CN1842539A - Use of hydrophobic-interaction-chromatography or hinge-region modifications for the production of homogeneous antibody-solutions - Google Patents

Use of hydrophobic-interaction-chromatography or hinge-region modifications for the production of homogeneous antibody-solutions Download PDF

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CN1842539A
CN1842539A CN 200480024576 CN200480024576A CN1842539A CN 1842539 A CN1842539 A CN 1842539A CN 200480024576 CN200480024576 CN 200480024576 CN 200480024576 A CN200480024576 A CN 200480024576A CN 1842539 A CN1842539 A CN 1842539A
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polypeptide
antibody
molecule
dimer
heavy chain
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CN100497390C (en
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加里·R·布拉斯拉夫斯基
斯科特·格拉泽
杨祖宏
珍妮弗·霍普
保罗·钦
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Biogen MA Inc
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Biogen Idec MA Inc
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Abstract

The instant invention describes methods of separating or preferentially synthesizing dimers which are linked via at least one interchain disulfide linkage from dimers which are not linked via at least one interchain disulfide linkage from a mixture comprising the two types of polypeptide dimers. These forms can be separated from each other using hydrophobic interaction chromatography. In addition, the invention pertains to connecting peptides that result in the preferential biosynthesis of dimers that are linked via at least one interchain disulfide linkage or that are not linked via at least one interchain disulfide linkage. The invention also pertains to compositions in which a majority of the dimers are linked via at least one interchain disulfide linkage or are not linked via at least one interchain disulfide linkage. The invention still further pertains to novel binding molecules, e.g., comprising connecting peptides of the invention.

Description

Hydrophobic-interaction-chromatography or hinge area are modified the purposes for the preparation of homogeneous antibody-solutions
Related application
The application requires the right of priority of the USSN60/483877 that is entitled as " purifying of polypeptide and preferred synthetic, " and the USSN 60/508,810 that is entitled as " purifying of antigen-binding polypeptides is with preferred synthetic, " that 2003-10-3 submits to of 2003-6-27 submission.This application requires also that 2003-10-28 submits to be entitled as " antibody molecule that comprises the modification of connection peptides; " USSN 60/515,351 and the right of priority of the USSN 60/516,030 that is entitled as " antibody molecule that comprises the modification of connection peptides, " that submits to of 2003-10-30.This application is also relevant with 200406028 USSNXX/XXXXXX that are entitled as " binding molecule that comprises the modification of connection peptides " that submit to.The full text content of these applications is included in this paper as a reference.
Background technology
Antibody is dimer molecule; Form this dimeric each monomer and comprise a light chain and a heavy chain.The solution of antibody molecule exists with the heterogeneous relevant form of hinge with two kinds.Utilization is analyzed the SDS-PAGE of the Mab MAb of purifying, and these two kinds of forms are as two protein bands i.e. a main band (the about 150-160kDa of MW) and a less important band (the about 75-80kDa of MW) and be observed usually.A kind of form in this back is observed after the SDS-PAGE to the IgG4 prepared product of purifying analyzes usually, but can comprise that MAb is identified (Angal etal.1993.Mol.Immunol.30:105 with much lower frequency in the reorganization of purifying at all IgG isotypes; Norderhaug et al.1990.Eur.J.Immunol.21:2370).The isotype of larger molecular weight is called the A type, contains to be positioned at corresponding Kabat numbering system position 239 and 242 (positions 226 and 229, the EU numbering system) covalency interchain disulfide bond (Kabat, E, Wu, TT, Perry, HM, Gottesman, KS, Foeller, C:Sequences of Proteins ofImmunological Interest.Bethesda, US Department of Health and people Services, NIH, 1991).Second kind of isotype Type B is considered to not comprise the interchain disulfide bond between covalently bound between two heavy chains and two the adjacent cysteine residues, confirms as the 75-80kDa seen in the irreducibility SDS-PAGE electrophoresis.Two heavy chains of inferring Type B are interacted by strong non-covalent (being ion) relevant with the CH3 structural domain zone of this molecule and connect.The mixture of A type and Type B is not present in the segmental solution of Mab, and described fragment contains complete hinge area, but lacks the CH3 district, such as F (ab) 2Fragment.Usually, F genetic modification or enzymic digestion (ab) 2The Mab prepared product lacks Type B, and this is to keep the required structural domain of noncovalent interaction (for example hydrogen bonded) because this molecule lacks.Yet, they are present in the Mab prepared product that contains the CH3 district, and such as IgG4, the Mab fragment of CH2 district disappearance (for example, described in 02/060955 A2) and miniantibody (for example seeing Hu et al.1996.CancerResearch 56:3055) and IgG4 molecule in.
The albumen renovation technique is applied to therapeutic antibodies design also produces multiple antibody pattern, it has change, has pharmacodynamics, bio distribution and the active collection of illustrative plates of improvement in some cases.In the antibody molecule of some changes, the many cysteine residues in the hinge area are reduced to one, with the gathering of enhancing antibody molecule, because cysteine residues is essential for forming single disulfide linkage only.This also provides the specific target (United States Patent (USP) 5,677,425) that hinge area is attached to another hinge area or effector molecule or reporter molecule.Cysteine residues number in the antibody hinge also increases (United States Patent (USP) 5,677,425).The antibody of other sudden change is fabricated, and wherein IgG1 hinge area and CH2 structural domain personnel selection IgG3 hinge area is replaced (WO 97/11370).These molecules contain 11 sulfydryls and are used for replacing a plurality of haptens via thiol group.
The molecular weight of the antibody of CH2 structural domain disappearance is about 120kDa, and demonstration compares with total length IgG, and its ability that penetrates tumour is obviously better.Miniantibody, it also lacks the CH2 structural domain, has similar character.The molecule of these structural domains disappearance in the gathering of tumor sites than other Mab fragment such as F (ab) ' 2More effective, but do not have being seen unfavorable pharmacodynamics collection of illustrative plates when utilizing complete IgG antibody.The antibody of CH2 structural domain disappearance is by forming with the lower section: VLCL light chain and VH1 heavy chain structural domain and the part hinge area (for example, going up and middle hinge) that merges (directly and via synthetic peptide spacer) with the CH3 domain gene.For example, the ddCC49's of recombinant C H2 structural domain disappearance is synthetic, discerns the antibody of the relevant antigenic structural domain disappearance of TAG72 of the tumour expressed on the various human cancers, produces A and the B isotype that distributed in about 50: 50 in cell culture.Transform the antibody of the CH2 structural domain disappearance of other form of expression as, described antibody is the antibody of tetravalence CH2 structural domain disappearance for example, miniantibody, or the cell of tetravalence miniantibody is also expressed A and B isotype and/or the monomer half-molecular mixture of body (mer).
Even after the Mab purifying, the A type separates with Type B is also extremely difficult, and this is because they are made up of identical amino acid, has identical molecular weight and similar physics and chemical property thus.They can not comprise the proteic standard gel filtration of reorganization MAb by being generally used for the antibody purification molecule, and affinity chromatography is separated with ion exchange chromatography.Present production technique abandons at least 50% of the total antibody that produces, and this has negative impact to overall yield.In addition, the existence of two kinds of isotypes increases the required effort of downstream processing.Therefore, the biosynthetic method of separating the method for A type and Type B or increasing the antibody of one or another kind of form is highly profitable.
Summary of the invention
The present invention to small part based on following discovery: comprising the dimer peptide molecule (molecule that comprises two heavy chains parts that contains different isotypes, wherein the part molecule comprises two heavy chain parts that connect via at least one interchain disulfide bond (A type), the part molecule comprises not two the heavy chains parts (Type B) that connect via at least one interchain disulfide bond) in the composition of mixture, a kind of form or another kind can preferably obtain, for example by utilizing hydrophobic interaction chromatography to separate or by comprising the preferred biosynthetic synthetic connection peptides that causes A type or Type B.Connection peptides of the present invention can be included in to be tended to form in any dimer molecule of A type and Type B, antibody molecule for example, the antibody molecule (for example lacking all or part CH2 structural domain) of structural domain disappearance, miniantibody, bivalent antibody, fusion rotein etc.In preferred embodiments, the formation of A type is enhanced.A type polypeptide dimer shows the enhanced bio distribution in vivo in external demonstration enhanced stability.
Therefore, on the one hand, the present invention relates to composition, it comprises the polypeptide dimer with at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain part and synthetic connection peptides, wherein surpass 50% described polypeptide dimer via at least one interchain disulfide bond connection.
In the embodiment, the described polypeptide dimer above 90% connects via at least one interchain disulfide bond.
In the embodiment, described polypeptide chain comprises via the connection peptides gene fusion in VL, the CH3 structural domain of VH or CH1.
In the embodiment, described polypeptide chain lacks all or part CH2 structural domain.
In the embodiment, described polypeptide dimer connects via two or more interchain disulfide bonds.
In the embodiment, described heavy chain partly is derived from the antibody isotype that is selected from down group: IgG1, IgG2, IgG3, or IgG4.
In the embodiment, described heavy chain partly comprises the aminoacid sequence that is derived from the hinge area that is selected from down group: γ 1 hinge, γ 2 hinges, γ 3 hinges, or γ 4 hinges.
In the embodiment, wherein said heavy chain partly comprises chimeric hinge.
In the embodiment, described synthetic connection peptides comprises to small part IgG1 hinge arrangement territory, to small part IgG3 hinge arrangement territory.
In the embodiment, described binding site independently is selected from following group: antigen binding site, the ligand binding moiety of acceptor, or the receptor binding moiety of part.
In the embodiment, described polypeptide dimer comprises four polypeptide chains.
In another embodiment, there are two to comprise at least one heavy chain part and synthetic connection peptides in the described polypeptide chain.
On the other hand, the present invention relates to composition, it comprises the polypeptide dimer with at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain part and synthetic connection peptides, wherein surpass about 50% described polypeptide dimer and connect via at least one interchain disulfide bond, wherein said connection peptides comprises the proline residue of the position 243 that is positioned at the Kabat numbering system.
In the embodiment, the described polypeptide dimer above 90% connects via at least one interchain disulfide bond.
In the embodiment, described synthetic connection peptides also comprises the position 239 that is positioned at the Kabat numbering system and 242 cysteine residues.
In the embodiment, at least one comprises via connection peptides and is connected in VL, the CH3 structural domain of VH and CH1 structural domain in the described polypeptide chain.
In the embodiment, described synthetic connection peptides also comprises the alanine residue of the position 244 that is positioned at the Kabat numbering system and the proline residue of position 245.
On the other hand, the present invention relates to treat experimenter's method, described experimenter will benefit from the treatment that utilizes binding molecule, and described method comprises that giving the experimenter with composition of the present invention makes treatment take place.
In the embodiment, described experimenter suffers from cancer.
In the embodiment, described experimenter suffers from lymphoma.
In the embodiment, described experimenter suffers from autoimmune disease or illness.
In the embodiment, described experimenter suffers from inflammatory diseases or illness.
In the embodiment, described binding site independently is selected from down group: antigen binding site, the ligand binding moiety of acceptor, or the receptor binding moiety of part.
On the other hand, the present invention relates to nucleic acid molecule, it comprises the nucleotide sequence of code book invention polypeptide chain.
On the other hand, the present invention relates to comprise the host cell of the nucleic acid molecule of claim 23.
In the embodiment, described polypeptide dimer comprises four polypeptide chains, and in the wherein said polypeptide chain two comprise at least one heavy chain part and synthetic connection peptides.
On the one hand, the present invention relates to connection peptides, it comprises the aminoacid sequence that is selected from SEQ ID NO:8-15 or 53 groups of forming.
On the one hand, the present invention relates to connection peptides, it is made up of the aminoacid sequence that is selected from the group that SEQ ID NO:8-15 or 53 forms.
On the one hand, the present invention relates to the nucleic acid molecule of coded polypeptide chain, wherein said nucleic acid molecule comprises the nucleotide sequence that is selected from down group: SEQ ID NO:16,20,21,38,42,46 or 47.
On the one hand, the present invention relates to the nucleic acid molecule of coded polypeptide chain, wherein said nucleic acid molecule comprises the nucleotide sequence that is selected from down group: SEQ ID NO:24 or 25.
On the one hand, the present invention relates to the antibody molecule of structural domain disappearance, it comprises the aminoacid sequence that is selected from down group: SEQ ID NO:18, and 22,23,40, or 44.
On the one hand, the present invention relates to the antibody molecule of structural domain disappearance, it comprises the aminoacid sequence that is selected from down group: SEQ ID NO:26 or 27.
On the one hand, the present invention relates to antibody molecule, it comprises aminoacid sequence SEQ ID NO:31.
On the one hand, the present invention relates to antibody molecule, it comprises aminoacid sequence SEQ ID NO:35.
On the one hand, the present invention relates to separate first kind and the dimeric method of second peptide species, wherein the first peptide species dimer comprises at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise the heavy chain part, wherein the first peptide species dimer connects via at least one disulfide linkage, the second peptide species dimer comprises at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise the heavy chain part, wherein the second peptide species dimer does not connect via at least one disulfide linkage, said method comprising the steps of:
I) will contain the dimeric mixture of first and second peptide species and the medium that separates described polypeptide dimer based on hydrophobic reactant contacts under the second peptide species dimer and the described medium bonded condition allowing; With
Ii) collect the first peptide species dimer, separate the first peptide species dimer thus, the collection step comprises makes described medium contact with the solution of about neutral pH, the about 120mS/cm of electric conductivity.
In the embodiment, the about 116mS/cm of described electric conductivity.
In the embodiment, described method also is included in step I i) before, described medium is contacted under about neutral pH with the solution of the electric conductivity with about 140mS/cm, make the described first and second peptide species dimers combine with described medium.
In the embodiment, described polypeptide chain comprises via synthetic connection peptides and is connected in VL, the CH3 structural domain of VH or CH1 structural domain.
In the embodiment, the dimeric heavy chain of described first and second peptide species partly is derived from the antibody isotype that is selected from down group: IgG1, IgG2, IgG3, or IgG4.
In the embodiment, described heavy chain partly comprises the aminoacid sequence from the hinge area that is selected from down group: γ 1 hinge, γ 2 hinges, γ 3 hinges, or γ 4 hinges.
In the embodiment, described binding site independently is selected from following group: antigen binding site, the ligand binding moiety of acceptor, or the receptor binding moiety of part.
In the embodiment, at least one binding site of polypeptide dimer comprises at least one CDR of antibody, and it is selected from: 2B8, and Lym 1, and Lym 2, LL2, Her2, B1, MB1, BH3, B4, B72.3, CC49, or 5E10.
In the embodiment, at least one binding site of polypeptide dimer comprises the receptor binding moiety of part.
In the embodiment, at least one binding site of polypeptide dimer comprises the ligand binding moiety of acceptor.
On the other hand, the present invention relates to treat the experimenter's that can benefit method, comprise that giving the experimenter with composition of the present invention makes treatment occur from the treatment that utilizes binding molecule.
In the embodiment, described polypeptide dimer comprises four polypeptide chains, has two to comprise at least one heavy chain part in the wherein said polypeptide chain.
On the other hand, the present invention relates to separate the first correct folding antibody molecule method from the second correct folding antibody molecule, each self-contained polypeptide chain of first and second antibody molecules wherein, wherein at least two chains comprise at least one heavy chain part, have at least two to comprise at least one light chain part in the described chain, said method comprising the steps of:
I) will comprise the mixture of first and second antibody molecules contacts with medium based on hydrophobic reactant separation antibody molecule;
Ii) described matrix is contacted under about neutral pH with the solution with about 120mS/cm electric conductivity, make described first antibody molecule not combine with medium, the second antibody molecule combines with described medium, separates described first and second antibody molecules thus.
In the embodiment, the present invention relates to respect to the dimeric amount of second peptide species, increase the method for the dimeric amount of first peptide species of cell generation, wherein the first and second peptide species dimers comprise at least two binding sites and at least two polypeptide chains, described at least two polypeptide chains comprise the heavy chain part, wherein first dimer connects via at least one disulfide linkage, and wherein second dimer is not to connect via at least one disulfide linkage, described method comprises transforms described polypeptide chain to comprise the step of synthetic connection peptides, and the dimeric amount of first peptide species that makes cell produce increases with respect to the dimeric amount of second peptide species.
In the embodiment, the present invention relates to composition, it comprises first polypeptide by method preparation of the present invention.
In the embodiment, described polypeptide dimer comprises four polypeptide chains, and wherein said polypeptide chain has chain to comprise at least one heavy chain part and synthetic connection peptides.
In the embodiment, the present invention relates to comprise the polypeptide of synthetic connection peptides, it comprises aminoacid sequence SEQ ID NO:37, and wherein said polypeptide is not naturally occurring IgG3 molecule.
In the embodiment, described molecule is the IgG4 molecule.
In the embodiment, described polypeptide is in conjunction with VLA-4.
On the one hand, the present invention relates to increase the method for the dimeric amount that connects by at least one disulfide linkage in the IgG4 molecule colony that produces in the cell, comprise the IgG4 molecule that causes cell expressing to comprise synthetic connection peptides, make the dimeric amount that connects by at least one disulfide linkage in the IgG4 molecule colony increase.
On the one hand, the present invention relates to composition, it comprises the polypeptide dimer with at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain part and lack all or part of CH2 structural domain, wherein surpass 50% polypeptide dimer and connect via at least one interchain disulfide bond.
On the one hand, the present invention relates to comprise the composition of claim 57, wherein said polypeptide dimer comprises four polypeptide chains, has two to comprise at least one heavy chain part in the wherein said polypeptide chain.
The accompanying drawing summary
In the antibody of Fig. 1 display structure territory disappearance, as the dimeric A type of 120kDa with as the monomeric Type B of 60kDa.
Fig. 2 A and B show the specific gravity hydrometer mapping of the non-reduced SDS-PAGE gel of ddCC49 (having the CH2 spacer) and ddCC49 (having the Gly/Ser spacer) respectively.
Fig. 3 shows G1/G3/Pro243Ala244Pro245+[Gly/Ser] (Fig. 3 D) and G1/G3/Pro243+[Gly/Ser] (Fig. 3 C) hinge causes mainly producing the huCC49 antibody of A type CH2 structural domain-disappearance, and Type B output is very low or can not detect basically.Otherwise the huCC49Cys242Ser:Pro243Ala244Pro245 (Fig. 3 B) of the huCC49Cys242Ser:Pro243 of CH2 structural domain-disappearance (Fig. 3 A) and CH2 structural domain-disappearance causes moderate to arrive the preference of tangible Type B isotype respectively.
Fig. 4 A (SEQ ID NO:16) shows the huCC49Pro243Ala244Pro245+[Gly/Ser of heavy chain CH2 structural domain-disappearance] the single stranded DNA sequence of hinge connection peptides.Fig. 4 B (SEQ IDNO:17) shows the single stranded DNA sequence of the huCC49 of light chain CH2 structural domain-disappearance.
Fig. 5 shows the SDS-PAGE gel of the huCC49 G1/G3/PAP only utilize the Protein G column purification, and its antibody that shows wash-out is basically as single peak, purity 〉=96%, and need not further HIC purifying.
Fig. 6 shows the SDS-PAGE gel of the huCC49 V2 G1/G3/PAP only utilize the Protein G column purification, and its antibody that shows wash-out is basically as single peak, purity 〉=96%, and need not further HIC purifying.
Fig. 7 shows the peptide drawing HPLC-MS of the HuCC49 Δ CH2 antibody of hinge-transformation.Figure A is that Endo Lys-C is non-reducing.Figure B is reduced form Endo Lys-C.Figure C is Tryptic.G1/G3:PAP fragment H146-208 shows the skew with respect to the shelf time of PAP and HuCC49 Δ CH2, and it is Leu → Met results of mutation due to the artificial PCR.This proofreaies and correct in all constructs subsequently.
Fig. 8 A (SEQ ID NO:18) shows the huCC49Pro243Ala244Pro245+[Gly/Ser of heavy chain CH2 structural domain-disappearance] aminoacid sequence of hinge connection peptides.Fig. 8 B (SEQ ID NO:19) shows the aminoacid sequence of the huCC49 of light chain CH2 structural domain-disappearance.
Fig. 9 (SEQ ID NO:20) shows the huCC49 G1/G3/Pro243+[Gly/Ser of heavy chain CH2 structural domain-disappearance] the single stranded DNA sequence of hinge connection peptides.
Figure 10 (SEQ ID NO:21) shows the single stranded DNA sequence of the huCC49 of heavy chain CH2 structural domain-disappearance, and it contains G1/G3/Pro243Ala244Pro245+[Gly/Ser] the hinge connection peptides.
Figure 11 (SEQ ID NO:22) shows the huCC49G1/G3/Pro243+[Gly/Ser of heavy chain CH2 structural domain-disappearance] aminoacid sequence of hinge connection peptides.
Figure 12 (SEQ ID NO:23) shows the aminoacid sequence of the huCC49 of heavy chain CH2 structural domain-disappearance, and it contains G1/G3/Pro243Ala244Pro245+[GlySer] the hinge connection peptides.
Figure 13 A (SEQ ID NO:24) shows the dna sequence dna of the huCC49 V2 of heavy chain CH2 structural domain-disappearance, and it contains G1/G3/Pro243Ala244Pro245+[GlySer] the hinge connection peptides.Figure 13 B (SEQ ID NO:25) shows the dna sequence dna of the huCC49 V2 of light chain CH2 structural domain-disappearance.
Figure 14 A (SEQ ID NO:26) shows the aminoacid sequence of the huCC49 V2 of heavy chain CH2 structural domain-disappearance, and it contains G1/G3/Pro243Ala244Pro245+[GlySer] the hinge connection peptides.Figure 14 B (SEQ ID NO:27) shows the aminoacid sequence of the huCC49 V2 of light chain CH2 structural domain-disappearance.
Figure 15 shows the chromatogram of the HIC purifying of the huCC49 A of CH2 structural domain-disappearance and B isotype.
Figure 16 shows the huCC49A type and the Type B of the CH2 structural domain-disappearance of purifying, and it is shown in respectively in swimming lane 3 and 4.
Figure 17 is presented at the huCC49 isotype that the preparation scope is separated two kinds of CH2 structural domain-disappearances.Two peaks that occur comprise the equipotential wash-out of A type earlier, and second peak shows the Type B of wash-out, and the 3rd peak contains impurity, and it can be removed from stationary phase in cleaning course.
Figure 18 demonstration utilizes 0.73M ammonium sulfate/20mM sodium phosphate, and the huCC49 fraction of the CH2 structural domain-disappearance of pH 4.0 (swimming lane 6-8) equipotential wash-out mainly comprises A type (purity＞90%).
Figure 19 A shows mouse CC49 (SEQ ID NO:28) variable region of light chain, and LEN (SEQ ID NO:29) humanized CC49 V1 (version 1) (SEQ ID NO:30) is with humanized CC49 V2 (version 2; Compare with humanized CC49 V1, it comprises the place's aminoacid replacement in the light chain, see underlined amino acid) comparison of (SEQ ID NO:31), Figure 19 B shows mouse CC49 (SEQ ID NO:32) variable region of heavy chain, 21/28 ' CL (SEQ ID NO:33), humanized CC49 V1 (SEQ ID NO:34), (it is compared with humanized CC49 with humanized CC49 V2, comprise two aminoacid replacement in the heavy chain, see the comparison of underlined amino acid (SEQ ID NO:35).
Mucinous CBA under Figure 20 demonstration and the ox jaw, time-resolved fluorescent immunoassay is passed through in the antigenic source of TAG-72.
Figure 21 shows in the comparison LS174T tumor xenogeneic graft 90Tumour-organ the ratio of the CC49 construct of Y/CHx-DTPA link coupled structural domain disappearance.
Figure 22 show comprise G1/G3/Pro243Ala244Pro245+[Gly/Ser] bio distribution of the people CC49V2 of connection peptides.
Figure 23 shows HuCC49[gly/ser] bio distribution.
Figure 24 show with the CC49 of structural domain disappearance with Gly-Ser knuckle joint compare the CC49 G1/G3/Pro243Ala244Pro245+[Gly/Ser of structural domain disappearance] (SEQ ID NO:9) more stable to gsh (GSH) reduction, as parental generation CC49.
Figure 25 A (SEQ ID NO:46) show contain G1/G3/Pro243Ala244Pro245+[GlySer] dna sequence dna of the anti-CD 20 IgG1 C2B8 of the heavy chain CH2 structural domain-disappearance of hinge connection peptides.Figure 25 B (SEQ ID NO:47) show contain G1/G3/Pro243Ala244Pro245+[GlySer] the corresponding aminoacid sequence of the heavy chain C2B8 of hinge connection peptides.
Figure 26 A (SEQ ID NO:48) shows the dna sequence dna of the anti-CD 20 IgG1C2B8 of light chain CH2 structural domain-disappearance.Figure 26 B (SEQ ID NO:49) shows the corresponding aminoacid sequence of light chain C2B8.
Figure 27 shows G1/G3/Pro243Ala244Pro245+[Gly/Ser] to be included in what cause in the different antibody of specificity (the C2B8 antibody of CH2 structural domain-disappearance of this paper) producing all be A type antibody to connection peptides (SEQ ID NO:9) basically, maybe can not detect (swimming lane 3) and Type B is less.
Figure 28 A (SEQ ID NO:38) show contain G1/G3/Pro243Ala244Pro245+[GlySer] dna sequence dna of the resisting of the heavy chain CH2 structural domain-disappearance of hinge connection peptides-CD23 Ab 5E8.Figure 28 B (SEQ ID NO:39) shows the dna sequence dna of the anti--CD23 5E8 of light chain CH2 structural domain-disappearance.
Figure 29 A (SEQ ID NO:40) show contain G1/G3/Pro243Ala244Pro245+[GlySer] aminoacid sequence of the resisting of the heavy chain CH2 structural domain-disappearance of hinge connection peptides-CD23 antibody 5E8.Figure 29 B (SEQ ID NO:41) shows the aminoacid sequence of the anti--CD23 antibody 5E8 of light chain CH2 structural domain-disappearance.
Figure 30 shows G1/G3/Pro243Ala244Pro245+[Gly/Ser] to be incorporated into what cause in the different antibody of specificity (the 5E8 antibody of CH2 structural domain-disappearance of this paper) producing all be A type antibody to connection peptides (SEQ ID NO:9) basically, maybe can not detect (swimming lane 2) and Type B is less.
Figure 31 shows the result by the CBA of time-resolved fluorescent immunoassay and solvable CD23.
Figure 32 A (SEQ ID NO:42) show contain G1/G3/Pro243Ala244Pro245+[GlySer] dna sequence dna of the chB3F6 of the heavy chain CH2 structural domain-disappearance of hinge connection peptides.Figure 32 B (SEQ IDNO:43) shows the dna sequence dna of the chB3F6 of light chain CH2 structural domain-disappearance.
Figure 33 A (SEQ ID NO:44) show contain G1/G3/Pro243Ala244Pro245+[GlySer] aminoacid sequence of the chB3F6 of the heavy chain CH2 structural domain-disappearance of hinge connection peptides.Figure 33 B (SEQ IDNO:45) shows the aminoacid sequence of the chB3F6 of light chain CH2 structural domain-disappearance.
Figure 34 shows G1/G3/Pro243Ala244Pro245+[Gly/Ser] (it all is A type antibody that SEQ ID NO:9 is incorporated into what cause in the different antibody of specificity (the chB3F6 antibody of CH2 structural domain-disappearance of this paper) producing to connection peptides basically, maybe can not detect (swimming lane 4) and Type B is less.
Figure 35 shows chimeric B3F6 (chB3F6) and the equal competition of antibody of chimeric B3F6 structural domain disappearance and combining of GEO tumour cell that contains connection peptides (B3F6 Δ CH2 G1/G3/Pro243Ala244Pro245).
Detailed Description Of The Invention
People (Igs) comprises monoclonal antibody (MAb), can exist with two kinds of forms relevant with the hinge heterologous. In natural solution, these forms all exist as dimer protein (each monomer comprises a heavy chain and a light chain). An immunoglobulin molecules comprises the four chain building bodies of stablizing of about 150-160kDa, wherein dimer links together by interchain heavy chain disulfide bond (A type), and one comprises the form (Type B) that dimer does not wherein connect by interchain disulfide bond. Type B also forms under natural endowment and stablizes dimer, but can identify under the non-reduced condition of sex change, and wherein heavy chain dissociates and produces the 75-80kDa molecule. The extremely difficult separation of these forms, in addition still like this after Mab affinity purifying.
The frequency that Type B occurs in the various complete IgG isotypes is owing to but be not limited to the architectural difference relevant with the hinge area isotype of MAb molecule. In fact, the replacement of the single amino acids in the hinge area of human IgG 4 hinges can obviously reduce the appearance (Angal et al.1993.Molecular Immunology 30:105) of Type B to the level of utilizing human IgG1's hinge usually to observe. Yet, the MAb fragment that has wherein kept the CH3 domain is carried out the generation that identical 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor can not be eliminated Type B. Usually, the antibody of all recombinant C H2 domain disappearances that produce during cell is cultivated causes the hinge heterologous usually, and it can not be proofreaied and correct via molecular mutation similar in the hinge.
