucleotide sequence from a carrier colony to the transfer of another carrier colony and do like this and can shift and need not single purpose DNA is separated each DNA simultaneously.Such carrier colony comprises for example library of purpose variable region, promotor, leader sequence or enhancing element.Then these sequences be need not to separate in advance and can transfer on the mammalian expression vector from phage vector.In particular for antibody sequence, this technology has been guaranteed V _HAnd V _LBeing connected between the diversity do not lost the library when transferring to mammalian expression vector from for example selecting carrier (for example Vector for Phage Display).So initial V _HAnd V _LPairing is retained.

Term " transfection " is herein as the broad terms with the foreign DNA transfered cell.Described term also comprises other method with the functional equivalent of foreign DNA transfered cell, for example transforms, infects, the fusion of transduction or donorcells and recipient cell.

Term " selection " is used for described method, and cell obtains certain feature in the described method, and this feature can make it come out from the cellular segregation that those do not obtain this feature.This category feature can be the resistance of pair cell virulence factor or produce basic nutrition thing, enzyme or color.

Term " but selectable marker gene ", " selectable marker gene ", " selection gene " and " marker gene " but be used to describe with goal gene altogether the gene of the coding selective marker of transfered cell (for example give to some cytotoxic drug for example some microbiotic with the gene of resistance, can be created in the gene of the basic nutrition thing that can exhaust on the growth medium, coding produces the gene of enzyme of analyzable metabolite or coding chromoprotein for example can be according to the proteinic gene of FACS classification).

The expression vector cells transfected that term " recombinant protein " is used for describing with comprising described proteic encoding sequence is an expressed protein.

As used herein, term " connection effectively " is meant fragment is connected with another fragment, keeps functional relationship between them simultaneously.For example, participate in the leading peptide of polypeptide, can think that then it is connected on the DNA of this polypeptide of coding effectively to the transfer of endoplasmic reticulum if the DNA of coded signal sequence is expressed as.In addition, if transcribing of promotor or enhanser stimulus coding sequence can think that then it is connected on this encoding sequence effectively.

Term " most of cell individual " is meant the per-cent of described cell, for example surpasses 80%, preferably surpasses 85%, more preferably surpasses 90%, 95%, or even 99% or higher.

As used herein, term " genome " is not literally to go up only to be interpreted as to be present in intracellular normal complete karyomit(e), component outside the karyomit(e) that also refers to be imported into and to keep in cell.The outer component of this chromosomoid can include but not limited to minichromosome, YACs (yeast artificial chromosome), MACs (mouse artificial chromosome) or HACs (human artificial chromosome).

Term " promotor " is meant to relate to and combines the DNA zone of transcribing with startup with RNA polymerase.

Term " promotor (head-to-head promoter) head to head " is meant that to be placed in very in-plant promotor right, takes place so that transcribed with opposite direction by two gene fragments of described promoters driven.Head to head promotor also can make up with the weighting material of uncorrelated nucleic acid between two promotors.This weighting material can easily contain and surpass 500 Nucleotide.

" antibiotics resistance gene " is meant that coding overcomes the inhibition of microbiotic pair cell or the proteic gene of toxic effect, thereby guarantees cell survival and continue propagation under the situation that has microbiotic to exist.

Term " internal ribosome entry site " or " IRES " describe the structure of the 5 ' cap sequence that is different from normal mRNA.Two structures can be discerned to begin scanning by rrna and seek the AUG codon with initial translation.By using a promoter sequence and two initial AUG, can translate first and second peptide sequences from single mRNA.Therefore in order to translate first and second polynucleotide sequences altogether from single bicistronic mRNA, the described first and second polynucleotide sequences can be transcribed fusion by the connexon sequence, and described connexon sequence comprises the IRES sequence that can translate IRES sequence downstream polynucleotide sequence.In this case, the bicistronic mRNA RNA molecule of transcribing out all can be translated in its inside IRES sequence of attaching the names of pre-determined candidates 5 ' end and this bicistronic mRNA RNA molecule, produces first and second polypeptide thus.

Term " but abduction delivering " is used for describing such expression, the interaction that needs inductor molecule and regulation protein of this expression, or check the release of molecule from the regulation protein altogether.

Term " constitutive expression " is meant it is not derivable expression usually.

Term " recombinase " is meant the enzyme of recombinating between the two or more recombination sites of catalysis.The recombinase that can use in invention is recombinated in specific recombination site catalysis, and described recombination site is the specific nucleic acid sequence by specific recombinase identification.

Term " mixes (scrambling) " and describes the situation of expressing the proteinic different members of two or more polyclones from single cell, and described polyclone protein comprises two different polypeptide chains, for example from described immunoglobulin superfamily.On its genome, integrated when described single cell and can produce this situation when surpassing a pair of gene fragment, wherein every pair of gene fragment proteinic different members of described polyclone of encoding.Can produce in this case from the unexpected combination of the polypeptide chain of described gene fragment expression.The unexpected combination of these polypeptide chains may be without any result of treatment.

Term " H _H-V _LChain mixes " be miscellaneous example defined above.In this example, V _HAnd V _LThe encoding gene fragment has constituted a pair of gene fragment.As unexpected V _HAnd V _LWhen polypeptides in combination produces from cell, just occurred mixing, wherein in described cell, two different V _HAnd V _LThe encoding gene fragment is to being integrated in the same cell.This type of miscellaneous antibody molecule unlikely keeps original specificity, therefore may not have any result of treatment.

Term " recombinant polyclonal type of production clone " is meant the protein expression cells colony with the library transfection of various objectives nucleotide sequence, the described single cell of wherein forming recombinant polyclonal type of production clone together only carries the different purpose nucleotide sequence of a copy, and the proteinic member of the described purpose recombinant polyclonal of described purpose nucleic acid sequence encoding and each copy are incorporated into the same loci of each cellular genome.The ability that the various objectives nucleotide sequence of integrating according to their reservations copies (for example selecting by microbiotic) selects to form the described cell of recombinant polyclonal type of production clone.The cell that can form this type of production clone can be bacterium, fungi, eukaryotic cell (for example yeast, insect cell or a mammalian cell for example, particularly the mammalian cell of immortalization for example Chinese hamster ovary celI, COS cell, bhk cell, myeloma cell (Sp2/0 cell for example, NS0), the human cell of NIH 3T3, YB2/0 and immortalization for example HeLa cell, NEK293 cell or PER.C6.

" focus (hot spot) " in the term " focus clone " is meant the seat of establishing in advance in the genome of described cell, described seat be selected or produce and have a feature of after expression vector being incorporated on this seat, efficiently transcribing the purpose nucleotide sequence of integration.

Term " difference (bias) " is used for representing the phenomenon of recombinant polyclonal protein production, wherein said polyclone carrier, polyclone clone or the proteinic composition of polyclone since at random between genetic mutation, the single cell between the difference of growth kinetics, the different table expression constructs sequence difference of expression level or the difference of dna clone efficient cause in for some time, changing.

Term " RLFP " is meant " restriction fragment length polymorphism ", is a kind of method, wherein with Restriction Enzyme cutting back nucleic acid fragment mobility spectrum on the gel is analyzed.

Term " HDS " is meant the high-density screening method, in described method many discrete molecule parllels is tested on film, so just can screen to find out to a large amount of test compounds simultaneously to have given active compound.

As used herein, " TaqMan PCR " is meant people such as Holland, P.M., and the PCR based on the TaqMan system that describes among the Proc.Natl.Acad.Sci.U.S.A.88:7276-7280 (1991) detects.

Term " 5 ' UTR " is meant 5 ' the untranslated zone of mRNA.

Term " Pfu PCR " is meant the PCR reaction of using Pfu archaeal dna polymerase (isolating from fireball bacterium (Pyrococcusfuriosus)) to carry out, uses described Pfu archaeal dna polymerase to be because it has high frequency high fidelity in known thermostability polysaccharase.

Abbreviation: " CMV "=(people) cytomegalovirus (Cytomegalo Virus)." MSPSV "=bone marrow proliferation sarcoma virus (Myeloproliferative sarcoma virus)." AdMLP "=adenovirus major late promoter.SV40 poly A=simian virus 40 polyadenylation signal sequences.The GFP=green fluorescent protein.The PVDF=poly(vinylidene fluoride).The TcR=T lymphocyte receptor.The absorption of ELISA=enzyme linked immunological detects.LTR=is long terminal repetition.

Accompanying drawing is described

Fig. 1: the illustrative of " promotor head to head " expression vector, described expression vector comprises following elements: Amp pro=allows to express the promotor of ampicillin resistance gene.The Amp=ampicillin resistance gene.PUC origin=pUC replication origin.Restriction enzyme site: NotI and EcoRI.Promoter A/Promoter B=comprises the box of promotor head to head of leader sequence (for example CMV/MPSV).The sequence of V Heavy=coding GOI variable region of heavy chain.The sequence of C Heavy Chain=encoding heavy chain (for example mouse IgG1 or IgG2B CH sequence) constant region.R-B-globin pA=rabbit betaglobulin poly A sequence.BGH polyA=Trobest poly A sequence.The sequence of the variable kappa of V Kappa=coding GOI.The sequence of C Kappa chain=coding kappa chain constant region.FRT site=FRT recombination site.The gene of Hygromycin=coding hygromycin resistance.SV40 poly A=polyadenylation signal sequence.

Fig. 2: the expression vector illustrative of unidirectional expression, described expression vector comprises following element: Amp pro=allows to express the promotor of ampicillin resistance gene.The Amp=ampicillin resistance gene.PUC ori=pUC replication origin.Promoter A=the comprise mammalian promoter of leader sequence (for example AdMLP).The sequence of V Heavy=coding GOI variable region of heavy chain.The sequence of C Heavy Chain=encoding heavy chain (for example mouse IgG1 CH sequence) constant region.HGH polyA=human growth hormone poly A sequence.BGH polyA=Trobest poly A sequence.The sequence of the variable kappa light chain of V Kappa=coding GOI.The sequence of CKappa=coding kappa chain constant region.The FRT=FRT recombination site.The gene of Hygromycin=coding hygromycin resistance.SV40 poly A=polyadenylation signal sequence.The sequence of hGH poly A and promotor A can be replaced by the IRES structure.

Fig. 3: the schema that general introduction recombinant polyclonal type of production clone produces and recombinant polyclonal protein produces.1) whole transfection strategy is described; 2) semi-monolithic transfection strategy and 3 is described) individual transfection strategy is described.A) diagram vector library (sea line), arrow are represented the carrier grouping.Go into one at carrier branch described in the strategy 1 and organize greatly, during they are divided into than group in strategy 2 (semi-monolithic), and the state (individuality) that their maintenances are dispersed mutually in strategy 3.B) diagram transfection, wherein the number of test tube depends on the grouping of the carrier of forming the library.C) cell is selected in diagram, and described cell locus specificity ground is incorporated into GOI in the described host cell gene group, and the D) generation of diagram polyclone GOI library mother liquor is with the freezing storage of cell in the described integration of the composition that chooses library.Monospecific polyclonal can be randomly stored and maybe merging will be cloned.E) beginning of diagram production phase, will melt from the clone in the mother liquor herein (can be single, come the less grouping of rotation or from the merging group) and make it to adapt to and in suspension, grow.In screening stage or this stage, its adaptation is grown on serum free medium.F) the diagram production phase, bred to inoculate bigger bio-reactor (although not explanation also can be carried out the intermediary inoculation step) in described polyclone clone of described production phase.In strategy 2 and 3, this stage is: described polyclone cell clone mother liquor no longer remains monospecific polyclonal or semi-monolithic grouping, but is merged into cell aggregation, forms recombinant polyclonal type of production clone.G) diagram is from the finished product of bio-reactor production acquisition.After the production phase, gather in the crops described polyclone protein composition with purified product and description product feature.

Fig. 4: the schema that mammalian expression vector produces is described.

(A) illustrative of phage vector pSymvc10, described carrier are carried coding GOI member's sequence.P tac and P lacZ=bacterium be the promotor box head to head.The sequence of the variable kappa light chain of V Kappa=coding GOI.The sequence of the constant kappa light chain of C Kappa chain=encoding murine.The sequence of V Heavy=coding GOI variable heavy chain.The encode sequence of constant heavy chain CH1 structural domain of C Heavy Chain=.Restriction enzyme site: EcoRI, NotI, SacI and XhoI.CpIII=phage protein I II.Amp pro=allows to express the promotor of ampicillin resistance gene.The Amp=ampicillin resistance gene.PUC Ori=pUC replication origin.

Step 1: by carrying out restrictive diges-tion with SacI and XhoI, described bacterium promotor scales off and replaces (B) by connection with the mammalian promoter box from pSymvc 10 and replaces, and described mammalian promoter box also is to come by carry out the restrictive diges-tion preparation with SacI and XhoI.

(C) illustrative of phage vector pSymvc 12 is in the sequence of carrying with the described carrier in the Mammals back of promotor box exchange head to head from GOI.The selected promotor head to head of Promoter A/Promoter B=box (for example CMV/MPSV).The sequence of the variable kappa light chain of V Kappa=coding GOI.The sequence of the constant kappa light chain of C Kappa chain=encoding murine.The sequence of V Heavy=coding GOI variable heavy chain.The encode sequence of constant heavy chain CH1 structural domain of C Heavy Chain=.Restriction enzyme site: NotI, SacI, XhoI and EcoRI.CpIII=phage protein I II.Amp pro=allows to express the promotor of ampicillin resistance gene.The Amp=ampicillin resistance gene.PUC Ori=pUC replication origin.

Step 2: by using EcoRI and NotI restrictive diges-tion pSymvc12, can downcut from pSymvc12 and comprise the nucleic acid fragment of complete kappa, promotor box and V heavy chain and be connected to for example pSymvc20 of isotype code carrier, described pSymvc20 also is by using the preparation of EcoRI and NotI restrictive diges-tion, therefore producing described mammalian expression vector pSymvc21 (E).

(E) be used for the illustrative of the mammalian expression vector pSymvc21 of antibody expression, described carrier has from the variable heavy chain of GOI and kappa zone.This mammalian expression vector comprises following element: Amp pro=allows to express the promotor of ampicillin resistance gene.The Amp=ampicillin resistance gene.PUC ori=pUC replication origin.Restriction enzyme site: EcoRI and NotI.The selected promotor head to head of Promoter A/Promoter B=box (for example CMV/MPSV).The V Kappa sequence of the variable kappa light chain of V Kappa=coding GOI.The sequence of the constant kappa light chain of C Kappa chain=encoding mammalian (for example constant kappa chain of mouse).The V sequence of heavy chain of V Heavy=coding GOI variable heavy chain.The sequence of the constant heavy chain of C Heavy Chain=encoding mammalian (for example sequence of mouse IgG1 or the constant heavy chain of IgG2B).R-B-globin pA=rabbit betaglobulin poly A sequence.BGH polyA=Trobest poly A sequence.FRT site=FRT recombination site.The gene of Hygromycin=coding hygromycin resistance.SV40 poly A=polyadenylation signal sequence.

In essence, step 1 and 2 order can be reversed, to from pSymvc10, downcut from the fragment of pSymvc10 by utilizing EcoRI and NotI to carry out restrictive diges-tion like this, then can be connected on the isotype code carrier for example pSymvc20, described fragment from pSymvc10 comprises whole kappa, bacterium promotor box and V heavy chain.Then by utilizing SacI to be connected (being similar to Fig. 4 B) pSymvc20 is carried out the promotor exchange with XhoI restrictive diges-tion pSymvc20 and with the mammalian promoter box fragment of SacI+XhoI digestion.

Fig. 5: show the histogram that the genotype in the TG1 cell that transforms with plasmid prepared product 1 distributes.Em 223-228 represent respectively to have the anti-B2M of coding (anti--B2M), anti--alkaline phosphatase (anti--AP), antiovalbumin (anti--OVA), anti--Factor IX (anti--FVIII), anti--N,O-Diacetylmuramidase (anti--LYS), anti--haptoglobin (carrier of anti--HAP) promotor.Em223-228 is the carrier of pSymvc10 type.The Id number that the fragment collection of illustrative plates of being determined by RFLP is released is corresponding to the number of the individual bacterium colony in the sum of the bacterium colony of sorting out.

Fig. 6: the histogram that shows the TG1 cytogene type distribution that transforms with plasmid prepared product 2.Em 229-234 represent respectively to have the anti-B2M of coding (anti--B2M), anti--alkaline phosphatase (anti--AP), antiovalbumin (anti--OVA), anti--Factor IX (anti--FVIII), anti--N,O-Diacetylmuramidase (anti--LYS), anti--haptoglobin (carrier of anti--HAP) mammalian promoter (CMV/MPSV).Em229-234 is the carrier of pSymvc12 type.Clone's number represents to have and number by the similar clone of the definite sequence map of the RFLP of this Em type (complete sequential analysis is not carried out) through observation.