The present invention is by providing the method for for example separating first kind of dimer polypeptide and second dimer polypeptide that prior art is made contributions, wherein first kind and second polypeptide comprise at least two polypeptide chains, have at least two to comprise at least one heavy chain part in the described polypeptide chain. In the embodiment, polypeptide of the present invention lacks all or part CH2 domain. Described monomer connects (this paper is called " A type ") via at least one interchain disulfide bond, and the monomer of second polypeptide does not connect via at least one interchain disulfide bond (this paper is called " Type B "). These forms can utilize hydrophobic interaction chromatography disconnected from each other. In addition, the present invention relates to comprise the polypeptide that connects peptide. Comprise that concrete connection peptide causes the polypeptide dimer that connects via at least one interchain disulfide bond or the polypeptide dimer that do not connect via at least one interchain disulfide bond preferred synthetic.
Further describe before the present invention, for convenient, describe hereinafter some terms:
I. definition
Polypeptide of the present invention is binding molecule, that is, peptide molecule or their nucleic acid molecules of encoding, it comprises at least one binding structural domain, and described domain comprises the binding site with target molecule (such as antigen or binding partners) specific binding. For example, in the embodiment, binding molecule of the present invention comprises the acceptor molecule part of immunoglobulin (Ig) antigen binding site or responsible ligand binding or the ligand molecular part of responsible receptors bind. Binding molecule of the present invention is polypeptide or their nucleic acid molecules of encoding.
In the embodiment, described binding molecule comprises at least two binding sites. In the embodiment, described binding molecule comprises two binding sites. In the embodiment, described binding molecule comprises three binding sites. In another embodiment, described binding molecule comprises the four combinations site.
Polypeptide of the present invention is polymer. For example, in the embodiment, polypeptide of the present invention is dimer. In the embodiment, polypeptide of the present invention is homodimer, comprises two identical monomer subunits. In another embodiment, dimer of the present invention is heterodimer, comprises two not identical monomer subunits. Described dimeric subunit comprises one or more polypeptide chain. For example, in the embodiment, described dimer comprises at least two polypeptide chains. In the embodiment, described dimer comprises two polypeptide chains. In another embodiment, described dimer comprises four polypeptide chains (for example, in the situation of antibody molecule).
Polypeptide of the present invention comprises at least a amino acid sequence from immunoglobulin domains. " be derived from " polypeptide of protein of appointment or the source that amino acid sequence refers to polypeptide. Preferably, polypeptide or amino acid sequence from concrete initial polypeptide or amino acid sequence have the amino acid sequence substantially the same with homing sequence, or its part, wherein said part is by 10-20 amino acid at least, preferred 20-30 amino acid at least, more preferably 30-50 amino acid forms at least, or described polypeptide or amino acid sequence can be accredited as it by those skilled in the art and derive from described homing sequence.
Preferred Binding peptide comprises the amino acid sequence from the human amino acid sequence. Yet Binding peptide can comprise one or more amino acid from another mammalian species. For example, primate heavy chain part, hinge fraction, or binding site can be included in tested Binding peptide and/or the connection polypeptide. Optional, one or more mouse amino acid can be present in the Binding peptide, for example, and in the antigen binding site of binding molecule. The preferred binding molecule of the present invention is not immunogenic.
It will be understood by those skilled in the art that binding molecule of the present invention (for example, the heavy chain of tested polypeptide or light chain part or bound fraction) can be modified so that their amino acid sequence is different from its naturally occurring immunoglobulin molecules of originating. For example, can cause the conservative nucleotides that replaces or 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor or in the change of " nonessential " amino acid residue.
Coding source from immunoglobulin (Ig) (for example, heavy chain immunoglobulin part or light chain part) the nucleic acid molecules of separation of non-natural variant of polypeptide can be by one or more nucleotides be replaced, interpolation or disappearance import the nucleotide sequence of immunoglobulin (Ig) so that one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor adds or lack the protein that is imported into coding producing. Sudden change can import by standard technique, such as the mutagenesis of direct mutagenesis and PCR mediation. Preferably, conserved amino acid is substituted in one or more nonessential amino acid residue and carries out. " conserved amino acid replacement " is that wherein the amino acid residue of amino acid residue with similar side chain replaces. Family with amino acid residue of similar side chain can determine in the present invention, comprises basic side chain (lysine for example, arginine, histidine), acid side-chain (for example aspartic acid, glutamic acid), uncharged acute side chain amino acid (glycine for example, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar sidechain (alanine for example, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and aromatic side chain (tyrosine for example, phenylalanine, tryptophan, histidine). Therefore, the nonessential amino acid in the immunoglobulin polypeptides is preferably replaced by another amino acid residue from identical side chain family. In another embodiment, a string amino acid can and/or form difference but the similar amino acid string replacement of structure by order among the side chain family member.
Optional, in another embodiment, sudden change can be introduced at random along all or part immunoglobulin coding sequence, and such as passing through saturation mutagenesis, the mutant of generation can mix polypeptide of the present invention, and screens them in conjunction with the ability of required antigen.
This paper term " heavy chain part " comprises the amino acid sequence from heavy chain immunoglobulin. The polypeptide that comprises heavily the connection part comprises at least one in the following domain: the CH1 domain, hinge (for example, on, in, and/or lower hinge area) domain, CH2 domain, CH3 domain, or its variant or fragment. In the embodiment, polypeptide of the present invention comprises polypeptide chain, and described polypeptide chain comprises the CH1 domain, at least part of hinge arrangement territory, and CH2 domain. In another embodiment, polypeptide of the present invention comprises polypeptide chain, and described polypeptide chain comprises CH1 domain and CH3 domain. In another embodiment, polypeptide of the present invention comprises polypeptide chain, and described polypeptide chain comprises the CH1 domain, at least part of hinge arrangement territory, and CH3 domain. In another embodiment, polypeptide of the present invention comprises the polypeptide chain that contains the CH3 domain. In the embodiment, polypeptide of the present invention lacks at least part of CH2 domain (for example, all or part CH2 domain). In another embodiment, polypeptide of the present invention comprises complete Ig heavy chain. As mentioned above, those skilled in the art understand these domains (for example, described heavy chain part) and can be modified so that their amino acid sequence is different from naturally occurring immunoglobulin molecules.
In the embodiment, at least two polypeptide chains of binding molecule of the present invention comprise at least one heavy chain part that is derived from antibody or immunoglobulin molecules. In the embodiment, at least two heavy chains of polypeptide of the present invention partly are present on the different polypeptide chains and for example form dimer protein via at least one disulfide bond interaction (A type) or via noncovalent interaction (Type B), and dimeric each monomer comprises at least one heavy chain part.
In the embodiment, the heavy chain of a polypeptide chain of dimer part is identical with on another polypeptide chain of dimer those. In the embodiment, dimeric monomer of the present invention (or halfbody) is identical. In another embodiment, they are not identical. For example, each monomer can comprise different target binding sites.
In the embodiment, dimer of the present invention for example passes through covalent interaction, and disulfide bond links together. In the embodiment, dimer of the present invention links together by one or more disulfide bond. In another embodiment, dimer of the present invention is by one or more, preferred two disulfide bond link together. in another embodiment, dimer of the present invention is by one or more, preferred three disulfide bond link together. in another embodiment, dimer of the present invention is by one or more, and preferred four disulfide bond link together. In another embodiment, dimer of the present invention is by one or more, and preferred five disulfide bond link together. In another embodiment, dimer of the present invention is by one or more, and preferred six disulfide bond link together. In another embodiment, dimer of the present invention is by one or more, and preferred seven disulfide bond link together. In another embodiment, dimer of the present invention is by one or more, and preferred eight disulfide bond link together. In another embodiment, dimer of the present invention is by one or more, and preferred nine disulfide bond link together. In another embodiment, dimer of the present invention is by one or more, and preferred ten disulfide bond link together. In another embodiment, dimer of the present invention does not pass through disulfide bond, but for example, links together by non-covalent reaction.
The heavy chain part of polypeptide can be from different immunoglobulin molecules. For example, the heavy chain of polypeptide part can comprise the CH1 domain that is derived from the IgG1 molecule and the hinge area that is derived from the IgG3 molecule. In another example, the heavy chain part can comprise hinge area, and its part is derived from the IgG1 molecular moiety and is derived from the IgG3 molecule. In another example, the heavy chain part can comprise chimeric hinge, and its part is derived from the IgG1 molecule, and part is derived from the IgG4 molecule.
This paper term " light chain part " comprises the amino acid sequence that is derived from light chain immunoglobulin. Preferably, described light chain partly comprises at least a of VL or CL domain.
In the embodiment, polypeptide of the present invention comprises amino acid sequence or one or more is not derived from the part of Ig molecule. Exemplary being modified at hereinafter described in detail. For example, in the embodiment, polypeptide of the present invention can comprise the flexible joint sequence. In another embodiment, polypeptide can be modified to add funtion part (for example, PEG, medicine, or mark).
In the embodiment, Binding peptide of the present invention is fusion. Fusion is chimeric molecule, and it comprises binding structural domain, and described binding structural domain comprises at least one target binding site and at least one heavy chain part. In the embodiment, fusion also comprises the synthetic peptide that connects.
" chimeric " albumen comprises first amino acid sequence, and it links to each other with natural unconnected second amino acid sequence. Described amino acid sequence is present in the protein of the separation that forms together fused protein usually, and perhaps they usually are present in the same protein but rearrange in fused polypeptide. Chimeric protein or son can produce by for example chemical synthesis or generation and the new polynucleotides of translation, and wherein said peptide district is with required relation coding. Exemplary chimeric polyeptides comprises fusion of the present invention and chimeric chain connection peptide.
Term " allos (heterologous) " is used for polynucleotides and polypeptide, refers to the different entity of the remainder of the entity that described polynucleotides or peptide source are compared from genotype and its. For example, heterologous polynucleotide or antigen can be derived from different plant species source, the same cell type of different cell types or Different Individual.
This paper term " ligand binding domains " or " ligand binding moiety " refer to that any natural receptor (for example, cell surface receptor) or its keep any district or the derivative of at least qualitative ligand binding capacity, the biologically active of preferred corresponding natural receptor.
This paper term " receptors bind domain " or " receptor binding moiety " refer to that any native ligand or its keep bioactive any district or the derivative of the preferred corresponding native ligand of at least qualitative receptor binding capacity.
In the embodiment, binding molecule of the present invention is fusion. Fusion of the present invention is chimeric molecule, and it comprises binding structural domain (it comprises at least one binding site) and Dimerized domain (it comprises at least one heavy chain part). Described heavy chain part can be derived from any immunoglobulin (Ig), such as IgG1, and IgG2, IgG3, or IgG4 hypotype, IgA, IgE, IgD or IgM.
In another embodiment of the present invention, binding molecule is " antibody-fusion chimera ". Described molecule comprises the molecule that at least one binding structural domain of binding antibody is combined with at least one fusion. Preferably, the interface between two peptide species is the CH3 domain of immunoglobulin molecules.
In the embodiment, binding molecule of the present invention is " antibody " or " immunoglobulin (Ig) " molecule, for example, the antibody molecule of naturally occurring antibody or immunoglobulin molecules or genetic modification, it is with the mode conjugated antigen of similar antibody molecule. This paper term " immunoglobulin (Ig) " comprises polypeptide, and it has the combination of two heavy chains and two light chains, and no matter whether it has relevant Immunel response. " antibody " refers to have the aggregate to the obvious known specific immune response activity of purpose antigen (for example tumor associated antigen). Antibody and immunoglobulin (Ig) comprise light and heavy chain, and it is covalently bound to have and do not have interchain therebetween. Basic immunoglobulin structure is relatively known in the vertebrate system.
As will be explained below, hereditary term " immunoglobulin (Ig) " comprises five kinds of dissimilar antibody, and it can be distinguished by biochemical method. All species antibody all obviously comprise within the scope of the present invention, and the IgG kind that relates in general to immunoglobulin molecules below is discussed. For IgG, immunoglobulin (Ig) comprises two identical light polypeptide chains, about 23,000 dalton of its molecular weight, and molecular weight 53,000-70, two identical heavy chains of 000. Described four chains connect with " Y " configuration by disulfide bond, and wherein light chain encases heavy chain at the opening part of " Y ", and is extended to whole variable region.
The district that light chain and heavy chain is divided into the 26S Proteasome Structure and Function homology. Term " constant " and " variable " are used in reference to function. In this respect, will understand variable region decision antigen recognizing and the specificity of light chain (VL) and heavy chain (VH) part. Otherwise the constant region of light chain (CL) and heavy chain (CH1, CH2 and CH3) shows important biological property, such as secretion, strides the placenta energy, the Fc receptors bind, and the complement combination, etc. Usually with antigen binding site or the aminoterminal of constant region away from antibody, the number of constant region increases. The N end is the variable region, and the C end is constant region; CH3 and CL domain reality comprise respectively the carboxyl terminal of heavy chain and light chain.
Light chain is categorized as kappa or lambda (κ, λ). Each heavy chain kind can be combined with kappa or lambda light chain. Usually, when immunoglobulin (Ig) by hybridoma, when the host cell of B cell and genetic modification produced, light chain and heavy chain were mutually covalently bound, " tail " part of two heavy chains by covalent disulfide bonds be connected connection and mutually combine. In heavy chain, amino acid sequence begins to the C end of every chain bottom from the N end of the furcated end that is positioned at the Y configuration. It will be appreciated by those skilled in the art that heavy chain is categorized as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) wherein also have some subclass (for example, γ 1.-γ 4). The character of this chain has determined antibody " kind " to be respectively IgG, IgM, IgA IgG or IgE. Immunoglobulin subclass (isotype) for example, IgG1，IgG 2，IgG 3，IgG 4，IgA 1Etc. character known and known itself so that functional specialization. The version of the modification of each of these kinds and isotype in conjunction with instruction of the present invention, is can be gently intelligible to those skilled in the art, therefore, is included within the scope of the present invention.
As mentioned above, the variable region allows the epi-position on the selective identification of antibody and the specific binding antigen. That is, the V of antibodyLDomain and VHDomain is in conjunction with forming the variable region that limits three-dimensional antigen binding site. This quaternary antibody structure forms the antigen binding site of each the arm end that has Y. More specifically, described antigen binding site passes through VHAnd VLThree complementary determining regions (CDRs) on each bar of chain limit.
This paper term " binding site " or " binding structural domain " comprise the zone of being responsible for the polypeptide of purpose target molecule (for example antigen, part, acceptor, substrate or mortifier) selective binding. The exemplary combination domain comprises antibody variable region, the receptors bind domain of part, the ligand binding domains of acceptor or enzymatic structure territory.
In the embodiment, described binding molecule has at least one binding site, its molecular specific to being reduced or eliminated by target, for example, cell surface antigen or soluble antigen.
In the preferred embodiment, described binding structural domain is antigen binding site. Antigen binding site forms by the variable region, described variable region in a polypeptide from another polypeptide in different. Polypeptide of the present invention comprises at least two antigen binding sites. This paper term " antigen binding site " comprises the site of specific binding (immune response takes place with it) antigen (for example, cell surface or soluble antigen). Antigen binding site comprises heavy chain immunoglobulin and variable region of light chain, and the binding site that these variable regions form determines the antibody specificity. In the embodiment, antigen binding molecules of the present invention comprises at least one heavy chain or the light chain CDR (for example, its sequence is known in the art or described herein) of antibody molecule. In another embodiment, antigen binding molecules of the present invention comprises at least two CDR of one or more antibody molecule. In another embodiment, antigen binding molecules of the present invention comprises at least three CDR of one or more antibody molecule. In another embodiment, antigen binding molecules of the present invention comprises at least four CDR of one or more antibody molecule. In another embodiment, antigen binding molecules of the present invention comprises at least five CDR of one or more antibody molecule. In another embodiment, antigen binding molecules of the present invention comprises at least six CDR of one or more antibody molecule. The exemplary antibodies molecule comprises at least one CDR, and it can be included in the tested antigen binding molecules, is known in the art, example molecule described herein.
Described polypeptide comprises two heavy chain parts disclosed herein, and it can be connected to form the Y of two combinations, so that the four combinations site forms " tetravalence " molecule (for example seeing, WO02/096948A2)). In another embodiment, can prepare the antibody of tetravalence miniantibody or domain disappearance.
Term " specificity " comprises possible binding site number, the given target of its specific binding (immune response for example takes place with it). Polypeptide can be monospecific and comprises one or more binding site, and described locus specificity is in conjunction with target, or polypeptide can be polyspecific and contains the binding site of the identical or different target of two or more specific bindings.
In the embodiment, binding molecule of the present invention is bispecific molecule (for example, antibody, miniantibody, the antibody of domain disappearance, or fusion, its molecule to more than one has binding specificity, for example more than one epi-position on more than one antigen or the same antigen. In the embodiment, described bispecific molecule has at least one target binding site, its to by target with the molecule that is lowered or eliminates and the targeting type molecular specific on the cell. In another embodiment, described bispecific molecule has at least one target binding site that is specific to the molecule that is reduced by target or eliminate and the target binding site that at least one is specific to medicine. In another embodiment, described bispecific molecule has at least one target binding site that is specific to the molecule that is reduced by target or eliminate and the target binding site that at least one is specific to prodrug. In the preferred embodiment, described bispecific molecule is tetravalent antibody, and it has two and is specific to the target binding site that a target and two target binding sites are specific to second target. The tetravalence bispecific molecule can be divalence for every species specificity. Further describing of bispecific molecule is as follows.
This paper term " valency " refers to the number of potential target binding site in the polypeptide. Specificity site on target molecule of each target binding site specific binding or the target molecule. When polypeptide comprises more than one target binding site, but the identical or different molecule of each target binding site specific binding (for example, can be in conjunction with different ligands or synantigen not, or the different epi-positions on the same antigen).
In the naturally occurring antibody, six CDR that exist on each monomeric igg are short, discontinuous amino acid sequence, and it has specific position to form antigen binding site, because antibody is its 3-d modelling in aqueous environment. The intermolecular variation of the remainder displaying acid sequence of heavy chain and light chain variable domain is littler, and is called framework region. The most of beta sheet configuration that adopts of framework region, CDR forms ring, its connection, forming section beta sheet structure under the certain situation. Therefore, these these framework regions are done in order to forming framework, its by the interchain noncovalent interaction so that six CDR location in the right direction. The antigen binding site that the CDR of location forms define with immunoreactivity antigen on the surface of epi-position complementation. This complementary surface enhancing antibody and the non-covalent combination of immunoreactivity epitope. The non-covalent combination of this complementary surface enhancing antibody and immunoreactivity epitope. The position of CDR can be identified easily by those skilled in the art.
As previously mentioned, the subunit structure of the constant region of various immunoglobulin (Ig) kinds and 3-d modelling are known. This paper term " VH domain " comprises the amino terminal variable domains of heavy chain immunoglobulin, and term " CH1 domain " comprises first (most of amino terminal) constant region domain of heavy chain immunoglobulin. The CH1 domain is in abutting connection with the VH domain and be positioned at the amino terminal of heavy chain immunoglobulin molecule hinge area.
This paper term " CH2 domain " comprises the part heavy chain molecule, utilizes conventional numbering plan, and its residue 244 from for example antibody expands to residue 360 (residue 244-360, Kabat numbering system; With residue 231-340, the EU numbering system; With Kabat EA et al.Sequences of Proteins of Immunological Interest.Bethesda, US Department of Health and Human Services, NIH.1991). The unique distinction of CH2 domain is that itself and another domain is not closely to match. And branch's carbohydrate chain of two N connections places between two CH2 domains of complete natural IgG molecule. Also known CH3 domain extends to the C end of complete natural IgG molecule and comprises about 108 residues from the CH2 domain.
This paper term " hinge area " comprises the part heavy chain molecule, and it is connected in the CH2 domain with the CH1 domain. This hinge area comprises about 25 residues and has flexibility, allows thus the terminal antigen binding domain of two N independently mobile. Hinge area can be subdivided into three different domains: hinge arrangement territory, upper, middle and lower (Roux et al.J.Immunol.1998 161:4083).
In the embodiment, binding molecule of the present invention comprises the connection peptide. Connection peptide of the present invention synthesizes. This paper term " synthetic " that relates to polypeptide comprises the polypeptide of the amino acid sequence that contains the non-natural existence. For example, for the non-natural polypeptide (for example comprise sudden change such as interpolation, replace or disappearance) of the modified forms of natural polypeptides or comprise first amino acid sequence (it can be or can not be naturally occurring) that links to each other by natural unconnected second amino acid sequence of linear amino acid sequence and its (its can be or can not for naturally occurring).
Connection polypeptide of the present invention connects two domains (for example, binding structural domain and Dimerized domain) of binding molecule of the present invention. For example, connect peptide and heavy chain partly is connected in the binding structural domain that comprises binding site. In the embodiment, two CH domains in the linear amino acid sequence of connection peptide connection polypeptide chain are such as CH1 and CH2 domain; CH1 and CH3 domain; Hinge and CH1 domain; Hinge and CH3 domain; VH and hinge arrangement territory, or CH3 domain and non--immunoglobulin polypeptides). Preferably, described connection peptide provides flexible for peptide molecule and promotes Dimerized via disulfide bond. Connection peptide of the present invention is used for (for example replacing one or more heavy chain domain, at least part of constant region domain in the construct of domain disappearance (for example, at least part of CH2 domain) and/or at least part of hinge area (for example, at least part of low hinge area domain)). For example, in the embodiment, the VH domain is blended in CH3 domain (connect PEPC-end and be attached to CH3 domain N-end, connect peptide N-end and be attached to VH domain C-end) via connecting peptide. In another embodiment, the VL domain is blended in CH3 domain (connect PEPC-end and be attached to CH3 domain N-end, connect peptide N-end and be attached to VL domain C-end) via connecting peptide. In another embodiment, the CH1 domain is blended in CH3 domain (connect PEPC-end and be attached to CH3 domain N-end, connect peptide N-end and be attached to CH1 domain C-end) via connecting peptide.
In the embodiment, the synthetic peptide that connects comprises part constant region domain. For example, in the embodiment, the connection peptide that replaces the CH2 domain can comprise part CH2 domain.
In the embodiment, connect peptide and comprise or formed by the gly-ser joint. This paper term " gly-ser joint " refers to a kind of peptide, and it is made up of glycine and serine residue. Exemplary gly/ser joint comprises amino acid sequence GGGSSGGGSG (SEQ ID NO:1). In the embodiment, connection peptide of the present invention (for example comprises at least part of upper hinge district, be derived from IgG1, IgG3 or IgG4 molecule), hinge area (for example, is derived from IgG1, IgG3 at least part of, or IgG4 molecule) and a series of gly/ser amino acid residue (for example, gly/ser joint such as GGGSSGGGSG (SEQ ID NO:1)). In the embodiment, compare with naturally occurring IgG1 or IgG3 hinge area, described connection peptide comprises one or more 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor. In another embodiment, this connection peptide comprises the amino acid sequence described in WO 02/060955. Connecting peptide describes in detail hereinafter.
This paper term " disulfide bond " comprises the covalent bond that forms between two sulphur atoms. Cysteine comprises thiol group, its can form disulfide bond or with second thiol group bridge joint. In most of naturally occurring IgG molecules, CH1 links to each other by disulfide bond with the CL district, and two heavy chains link to each other by two disulfide bond, utilize the Kabat numbering system, corresponding 239 and 242 (position 226 or 229, the EU numbering systems) in the position of described disulfide bond.
Known in the art, constant region mediates multiple effector function. For example, C1 component and antibody in conjunction with the complement activation system. The activation of complement is important to conditioning and the dissolving of cytopathy substance. Complement activation also stimulates inflammatory reaction and also participates in self hypersensitivity. In addition, in conjunction with cell, wherein the Fc acceptor site in the antibody Fc district is in conjunction with the Fc acceptor (FcR) on the cell via the Fc district for antibody. The multiple Fc acceptor special to different types of antibody arranged, comprise IgG (gamma acceptor), IgE (epsilon acceptor), IgA (alpha acceptor) and IgM (mu acceptor). The combination of antibody and cell surface Fc acceptor excites multiple important and various biological respinse, comprise the particle of engulfing and destroying antibody sandwich, remove immune complex, by the coated target of killer cell lytic antibody (cell toxicant or the ADCC that are called the antibody dependent cellular mediation), discharge immune-mediated thing, the placenta of immunoglobulin (Ig) shifts and generates control.
In the embodiment, described Fc part can utilize technology known in the art to suddenly change to reduce effector function. For example the disappearance of constant region domain or inactivation (by point mutation or other method) thus the Fc receptors bind that can reduce the modified antibody of circulation increases tumor-localizing. In other situation, constant region is modified can be consistent with appropriate complement combination of the present invention, reduces thus the cytotoxic non-specific binding of serum half life and coupling. Other modification of constant region can be used for modifying disulfide bond or oligosaccharides part, and it allows because the flexible location that causes that increases of antigentic specificity or antibody increases. More generally, the antibody that those skilled in the art will recognize that the modification of this paper can show the multiple delicate effect that can or can not recognize easily. Yet the physiology collection of illustrative plates that produces, other biochemical effect of bioavilability and modification such as tumor-localizing, bio distribution and serum half life, can utilize known immunological technique to need not quantitatively that unsuitable test is measured easily and quantitatively.
In the embodiment, the antibody of modified forms can utilize technology preparation known in the art from complete precursor or parental antibody. Exemplary techniques more is discussed in detail hereinafter. In the concrete preferred embodiment, the variable region of polypeptide of the present invention and constant region all are the people. In the embodiment, fully human antibodies can utilize technology preparation known in the art. For example the fully human antibodies of anti-specific antigen can be by preparing antigen administration transgenic animals, and described animal via is modified and responded to the antigen attack and produce described antibody, but its endogenous gene seat is inactivation. For the preparation of the exemplary techniques of antibody at United States Patent (USP): 6,150,584; 6,458,592; Describe in 6,420,140. Other technology is known in the art.
The polypeptide that comprises the heavy chain part can comprise or not comprise other amino acid sequence or the part that is not derived from immunoglobulin molecules. Described being modified at hereinafter described in detail. For example, in the embodiment, polypeptide of the present invention can comprise the flexible joint sequence. In another embodiment, polypeptide can be modified to add funtion part such as PEG.
Polypeptide of the present invention comprises at least two binding sites, itself so that polypeptide be combined with selected target molecule.
In the embodiment, binding molecule of the present invention comprises antibody molecule, for example, and complete antibody molecule, or the fragment of antibody molecule. In another embodiment, binding molecule of the present invention is that modify or synthetic antibody molecule. In the embodiment, binding molecule of the present invention comprises monoclonal antibody, humanized antibody, chimeric antibody, or all or part (for example, at least one antigen binding site of the antibody of restructuring preparation, at least one CDR, or at least one heavy chain part).
That described antigen binding site and described heavy chain partly need not to be derived from identical immunoglobulin molecules in the embodiment of antibody of antibody or modification at described binding molecule. In this respect, the variable region can be derived from the animal of any type, and it can be induced and excite humoral response and produce immunoglobulin (Ig) for described antigen. Thus, the variable region of polypeptide can for example can be the people for example from mammal, mouse, and non-human primates (such as machin, macaque etc.), wolf, the camel class (for example, from camel, vigone (llamas) and relative species) source. In another embodiment, described variable region can be from cartilage class animal (condricthoid) (for example, from shark).
Polypeptide of the present invention can utilize technology preparation known in the art. In the embodiment, polypeptide of the present invention is " restructuring preparation " antibody molecule, namely utilizes recombinant DNA technology to prepare. The exemplary techniques of Dispersal risk molecule is described hereinafter in more detail.
In the embodiment, polypeptide of the present invention is the antibody of modifying. This paper term " antibody of modification " comprises the antibody of synthetic form, and it is changed so that they are not naturally occurring. For example comprise at least two heavy chains part rather than two complete heavy chains antibody (such as, antibody or the miniantibody of domain disappearance); Multi-specificity antibody (for example, bispecific, tri-specific etc.), it is changed with in conjunction with two or more not synantigens or in conjunction with the different epi-positions on the single antigen; Be connected in the heavy chain molecule of scFv molecule etc. The ScFv molecule is known in the art and for example, and United States Patent (USP) 5,892 is described in 019. In addition, term " antibody of modification " comprises the antibody (for example, trivalent, the antibody such as tetravalence, it is in conjunction with the same antigen of three or more copies) of multivalence form. In another embodiment, binding molecule of the present invention is fusion, the polypeptide binding structural domain that it comprises at least one heavy chain part that lacks the CH2 domain and comprises the bound fraction that contains a right member of receptors ligand.
In the embodiment, term of the present invention " antibody of modification " comprises immunoglobulin (Ig), antibody, or immunoreactivity fragment or its recombinant, wherein at least a portion of one or more constant region domain is lacked or is changed to provide required biochemical characteristic, such as the ability that non-covalent dimerization takes place, be positioned the ability of the increase of tumor sites, or compare the serum half life of reduction with the roughly the same complete unaltered antibody of immunogenicity. In the preferred embodiment, polypeptide of the present invention is the antibody of domain disappearance, and it comprises the polypeptide chain that is similar to heavy chain immunoglobulin, but it lacks at least a portion of one or more heavy chain domain. More preferably, the antibody constant region of modification complete structure territory will by disappearance and more preferably all or part CH2 domain will be lacked.
In the preferred embodiment, polypeptide of the present invention is with the harmful immune response in the exciting human. The constant region compatible with the present invention modified one or more the amino acid whose interpolation that comprises in one or more domain, disappearance or replacement. That is, polypeptide of the present invention can comprise in three heavy chain constant domain (CH1, CH2 or CH3) one or more and/or to change or the modification of constant region of light chain domain (CL).
For example, in the embodiment, the present invention relates to humanization CC49 (huCC49 version 2 (V2)), have amino acid difference between itself and the previously known humanization CC49. Figure 19 A and B show the light and variable region of heavy chain of mouse CC49, people's antibody LEN or 21/28 ' CL, humanized CC49, (it is compared with humanized CC49 with humanized CC49V2, comprise place's 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor in the light chain and two place's 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factors in the heavy chain, take a favourable turn position 43 and the position 69 of heavy chain and 93 the underlined amino acid of chain, Kabat numbering system) other comparison of branch.
In the embodiment, the present invention relates to humanized CC49, it comprises new connection peptide of the present invention. For example, Figure 13 A (SEQ ID NO:24) shows the dna sequence dna of the huCC49 V2 of the heavy chain CH2 domain contain G1/G3/Pro243Ala244Pro245+[Gly/Ser] chain connection peptide-disappearance. Figure 13 B (SEQ ID NO:25) shows the dna sequence dna of the huCC49 V2 of light chain CH2 domain-disappearance. Figure 14 A (SEQ ID NO:26) show contain G1/G3/Pro243Ala244Pro245+[Gly/Ser] amino acid sequence of the huCC49 V2 of the heavy chain CH2 domain of chain connection peptide-disappearance. Figure 14 B (SEQ ID NO:27) shows the amino acid sequence of the huCC49 V2 of light chain CH2 domain-disappearance.
In the embodiment, polypeptide of the present invention can utilize technology known in the art to modify to reduce their immunogenicity.For example, antibody of the present invention or polypeptide can be humanized, go the immunity, maybe can prepare chimeric antibody.The antibody of these types is derived from non--people's antibody is generally murine antibody, its maintenance or keep the antigen of parental antibody-substantially in conjunction with character, but its immunogenicity in the people is lower.This can realize by the whole bag of tricks, comprise (a) with whole non--people's variable domains is transplanted on the human constant region to generate chimeric antibody; (b) at least a portion of one or more inhuman complementary determining region (CDR) is transplanted in people's framework and the constant region, is kept or do not keep crucial framework residue; Or (c) transplant whole non--people's variable domains, but by replace surface residue with the proper manners secretion " cover (cloak) " they.Described method is disclosed in Morrison et al., Proc.Natl.Acad.Sci.81:6851-5 (1984); Morrison et al., Adv.Immunol.44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan, Molec.Immun.28:489-498 (1991); Padlan, Molec.Immun.31:169-217 (1994) and United States Patent (USP) 5,585,089,5,693,761 and 5,693,762, its full text content is included in this as a reference.
Go immunity to can be used for reducing antibody mediated immunity originality.This paper term " go immunity " and comprise to change antibody with modify t cell epitope (for example see, WO9852976A1, WO0034317A2).For example, the VH of initial antibody and VL sequence are analyzed, and human T-cell's epi-position " collection of illustrative plates " in each V district shows the location with complementary determining region (CDR) associated epitope, and other the important residue in the described sequence.Each t cell epitope of analyzing the t cell epitope collection of illustrative plates is to identify that changing the less optional amino acid of final antibody activity possibility replaces.Plurality of optional VH and VL sequences Design make up for comprising aminoacid replacement, and these sequences are impregnated in the polypeptide of the present invention of multiple its function to be determined subsequently.Usually, produce 12-24 kind variant antibody and detection.Comprise the V of modification and the heavy fully and light chain gene in people C district and be cloned into expression vector subsequently, and be imported into the plasmid that clone is used to prepare complete antibody subsequently.Described antibody compares at suitable biochemistry and bioassay method kind subsequently, identifies optimum variant.
In the embodiment, described binding molecule comprises chimeric antibody.In background of the present invention, finger any wherein immunoreactivity zone of term " chimeric antibody " or site derive from or be derived from first species and constant region (it can be complete, part or modify according to the present invention) available from the antibody of second kind of species.In the embodiment preferred, described target land or site are from inhuman source (for example mouse), and constant region is a human constant region.Preferably, heavy chain and constant region of light chain replace one or more CDR by part and change by replacement of part frame district and sequence if desired.Although CDRs can be derived from the antibody in framework region source of the same race or even with the antibody of subspecies, expection CDR will be derived from different types of antibody and preferred from the antibody that is derived from different plant species.Need not to be used for complete CDR from the donor variable region replaces all CDR and the antigen binding capacity of a variable region can be transferred to another.Yet, may only need to shift and keep the active required residue of target binding site.According to United States Patent (USP) 5,585,089,5,693,761 and 5,693,762 explanation, those skilled in the art can be by implementing routine test or obtaining the functional antibodies that immunogenicity reduces by test or error-detecting.
This paper term " correct folding polypeptide " comprises such polypeptide (for example, antigen binding molecules is such as antibody), wherein comprises the active difference of all functions structural domain of described polypeptide.This paper term " incorrect folding polypeptide " comprises the wherein polypeptide of at least one functional domain non-activity of polypeptide.In the embodiment, correct folding polypeptide comprises the polypeptide chain that connects by at least one disulfide linkage, otherwise incorrect folding polypeptide comprises the polypeptide chain that is not by at least one disulfide linkage connection.
This paper term " pernicious " refers to non-benign tumour or cancer.This paper term " cancer " comprises the malignant disease that is characterised in that cell growth imbalance or out of control.Exemplary cancer comprises: cancer, sarcoma, leukemia and lymphoma.Term " cancer " comprises primary malignant neoplasm (site of original tumor sites except for example its cell is not moved in the subject) and secondary malignant tumour (for example be derived from transfer, tumor cell migration is gone into the secondary site different with original tumor sites).
In the embodiment, binding molecule of the present invention is in conjunction with tumour cell.The exemplary antibodies that comprises in conjunction with the antigenic antigen binding site of tumor cells expression is known in the art, and one or more CDR of described antibody can be included in the binding molecule of the present invention.Exemplary antibodies comprises: 2B8, and Lym 1, and Lym 2, LL2, Her2, B1, MB1, BH3, B4, B72.3,5E8, B3F6 and 5E10.In the embodiment preferred, polypeptide of the present invention is the C2B8 antibody in conjunction with CD20.In another embodiment preferred, polypeptide of the present invention is the CC49 antibody of identification TAG72.
In the embodiment, binding molecule of the present invention is in conjunction with the molecule that can be used for treating autoimmunization or inflammatory diseases or illness.
This paper term " autoimmune disease or illness " refers to the disease among the experimenter, and wherein immune system attack body self cell causes disorganization.Autoimmunization also comprises the general autoimmune disease, and promptly wherein autoimmune response appears at multiple tissue simultaneously, or the organ specificity autoimmune disease, i.e. the single organ of autoimmune response target wherein.Can include, but are not limited to clone disease by the example of method and composition diagnosis of the present invention, prevention or the autoimmune disease for the treatment of; Inflammatory bowel (IBD); Systemic lupus erythematous; Ulcerative colitis; Rheumatoid arthritis; Goodpasture ' s syndrome; Grave ' s disease; Hashimoto (the thyroiditis of Hashimoto ' s); Pemphigus vulgaris; Myasthenia gravis; Scleroderma; Autoimmune hemolytic anemia; Autoimmune thrombocytopenic purpura; Polymyositis and dermatomyositis; Pernicious anemia; Sjogren syndrome; The competency spondylitis; Vasculitis; Type i diabetes; Sacred disease, multiple sclerosis, and the secondary disease that causes of autoimmune disease.
This paper term " inflammatory diseases or illness " comprises the disease that is caused or increased the weight of by inflammation to small part, the blood flow of Zeng Jiaing for example, oedema, the activation of immunocyte (propagation for example, cytokine generates, or engulfing of increasing).Exemplary disease comprises wherein inflammation or inflammatory factor or (for example, matrix metalloproteinase (MMPs), nitrogen protoxide (NO), TNF, interleukin, plasmosin matter, cell defense system, cytokine, lipid metabolism thing, proteolytic enzyme, toxicity free radical, plastosome, apoptosis, adhesion molecules etc.) participate in abnormal amount or be present in a certain zone, for example described amount can advantageously change, for example so that favourable to the experimenter.Inflammatory process is the reaction of biological tissue to damage.The reason of inflammation can be physical damnification, chemical substance, microorganism, tissue necrosis, cancer or other medicament.The acute inflammation time length is short, only continues a couple of days.If its lasting long period, can be called chronic inflammatory diseases so.
Inflammatory diseases comprises the acute inflammation disease, chronic inflammatory disease and recurrent inflammatory diseases.The common time-histories of acute inflammation disease is shorter, continues approximate number minute by about 1-2 days, but its also sustainable several weeks.The principal character of acute inflammation disease comprises that blood flow increases, and liquid and plasma albumin ooze out (oedema), and white corpuscle is moved out such as neutrophilic granulocyte.The common course of disease of chronic inflammatory disease is longer, for example several weeks to several months to several years or longer time, and the propagation with the existence of lymphocyte and scavenger cell and blood vessel and knot a kind of thick silk tissue is relevant on histology.The recurrent inflammatory diseases is included in the disease that recurs after for some time and has the periodically disease of the course of disease.The example of recurrent inflammatory diseases comprises asthma and multiple sclerosis.Some diseases can fall into one or more kind.
The feature of inflammatory diseases be usually heat, red, swollen, the pain and afunction.The example of the inflammatory diseases cause of disease includes but not limited to infected by microbes (bacterium for example, virus and fungi infestation), physical factor (for example burn radiates and wound), chemical factor (for example toxin and pathogenic material), tissue necrosis and various types of immune response.The example of inflammatory diseases includes but not limited to, osteoarthritis, rheumatoid arthritis, acute and chronic infection (bacterium, virus and fungi); Acute and chronic bronchitis, sinusitis paranasal sinusitis, and other respiratory infection comprise common cold; Acute and chronic gastroenteritis and colitis; Acute and chronic cystitis and urethritis; Adult respiratory distress syndrome, cystic fibrosis; Acute and chronic dermatitis; Acute and chronic conjunctivitis; Acute and chronic serositis (pericarditis, peritonitis, synovitis, pleuritis and tendinitis); Uremic pericarditis; Acute and chronic cholecystitis; Acute and chronic vaginitis; Acute and chronic uveitis; Drug reaction; And burn (heat, chemistry and).
" medium based on the hydrophobic reactant isolated polypeptide " comprises the medium that contains the hydrophobic ligand (for example alkyl and aryl) that is covalently attached to matrix to this paper term.Described medium can be used for based on the interaction isolated polypeptide between the hydrophobic ligand of the hydrotropy on polypeptide surface and come-at-able non-polar group and described medium.Exemplary media is phenyl 5PW-HR, and it can derive from Tosoh Bioscience.
This paper term " electric conductivity " comprises the electrical conductivity of solution of measuring with microSiemens/cm (being micromhos/cm in the past).The ion content of solution is high more, and electrical conductivity of solution is high more.Specific conductivity can utilize technology known in the art to measure (for example by measuring by the electric current between two electrodes) easily.
Separation method of the present invention can utilize the pH scope to arrive neutral solution for acidity, and for example approximately pH3.5 arrives approximately neutral.This paper term " approximately neutral pH " comprises about 7 pH.For example, in the embodiment, separation method of the present invention can utilize the solution (for example damping fluid) with following pH to carry out: about 3, about 4, about 5, about 6, about 7, or about 8.Preferably, described pH value of solution is about 6 or about 7.In the embodiment, described pH value of solution is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.
This paper term " affinity matrix " comprises such matrix, such as the agarose that is attached with affinity ligand, and the porose glass of control and poly (vinylbenzene divinyl (styrenedivinyl)) benzene.Described affinity ligand is in conjunction with required polypeptide, and contaminative polypeptide debond affinity ligand.Required polypeptide can utilize known arrangement from the affinity matrix wash-out.
This paper term " transformation " comprises by synthetic method (for example by recombinant technology, external peptide is synthetic, by the enzymatic of peptide and some combinations of chemical coupling and these technology) operation nucleic acid or peptide molecule.Preferably, binding molecule of the present invention is transformed, for example to express connection peptides of the present invention.
This paper term " connect, " " merge " or " fusions " is used interchangeably.Described term refers to by any method that comprises chemical coupling or recombination method two above elements be linked to each other with component." the frame endomixis " refers to two or more open reading frame (ORF) are connected to form long continuously ORF, the feasible correct frame that keeps original ORF of its mode.Therefore, the recombination fusion protein of generation is to contain two or more segmental single albumen, the corresponding original ORF encoded polypeptides (this fragment is not natural connection usually) of described fragment.Although continuously, described fragment can be by for example frame internal connection sequence physics or spatial isolation in the fragment of whole fusion for frame.
In the background of polypeptide, " linear order " or " sequence " is a series of amino acid from amino to the carboxyl direction in the polypeptide, and wherein adjacent residue is adjacent in the primary structure of polypeptide in the sequence.
" experimenter that can benefit from the administration of binding molecule " comprises such experimenter to this paper term, such as mammalian subject, it can benefit from the administration of used binding molecule, for example be used to detect antigenic (for example being used for diagnostic method) that binding molecule is discerned, and/or from being used to reduce or remove the treatment benefit of utilizing binding molecule of the target of described binding molecule identification.For example, in the embodiment, the benefit that reduces or eliminates that described experimenter can be from solubility or concrete molecule (for example toxin or pathogenic agent) from circulation or serum, or from the minimizing of the cell colony (for example tumour cell) of expressing described target or remove and benefit.As preceding specifically as described in, described binding molecule can with non-link coupled form use or the link coupled form for example with medicine, prodrug, or isotype link coupled form is used.
II. synthetic connection peptides
Dimeric at least one polypeptide chain of the present invention can comprise synthetic connection peptides of the present invention.In the embodiment, dimeric at least two chains of the present invention comprise connection peptides.In the embodiment preferred, dimeric two chains of the present invention comprise connection peptides.
In the embodiment, connection peptides is used in two heavy chain parts of the interior connection of frame of single polypeptide chain.For example, in the embodiment, connection peptides of the present invention can be used for CH3 structural domain (or synthetic CH3 structural domain) is blended in hinge area (or synthetic hinge area).In another embodiment, connection peptides of the present invention can be used for CH3 structural domain (or synthetic CH3 structural domain) is blended in CH1 structural domain (or synthetic CH1 structural domain).In another embodiment, connection peptides can be used as the spacer between hinge area (or synthetic hinge area) and the CH2 structural domain (or synthetic CH2 structural domain).
In another embodiment, the CH3 structural domain (for example can be blended in the extracellular protein structural domain, VL structural domain (or composite structure territory), VH structural domain (or composite structure territory), CH1 structural domain (or composite structure territory), hinge arrangement territory (or synthetic hinge), or be blended in the ligand binding moiety of acceptor or the receptor binding moiety of part).For example, in the embodiment, VH or VL structural domain are blended in CH3 structural domain (the C-end of this connection peptides is attached to the N-end of CH3 structural domain, and the N-end of this connection peptides is attached to the C-end of VH or VL structural domain) via connection peptides.In another embodiment, the CH1 structural domain is blended in CH3 structural domain (the C-end of this connection peptides is attached to the N-end of CH3 structural domain, and the N-end of this connection peptides is attached to the C-end of CH1 structural domain) via connection peptides.In another embodiment, connection peptides of the present invention can be used for CH3 structural domain (or synthetic CH3 structural domain) is blended in hinge area (or synthetic hinge area) or its part.In another embodiment, connection peptides can be used as the spacer between hinge area (or synthetic hinge area) and the CH2 structural domain (or synthetic CH2 structural domain).
In the embodiment, connection peptides can comprise or be made up of the gly/ser spacer.For example, having the CC49 construct that the structural domain of the short amino acid spacer GGSSGGGGSG (SEQ.ID No.1) that replaces the CH2 structural domain and rudimentary hinge area (CC49. Δ CH2[gly/ser]) lacks can be used.In another embodiment, connection peptides comprises aminoacid sequence IGKTISKKAK (SEQ ID NO:36).
In another embodiment, connection peptides can comprise partial immunity sphaeroprotein hinge area at least.For example, can make up the chimeric hinge arrangement territory that hinge components wherein is derived from the different antibodies isotype.In the embodiment, connection peptides comprises the hinge area to small part IgG1.In another embodiment, connection peptides can comprise the hinge area to small part IgG3.In another embodiment, connection peptides can comprise to small part IgG1 hinge area with to small part IgG3 hinge area.In the embodiment, connection peptides can comprise on the IgG1 and hinge repetition motif among middle hinge and the single IgG3.
Because described comprising can be according to the length variations of described connection peptides from the numbering of the single amino acids in the connection peptides of the aminoacid sequence of immunoglobulin hinge region, the numbering of amino acid position utilizes the Kabat numbering to provide in these molecules, referring to table 2.Table 1 shows IgG1, the naturally occurring hinge sequence of IgG3 and IgG4 molecule.Table 2 shows that the Kabat of the part of these hinge molecules numbers and also show the Kabat numbering of the connection peptides amino-acid residue in this table.
In the embodiment, connection peptides of the present invention comprises non--naturally occurring immunoglobulin hinge region structural domain, for example, thus not naturally be present in the hinge area structural domain in the polypeptide that comprises the hinge area structural domain and/or be changed the hinge area structural domain different with the aminoacid sequence of naturally occurring immunoglobulin hinge region structural domain.In the embodiment, can make the sudden change of hinge area structural domain to produce connection peptides of the present invention.In the embodiment, connection peptides of the present invention comprises and do not conform to the natural hinge arrangement territory that has a number halfcystine, the halfcystine number that promptly described connection peptides comprises be less than or unnecessary naturally occurring hinge molecule in the number of halfcystine.In the embodiment preferred, connection peptides is mixed polypeptide produce such composition, wherein surpass 50%, 60%, 70%, 80% or 90% dimer molecule and exist via the form that connects at least one interchain disulfide bond with two heavy chains parts wherein.
In one embodiment of the invention, connection peptides comprises the hinge area structural domain, and described hinge area structural domain comprises proline residue, and its amino acid position is corresponding to the amino acid position in the Kabat numbering system 243 (position 230, EU numbering system).In the embodiment, connection peptides comprises alanine residue, and its amino acid position is corresponding to position 244, Kabat numbering system (position 246, EU numbering system).In another embodiment, connection peptides of the present invention comprises proline residue, and its amino acid position is corresponding to position 245 (Kabat numbering system; Position 247, the EU numbering system)).In the embodiment, connection peptides comprises cysteine residues, and its amino position is corresponding to position 239, Kabat numbering system (position 226, EU numbering system).In the embodiment, connection peptides comprises serine residue, and its amino position is corresponding to position 239, Kabat numbering system (position 226, EU numbering system).In the embodiment, connection peptides comprises cysteine residues, and its amino position is corresponding to position 242, Kabat numbering system (position 229, EU numbering system).In the embodiment, connection peptides comprises serine residue, and its amino position is corresponding to position 242, Kabat numbering system (position 229, EU numbering system).
In the embodiment, described connection peptides can be selected as causing the preferential synthetic of the concrete isotype of polypeptide, and for example, wherein two heavy chain parts are via connecting disulfide linkage or not connecting via disulfide linkage.For example, described in the embodiment of the invention, G1/G3/Pro243+[gly/ser] joint (SEQ ID NO:8), G1/G3/Pro243Ala244Pro245+[gly/ser] joint (SEQ ID NO:9), Pro243+[gly/ser] joint (SEQ ID NO:15), and Pro243Ala244Pro245+[gly/ser] joint (SEQID NO:14), connection peptides causes only producing the antibody of A type CH2 structural domain-disappearance and does not have detectable Type B.On the contrary, the Cys242Ser:Pro243Ala244Pro245 (SEQ ID NO:13) of the Cys242Ser:Pro243 of CH2 structural domain-disappearance (SEQ ID NO:12) and CH2 structural domain-disappearance all causes the preference to the B isotype.These synthetic hinge area connection peptides will can be used for helping the synthetic of A or B isotype thus.This is all like this for any antibody isotype (for example, IgG1, IgG2, IgG3, or IgG4), and it is based on the high homology between the CH3 structural domain of all four kinds of people's isotypes.(comprise identical and conservative amino-acid residue, IgG1 CH3 structural domain and IgG2 CH398.13% homology are with IgG3 CH397.20% homology, with IgG4 CH396.26% homology).The parenthesis that are used for connection peptides of the present invention and various binding molecules are represented equivalent terms, except as otherwise noted.
In the embodiment, it is flexible gly/ser joint then that connection peptides of the present invention comprises the hinge area structural domain.Exemplary connection peptides is shown in table 2 and SEQ ID NO:8-15,37 and 53.The various forms of understanding these exemplary connection peptides can be replaced by importing one or more Nucleotide, add or lack making one or more aminoacid replacement in the nucleotide sequence of coding connection peptides, add or disappearance is imported into connection peptides and produces.For example, sudden change can import by standard technique, such as site-directed mutagenesis and PCR-mediated mutagenesis.Preferably, conserved amino acid is substituted in one or more non--indispensable amino acid residue to make that connection peptides preferably improves the ability of A type or Type B constant." conserved amino acid replacement " is the amino acid that wherein amino-acid residue is had the aminoacid replacement of similar side chain.Family with amino-acid residue of similar side chain defines at this paper, and it comprises basic side chain (for example Methionin, arginine, Histidine), acid side-chain (for example aspartic acid, L-glutamic acid), uncharged polar side chain is (for example, glycine, l-asparagine, glutamine, Serine, Threonine, tyrosine, halfcystine), non-polar sidechain (for example, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), beta-ramose side chain (for example, Threonine, Xie Ansuan, Isoleucine) and aromatic side chain (for example, tyrosine, phenylalanine, tryptophane, Histidine).Therefore, the amino-acid residue in the immunoglobulin polypeptides is preferably by another aminoacid replacement from identical side chain family.In another embodiment, a string amino acid can be by similar but different amino acid string of order and/or side chain family member's combination replace.
Connection peptides of the present invention can be multiple length.In the embodiment, about 50 amino acid of the about 15-of the length of connection peptides of the present invention.In another embodiment, the length of connection peptides of the present invention is about 45 amino acid of about 20-.In another embodiment, connection peptides length of the present invention is about 40 amino acid of about 25-.In another embodiment, the length of connection peptides of the present invention is about 35 amino acid of about 30-.In another embodiment, connection peptides length of the present invention is about 27 amino acid of about 24-.In another embodiment, connection peptides length of the present invention is about 42 amino acid of about 40-.
Connection peptides can utilize technology known in the art to import peptide sequence.For example, in the embodiment, can utilize by Overlap Extension (SOE) method (Horton, R.M.1993 Methods inMolecular Biology, Vol 15:PCR Protocols:Current Methods and applications.Ed.B.A.White) splicing (Splicing).Modification can confirm by dna sequence analysis.Plasmid DNA can be used for transformed host cell with the stable polypeptide that generates that produces.
In the embodiment, one of purpose connection peptides is mixed polypeptide produce the composition that comprises peptide molecule, wherein said peptide molecule has at least two binding sites and at least two polypeptide chains, wherein at least two described polypeptide chains comprise synthetic connection peptides, wherein surpass 50% molecule and exist via the form that connects at least one interchain disulfide bond with two heavy chains parts wherein.In another embodiment, surpass 60% molecule and exist via the form that connects at least one interchain disulfide bond with two heavy chains parts wherein.In another embodiment, surpass 70% molecule and exist via the form that connects at least one interchain disulfide bond with two heavy chains parts wherein.In another embodiment, surpass 80% molecule and exist via the form that connects at least one interchain disulfide bond with two heavy chains parts wherein.In another embodiment, surpass 90% molecule and exist via the form that connects at least one interchain disulfide bond with two heavy chains parts wherein.
III. binding molecule
A. antibody or its part
In the embodiment, binding molecule of the present invention is an antibody molecule.Utilize scheme known in the art, for example, antibody is preferably by repeatedly exciting through subcutaneous or intraperitoneal injection related antigen (for example, the tumor associated antigen of purifying or comprise described antigenic cell or cell extract) and adjuvant in Mammals.Immunity like this is challenge usually, comprises from activatory splenocyte or lymphocyte producing antigen reactivity antibody.Though the antibody that is generated can need be gathered in the crops individual lymphocyte so that the homogeneous prepared product of monoclonal antibody (MAb) to be provided from spleen, lymphoglandula or peripheral blood usually from animal serum results so that the polyclone prepared product to be provided.Preferably, described lymphocyte is available from spleen.
(Kohler et al. in this known method, Nature, 256:495 (1975)) antigenic mammiferous short-lived relatively from having injected, or lymphocyte that will certainly die and immortalization tumor cell line (for example myeloma cell line) fusion, produce hybrid cell or " hybridoma " thus, it is not only immortalization and antibody that can produce the genetic coding of B cell.The heterozygote that produces is separated into the single bacterial strain that belongs to together by selection, dilution and regrowth, and each individual bacterial strain comprises the specific gene of single individual information.Their produce at required antigenic homogeneous antibody, and the pure source that belongs to together according to them is called " monoclonal ".
So the hybridoma of preparation is inoculated and is grown in the appropriate media, and it preferably contains one or more and suppresses fusion, parent myeloma cell's growth or the material of survival.The reagent, clone and the medium that it will be understood by those skilled in the art that the formation that is used for hybridoma, selection, growth can be available from multiple sources, and standard scheme is to determine.Usually, measure wherein growth and have in the substratum of hybridoma generation at required antigenic monoclonal antibody.Preferably, the binding specificity of the monoclonal antibody of hybridoma generation is by immunoprecipitation or external test method such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) mensuration.Hybridoma is accredited as to produce has after required specificity, affinity and/or the active antibody, described clone can be by restriction dilution method subclone and by standard method growth (Goding, Monoclonal Antibodies:Principles and Practice, pp59-103 (Academic Press, 1986)).Should be further understood that described subclone excretory monoclonal antibody can be by conventional method of purification such as for example protein-A, hydroxyapatite, gel electrophoresis, dialysis or affinity chromatography are separated from substratum.
In another embodiment, the DNA of the required monoclonal antibody of encoding can utilize ordinary method delamination and order-checking (for example by utilize can specificity in conjunction with the oligonucleotide probe of the gene of encode murine antibody heavy chain and light chain).The hybridoma of isolating and subclone is as the preferred source of described DNA.In case separate, DNA can place expression vector, subsequently expression vector is transfected into otherwise does not produce the protokaryon of immunoglobulin (Ig) or eukaryotic host cell such as Bacillus coli cells, monkey COS cell, Chinese hamster ovary (CHO) cell or myeloma cell.More specifically, separated DNA (as described herein its can be synthetic) can be used for cloning constant and variable region sequences is used for the al. as Newman et, the United States Patent (USP) 5,658 that 1995-1-25 submits to, 570 described preparation antibody, described document is included in this paper as a reference.In fact, this need change into cDNA from selected cell extraction RNA, and utilizes the Ig Auele Specific Primer to pass through pcr amplification.The suitable primer that is used for this purpose is also at United States Patent (USP) 5,658, describes in 570.Following stationery body is described, expresses can growing so that clinical and commercial required immunoglobulin (Ig) to be provided with relatively large amount through cell transformed of required antibody.
It will be appreciated by those skilled in the art that (for example, antigen binding site) DNA also can be derived from the antibody phage storehouse, for example utilizes pd phage or Fd phasmid technology for encoding antibody or antibody fragment.Illustrative methods is for example seen, EP 368 684 B1; United States Patent (USP) 5,969,108, Hoogenboom, H.R. and Chames.2000.Immunol.Today 21:371; Nagy et al.2002.Nat.Med.8:801; Huie etal.2001.Proc.Natl.Acad.Sci.USA 98:2682; Lui et al.2002.J.Mol.Biol.315:1063, each piece of writing is included in this paper as a reference.Some open source literatures (for example, Marks et al.Bio/Technology 10:779-783 (1992)) have been described by reorganization in chain reorganization and combination infection and the body and have been prepared high affinity human antibody as the strategy that makes up big phage library.In another embodiment, ribosomal display can be used for replacing phage and (for example sees Hanes et al.2000.Nat.Biotechnol.18:1287 as display platform; Wilson et al.2001.Proc.Natl.Acad.Sci.USA98:3750; Or in another embodiment of Irving et al.2001 J.Immunol.Methods 248:31., can screen antibody (the Boder et al.2000.Proc.Natl.Acad.Sci.USA 97:10701 in the cell surface library; Daugherty et al.2000 J.Immunol.Methods 243:211.Described method provides other selection of monoclonal antibody separation and clone's subsequently conventional hybridization knurl technology.
Another embodiment of the present invention is included in and produces people's antibody or people's antibody basically in the transgenic animal (for example mouse), and described animal can not produce endogenous immunoglobulin and (for example see United States Patent (USP) 6,075,181,5,939,598,5,591,669 and 5,589,369, every piece of document is included in this paper as a reference).For example, the inhibition fully that the homozygous deletion of heavy chain of antibody association area in chimeric and germ line mutation mouse causes endogenous antibody to produce has been described.Human immunoglobulin gene's array is transferred to described germ line mutation mouse will be caused producing people's antibody under antigen is attacked.Another preferred method that utilizes the SCID mouse to produce people's antibody is disclosed in U.S.Pat.No.5,811,524 its be included in this paper as a reference.To separate as described herein and operate understanding the described genetic material relevant with these people's antibody.