Fig. 7: the histogram that shows the TG1 cytogene type distribution that transforms with plasmid prepared product 3.Em 235-240 respectively the anti-B2M of presentation code (anti--B2M), anti--alkaline phosphatase (anti--AP), antiovalbumin (anti--OVA), anti--Factor IX (anti--FVIII), anti--N,O-Diacetylmuramidase (anti--LYS), anti--haptoglobin (anti--HAP) mouse IgG1 mammalian expression vector (comprising rabbit beta Globulin polyadenylation signal).Em235-240 is the carrier of pSymvc21 type.Clone's number represents to have and number by the similar clone of the definite sequence map of the RFLP of this Em type (complete sequential analysis is not carried out) through observation.

Fig. 8: show the histogram that TG1 cytogene type distributes, described cell with two enzymic digestions/connection plasmid prepared product transform (plasmid prepared product 1 step afterwards global transfer in mammalian expression vector and do not need in intestinal bacteria, to carry out DNA cloning).

Fig. 9: show that mixture with the mammalian expression vector of six goal gene of coding transforms (A) 16 days and (B) histogram that distributes of the genotype of the CHO-Flp-In cell after 34 days.

Figure 10: from 34 days the antigen-specific ELISA of supernatant liquor of CHO-Flp-In cell after the transfection, described cell carries out whole transfection with the expression vector mixture of six goal gene of coding.

Figure 11: merge the anti-kappa coating ELISA of the supernatant liquor of thing from 34 days CHO-Flp-In cell after the transfection, described cell carries out transfection with the encode single expression vector of a goal gene or the expression vector mixture of six goal gene of encoding.

Figure 12: from 17,31,45,59 and 73 days CHO-Flp-In clone 019 the quantitative antigen-specific ELISA of supernatant liquor after the transfection, described cell carries out whole transfection with the expression vector mixture of six goal gene of encoding.A, B represent three different transfection experiments with C.

Figure 13: from the antigen-specific ELISA of the supernatant liquor that mixes the CHO-Flp-In cell culture of back after 8,17,30,45,57,72 and 85 days, described mixing is the mixing of expressing the single member's of mini sixlibrary CHO-Flp-In clone.The result is shown as the mean value ± SD of three independent experiments.

Detailed Description Of The Invention

Recombinant polyclonal protein expression system

The invention provides for steady production recombinant polyclonal method of protein, described restructuring Polyclone protein preferably is selected from immunoglobulin superfamily, has the similar knot of immunoglobulin (Ig) The protein families in structure territory. Most of described members relate to the cell surface recognition reaction. Sequence together Source property shows antibody, φt cell receptor, MHC molecule, some CAM and cell factor Acceptor has close homology. Especially, this family of containing the variable region according to the present invention The member of family is suitable for producing recombinant polyclonal protein. This class members comprises that antibody, film are in conjunction with anti-Body (B-cell receptor), Fab fragment, Fv fragment, scFv (scFv) fragment, T are thin Born of the same parents' acceptor (TcRs), soluble T cRs, TcR variable domains fragment, connect by polypeptide The TcR variable domains fragment of sub-connection or other antibody or TcR derived fragment. Especially, Can expect that the present invention may be used for large-scale production and manufacturing comprises restructuring therapeutic Anti-TNF-α The new class therapeutic agent of body or TcRs.

A main favourable aspect of production method of the present invention is to form described recombinant polyclonal albumen All members of matter can produce to about 10 bioreactors or its equivalent at 1. Advance In one step, described recombinant polyclonal protein compositions can be with single prepared product from described reactor Purifying and do not need in described process, will form single member's branch of described recombinant polyclonal protein From coming out. On the contrary, if there is the people to wish (for example to exist by the monoclonal antibody of mixing purifying Propose among the WO91/16074) with simulation recombinant polyclonal antibody mixture, will need waiting to wrap Each antibody of drawing together in composition is produced in bioreactor respectively, and probably also wants With the independent purifying of described antibody. With Restruction polyclonal antibody described herein or other many grams Grand method of protein is compared, and such monoclonal antibody mixture production will be very expensive expense The time take the space. So, cause being included in mixing such as the method described in the WO91/16074 The physical constraints of the number of the monoclonal antibody in the thing is lower than 10 probably, and described herein Technology can be produced the polyclonal antibody with the many single members that want usually.

For obtain from the recombinant polyclonal protein of recombinant polyclonal type of production clone can Expection is expressed, and should quite at large understand the modulating properties of genomic integration site.

Use traditional transfection and the expression of recombinant proteins technology of random integration that Restruction is restrained more Grand protein is undesirable, because the randomness essence of this method will cause described integration The position of nucleotide sequence changes at different iuntercellulars with number. So, if by this Produced in conventional processes recombinant polyclonal protein may cause having polyclone protein single Member's difference is expressed foreign cell culture and because the position of the DNA of described integration of speed The genetic instability that effect causes. This will cause forming described polyclone protein probably Member's differential expression.

Therefore need to be introduced into predetermined genomic locus, this in principle can be heavy by homology Group realizes. Yet because unconventional recombination event dominate, therefore, homology is heavy The efficient of group is low-down.

In addition, when described polyclone protein is antibody or φt cell receptor (TcR), use Traditional transfection method that causes random integration produces another problem. Antibody and TcRs are by in pairs The separate gene fragment coding, namely antibody is by light chain and heavy chain coded sequence coding, and TcRs By α and β chain or δ and the sequential coding of γ chain encoding. Polypeptide product from these genetic fragments Covalently bound in the process in the cell of antibody molecule or TcR. Cause random integration Traditional rotaring dyeing technology cause with the different heavy chains of several copies and light chain or α and β chain import with A cell, this causes the random combine of heavy chain and light chain, i.e. so-called V_H-V _LChain mixes or α-β chain mixes. As a result, the performance that this has damaged antibody or the TcRs of described expression causes the parent Affinity and/or specificly lose, possible new specificity and/or the activity specific that reduces Generation.

In order to overcome these problems, expression system of the present invention uses site-specific integration to single In the genome of one host cell. System of the present invention guarantees to comprise described different purpose nucleic acid sheet The cassete exchange method of the purpose vector library of section by recombinase-mediated is inserted into the precognition feature On the chromosomal loci, produce thus clone, wherein individual cells is expressed described recombinant polyclonal The single different members of destination protein. As described below, form recombinant polyclonal at some Many places may take place in the cell of type of production clone to be integrated. Yet, as long as most cells is individual Body surface reaches the single different members of described recombinant polyclonal protein, this just not one of Cheng Qiwei ask Topic.

Can use recombinase for example Cre, Flp, β-recombinase, Gin, Pin, PinB, PinD, R/RS, lambda integrase or bacteriophage Ф C31 integrase. Be used for being incorporated into described chromosomal loci Suitable integrase can provide in the following manner: (i) express the genome from cell self, Described genome introgression described nucleotide sequence, (ii) had by the nucleotide sequence that is inserted into cell Effect ground coding (iii) is expressed from second nucleic acid molecules or (iv) as albumen. Preferably In the embodiment, the different IPs acid sequence that is included in the described purpose carrier is incorporated into the mediation purpose The seat (so-called " focus ") of nucleotide sequence high level transcript and expression.

Employed host cell is preferably to comprise these to be typically used as bio-pharmaceutical protein The mammal cell line of expressing, for example Chinese hamster ovary celI, COS cell, bhk cell, myeloma Cell (Sp2/0 cell for example, NS0), YB2/0, NIH 3T3 and Immortalized be human thin Born of the same parents are HeLa cell, HEK293 cell or PER.C6 for example. Used in the present invention CHO thin Born of the same parents. Yet those of ordinary skills can get with described other mammalian cell at an easy rate For Chinese hamster ovary celI, or even use the cell of other type, comprise plant cell, yeast cells, Insect cell, fungus and bacterium. So the selection of cell type can not limit the present invention. Excellent In the embodiment of choosing, contain focus, the mediation restructuring purpose polyclone protein of predicting feature The mammalian cell of high level expression is used to produce.

In the further embodiment of the present invention, utilize the same chromosome in the host cell whole The co-bit different purpose nucleotide sequences of naming a person for a particular job are integrated in the locus specificity mode. This single spy The opposite sex site integration reduced to greatest extent position effect, and described position effect can With observing under the situation that a plurality of sites are integrated on random integration or the genome. Especially, work as table Reach and have for carrying out at genome when surpassing the polyclone protein that a polypeptide chain forms The Single locus of site-specific integration is more useful. If this is because single cell is expressed Surpassing an integration thing just may mix in its subunit.

In the locus specificity integration system, described single host cell is expressed substantially identical egg The white matter structure, but exist different, for example anti-in the variable region of recombinant polyclonal destination protein The antigen binding domain of body or TcRs. Therefore, the most cells in this class cell aggregation for Productivity ratio and genetic stability should demonstrate similar feature, and therefore this technology provides Control recombinant polyclonal protein for example recombinant polyclonal antibody or TcRs manufacturing feasibility.

Recombinant polyclonal protein of the present invention is intended to cover and comprises different but the albumen of homology The protein compositions of matter molecule, described protein molecule are natural variable, and the meaning is, In the preferred embodiment, the library that different nucleic acid consist of comprises the diversity of natural generation. In Be, other molecule homology in each protein molecular and the described composition, but also contain one or many Individual variable peptide sequence fragment, described variable peptide sequence fragment is characterised in that described polyclone Amino acid sequence difference between the single member of protein. The ammonia that forms described variable peptide sequence Difference on the base acid sequence may be little of an amino acid. The difference on the amino acid sequence preferably Surpass an amino acid.

Normally, the natural changeability of polyclonal antibody or TcR is considered to be positioned at described polypeptide chain So-called variable region or V district.

In one aspect of the invention, the single member of polyclone protein comprises near 80 and arrives The variable region of 120 amino acid lengths. Super variable domains, example may be contained in described variable region Such as complementary determining region (CDR).

In the TcRs of natural generation, there are 4 kinds of CDRs in each variable region. In natural generation Heavy chain of antibody on have 3 kinds of CDRs, have 3 kinds of CDRs at light chain.

In additional aspects of the present invention, the single member's of described polyclone protein variable region contains At least one super variable domains is arranged, and described super varistructure length of field is at 1 and 26 amino acid Between, preferably between 4 and 16 amino acid. This super variable domains can corresponding to The CDR3 zone. For antibody, each variable region preferably forms 3 super variable domains. These Can be corresponding to CDR1, CDR2 and CDR3. For TcRs, each variable region preferably forms 4 Super variable domains. These can be corresponding to CDR1, CDR2, CDR3 and CDR4. Super variable knot The structure territory may be formed in alone the variable order in the variable region of recombinant polyclonal protein of the present invention Row.

In the context of the present invention, should be appreciated that the changeability (polyclone) of peptide sequence The monospecific antibody branch in the C district that is positioned at so-called constant region or described antibody polypeptides chain also can be described Difference between the son is for example containing the mixtures of antibodies of 2 or a plurality of different antibodies isotypes Situation under (for example people's isotype IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, Or mouse isotype IgG1, IgG2a, IgG2b, IgG3 and IgA). So, recombinant polyclonal Antibody can comprise with between the monospecific antibody in the variable region (V district) or constant region (C district) or this The sequence difference in two zones is the antibody molecule of feature.

For the different IPs acid sequence of the coding albumen of being combined with specific antigen is provided, can use perhaps Many methods known in the art. Generally, the present invention will benefit from use and can identify and/or separate volume The screening technique of the nucleic acid of the albumen that code is combined with specific antigen. Several these class methods comprise what is called Biological screening step (biopanning step), can know in technology phage display for example Technology (Kang, the people such as A.S., 1991.Proc Natl Acad Sci USA 88,4363-4366), Ribosomal display (Schaffitzel, the people such as C., 1999.J.Immunol.Methods 231,119-135), DNA shows (Cull, the people such as M.G., 1992.Proc Natl Acad Sci USA 89,1865-1869), the RNA-peptide shows (Roberts, R.W., Szostak, J.W., 1997. Proc Natl Acad Sci USA 94,12297-12302), covalency show (WO98/37186), Bacterium surface displaying (1991.Biotechnology 9 for Fuchs, the people such as P., 1369-1372), Yeast surface display (Boder, E.T., Wittrup, K.D., 1997.Nat Biotechnol 15,553-537) show (Grabherr, R., Ernst, W., 2001.Comb. with eucaryon virus Chem.High Throughput.Screen.4,185-192), this area is known side all Method and all methods all have help in putting into practice the present invention. FACS and anti-by usage flag Former magnetic bead sorting also can be applicable to enrichment (screening) purpose. Immune detection test case such as ELISA (Dreher, the people such as M.L., 1991.J.Immunol.Methods.139,197-205) And ELISPOT (Czerkinsky, the people such as C.C., 1983, J.Immunol.Methods. 65,109-21) also can after the biological screening step, use or use separately.

Restructuring purpose polyclone protein compositions comprises definite protein subclass, described albumen The matter subclass defines by common trait, total in conjunction with active to the target wanted for example, example As in the situation of polyclonal antibody to the purpose antigen wanted. Polyclone protein group usually Compound has at least 3,4,5,10,20,50,100,1000,10⁴、10 ⁵, or 10⁶Individual distinct member. The number of needed different members in the polyclone protein compositions Order will depend on the complexity of described target. In the situation of antibody, the complexity of targeting antigen Number with necessary distinct member in the described polyclonal antibody composition of impact. Right In little or very not complicated target small protein for example, comprise 3 to 100 different members The polyclonal antibody composition is just enough, and preferably the number of different members is no more than 90 Individual or even 80 or 70. In many cases, the number of different members be no more than 60 or 50, and preferably the number of different members between 5 and 40, for example 5 and 30 Between individual. And for more complicated target, for example have complicated or commutative surface protein or The virus that comprises several virus subtypes comprises the polyclonal antibody group of 20 to 500 different members Compound is just enough. For very complicated target (wherein antigen comprises many different moleculars), May require to comprise the polyclonal antibody composition of 50 to 10,000 different members.

The known example of the polyclone protein of several natural generations is arranged in mammal, described Polyclone protein has for example antibody or the immunoglobulin molecules that freely circulates in blood, Or be present in for example φt cell receptor and the B-cell receptor of cell surface. Some mammal In, carry out Genetic Recombination by the gene to these protein variable regions of encoding and can obtain these days The right polyclone albumen qualitative diversity that takes place. Know that further antibody increases by somatic mutation Add its diversity. Be responsible for described diversity (for example immunoglobulin molecules or TcRs by separating Variable domains or CDR district) sequence and produce the library from it, the present invention can utilize these Natural diversity. To the protein by two separate gene fragment codings, for example antibody variable is heavy Chain and variable light chain, TcR α chain and β chain or TcR δ chain and γ chain, each carrier in the library The coded sequence pair of these variable regions will be formed. The coded sequence of variable region is created in the library This area is known.

The multifarious library that comprises natural generation is the combinatorial libraries (code sequence of variable region for example The row random pair) (coded sequence from homocellular variable region is joined with related pairing library Right). The library that further produces from the CDR genetic fragment of separating also can be used for the present invention, its Described in fragment be incorporated in the suitable framework district (Soderlind for example, the people such as E., 2000. Nat.Blotechnol.18,852-856) for example antibody or TcR variable region. Preferably sieve Select described library to obtain to have the specific inferior library (purpose library) of wanting.

The albumen qualitative diversity also may obtain by manual type, for example synthesizes or passes through and suddenly change. Sudden change can be rite-directed mutagenesis at random or that encode single nucleic acid sequences to proteins, produces thus Give birth to the polyclonal population of described single albumen. Another example that produces the artificial antibody library is described In EP 0 859 841, the method based on produce can be with other CDRs library combination variable The method in framework library, district.

In the preferred embodiment of the invention, described recombinant polyclonal protein is the many grams of restructuring Grand antibody or antibody fragment.

In another preferred embodiment of the present invention, described recombinant polyclonal protein is heavy Group polyclone TcR or TcR fragment.

Except the diversity that obtains by the heredity in so-called variable region and body cell restructuring Outward, the immunoglobulin (Ig) of the different isotypes of with good grounds heavy chain definition also. Main isotype is IgM, IgG, IgA, IgD and IgE.

Recombinant polyclonal protein of the present invention therefore also can be by different isotypes or preferred Form with subclass. The polyclone of immunoglobulin (Ig) can occur in described immunoglobulin molecules Constant portion or variable domains, or occur in simultaneously constant portion and variable domains.

Therapeutic for antibody is used, in the so-called constant region (the particularly heavy chain of antibody) Polyclone be interesting. The isotype of various immunoglobulin (Ig)s has different lifes Thing is learned function (being summarized in table 1), described various immune globulins when using antibody to be used for the treatment of White isotype may carry out suitable combination, because the different isotypes of immunoglobulin (Ig) can relate to And to innate immunity (Canfield and Morrison 1991.J.Exp.Med.173, 1483-91; The people such as Kumpel, 2002.Transfus.Clin.Biol.9,45.-53; The people such as Stirnadel, 2000.Epidemiol.Infect.124,153-162) difference The aspect.

Table 1: the biological function of human immunoglobulin(HIg) isotype

	Human immunoglobulin(HIg)
										IgG1	IgG2	IgG3	IgG4	IgA1	IgA2	IgM	IgD	IgE
	Classical complement activation	+++	++	++++	+	-	-	++++	-										-
Substituting complement activation										+	+	+	+++	+	-	-	+	-
	Placenta shifts	+	++	+	++	-	-	-	-										-
Bacteria lysis										+	+	+	+	+++	+++	+	？	？
	Macrophage/other phagocyte combination	+	-	+	+	+	+	-	-										-
Mast cell/basophilic granulocyte associativity										-	-	-	-	-	-	-	-	-
	The staphylococcal protein A reactivity	+	+	-	+	-	-	-	-										-

Further embodiment of the present invention is the polyclone type of production clone of restructuring, and is described thin Born of the same parents system comprises the cell aggregation of the library transfection that consists of with the different IPs acid sequence, wherein said set In each cell with member's transfection in the described library and can express this member, this member's coding The different members of the polyclone protein of being combined with specific antigen and be arranged in described set cell Identical single site in the genes of individuals group, wherein said nucleotide sequence are not natively with described Cell in the set is relevant.

In the additional embodiment of above-mentioned embodiment, described coding polyclone protein is (excellent Selection of land is from immunoglobulin superfamily) the different IPs acid sequence all from naturally occurring order Row for example separate from donor.

WO 02/44361 has described the cell composition that contains different nucleic acid, described nucleic acid position Genomic single specificity site in each cell. This piece document discloses the use cell and has identified Method with molecule of wanting characteristic, but the document does not relate to and production system is provided or provides Polyclone protein take specific binding antigen as feature.

Clonal diversity

One of feature of polyclone protein is that it is made up of many single protein molecules, wherein Other molecule homology of each protein molecular and described polyclone protein but still have changeability, institute State changeability and be take the difference of amino acid sequence between the single member of described polyclone protein as Feature. Preferably, described difference is limited to the distinct zones of described polyclone protein general structure The territory. This class zone is the variable region of antibody or TcR for example, and may be limited to further this The CDR district in a little zones. This changeability also can be described as diversity, can be at nucleic acid and albumen (for example on the specificity and affinity difference to target) is to described many on two levels of matter function Sample is identified.

By the separation clone from the cell aggregation of expressing recombinant polyclonal protein is used RFLP (or (RT)-order-checking of PCR product) can the clonal diversity of described clone be carried out Analyze. Also can be by the recombinant polyclonal protein that is produced by described clone being carried out the function inspection Survey (for example ELISA) and analyze described diversity.

Clone's otherness (the gradually changing of monospecific antibody content that namely forms polyclonal antibody), If exist, the clonal diversity in initial library that can be by relatively being used for transfection with express described The diversity of finding in the cell of polyclone protein (clone) set and estimating.

Clonal diversity by the polyclone protein of expression of cell lines can be estimated as described polyclone The target of protein covers. In this case, when the purpose branch of wanting near 25-100% Son then can be thought to have obtained enough diversity by described polyclone protein bound. For example In the example of polyclonal antibody, non-uniform on antibody and at least 25% the purpose antigenic surface The epitope combination then provides enough diversity in composition. Preferably, by target The clonal diversity of cover determining is 50% at least, and even more preferably at least 75%. Right In antibody, this target covers and can for example estimate by epitope mapping.

Alternatively, clonal diversity can be estimated as the single member of described polyclone composition Distribution. This distribution can be estimated as: with import at first described clone during the transfection The number of different coding sequence compare, different in described final polyclone protein compositions Single member's sum. In this case, when at least 50% and preferably at least 75% The coded sequence that is applied to transfection be accredited as the different single of described final polyclone protein During the member, can think to obtain enough diversity.

The single member's of described polyclone composition distribution also can be estimated as described single one-tenth Mutual distribution between the member. In this case, if there is not single member's group of described composition Become above 75% of single member's sum in the final polyclone composition, just think to have obtained enough Clonal diversity. Preferably, there is not single member to surpass 50% even preferred 25% Single member's sum with most preferred 10% final polyclone composition. By rflp analysis, (example is used for definite polyclone composition feature as described later for sequence analysis and analysis of protein Method) carries out estimating based on the clonal diversity of single member's distribution in the polyclone composition.

Clone's difference may cause the minimizing of clonal diversity, and described clone's difference can result from a) In clone's process, the result who b) changes as cell proliferation, or c) mixing by a plurality of integrons Assorted. If this species diversity produces, by being carried out littler change, method described herein just can Retrieving easily the clonal diversity in each this source loses.

In order to limit owing to variable domains is cloned into the difference that causes on the suitable carrier, can Design shifts to limit clone's difference to the genes of interest of another carrier from a carrier. Whole Body transfer techniques and the Escherichia coli of meticulously selecting to be used for amplification can reduce described clone's difference. In addition A possibility is to carry out the single transfer of each polynucleotide between carrier of the present invention, and is described The single member of the described polyclone protein of polymerized nucleoside acid encoding.

Might be in long period of time, the cell proliferation speed of single cell in the clone Variation difference can be incorporated in the described recombinant polyclonal protein expression, increase or reduce Existence by some member of the recombinant protein of expression of cell lines. This multiplication rate changes Reason is that the cell colony that is configured for the initiator cell system of initial transfection is heterogeneous. Single cell can the difference growth after the long time in the known clone. In order to ensure homogeneity more Parent material, by using the described cell of restricted dilution to be tied to the single cell level and with each Single cell is cultivated into new cell colony (the so-called inferior gram of cell that is undertaken by restricted dilution Grand) before with the transfection of purpose library, clone is carried out subclone. Then based on its breeding and Expression characterization is selected one or more such cell bodies as parent material.

Further, be used for guaranteeing to only have the cell of those acceptor site specificitys integration just can deposit The selection pressure of living may affect the proliferative speed of single cell in the multi-clone cell line. This can Can be owing to the preference that stands the cell of certain hereditary change in order to adapt to described selection pressure causes . So the selection of selected marker also can affect the difference that proliferative speed causes. If this Situation takes place, and should test different selected markers. In some cases, if choosing Select being based on the virulent material of cell, should test optimal concentration meticulously and selection is No interim when whole production all be essential or only at the incipient stage needs.

Another method of guaranteeing the good cell colony that limits is to make after transfection and selection course With fluorescence-activated cell sorting (FACS). FLA can be used for the cell of enrichment high yield, Described cell use by oneself the transfection of IgG construct cell aggregation (people such as Brezinsky, J.2003. Immunol Methods 277,141-155). This method also can be used for sorting and express phase Like the cell of the immunoglobulin (Ig) of level, be created in thus the cell colony of homogeneous on the productivity ratio. Equally, by using with fluorescent dye 5 6-Fluoresceincarboxylic acid oxalic acid succinimide ester (CFSE) mark can be selected the cell that shows similar proliferative speed by the FACS method.

Even the difference that causes when proliferative speed determines to have taken place, single member's loss or excessively Presenting may be not necessarily important, and this depends on described final recombinant polyclonal protein Diversity require and interior multifarious stability of a period of time.

In the single integrate body of locus specificity, described cell is variable at the antibody that will express only Variant on the region sequence. Therefore, by the different differences that impose with gene regulatory elements of integration site Impact cell is eliminated and cell proliferation speed is only had the impact of minimum degree. Mix with many The site is integrated and all can not be had problems at the proliferative speed of described type of production clone, because These are rare events. Random integration is usually with near 10^-5Efficient take place, site-specific integration is then with near 10^-3Efficient take place. If multidigit desirable other places o'clock sharp has produced problem, Selectable method is to repeat with the transfection of purpose vector library, can because described event is recurrent The energy property is very little, as described above. Among another replacement method embodiment 3 below Be described.

Another method of controlling undesirable clone's difference is with whole purpose vector library branch Become several subgroups to carry out transfection or on transfection time point early, be divided into described cell mass several Subgroup. In this, difference is should be also not obvious and should obtain statistically Lack the subgroup with undesirable breeding advantage. Consequent undesirable clone's Eliminating must be satisfied the multifarious requirement of final recombinant polyclonal protein. From statistics Consider that the whole transfection of a large amount of cells has also formed the method that overcomes undesired clone's difference. In this method, the whole transfection host cell in library that consists of with the different IPs acid sequence is. This The library construction of sample numerous copies of variant member in described library. These copies should be excellent Selection of land is incorporated in a large amount of host cells. The library that preferably consists of with the different IPs acid sequence The multicopy different members is advanced at least 100,1000,10000 or 100000 independent cells The row transfection. So, if the library that the different IPs acid sequence consists of is integrated into separately by 1000 Unique member in 1000 independent cells consists of, and should produce 10 so from transfection⁶Individual The clone who contains the GOI of site-specific integration. By this method, single cell times speedup The shallow type of the Gauss of rate will only affect whole colony with very little degree. This will increase along with the time Passage keeps the constant probability of clone's composition, even the described type of production cell exhibition of low percentage Now unusual growth and/or expression characterization.

Alternatively, the purpose vector library can be divided into and contain about 5 to 50 described literary compositions The part of the single carrier in storehouse. Preferably, the part in described library consists of 10 to 15 lists One carrier. Then each several part is transfected in the equal portions cell. Then allow described each equal portions cell warp Whether any one clones difference in them to observe after a while. If take place, Before remaining equal portions cell compiled the reconstituted cell set this equal portions cell is removed. Appoint Selection of land keeps separating at equal portions cell described in the whole production process, and described Anti-TNF-α The body composition is to form by the product that makes up each equal portions thing, rather than by before production With the equal portions groups of cells altogether. The manipulable number that compiles body is expected between 5 to 10 (referring to before to the description of monoclonal antibody).

Alternatively, can implement high throughput method, in described high-energy method, use the carrier branch Other transfectional cell, cell is based on from the monospecific polyclonal in the initial library of described purpose carrier and select. This will eliminate any possible sequence difference between transfection and integration period. At random, can determine If the genotype of single transfectant and suitable can be just combination before production or more early the time Go out abundant multifarious cell aggregation. As selection, will be single before producing polyclone protein When solely the cell of transfection compiled, (generation had the different IPs acid sequence in the independent transfection of a large amount of cells Many clones of unique member in the library that consists of) can produce the statistics identical with whole transfection On favourable aspect.

Host cell

Suitable host cell comprises one or more suitable restructuring positions at its genome area Point, the nucleotide sequence of namely being identified by one or more recombinases. For selecting integron, (namely Integrated the cell of purpose nucleotide sequence copy at integration site), with recombination site effectively Be connected to first Select gene of being positioned at described integration site 3 ' (antibiotic resistance base for example Because of). In addition, weak promoter (for example SV40 early promoter of brachymemma) and transcription initiation are close Numeral can be positioned at constitutive resistance mark-coding region integration site recombination site 5 '. So, before the library transfection with the described polyclone protein expression carrier of coding, described The transcription initiation codon starts transcribing of described Select gene in host cell.

The host cell that is used for as described above site-specific integration can be from integrating DNA To its chromosome or keep outside the chromosome component for example minichromosome, YACs (yeast manually dyes Colour solid), MACs (mouse artificial chromosome) or HAC's (human artificial chromosome) is any Produce in the cell. In WO97/40183, describe MACs and HACs in detail, quote work herein Be reference. Preferably use mammalian cell for example Chinese hamster ovary celI, COS cell, bhk cell, Myeloma cell's (for example Sp2/0 or NS0 cell), fibroblast for example NIH 3T3 and The Immortalized human cell is HeLa cell, HEK293 cell or PER.C6 for example. Yet also can To use nonmammalian eukaryotic or prokaryotic, for example plant cell, insect cell, Yeast cells, fungi, Escherichia coli etc.

In one embodiment of the invention, by carrying out so-called restricted dilution with cell System is diluted to the individual cells level, follows before with the library transfection of purpose expression vector single thin Born of the same parents cultivate into new cell colony, come the clone as parent material is carried out subclone. As Fruit needs, and carries out in the clone process that this subclone also can be selected to be fit to afterwards.

Can obtain for the host of site-specific integration thin by the plasmid transfection with random integration Born of the same parents, described plasmid comprises weak promoter (for example SV40 early promoter of brachymemma), has transcribed The beginning codon, be positioned at the recombination site of described initiation codon 3 '. Preferably, described integration Plasmid also comprises the marker gene that is connected with first Select gene. One of this integrated plasmid Example is the pFRT/LacZeo2 from Invitrogen (Carlsbad, CA). Described mark Gene can be used to estimate the relative expression's intensity for the genomic locus that inserts purpose nucleic acid. Can With marker gene (for example beta galactosidase (LacZ), green fluorescent protein (GFP) or Cell surface marker) with the form of fusion or by IRES (internal ribosome entry site) Be connected on first Select gene to transcribing property, so that first Select gene and marker gene Coexpression. Being used in combination of Select gene and mark is to guarantee to keep in genome described integration The effective ways of the high yield type cell of plasmid, described Select gene is set up survival pressure to cell (for example drug resistance or nutrition loss), described mark allow to estimate to be tied to clone from cell Relative expression's level. Cell with recombination sequence will cause for example GFP of described marker gene Or the expression of the height of LacZ, wherein said recombination sequence is inserted into the position with remarkable transcriptional activity The point on. Cell sorting (FACS) by fluorescence-activation is selected the high expressed person and is cloned. Whether at this moment also should analyze described integron is single integron. This can pass through PCR in real time Analyze with the Southern blotting.

Relative expression's water of the cell of another integrated plasmid transfection of be used for estimating to use by oneself Flat method is that the cell that produces is as described above carried out extra integration-excision step. Again With plasmid and second plasmid transfection of coding corresponding to the recombinase of described integrated plasmid recombination site This selected cell aggregation, described second plasmid contain second choosing with initiation codon Selecting mark, was equally corresponding to first integrated plasmid before the code area of second selected marker Recombination sequence. This second plasmid also contains coding fluorescence labelled protein (mammal for example The GFP of promoters driven (or equal fluorescin)) sequence. This germplasm of recombinase-mediated Grain is incorporated on the host cell gene group, will similarly recombinate by integrated plasmid before this Sequence is inserted in the described host cell gene group. Have to be inserted in and have remarkable transcriptional activity position The cell of the recombination sequence on the point will cause the height of described fluorescin to be expressed. Swash by fluorescence Living cells sorting (FACS) is selected the high expressed person and is cloned. With recombinase transfection and logical Crossing first selected marker selects to have consistent high expressed and contains described insertion plasmid of copy The clone identifies the cell removed of reorganized enzyme of second plasmid sequence wherein, makes described first Selected marker is reworked. These cells still contain to be inserted into transcribes the restructuring of first on the focus Sequence and can be used for the expression genes of interest now.

Behind single copy plasmid integration, obtain clone that described marker gene height expresses be used for into The transfection of row genes of interest. Recombination site in the host cell is preferably located in has remarkable expression Active gene or zone namely are positioned at so-called focus.

The carrier that is used for site-specific integration

Suitable carriers contains the suitable recombination site that is connected on the suitable selection gene, and described selection gene is different from the selection gene that is used to make up host cell.The gene of suitable selection gene that is used for mammalian cell expression, for example Thymine deoxyriboside kinase gene (TK), glutamine synthetase gene (GS), tryptophan synthetase gene (trpB) or group ammonia alcohol dehydrogenase gene (hisD) including, but not limited to allowing nutrition to select.Further, selective marker is to give the anti-metabolic thing resistant gene of drug resistance, and for example available xanthoglobulin and Thymine deoxyriboside lack the dihydrofolate reductase gene (dhfr) that substratum is selected and further selected with methotrexate, the xanthine-guanine phosphoribosyl transferase gene that available mycophenolic acid is selected, in eukaryotic cell with G418 and the neomycin phosphotransferase gene (neo) that available Xin Meisu or kantlex are selected in prokaryotic cell prokaryocyte, hygromycin B phosphotransferase (the hyg that available Totomycin is selected, hph, hpt) gene, the blasticidin S deaminase gene (Bsd) that tetracycline N-acetyl-transferase gene (pac) that available tetracycline is selected or available blasticidin are selected.At last, coding can also can be used as selective marker by the proteic gene of for example selected by flow cytometry apoptosis, for example green fluorescent protein (GFP), trk C (NGFR) or other membranin or beta-galactosidase enzymes (LacZ).

Promptly be not activated son before one aspect of the present invention, described selection gene and also be not furnished with translation initiation codon.Provide promotor and ATG codon at selected locus specificity recombination site.If the position of this vector integration is not the recombination site of selecting in the host genome, make this second to select gene not express owing to lacking promotor and initiator codon so.Occur in the recombination site of selecting in the host cell gene group if integrate, second selects genetic expression and first selects gene not express.For example integrate with Flp recombinase or its mutant from Saccharomyces cerevisiae (Saccharomycescerevisiae) by the so-called FRT site (5 '-gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3 ' (SEQ ID NO 1) and its variant) of using the genome neutralization to be used on the carrier of site-specific integration.Yet, other recombinase system makes well too, comprise those Cre recombinases and various lox site, for example from phage P1 or its variant or mutant (lox66, lox71, lox76, lox75, lox43, lox44 and lox511 (C.Gorman and C.Bullock for example, Curr.Opinion inBiotechnology 2000, loxP 11:455-460)) or by using phage intergrase Ф C31 or lambda integrase, described lambda integrase (the A.C that between attP and attB site, recombinates, people such as Groth, PNAS 2000,97:5995-6000).Further can be used for recombinase system of the present invention and be but be not limited to from bacterial plasmid pSM19035 β-recombinase-6 system, from the Gln-gix system of phage Mu or from the R-RS system of zygosaccharomycesrouxii.