The another kind of height effective means that is used to produce recombinant antibodies is disclosed in Newman, Biotechnology, 10:1455-1460 (1992).Particularly, this technology causes primates source production of antibodies, and described antibody contains monkey variable domains and people's constant series.The document is included in this paper as a reference in full.In addition, this technology is also at United States Patent (USP) 5,658, describes in 570,5,693,780 and 5,756,096, and every piece of document is included in this paper as a reference.
In another embodiment, lymphocyte can be selected by micrurgy and isolating variable gene.For example, the Mammals of the separable immunity of hanging oneself of peripheral blood lymphocytes and about 7 days in vitro culture.But satisfy the specific IgG of screening criteria in the screening and culturing thing.Cell from positive hole can be separated.Individual Ig generation type B cell can separate by FACS or by identifying in the haemolysis plaque test of complement-mediated.Ig can be produced the micrurgy of type B cell in test tube, and for example utilize, RT-PCR amplification VH and VL gene.VH and VL gene can be cloned into antibody expression vector and be transfected into cell (for example eucaryon or prokaryotic cell prokaryocyte) and be used for expressing.
In addition, being used to produce the genetic sequence of polypeptide of the present invention can be available from multiple different sources.For example, as indicated above, the various human antibody gene can derive from the obtainable preservation of the public.The gene of many antibody sequences and encoding antibody is open, and can utilize known technology from the suitable antibody gene of these sequence chemosynthesis.The oligonucleotide synthetic technology compatible with this aspect of the present invention is well known by persons skilled in the art and can utilizes the multiple automatic DNA synthesizer DNA of buying to carry out.In addition, the dna sequence dna of polytype heavy chain of coding described herein and light chain can obtain by the service of the synthetic retailer of commercialization DNA.That utilize that genetic material that any preceding method obtains can be for a change or synthetic is to obtain polypeptide of the present invention.
Optional, antibody-producting cell system can utilize the choice of technology well known by persons skilled in the art and cultivation.Described technology is described in kinds of experiments chamber handbook and main publication.In aspect this, followingly be suitable for technical description of the present invention in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, NewYork (1991), its in full and appendix be included among the present invention as a reference.
Further comprise the allelotrope of all antigens in conjunction with dna sequence dna, variant and mutant with understanding scope of the present invention.
Known RNA can separate from original hybridoma or other cell transformed by standard technique, and extracting and precipitate such as guanidinium isothiocyanate (guanidinium isothiocyanate) is centrifugal or chromatography then.When needing, mRNA can separate from total RNA by standard technique such as chromatography on few dT Mierocrystalline cellulose.Appropriate technology is that those skilled in the art are familiar with.
In the embodiment, the cDNA of encoding antibody light chain and heavy chain can utilize reversed transcriptive enzyme and archaeal dna polymerase, prepares at the same time or separately according to known method.PCR can be by total constant region primer or by more specific primer, based on disclosed light chain and heavy chain DNA and aminoacid sequence startup.As mentioned above, PCR also can be used for separating the dna clone of encoding antibody light chain and heavy chain.In this case, can screen described library such as mouse constant region probe by total primer or bigger homologous probe.
DNA, normally plasmid DNA can utilize technical point known in the art from from cell, limits drawing and order-checking according to the standard known technology that for example describes in detail in aforementioned content about recombinant DNA technology.Certainly, any point in sepn process or analysis subsequently, according to the present invention, described DNA can be synthetic.
B. the antibody of Xiu Shiing
In the embodiment, binding molecule or antigen binding molecules be of the present invention comprise the synthetic constant region wherein one or more structural domain be (" antibody of structural domain-disappearance ") that partially or completely lacks.In the concrete embodiment preferred, the antibody of the modification that is fit to will comprise the construct or the variant of structural domain disappearance, and wherein whole C H2 structural domain is removed (Δ CH2 construct).For other embodiment preferred, available short connection peptides replaces the structural domain of disappearance with flexibility that the variable region is provided and the degree of freedom that moves.It will be understood by those skilled in the art that the accommodation property owing to CH2 structural domain antagonist rate of decomposition, described construct is particularly preferred.
In the embodiment, the antibody of modification is of the present invention to be miniantibody.Miniantibody can utilize this area described method preparation (for example see, for example see, United States Patent (USP) 5,837,821 or WO94/09817A1).
In another embodiment, the antibody of modification of the present invention is the antibody of CH2 structural domain disappearance, and it is known in the art.The construct of structural domain disappearance can utilize coding IgG 1The carrier of people's constant domain (for example, available from IDEC Pharmaceuticals, San Diego) derive (for example seeing WO 02/060955A2 and WO02/096948A2).IgG to lack the CH2 structural domain and to provide the expression structure territory to lack can be provided this exemplary carrier 1The synthetic vectors of constant region.Coding C2B8 antibody, 5E8 antibody, the mouse variable region of B3F6 antibody or the gene of humanized CC49 antibody variable region can be inserted into described synthetic vectors subsequently and be cloned.When expressing in cell transformed, these carriers provide C2B8. Δ CH2,5E8. Δ CH2, B3F6. Δ CH2 or huCC49. Δ CH2 or provide respectively.These constructs show multiple character, make that they are attractive especially material standed fors for the monomer subunit.
Transformed the CH3 structural domain directly is blended in the hinge area of each polypeptide of the present invention noticing these exemplary constructions bodies.In other construct, between hinge area and synthetic CH2 and/or CH3 structural domain, provide the peptide spacer to need.For example, suitable construct can be expressed, and wherein the CH2 structural domain has been lacked, and remaining CH3 structural domain (synthetic or nonsynthetic) utilizes 5-20 amino acid whose spacer to be connected in hinge area.Described spacer can be added, for example with guarantee the constant region regulatory element keep free and can near and hinge area keep flexible.For example, replace the CH2 structural domain with short amino acid spacer GGSSGGGGSG (SEQ.ID No.1) in the CC49 construct of structural domain disappearance, and can use rudimentary hinge area (CC49. Δ CH2[gly/ser]).Other exemplary connection peptides is shown in table 2.These connection peptides can be used for polypeptide of the present invention.Preferably, described connection peptides is used with the polypeptide that lacks CH2 heavy chain structural domain.Preferably, anyly be suitable for joint of the present invention and will be the non-covalent combination that does not suppress polypeptide of the present invention of non-immunogenic relatively.
In the embodiment, even polypeptide of the present invention comprises the heavy chain immunoglobulin that has some single amino acids disappearances or replace, as long as it allows the required covalently or non-covalently combination between the monomer subunit.For example, the sudden change of the single amino acids in institute's favored area of CH2 structural domain is enough to or reduces Fc basically in conjunction with also increasing tumor-localizing thus.Similarly, it is required only needing to lack the part of controlling effector function to be regulated (for example complement combination) in one or more constant region structural domain.The selected character (serum half life) that the disappearance of the described part of constant region can be improved antibody keeps other and the relevant telotism of purpose constant region structural domain simultaneously.In addition, as mentioned above, the constant region of disclosed antibody can be a synthetic by suddenling change or replacing one or more amino acid that strengthens gained construct collection of illustrative plates.In aspect this, can destroy the activity of conservative binding site (for example Fc in conjunction with), keep the structure picture and the immunology collection of illustrative plates of modified antibody simultaneously.Another preferred embodiment can comprise one or more connection of amino acid adding constant region to improve desired characteristic such as effector function or more cytotoxin or carbohydrate are provided.In described embodiment, need insert or duplicate specific sequence from selected constant region structural domain.
C. fusion rotein
The invention still further relates to binding molecule, it comprises one or more immunoglobulin domains.Fusion rotein of the present invention comprises binding domains (it comprises at least one binding site) and Dimerized structural domain (it comprises at least one heavy chain part).The purpose fusion rotein can be dual specific (have one at first target in conjunction with the position with at second binding site of second target) or can be polyvalent (having two binding sites) at same target.
The exemplary fused albumen of reporting in the document comprises TXi Baoshouti fusions (Gascoigne et al., Proc.Natl.Acad.Sci.USA 84:2936-2940 (1987)); CD4 (Capon et al., Nature337:525-531 (1989); Traunecker et al., Nature 339:68-70 (1989); Zettmeissl et al., DNA Cell Biol.USA 9:347-353 (1990); With Byrn et al., Nature344:667-670 (1990)); L-selectin (returning the nest acceptor) (Watson et al., J.Cell.Biol.110:2221-2229 (1990); With Watson et al., Nature 349:164-167 (1991)); CD44 (Aruffo et al., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley et al., J.Exp.Med.173:721-730 (1991)); CTLA-4 (Lisley et al., J.Exp.Med.174:561-569 (1991)); CD22 (Stamenkovic et al., Cell 66:1133-1144 (1991)); TNF acceptor (Ashkenazi et al., Proc.Natl.Acad.Sci.USA 88:10535-10539 (1991); Lesslauer et al., Eur.J.Immunol.27:2883-2886 (1991); With Peppel et al., J.Exp.Med.174:1483-1489 (1991)); With IgE acceptor a (Ridgway and Gorman, J.Cell.Biol.Vol.115, Abstract No.1448 (1991)).
In the embodiment, fusion rotein combines the binding domains (for example receptor extracellular structural domain (ECD)) of part or acceptor with at least one heavy chain structural domain and synthetic connection peptides.In the embodiment, when preparation fusion rotein of the present invention, the C-terminal of the nucleic acid of the binding domains of coding part or receptor domain can be blended in the nucleic acid of coding constant region for immunoglobulin sequence N-end.In the embodiment, fusion rotein comprises CH2 and CH3 structural domain.Also can be at constant domain Fc partial C-end, or N-terminal place or the light chain respective regions of the CH1 of next-door neighbour's heavy chain merge.
In the embodiment, the sequence of part or receptors bind structural domain is blended in the N-end of immunoglobulin molecules Fc structural domain.Also complete CH may be blended in part or receptors bind structural domain sequence.In the embodiment, the sequence that originates in the upstream, similar site that is arranged in papoid cracking site or other immunoglobulin (Ig) in the hinge area just is used for merging, described papoid cracking site is chemically limiting IgG Fc (be residue 216, first residue of heavy chain is defined as 114).The accurate site that fusion takes place is unimportant; Concrete site is known and can be selected so that optimize the biological activity of molecule, secretion or binding characteristic.The method for preparing fusion rotein is known in the art.
For bispecific fusion protein, it is polymer that fusion rotein is assembled, and is specially the heterodimer or the different tetramer.Usually, these accumulative immunoglobulin (Ig)s will have known subunit structure.Four basic chain structure subunits are wherein to have IgG, the form of IgD and IgE.Four chain subunits repeat in the high molecular immunoglobulin (Ig); IgM is usually as four tetramers that basic subunit forms by disulfide bonds.The IgA sphaeroprotein also has the IgG sphaeroprotein sometimes, also can the polymer form be present in the serum.Under polymeric situation, each of four subunits can be identical or different.
Other the exemplary part and the acceptor thereof that can be included in the fusion rotein comprise:
Cytokine and cytokine receptor
Cytokine is to lymphocytic propagation, and differentiation and functional activation have multi-directional effect.Various cytokines, or its receptor binding moiety can be used in the fusion rotein of the present invention.The exemplary cells factor comprises interleukin (IL-1 for example, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, and IL-18), G CFS (CSF) (for example granulocyte CSF (G-CSF), granulocyte-macrophage CSF (GM-CSF) and monocyte scavenger cell CSF (M-CSF)), tumour necrosis factor (TNF) alpha and beta, and Interferon, rabbit is such as interferon-' alpha ', β, or γ (United States Patent(USP) Nos. 4,925,793 and 4,929,554).
Cytokine receptor is made up of part-specificity alpha chain and common beta chain usually.The exemplary cells factor acceptor comprises the GM-CSF acceptor, IL-3 acceptor (U.S. Patent No. 5,639,605), IL-4 acceptor (U.S. Patent No. 5,599,905), IL-5 acceptor (U.S. Patent No. 5,453,491), IFN γ acceptor (EP0240975) and TNF receptor family are (for example, (for example (EP 417 for TNFR-1 for TNF α, 563), TNFR-2 (EP 417,014) lymphotoxin beta acceptor).
Attachment proteins
Adhesion molecule is membrane-bound albumen, and it allows cell interaction.Various attachment proteinses comprise that white corpuscle returns nest acceptor and cell adhesion molecule, or its receptor binding moiety, can be included in the fusion rotein of the present invention.White corpuscle returns the nest acceptor and be expressed in cell surface in inflammatory process, and comprise β-1 integrin (VLA-1 for example, 2,3,4,5, with 6) (its mediation and the combining of extracellular matrix components), and β 2-integrin (LFA-1 for example, LPAM-1, CR3, and CR4) (it is in conjunction with the cell adhesion molecule on the blood vessel endothelium (CAM)).Exemplary CAM comprises ICAM-1, ICAM-2, VCAM-1, and MAdCAM-1.Other CAM comprises the member of selectin family, comprises E-selectin, L-selectin, P-selectin.
Chemokine
Chemokine stimulates the chemotactic protein of white corpuscle to the sites of infection migration, also can be included in the fusion rotein of the present invention.Exemplary chemokine comprises macrophage inflammatory protein (MIP-1-α and MIP-1-β), neutrophilic chemotactic factor, and RANTES (regulate activation, be generally the T-cell expressing and excretory).
Somatomedin and growth receptors
Somatomedin or its acceptor (or receptors bind or its ligand binding moiety) can be included in the fusion rotein of the present invention.Exemplary somatomedin comprises vascular endothelial growth (VEGF) and isotype (U.S.Pat.No.5,194,596) thereof; Fiber mother cell growth factor (FGF) comprises FGF and bFGF; The atrium sodium diuresis factor (ANF); Liver growth factor (HGFs; United States Patent(USP) Nos. 5,227,158 and 6,099,841), the neurotrophic factor (BDNF) in neurotrophic factor such as bone source, neurotrophin-3 ,-4 ,-5, or-6 (NT-3, NT-4, NT-5, or NT-6), or somatomedin (PDGF) (United States Patent (USP) 4,889,919,4 in nerve growth factor such as NGF-β thrombocyte source, 845,075,5,910,574 and 5,877,016); Transforming growth factor (TGF) such as TGF-alpha and TGF-beta (WO 90/14359), bone-inducing factor comprises Delicious peptide (BMP); Insulin like growth factor-1 and-II (IGF-I and IGF-II; United States Patent(USP) Nos. 6,403,764 and 6,506,874); Erythropoietin (EPO); STEM CELL FACTOR (SCF), thrombopoietin (thrombopoietin) (c-Mpl part) and Wnt polypeptide (U.S. Patent No. 6,159,462).
Exemplary growth factor receptors, it can be used to target receptor domain of the present invention, comprises the EGF acceptor; Vegf receptor (for example Flt1 or Flk1/KDR), pdgf receptor (WO 90/14425); (the avidity acceptor (LNGFR) that comprises is also referred to as p75 for HGF acceptor (United States Patent(USP) Nos. 5,648,273 and 5,686,292) and neurotrophy acceptor NTROr p75, it is in conjunction with NGF, BDNF, and NT-3), and high-affinity receptor, it is member's (for example trkA, trkB (EP 455,460), trkC (EP 522,530)) of receptor tyrosine kinase trk family.
E) hormone
Exemplary tethelin as the target reagent in the fusion rotein of the present invention comprises feritin, human growth hormone (HGH; U.S. Patent No. 5,834,598), N-methionyl human growth hormone; Trobest; Somatotropin releasing factor; Rat parathyroid hormone 1-34 (PTH); Thyrotropin (TSH); Thyroxine; Proinsulin and Regular Insulin (United States Patent(USP) Nos. 5,157,021 and 6,576,608); Folliculus stimulates hormone (FSH), thyrocalcitonin, short corpus luteum (luteinizing) hormone (LH), leptin, hyperglycemic-glycogenolytic factor; Magainin; Tethelin; Miller pipe (mullerian)-inhibition; Relaxin (relaxin) and former Relaxin (prorelaxin); The gonad-stimulating hormone related peptides; Prolactin (prolactin), human placental lactogen (lactogen); OB albumen; Or Miller pipe inhibition.
Thrombin
Exemplary thrombin as the target agent in the fusion rotein of the present invention comprises thrombin (for example, factor V, VII, VIII, X, IX, XI, XII and XIII, the von Willebrand factor); Tissue factor (United States Patent (USP) 5,346,991,5,349,991,5,726,147 and 6,596,84); Zymoplasm and thrombogen; Fibrinogen and Parenogen; Plasmin and Profibrinolysin; Plasminogen activator is such as urokinase or people's urine or tissue-type plasminogen activator (t-PA).
Other exemplary fused albumen is instructed among WO0069913A1 and the WO0040615A2 for example.Other example molecule that can be included in fusion rotein of the present invention is IGSF9.
Fusion rotein can utilize means known in the art preparations (seeing for example United States Patent (USP) 5,116,964 and 5,225,538).Usually, part or part binding partners are blended in CH (or heavy chain part) N-terminal at C-terminal, and replace the variable region.Any part bind receptor stride film district or lipid or phosphatide grappling recognition sequence preferably before fusion be inactivation or the disappearance.The DNA of coding part or part binding partners by restriction enzyme or close on 5 ' and 3 ' the terminal cracking of the segmental DNA of the required ORF of coding.The dna fragmentation that produces easily inserts among the DNA of encoding heavy chain constant region subsequently.The accurate site of merging can rule of thumb be selected with the secretion of optimizing the melt-moldable hop protein or in conjunction with character.The DNA of encoding fusion protein is transfected into host cell subsequently and expresses.
In the embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO16) that is shown in Fig. 4 A.In another embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:17) that is shown in Fig. 4 B.In the embodiment, peptide molecule of the present invention comprises peptide sequence, and it is by nucleic acid molecule encoding, and described nucleic acid molecule comprises the nucleotide sequence (SEQ IDNO:16) that is shown in Fig. 4 A.In another embodiment, peptide molecule of the present invention comprises peptide sequence, and it is by nucleic acid molecule encoding, and described nucleic acid molecule has the nucleotide sequence (SEQ ID NO:17) that is shown in Fig. 4 B.
In the embodiment, peptide molecule of the present invention comprises the aminoacid sequence (SEQ ID NO:18) that is shown in Fig. 8 A.In another embodiment, peptide molecule of the present invention comprises the aminoacid sequence (SEQ ID NO:19) that is shown in Fig. 8 B.
In another embodiment, polypeptide of the present invention comprises the nucleotide sequence coded aminoacid sequence (SEQ ID NO:20) that is shown in Fig. 9.In another embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:20) that is shown in Fig. 9.
In another embodiment, polypeptide of the present invention comprises the nucleotide sequence coded aminoacid sequence (SEQ ID NO:21) that is shown in Figure 10.In another embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:21) that is shown in Figure 10.
In another embodiment, polypeptide of the present invention comprises the nucleotide sequence coded aminoacid sequence (SEQ ID NO:38) that is shown in Figure 28 A.In another embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:38) that is shown in Figure 28 A.
In another embodiment, polypeptide of the present invention comprises the nucleotide sequence coded aminoacid sequence (SEQ ID NO:39) that is shown in Figure 28 B.In the embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:39) that is shown in Figure 28 B.
In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQ IDNO:22) that is shown in Figure 11.
In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQ IDNO:23) that is shown in Figure 12.
In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQID NO:40) that is shown in Figure 29 A.In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQ ID NO:41) that is shown in Figure 29 B.
In another embodiment, polypeptide of the present invention comprises the nucleotide sequence coded aminoacid sequence (SEQ ID NO:42) that is shown in Figure 32 A.In another embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:42) that is shown in Figure 32 A.In another embodiment, polypeptide of the present invention comprises the nucleotide sequence coded aminoacid sequence (SEQ ID NO:43) that is shown in Figure 32 B.In the embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:43) that is shown in Figure 32 B.
In another embodiment, polypeptide of the present invention comprises the nucleotide sequence coded aminoacid sequence (SEQ ID NO:24) that is shown in Figure 13 A.In the embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:24) that is shown in Figure 13 A.In another embodiment, polypeptide of the present invention comprises the nucleotide sequence coded aminoacid sequence (SEQ ID NO:25) that is shown in Figure 13 B.In the embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ IDNO:25) that is shown in Figure 13 B.
In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQID NO:26) that is shown in Figure 14 A.In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQ ID NO:27) that is shown in Figure 14 B.
In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQID NO:44) that is shown in Figure 33 A.In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQ ID NO:45) that is shown in Figure 33 B.
In another embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:46) that is shown in Figure 25 A.In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQ ID NO:47) that is shown in Figure 25 B.
In another embodiment, nucleic acid molecule of the present invention comprises the nucleotide sequence (SEQ ID NO:48) that is shown in Figure 26 A.In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQ ID NO:49) that is shown in Figure 26 B.
In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQ IDNO:31) that is shown in Figure 12.
In another embodiment, polypeptide of the present invention comprises the aminoacid sequence (SEQ IDNO:35) that is shown in Figure 12.
Disclosed other nucleic acid and aminoacid sequence are also contained within the scope of the present invention in sequence table of the present invention and the accompanying drawing.
D. polypeptide expression
Operate isolating genetic material with after the above-mentioned polypeptide of the present invention is provided, gene is inserted expression vector in a usual manner to import host cell, this host cell can be used for preparing the polypeptide of aequum, thereby the polypeptide of claim is provided.
, be the purpose of specification sheets and claims herein, term " carrier " or " expression vector " are meant according to the present invention as carrier required gene (desired gene) transfered cell and the carrier of expressing.Just as is known to the person skilled in the art, this carrier can be selected from the group that plasmid, phage, virus and retrovirus are formed at an easy rate.In a word, be applicable to that carrier of the present invention will have selected marker, helps the clone's of required gene suitable restriction site, the ability that enters eukaryotic cell or prokaryotic cell prokaryocyte and/or duplicate therein.
To achieve the object of the present invention, may use many expression vector systems.For example, one type carrier utilization comes from the DNA element of animal virus such as bovine papilloma virus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV or MOMLV) or SV40 virus.Other relates to the application of the polycistron system with internal ribosome binding site.In addition, can select the marker of the host cell of transfection, select those DNA to be incorporated into self chromosomal cell by importing one or more permissions.Described mark can be the auxotrophy host prototroph is provided, sterilant (for example microbiotic) resistance or to the resistance of heavy metal such as copper.The selected marker can directly connect (linked) and arrive dna sequence dna so that express, and also can import same cell by cotransformation.It is required that other element is also optimized synthetic mRNA.These elements can comprise the signal sequence splicing signal, and transcripting promoter, enhanser and termination signal.In preferred specific embodiments,,, insert expression vector together with the synthetic heavy chain of preamble discussion and the constant region gene of light chain (the preferred mankind's) with clone's variable region gene.Preferably, the patent expression vector that adopts IDEC company is effectively, and its name is called NEOSPLA (U.S. patent 6,159,730).This carrier contains cytomegalovirus promoter/enhanser, the main promotor of mouse beta Globulin (major promoter), SV40 replication orgin, Trobest polyadenylation sequence, neomycin phosphotransferase exons 1 and exon 2, dihydrofolate reductase gene and leader sequence.As example finding hereinafter, found this carrier mix variable region and constant region gene, transfection in Chinese hamster ovary celI, substratum through containing G418 is selected and the Rheumatrex amplification after, cause the expression of the high level of antibody.Carrier system is at United States Patent (USP) the 5th, 736, also does explanation in 137 and 5,658,570, and said two devices all is embodied in herein as a reference in full.This system provides high expression level, as＞30pg/ cell/sky.Other representative carrier system also is disclosed, as United States Patent (USP) the 6th, 413, in 777.
In other embodiment preferred, polypeptide of the present invention can utilize the polycistron construct, the 60/331st, No. 481 those disclosed of common unsettled U.S. Provisional Application that described construct such as those November 16 calendar year 2001 submit to, this patent also are embodied in herein as a reference in full.In these expression systems newly developed, the heavy chain of interested polygene product such as antibody and light chain can be produced by single polycistron structure.These systems advantageously use internal ribosome entry site, and (internal ribosome entrysite IRES), provides high-caliber relatively polypeptide of the present invention in eukaryotic host cell.Suitable IRES sequence is at United States Patent (USP) the 6th, 193, announces that this patent is embodied in herein in No. 980.Those skilled in the art can understand, and this expression system can be used to disclosed all polypeptide among production the application effectively.
More generally, in case the carrier or the dna sequence dna of the monomer subunit of coded polypeptide (as the antibody after the modification) are prepared, expression vector can be imported into proper host cell.That is, described host cell can be transformed.Plasmid is imported the multiple technologies that host cell can adopt these those skilled in the art to know to be finished, these technology comprise transfection (comprising electrophoresis and electroporation), protoplastis fusion method, calcium phosphate precipitation method, are infected method with bag by the cytogamy of DNA, microinjection and intact virus, but are not limited to aforesaid method.Consult Ridgway, A.A.G. shows " (Mammalian Expression Vectors) " the 24th chapter .2 joint, 470-472 page or leaf " carrier ", and Rodriguez and Denhardt write (Eds).(Butterworths，Boston，Mass.1988)。More preferably, by electroporation plasmid is imported the host.Transformant is grown under the condition that light chain and heavy chain generate being fit to, and measures its heavy chain and/or light chain protein synthetic (amount).Representative analytical technology comprise enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), fluorescence-activation the cell sorting analytical method (flourescence-activated cell sorter analysis, FACS), immunohistochemical method etc.
Therefore term used herein " conversion " broadly relates to DNA is imported the acceptor host cell, changes its genotype and causes any importing that recipient cell changes.
In like manner (Along those same lines), " host cell " relate to the carrier that is utilized the recombinant DNA technology structure and transform, and the cell of the foreign gene of encoding at least.At explanation antibody during from the recombinant host separating process, term " cell " and " cell cultures " but alternate application is all represented antibody sources, unless clearly emphasize to refer else.In other words, reclaim polypeptide from " cell " and can refer to reclaim, also can refer to from the cell cultures of the cell that comprises substratum or suspendible, reclaim from (spun down) intact cell of centrifugal gained.
The cell that is used for the first-selected Mammals of the host cell source of protein expression be sure of that those skilled in the art have that preferred decision is the most suitable will express the ability of the concrete clone of required gene product therein.Representative host cell strain comprises: and DG44 and DUXB11 (Chinese hamster ovary line, DHFR-), HELA (human cervical carcinoma), CVI (monkey-kidney cells system), COS (with SV40 T antigen by CVI deutero-clone), R1610 (Chinese hamster fibroblast), BALBC/3T3 (mouse fibroblast), HAK (hamster kidney cell line), SP2/O (mouse myeloma), P3.times.63-Ag3.653 (mouse myeloma), BFA-1c1BPT (ox endotheliocyte), RAJI (human lymphocyte) and 293 (human kidney cells).Preferred Chinese hamster ovary celI.Host cell strain is usually available from commerce services, American type culture collection or the article of having delivered.
Experiment in vitro allows the expansion scale to produce the polypeptide of a large amount of expections.Mammalian cell culture technique under the conditions of tissue culture is known in the art, cultivate (homogeneous suspension culture) as in air transport reactor or lasting stirred reactor comprising the homogeneous suspension, or immobilization (immobilized) or (entrapped) cell cultures of catching are as in tubular fibre, microcapsule (microcapsules), on sepharose 4B (microbeads) or pottery tube.If necessary and/or need, polypeptide solution can be by purification by chromatography commonly used, as gel filtration method, ion exchange chromatography, DEAE chromatography or (immunity-) affinity chromatography, for example, after the preferred biosynthesizing of synthetic hinge area polypeptide, or before or after HIC chromatographic step described herein.
The gene of code book invention polypeptide also can be expressed in nonmammalian cell such as bacterium, yeast or vegetable cell.Be appreciated that from this aspect different nonmammalian unicellular microorganisms such as bacterium also can be transformed, i.e. those microorganisms that can in culture and fermented product, grow.The bacterium that is easy to transform comprises for example bacterial strain of intestinal bacteria or Salmonellas of enterobacteriaceae member; Bacillaceae (Bacillaceae) is as subtilis; Streptococcus pneumoniae (Pneumococcus); Suis (Streptococcus) and hemophilus influenzae (Haemophilus influenzae).Also can further understand, when expressing in bacterium, polypeptide becomes the part of inclusion body usually.The molecule that this polypeptide must be separated, purifying is assembled into function then.If expect the tetravalence form of antibody, subunit can the oneself be assembled into tetravalent antibody (WO02/096948A2).
Except that prokaryotic organism, also can utilize eukaryotic microorganisms.Yeast saccharomyces cerevisiae (Saccharomycescerevisiae) or common bread yeast (common baker ' s yeast) are the eukaryotic microorganisms of normal application, although some other bacterial strain is also available usually.For in sugar yeast (Saccharomyces), expressing carrier such as plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979) commonly used; Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)).This plasmid has had the TRP1 gene, and this gene provides selected marker for the yeast mutant strain that lacks the ability of growing in tryptophane, and described bacterial strain is ATCC No.44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)) for example.The existence of trp1 infringement by growing, provides effective environmental for detecting to transform as the characteristic of this yeast host cell gene group under the condition that lacks tryptophane.