The further change of locus specificity reorganization is to use the non-homology recombination site.In this system, two inconsistent recombination sites are imported in the host genome to produce specificity purpose site.Recombination site corresponding to both sides, purpose site sequence also is positioned at the construct both sides of containing goal gene.Described this system in WO 99/25854, it is incorporated herein by reference in full.The use of non-homology recombination site can suppress GOI and excise from karyomit(e).Described non-consistent recombination site can be made up of any above-described recombination site, as long as corresponding recombinase is provided.For example, the nonuniformity recombination site can be formed or utilized the FRT site and the loxP site of Flp and the integration of Cre recombinase to form by FRT site that utilizes the Flp recombinase to integrate and mutant FRT site.

Further, at people's such as Verhoeyen Hum.Gene Ther.2001 12, the system of using two kinds of different FRT sites has been described among the 933-44.In this method, change described integrated plasmid over to host cell by retroviral infection.Described plasmid is by the reporter gene and first selectable marker gene and infect needed retrovirus combination of elements and constitute.Described retrovirus 3 ' LTR contains 2 different FRT sites.Non-functional second selectable marker gene that lacks promotor and translation initiation codon is positioned at 3 ' of these sites.3 ' LTR sequence is copied to 5 ' LTR in the retroviral infection process.This causes the reporter gene and the first selectable marker gene both sides respectively is a different FRT site.Can exchange with the GOI under strong promoter control in the sequence between the FRT site, the outside.The both sides of box of containing GOI are by same set of FRT site side joint.Described reaction is by the catalysis of Flp recombinase.IRES element and translation initiation codon further are positioned at the downstream of described GOI in the exchange plasmid of transfection.After replacing described integration box, the translation initiation codon that is provided by GOI structure box activates described no function selectable marker gene, and described gene is positioned at 3 ' the LTR sequence that is in the outside, described FRT site.If negative selectable marker (for example Thymine deoxyriboside kinases) is present on the described integrative vector, the exchange situation can further be strengthened.

Also can change integrative vector over to described host cell by the standard transfection.Described in this case integration box is 5 ' terminal 3 ' terminal by different FRT ' site side joints at it by the FRT side joint at it.Second resistant maker gene that lacks ATG is positioned at the more downstream in 3 ' FRT ' site.As the described method of retrovirus system is exchanged GOI.

Another stops GOI is described Ф C31 intergrase in its site-specific integration cut system behind the karyomit(e), also mentions in the above.At large described this individual system in patent application WO01/07572 and WO02/08409, it is incorporated herein by reference in full.

Of the present invention further aspect, the carrier that is used for the goal gene site-specific integration further comprises the proteinic member's of coding reorganization purpose polyclone DNA, adds before described DNA that randomly the mammalian promoter of himself instructs described protein expression.If containing, the proteinic member of purpose polyclone surpasses a protein chain, if for example the member is antibody or TXi Baoshouti, the mammalian promoter that added himself before the DNA of code for said proteins chain instructs each chain high level expression (two-way or unidirectional, respectively referring to Fig. 1 and 2).In two-way expression, can in expression vector, use promotor configuration head to head, and for unidirectional expression can use two promotors or combination for example the promotor of IRES sequence express.The suitable configuration of promotor head to head for example for but be not limited to AdMLP promotor and mouse metallothionein(MT)-1 promotor, the AdMLP promotor of both direction and the CMV promotor and the MPSV promotor of elongation factor-1 promotor or both direction of both direction.

The nucleotide sequence that expression vector can comprise the encoding function leader sequence is to instruct described gene product specific position organoid for example in endoplasmic reticulum or the cell.Strong polyadenylation signal can be positioned at coded protein dna sequence dna 3 '.Polyadenylation signal is guaranteed the termination and polyadenylic acidization and relevant with courier's stability of nascent RNA transcript.For example, the member's of coding recombinant polyclonal target protein DNA is the heavy chain and the light chain of encoding antibody or antibody fragment simultaneously, and each gene order randomly adds the mammalian promoter element of himself and/or connecting the high level expression that strong Poly a-signal instructs each chain in two chains thereafter in its front.

The expression vector portability that is used for site-specific integration is used to strengthen the other transcriptional regulatory element that integration site is expressed, for example enhanser or UCOE (the open element of omnipresence chromatin).Enhanser is to participate in the nucleotide sequence of the protein interaction transcribe specifically with in the cell.Thereby UCOE opens chromatin or keep the chromatin open state to promote the gene (more detailed description is arranged among the WO00/05393, and it is quoted as a reference in full) that effectively connects repeatedly to express herein.When being incorporated into host cell chromosome, one or more above-described controlling elements just claims that they are the allos controlling element.

Structure is used for the expression system of protein high level expression

Be used for the method for nucleotide sequence transfered cell is known in this area.These methods generally include uses dna vector with aim sequence transfered cell, genome or extra-chromosomal element.Can realize the transfection of pair cell comprising calcium phosphate precipitation, electroporation, microinjection, liposome fusion, the fusion of RBC blood shadow, protoplastis fusion etc. by many methods well-known to those skilled in the art.

Be transfection host cell system, the application target vector library, wherein each carrier only comprises the proteinic member's of coding reorganization purpose polyclone of a copy nucleotide sequence.The described reorganization purpose polyclone protein of the common coding in this purpose expression vector library.Part has been described the suitable carrier that is used for site-specific integration in front.The single carrier that constitutes various objectives nucleotide sequence library can mix and form single composition, and each library member's that perhaps encodes single carrier also can remain independent composition or form composition by about 5 to 50 single carrier library mixtures.

Can obtain recombinant polyclonal type of production clone and from the proteinic production of the recombinant polyclonal of this clone by several different transfections and production strategy.Fig. 3 has summarized these strategies.

A method is to utilize to mix the expression vector library transfection host cell system that forms single composition.This method is called whole transfection.Common previously described carrier and host cell design will be guaranteed can obtain polyclone clone by suitable selection.Most of cell individual is incorporated into the nucleic acid molecule of a copy from purpose nucleotide sequence library in the genome in this clone, the proteinic different members of described nucleic acid molecule encoding recombinant polyclonal.The nucleotide sequence of described single copy is incorporated into the single specificity site of each cellular genome in the cell aggregation, has produced the polyclone clone that is made of the cell individual of expressing the single member of described purpose polyclone protein thus.The refrigerated storage thing of the described polyclone clone of preparation before beginning recombinant polyclonal protein production.

Another method is to utilize the vector library that is divided into small portion to carry out transfection, and the vector library of described small portion contains the single carrier in about 5 to 50 described libraries in composition.Preferably, the library of described small portion is made of 10 to 20 single carriers.Then with each composition transfection in the equal portions host cell.This method is called the semi-monolithic transfection.The quantity of the equal portions cell of transfection will depend on the number of single carrier in the size in library and each small portion.If for example the library comprises 100 different members, described library is divided into the small portion that contains 20 different members in composition, need carry out transfection with the library composition of the different piece that constitutes former library to 5 equal portions host cells.Select described equal portions host cell to carry out site-specific integration.Preferably, different equal portions are separately to select.Yet they also can compile before selection.Can analyze and have only those to have the abundant multifarious polyclone GOI library stock that just can be used for producing the clonal diversity of described equal portions.For the polyclone clone that is used to produce that obtains to want, described equal portions can be taken out from stock afterwards or mix after of short duration propagation and adaptation time at them before producing the refrigerated storage thing.Randomly, described equal portions cell is kept separating in whole production, and mix to form described polyclone protein composition by product (rather than producing preceding equal portions cell) with each equal portions.

The third method is a high throughput method, and host cell carries out transfection with the single carrier that constitutes the purpose library respectively in described method.This method is called individual transfection.The host cell of described individual transfection is preferably selected according to site-specific integration respectively.Yet, before selection, also they can be compiled.The breeding time and the integration mode of cloning according to the single cell of selecting to produce are analyzed, and preferably, those cell clones with similar growth velocity and single site-specific integration are used to produce polyclone GOI library stock.Before producing stock, after just from stock, taking out or after, described single cell clone can be mixed the polyclone clone of wanting to obtain through of short duration breeding and adaptation time.

This method can be eliminated any possible residual sequence difference during transfection, integration and selection.Perhaps, the host cell with described independent transfection before selecting mixes, and this will guarantee the control to the sequence difference that is caused by transfection.

The denominator of the production strategy of summarizing above is can produce all in one or finite population (maximum about 10 s') bio-reactor to constitute the proteinic single members of recombinant polyclonal.Onlyly be not both the stage that people select to produce the cell aggregation that constitutes recombinant polyclonal type of production clone.

Being used for expressing and producing the proteinic host cell cording of reorganization purpose polyclone has one or more by recombinase (for example, as US5, having of describing in 677,177 is arranged in the previously prepared cell that genome pre-determines the FRT site on the seat) nucleic acid molecule of identification.

The carrier that is used for site-specific integration preferably is incorporated into the genome seat of predetermined mediation high level expression (so-called focus).

Increase expression level if desired, can utilize gene amplification is carried out in the selection of DHFR gene or glutamine synthetase (GS) gene.This requires to use the carrier that contains this selective marker.

In order to produce polyclone protein (wherein each protein member constitutes by surpassing two polypeptide chains), the combination of described chain has vital role to proteinic avidity, specificity and the activity of its formation.This can be found out from for example antibody and TcRs.For example, the combined effect of known antibodies variable heavy chain and variable light chain is by the avidity and the specificity of the antibody of described chain formation.So,, need guarantee the combination of variable heavy chain and light chain in this final product of correspondence when selecting to produce and certain object when having the antibody coding sequence library of antibody of avidity.Therefore the polypeptide chain that will constitute the proteinic single member of polyclone is put into the same vehicle that is used for integrating, and guarantees that thus they all the time together in whole process.

Following description is an example that how to obtain recombinant polyclonal antibody express cell system, and mixing of wherein said chain is minimum if present.

Structure has the sub-box of generally starting of the constitutive expression of two promotors that place opposite transcriptional orientation, the head to head configuration that surrounds by variable heavy chain and whole kappa light chain for example, to allow changing whole construct over to be used for site-specific integration carrier, described carrier comprises FRT site and hygromycin gene and CH.Can expect the promotor box that also can use abduction delivering.In addition, can tail promotor being placed on tail ground rightaboutly transcribes or places to the mode of transcribing with tail enemy's folk prescription to cause.Use in this experiment can stably express lacZ-Zeocin fusion gene the CHO-Flp-In cell (Invitrogen, Carlsbad CA), make described cell resistance microbiotic Zeocin.Described cell maintains on the substratum that contains Zeocin.Described cell carries out whole transfection with the vector library that is used for site-specific integration of coding polyclonal antibody and different choice mark (hygromix phosphotransferase) together with the plasmid of expressing the Flp recombinase.Inducible promoter also can be used to control and expresses.After the transfection, described cell is cultivated under the situation that has Totomycin to exist.There is the cell of resistance to be grown in subsequently in the different culture systems to Totomycin, for example traditional a small amount of is cultivated narrow-necked bottle, nucleic acid multi-layered cell factory, (MiniPerm INTEGRA-CELLine) arrives hollow-fiber bioreactor with rotary flask to little high yield bio-reactor.Detect the antibody production of described cell with ELISA.With the long-term suspension culture of polyclone clone on the serum free medium of no selective pressure to select its viability.The clone stock is cultivated under the situation that has Totomycin to exist.

Estimate the maintenance of polyclone in the expression system

According to the present invention, guarantee usually that aborning the polyclone of expression system does not take place by serious the change is important, when having changed really, polyclone just stops production so possibly.Guarantee this point according to the present invention by relative expression's level of monitoring various nucleotide sequences.For example described expression level can be by for example using RLFP analysis, array or PCR in real time on the mRNA level or use that for example two-phase polyacrylamide gel electrophoresis, mass spectrum or various chromatographic technique are monitored on protein level.Just may establish basic values by these technology to many all individual expression levels, take a sample from culture at production period then and whether changes to measure expression level (overall with relative two aspect).In normal practice of the present invention, can establish numerical range, and if find that relative expression's level goes beyond the scope with regard to termination production around basic value.

For stability and the productivity that can estimate expression system, the preparation coding has anti-chicken egg white, ox alkaline phosphatase (AP), people β ₂-microglobulin (β ₂M), six segmental carriers of different Fab (mini six library) of people's haptoglobin (HAP), human factor VII I (FVIII) and egg albumen lysozyme activity.Described different Fab fragment coding sequence is inconsistent so shows different RFLP collection of illustrative plates that therefore available rflp analysis genotype is formed.

By the expression vector transfection of promotor box imports the CHO-Flp-In cell with described mini sixlibrary with having head to head.Both can use the whole transfection CHO-Flp-In of the mixture cell of the purpose expression vector of six different antibodies of coding, express the polyclone clone of described six antibody to produce by known combination, also can use single ground of a member transfectional cell of purpose expression library, then mix cells transfected, produce the recombinant polyclonal antibody express cell system of expressing described six antibody by known combination.Just may detect mammalian cell in this way whether transfection takes place, and can be to the clone's of one or several described recombinant polyclonal antibody express cell system generation difference.In addition, may be used to check breeding difference or the difference that causes by the purifying of polyclonal antibody composition.

Make up antiovalbumin recombinant polyclonal antibody type of production clone

By using phage display and ELISA to identify that relevant clone is to select ovalbumin in conjunction with phage clone.Two kinds of devices are used for identifying from the antibody of ovalbumin in conjunction with the clone, promptly based on the elisa plate that covers with ovalbumin or based on the high-density screening method (HDS) that ovalbumin is fixed on the pvdf membrane.Obtain the antibody group in this way, some identifications wherein are fixed on the ovalbumin on the elisa plate, and other then discerns the ovalbumin that is fixed on the pvdf membrane.

The ovalbumin of selecting may have it in conjunction with phage clone and be connected to variable heavy chain and kappa chain DNA sequence on the mammalian promoter, and forward to be used for antibody expression pSymvc20 type (Fig. 4 D) carrier to produce clone's set of pSymvc21 type (Fig. 4 E).Described CHO-Flp-In cell both can be cloned mixture with pSymvc21 and be carried out whole transfection, can carry out individual transfection with single pSymvc21 antibody expression plasmid again, mixes the transfectional cell of expressing other ovalbumin binding antibody then.Can be by the monospecific antibody express cell being carried out dna sequencing, TaqMan PCR and rflp analysis and the mixtures of antibodies that produces being carried out the analyses of ELISA, two-phase (2D) liquid chromatography (LC) (LC) and mass spectrum (MS) monitor the process of making antiovalbumin polyclonal antibody type of production clone.

Culturing cell and production recombinant polyclonal antibody

The polyclone clone that produces is as described above cultivated in suitable medium being suitable for expressing under the proteinic condition of described purpose polyclone, and described purpose polyclone protein is by the different nucleic acid sequence encodings that are inserted in the cellular genome.Can carry out cell cultures by several steps.In a first step polyclone clone is selected site-specific integration.When using mammalian cell, preferably make selected cell adapted suspension culture and serum-free condition.This can carry out by one or two step and being with or without under the situation of selective pressure.After adapting to conditions suitable, described polyclone clone can begin amplification test.At this moment the working cardial cell stock can thaw.Preferably use the bio-reactor between 30 and 100 liters, but also can use littler or bigger bio-reactor.The production time that is fit to and the selection of bio-reactor size are depended on the protein output wanted from this batch and from the expression level of described clone.The variation in reaction times can be from several days to three months.The recombinant polyclonal protein of expressing is separated from cell or supernatant liquor.According to method well-known to those skilled in the art described recombinant protein is carried out purifying and evaluation.List the example of purifying and authentication method below.

Purification of Recombinant polyclone protein from culture supernatant

It is possible using various chromatographic techniques that specific protein is separated from culture supernatants, described chromatographic technique is a difference of utilizing the protein physics-chem characteristic, for example molecular weight, static charge, hydrophobicity or to the difference of specific ligand or proteinic avidity.So can use gel permeation chromatography or use ion-exchange (anionic/cationic) chromatography or use chromatofocusing isolated protein according to molecular weight according to static charge.Similarly, use hydrophobic interaction chromatography or the separable protein of affinity chromatography according to hydrophobicity, described affinity chromatography is the difference of utilizing particular fixed part or proteinic avidity.So the combination by a series of various chromatographic theories can obtain the separation to the protein complex mixture.So use ion exchange chromatography at first protein mixture to be separated according to static charge for example, and the protein with similar static charge then uses gel permeation chromatography to separate according to molecular weight or uses the interaction chromatography to separate according to hydrophobicity under the situation that high density selects salt to exist.

For example ion exchange chromatography, hydrophobic interaction and gel-filtration have been usually used in from different material sources for example IgG purification (polyclonal and monoclonal) and TcR ascites liquid, cell culture supernatant and the serum affinity chromatography in conjunction with the subsequent purification step.Protein affinity purification is kind the easy method of row fast, wherein separates to be based on described protein and to be connected to reversible interaction between the specific ligand of chromatography substrate, and it can provide highly selective, high yield and be concentrated in the smaller volume.A-protein and protein G are two kinds of bacterial cell surface proteins, the Fc zone there is very high avidity, and its immobilization form has been used for the application of many routines, comprises polyclone IgG and subclass and absorption and the purifying immunocomplex of purifying from each species.