IV. from the polypeptide that lacks interchain disulfide bond, separate the polypeptide that contains an interchain disulfide bond at least.
On the one hand, the present invention relates to adopt the molecule of hydrophobic interaction chromatography two heavy chain parts of decoupled band from mixture, two heavy chains of this mixture middle part fractionated molecule connect with at least one interchain disulfide bond, also have the part molecule not connect by at least one disulfide linkage.
Hydrophobic interaction chromatography is to grow up after can being fixed on the avidity gel observing the protein that contains hydrocarbon polymer spacerarm (hydrocarbonspacer arms) but lack affinity ligand the earliest.Can change solvent, pH, ionic strength or add chaotropic material or organism properties-correcting agent (organic modifier) is realized after as ethylene glycol or propylene glycol from the wash-out of HIC upholder.Can be about the explanation of the rule of hydrophobic interaction chromatography at United States Patent (USP) as 3,917, find in 527 and 4,000,098.HIC in high performance liquid chromatography (HPLC) background is used in a single stage scheme, is used for separating the antibody fragment that lacks the heavy chain part (as, F (ab ') from complete antibody molecule 2).
Separation method of the present invention can be applicable to not purified polypeptide (as culture supernatant or prepared product or separate the polypeptide formulations of preparation from the prokaryotic organism inclusion body).As one of selecting, this separation method also can be used for the polypeptide mixture that obtains through one or more initial purification steps, for example at the prepared product that comprises A type and Type B behind the affinity matrix wash-out.
In a kind of embodiment, the binding molecule of using the HIC chromatography comprises connection peptides of the present invention.
In preferred embodiments, HIC can be used for passing through the partially purified mixture of other method of purifying protein.Term used herein " partial purification " comprises such protein Preparation thing, and wherein target protein accounts for 5% weight at least, more preferably accounts for 10% weight at least, preferably preferably accounts for 45% weight at least.Initial or subsequent purification step can be used for removing following material, as immunoglobulin (Ig) aggregation, malfolding kind (misfolded species), host cell proteins, and from the residuals (such as a-protein, condition is when being applied to this albumen) of before chromatographic separation step.In a kind of embodiment, HIC can carry out the polypeptide that separation comprises connection peptides of the present invention.Correspondingly, also be appreciated that under the background of complete purification scheme (protocol) and use HIC.Can comprise prior to the purification step of HIC or the illustrative of using after HIC: affinity chromatography (for example, comprise PROSEP-A  (BioProcessing Ltd. with the albumin A of controlled porose glass covalent coupling, U.K.), or albumin A EPHAROSE  FastFlow (Pharmacia) or TOYOPEARL 650M albumin A (TosoHaas)).To people γ 1, γ 2, or γ 4 heavy chain optimization protein A, to mouse isotype optimization protein G.If described molecule contains CH3 zone (structural domain) then can use Bakerbond ABXtm resin.Also can use ion exchange chromatography as a supplement or alternative.Under this consideration, various negatively charged ion or cationic substituent can be adsorbed on matrix to form required negatively charged ion or the positively charged ion upholder of chromatography.The anionresin substituting group comprise the diethylamino ethyl (diethylaminoethyl, DEAE), quaternary aminoethyl (quaternaryaminoethyl, QAE) and quaternary amine (quaternary amine, Q) etc.The cation replacement substituting group comprise carboxymethyl (carboxymethyl, CM), sulfoethyl (sulfoethyl, SE), sulfopropyl (sulfopropyl, SP), phosphate (phosphate, P) and sulfonic group (sulfonate, S).Cellulose ion exchange resin such as DE23, DE32, DE52, CM-23, CM-32 and CM-52 can be available from Britain Maidstone, Kent, (the Whatman Ltd.Maidstone of Ward door company limited, Kent, U.K.), based on SEPHADEX  with-ion-exchange thing (locross-linked ion exchangers) that locross-is connected also is known.For example, DEAE-, QAE-, CM-, SP-SEPHADEX , DEAE-, Q-, CM-, S-SEPHAROSE  and SEPHAROSE  Fast Flow's available from PharmaciaAB.In addition, DEAE and CM deutero-ethylene glycol-alkylmethacrylate polymer (ethyleneglycol-methacrylate copolymer) all can derive from Toso Haas Co. as TOYOPEARL DEAE-650S or M and TOYOPEARL CM-650S or M, Philadelphia, Pa.Because wash-out need add salt usually from the ion-exchange upholder, and, be preferred with salt so after ion exchange chromatography step or other are the purification step of medium, adopt the HIC step because HIC is strengthened under the situation of the salt concn that increases.Additional purification scheme can comprise the steps, but need not be confined to this: further ion exchange chromatography, size exclusion chromatography, inactivation of virus, concentrate, lyophilize, hydroxyapatite chromatography method, gel electrophoresis, dialysis, ethanol sedimentation, reversed-phase HPLC, silicon-dioxide chromatography, heparin SEQHAROSE TM chromatography, chromatofocusing or ammonium sulfate precipitation.
Before adopting described method purifying, contain the composition of wanting the isolated polypeptide mixture and preferably be placed in the damping fluid of acidity or nearly neutral pH value.For example, can finish as follows: add spissated damping fluid, sample is resuspended in the damping fluid, exchange buffering liquid (as adopting dialysis or ultrafiltration).The pH that also can adjust sample buffer simply makes it to remain on required scope.
Hydrophobic interaction reaches the strongest when high ion concentration, and therefore, this unpack format carries out after salt precipitation or ion-exchange step easily.High salt concentration makes the easier HIC of the being adsorbed in chromatography column of albumen, but according to the difference of proteic characteristic and selected HIC part, actual salt concn can change in a very wide scope.Various ions can be with so-called soluphobic serial arrangement, and different ions is prepared in the decay whether this their basis strengthens hydrophobic interaction (salting-out effect) or destroy water structure (chaotropic effect) and cause hydrophobic interaction.According to the tactic positively charged ion of salting-out effect enhanced is Ba ++＜Ca ++＜Mg ++＜Li +＜Cs +＜Na +＜K +＜Rb +＜NH4 +, be PO according to the tactic negatively charged ion of chaotropic effect enhanced ---＜SO 4 --＜CH 3COOO -＜Cl -＜Br -＜NO 3 -＜ClO 4 -＜I -＜SCN -
In general, Na, K or NH 4Vitriol can strengthen part among the HIC-proteic interaction effectively.Salt is illustrated by following listed relation the influence of interaction strength: (NH 4) 2SO 4＞Na 2SO 4＞NaCl＞NH 4Cl＞NaBr＞NaSCN.In general, the NaCL concentration between the ammonium sulfate concentrations between about 0.75 to about 2M and about 1 to 4M is useful.
Will use some chromatography upholders when preparation HIC chromatography column, most widely used is agarose, silicon-dioxide, organic polymer or interpolymer resin.The hydrophobic interaction material is generally base matrix (as hydrophobic carbon hydrate (as crosslinked agarose) or synthetic interpolymer material), and its coupling has hydrophobic ligand.Preferred HIC material comprises the agarose resin that is replaced by phenyl group.Representative HIC material comprises: phenyl SEPHAROSE TM, have low or high replacement FAST FLOW (Pharmacia LKBBiotechnology, AB, Sweden); Phenyl SEPHAROSE TMHigh performance column; Phenyl or butyl-SEPHAROSE  CL-4B, butyl-SEPHAROSE  FF, octyl group-SEPHAROSE  FF and phenyl-SEPHAROSE  FF (Pharmacia LKB Biotechnology AB, Sweden); FractogelTM EMD propyl group or FRACTOGEL TMEMC phenyl post (E.Merck, Germany); MACROPREP TMMethyl or MACRO-PREP TMT-butyl upholder (Bio-Rad, California); WP HI-Propyl (C3) TMThe exemplary HIC material of post (J.T.Baker, New Jersey) also can derive from Tokyo Tosoh company, name of product is a TOYOPEARL ether 650, phenyl 650, butyl 650 (Fractogel), ether-5PW-HR, or phenyl-5PW-HR; From Miles-Yeda, Rehovot, Israel, name of product are alkyl-agarose, and wherein alkyls comprises 2-10 carbon atom, and J.T.Baker, and Phillipsburg, N.J., name of product are Bakerbond WP-HI-propyl group.It also is possible adopting conventional chemical process to prepare required HIC chromatography column.(Sa: for example, Er-el.Z.gl all, Biochem.Biophys.Res.Comm.49:383 (1972) or Ulbrich, V.rd gL Coll.Czech.Chem.Commum.9:1466 (1964)).
The selection of concrete gel can be determined by those skilled in the art.In general, the action intensity between albumen and the HIC part is suitable but contain 4 parts to about 8 carbon atoms of having an appointment for most of isolated polypeptides along with the chain length of this alkyl part increases and increases.Phenyl group has similar hydrophobicity to amyl group, but its selectivity can owing to the protein aromatic group between may have the pi-pi rail interaction and different.Selectivity also may be subjected to supporting the influence of the chemical property of resin.
Ligand density is an important parameter, not only influences interaction strength, also influences the capacity of chromatography column.The ligand density of business-like phenyl or octyl phenyl gel is generally 40 picomole/milliliter (pmoles/ml) gel beds.Gel capacity is the kind of specific purposes albumen and pH value, temperature and salt and the function of concentration, but is generally expected that in the scope of 3-20mg/ml gel.
In general, the reduction of temperature can cause the interactional decline of HIC storeroom.Yet any benefit that elevated temperature obtains also must may be weighed the detrimental action that protein stability exists mutually with it.
In a kind of embodiment, polypeptide of the present invention can be by synchronous (isocratically) wash-out.In synchronous wash-out, all compounds begin migration from initiating terminal and pass through post.Yet every kind of compound causes very fast or slower elution speed with different speed migrations, and for example, as illustration in this paper example, A type albumen can flow by chromatography column and by wash-out along with liquid.
In another embodiment, one or more polypeptide of the present invention can be incorporated into chromatography column and wash-out, for example adopt progressively wash-out (stepwise elution) or gradient elution (gradient elution).No matter be progressively wash-out or gradient elution, all can finish by different approaches.(a) by changing salt concn, (b) by the change solvent polarity or (c) by adding stain remover.By reducing salt concn, the order that the protein of absorption increases according to hydrophobicity is by wash-out.Polar changes the influence that can be subjected to adding solvent such as ethylene glycol, propylene glycol or (different) propyl alcohol, thereby reduces the intensity of hydrophobic interaction.The function of stain remover is as proteinic displacer, is mainly used in the purifying of membranin.
Carry out in the separating process, polypeptide mixture can contact with the HIC material, for example adopts purification technique or employing post in batch.Before the HIC purifying, may wish to remove any chaotropic reagent or the strong material of hydrophobicity, for example be undertaken by making mixture pass through pre-chromatography column (precolumn).
For example, for purifying (batch purification) in batch, the HIC material prepares or balance in required initial damping fluid.Obtain the slurry (slurry) of HIC material.Polypeptide solution contacts with slurry, and at least a polypeptide to be separated is adsorbed on the HIC material.Do not contain and from described slurry, separate, for example, remove supernatant liquor by allowing described slurry precipitation with the solution of HIC material bonded polypeptide.Described slurry is adopted one or more washing steps.If desired, packing material can combine with the lower solution of specific conductivity so that with HIC material bonded polypeptide desorption.Be the polypeptide of elution of bound, can reduce salt concn.
In a kind of embodiment, the HIC material can be filled in the chromatography column.The mixture that contains polypeptide to be separated can be splined on chromatography column, allow at least a polypeptide to be separated to be adsorbed onto on the chromatography column.Be not adsorbed onto flow through chromatography column and being collected of polypeptide on the chromatography column.Be the polypeptide of elution of bound, can reduce salt concn, for example adopt progressively mode or utilize the salt concn gradient.
Because Type B is strong than A type hydrophobicity, it irreversibly is adsorbed in stationary phase (stationaryphase), adopts about 0.7M (as 0.73M) ammonium sulfate/20mM sodium phosphate, and pH value 4 to 8 is as moving phase (mobile phase).The A type is low with stationary phase bonded degree under these conditions, therefore by synchronous wash-out, promptly leaves chromatography column with the part of the chromatography column of flowing through.Behind the wash-out A type, remove ammonium sulfate from moving phase and make the Type B desorption synchronously.
In exemplary purification scheme, the HIC material makes specific conductivity about 160 between about 110Ms/cm at institute's salt concentration, more preferably from about 140 arrive between about 110Ms/cm, more preferably about 130 or about 120Ms/cm in the damping fluid of about 117Ms/cm, reach balance.For example, representative starting soln salt concentration for about 1M to 0.7M, arrive 0.7M ammonium sulfate as 1M.In preferred embodiments, the solution that contains polypeptide mixture to be separated also has identical or roughly the same specific conductivity (as adopting spissated salt mother liquor).Under these conditions, the A type when specific conductivity is about 120mS/cm from the chromatography column wash-out.Be the wash-out Type B, with step by step or the ammonium sulfate inclusion that descends of linear gradient add to chromatography column.Wash-out when Type B is about 115 to about 100mS/cm in specific conductivity.
In a kind of embodiment, described purification process produces the composition that contains peptide molecule, this peptide molecule contains at least two binding sites and two heavy chain parts, but heavy chain partly lacks the CH2 zone, and the existence form that wherein surpasses the molecule more than 50% is that two heavy chain parts connect with at least one interchain disulfide bond.In the another embodiment, the existence form that surpasses the molecule more than 60% is that two heavy chain parts connect with at least one interchain disulfide bond.In the another embodiment, the existence form that surpasses the molecule more than 70% is that two heavy chain parts connect with at least one interchain disulfide bond.In the another embodiment, the existence form that surpasses the molecule more than 80% is that two heavy chain parts connect with at least one interchain disulfide bond.In the another embodiment, the existence form that surpasses the molecule more than 90% is that two heavy chain parts connect with at least one interchain disulfide bond.
In a kind of embodiment, this material purification process produces the composition that contains the recombinant polypeptide molecule, this recombinant polypeptide molecule contains at least two binding sites and two heavy chain parts, and wherein the existence form of the molecule more than 99% is that two heavy chain parts connect with at least one interchain disulfide bond.
In a kind of embodiment, this material purification process produces the composition that contains peptide molecule, this peptide molecule contains at least two binding sites and two heavy chain parts, wherein the existence form of the molecule more than 95% is that two heavy chain parts connect with at least one interchain disulfide bond, and the heavy chain of polypeptide part is from IgG4 isotype (isotype) antibody.
In a kind of embodiment, this material purification process produces the composition that contains peptide molecule, this peptide molecule contains two light chain parts and two heavy chain parts, but heavy chain partly lacks the CH2 zone, and wherein the existence form of the molecule more than 80% is that two heavy chains partly do not connect with at least one interchain disulfide bond.
On the other hand, the present invention also provides the method for measuring purifying and/or preferred biosynthetic result, comprises the relative quantity of measuring A type and Type B in the composition.The following method of passing through of A type and Type B is measured: non-reduced sds polyacrylamide gel electrophoresis for example described herein or mass spectrometry.
V. the mark of polypeptide or coupling
Peptide molecule among the present invention can be non-the link coupled form use, also can with at least a molecule coupling, for example, the facilitation target detects or is used for patient's video picture or treatment.Polypeptide of the present invention can be labeled before or after purifying or coupling.Specifically, polypeptide of the present invention can be coupled to cytotoxin (as radio isotope, cell toxicity medicament or toxin) therapeutical agent, inhibition of cell proliferation, biotoxin, prodrug, peptide, protein, enzyme, virus, lipid, biological answer-reply instrumentality, pharmaceutical formulations, immunocompetence part (as lymphokine or other antibody, wherein the gained molecule both can be coupled to tumour cell, also can be coupled to effector cell such as T cell) or PEG.In the another embodiment, polypeptide of the present invention can be coupled to and reduce the molecule that tumor vessel forms.In other embodiment, disclosed composition can comprise and medicine or prodrug link coupled polypeptide of the present invention.Other embodiment of the present invention comprises the purposes with concrete biotoxin or its cell toxicant fragment such as Ricin, gelonin, Pseudomonas exotoxin or diphtheria toxin link coupled polypeptide of the present invention.The selection of coupling or non-link coupled polypeptide depend on the type of cancer and by stages, the application (as chemotherapy or external radiotherapy) of assisting therapy and patient's situation.Be appreciated that and consider content teaching herein, those skilled in the art can make this selection easily.
Be appreciated that also in the previous research that in animal model and the human case of part, isotope-labeled anti-tumour antibody has been successfully used to destroy solid tumor and lymphoma/leukemic oncocyte.Common isotropic substance comprises: 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re.Radionuclide works by producing ionizing rays, and ionizing rays can cause examining the fracture of DNA chain many places, thereby causes necrocytosis.The isotropic substance that is used to produce the treatment conjugate can produce high-energy α-or beta particle usually, and this particle has short path length (short path length).This radionuclide kills in-plant cell, and for example conjugate has adhered to or entered wherein tumour cell.They seldom or not act on non local cell.Radionuclide right and wrong basically is immunogenic.
About the application of the radiolabeled conjugate relevant with the present invention, polypeptide of the present invention can be by direct mark (as passing through iodization), or by using the sequestrant indirect labelling.Term used herein " indirect labelling " and " indirect labelling approach " all refer to sequestrant and binding molecule covalent attachment, and at least a radionuclide links to each other with this sequestrant.This sequestrant is often referred to difunctional inner complex, and they had both combined also with polypeptide and have combined with radio isotope.Concrete preferred sequestrant comprises 1-isothiocyanato benzyl-3-methyl diethylene triaminepentaacetic acid(DTPA) (1-isothiocycmatobenzyl-3-methyldiothelenetriaminepentaac etic acid) (" MX-DTPA ") and cyclohexyl diethylene triaminepentaacetic acid(DTPA) (cyclohexyl diethylenetriamine pentaacetic acid) (" CHX-DTPA ") derivative.Other sequestrant comprises P-DOTA and EDTA derivative.The radionuclide that specifically is preferred for indirect labelling comprises 111In and 90Y.
This paper term " directly mark " and " directly mark approach " all refer to inner complex and the direct covalent attachment of polypeptide (be typically and pass through amino-acid residue).More specifically, these combining method are learned and are comprised random labelling and site-directed mark (site-directed labeling).For the latter, mark is at the specific site of polypeptide, as exists only in the saccharide residue that the N end of conjugate Fc part connects.In addition, different direct labeling technique and schemes are applicable to the present invention.For example, technetium-99 m labeled polypeptide can prepare by the following method: the ligand exchange method, and by using the solution reduction Sodium pertechnetate-99Tc (TcO that contains divalent tin ion 4), the reductive technetium is sequestered on the Sephadex post, and polypeptide is loaded on this post; Or batch labeling technique (batchlabeling techniques), for example, by incubation Sodium pertechnetate-99Tc, reductive agent such as SnCL2, damping fluid such as sodium phthalate-potassium and antibody.In any case the radionuclide that is preferred for direct traget antibody is well known in the art, the radionuclide that specifically is preferred for direct mark is covalently bound by tyrosine residues 131I.Polypeptide according to the present invention can, for example, utilize following material to derive: radio-iodidesodium or potassiumiodide and chemical oxidizing agent such as clorox, chloramine-T etc., or oxydasis agent such as lactoperoxidase (lactoperoxidase), glucose oxidase and glucose.Yet to achieve the object of the present invention, the indirect labelling method is particularly preferred.
The patent relevant with the sequestrant conjugate with sequestrant is well known in the art.For example, the United States Patent (USP) of Gansow is for the 4th, 831, No. 175 about polysubstituted diethylene triaminepentaacetic acid(DTPA) inner complex, contains the protein conjugate of diethylene-triamine pentaacetic acid, and the preparation method of these materials.The United States Patent (USP) the 5th, 099,069,5,246,692,5,286,850,5,434,287 and 5,124 of Gansow, No. 471 also is about polysubstituted DTPA inner complex.These full patent texts are embodied in herein as a reference.Other example of the metal chelator that is suitable for is Calcium Disodium Edetate acid (ethylenediaminetetraacetic acid, EDTA), diethylene-triamine pentaacetic acid (diethylenetriaminepentaacetic acid, DPTA), 1,4,8,11-four azepines, four decane (tetraazatetradecane), 1,4,8,11-four azepine four decane-1,4,8,11-tetraacethyl, 1- (oxa)-4,7,12,15-four azepine heptadecanes (tetraazaheptadecane)-4,7,12,15-tetraacethyl etc.Cyclohexyl-DTPA or CHX-DTPA are concrete preferred, and specify hereinafter.Also have other sequestrant that is suitable for, comprise that those are still undiscovered, those skilled in the art can distinguish out that they obviously belong to scope of the present invention easily.
The sequestrant that is suitable for, be included in common pending application series the 08/475th, 813,08/475,815 and 08/478, be used to promote the concrete bifunctional chelating agent of sequestering action in No. 967, preferred selectedly think that trivalent metal provides high-affinity, show that the tumour-non-tumour ratio (tumour-to-non-tumour ratios) that increases, the skeletal bones of reduction and radionuclide store in target site is the more body in B cell lymphoma site.Yet, other have or not the bifunctional chelating agent of all these characteristics of tool be known in this area, they also may be useful to oncotherapy.Also be appreciated that technology according to this paper, polypeptide can with different radioactively labelled substance couplings be used for the diagnosis and therapeutic purpose.For this purpose, all be embodied in full in the reference of this paper, aforesaid application undetermined, disclosed before drug treatment antibody, be used for the radiolabeled treatment conjugate of diagnosing tumor " video picture "." In2B8 " conjugate contains the mouse monoclonal antibody 2B8 of specificity at people CD20, this monoclonal antibody by bifunctional chelating agent be MX-DTPA (diethylenetriaminepentaacetic acid) with 111The In combination, MX-DTPA is 1-isothiocyanato benzyl-3-methyl D TPA (1-isothiocyanato-benzyl-3-methyl-DTPA) and 1-methyl-3-isothiocyanato benzyl-DTPA (1-methyl-3-isothiocyanatobenzyl-DTPA) mixture in 1: 1 ratio.Concrete preferred 111In uses radionuclide as diagnosis, but because its safely use and the toxicity that can not detect between about 1 to 10mCi scope, and image data is to subsequently 90The distribution of Y-traget antibody has the indication meaning usually.Most iconography research and utilization 5mCi 111The antibody of In-mark because both safety of this dosage, and is compared the imaging results that increase is arranged with lower dosage, gives antibody and occurs best video picture after three to six days.See also, for example, Murray, J.Nuc.Med.26:3328 (1985) and Carraguillo etal., J.Nuc.Med.26:67 (1985).67(1985)。
Shown in above, different radionuclides can be applicable to the present invention, and those skilled in the art are easy to distinguish which kind of radionuclide is the most suitable under different situations.For example, 131I is the radionuclide of the immunotherapy that is used for target that is widely known by the people.Yet, 1318The clinical application of I can be limit by following Several Factors: eight days physical half life, iodinating antibody are in blood and the dehalogenation effect of tumor sites, radioactive nature (for example a large amount of gamma compositions), and it piles up (deposition) for the local dose in tumour may be suboptimum.Along with the appearance of better sequestrant, with the chance of metal-chelating group and protein bound, increased use other radionuclide as 111In and 90The possibility of Y.In using, radioimmunotherapy utilizes 90Y has some benefit: 90Y64 hour transformation period is long enough to make antibody to accumulate in tumour, and unlike 131I, 90Y is high-octane pure radiator beta-ray, and without the gamma radiation, the scope in tissue is 100 to 1000 cell dias in the attenuation process.In addition, the penetrating radiation of minimum allows the out-patient is used 90The antibody of Y-mark.In addition, do not need the internalization of the antibody of mark to come cell killing, the part radiation of ionizing rays should be fatal to the tumour cell that closes on that lacks target molecule.
It will be understood by those skilled in the art that the conjugate of these no radioactivities, also can adopt different technical combinations to get up according to the selected link coupled material of wanting.For example, the conjugate of band vitamin H can prepare by the following method: for example, the active ester of polypeptide and vitamin H such as vitamin H N-hydroxy-succinamide ester are reacted.Equally, can under the situation that coupling agent exists, prepare with fluorescently-labeled conjugate, for example described coupling agent listed as mentioned those; Perhaps with the preferred fluorescein isothiocyanate reaction of lsothiocyanates.The conjugate of polypeptide of the present invention and cell growth-inhibiting/cytotoxic substance and metallo-chelate can take similar mode to prepare.
The preparation of using among the present invention is cell toxicity medicament preferably, refers to that specifically those are applied to the medicine of cancer therapy.Herein, used " cytotoxin or cytotoxic substance " is meant that cell growth and propagation are harmful to, and may reduce, suppresses or destroy any material of cell or malignant tumour.Representative cytotoxin includes but not limited to: radionuclide, biotoxin, enzyme activity toxin, cell growth-inhibiting or cytotoxic treatments material, prodrug, immunocompetence part and biological response modifier such as cytokine.Any cytotoxin that stops or delay the growth of immunologically competent cell or malignant cell is all in category of the present invention.
In general, the exemplary cells toxin comprises: cytostatic agent, alkylating agent, metabolic antagonist, antiproliferative, tubulin binding substances, hormones and hormone antagonist etc.Be applicable to that representative cytostatic agent of the present invention comprises: alkylating agent such as mustargen (mechlorethamine), thiotrithylene phosphoramide (triethylenephosphoramide), endoxan (cyclophosphamide), ifosfamide (ifosfamide), Chlorambucil (chlorambucil), busulfan (busulfan), melphalan (melphalan) or triaziquone (triaziquone), and nitroso-urea compounds (nitrosoureacompounds) is as carmustine (carmustine), lomustine (lomustine) or Me-CCNU (semustine).Other preferred classes of cytotoxic substance comprises, for example the medicine of CHROMATOGRAPHIC FRACTIONATION AND MASS (maytosinoid) family.Other preferred classes of cytotoxic substance comprises: for example medicine of anthracycline antibiotics (anthracycline) family, Vinca class (vinca) medicine, mitomycin (mitomycins), bleomycin (bleomycins), cytotoxicity nucleosides, pteridine class medicine, diine base class (diynenes) and podophyllotoxin (podophyllotoxins).Concrete useful composition comprises in these classifications: for example, and Zorubicin (adriamycin), Carubicin (carminomycin), daunorubicin (daunorubicin) (daunomycin (daunomycin)), Dx (doxorubicin), aminopterin (aminopterin), methotrexate (methotrexate), methopterin (methopterin), Plicamycin (mithramycin), streptonigrin (streptonigrin), dichloromethane aminopterinum (dichloromethotrexate), ametycin (mitomycin C), dactinomycin (actinomycin-D), porfiromycin (porfiromycin), 5-fluor-uracil (5-fluorouracil), fluorodeoxyuridine (floxuridine), Tegafur (ftorafur), Ismipur (mercaptopurine), cytosine arabinoside (cytarabine), cytosine arabinoside cytosine(Cyt) (cytosine arabinoside), podophyllotoxin (podophyllotoxin) or podophyllotoxin derivative such as etoposide (etoposide) or phosphoric acid Etoposide, melphalan, vincaleucoblastine (vinblastin), vincristine(VCR) (vincristine), leurosidine (leurosidine), vindesine (vindesine) and vinleurosine (leurosine) etc.Be applicable to other cytotoxin in addition that the present invention instructs, comprise taxol (taxol), Taxan (taxane), Cytochalasin B (cytochalasin B), Gramicidin D (gramicidin D), ethidium bromide (ethidium bromide), Hemometine (emetine), tenoposide, colchicine (colchicin), dihydroxyl anthracin diketone (dihydroxy anthracin dione), mitoxantrone (mitoxantrone), PROCAINE HCL, PHARMA GRADE (procaine), tetracaine (tetracaine), lignocaine (lidocaine), propranolol (propranolol), tetracycline (puromycin) and analogue or homologue.Hormone and hormone antagonist also are applicable to instruction of the present invention, for example cortin such as prednisone (prednisone), Progesterone (progestin) is as hydroxyprogesterone (hydroxyprogesterone) or U.S. holder progesterone (medroprogesterone), oestrogenic hormon such as stilboestrol (diethylstilbestrol), antiestrogen such as Tamoxifen (tamoxifen), male sex hormone such as testosterone and aromatase inhibitor such as aminogluthetimide.As mentioned above, those skilled in the art can carry out chemically modified to required compound, make the reaction of this compound be more conducive to prepare conjugate of the present invention.
Preferred cytotoxic specific examples comprises the member or the derivative of enediyne family in the antitumor antibiotics, comprises calicheamycin (calicheamicin), esperamicins or anthracycline antibiotics (dynemicins).These toxin are extremely potent, cause necrocytosis by making the nuclear dna break.Do not resemble archon, in vivo can the many non-activities of cleaved generation but immunogenic polypeptide fragment is arranged, toxin such as calicheamycin, esperamicins and other enediynes are small molecules, essentially no immunogenicity.These non-peptide toxoids are connected in the dimer and the tetramer by the technology chemistry that before once was used to labeled monoclonal antibody and other molecule.These interconnection techniques comprise that the locus specificity of the saccharide residue that connects by the N end that exists only in construct Fc part connects.The advantage of this fixed point method of attachment is to have reduced to connect the issuable influence of the binding characteristic of this structure.
As mentioned before, the cytotoxin of Shi Yonging can comprise prodrug (prodrug).Term used herein " prodrug " is meant the precursor or the derivative of pharmaceutically active substance, and it is less to the cytotoxicity of tumour cell with respect to parent's medicine, and can be activated or be converted into active stronger parent's form (parent form) by enzyme.Be applicable to that prodrug of the present invention includes but not limited to: the prodrug of the prodrug of phosphorous hydrochlorate (ester), the prodrug that contains sulfo-phosphoric acid salt (ester), sulfur-bearing hydrochlorate (ester), contain the propeptide medicine, contain the prodrug of beta-lactam, the prodrug that contains phenoxy-acetamide (phenoxyacetamide) that replaces arbitrarily, the prodrug that contains phenylacetamide, 5-flurocytosine and other 5-fluor-uracil prodrug that replace arbitrarily, these prodrugs can be converted into the medicine of active stronger no cytotoxicity.Other example that can be derived to the cytotoxic drug that is applied to prodrug form of the present invention comprises above-mentioned those chemotherapeutics.
In other cytotoxin, be appreciated that polypeptide also can be connected with biotoxin such as Ricin subunit A, abrin (abrin), diphtheria toxin, novain (botulinum), cyanginosins, shellfish poison (saxitoxin), Shigellae toxin (shigatoxin), tetanus toxin (tetanus), tetraodotoxin (tetrodotoxin), trichothecene (trichothecene), verrucologen or toxicity enzyme.Preferably, adopt the genetic modification technology that allows antibody-toxin construct directly to express to prepare this construct.Other biological answer-reply instrumentality that can be connected with polypeptide of the present invention comprises cytokine such as lymphokine and Interferon, rabbit.According to of the present invention open, those skilled in the art can adopt the synthetic easily this structure of routine techniques.
That can be connected with polypeptide disclosed by the invention, suitable cytotoxic other classification is a radiosensitizer, and this radiosensitizer can be by effectively at tumour or immunoreactive cell.This medicine has strengthened the susceptibility to ionizing rays, has therefore increased the effect of radiotherapy.Discharged radiosensitizer by the antibody coupling matter of tumour cell internalization near nucleus, it is maximum that the radiation sensibilized meeting at this position reaches.The polypeptide that the uncombined radiosensitizer of the present invention connects can be removed from blood very soon, and remaining radiosensitizer is positioned in the target tumour, makes that the picked-up of healthy tissues is minimum.After removing fast from blood, auxiliary radiotherapy meeting is carried out with one of following three kinds of approach: 1) specificity is at the external-beam of tumour, 2) the direct radioactivity of implantation tumour, 3) utilize the whole body of identical targeting type antibody to put to exempt from treatment.It will be that the immune conjugate of treatment with radio isotope and radiosensitizer combined that this approach a kind of has the variation of potential magnetism, so provide need only use the facility of single medicine to the patient.
In a kind of embodiment, the part (moiety) that strengthens polypeptide stability or usefulness can be by coupling.For example, in a kind of embodiment, PEG can be coupled on the polypeptide of the present invention to prolong its transformation period in vivo.Leong, S.R., et al.2001.Cytokine 16:106; 2002; Adv.in Drug Deliv.Rev.54:531; Or Weir et al.2002.Biochem.Soc.Transactions 30:512.
VI. polypeptide uses
The method for preparing polypeptide of the present invention and use to the patient is known to those skilled in the art and is determined easily.The route of administration of polypeptide of the present invention comprises oral administration, passes through the parenteral admin of suction or topical application.Term used herein " parenteral " comprises through intravenously, intra-arterial, intraperitoneal, intramuscular, subcutaneous, internal rectum or intravaginal to be used.Usually preferred non-enteron aisle form of administration is through intravenously, intra-arterial, subcutaneous and intramuscular administration formulation.For all these formulations in the category of the present invention, form of administration will be an injection solution, specifically through the formulation of vein or intra-arterial injection or instillation.Usually, the pharmaceutical composition that is applicable to injection comprises damping fluid (as acetate, phosphoric acid salt or citrate buffer), tensio-active agent (as multi-sorbitol ester (polysorbate)), optional stablizer (as human albumin) etc.Yet in being applicable to other method that this paper says, this polypeptide can directly be delivered to harmful position, cell mass place, therefore increases the exposure of illing tissue to medicine.
The prepared product of non-enteron aisle form of administration comprises sterilized water or non-aqueous solution, suspension and emulsion.Examples of non-aqueous is propylene glycol, polyoxyethylene glycol, vegetables oil such as sweet oil and injectable organic ester such as ethyl oleate.Aqueous carrier comprises water, contains the solution of ethanol/water, emulsion or suspension, comprises salt and buffering medium.Among the present invention, pharmaceutically acceptable carrier includes but not limited to: 0.01-0.1M and preferred 0.05M phosphate buffered saline buffer or 0.8% salt solution.Other common parenteral carrier comprises phosphate solution, woods Ge Shi glucose, glucose and sodium-chlor, Lactated Ringer'S Solution or fixed oil.Intravenous vehicles comprise liquid, nutritional supplement, electrolyte supplements such as those based on woods Ge Shi glucose or the like.Also have sanitas and other additive, as antiseptic-germicide, antioxidant, sequestrant and rare gas element etc.More specifically say, be applicable to that the pharmaceutical compositions of injection comprises aseptic aqueous solution (water-soluble) or colloidal solution and the sterilized powder that is used for preparing immediately aseptic injectable solution or colloidal solution.In these situations, said composition must be aseptic, and is the liquid of easily injecting.It should be stable under processing and holding conditions, preferably preserves in antimicrobial container as bacterium and fungal contamination.Carrier can be solvent or dispersion medium, contains, for example water, ethanol, polyol (as glycerine, propylene glycol and liquid macrogol etc.) and suitable said mixture.Suitable flowability can keep by the following method, for example, and by utilizing the particle size required in Yelkin TTS bag quilt, the maintenance dispersion system and the application of tensio-active agent.Prophylaxis of microbial is polluted and can be realized that described antimicrobial drug and antifungal drug be metagin (parabens), trichloro-butyl alcohol (chlorobutanol), phenol (phenol), xitix, thimerosal (thimerosal) etc. for example by using various antimicrobial drugs and antifungal drug.In many situations, preferably include isotonic agent in the described composition, for example sugar, polyvalent alcohol such as N.F,USP MANNITOL, sorbyl alcohol or sodium-chlor.Can prolong medicament such as aluminum monostearate and the gelatinum that absorbs by in composition, containing, thus the absorption that prolongs Injectable composition.
In any situation, aseptic injectable solution can prepare by the following method: with the active compound (polypeptide itself or its combine with other active substance) of required dosage, in appropriate solvent,, carry out filtration sterilization as required subsequently herein with a kind of composition enumerated or the combined hybrid of multiple composition.Generally, dispersion system prepares by the following method: active compound and the sterile carrier that contains basic dispersion medium are reached other required composition of above enumerating mix.To being used to prepare the sterilized powder of aseptic parenteral solution, preferred manufacturing procedure is: vacuum-drying and lyophilize make to produce the powder that activeconstituents adds additional required composition from the solution of its previous sterile filtration.Injection preparation is treated, packs in container such as ampoule, sack, bottle, syringe or the phial under aseptic condition and seals according to methods known in the art.Next step, preparation can be with such as common pending application U.S.S.N.09/259, the packaged of the test kit of explanation and selling in 337 and U.S.S.N.09/259,338, above-mentioned two pieces of indexed documents are in this paper reference.This goods preferably have trade mark or package insert (package inserts), and the composition that prompting is relevant can be used for treating the patient who suffers from or easily suffer from autoimmune disease or tumour.The effective dose of the above-mentioned situation of combination treatment of the present invention, have difference according to many different factors, these factors comprise that medication, target site, patient's physiological status, patient are human or animal, used other medicines, and to handle be preventative or curative.Usually, the patient is human, also can be treated but non-human mammal comprises transgene mammal.Therapeutic dose can adopt ordinary method titration well known by persons skilled in the art, so that security and validity reach optimization.For using the antibody passive immunization, dosage range can, for example, 0.0001 between 100mg/kg host's body weight, and more common is to (0.02mg/kg, 0.25mg/kg, 0.5mg/kg, 0.75mg/kg, 1mg/kg, 2mg/kg etc.) between 5mg/kg host's body weight 0.01.For example, dosage can be 1mg/kg body weight or 10mg/kg body weight or in the scope of 1-10mg/kg body weight, preferably 1mg/kg body weight at least.Also be intended in scope of the present invention between the dosage between the above-mentioned scope.But patient every day, or the next day (alternative day), or weekly, or the table At All Other Times of rule of thumb analyzing decision is accepted described dosage.Representative treatment is in the period that prolongs, as a plurality of dosage of administration at least six months.Another representative treatment plan (regimes) is to be administered once in per two weeks, or every month once, or per 3 to June is once.Representative drug dosage schedule table comprises 1-10mg/kg or the every order successive administration of 15mg/kg, and administration or 60mg/kg are weekly next day of 30mg/kg.In some method, can use the monoclonal antibody of the different binding specificities of two or more tools simultaneously, wherein the dosage of every kind of antibody is all in range of indication.
Polypeptide of the present invention can be used under multiple situation.The interval of single agent administration can be weekly, every month once or once a year.According to the mensuration prompting of polypeptide in patient's blood or target molecule level, dosing interval can be irregular.In some method, adjust dosage so that plasma polypeptide concentration reaches 1-1000 μ g/ml, some method then can reach 25-300 μ g/ml.Optional, binding molecule can be used as extended release preparation and uses, and wherein required administration frequency reduces.Dosage is different and different in the intravital transformation period of patient according to antibody with frequency.In general, the transformation period of people's antibody is the longest, secondly is chimera antibody and non-human antibody.In a kind of embodiment, binding molecule of the present invention can non-link coupled form be used, and in another embodiment, polypeptide of the present invention can the link coupled form repeatedly be used.In the embodiment, binding molecule of the present invention can be used with non-link coupled form earlier, uses with the link coupled form then, or uses with opposite order again.
Dosage and frequency can be preventative or therapeutic and different according to this treatment.In the prophylactic application, contain the composition of antibody of the present invention or its mixing element, give as yet not the patient in morbid state to increase its resistibility.This amount is defined as " prevention effective dose ".In this usage, exact dosage desired also depends on patient health state and general immunity power, but generally the scope of each dosage is 0.1 to 25mg, specifically is 0.5 to 2.5mg.In long-time, use relatively low dosage with frequent relatively interval.Some patient continued to receive treatment in its remaining years.
During therapeutic is used, sometimes need (for example use higher relatively dosage with relatively short interval, about 1 to 400mg/kg binding molecule every dose of antibody for example, usually arrive the dosage of 25mg for radioimmunity conjugate thing more commonly used 5, dosage is then higher for cytotoxic drugs link coupled molecule), slow down or stop up to progression of disease, last till that preferably the patient shows that disease symptoms partially or completely alleviates.After this, but administration patient's prevention scheme also.
In a kind of embodiment, can use the nucleic acid molecule treatment experimenter of code book invention polypeptide.The dosage range of nucleic acid encoding be every about 10ng of patient to 1g,, 100ng is to 100mg, 1 μ g to 10mg or 30-300 μ g DNA.The dosage of infectious expression vector is that every dose 10 to 100 virion does not wait or more.
Therapeutic preparation can pass through in parenteral route, part, intravenously, oral, subcutaneous, intra-arterial, encephalic, intraperitoneal, the nose or the intramuscular administration, is used for preventative or therapeutic treatment.The modal route of administration of immunogenicity medicine is through subcutaneous, but other approach is effective equally.Inferior approach commonly used is intramuscularly.Such injection is many to be carried out in the muscle of arm or leg.In some method, medicine directly injects settling (deposit) accumulative specifically to be organized, for example intracranial injection.Antibody use preferred intramuscularly or venoclysis.In some method, specific treatment antibody is injected directly into cranium.In some method, the antibody conduct continues the composition that discharges or installs such as Medipad TMDevice is used.
Medicament of the present invention can be chosen wantonly and other disease or coupling of illness efficacious agents to needs treatments (for example, preventative or therapeutic).
Of the present invention 90The single effective dose of the polypeptide of Y-mark (promptly treating effective dose) scope is between about 5 to about 75mCi, preferably between about 10 to about 40mCi. 131(non-marrow ablative) single dose therapeutically effective scope of the no marrow destruction of I-traget antibody is between about 5 to about 70mCi, preferably between about 5 to about 40mCi. 131(ablative) single dose therapeutically effective (promptly may need the autologous bons marrow transplantation value) scope that the marrow destruction is arranged of I-traget antibody is about 30 between about 600mCi, preferably about 50 arrive less than about 500mCi between.For chimeric antibody, because the murine antibody circulating half-life is longer, 131The single dose therapeutically effective scope of the no marrow destruction of the chimeric antibody of I-mark is between about 5 to about 40mCi, preferably less than about 30mCi.For example 131The imaging standard of I-mark is usually less than about 5mCi.
When 131I and 90After the application of Y had obtained a large amount of clinical experiences, other radio-labeling known in the art was used to similar purpose.Also have other radio isotope to be used to imaging.For example, be applicable to that other radio isotope of the present invention includes but not limited to: 123I, 125I, 32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 132I, 197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd, 153Sm, 188Re, 199Au, 225Ac, 211At reaches 213Bi.In this, α, γ and radiator beta-ray all are applicable to the present invention.In addition, according to of the present invention open, which kind of radionuclide those skilled in the art can determine be applicable to the selected course of disease of treatment easily, and do not need inappropriate experiment.For this purpose, other radionuclide that has been applied to clinical diagnosis comprises: 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga reaches 111In.Antibody is also with the different labelled with radioisotope (Peirersz et al.Immunol.Cell Biol.65:111-125 (1987)) that the immunotherapy that is applied to be used for target is arranged.These radionuclides comprise in small range 188Re, 186Re, 199Au, 67Cu, United States Patent (USP) 5,460,785 have provided these radioisotopic postscripts, and this patent is embodied in herein as a reference.
No matter whether polypeptide of the present invention is used with coupling or non-link coupled form, be appreciated that a main advantage of the present invention is that these polypeptide can be used for myelosuppressive patient, promptly those are accepting or are accepting the patient of assisting therapy such as radiotherapy or chemotherapy.That is, the useful collection of illustrative plates (beneficialde liver y profile) (the promptly short relatively serum residence time, high binding affinity and enhanced localization) of sending of this polypeptide makes them for treatment Red bone marrow deposit decline and more useful to the patient of bone marrow toxicity sensitivity.In this, this polypeptide uniqueness send collection of illustrative plates, make them very effective for using radiolabeled conjugate to myelosuppressive cancer patients.Equally, polypeptide of the present invention can be used for before accepting the patient of assisting therapy such as treatment of outside ray irradiation or chemotherapy with coupling or non-link coupled form.In preferred other embodiment, this polypeptide (with coupling or non-link coupled form) can be used for the combined treatment with chemotherapeutic agents.It will be understood by those skilled in the art that this treatment plan can comprise continuous administration, (simultaneous) and deposit (concurrent) or with prolonging the disclosed antibody of (coextensive) administration and one or more chemotherapeutics simultaneously of passing through.The concrete in this respect preferred embodiment of the present invention will comprise using of radiolabeled polypeptide.
Though this polypeptide can be used by just described mode above, must emphasize in other embodiments, coupling or non-link coupled polypeptide can be used as the first-line treatment medicament administration in otherwise be healthy patient.In these embodiments, this polypeptide can be applied to the Red bone marrow deposit with normal or mean level (ML) and/or not accept or be not the patient who is accepting assisting therapy such as treatment of outside ray irradiation or chemotherapy.
Yet just as discussed above, the embodiment that the present invention selects comprises uses polypeptide to the bone marrow depression patient, or is making up or coupling (being the treatment plan of coupling) with one or more assisting therapy such as radiotherapy or chemotherapy.Combine with assisting therapy herein or coupling gives polypeptide and is meant: this therapy and disclosed polypeptide continuous pass through, simultaneously, with prolonging and depositing, follow (concomitant) or the same period (contemporaneous) to give or use.The different integral parts that skilled person in the art will appreciate that the coupling treatment plan can be timed the whole validity of using or using with the enhancing treatment.For example, can in the known course of treatment of standard, use chemotherapeutic agents, within several weeks, use radioimmunity conjugate of the present invention then.On the contrary, the polypeptide of cytotoxin associating can be used the directed outside ray irradiation of tumour then through intravenous administration.Yet in other embodiments, this polypeptide can be used when once going to a doctor simultaneously with selected one or more chemotherapeutic agents.Those skilled in the art (for example experienced oncologist) can be according to the guidance of selected assisting therapy and this specification sheets, distinguishes effective combined treatment easily and does not need too much experiment.
Be appreciated that the combination of this polypeptide (having or do not have cytotoxin) and chemotherapeutic agents from this aspect, can be by any order or administration in the time window that the treatment benefit is being provided the patient arbitrarily.In other words, chemotherapeutic agents and polypeptide administration or administration simultaneously in any order.In the embodiment of selecting, polypeptide of the present invention can be applied to the patient who has before accepted chemotherapy.In another embodiment, this polypeptide and chemotherapeutic treatment can be substantially simultaneously or and deposit administration.For example, can in accepting the process of chemotherapy, the patient give binding molecule.In preferred embodiments, binding molecule should be used in any chemotherapeutic agents or treatment 1 year in administration.In other embodiment preferred, this polypeptide should be used in 10,8,6,4 or February of any chemotherapeutics or treatment.And in other embodiment preferred, this polypeptide should be used in 4,3,2 or 1 week of any chemotherapeutic agents or treatment.In other embodiments, this polypeptide should be used in selected chemotherapeutics or treatment 5,4,3,2 or 1 days.Can further understand, these two kinds of medicaments or treatment can (promptly simultaneously basic) be applied to the patient in a few hours or several minutes.
In addition, according to the present invention, the bone marrow depression patient should refer to any patient that cytometry reduces.It will be understood by those skilled in the art that several cytometry parameters are conventionally used as myelosuppressive clinical indices, can measure myelosuppressive degree among the patient in view of the above easily.The example that the bone marrow depression that this area is accepted is measured be absolute neutrophil leucocyte counting (Absolute Neutrophil Count, ANC) or platelet count.It can be the result of various biochemical illnesss or disease that this bone marrow depression or part marrow destroy (myeloablation), and described illness and disease are the results of previous chemotherapy or radiotherapy.In this respect, skilled person in the art will appreciate that the patient who accepted conventional chemotherapy can occur the Red bone marrow deposit usually and descend.As discussed above, owing to can cause mortality ratio or sickness rate side reaction such as anaemia or immunosuppression that increase, that be difficult to accept, this patient often can't use cytotoxin (the being radionuclide) treatment of optimum level.
Say that more specifically coupling of the present invention or non-link coupled polypeptide can be used for effectively treating ANC and be lower than about 2000/mm 3Or platelet count be lower than about 150,000/mm 3The patient.More preferably, polypeptide of the present invention can be used for treating ANC and is lower than about 1500/mm 3, be lower than about 1000/mm 3The patient, more preferably less than about 500/mm 3The patient.Equally, polypeptide of the present invention, can be used for treating platelet count be lower than about 75,000/mm 3, be lower than about 50,000/mm 3, even be lower than about 10,000/mm 3The patient.Say that more at large those skilled in the art can use the guide or the method for government's formulation and determine to exist myelosuppressive patient easily.
As mentioned, many bone marrow depression patients have accepted the multiple course of treatment, comprise chemotherapy, implant radiotherapy or the treatment of outside ray irradiation.For the latter, external radiation source is for malignant tumour is carried out partial irradiation.For implanting radiotherapy, the reactant of radioactivity is implanted in the malignant tumour by operation, so the position of elective irradiation disease.Howsoever, the polypeptide that the present invention announces can be used for treating the illness that has myelosuppressive patient, and no matter how cause myelosuppressive reason.
From this aspect, can further understand, polypeptide of the present invention can be used to eliminate, reduce, the associating or the combined therapy (the coupling treatment plan promptly is provided) of any chemotherapeutic agents of inhibition or the growth of control volume inner tumour cell.As discussed above, this medicine often causes the decline of Red bone marrow deposit.This decline can be remedied wholly or in part by the bone marrow toxicity that compound of the present invention has reduced, and this further treatment to this patient's tumour is favourable.In other embodiment preferred, radiolabeled immune conjugate disclosed herein can be used the radiosensitizer of the susceptibility of radionuclide with increasing tumour cell.For example, can from blood flow, have been swept off at the radio-labeled binding molecule but when tumour or tumor locus still keep the treatment level of significance, used the radiation sensitization compound.
Consider these aspects of the present invention, be applicable to that representative chemotherapeutics of the present invention comprises: alkylating agent, vinca alkaloids (for example vincristine(VCR) (vincristine) and vincaleucoblastine (vinblastine)), procarbazine (procarbazine), methotrexate (methotrexate) and prednisone (prednisone).The MOPP scheme (dichloromethyldiethylamine (mechlethamine) (mustargen), vincristine(VCR) (Oncovin), procarbazine and prednisone (prednisone)) of four medicines associating is very effective for the various types of lymphomas of treatment, and contains the preferred embodiment of the present invention.To the drug-fast patient of MOPP-, can use ABVD (is Zorubicin (adriamycin), bleomycin (bleomycin), vincaleucoblastine and dacarbazine (dacarbazine)), ChlVPP (Chlorambucil (chlorambucil), vincaleucoblastine, procarbazine and prednisone), CABS (lomustine (lomustine), Zorubicin (doxorubicin), bleomycin (bleomycin) and streptozocin (streptozotocin)), MOPP adds ABVD, MOPP adds ABV (Zorubicin, bleomycin and vincaleucoblastine) or BCVPP (carmustine (carmustine), endoxan (cyclophosphamide), vincaleucoblastine, procarbazine and prednisone) combination.Arnold S.Freedman and Lee M.Nadler, Malignant Lymphomas, inHarrison ' s Principles of Internal Medicine 1774-1788 (Kurt J.Isselbacher et al., 13th ed.1994) and V.T.DeVita et al. (1997) eds..The reference of wherein quoting has standard dose and treatment plan table.As described herein, these treatment plans can not add any change or by the change of concrete needs of patients, and with one or more polypeptide couplings of the present invention.
Other useful in the background of the present invention (context) scheme comprises: the application of single alkylating agent such as endoxan or Chlorambucil, combination is such as CVP (endoxan, vincristine(VCR) and prednisone), CHOP (CVP and Zorubicin (doxorubicin)), C-MOPP (endoxan, vincristine(VCR), prednisone and procarbazine), endoxan, vincristine(VCR), prednisone and procarbazine), CAP-BOP (CHOP methylate benzyl hydrazine and bleomycin), (CHOP adds methotrexate to m-BACOD, bleomycin and formyl tetrahydrofolic acid (leucovorin)), ProMACE-MOPP (prednisone, Rheumatrex, Zorubicin, endoxan, etoposide and formyl tetrahydrofolic acid add standard MOPP), ProMACE-CytaBOM (prednisone, Zorubicin, endoxan, etoposide, cytosine arabinoside (cytarabine), bleomycin, vincristine(VCR), methotrexate and formyl tetrahydrofolic acid) and MACOP-B (methotrexate, Zorubicin, endoxan, vincristine(VCR), the prednisone of fixed dosage, bleomycin and formyl tetrahydrofolic acid) application.Those skilled in the art can determine the dosage and the treatment plan of above-mentioned every kind of drug regimen easily.CHOP also can with bleomycin, methotrexate, procarbazine, mustargen, cytosine(Cyt) cytosine arabinoside (cytosine arabinoside) and etoposide applied in any combination.Other chemotherapeutics that is suitable for includes but not limited to: 2-chlorodeoxyadenosine (2-chlorodeoxyadenosine, 2-CDA), 2 '-deoxycoformycin (2 '-deoxycoformycin) and fludarabine (fludarabine).
The patient of being unrealized and alleviating or recur suffering from medium or senior NHL can adopt rescue therapy.Used medicine of rescue therapy such as cytosine(Cyt) cytosine arabinoside (cytosine arabinoside), cis-platinum (cisplatin), etoposide (etoposide) and ifosfamide (ifosfamide).Recurrence or progress form for certain knurl venereal disease change, the following treatment plan of normal employing: IMVP-16 (ifosfamide, methotrexate and etoposide), MIME (Methyl GAG (methyl-gag), ifosfamide, methotrexate and etoposide), DHAP (dexamethasone (dexamethasone), the cytosine arabinoside of high dosage and cis-platinum), ESHAP (etoposide, dexamethasone (methylpredisolone), the HD cytosine arabinoside, cis-platinum), CEPP (B) (endoxan, etoposide, procarbazine, prednisone and bleomycin) and CAMP (lomustine, methotrexate, cytosine arabinoside and prednisone), wherein every kind dose ratio and scheme all are known.
The consumption of chemotherapeutic agents during with polypeptide drug combination of the present invention, can be with different patients difference, or according to dosed administration known in the art.For example can consult: Bruce A Chabner et al., Antineoplastic Agents, in Goodman ﹠amp; Gilman ' s The Pharmacological Basis ofTherapeutics 1233-1287 ((Joel G.Hardman et al., eds., 9th ed.1996).
Discuss as preamble, polypeptide of the present invention, its immunoreactivity fragment or recombinant chou can be used according to pharmacy effective dose, to be used for the interior therapeutic of mammalian diseases.Select from this and to be appreciated that binding molecule disclosed by the invention can be mixed with the stability that can make things convenient for administration and increase promoting agent.Preferably, according to pharmaceutical compositions of the present invention, contain pharmaceutically acceptable, nontoxic sterile carrier for example physiological saline, nontoxic damping fluid, sanitas etc.For reaching the application's purpose, coupling or be not coupled to pharmacy effective dose polypeptide, its immunocompetence fragment or recombinant chou of medicine, refer to be enough to obtain the effective dosage that combines and benefit with target, for example improve symptom or the detection material or the cell of disease and illness.For tumour cell, this polypeptide preferably can interact with selected immune response antigen on tumour or the immunoreactive cell, and makes the death of those cells increase.Certainly, pharmaceutical compositions of the present invention can be with single or multiple dosed administrations, so that the pharmacy effective dose of this polypeptide to be provided.
Consistent with scope of the present invention, polypeptide of the present invention can be applied to the human or animal with competent dosage according to the aforementioned therapies method, so that treatment or preventive effect to be provided.Binding molecule of the present invention formulation (dosage form) routinely is used for these human and other animals, and this regular dosage form can be according to known technology, by antibody of the present invention and conventional, pharmaceutically acceptable carrier or thinner combination are prepared.Those skilled in the art can approve that the form of pharmaceutically acceptable carrier and thinner and characteristic are determined by amount, route of administration and other known variables with its bonded activeconstituents.Those skilled in the art can further understand, and contain one or more mixtures according to polypeptide of the present invention and will be proved effective especially.
VII. application method
Polypeptide of the present invention can be used for diagnosis or therapeutic purpose.The preferred embodiment of the invention is provided for diagnosing and treating compound, composition, test kit and the method for illness, for example needs the neoplastic disease of the mammalian subject of this treatment.Preferred experimenter is the people.
Polypeptide of the present invention can be useful in some different application.For example, in a kind of embodiment, being tried binding molecule will be to reducing or to remove the cell with target of being discerned by binding molecule of the present invention useful.In another embodiment, tried binding molecule to the concentration that reduces solubility target molecule in the circulation or to remove this antigen effective.
In a kind of embodiment, tumour size, the lifetime of suppressing tumor growth and/or prolongation tumor animal.Correspondingly, the present invention also relates in people or other animal, treat the method for tumour by the polypeptide of using effective and non-toxic for this human or animal.Those skilled in the art can rely on normal experiment, determine this polypeptide to be used for the treatment of the dosage malignant tumour purpose, effective and nontoxic.For example, the treatment effective dose of polypeptide can be different according to excite factor the ability of required reaction in the patient such as staging (for example: the I phase is to the IV phase), age, sex, medical science complication (as immunosuppressant illness or disease), weight in patients and this antibody.Can adjust dosage so that best therapeutic response to be provided.For example, every day, dosage can divide administration several times, or suggested according to the urgency level of treatment situation, reduced dosage in proportion.Yet in general, the expection effective dosage ranges is in about 0.05 to 100 milligram of per kilogram of body weight every day, more preferably from about 0.5 to 10 milligram of per kilogram of body weight every day.
For the purpose of clarification, " Mammals " relates to any Mammals person that is categorized as, and comprises that people, domestic and farm-animals, zoological park feeding animals, motion are with animal or pets animal, for example dog, horse, cat, ox etc.Preferred Mammals is the people.
" treatment " relates to two kinds of therapeutic treatment and prophylactic treatments.Need the curer to comprise take a disease disease or disorder, also comprise preventing disease or disorder.Therefore, this Mammals can be suffered from this disease or illness by diagnosis, perhaps has to suffer from this disease tendency or to this disease-susceptible humans.