After affinity chromatography, can carry out downstream chromatographic step for example ion-exchange and/or albumin A and the DNA of hydrophobic interaction chromatography to remove host cell proteins, seepage.

Gel-filtration as the final purification step can be used to remove depollute molecule for example dimer and other aggregate, and sample is changed in the storage buffer.Be necessary to comprise that according to source and expression condition extra purification step is to obtain the antibody purity of the level that required.So antibody that often uses hydrophobic interaction chromatography or ion exchange chromatography and a-protein and gel permeation chromatography to combine and be used for the treatment of with purifying.

For the antibody of other kind of purifying, must use other affinity chromatography medium, because a-protein and G do not combine with IgA and IgM.Can use immunoaffinity purification (will resist IgA or anti-IgM monoclonal antibody to be connected to the solid phase) or use the rapid purifying strategy of multistep that comprises ion-exchange and hydrophobic interaction chromatography.

Structural characterization

Because the complicacy (polyclone diversity and glycosylation) of described mixture, polyclone the protein for example structural characterization of antibody and TcRs require high resolving power.The traditional filtration of method example gel, ion exchange chromatography or electrophoresis may not have enough resolving power to distinguish single antibody.Used two-phase polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrum (MS) subsequently or liquid chromatography (LC)-MS that the protein mixture (for example protein group) of complexity is characterized.Very useful on polyclone, few clone and the monoclonal immunoglobulin of the 2D-PAGE of the verified separation method based on protein electric charge and quality in distinguishing serum sample.Yet this method has some limitation.How chromatographic technique particularly is used to the peptide mixt of Analysis of Complex with electrospray ionization MS link coupled kapillary and LC Chi Lai more.LC-MS has been used for identifying the feature of monoclonal antibody and also has been used for sign to the polyclonal antibody light chain in recent years.Analysis to unusual complex sample requires stronger chromatographic system resolving power, and described resolving power can obtain by separating with two-phase (or more heterogeneous).This method can combine with MS alternatively based on carrying out ion-exchange and carry out reverse phase chromatography (or hydrophobic interaction) mutually second in mutually first.

Function characterizes

Polyclone protein by for example with have to identical object specificity in conjunction with or the polyclone albumen of similar vigor compare research and identify feature on the function.This research can be carried out in vitro and in vivo.

It can be immunoprecipitation for example that the external function of polyclonal antibody characterizes, and described immunoprecipitation is to be used for slightly put forward the high degree of specificity technology of the target antibody analytical separation of lysate from cell.By combining of immunoprecipitation and other technology, for example then carry out protein staining (Coomassie brilliant blue, silver dye or biotin labeling) and/or immunoblotting after the SDS-PAGE, just may for example detect and quantitative antigen, so estimate some functional performance of described antibody.Although this method is not only estimated the number of antibody molecule but also do not estimate its binding affinity, it provides target protein matter visual with and specificity.This method may be used for monitoring antibody to the potential difference of antigen (integrity of clonal diversity) bonded during the expression process.

Function characterizes and can pass through for example infection research in the body of polyclonal antibody.Laboratory animal for example mouse can be used for example specific virus infection, and the polyclonal antibody of the described virus in pin ground has developed.Infect repressed degree and can show the function of described polyclonal antibody.

Therapeutic composition

In embodiments of the invention, contain the recombinant polyclonal protein that is selected from immunoglobulin superfamily and can be used to treatment or prevention Animal diseases, for example be selected from cancer, infection, inflammatory disease, allergy, asthma and other respiratory disease, autoimmune disorders, dysimmunity, cardiovascular disorder, central nervous system disease, metabolism and endocrinopathy, transplant rejection and the disease of the pregnancy do not expected etc. as the pharmaceutical composition of activeconstituents.Described Mammals is people, the animal of raising and train or pet preferably.

In the preferred embodiment of the invention, described pharmaceutical composition comprises that recombinant polyclonal antibody or antibody fragment are as activeconstituents and the acceptable vehicle of pharmacology.

In another preferred embodiment of the present invention, described pharmaceutical composition comprises recombinant polyclonal TXi Baoshouti or TXi Baoshouti fragment as active ingredient and the acceptable vehicle of pharmacology.

In order to treat or preventing infection, pharmaceutical composition according to the present invention contains the microbial reaction or the bonded purpose recombinant polyclonal protein that can for example be selected from bacterium, mycobacterium, virus, mycoplasma, Rickettsiae, spirochete, protozoon, fungi, worm and epizoon with infective micro-organisms.

Recombinant human polyclone protein can be with the unit dosage form dispenser in pharmacology acceptable diluent, carrier or vehicle.Traditional pharmacology practice can be used for providing appropriate formulation or composition, for example suffers because the patient of the disease that excessive cell proliferation causes so that described mixture is administered to.Dispenser may begin to carry out before patient has symptom.Can use any suitable route of administration, for example administration can be in in parenteral, intravenous, endarterial, subcutaneous, muscle, the head, in the socket of the eye, eye, intraventricular, the capsule, intravertebral, the pond, endoperitoneal, nose is interior, aerosol, suppository or oral dispenser.For example, the treatment preparation can be with liquor or form of suspension; For oral dispenser, preparation can be the form of tablet or capsule Chewing gum, paste, the composition that is fit to be applied to skin can be creme, ointment, washing lotion, gel, mat or other form, the composition that is fit to be applied to vagina or urogenital tract mucous membrane can be vaginal suppository (vagitories), gel or other form, for preparation in the nose then be powder, nose drips or aerosol form.

Pharmaceutical composition of the present invention prepares by known methods, for example, processes by traditional dissolving, freeze-drying, mixing, grinding or massecuite (confectioning).Learn according to conventional medicament and to put into practice (for example referring to, Remington:The Science and Practice ofPharmacy (20 ^ThEd.), ed.A.R.Gennaro, 2000, Lippincott Willianms﹠amp; Wilkins, Philadelphia, PA and Encyclopedia of PharmaceuticalTechnology, eds.J.Swarbrick and J.C.Boylan, 1988-1999, MarcelDekker, New York, NY) compounding pharmaceutical composition.

Preferably use active ingredient solution, also have suspension, particularly isotonic aqueous solution or suspension, for example only containing activeconstituents or for example under the situation of seminose, can prepare this class solution or suspension before use very much together with carrier.Described pharmaceutical composition may sterilize and/or may comprise vehicle, sanitas, stablizer, wetting agent and/or emulsifying agent, solubilizing agent, in order to regulate the salt and/or the damping fluid of osmotic pressure, and be prepared by known methods, for example by traditional dissolving or freeze-drying method for processing.Described solution or suspension can contain the material that increases viscosity, for example Xylo-Mucine, carboxymethyl cellulose, dextran, polyvinylpyrrolidone or gelatin.

Injectable composition is prepared by common mode at aseptic condition; Also pack in ampoule or the bottle described composition and sealed vessel with the same manner.

The pharmaceutical composition that is used for oral dispenser can obtain by activeconstituents is combined (if wanting the gained granulating mixture) with solid carrier, if want or necessity, after adding suitable vehicle, mixture is processed into tablet, drageeing (drage core) or capsule.Also can be integrated in the plastic carrier that allows activeconstituents to spread lentamente or discharge with quantitative manner.

Described pharmaceutical composition comprises from about 1% to about 95%, preferably from about activeconstituents of 20% to 90%.According to pharmaceutical composition of the present invention can be the form of unitary dose for example, for example with ampoule, bottle, suppository, drageeing, tablet or capsular form.

Described preparation can give patient's dispenser to provide treatment to disease or pathological condition by treatment effective dose (for example, preventing, eliminate or alleviate the dosage of pathological condition).The preferred dose that is used for the medicine of dispenser may depend on this class parameter, as the total healthy state of the kind of disease and degree, concrete patient, preparation and its drug delivery route of compound vehicle.

If desired, use treatment more traditional therapy combinations together of recombinant human polyclonal antibody.For example in treatment for cancer, this combined therapy can be taked to perform the operation or use the form of chemotherapeutic or other cancer therapy drug.

In another embodiment of the invention, pharmaceutical composition according to the present invention comprises microbial reaction or the bonded purpose recombinant polyclonal protein that for example is selected from bacterium, mycobacterium, virus, mycoplasma, Rickettsiae, spirochete, protozoon, fungi, worm and epizoon with infective micro-organisms.

The treatment of composition according to the present invention is used

Pharmaceutical composition according to the present invention can be used for the treatment of, improves or prevent mammalian diseases.Can comprise cancer, infectious diseases, inflammatory disease, allergy, asthma and other respiratory disease, autoimmune disorders, dysimmunity, cardiovascular disorder, central nervous system disease, metabolism and endocrinopathy, transplant rejection and the pregnancy of not expecting with the disease of this medicine composite for curing.

One aspect of the present invention is the method that is used for the treatment of, improves or prevent disease in the animal, wherein uses the described recombinant polyclonal antibody or the antibody fragment of effective dose.Use the described recombinant polyclonal TXi Baoshouti or the TXi Baoshouti fragment of effective dose further.

The other aspect of the present invention is to utilize the produced in fragments of recombinant polyclonal antibody or recombinant polyclonal TXi Baoshouti or antibody or TXi Baoshouti to be used for the treatment of the composition of disease, the pregnancy that described disease is selected from cancer, infection, inflammatory disease, allergy, asthma or other respiratory disease, autoimmune disorders, dysimmunity, cardiovascular disorder, central nervous system disease, metabolic trouble, endocrinopathy, transplant rejection and does not expect.

Diagnostic use and environmental surveillance applications

The method that another embodiment of the invention relates to diagnostic kit and is used for the test kit of environmental surveillance applications and uses these test kits.Test kit according to the present invention comprises recombinant polyclonal protein prepared in accordance with the present invention, wherein this protein can with detectable mark carry out mark or not mark carry out non-marked and detect.If mark, described recombinant polyclonal protein can join and suspect and contain in the sample of target molecule, and the existence of described mark or lack the existence that shows described target molecule or lack.Detected sample can be a humoral sample, and for example blood, serum, blood plasma, spinal fluid, lymph liquid or urine or nonmammalian sample are for example suspected the sample from the environment source that contains pollutent.The nonmammalian sample can be water, air or Contaminated soil.Non-marked detects and comprises that the refractive of measuring in conjunction with back BIAcore changes, and wherein said recombinant polyclonal protein is used for the acquisition target molecule.

Embodiment

Following embodiment has described how to express and to produce recombinant polyclonal antibody in high yield person clone, and wherein goal gene/carrier has been inserted into karyomit(e) " focus " position of identifying in advance by site-specific integration.

In described embodiment, Chinese hamster ovary celI is used as host cell.Its favourable aspect comprises easy acquisition proper growth substratum, it grows up to highdensity ability and its effectively with the formal representation mammalian proteins matter of the biologic activity ability of antibody for example in cultivation.

Usually, carry out transfection according to traditional method according to colibacillary conversion of the present invention and mammalian cell.For promoting the understanding of the present invention, the structure of having described carrier as an example in the following embodiments is used for the application of the proteinic recombinant polyclonal type of production of express recombinant polyclone clone with it in production.

The following example is set forth the present invention, but should not be considered to the restriction to scope of the present invention.

Embodiment 1

Site-specific integration contrast random integration

For carrying out following transfection experiment, use the CHO-Flp-In cell (Invitrogen, Carlsbad, CA).Personnel selection secretor type alkaline phosphatase (SEAP) detects the efficient of described system as reporter gene.Prepare two plasmid construction bodies:

1. (Invitrogen, Carlsbad CA) (are used for random integration) SEAP to be inserted pcDNA3.1 hygro+

2. (Invitrogen, Carlsbad CA) (are used for site-specific integration) SEAP to be inserted pcDNA5/FRT

Two plasmid construction bodies are closely similar aspect controlling element (being promotor, polyadenylic acidization etc.), and what this make to use that described plasmid carries out random integration and site-specific integration relatively becomes possibility.

Use plasmid construction body 1 separately or use plasmid construction body 2 in conjunction with recombinase coding plasmid pOG44 transfection CHO Flp-In cell according to the method that Invitrogen describes.Use Totomycin to select the transfection body, and measure output from the SEAP of transfection body set.

Produce the cell that approximately infects by the site-specific integration cells transfected and Duo 6 times SEAP, proved the validity of described system and described clone than random integration.

Embodiment 2

Design and preparation are used for the expression vector at the host cell site-specific integration

Being suitable for site-specific integration can be assembled by following DNA element to the expression vector of host cell focus chromosomal region:

A) the FRT recombination site of connection hygromycin gene,

B) pUC duplicates super beginning site,

C) ampicillin resistance gene (bla),

D) the bla promotor that allows penbritin (bla) resistant gene to express,

E) gene, coding target protein matter (GOIs),

F) allow described GOI expression promoter and

G) randomly, additional transcription or translational control element, for example enhanser or UCOE ' s are to be increased in the expression of integration site or IRES.

In order better to understand the structure of expression vector, give each element more detailed description:

A) clone of having used the FRT recombination site that connects hygromycin gene to have most of single integrons with the integration that is used for the Flp recombinase-mediated and selection.Be not activated son before the described hygromycin gene and also do not assemble the transcription initiation codon, only add polyadenylation signal at 3 ' of gene.Employed FRT site is 5 '-gaagttcctattccgaagttcctattctctagaaagtataggaacttc-3 ' (SEQ ID NO 1).

B) comprised the permission pUC replication origin that the high copy number amount is duplicated in e. coli host cell.

C) comprised penbritin (bla) resistant gene (β-Nei Xiananmei) that allows to select the intestinal bacteria transformant.

D) allow penbritin (bla) resistant gene in the bla-of expression in escherichia coli promotor.

E) coding target protein the matter for example nucleotide sequence and all or part control antibody molecule regulation and control nucleotide sequence of expressing randomly of the GOI of the heavy chain of recombinant polyclonal protein, antibody, antibody and light chain and coding all or part antibody molecule constant region or variable region have been comprised.

The immunoglobulin (Ig) seat of heavy chain includes but not limited to that V, D, J and transition zone (comprise intervening sequence, be also referred to as intron) and with specific CH gene-correlation or adjacent flanking sequence, it also can comprise the zone (comprising intron) that is positioned at constant region or downstream.

Light chain immunoglobulin (Ig) seat can include but not limited to V and J district, its upstream flanking sequence and with described constant region of light chain gene-correlation or adjacent intervening sequence (intron), and it can comprise the zone (comprising intron) that is positioned at described constant region or downstream.

Modification for the constant region of all or part antibody, modification sequence of the present invention can include but not limited to have specific effect thing function, kind and/or source (for example, the immunoglobulin IgG of people or any other kind, IgA, IgM, IgD or IgE constant region) antibody constant region or modified the part constant region of antibody constant region activity or character; And coding is given the antibody the modified gene with other molecule of some new function, for example enzyme, toxin etc.

The gene of coding target protein matter can be connected to the nucleotide sequence that coding instructs the leader sequence that function arranged of gene product in the Secretory Pathway effectively.

In addition, coding target protein matter for example contain heavy chain and light chain polyclonal antibody GOI 3 ' can have strong polyadenylation signal.The application of mouse isotype IgG1 is not meaned and will be limit scope of the present invention as illustration purpose in the example below.

F) provide permission GOI expression promoter.Therefore, described and contained the box that is useful on expression promoter and enhancer element.In described expression vector, the mammalian promoter element of each chain high level expression in its oneself two chains of guidance is arranged before each antibody gene, unidirectional, two-way or tail is transcribed box and all can be used the tail direction.

In two-way expression, can use head to head promotor configuration (at US Patent No.5, describe the structure of this system in 789,208 in detail, it is incorporated by reference in this text examines).In unidirectional expression system, two promotors or a promotor also can be used for expressing in conjunction with for example IRES sequence.

In order to make up promotor head to head, respectively promotor is carried out the Pfu pcr amplification.5 '-primer will be from 5 ' the base of described promotor, and 3 ' end primer will comprise single restriction site, for example the XbaI site.After pcr amplification, described fragment is separated with sepharose and it is separated from glue with QiaQuick post (Qiagen).After this with the XbaI restrictive diges-tion, under 65 ℃, carry out 20 minutes heat inactivations, and fragment is carried out column purification with QiaQuick.Then described fragment is mixed and connect with intestinal bacteria ligase enzyme (New EnglandBiolabs (NEB)), described enzyme preferably connects sticky end.The connection mixture carries out pcr amplification to produce complete promotor head to head (promotor A/ promotor B) fragment with 5 ' primer of each promotor.This fragment (NEB) is carried out phosphorylation with T4 polynueleotide kinase (PNK), described enzyme carries out 20 minutes heat inactivations under 65 ℃, and described fragment is connected (the flat terminal) (pSymvc10 of pcr amplification (Fig. 4) fragment to the purpose carrier with T4 ligase enzyme (NEB), the primer and respectively the holding of promoter region that wherein are used to increase are annealed the All Ranges of amplification except that promotor).