As discussed above, polypeptide of the present invention can combine with one or more tumour molecules or the molecule relevant with dysimmunity.In the embodiment, for neoplastic disease, the antigen binding site of polypeptide disclosed by the invention (being its variable region or immunoreactivity fragment or recombinant chou) combines with selected tumour associated molecule at the malignant tumour position.Equally, in immunity (comprising autoimmunization) disease, polypeptide disclosed by the invention can combine with the selected mark of invasion cell (offending cells).According to the number of the molecule of having reported relevant, it will be understood to those of skill in the art that polypeptide disclosed by the invention can be derived from any one of multiple complete antibody with tumour or dysimmunity.Say that more at large useful polypeptide can derive from or originate from any antibody (comprising what previous bibliographical information was crossed) that the target relevant with selected condition or mark react among the present invention.In addition, be used to produce the parental antibody of polypeptide disclosed by the invention or precursor antibody, its fragment, can be mouse, people, chimeric, humanized, non-human primates or the long sourceization of spirit.In other embodiment preferred, that polypeptide disclosed by the invention can contain is as described herein, the single-chain antibody construct (such as United States Patent (USP) 5,892,019 is disclosed, is included in this paper as a reference) of the reformed constant region of tool.Therefore, according in the synthetic antibody of these types of this paper instruction any one, all be applicable to the present invention.
" tumour associated molecule " used herein is meant usually any antigen or the target molecule relevant with tumour cell, promptly compare with normal cell appear at identical or wider.More commonly, the tumour associated molecule comprises makes immunoreactivity antibody be positioned at any molecule of tumour cell, and does not consider its expression on non-malignant cell.This molecule is relative tumour-specific, and its expression is limited to the malignant cell surface.As a kind of selection, this molecule all can be found on pernicious and non-malignant cell.For example, CD20 is general (pan) B antigen of finding at pernicious and non-Malignant B cell surface, and it has been proved to be the very effectively target of the immunotherapy antibody of treatment non-Hodgkin lymphoma.From this respect, general T cell antigen such as CD2, CD3, CD5, CD6 and CD7 also contain the tumour associated molecule in the range of definition of the present invention.Other representative tumour associated molecule includes but not limited to: MAGE-1, MAGE-3, MUC-1, HPV 16, HPV E6 ﹠amp; E7, TAG-72, CEA, L6-antigen, CD19, CD22, CD37, CD52, HLA-DR, EGF acceptor and HER2 acceptor.In many situations, the existing bibliographical information mistake of any one immunoreactivity antibody in these antigens.Skilled person in the art will appreciate that according to the present invention any one of these antibody all can be used as the precursor of polypeptide of the present invention.
Polypeptide of the present invention is preferably with above-mentioned tumour or immunity related molecular links to each other or combination.Therefore, describe in detail as some parts hereinafter is concrete, polypeptide of the present invention can be derived from, and produces from any in the antibody of multiple and the reaction of tumour associated molecule of, preparation.In the preferred embodiment, described polypeptide is the antibody of synthetic or structural domain disappearance, it can utilize the genetic modification derives from technology, and at least a portion of one or more constant region structural domain is lacked or changes thus, to provide required biochemical property such as the half life that shortens.More specifically, as follows, but the genetic sequence of the variable and/or constant region of those skilled in the art's delamination correspondence purpose antibody and disappearance or change suit Nucleotide so that polypeptide of the present invention to be provided, and it is used as the monomer subunit according to the present invention.To understand compatible polypeptide of the present invention can utilize established scheme to express or preparation in clinical or commodity coverage.
Report in the past and antibody tumour associated molecule reaction can be as described herein and be changed, so that polypeptide of the present invention to be provided.Can be used for providing antigen binding domain to include, but are not limited to 2B8 and C2B8 (Zevalin with the exemplary antibodies that produces or drive disclosed polypeptide And Rituxan , IDECPharmaceuticals Corp., San Diego), Lym 1 and Lym 2 (Techniclone), LL2 (Immunomedics Corp., New Jersey), HER2 (Herceptin , Genentech Inc., South San Francisco), B1 (Bexxar , Coulter Pharm., San Francisco), Campath (Millennium Pharmaceuticals, Cambridge) MB1, BH3, B4, B72.3 (Cytogen Corp.), CC49 (National cancer Institute) and 5E10 (University ofIowa).In the embodiment preferred, polypeptide of the present invention will be incorporated into the identical tumour conjugated antigen of above enumerating of antibody.In the concrete embodiment preferred, described polypeptide will be derived from or in conjunction with identical antigen such as 2B8, C2B8, and CC49 and C5E10, even more preferably, will comprise the antibody (that is Δ CH2 antibody) of structural domain disappearance.
In first preferred embodiment, described polypeptide will be in conjunction with identical tumor associated antigen such as Rituxan Rituxan (also being known as rituximab, IDEC-C2B8 and C2B8) is that first kind of FDA approves that being used for the treatment of the lymphadenomatous monoclonal antibody of human B cell (sees United States Patent(USP) Nos. 5,843,439; 5,776,456 and 5,736,137, every piece of document is included in this paper as a reference).Y2B8 (the 2B8 of 90Y mark; Zevalin ; Ibritumomab tiuxetan) is mouse C2B8 parent.Rituxan Be chimeric anti-CD 20 monoclonal antibody, it suppresses growth and it is reported the lymphoma cell line that sensitization is specific, with at external apoptosis by chemical agent.Described antibody has strong FcR combination effectively in conjunction with people's complement, and can be machine-processed at the external human lymphocyte (Reff et al., blood 83:435-445 (1994)) that effectively kills via complement-dependent (CDC) and antibody-dependency (ADCC).It will be appreciated by those skilled in the art that the dimer variant (homodimer or heterodimer) of according to the present invention synthetic C2B8 or 2B8, can use the patient that suffer from the CD20+ malignant disease with effective treatment with link coupled or non-link coupled form.More commonly, must reaffirm that polypeptide of the present invention can use any with in the multiple disease of effective treatment with " exposed " or non-link coupled state or with cell toxicant medicament link coupled state.
In other preferred embodiment of the present invention, polypeptide of the present invention will be derived from or be incorporated into the tumor associated antigen identical with CC49.As mentioned previously, CC49 is in conjunction with people's tumor associated antigen TAG-72, and it is relevant with people source specific tumors cell surface, specifically is the LS174T tumor cell line.LS174T[American type culture collection (this paper ATCC) No.CL 188] be the variant of LS180 (ATCC No.CL 187) colon adenocarcinoma cell system.Will be further understood that multiple mouse monoclonal antibody is developed, its specificity is in conjunction with TAG-72.A kind of such monoclonal antibody B72.3 is hybridoma B72.3 (the mouse IgG1 that ATCC No.HB-8108 produces.B72.3 utilizes the human breast carcinoma extract (to see Colcher et al., Proc.Natl.Acad.Sci. (USA), 78:3199-3203 (1981) as the first-generation monoclonal antibody of immunogen exploitation; With United States Patent (USP) 4,522,918 and 4,612,282, every piece of document is included in this paper as a reference).Other monoclonal antibody at TAG-72 is called " CC " (for colon cancer).Of Schlom etal. (U.S.Pat.No.5,512,443 its be included in this paper as a reference), the CC monoclonal antibody is the s-generation mouse monoclonal antibody that utilizes by the TAG-72 preparation of B72.3 purifying.Because they are the TAG-72 binding affinity relatively preferably, following CC antibody is deposited in ATCC, can require to obtain with limitation: CC49 (ATCC No.HB 9459); CC 83 (ATCC No.HB 9453); CC46 (ATCC No.HB 9458); CC92 (ATTCC No.HB 9454); CC30 (ATCC No.HB 9457); CC11 (ATCC No.9455); And CC15 (ATCC No.HB 9460).U.S.P.N.5,512,443 have further instructed disclosed antibody to replace the chimeric form that the mouse constant region changes over described antibody with for example human constant region (Fc) structural domain by utilizing technology recombinant DNA known in the art.Except openly mouse and inosculating antibody resist-TAG-72 antibody, Schlom et al. has also prepared as the variant of disclosed humanized CC49 antibody among the PCT/US99/25552 and United States Patent (USP) 5,892,019 disclosed strand constructs, each piece of writing is included in this paper as a reference.It will be appreciated by those skilled in the art that every kind of aforesaid antibody, construct or recombinant chou, and their variant can be synthesized and is used to provide polypeptide of the present invention.
Resist-TAG-72 antibody except above-mentioned, each group has reported that structure and the part of the CC49 of structural domain disappearance and B72.3 antibody is qualitative (for example, Calvo et al.Cancer Biotherapy, 8 (1): 95-109 (1993), Slavin-Chiorini et al.Int.J.Cancer 53:97-103 (1993) andSlavin-Chiorini et al.Cancer.Res.55:5957-5967 (1995).
In the embodiment, binding molecule of the present invention is in conjunction with CD23 (United States Patent (USP) 6,011,138).In the embodiment preferred, binding molecule bonded epi-position of the present invention is identical with the epi-position of 5E8 antibodies.In another embodiment, binding molecule of the present invention comprises from anti--CD23 antibody, for example, and at least one CDR of 5E8 antibody.
In the embodiment, binding molecule of the present invention is in conjunction with CRIPTO-I antigen (WO02/088170A2 or WO03/083041A2).In the embodiment preferred, binding molecule bonded epi-position of the present invention is identical with the epi-position of B3F6 antibodies.In another embodiment, binding molecule of the present invention comprises from anti--CRIPTO-I antibody, for example, and at least one CDR of the B3F6 antibody.
Other embodiment preferred of the present invention comprises the antibody of modification, the tumor associated antigen that it is derived from or combination is identical with C5E10.Of common pending application 09/104,717, C5E10 is the antibody of the glycoprotein determinant of the about 115kDa of identification, and it shows prostate tumor cells system (for example DU145, PC3, or ND1) special.Therefore, in conjunction with the present invention, specificity can be produced and use with the treatment tumor disease with coupling or non-link coupled form in conjunction with the polypeptide (for example antibody of CH2 structural domain-disappearance) of the tumor associated antigen that C5E10 antibody is discerned.In the concrete embodiment preferred, described binding molecule will be derived from or comprise all or part antigen binding domain from the hybridoma cell line excretory C5E10 of ATCC preserving number PTA-865 antibody.The binding molecule that produces is with the radionuclide that is coupled to as described below, and the method according to this invention suffers from the patient of prostate cancer.
Generally speaking, invention disclosed can be used for preventing or any any tumour that comprises the mark that allows described binding molecule target cancerous cells of curing the disease property treatment.Medicable exemplary cancer includes, but not limited to prostate cancer, and the stomach cancer is such as colorectal carcinoma, skin carcinoma, mammary cancer, ovarian cancer, lung cancer and carcinoma of the pancreas.More specifically, antibody of the present invention can be used for treating Ka Boqi sarcoma (Kaposi ' s sarcoma), cns tumor (kapillary hemangioblastoma (capillary hemangioblastomas), meningioma and brain shift), melanoma, stomach and intestine and sarcoma of kidney, rhabdosarcoma, glioblastoma multiforme (preferred glioblastoma multiforme (glioblastoma multiforme)), leiomyosarcoma, retinoblastoma, papillarycystadenocarcinomaofovary (papillary cystadenocarcinoma of the ovary), Wilm ' s knurl or small cell lung cancer.To understand each relevant tumour associated molecule that suitable polypeptide can stem from aforementioned tumour, and openly need not inappropriate test according to of the present invention.
Can comprise He Jiejin lymphomas and non_hodgkin lymphoma and leukemia according to the exemplary blood malignant disease of open treatment of the present invention, comprise ALL-L3 (Burkitt type leukemia), lymphocytic leukemia (CLL) and monocytic leukemia.It is especially effective for the multiple B-cell lymphoma of treatment to understand Compounds and methods for of the present invention, comprise rudimentary/folliculus non--He Jiejin lymphomas (NHL), cell lymphoma (FCC), lymphoma mantle cell (MCL), diffuse large cell lymphoma (DLCL), small lymphocyte (SL) NHL, intermediate stage/folliculus NHL, intermediate stage diffusivity NHL, senior immunoblast (high grade immunoblastic) NHL, senior lymphoblast (high gradelymphoblastic) NHL, senior little non-depression cell (high grade small non-cleavedcell) NHL, sick NHL of bulky and Waldenstrom ' s macroglobulinemia.Those skilled in the art should know because these lymphomas of change of categorizing system have different titles usually, and suffering from the lymphadenomatous patient who is categorized as different titles can benefit from combined treatment of the present invention.Except above-mentioned tumor disease, but should understand other malignant disease that the present invention's preferred therapeutic is carried suitable tumour associated molecule.
Except tumor disease, polypeptide of the present invention is especially effective for treatment autoimmune disease and abnormal immune reaction.In this regard, will understand polypeptide of the present invention and can be used for control, suppress, regulate or eliminate unwanted immune response outside and self antigen.For example, in the embodiment, described antigen is autoantigen.In another embodiment, described antigen is anaphylactogen.In another embodiment, described antigen is isoantigen or heterologous antigen.It is particularly useful in transplanting to the immune response of of the same race or heterologous antigen to utilize disclosed polypeptide to reduce, and for example is used to suppress transplant recipient to for example repulsion of tissue or organ graft or bone marrow graft of acceptor graft.In addition, acceptor T cell inhibiting or the elimination in the bone marrow graft can be used for the inhibition of transplant versus-host disease.
In another embodiment, polypeptide of the present invention can be used for treating Immunological diseases, includes but not limited to allergic bronchopulmonary aspergillosis (allergic bronchopulmonary aspergillosis); Allergic rhinitis; Autoimmune hemolytic anemia; Acanthosis nigricans (Acanthosis nigricans); Allergic contact dermatitis (Allergic contact dermatitis); The AddisonShi disease; Atopic dermatitis; Alopecia areata (Alopeciaareata); General alopecia (Alopecia universalis); Amyloidosis (Amyloidosis); Anaphylactoid purpura (Anaphylactoid purpura); Anaphylactoid reaction (Anaphylactoid reaction); Aplastic anemia; Hereditary angiodysplasia (Angioedema, hereditary); The special property sent out angioedema (Angioedema, idiopathic); Ankylosing spondylitis (Ankylosing spondylitis); Cerebral arteritis (Arteritis, cranial); Giant cell arteritis (Arteritis, giant cell); Takayasu arteritis; Temporal arteritis; Asthma; Ji imbalance telangiectasis (Ataxia-telangiectasia); Autoimmune oophoritis (Autoimmune oophoritis); Autoimmunity orchitis (Autoimmune orchitis); The many endocrinasthenias of autoimmunity (Autoimmune polyendocrine failure); Behcet's disease (Behcet ' sdisease; The Berger disease; The Buerger disease; Bronchitis (bronchitis); BP (Bullouspemphigus); Chronic mucocutaneous moniliosis (candidiasis, chronic mucocutaneous); Caplan syndrome; Postmyocardial infarction syndrome; Postpericardiotomy syndrome (Post-pericardiotomy syndrome); Carditis (Carditis); Sprue (Celiac sprue); The Chagas disease; Chediak-Higashi syndrome; The Churg-Strauss disease; Cogan syndrome; Cold agglutinin disease (Cold agglutinin disease); CREST syndrome; The CrohnShi disease; Cryoglobulinemia; CFA (Cryptogenic fibrosing alveolitis); Dermatitis herpetifomis; Dermatomyositis; Diabetes; Diamond-Blackfan syndrome; DiGeorge syndrome; Discoid lupus erythematosus (Discoid lupus erythematosus); Eosinophilic fasciitis (Eosinophilic fasciitis); Episcleritis (Episcleritis); Drythema elevatum diutinum; Erythema marginatum (Erythemamarginatum); Erythema multiforme (Erythema multiforme); Erythema nodosum (Erythemanodosum); Familial Mediterranean fever (Familial Mediterranean fever); Felty ' s syndrome; Pulmonary fibrosis; Irritated sample glomerulonephritis (Glomerulonephritis, anaphylactoid); The autoimmunity glomerulonephritis (Glomerulonephritis, autoimmune); Glomerulonephritis behind the suis (Glomerulonephritis, post-streptococcal); Glomerulonephritis after transplanting (Glomerulonephritis, post-transplantation); Film renal glomerulus ephrosis (Glomerulopathy, membranous); Goodpasture ' s syndrome; Immune-mediated granulopenia (Granulocytopenia, immune-mediated); Annular granuloma (Granuloma annulare); Allergic granulomatosis (Granulomatosis, allergic); Granulomatous dermatomyositis (Granulomatousmyositis); The Grave disease; Struma lymphomatosa (Hashimoto ' s thyroiditis); Newborn hemolytic disease (Hemolytic disease of the newborn); The special property sent out hemochrome disease (Hemochromatosis, idiopathic); The Henoch-Schoenlein purpura; Chronic active hepatitis and chronic progressivity hepatitis; Histiocytosis X; High acidophilia (Hypereosinophilic) syndrome; Idiopathic thrombocytopenic purpura (Idiopathic thrombocytopenic purpura); Job ' s syndrome; JDMS (Juvenile dermatomyositis); Juvenile rheumatoid arthritis (Juvenile rheumatoidarthritis) (juvenile chronic arthritis); The Kawasaki disease; Keratitis; Keratoconjunctivitis sicca; Landry-Guillain-Barre-Strohl syndrome; The lepromatous leprosy disease (Leprosy, lepromatous); Loeffler ' s syndrome; Lupus; Lyell ' s syndrome; Lyme disease (Lyme disease); Lymphomatoid granulomatosis; Systemic mastocytosis (Mastocytosis, systemic); MCTD; Mononeuritis multiplex (Mononeuritis multiplex); Muckle-Wells syndrome; Mucocutaneous lymphoglandula (Mucocutaneous lymph node) syndrome; Mucocutaneous lymphoglandula (Mucocutaneouslymph node) syndrome; Multicentric reticulohistiocytosis (Multicentricreticulohistiocytosis); Multiple sclerosis; Myasthenia gravis; Mycosis fungoides (Mycosisfungoides); Systemic necrotizing vasculitis (Necrotizing vasculitis, systemic); Nephrotic syndrome; Overlapping (Overlap) syndrome; Pimelitis (Panniculitis); Paroxysmal cold hemoglobinuria (Paroxysmal cold hemoglobinuria); Paroxysmal nocturnal hemoglobinuria (Paroxysmalnocturnal hemoglobinuria); Pemphigoid (Pemphigoid); Pemphigus (Pemphigus); Pemphigus erythematosus (Pemphigus erythematosus); Pemphigus foliaceus (Pemphigus foliaceus); Pemphigus vulgaris (Pemphigus vulgaris); Pigeon alimenter disease (Pigeon breeder ' s disease); The hypersensitivity pneumonia (Pneumonitis, hypersensitivity); Polyarteritis nodosa (Polyarteritisnodosa); Rheumatic polymyositis (Polymyalgia rheumatic); Polymyositis (Polymyositis); IP (Polyneuritis, idiopathic); Portugal's familial polyneuropathy (Portuguese familial polyneuropathies); Preeclampsia (Pre-eclampsia)/eclampsia (eclampsia); Primary biliary cirrhosis (Primary biliary cirrhosis); Progressivity systemic sclerosis (Progressive systemic sclerosis) (scleroderma); Psoriatic; Psoriatic arthritis; Pulmonary alveolar proteinosis (Pulmonary alveolarproteinosis); Pulmonary fibrosis (Pulmonary fibrosis), Raynaud's phenomenon (Raynaud ' s phenomenon)/syndrome; Reidel ' s thyroiditis; Reiter ' s syndrome, recurrent polychondritis (Relapsing polychrondritis); Rheumatic fever (Rheumatic fever); Rheumatoid arthritis (Rheumatoid arthritis); Sarcoidosis (Sarcoidosis); Scleritis (Scleritis); Sclerosing cholangitis (Sclerosing cholangitis); Serum sickness (Serum sickness); Sezary syndrome; Sjogren ' s syndrome; Stevens-Johnson syndrome; The Still disease; Subacute sclerosing panencephalitis (Subacute sclerosing panencephalitis); Sympathetic ophthalmia (Sympatheticophthalmia); Systemic lupus erythematous (Systemic lupus erythematosus); Transplant rejection (Transplant rejection); Ulcerative colitis (Ulcerative colitis); The connective tissue disease (CTD) of undifferentiated (Undifferentiated connective tissue disease); Chronic urticaria (Urticaria, chronic); Urticaria, cold; Uveitis (Uveitis); Vitiligo (Vitiligo); The Weber-Christian disease; Wegener granulomatosis and Wiskott-Aldrich syndrome.
Also by following examples explanation, described embodiment should not be interpreted as limitation of the present invention in the present invention.All reference that this paper quotes, patent, the content of disclosed patent application is included in this paper as a reference.
Embodiment
Embodiment 1: identify A and B isotype
Antibody molecule solution comprises two kinds of different isotypes.A kind of A type wherein comprises the heavy chain molecule that connects via at least one disulfide linkage.Another kind of Type B comprises the heavy chain molecule that does not connect via at least one disulfide linkage.At complete gamma 1 MAb, in Rituxan , Type B do not occur or frequency very low.Yet compare with (dd) construct of the structural domain disappearance with similar hinge, the frequency of Type B is much higher.These forms can utilize the non-reduced SDS page of sex change type to distinguish.In the antibody preparations of structural domain disappearance, the A type is a 120kDa dimer and Type B is 60kDa monomer (Fig. 1).Fig. 2 A and 2B show the specific gravity hydrometer mapping (densitometer plot) of non--reduction SDS-PAGE gel of ddCC49 (CH2) and ddCC49 (Gly/Ser) respectively.
Embodiment 2: the hinge area heterology in the Mab fragment of evaluation CH2 structural domain disappearance
The hinge arrangement territory can be divided into three different districts again: on, in and following hinge area (Roux et al.J.Immunol.1998 161:4083).For IgG1 and IgG3 hinge, the peptide sequence that comprises these districts is shown in table 1.15 amino acid whose motifs of triplicate are also contained in the district in the IgG3 hinge except containing two conserved cysteine residue.Aminoacid sequence from these districts is used to design synthetic IgG1/IgG3 connection peptides.These are grouped into by following one-tenth: corresponding to the IgG1 upper hinge residue of position 226-238, corresponding to hinge among the IgG1 of position 239-241, with repeat motif corresponding to hinge among the single IgG3 of position 241EE-242, the other proline(Pro) of its binding site 243 or lay respectively at position 243,244, with the proline(Pro) of the adding of 245 (Kabat numbering systems), L-Ala, proline(Pro) are flexible Gly/Ser spacer (table 2) then.In addition, new connection peptides is designed to by forming with the lower section: the proline(Pro) of the interpolation of the serine residue binding site 243 of the position of substitution 239 or 242 halfcystine and lay respectively at position 243,244, the proline(Pro) of 245 interpolation, L-Ala, proline(Pro) (Kabat numbering system).Pro243Ala244Pro245 and Pro 243 connection peptides have also been prepared.The aminoacid sequence of the humanized CC49 connection peptides of parent CH2 structural domain disappearance starts from first residue (position 226 of IgG1 hinge, the Kabat numbering system), and being extended to second residue of hinge/GlySer connection peptides, described aminoacid sequence is shown in table 2.By comparing, various connection peptides designs have also been shown with CC49 with cysteine residues position shown in the Kabat numbering system.
Table 1:IgG1, IgG3 and IgG4 hinge area
IgG Upper hinge Middle hinge Following hinge
IgG1 IgG3 IgG4 EPKSCDKTHT (SEQ ID NO：2) ELKTPLGDTTHT (SEQ ID NO：5) ESKYGPP (SEQ ID NO：50) CPPCP (SEQ ID NO：3) CPRCP(EPKSCDTPPPCPRCP)3 (SEQ ID NO：6) CPSCP (SEQ ID NO：51) APELLGGP (SEQ ID NO：4) APELLGGP (SEQ ID NO：4) APEFLGGP (SEQ ID NO：52)
Table 2: hinge area connection peptides sequence
Figure A20048002457600831
Embodiment 3. connects the structure and the preferred of isotype of polypeptide and synthesizes
Utilize splicing (SOE) method (Horton by overlapping extension, R.M.1993 Methods inMolecular Biology, Vol 15:PCR Protocols:Current Methods and applications.Ed.B.A.White), the nucleotide sequence that coding is shown in the hinge area connection peptides of table 2 imports the huCC49 gene order of CH2 structural domain disappearance.Correct modification to hinge area confirms by dna sequence analysis.Plasmid DNA is used to transform CHO DG44 cell, with stable preparation antibody protein.
Structure contains the huCC49 antibody of CH2 structural domain disappearance of the synthetic connection peptides of 8 kinds of designs shown in the table 2, and produces antibody in CHO DG44 cell.Supernatant liquor is collected the clone of self-separation, and the antibody concentration in the culture supernatant is determined by immunoassay.The supernatant of every kind of clone that contains the antibody of the total antibody protein of 0-30ng utilizes non-reduced SDS-PAGE electrophoretic analysis, utilizes Anti-Human kappa HRP link coupled antibody to carry out the WesteRN trace to detect the huCC49 A type and the Type B isotype of CH2 structural domain disappearance then.Under these conditions, the A type moves as single 120kDa homodimer, and Type B moves as 60kDa binary (doublet).Be also shown in kappa chain monomer and dimer.SEQ ID NO:8,9,14 and 15 connection peptides all increases the ratio of A type.Example results is shown in Fig. 3.These results show G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) (Fig. 3 D) and G1/G3/Pro243+[Gly/Ser] (SEQ ID NO:8) (Fig. 3 C) hinge causes producing mainly even fully the huCC49 antibody of A type CH2 structural domain-disappearance, and Type B almost maybe can not detect.On the contrary, the huCC49 Cys242Ser:Pro243Ala244Pro245 (SEQ ID NO:13) (Fig. 3 B) of the huCC49 Cys242Ser:Pro243 of CH2 structural domain-disappearance (SEQ ID NO:12) (Fig. 3 A) and CH2 structural domain-disappearance causes respectively the moderate of Type B isotype is arrived obvious preference.
Contain the Pro243Ala244Pro245+[Gly/Ser that imports the huCC49 antibody sequence] (SEQ IDNO:14) and G1/G3/Pro243Ala244Pro245+[Gly/Ser] clone of (SEQ ID NO:9) connection peptides is used for Antibody Preparation.Pro243Ala244Pro245+[Gly/Ser] and G1/G3/Pro243Ala244Pro245+[Gly/Ser] connection peptides also is imported into huCC49 V2 antibody sequence, (humanized CC49 version 2 sequence description is in embodiment 8 to produce clone; See among Figure 19 A and the B comparison) to light chain and heavy chain huCC49 and huCC49 V2 sequence.Antibody produces from CHO DG44 cell, and utilizes the method purifying described in the embodiment 4.The output of A type isotype after Protein G and HIC step is reported in the table 3.From these results obviously as seen, the modification in the antibody hinge region of importing CH2 structural domain disappearance causes the preferred synthetic of A isotype.Behind the embodiment 4 described HIC purification techniques, the HuCC49 Pro230Ala231Pro232 of purifying and HuCC49 V2 Pro230Ala231Pro232 A type material obtain to surpass 98% value.Only utilize the HuCC49G1/G3/Pro243Ala244Pro245 and the HuCC49 V2 G1/G3/Pro243Ala244Pro245 A type material of Protein G column purification, as the single peak wash-out of purity 〉=96% and need not further HIC purifying (Fig. 5 and Fig. 6).All antibody utilize the size exclusion chromatography check and as single peak wash-out, show not significantly gathering or the decomposition of antibody product.
Utilize peptide to draw and measure the HuCC49 of CH2 structural domain disappearance, the integrity that disulfide linkage forms in the heavy chain hinge area of HuCC49 PAP and HuCC49G1/G3/PAP antibody.The CC49 antibody sample of CH2 structural domain disappearance is reduced and utilizes the following digestion of trypsinase by sex change: aliquots containig 150 μ g are diluted and at the 6M Guanidinium hydrochloride sex change among the 50mM Tris pH 8.0 in 100ml HPLC water.Sample is by adding 20mM DTT reduction and being incubated 30 minutes at 37 ℃.The reductive sample utilizes the 50mM iodoacetic acid 37 ℃ of insulations 30 minutes.Alkylation reaction is by adding excessive DTT quencher.Reductive and alkylating sample utilize the exchange of PD-10 post buffering to go into 25mM TRIS, 20mM CaCl2, and pH 7.5.The ratio of trypsinase with 1: 15 (w/w) added each sample and be incubated 4 hours at 37 ℃.Digestion stops by adding trifluoroacetic acid (TFA) to final concentration 0.1%.Analyze the sample (15 μ g) of tryptic digestion subsequently according to following chromatography condition.
The CC49 antibody sample of CH2 structural domain disappearance digests by endo-protease Lys-C to be analyzed.Sex change and reductive sample are by adding final concentration 4M guanidine HCl and the 25mM DTT specimen preparation to 1.5mg/mL.Non-reducing sample is by adding final concentration 4M guanidine HCl the specimen preparation of 1.5mg/mL.Sample was 37 ℃ of insulations 2 hours.Digestion damping fluid (50mM Tris, pH 7.0 and 0.062AU/ml endo-protease Lys-C) adds sample with 1: 1 (v/v) subsequently, and sample was 37 ℃ of insulations 15 hours.At the 15th hour, with second part of enzyme aliquots containig (0.29mAU: μ g antibody) add, and sample is incubated 6 hours again at 37 ℃.Be the quencher reaction, add TFA with final concentration 0.1%.The non-reducing sample (12 μ g) that digests with reductive endo-protease Lys-C is subsequently according to following methods analyst.