Fig. 1 and 2 demonstration comprises the expression vector that is used for two-way and unidirectional expression promoter respectively.These promotors are used to illustrate, but do not limit the selection of promotor among the present invention.

G) in order to increase the expression of integration site and/or IRES, described expression vector can carry other transcribing and/or the translational control element, for example enhanser and/or UCOE ' s.

Embodiment 3

Appraisal to the polyclone preservation in the production system of exploitation

In order to estimate the stable and repeated of production system, the clone of the polyclone composition of different antibodies is expressed in preparation with known combination.Described polyclonal antibody composition is called the minisix composition.The encode nucleotide sequence library of described mini six composition is called mini sixlibrary.

(a) clone's starting point

Use the sequence of the Fab fragment (goal gene) of following coding and antigen 1-6 reactions in this embodiment:

1. ovalbumin (OVA).Described Fab encode fragment is selected from mouse-anti OVA phage display library.

2. alkaline phosphatase (AP).Described Fab encode fragment is selected from mouse-anti AP phage display library.

3. β ₂-microglobulin (β ₂M).(given by Dr.L. Ф .Pedersen, Denmark), it is at β from hybridoma BBM.1 for described Fab encode fragment clone ₂M and producing.

4. people's haptoglobin (HAP).The Fab encode fragment is selected from mouse-anti people haptoglobin phage display library.

5. Factor IX (FVIII).The segmental parent's monoclonal antibody of this Fab is that the FVIIIF25 monoclonal antibody (is given by Novo Nordisk, Denmark).The segmental V of this Fab will encode _HWith the DNA subclone of complete Kappa chain in phage, then the prokaryotic promoter box is inserted in the construct.

6. hen's egg-white lysozyme (LYS).This construct originates from D1.3scFv clone (Boulot, people such as G., J.Mol.Biol., 213 (4) (1990) 617-619), by to V _HAnd V _κSegmental pcr amplification also is cloned in the phage.

The e. coli strains that described phage clone is present in conversion is (being kept at-80 ℃) or exist as the phage DNA prepared product in the glycerine stock of TG1.

(b) rflp analysis of mini six library and dna sequencing.

Following by the encode nucleotide sequence of described Fab fragment heavy chain of rflp analysis: as to detect the band collection of illustrative plates that obtains by the fragment that produces with NlaIII and Hinf I enzymic digestion PCR.Different Fab fragment coding sequences represents collection of illustrative plates very different and that easily distinguish.To coding V _HAnd V _LSegmental nucleotide sequence order-checking and find out sequence corresponding to the RFLP collection of illustrative plates.In addition, described nucleic acid sequence encoding open reading frame and can translate into clearly the polypeptide of definition.

(c) elisa assay of mini six composition

With the described Fab fragment of elisa assay, wherein analyze all Fab fragments to all antigenic reactivities by clonal expression.Expression with anti-kappa ELISA monitoring Fab.All Fab fragments are carried out the secondary repeated test with ELISA.All clones express the Fab fragment, and the relative antigen of described Fab fragments specific reacts.In elisa assay, do not find background problems.

The e. coli strains that described 6 phage clones are present in conversion respectively is in the TG1 glycerine stock, uses described clone at the modular system that is used for inoculation as described below.

(d) design has the polyclone modular system of 6 different antibodies of known combination.

By Fab fragment that detect to express the reactivity of related antigen is determined that the Fab cloning by expression of described 6 selections (expresses anti-OVA, anti-AP, anti-β ₂The segmental clone of Fab of m, anti-HAP, the anti-FVIII and anti-LYS) characteristic.These clones have formed the part of polyclone modular system, and described modular system is used to test the expression and the output of 6 different antibodies of known combination (described mini six composition).All Fab coding nucleotide sequences (described minisix library) are transferred to phage vector (shown in pSymvc10, Fig. 4 A).

(d.1) GOI shifts from the individuality of phage vector to the carrier that is used for the Mammals expression.

In this embodiment, goal gene (described mini six library) is undertaken by 2 footworks to the transfer of the carrier that is used for the Mammals expression from phage vector.The first step is to use the mammalian promoter box replacement prokaryotic promoter of direction head to head.Be that variable region, promotor box and constant kappa with GOI ' s transfers in the expression vector as described below after this step, and in Fig. 4, illustrate.

By using SacI/XhoI digestion, then cause changing into from mammiferous promotor, so head to head promotor box (promotor A/ promotor B) is inserted on each clone's the phage vector from the promotor of bacterium by connection.Then with EcoRI and NotI digestion with variable heavy chain, head to head promotor box (promotor A/ promotor B) and complete kappa chain (EcoRI/NotI fragment) move on on the expression vector from phage vector.

Schema at Fig. 4 has provided the individual example that shifts of each clone.This figure shows plasmid pSymvc10, wherein purpose heavy chain and kappa encoding sequence are (for example, gc032 OVA) is positioned on the phage vector, mammalian promoter box construct is connected on the described phage vector to replace the bacterium promotor, generation pSymvc12 by using the SacI/XhoI fragment to shift head to head.

From this construct, will comprise that by using NotI/EcoRI to shift the variable heavy chain encoding sequence of promotor box and complete kappa chain encoding sequence forwards on the Mammals isotype code carrier (pSymvc20).The final carrier (pSymvc21) that obtains is expressed purpose mouse antibodies (for example anti-OVA IgG1 antibody).

To shift from variable heavy chain encoding sequence, mammalian promoter box and the complete kappa chain encoding sequence of each clone among six clones respectively by the NotI/EcoRI transfer, produce mammalian expression vector pSymvc21, its antibody sequence of expressing each GOI coding is as mouse IgG1 antibody.

6 single pSymvc21 clones that contain described 6 GOIs save as TG1 glycerine stock.

In order to be transfected into CHO Flp-In cell, described TG1 stock is bred respectively, and at OD ₆₀₀After proofreading and correct the intestinal bacteria number, described 6 cultures are mixed and are used for the plasmid preparation.This plasmid prepared product (mini six library) that will comprise described 6 GOIs is used for the whole transfection to mammalian cell, and described mammalian cell is used for recombinant polyclonal antibody expresses.

(d.2) with described GOI ' s from the phage vector global transfer to being used for the carrier that Mammals is expressed.

Phage vector be will be arranged in and 6 different Fab fragments (anti-OVA, anti-AP, anti-β encoded ₂M, anti-HAP, the anti-FVIII and anti-LYS) GOIs (described mini six library) (herein for EcoRI/NotI fragment) with the mixture global transfer of described 6 vector construction bodies to being used for the carrier that Mammals is expressed, to produce the mixture of 6 different expression vectors.

It is the same with method described in (d.1) to relate to the experimental technique of global transfer, except it carries out with integral way, soon all 6 GOIs (the coding variable heavy chain comprises head to head promotor box and complete kappa chain) shift simultaneously with the form of 6 phage vector mixtures.

The preparation of mini six library plasmid

Plasmid prepared product 1 is meant described 6 kinds of phage vectors (having the antibody coding sequence that is contained on the carrier pSymvc10) blended plasmid prepared product.

Plasmid prepared product 2 is meant the plasmid prepared product that has 6 kinds of phage vectors of the encoding sequence that is contained on the carrier pSymvc12 in global transfer step 1 (referring to Fig. 4 C) afterwards, and described step 1 causes the exchange of prokaryotic promoter and mammalian promoter box construct.

Plasmid prepared product 3 is meant the plasmid prepared product after global transfer step 2 (referring to Fig. 4 D), described step 2 causes variable heavy chain, head to head promotor box and complete kappa chain exchange to mammalian expression vector (pSymvc21) from pSymvc12, so allow the antibody of 6 selections to express as total length mouse IgG1 antibody.

Determine the genotype of the TG1 cell that the plasmid prepared product that uses in the global transfer transforms

Transform the TG1 cell and on 2xYT (Sigma Y 2627) plate, after the incubated overnight, single clone is chosen mini six library is whole with electroporation.Choosing 180 in each experiment clones and hatched in the 2xYT liquid nutrient medium 4 hours in 96 well culture plates.Dilute with water equal portions culture, sex change also are used as pcr template.In all experiments, variable heavy chain is increased.The primer that is used for phage vector (pSymvc10-type) is:

5 '-GCATTGACAGGAGGTTGAGGC-3 ' (SEQ ID NO 2) and

5’-GCTGCCGACCGCTGCTGCTGGTC-3’(SEQ?ID?NO?3)

The primer that is used to have the carrier of mammalian promoter box is (a pSymvc12-type):

5 '-GCATTGACAGGAGGTTGAGGC-3 ' (SEQ ID NO 4) and

5’-GTGTCCACTCTGAGGTTCAG-3’(SEQ?ID?NO?5)

The primer that is used for the pSymvc21 construct is:

5 '-CAAATAGCCCTTGACCAGGC-3 ' (SEQ ID NO 6) and

5’-GTGTCCACTCTGAGGTTCAG-3’(SEQ?ID?NO?7)

Digest all PCR products to guarantee clear and definite gene type with NlaIII and HinfI simultaneously.Analyze described digestion fragment and show band with agarose gel electrophoresis with ethidium bromide staining.The fragment collection of illustrative plates that RFLP determines identifies Id number, and it is corresponding to the number of the single clone of each antibody in described 6 antibody of representative in clone's overall number of selecting at all.

(d.2a) integrally forward it to the Mammals carrier (two step TRAP) from phage vector after DNA increases in intestinal bacteria.

From described 6 e. coli tg1 glycerine stocks each prepares described plasmid prepared product 1, and described each stock comprises in 6 kinds of phage vectors that constitute mini six library.Stock is bred respectively, and at OD ₆₀₀After proofreading and correct the intestinal bacteria number, will described 6 kinds of culture balanced mix and be used for the plasmid preparation, formation plasmid prepared product 1.Distribute with the genotype that detects 6 kinds of phage vectors in plasmid prepared product 1 by TG1 cell that it is transformed into the electroreception attitude and the rflp analysis that carries out subsequently.Fig. 5 shows the distribution of different genotype in the TG1 cell.

Digest the plasmid prepared product 1 that contains the polyclone phage vector with SacI/XhoI, described phage vector is expressed the equal amount of mixture of the Fab fragment gene type of 6 selections.Head to head promotor box (CMV promotor/MPSV promotor) inserts by connecting then.Using the genotype distribution that in the TG1 cell, detects carrier promotor exchange back carrier from the DNA conversion back of Connection Step (Fig. 6).

Be coated onto described cell on the plate and be grown on big (245mmx245mm) 2xYT agarose plate, and preparation plasmid prepared product 2 comprises the phage vector of promotor box (pSymvc12) head to head with generation.

From plasmid prepared product 2, the variable heavy chain encoding sequence that will comprise promotor box and complete kappa chain with NotI/EcoRI digestion downcuts from phage vector and forwards in the carrier (pSymvc20) that contains mouse IgG1 constant region structural domain.This has formed the pSymvc21 collection of vectors, and described collection of vectors is expressed the variable region as 6 selected antibody clonings of total length mouse IgG1 antibody.

Perhaps described promotor shifts and can carry out in the Mammals carrier of coding isotype, the reversed order of restriction enzyme digestion, begin to digest target DNA is transferred in the Mammals carrier, be inserted into promoter region with SacI/XhoI restrictive diges-tion fragment then with NotI/EcoRI.

After changing variable heavy chain encoding sequence, promotor box and whole kappa chain encoding sequence over to expression vector, transform the TG1 cell to detect genotypic distribution by using DNA from second Connection Step (Fig. 7).Be coated in cell on big (245mmx245mm) 2xYT agarose plate and preparation (pSymvc21) plasmid prepared product 3, wherein in mouse IgG1 antibody framework region, express described 6 clones' variable region.

Described plasmid prepared product 3 can be used for that mammalian cell is carried out whole transfection and is used for the recombinant polyclonal type of production clone that recombinant polyclonal antibody is expressed with generation.

After DNA increased in Bacillus coli cells, the result of the global transfer from phage vector to isotype encoding mammalian carrier showed: 6 kinds of vector construction bodies breed respectively and mix (plasmid prepared product 1, Fig. 5) after, might obtain its equiblibrium mass distribution.The promotor box (plasmid prepared product 2, Fig. 6) after the exchange and insert mouse IgG1 isotype code carrier (plasmid prepared product 3, Fig. 7) after, can detect described 6 kinds of constructs with comparable level.

(d.2b) after plasmid prepared product 1 step, need not to carry out in the intestinal bacteria DNA cloning promptly from the phage vector global transfer to being used for the carrier (a step amplification method) that Mammals is expressed.

In order to exchange promotor, with the DNA of SacI/XhoI digestion from plasmid prepared product 1 (using 25 μ g herein), described DNA comprises the polyclone phage vector (pSymvc10) of the equal amount of mixture of expressing 6 kinds of Fab fragment gene types of selecting.The described SacI/XhoI carrier segments of purifying also is connected with promotor box (CMV promotor/MPSV promotor) head to head.Do not carry out any amplification after the exchange promotor, has the carrier of CMV/MPSV promotor box from phage vector, to cut out whole zone with NotI/EcoRI digestion with variable heavy chain encoding sequence, described variable heavy chain encoding sequence comprises promotor box and complete kappa chain encoding sequence, again with its global transfer to being used for the carrier that Mammals is expressed.In the variable heavy chain of will encoding, after the NotI/EcoRI fragment of described promotor box and kappa chain changes in the mouse IgG1 code carrier (pSymvc20), 6 expression vectors of selecting clone's variable region have been obtained under mouse IgG1 full length antibody environment, to express.Fig. 1 illustrates the composition of this expression vector.

Behind the carrier that is used for mammalian cell expression, detect genotypic distribution at the global transfer that causes promotor exchange and the nucleotide sequence that shifts coding variable heavy chain district, promotor box and complete kappa chain by using plasmid from second Connection Step to transform the TG1 cell.Be coated in described cell on big (245mmx245mm) 2xYT agarose plate and prepare plasmid prepared product (the carrier pSymvc21 of this double digestion/be connected to form, wherein under mouse IgG1 antibody background, express 6 kinds of clones' variable region, corresponding to from d.2a plasmid prepared product 3).Fig. 8 is presented at and uses the genotype of the TG1 cell that transforms from a plasmid prepared product that goes on foot TRAP to distribute.

Because a described step TRAP can be owing to produce undesired connection product, thereby may in from the heavy chain of described 6 kinds of Fab encoding sequences and light chain, introduce some and mix, in intestinal bacteria, can avoid producing described mixing during the amplification under the normal circumstances.Yet,, can introduce the screening step to guarantee to keep enough clonal diversities if this specific admixture takes place.

(d.2c) promotor exchange back direct transfection mammalian cell

For the express recombinant polyclonal antibody, product from the plasmid prepared product of described mini six library can be used to direct whole transfection mammalian cell, and the plasmid prepared product of described mini sixlibrary not only can be from two step TRAP but also can be from a step TRAP.

(d.3) detect mammalian cells transfected

The combination of the known antibodies of described mini six polyclone modular system can be used to detect and guarantees the transfer and the transfection of mammalian cell are taken place by this way: in transfection with subsequently in the culturing process, kept clonal diversity and can't introduce difference in described antibody variable sequence gene type composition.Global transfer, whole transfection and Mammals are expressed and are used to monitor the method that genotype forms in the whole process and comprise as follows:

The dna sequencing of-separating clone

-single clone's rflp analysis

The elisa assay of the mixtures of antibodies of-described generation

The mass spectroscopy of the mixtures of antibodies of-described generation

The TaqmanPCR of-genome sequence and the relative composition of the mRNA that expresses different heavy chains and light chain

Deviation on the genotype introduced in transfection process is formed (if generation) since random integration or many integrons cause.As described in describe in detail, when use design in advance be used for the host cell of site-specific integration the time unlikely cause this problem.Yet available several different methods is controlled this deviation.Use very small amount of DNA for example when carrying out transfection with Lipofectamine DNA be 1 μ g/10 ⁷Cell maybe when with electroporation transfection DNA be 0.2 μ g/10 ⁷Cell can select to remove the integration on random gene group seat (except the seat at locus specificity seat).Also have, also can provide the DNA of the superhelix form that is unfavorable for random integration to integrate;

Eliminate by the single or multiple integration that negative selection will be positioned at outside the target site that designs in advance,, be not activated son or initiator codon because selectable mark will be arranged in genome.So these random integration incidents the recipient can not survive in chosen process.

Having many places integrates under the transformant of (wherein having a recombination event to occur in described target site) can survive in chosen process; Yet the probability of this many places integrated curriculum types is extremely low.So this incident causes minimized polyclone protein to mix.In addition, owing to there are 100 to 1000 proteinic other clones of the identical recombinant polyclonal of coding,,, many places take place then described mixing less than about 1% if integrating even ectopic expression is very high.As previously mentioned, the probability of ectopic integration can be reduced by employed DNA amount in the minimizing method.

Least possible incident is to incorporate in series a plurality of of target site.By using this incident of Flp recombinase system described herein, because its nicking activity on karyomit(e) is active apparently higher than integrating with rare.Yet, take place if incorporate in series with unacceptable high frequency, with the system that wherein can only insert single copy (referring to, for example, WO99/25854; Quote as a reference) the described Flp of replacing system.

(e) 6 kinds of different antibodies of expression known combination in mammalian cell

Usually in order to minimize the difference of proliferative speed, preferably each special GOI (being each single member of described mini six library in this example) is incorporated at least 100, preferably in 1000 and most preferred 10000 cells.Contain the less influence that is subjected to individual cells different reproductive speed of polyclone clone expection of the individual cells of the identical GOI of great expression (to all GOI ' s), and in final polyclone protein composition, can reduce the probability of difference.

Further, guarantee that the host cell that is used to express is that homology is favourable.This can realize by the described host cell of subclone before transfection system.In about the paragraph of clonal diversity, this process is described.

For hope to the further polyclone library of control of difference wherein, by introduce the final composition of the described polyclone protein of derivable transcriptional control element may command to described expression vector platform.Suitable induced controlling elements comprises, for example BD Tet on/off (BDBioscience, Franklin Lakes, NJ) and GeneSwitch (Invitrogen, Carlsbad, CA).These transcriptional switchings can be induced to minimize because any breeding difference that the difference of genetic expression or protein causes proper time point (for example, when cell aggregation fully increases).Do not use such controlling elements carry out this experiment.

No matter be as described in (d.1) individually or as described in (d.2) integrally with as described in after the GOI of 6 kinds of selections transfers on the mammalian expression vector from phage vector, by use site-specific integration with described mammalian expression vector transfection in the focus of CHO-Flp-In clone with 6 kinds of different antibody of expression as described below.

The GOI (d.1) that shifts for individuality is with plasmid DNA ' s breed respectively and individually transfection in the CHO-Flp-In cell or with described TG1 stock, breed respectively, and at OD ₆₀₀Behind the calibration intestinal bacteria number, described 6 kinds of cultures are mixed and are used for the plasmid preparation.With this whole transfection mammalian cell of plasmid prepared product that contains described 6 kinds of goal gene, to be used for the express recombinant polyclonal antibody.

For the GOI of global transfer (d.2a or d.2b), with plasmid DNA ' s is transfected into the CHO-Flp-In cell or is used for whole transfection then according to method amplification and purifying (plasmid prepared product 3) that (d.2a or d2.b) describes.

(e.1) cell cultures

With described CHO Flp-In host cell (Invitrogen, carlsbad CA) cultivates on Ham ' s F-12 substratum, and described substratum is added with glutamine (2mM) and FCS (10%) and 100 μ g/ml Zeocin (Invitrogen, Carlsbad, CA).For subculture, with trypsinase described cell is taken off wall and separate according to manufacturers instruction.Cell is grown in 5% carbonic acid gas, 37 ℃.Substratum and medium additives are from Gibco.

This clone is stably expressed the lacZ-Zeocin fusion gene, makes described cell can resist microbiotic Zeocin, and this resistance will be lost after external source gene locus specificity is integrated.Described cell contains Flp reorganization target (FRT) site of single copy, then be suitable as the host cell system that carries out site-directed integration, described site-directed integration used the Flp-In system (Invitrogen, Carlsbad, CA).

(e.2) transfection CHO cell

Inoculate 4.0x10 in each hole of 6 hole tissue culturing plates ⁵Individual CHO-Flp-In cell, and under O/N 37 ℃/5% carbon dioxide conditions, hatch.Carry out the transfection of these cells according to manufacturers instruction and use FuGENE with test ^TM6 (Roche), Lipofectine ^TM, LipofectAmine ^TMOr LipofectAMINE 2000 ^TM(Gibco) different transfection methods.In this experiment, LipofectAMINE 2000 ^TMAs transfection reagent.Tout court, in transfection the day before yesterday, the CHO-Flp-In cell that will be exponential growth is as described above inoculated and is hatched under O/N 37 ℃/5% carbon dioxide conditions.Have the hole that the 85-95% cell converges and be used for cotransfection.

Preparation contains two pipes of following inclusion:

Pipe 1: single expression vector (pSymvc21 that for example has OVA)+4.5 μ g mp040 (the maximum prepared product of pOG44) (plasmid of express recombinant enzyme Flp) that 0.5 μ g is had (d.1) described GOI joins the Optimem (the Eppendorf pipe of 1.5ml) of 250 μ l.

Pipe 2: with 7.5 μ l LipofectAMINE 2000 ^TMJoin the Optimem (1.5ml Eppendorf pipe) of 250 μ l and at room temperature (RT) hatched 5 minutes.

In pipe 1, then ovum was educated 20 minutes under RT with the substance transfer in the pipe 2.

According to manufacturers instruction described DNA-Lipofectamine mixture is transferred in the hole with cell.

After 24 hours, with the trypsinase described cell that comes off, separate (1: 3) and be assigned to the T-25 flask and the 100mm culture dish in, and cultivate in containing the fresh nutritional blend F-12 Ham+10%FCS+2mM L-glutaminate substratum of 900 μ g hygromycin B/ml as selective pressure.

(e.3) select site-specific integration and subculture transfectional cell

Cell was cultivated for 2 to 3 weeks under the hygromycin B selective pressure, changed the new substratum of the selective agent that contains same concentrations per 2 to 4 days of this period.To separate in the set of the survivaling cell in T-25 flask and the culture dish with trypsinase, and separate (1: 6) and be assigned in the T-flask further breeding under the selective pressure of mentioning in the above.Some single clones of picking from the culture dish that contains transfectant (using so-called clone's post (cloning cylinder)), and it is forwarded in the new hole to breeding and be used for expression level research, described transfectant produces according to method of describing in (d.1).

Can resist the hygromycin B concentration threshold each cell aggregation or single be cloned in to grow up on 6 orifice plates converge, again it is applied to and has corresponding burning base and add in culture dish, T-25, T-80 and the T-175 culturing bottle of hygromycin B.When index growthing cell grows into 80% converge in the T8-tissue culture flasks, that the bottle of each clone is freezing and be stored in liquid nitrogen N ₂(L) in the refrigerator.

In order to use plasmid mixture (the mini six library) transfection that contains 6 kinds of goal gene, at OD ₆₀₀The colibacillary number of the described 6 kinds of single cultures of place calibration is with its mixing and be used to prepare the polyclone plasmid prepared product that contains described 6 kinds of goal gene.Use 7.5 times of above-mentioned reagent and cell to carry out transfection, produce the recombinant polyclonal clone of the mixture of expressing described 6 kinds of different antibodies.

Cultivating and between nursery stage, the antibody production of the clone of described 6 clones of expressing single selected antibody member and the mixture of expressing described 6 different antibodies is being detected by using antigen-specific ELISA.

(f) composition of monitoring polyclone clone, 6 kinds of different antibodies of described expression of cell lines known combination.

Mixture by producing 6 kinds of selected goal gene then with its by whole transfection and site-specific integration in the CHO-Flp-In cell, produce the polyclone clone of expressing 6 kinds of different antibodies, described goal gene is positioned on the expression vector.

Genotype distribution, antibody expression and proliferative speed to described clone in ensuing 34 days are identified.The result is described below:

(f.1) genotype at the goal gene of 6 kinds of selections described in the CHO-Flp-In cell distributes, and wherein said CHO-Flp-In cell is used from OD ₆₀₀The plasmid prepared product transfection of the cell mixture of calibration.

Described polyclone CHO-Flp-In clone is diluted to 10 cell/ml with trypsin treatment and with cell suspending liquid.After this, 200 μ l are forwarded in each hole of 10 96 orifice plates altogether.After about 10 days, has single clone's hole by microscopic examination.The hole that has a single clone with PBS flushing once and add the water of 50 μ l.Dull and stereotyped hatching at 80 ℃ transferred to lysate in the 96 other orifice plates in 10 minutes.10 μ l lysates and following primer are used for 25 μ l OneStep RT-PCR (Qiagen).

5 '-CAAATAGCCCTTGACCAGGC-3 ' (SEQ ID NO 6) and

5’-GTGTCCACTCTGAGGTTCAG-3’(SEQ?ID?NO?7)

With HinfI and NlaIII the RT-PCR mixture of 10 μ l is carried out RFLP in 15 μ l reactive systems, described reactive system was hatched 2 hours in 37 ℃.Described digestion fragment is followed with ethidium bromide gel-colored demonstration with agarose gel electrophoresis.Anti-OVA, anti-AP, anti-β are determined to produce in for some time (after the transfection the 16th day and 34 days) back ₂The genotype of the cell of m, anti-HAP, the anti-FVIII and anti-LYS distributes, referring to Fig. 9.

(f.2) derive from the ELISA of the sample of CHO-Flp-In cell, described CHO-Flp-In cell is to use from OD ₆₀₀The plasmid prepared product transfection of intestinal bacteria (d.1) mixture of calibration.

With the described polyclone CHO-Flp-In of trypsin treatment clone (e.3) and with 3x10 ⁶Individual cell is applied in the T-75 culturing bottle that F-12 HAM+10%FCS+2mM L-glutaminate and 900 μ g Totomycin are housed.Every other day change a subculture and carry out ELISA at the 3rd day collection supernatant liquor.(B2M (being given by Copenhagen university), alkaline phosphatase (Sigma), ovalbumin (Sigma), Factor IX (Novo Nordisk gives, Denmark), hen's egg-white lysozyme (Sigma) and haptoglobin (Sigma)) are diluted to 10 μ g/ml in the 50mM carbonate buffer solution with antigen.Cover elisa plate and under 4 ℃, hatch O/N with antigen (each hole 50 μ l).Sealed 1 hour with dcq buffer liquid (1xPBS/0.05%Tween 20) flushing port 4 times and with 2% skim-milk (each hole 100 μ l) that dcq buffer liquid is prepared.50 μ l samples are joined in the hole and with plate under RT, hatched 1 hour.With plate flushing 4 times and add second antibody (goat anti-mouse igg/HRP conjugate (Sigma)) and carry out then washing in 1 hour 4 times.ELISA is with tmb substrate (each hole 50 μ l, DAKO S1600) colour developing 5 minutes and by adding 50 μ l 1M sulfuric acid stopped reaction.Immediately with plate in 450nm place reading.Figure 10 shows and to show all 6 data that purpose antibody is all expressed in the lysate, with obtaining described data after the expression vector mixture transfection of described 6 goal gene of encoding on the 34th day.Should be noted that: because the data that Figure 10 shows are from 6 different antigen-specific elisa assay, so can not directly compare the OD450 reading aspect the amount of antibody.

Further analyze by the ELISA antagonist expression level that covers with anti-kappa in the CHO-Flp-In cell aggregation, wherein the mixture with each single GOI or described 6 GOI has carried out transfection to described CHO-Flp-In cell.Figure 11 display result.This is presented at single cells transfected system and (for example, compares with carrier cells transfected system with the anti-AP antibody of coding, with the anti-β that encodes ₂The antibody that comparable measure has been expressed by m carrier cells transfected system) the antibody expression level is comparable between.Use term " with the CHO-Flp-In cell aggregation of single GOI transfection " herein, the clone's set come freely e.3 because described individual cells system is not from single clone described in.

(g) experiment conclusion

At first, these estimations to polyclone preservation in the production system (test) show not to be introduced selection or is breeding under the situation of difference (Fig. 7), will be from described mini six library (the anti-OVA that encodes, anti-AP, anti-β ₂M, anti-HAP, the anti-FVIII and anti-LYS) the goal gene of 6 selections to transfer to the mammalian expression vector from phage vector be possible, so the frequency of the goal gene of final described 6 selections is suitable.

The second, also cause the distribution of construct comparable in isolating mammalian cell with the whole transfection CHO-Flp-In of the construct mixture cell of the goal gene that contains described 6 selections.In for some time (after the transfection the 16th day and 34 days), it also is similar (Fig. 9) that the genotype of the goal gene of described 6 selections distributes, show that the 34th day described expression system still keep described six genotypic primary, identical distribution, and do not introduce breeding difference.

The 3rd, show that with the whole cells transfected of the mixture of described 6 goal gene all 6 antibody all express, as to carry out the anti-different in nature ELISA of antigen (Figure 10) as shown in checking from the 34th day the supernatant liquor of cell after the transfection.Because different binding affinities can not directly compare the antigenic ELISA result of difference aspect the amount of antibody.Yet, to from the CHO-Flp-In clone of a) described 6 individual transfections supernatant liquor and the b of (as with (d.1) described preparing carriers deposits yields)) to the supernatant liquor of the polyclone clone of the goal gene of 6 selections as described in expressing carry out based on be coated with goat-anti mouse kappa chain antibody catch ELISA show as described in have comparable antibody expression level in 6 genotype.

Generally speaking, shown that it is feasible that polyclone GOI is integrally transferred to mammalian expression vector from phage vector.This is being described by Sharon (US 5,789,208) before.In addition, the transfection integrally of the mixture of available Mammals expression construct still kept in mammalian cell and at least comparable frequency in 34 days after transfection.

Embodiment 3A

Estimate the polyclone preservation in the cell culture, described cell culture is produced by the subclone from CHO-Flp-In clone with the whole transfection of minisix library.

(a) subclone of " initial " CHO-Flp-In clone

As (the e.1 part of embodiment 3) the initial CHO-Flp-In clone of described cultivation (Invitrogen).After trypsinized, cell counting is also put into 1 cell in each hole on 96 well culture plates.Identify 20 holes after about 14 days with single clone, and with cell with trypsin treatment and transfer in 24 orifice plates.After identifying growth behavior feature and expression level, select one of them subclone CHO-Flp-In clone 019 to be used for further research.

(b) CHO-Flp-In clones the whole transfection and the selection of 019 cell

E.2 and e.3 carry out three multiple transfections, and basically as (embodiment 3, part) are described CHO-Flp-In is cloned 019 cell selects, except among pSymvc21 (Fig. 4 .e) with Xin Meisu selective marker replacement Totomycin mark.Thereby, between cell selection and incubation period, replace hygromycin B with Geniticin (450 μ g/ml).

(c) clone the ELISA of the sample that obtains 019 cell from CHO-Flp-In, described cell carries out whole transfection with mini six library.

Described cell after transfection, cultivated 73 days and the 17th day, 31 days, 45 days, 59 days and 73 days to being used for the sample sampling of ELISA.As (embodiment 3, f.2 part) the described quantitative ELISA (the mini six antibody that uses the difference purifying is as standard) that carries out.Figure 12 shows the result from 3 independent transfections.In different transfections, observe different expression characterizations.Yet, in all experiments, all detected all 6 kinds of antibody by the 59th day.

(d) experiment conclusion

Cloning 019 cell at CHO-Flp-In uses the experiment of Xin Meisu selective system to show that whole transfection has still kept single batch expression characteristic (Figure 12) after two months preferably.Be enough to satisfy the production purpose between such stationary phase.By a large amount of freezing stock that before production, produces and store single batch can solve viewed batch with criticize between variation.

Embodiment 3B

The preservation of appraisal polyclone in cell culture, described cell culture mix individual transfection after selection CHO-Flp-In cell produces.

(a) the individual transfection and the selection of CHO-Flp-In cell

The experimental technique that produces 6 clones of single member among the described expression mini sixlibrary is being described in (embodiment 3, e.3 part) before.Equivalent (each clone 5x10 at once after selection ⁵) mix to express 6 clone of single member among the described mini six library and the blended cell colony was cultivated 85 days.From described individual cells is to prepare three mixtures respectively.

(b) ELISA of the sample that from the polyclone cell culture that results from (a), obtains

Every fortnight is taken a sample and is passed through as the definite antibody composition of expressing from described mini six library of (embodiment 3, f.2 part) described ELISA.Mix the back and carried out ELISA at the 8th day, 17 days, 30 days, 45 days, 57 days, 72 days and 85 days.Figure 13 shows the described result (mean value of three repeated experiments ± SD).All antibody can detect after mixing on the 85th day.(embodiment 3, f.2 part) as previously mentioned, the reading of described different antibodies can not directly compare, but the data presentation of showing among Figure 13 still had metastable expression characterization on the 45th day at least after mixing, observe output afterwards and totally descend.In addition, the result who obtains from 3 independent mixtures relatively shows: described mini six antibody has similar expression characterization in for some time, shows that the result is repeatably.

(c) experiment conclusion

The polyclone cell culture of being made up of cell mixture shows that it is formed and can be retained to after the mixing at least 45 days, and wherein said cell is with the individually transfection of different members of described mini six library.45 days composition keeps the phase in most of the cases to be enough to satisfy the production purpose.In addition, the experiment of three multiple has provided similar result, therefore show with mix with the single cells transfected of different constructs cause the blended cell culture have lower batch and criticize between variation.

Embodiment 4

Set up antiovalbumin recombinant polyclonal antibody type of production clone

(a) express antiovalbumin polyclonal antibody composition

The ovalbumin that following evaluation characterizes is fully gathered in conjunction with phage clone.With the female BALA/C mouse of the OVA in the 50 μ g complete Freund's adjuvants by abdominal injection and four 8 all sizes of subcutaneous injection immunity, and the OVA booster immunization in the 21st day and the 42 days full freund's adjuvant that toos many or too much for use in immunity (the 0th day) back, and determine that all animals have serum and are converted to anti-OVA, this measures by antigen-specific ELISA.Obtain spleen the 31st day and 52 days from mouse with optimum response.Use phage vector (Symvc10) to produce Fab as described previously and show phage library from spleen RNA.The final library that produces contains 106 independent clonings of having an appointment.By making 5x10 ¹¹Individual Fab shows that phage and these libraries of OVA reaction pair that cover on the NUNC immunity pipe select, then flushing and take off in conjunction with phage with pickling.Because the OVA that contains significant proportion from the eluate of first round screening is in conjunction with the person, therefore from taking turns the eluate of screening screening OVA in conjunction with phage clone from first and second.

At first, identify the reactive phage clone of OVA by ELISA.Tout court, also react the second antibody reaction that then is connected with it with HRP with phage display Fabs with OVA coating elisa plate.As negative control, irrelevant antigen (BSA) or irrelevant phage display Fabs (anti-AP) have been used.

In addition, set up based on the HDS method that OVA is fixed on the pvdf membrane.These two kinds of methods have caused identifying different clone's subclass, another OVA in testing of the OVA in experiments of some clone identifications (set-up) and nonrecognition, and vice versa.Show phage clone for the Fab that reacts by ELISA or HDS and OVA, by dna sequencing to coding V _HThe nucleotide sequence of variable domains is determined, and is used Vector NTI software package to take place to analyze by system genetic diversity is estimated.The final group that obtains comprises 127 OVA in conjunction with the clone, all these clones' V _HThe nucleotide sequence of variable part is all determined.

All all have ovalbumin bonded ability with natural or denatured form by described 127 OVA-in conjunction with the Fab fragment of clonal expression.We have identified that about 30 are contained in for example clone of the difference among the pSymvc10 of phage vector, are used for global transfer and Mammals and express according to this technology.These antibody are with the formal representation of mouse IgA, IgG2A or IgG2B antibody.Because we have identified fully that these antibody produce the feature of clone's dna sequence dna, so we can monitor genotypic distribution in global transfer and Mammals expression process, method therefor be used for as embodiment 3 describe have as described in the method for modular system of 6 different antibodies identical.

(b) with OVA specific antibody sequence global transfer to being used for the carrier that Mammals is expressed

From phage vector goal gene is transferred to expression vector 2 steps (illustrating Fig. 4) are arranged, wherein the first step be with have head to head towards the promotor box of mammalian promoter of selection carry out the promotor exchange, after this variable region and the promotor box of goal gene are transferred on the expression vector.Use SacI/XhoI digestion then to connect, head to head promotor box (promotor A/ promotor B) is inserted in each phage vector in cloning, and has caused from the exchange of bacterium promotor and mammalian promoter (pSymvc12).

Then the digestion of EcoRI and NotI will be the coding variable heavy chain, head to head the sequence of promotor box (promotor A/ promotor B) and complete kappa chain moves on to the isotype code carrier pSymvc20 from phage vector (pSymvc12).Described pSymvc20 carrier can be accepted any NotI/EcoRI fragment from phage vector.This fragment will shift the constant sequence of heavy chain be connected on the pSymvc20 to the sequence of coding variable heavy chain and the sequence of whole coding kappa chain will be connected with bGH PolyA sequence.This global transfer will produce the expression vector that shows as Fig. 1, and described carrier is expressed after global transfer as the variable heavy chain district of mouse IgG2B antibody and whole kappa chain.

Described carrier pSymvc20 contains the murine heavy chain constant region of IgA, IgG2A, IgG2B, IgE or IgG1 gene, so can express any relevant purpose mouse immuning ball protein isotype.

(c) express the antiovalbumin recombinant polyclonal antibody

By global transfer, the sequence of encoding heavy chain variable region, promotor and complete kappa chain is moved on on the isotype code carrier from the phage vector library, formed polyclone mammalian expression vector composition.Be incorporated in the CHO-Flp-In clone by transfection and site-specific integration after this, produced recombinant polyclonal antibody type of production clone.The latter produces to the genomic phase homospecificity of each transfectional cell seat by proteinic each member's of described recombinant polyclonal that will encode goal gene targeted integration, wherein has only the expression construct that contains described nucleotide sequence of a copy to be incorporated in each cells transfected at one time.

The method of described cell cultures and transfection and selection is identical with embodiment 3 (e.1-e.3) description.

(d) stability of monitoring composition

Can aspect the clone between the single clone, expression and diversity, not introduce difference in order to ensure global transfer with when being transfected into mammalian cell, therefore can be with the process of following method monitoring global transfer and Mammals expression:

1) analyze generation time from the cell aggregation of each transfection construct,

2) analyze expression level from the cell aggregation of each transfection construct,

3) individual cells is carried out rflp analysis,

4) mixtures of antibodies that produces is carried out ELISA,

5) mixtures of antibodies that produces is carried out mass spectroscopy,

6) use V district Auele Specific Primer definite batch size to be analyzed ratio with definite variant clone by Taqman PCR (PCR in real time), or

7) use following parameters in for some time with and without batch analyzing that selective pressure (Totomycin) is cultivated:

A) clone distributes

B) protein expression level (quantity and distribution)

C) genome stability

D) to the adaptive influence of serum free medium

E) production of antiovalbumin recombinant polyclonal antibody composition

Recombinant polyclonal antibody generation type CHO-Flp-In clone is cultivated in different culture systems, comprise traditional little culturing bottle, Nunc multi-layered cell factory and small-sized high yield bio-reactor (MiniPerm, INTEGRA-CELLine).In addition, make described clone adapt to the serum-free suspension culture in revolving bottle, tubular fibre and bio-reactor, to cultivate subsequently.

The substratum of the clone selected of being used to grow is as manufacturer (Invitrogen, B﹠amp; D, Hyclone) substratum that knows of the serum-free of Tui Jianing, no albumen or chemical ingredients.

Collect the supernatant liquor of cultivating there not being the cell of selecting the adherent or suspension under (Totomycin) pressure.Characteristic as (3f) described analysis and supernatant liquor that identify to collect.Cell growing period or afterwards output, function and the quality of the antibody of its generation detected under fed-batch or perfusion condition.The cell of suspension growth is used for bigger revolving bottle or bio-reactor inoculation.

Purifying from the polyclonal antibody of the supernatant liquor of collecting in experimentation on animals, using later on.

Disclosed from here specification sheets of the present invention and practice consider that other embodiment of the present invention and application are apparent to those skilled in the art.That is to say that described specification sheets and embodiment only are considered to as an example, following claim shows true scope of the present invention and spirit.

Sequence table

<110>Symphogen?A/S

＜120〉be used to produce the recombinant polyclonal method of protein

<130>15685PCT00

<160>7

<170>PatentIn?version?3.2

<210>1

<211>48

<212>DNA

＜213〉Saccharomyces cerevisiae

<400>1

gaagttccta?ttccgaagtt cctattctct?agaaagtata?ggaacttc 48

<210>2

<211>21

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic PCR primer

<400>2

gcattgacag?gaggttgagg?c 21

<210>3

<211>23

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic PCR primer

<400>3

gctgccgacc?gctgctgctg?gtc 23

<210>4

<211>21

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic PCR primer

<400>4

gcattgacag?gaggttgagg?c 21

<210>5

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic PCR primer

<400>5

gtgtccactc?tgaggttcag 20

<210>6

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic PCR primer

<400>6

caaatagccc?ttgaccaggc 20

<210>7

<211>20

<212>DNA

＜213〉artificial

<220>

＜223〉synthetic PCR primer

<400>7

gtgtccactc?tgaggttcag 20

Claims (47)

1. produce the method for cell aggregation, described cell aggregation is suitable as recombinant polyclonal type of production clone, and described method comprises:

A) provide vector library, described vector library comprises the colony of different IPs acid sequence, wherein each described carrier comprises: 1) the unique nucleic acid sequence of single copy, the proteinic unique member of described nucleic acid sequence encoding polyclone, described polyclone protein comprises and specific antigen bonded different members; With 2) one or more recombinase recognition sequences;

B) described vector library is imported host cell system, the genome of each individual cells that wherein said host cell is comprises the recombinase recognition sequence, described recombinase recognition sequence is positioned at its genomic single specificity site, and with carrier on recombinase recognition sequence coupling.

C) guarantee in described cell, to exist one or more recombinases, so that the different IPs acid sequence locus specificity in the step (a) be incorporated in the cell of host cell system, wherein said one or more recombinase is i) by the described cell expressing that has imported described nucleotide sequence; Ii) encode effectively by the carrier among the step a; Iii) provide by second vector expression; Or iv) offer described cell as protein; With

D) select cell, described cell contains the copy from the integration in the library of described different IPs acid sequence.

2. the process of claim 1 wherein that described polyclone protein and described cell aggregation are not natural relevant.

3. claim 1 or 2 method, wherein said polyclone protein is polyclonal antibody or antibody fragment.

4. claim 1 or 2 method, wherein said polyclone protein is polyclone TXi Baoshouti or TXi Baoshouti fragment.

5. each method during aforesaid right requires wherein by with the described host cell set of the whole transfection of described vector library described vector library being imported to described host cell is.

6. each method in the claim 1 to 4, wherein the part that comprises the single carrier of 5 to 50 described vector library by use is come the equal portions thing of the described host cell of semi-monolithic transfection, thereby described vector library is imported in the described host cell system, and before or after step (d) is selected, described cell is merged the cell aggregation that formation is suitable as recombinant polyclonal type of production clone.

7. each method in the claim 1 to 4, the described vector library that wherein will be used for site-specific integration by the following method imports described host cell and is: use the described host cell of single member's transfection of described vector library respectively, and before or after step (d) is selected described cell is merged the cell aggregation that formation is suitable as recombinant polyclonal type of production clone.

8. the method for each during aforesaid right requires, wherein by the colony of the different nucleic acid in screening method separation or the authentication step (a), coding and described specific antigen bonded nucleic acid can be identified and/or separate to described screening method.

9. the method for claim 8, wherein said screening method comprises biological screening step and/or immunodetection analysis.

10. claim 8 or 9 method, wherein said screening method is selected from phage display, ribosomal display, DNA displaying, the displaying of RNA-peptide, covalency displaying, bacterium surface displaying, yeast surface display, the displaying of eucaryon virus, ELISA and ELISPOT.

Each method during 11. aforesaid right requires, the library of wherein said different IPs acid sequence comprises at least 3 different nucleotide sequences.

Each method during 12. aforesaid right requires, the single member in the library of wherein said different IPs acid sequence is incorporated on the single predetermined genome seat of cell individual in the described cell aggregation, and described seat can mediate described each member's of recombinant polyclonal protein high level expression.

Each method during 13. aforesaid right requires, wherein variant nucleotide sequence comprises paired gene fragment, the proteinic member of polyclone that described paired gene fragment coding is made of 2 different polypeptide chains.

14. the method for claim 13, wherein said paired gene fragment comprises antibody heavy chain variable region encoding sequence and antibody chain variable region encoding sequence.

15. the method for claim 13, wherein said paired gene fragment comprise TXi Baoshouti α chain variable region encoding sequence and TXi Baoshouti β chain variable region encoding sequence.

16. the method for claim 13, wherein said paired gene fragment comprise TXi Baoshouti γ chain variable region encoding sequence and TXi Baoshouti δ chain variable region encoding sequence.

Each method during 17. aforesaid right requires, the library of wherein said different IPs acid sequence comprises the diversity of the natural generation that is positioned at the different IPs acid sequence.

18. the method for claim 17, wherein the diversity of natural generation is arranged in the CDR zone of described different IPs acid sequence.

Each method during 19. aforesaid right requires, wherein said cell aggregation derives from mammal cell line or cell type.

20. the method for claim 19, wherein said mammal cell line are selected from Chinese hamster ovary (CHO) cell, COS cell, bhk cell, YB2/0, NIH3T3, myeloma cell, inoblast, HeLa cell, HEK293 cell, PER.C6 cell and its deutero-clone.

21. be used to produce the polyclone method of protein, wherein said polyclone protein comprises and specific antigen bonded different members, described method comprises:

A) provide the cell aggregation in the library of containing the different IPs acid sequence, the proteinic different members of the described polyclone of each described nucleic acid sequence encoding wherein, and wherein each described nucleotide sequence is incorporated in the described cell aggregation on the identical single site on each cell individual genome.

B) under the condition that helps described polyclone protein expression, cultivate described cell aggregation; With

C) cell from cell culture or the supernatant liquor in the cell culture reclaim the polyclone protein of described expression.

22. the method for claim 21, wherein the described cell aggregation in the step (a) is to produce according to each method among the claim 1-20.

23. the method for claim 21 or 22, wherein said polyclone protein is not natural relevant with described cell aggregation.

24. each method in the claim 21 to 23, wherein in step early, separate or authentication step (a) in the library of described different nucleic acid, described separation or identify by means of identifying and/or the screening method of separation energy coding and the proteic nucleic acid of specific antigen bonded.

25. the method for claim 24, wherein said screening method comprise biological screening step and/or immunodetection analysis.

26. the method for claim 24 or 25, wherein said screening method are selected from phage display, ribosomal display, DNA displaying, the displaying of RNA-peptide, covalency displaying, bacterium surface displaying, yeast surface display, the displaying of eucaryon virus, ELISA and ELISPOT.

27. each method in the claim 21 to 26, wherein said polyclone protein is polyclonal antibody or antibody fragment.

28. each method in the claim 21 to 26, wherein said polyclone protein are polyclone TXi Baoshouti or TXi Baoshouti fragment.

29. each method in the claim 21 to 28 is wherein monitored relative expression's level of described different IPs acid sequence.

30. the method for claim 29, the wherein described expression level of monitoring on mRNA level and/or protein level.

31. the method for claim 29 or 30, the wherein cultivation in the end step (b) when described relative expression's level exceeds predetermined scope at the latest.

32. recombinant polyclonal type of production clone, described clone comprises the library cells transfected set with the different IPs acid sequence, each cell in the wherein said set is with member's transfection in the described library and can express this member, described member encode can with the proteinic different members of specific antigen bonded polyclone, and be arranged on the genomic identical single site of described set cell individual, wherein said nucleotide sequence is not natural relevant with cell described in the described set.

33. the recombinant polyclonal type of production clone of claim 32, the library coding polyclonal antibody or the antibody fragment of wherein said different IPs acid sequence, wherein said polyclonal antibody or antibody fragment have the diversity of natural generation between its member's individuality.

34. the recombinant polyclonal type of production clone of claim 32, the library coding polyclone TXi Baoshouti or the TXi Baoshouti fragment of wherein said different IPs acid sequence, described polyclone TXi Baoshouti or TXi Baoshouti fragment have the diversity of natural generation between its member's individuality.

35. each recombinant polyclonal type of production clone in the claim 32 to 34, wherein said cell aggregation derives from mammal cell line or cell type.

36. the recombinant polyclonal type of production clone of claim 35, wherein said mammal cell line are selected from Chinese hamster ovary (CHO) cell, COS cell, bhk cell, YB2/0, NIH3T3, myeloma cell, inoblast, HeLa cell, HEK293 cell, PER.C6 cell and its deutero-clone.

37. be used for the vector library of site-specific integration, described vector library comprises the colony of the different IPs acid sequence of natural generation, and wherein each described carrier comprises 1) coding of a copy and the unique nucleic acid sequence and 2 of the proteinic different members of specific antigen bonded polyclone) one or more recombinase recognition sequences.

38. the library of claim 37, the colony's coding polyclonal antibody or the antibody fragment of the different IPs acid sequence of wherein said natural generation.

39. the library of claim 37, the colony's coding polyclone TXi Baoshouti or the TXi Baoshouti fragment of the different IPs acid sequence of wherein said natural generation.

40. each library in the claim 37 to 39, each member in the wherein said vector library is further contained the recombinase nucleic acid sequence encoding.

41. pass through recombinant polyclonal antibody or antibody fragment that the method for claim 27 obtains.

42. pass through recombinant polyclonal TXi Baoshouti or its polyclone fragment that the method for claim 28 obtains.

43. be used for the treatment of, improve or prevent the method for the disease in the animal, wherein use the recombinant polyclonal antibody or the antibody fragment of the claim 41 of effective dose.

44. be used for the treatment of, improve or prevent the method for disease in the animal, wherein use the recombinant polyclonal TXi Baoshouti or the TXi Baoshouti fragment of the claim 42 of effective dose.

45. the application of the fragment of recombinant polyclonal antibody or recombinant polyclonal TXi Baoshouti or antibody or TXi Baoshouti in the composition of preparation treatment disease, the pregnancy that described disease is selected from cancer, infection, inflammatory diseases, allergy, asthma or other respiratory disease, autoimmune disorders, dysimmunity, cardiovascular disorder, central nervous system disease, metabolic trouble, endocrinopathy, transplant rejection and does not expect.

46. a pharmaceutical composition, it contains the recombinant polyclonal antibody that obtained by the method for claim 27 or antibody fragment as activeconstituents, and pharmaceutically acceptable vehicle.

47. a pharmaceutical composition, it contains the recombinant polyclonal TXi Baoshouti that obtained by the method for claim 28 or TXi Baoshouti fragment as activeconstituents, and pharmaceutically acceptable vehicle.

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