HPLC/ mass spectroscopy sample is analyzed in the Agilent1100HPLC system that is connected in the single quadrupole mass spectrometer of Agilent MSD.Anti-phase C18 post (Vydac classification number 218TP52) and water/0.1%TFA (v/v) (buffer A) and acetonitrile/0.1%TFA (v/v) (buffer B) elution system divide use together with flow velocity 0.2mL/.Post (post column) " TFA fixing agent " solution acetonitrile and acetate (1: 1v/v) divide adding to improve ionization with 0.1mL/.Described column temperature is controlled at 45 ℃, and the wash-out collection of illustrative plates is 215 and 280nm monitoring.Total ion chromatography figure monitors in positive ion mode.Sample injects on the post and gradient is remained on 0% buffer B, 5 minutes.Utilize the linear gradient, 125 minutes of 0-50% buffer B, utilize the washing of 75% buffer B to surpass 10 fens then, and assign to realize wash-out above 30 with 0% buffer B reequilibrate.
In inscribe Lys-C reductive is analyzed, all do not detect fragment (L52-109) (Fig. 7 B) in all samples.This fragment is very hydrophobic and because strong reaction may not be from base for post matter wash-out.Corresponding trypsinase fragment (L68-109) does not detect (Fig. 7 C) yet in all samples.Because these fragments contain a large amount of amino acid, per-cent amino acid identity is dropped to～89% identity.In addition, fragment (L68-119) does not detect in the inscribe Lys-C of G1/G3/PAP analyzes, make identity be reduced to～79%.
Inscribe Lys-C is non--and the reductive analysis provides much better result (Fig. 7 A).Fragment (L52-109) detects in all samples as the disulfide linkage with fragment (L1-24).All other disulfide linkage are detected, and are～99% for the overall % amino acid of all samples identity.The G1/G3/PAP sample shows other heavy chain-heavy chain disulfide linkage in the fragment (H232-275), is lower than primary (H224-227) CPPC hinge area.Original and the observed molecular weight of the hinge area peptide of transforming is shown in table 4.HuCC49 Δ CH2 hinge inscribe Lys-C is non--and the MW of the observation of reductive peptide (residue H221-257) is 7419.4, and is consistent well with the calculating molecular weight 7419.4g/mol of the hinge of the connection that contains two interchain disulphide bridgeses.HuCC49 Δ CH2 PAP hinge inscribe Lys-C is non--and the MW of the observation of reductive peptide (residue H221-260) is 7949.7, and is also consistent well with the calculating molecular weight 7949.8g/mol of the hinge of the connection that contains two interchain disulphide bridgeses.Two hinges are non--and the reductive peptide fragment produces from the digestion of inscribe Lys-C to HuCC49 Δ CH2 G1/G3/PAP, and this is owing to there is the lysine residue that is positioned at Kabat position 241II in fifteen amino acid γ 3 motifs.The MW of the observation of peptide fragment THTCPPCPEPK (residue H221-231) and SCDTPPPCPRCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTK (residue H232-275) is 2414.3 and 8782.6, and is consistent well with the molecular weight 2413.0 and the 8782.0g/mol that calculate respectively.The THTCPPCPEPK peptide (residue H221-231) that this molecular weight data support is derived from HuCC49 H2 G1/G3/PAP contains inferring of two interchain disulphide bridgeses.Importantly, SCDTPPPCPRCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTK peptide (residue H232-275) contains at least one interchain disulphide bridges, and participating in the formation of two above disulphide bridgeses with chimeric G1/G3/PAP hinge, this is identical of views.These are analyzed and show that HuCC49 Δ CH2 PAP hinge forms two heavy chain interchain disulfide bonds.HuCC49 Δ CH2 G1/G3/PAP hinge forms at least three heavy chain interchain disulfide bonds, but also may be 5.Can affirm that fragment HuCC49 Δ CH2 G1/G3/PAP residue H232-275 contains at least one interchain disulfide bond, but can not distinguish hinge area that contains three interchain disulfide bonds and the molecular weight difference that contains the hinge area of single interchain and two interchain disulfide bonds.
Table 3. affinity chromatography (protein G) per-cent of A type antibody afterwards and after the HIC purifying
The antibody of CH2 structural domain disappearance After the %A type antibody protein G after the HIC purifying
HuCC49 (connection peptides SEQ ID NO:7) 60 98
HuCC49 PAP (connection peptides SEQ ID NO:14) 83 98
HuCC49 V2 PAP (connection peptides SEQ ID NO:14) 90 99
HuCC49 G1/G3/PAP (connection peptides SEQ ID NO:9) 98 Do not carry out
HuCC49 V2 G1/G3/PAP (connection peptides SEQ ID NO:9) 96 Do not carry out
Table 4
The peptide of the HuCC49 Δ CH2 antibody hinge region peptide of transforming is drawn
Sample The theoretical MW of fragment # A.A# The theoretical MW that connects The observed molecular weight reductive Observed molecular weight is non-reducing
HuCC49ΔCH2 PAP G1/G3：PAP HuCC49ΔCH2 PAP G1/G3：PAP Inscribe Lys-C fragment 29 (H221-257) 3711.7 THTCPPCGGGSSGGGSGGQPREPQVYTLPPSRDELTK inscribe Lys-C fragments 29 (H221-260) 3976.9 THTCPPCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTK inscribe Lys-C fragments 29 (H221-231) 1208.5 THTCPPCPEPK inscribe Lys-C fragments 30 (H232-275) 4394.0 SCDTPPPCPRCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTK trypsase fragments 39 (H221-241) 1971.8 THTCPPCGGGSSGGGSGGQPR trypsase fragments 39 (H221-244) 2237.9 THTCPPCPAPGGGSSGGGSGGQPR trypsase fragments 39 (H221-231) 1324.5 THTCPPCPEPK trypsase fragments 40 (H232-241) 1187.5 SCDTPPPCPR trypsase fragments 41 (H242-259) 1542.6 CPAPGGGSSGGGSGGQPR 7419.4 7949.8 2413.0 8782.0 NA NA NA NA NA 3711.5 3976.8 1208.9 4394.4 1972.6 2237.8 1325.1 1187.5 1542.9 7419.1 7949.7 2414.3 8782.6 NA NA NA NA NA
These data presentation, the synthetic hinge area connection peptides of new transformation can be used for preferred promote to form A or B isotype.These researchs have also disclosed the importance of cysteine residues in the antibody A type isotype of synthetic CH2 structural domain-disappearance of position 242 (Kabat numbering system).Therefore, in the embodiment, connection peptides of the present invention comprises and is arranged in position 239 or 242 at least one halfcystines.Make the biosynthesizing of antibody of CH2 structural domain-disappearance move to the Type B isotype with the halfcystine of Serine the position of substitution 239 or 242 (for example, utilize and be shown in SEQ ID NO:0,11,12, or 13 connection peptides).The use that increases the connection peptides of A type ratio causes processing, the favourable improvement of output and/or stability.The use of these synthetic hinge area connection peptides for example helps synthetic any antibody isotype, IgG1, and IgG2, IgG3, or the antibody A type isotype of the CH2 structural domain of IgG4 disappearance, it is based on the high homology of all four kinds of people's isotype CH3 structural domains.Comprise identical and conservative amino-acid residue, IgG1 CH3 structural domain and IgG2 CH3 have 98.13% homology, with IgG3 CH3 97.20% homology are arranged, and with IgG4 CH3 96.26% homology are arranged.
Embodiment 4: from containing the monoclonal antibody purifying mixture A type and the Type B of two kinds of isotypes
10mL ddCC49 supernatant uses 1M Tris pH 9.0 titration to whole pH 7.5.This material is through the membrane filtration of a series of Sol-Vac 0.8 μ and 0.4 μ.100mL XK50 Protein G post uses 1xPBS with flow velocity 80ml/min pre-equilibration.Titrating filtering supernatant is gone up sample to post with 80ml/min.Bonded albumen utilizes the level pad washing of twice column volume, uses 100mM glycine, pH 3.0 wash-outs then.Collection contains the fraction at ddCC49 peak and uses 1M Tris pH 9.0 titres to whole pH 7.0 at once.
Toso Biosep phenyl 5PW-HR post 20mM phosphoric acid salt pH 7.2; 1M vitriol pre-equilibration.The Protein G eluate utilizes 3.5M ammonium sulfate pH 7.2 mother liquors to be titrated to 1M ammonium sulfate and with sample on the concentration 2mg/ml gel bed.Bonded albumen utilizes 20mM phosphoric acid salt pH 4 or 7.2 ammonium sulfate wash-outs to regulate electric conductivity to 116.4mS/cm.The about 120kD of the apparent molecular weight of the material of condition wash-out on non-reduced SDS-PAGE (A type) since then.Reduction ammonium sulfate material linear gradient elution during remaining bonded antibody is used in the phosphate buffered saline buffer.Disulfide linkage and its molecular weight that the antibody of a kind of wash-out in back obviously lacks between the heavy chain are about 60kDa (Type B).Figure 15 shows the tomographic map of HIC purifying.The A of purifying and Type B are shown in the swimming lane 3 and 4 of Figure 16 respectively.
The material of above purifying all can be by being adjusted to ammonium sulfate concentrations 1M and it being gone up sample again catch to the phenyl 5PW-HR post of cleaning again.Bonded albumen utilizes 20mM phosphoric acid salt pH 7.2 wash-outs and dialysis to go into 1xPBS.
Embodiment 5. compares the stability of A type and Type B
The biological activity of A and Type B (as for example measuring, utilizing directly combination or competition research in trial test) discloses the A type and has similar biological activity with Type B.
The stability that compares A type and Type B.The ddCC49 molecule of purifying is concentrated to about 5mg/ml by the Amicon concentrating instrument of being furnished with YM30 film (Millipore).For each isotype, spissated material is divided into four parts, each fraction be placed in the 10K dialysis cassette (Pierce, cat#66410) in 16h, in following damping fluid, dialyse: 1) 10mM sodium phosphate, pH3; 2) 10mM sodium acetate, pH 5; 3) 10mM sodium phosphate, pH 7; With 4) the 10mM Sodium Tetraborate, pH 9.After the dialysis, the protein concentration of every kind of solution is adjusted to 3mg/ml.Except pure A and B isotype solution, mix the part A of every kind of pH and B solution contains 50% every kind of isotype with generation mixture.Produce 12 kinds of preparatons (four kinds of pH levels multiply by 3 kinds of antibody-solutions) altogether.Filter described solution and be filled in the 3ml 1 type glass serum bottle (West Pharmaceuticals) with gray butyl stopper.
Select three kinds of temperature, 2-8 ℃, preserve albumen to detect stability for 20-25 ℃ and 38-42 ℃.Before the preservation, take out 500 μ l samples and be used for physics and chemical molecular from every kind of preparaton, these 0 time point data are with comparing.In case preserve, at following time sampling: 2 weeks, 1 month, 2 months, 3 months, and censorship immediately.
Physics and chemical stability in order to assess two kinds of isotypes, adopt following method: measure turbidity at OD320, non--reduction SDS-PAGE, and size exclusion chromatography.
To being kept at 2-8 ℃, the 20-25 ℃ of sample with 38-42 ℃ carries out non--reduction SDS-PAGE in different time points.When preserving for 2-8 ℃, A type and Type B are all relatively stable.Yet but in pH7 and 9 whens preparation, A type and Type B show degraded, as less than original main band (the A type be 120kDa, Type B be 60kDa) the number increase of band shown in.Notice that specifically for the sample of the pH 7 that is kept at low temperature and medium temperature and 9, the intensity and the quantity that are lower than the band of 55kDa are higher than the A type in Type B.This shows that the A isotype is more stable than B isotype under these conditions.Yet, for pH5 and be kept at for 20-25 ℃ the A isotype really not so.As if the fragment that this sample has more than the B isotype.This looks it is because the artifacts that microbial contamination (describing in detail hereinafter) causes.Under high storage temperature, two kinds of forms obvious degradation all during pH9, and the gel pattern of sample does not almost have difference.Under this condition, the pollution zone of trace appears at the gel top, and it shows and has formed aggregation.Because aggregation can dissolve by SDS, the method research that described aggregation utilizes the present invention to describe with the lower section.
Table 5A-table 5C has listed the turbidity data of the ddCC49 that is kept at three kinds of differing tempss.Turbidity measurement solvable and non--solvable aggregation, and it is based on the amount of the light of these particle scatterings.When existing, aggregation is with scattered light and cause A320 to increase.As show shown in the 5A-C, for A and B isotype, the turbidity that is kept at 2-8 ℃ ddCC49 molecule increases with the increase of pH, and the former turbidity is lower than the latter's turbidity.This trend is correct for preserve the sample that is less than one month at higher temperature (20-25 ℃ and 38-40 ℃) for.Shelf time, the turbidity of the sample of high pH and temperature obviously increased when reaching 3 months, and the difference between A isotype and the B isotype reduces.These results are parallel with the result of SDS-PAGE, show two kinds of isotypes pH 3 and 5 all relatively stable (with regard to not forming aggregation), and the A isotype is lower than the B isotype for the accumulative susceptibility.
Table 5A. is kept at 2-8 ℃ ddCC49 sample turbidity what A320 measured
The A-isotype The B-isotype Mixture
Time (moon) pH＝3 5 7 9 3 5 7 9 3 5 7 9
0 0.030 0.038 0.044 0.056 0.034 0.042 0.046 0.066 0.036 0.042 0.051 0.061
0.029 0.029 0.046 0.045 0.030 0.038 0.048 0.058 0.034 0.033 0.043 0.055
1 0.033 0.039 0.035 0.055 0.033 0.035 0.044 0.059 0.032 0.040 0.039 0.066
2 0.042 0.022 0.042 0.044 0.039 0.037 0.055 0.067 0.042 0.024 0.040 0.058
3 0.035 0.047 0.051 0.050 0.038 0.041 0.066 0.081 0.027 0.048 0.051 0.065
Table 5B. is kept at 20-25 ℃ ddCC49 sample turbidity what A320 measured
The A-isotype The B-isotype Mixture
Time (moon) pH＝3 5 7 9 3 5 7 9 3 5 7 9
0.031 0.032 0.056 0.066 0.039 0.034 0.064 0.083 0.034 0.039 0.060 0.071
1 0.025 0.043 0.055 0.090 0.034 0.042 0.070 0.084 0.028 0.039 0.055 0.094
2 0.034 0.053 0.077 0.113 0.046 0.032 0.090 0.087 0.037 0.038 0.066 0.108
3 0.036 0.056 0.156 0.143 0.029 0.060 0.121 0.125 0.044 0.050 0.101 0.142
Table 5C. is kept at 38-42 ℃ ddCC49 sample turbidity what A320 measured
The A-isotype The B-isotype Mixture
Time (moon) pH＝3 5 7 9 3 5 7 9 3 5 7 9
0.041 0.042 0.068 0.063 0.041 0.044 0.080 0.067 0.041 0.039 0.070 0.064
1 0.041 0.043 0.071 0.065 0.036 0.040 0.079 0.069 0.032 0.048 0.078 0.070
2 0.047 0.030 0.066 0.060 0.046 0.045 0.087 0.082 0.051 0.034 0.078 0.079
3 0.058 0.051 0.098 0.105 0.046 0.057 0.101 0.157 0.056 0.057 0.101 0.126
Size exclusion chromatography (SEC) is the effective ways of per-cent that show the product (fragment and solvable aggregation) of complete molecule and degraded, and height can repeat.In table 5A-C, be kept at the A-isotype of differing temps, the complete monomer of B-isotype and mixture is listed.For the sample that is kept at 2-8 ℃, clearly the monomer per-cent of A type is higher than Type B, and the described per-cent of A type and Type B mixture is between the two aforementioned.At this storage temperature, two kinds of forms continue about three months at pH 3,5 and 7 relatively stable (pH 5 is the most stable conditions).Yet at pH9, the monomeric per-cent of Type B obviously reduces, and the only reduction of A type seldom.
At elevated temperatures, all samples displaying monomer per-cent increased and obviously increases with the shelf time; The increase of A isotype surpasses the B isotype.Yet, exception is also arranged, under similar preservation condition, the sample that is kept at the A isotype of room temperature shows more than the degraded of B isotype or its mixture.The check of this specific A isotype bottle shows that from the data of SDS-PAGE, the SEC of sample prompting microbial contamination may cause this unexpected result.At first, the degraded that SEC and SDS-PAGE result show this sample to fragmentation increase take the main responsibility, inferring is because microorganism digestion, otherwise expection accumulates in to a certain extent and increases.Secondly, contain each biased sample of 50% of A and B isotype and show, show that more stable A isotype has contribution to monomeric high per-cent than the better stable collection of illustrative plates of B isotype.At last, the A isotype that is kept at 2-8 ℃ and 38-42 ℃, pH5 all shows the monomer per-cent of the B isotype that is higher than under the simulated condition.Therefore, middle storage temperature should produce similar result.Because sample size is limited, can not carry out the microbial contamination test.
Be also noted that monomer reduces to about 30% for the IDEC-159 isotype that is kept at high pH (9) and 40 ℃.Under these exacting terms, the stability difference between two kinds of isotypes disappears.SEC result is consistent with the result who utilizes SDS-PAGE.Two results show that although have some chemistry and physical properties differences between two isotypes, the degradation mechanism and the by product of two kinds of isotypes are similar, or identical.
In a word, SEC result shows that A and B isotype have about 5 best pH, and the A isotype is with regard to keeping the complete monomer of high per-cent under similar preservation condition, more stable than B isotype.
Table 6A. is kept at the monomer per-cent of 2-8 ℃ ddCC49
The A-isotype The B-isotype Mixture
Time (moon) pH＝3 5 7 9 3 5 7 9 3 5 7 9
0 98.81 99.13 98.16 97.93 97.02 97.70 96.88 93.51 97.83 98.27 97.44 95.81
98.98 99.16 98.25 98.00 97.15 97.87 96.96 91.95 98.15 98.49 97.68 95.59
1 98.80 99.20 97.99 97.11 97.02 97.81 96.62 88.99 98.04 98.45 97.41 94.45
2 98.74 99.01 98.00 95.67 97.15 97.69 95.50 84.84 98.06 98.34 96.81 92.17
3 98.28 98.89 97.88 95.31 96.69 98.14 95.37 85.98 97.61 98.15 96.65 89.90
Table 6B. is kept at the monomer per-cent of 20-25 ℃ ddCC49
The A-isotype The B-isotype Mixture
Time (moon) pH＝3 5 7 9 3 5 7 9 3 5 7 9
97.83 99.04 97.12 93.65 95.84 97.62 93.71 79.61 96.75 98.30 95.37 87.67
1 96.60 96.63 95.65 88.09 94.38 97.23 90.69 72.26 95.36 97.99 93.05 80.92
2 93.62 92.79 93.17 80.06 91.71 96.96 85.51 66.53 92.78 97.51 89.33 73.91
3 92.81 89.56 x 74.31 89.30 96.04 82.57 63.25 90.46 97.02 86.80 69.36
Table 6C. is kept at the monomer per-cent of 38-42 ℃ ddCC49
The A-isotype The B-isotype Mixture
Time (moon) pH＝3 5 7 9 3 5 7 9 3 5 7 9
86.31 97.50 85.06 66.42 79.85 94.29 69.68 63.64 82.09 95.70 76.24 63.95
1 78.71 95.19 73.77 51.55 66.73 89.37 54.70 50.10 68.53 92.02 62.93 49.28
2 66.64 91.63 60.45 38.43 60.29 81.08 42.98 37.09 61.33 85.81 51.08 36.68
3 57.87 86.99 52.82 30.81 43.61 74.23 36.68 29.73 46.75 80.93 44.35 30.18
The preparation purifying of embodiment 7.A type and Type B
IDEC-159 (ddCC49) is the monoclonal antibody at the antigenic CH2 structural domain disappearance of the TAG-72 that is expressed in tumor surface.IDEC-159 contains two kinds of antibody isotypes, is called A type and Type B.Being used for the A type that the cell culture processes of IDEC-159 product produces and the ratio of Type B at present is about 50: 50.A type isotype is the antibody that has the CH2 district of disappearance in the heavy chain FC part.Except having the CH2 structural domain of disappearance, Type B also lacks the disulfide linkage of striding the Fc district and connects, and only is connected with salt bridge by hydrophobic reactant.
In the IDEC-159 method of purification the 3rd step is developed two isotypes that are used to separate IDEC-159 with last chromatographic step.Described separation utilizes phenyl TSKgel 5PW-HR sorbent material to carry out by hydrophobic reactant chromatography (HIC).Because the hydrophobicity of Type B is higher than the A type, its irreversible adsorption is wherein utilized about 0.73M ammonium sulfate/20mM sodium phosphate in stationary phase, and pH 4.0-pH 7.0 is as moving phase.The A type bonding force with stationary phase under these conditions is lower, and wash-out synchronously thus, and promptly it leaves pillar with the fraction that flows through post.After the synchronous wash-out of A type, remove ammonium sulfate from stationary phase and can make the Type B desorption.Following method is used to separate two isotypes of IDEC-159:
Described post utilization 〉=3CV 0.5N NaOH, with≤the 150cm/hr sterilization.
Described post utilization 〉=5CV 0.73M ammonium sulfate/20mM sodium phosphate, pH 4.0, with≤the 150cm/hr balance.
Described post is splined on room temperature TMAE Flowthrough, and it is adjusted to the 2.5M ammonium sulfate/20mM sodium phosphate that comprises 0.43 volume, pH 4.0 liquid mother liquors, the every ml resin of 5mg.Described antibody is at pH4.0, is splined on described post with≤100cm/hr.When the collection of antibody starts from effluent 280nm O.D. and reaches 10mAU.
Described post utilizes 15CV 0.73M ammonium sulfate/20mM sodium phosphate, and pH 4.0, with≤the 100cm/hr washing.Continue antibody in whole 15CV washing and collect, effluent goes out of use.
Described post utilization 〉=5CVs 20mM sodium phosphate, pH 4.0, peel off 6. described posts 〉=3CVs 0.5N NaOH with≤100cm/hr, clean with≤150cm/hr.
Described post usefulness 〉=3CVs 0.73M ammonium sulfate/20mM sodium phosphate, pH 4.0, with≤the 150cm/hr washing.
Described post is kept at 〉=3CV 20% ethanol, with 〉=150cm/hr.
Two kinds of forms are shown in Figure 17 (figure A and B) in the separation (5L column volume, total about 20g of IDEC-159 load) of preparation level.First peak comprises the synchronous eluate of A type, and second peak shows the Type B of wash-out, and the 3rd peak contains impurity, and it is removed from stationary phase in cleaning process.
This method also confirms by SDS PAGE in the ability of preparation horizontal separation A type and Type B.As shown in figure 18, utilize 0.73M ammonium sulfate/20mM sodium phosphate, the fraction (swimming lane 6-8) of pH 4.0 synchronous wash-outs mainly contains A type (impurity＞90%).
The humanization of embodiment 8. monoclonal antibody CC49
CC49 antibody is carried out multiple change to produce humanized CC49 version 2 (huCC49 V2).Figure 19 shows mouse CC49 light (Figure 19 A) and heavy chain (Figure 19 B) variable region, LEN or 21/28 ' CL, humanized CC49, the comparison of humanized CC49 V2 (it is compared with humanized CC49 and comprises one and be arranged in light aminoacid replacement and two aminoacid replacement that are arranged in heavy chain, sees underlined amino acid).For further reducing the potential immunogenicity of humanized CC49 Mab, people's framework residue that check exists the mouse residue in the described antibody also will be derived from people's receptor sequence LEN is used for light chain and replaces, and 21/28 ' CL is used for heavy chain and replaces.(Singer I I et al.，1993.Optimal Humanization of 1B4，anAnti-CD18 Murine Monoclonal Antibody，is Achieved by Correct Choice ofHuman V-Region Framework Sequences.J.Immunol.150：2844-2857.Padlan EA，1991.Possible Procedure For Reducing the Immunogenicity of AntibodyVariable Domains While Preserving Their Ligand-B inding Properties.MolecularImmunol.28：489-498)。
Be considered to only show that with the important framework residue of avidity some are different for the specificity that keeps binding site.In sequence of heavy chain, all choose residue Isoleucine and L-Ala of the residue of the expection embedding of position 69 (leucine) and 93 (Threonines) replaces respectively.In the sequence of light chain, most of residue (Serine) the personnel selection residue proline(Pro) that is embedded in position 43 of protecting of expection replaces.
The V2 CC49 antibody formation of preparation structural domain disappearance, connection peptides is inserted into huCC49 V2 sequence.Figure 13 A (SEQ ID NO:28) show contain G1/G3/Pro243Ala244Pro245+[Gly/Ser] dna sequence dna of the huCC49 V2 of the heavy chain CH2 structural domain-disappearance of hinge connection peptides.Figure 13 B (SEQ IDNO:29) shows the dna sequence dna of the huCC49 V2 of light chain CH2 structural domain-disappearance.Figure 14 A (SEQID NO:30) show contain G1/G3/Pro243Ala244Pro245+[Gly/Ser] the huCC49 V2 aminoacid sequence of the heavy chain CH2 structural domain-disappearance of hinge connection peptides.Figure 14 B (SEQ ID NO:31) shows the aminoacid sequence of the huCC49 V2 of light chain CH2 structural domain-disappearance.
Embodiment 9. comprises the enhanced bio distribution collection of illustrative plates of the antibody of new connection peptides
The antibody (having or do not have connection peptides) that detects various forms of structural domains disappearances in the competition binding assay is in conjunction with mucinous ability under the ox jaw of TAG-72 antigen source, described detection by time-the resolution fluorescence immunoassay utilizes Wallac 1420 Multilabel Counter Victor V (PerkinElmer) to carry out.The competition binding curve is shown in Figure 20.Assessment HuCC49 PAP (containing the connection peptides that is shown in SEQ ID NO:14), HuCC49 V2 PAP (containing the connection peptides that is shown in SEQ ID NO:14), HuCC49 G1/G3:PAP (containing the connection peptides that is shown in SEQ ID NO:9), HuCC49 V2 G1/G3/PAP (containing the connection peptides that is shown in SEQ ID NO:9) and contrast parent HuCC49 antibody.The antibody of all three kinds of hinge transformations in conjunction with active can not distinguish or the 2-3 of contrast parent CC49 antibody doubly within.
90Y-2-(p-isothiocyanato benzyl) (p-SCN-Bz)-cyclohexyl diethylene triaminepentaacetic acid(DTPA) part (CHx-DTPA) link coupled HuCC49 V2 PAP (containing the connection peptides that is shown in SEQ ID NO:14) and contrast parent HuCC49 antibody by wash-out and in the athymic mouse of suffering from LS-174T people's tumor xenogeneic graft its bio distribution relatively. 90The dosage (%ID) of the per-cent injection of every gram tumour of the radiolabeled antibody of Y or healthy tissues measured and was shown in table 7 at 3 and 24 hours.
Table 7.
7 mouse/groups
Blood Spleen Kidney Liver Tumour
HuCC49
3hrs 20.1±3.5 6.1±1.6 11.7±1.7 10.1±1.8 9.3±2.0
24hrs 0.7±0.2 9.5±4.0 11.0±2.0 12.0±1.5 12.7±7.1
HuCC49 V2 PAP
3hrs 24.6±3.0 4.6±2.2 10.0±1.4 8.4±1.0 16.1±5.0
24hrs 2.0±0.6 7.7±1.8 6.7±0.4 ** 11.2±2.2 21.3±4.8 *
Data representation mean value+/-standard deviation.
* compare with 24hr time point HuCC49 in the tumour, p＜0.05 is t check in pairs not
* compares with 24hr time point HuCC49 in the kidney, and p＜0.001 is t check in pairs not
Surprisingly, be absorbed in the tumour apparently higher than contrast HuCC49 antibody (p＜0.05 is t check in pairs), otherwise and be lower than contrast HuCC49 antibody at kidney middle kidney (p＜0.01) at 24 hours time point HuCC49 V2 PAP.When the tumour organ of these antibody relatively than the time, for except all organs of blood, HuCC49 V2 PAP causes the tumors of higher organ than (Figure 21).
These new hinges of these results suggest are given antibody structure and are changed the absorption that it has positive effect (positive effect) and reduce normal organ such as kidney tumor-localizing.Thus, when mixing therapeutic antibodies, these new hinges are particularly useful.
Embodiment 10. comprises the enhanced bio distribution collection of illustrative plates of the antibody of new connection peptides: detailed time time-histories
This embodiment confirmation has also been extended the result shown in the embodiment 9.Utilize the AmiconCentricon 30 of pre-rinse, antibody human CC49 V2 G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQID NO:9) and HuCC49 (gly/ser) diafiltration and be concentrated into low metal 5mM sodium acetate buffer, pH 5 (LMB).Centricon's is centrifugal in the fixed angle rotator, carries out in 5000xg, 2-8 ℃.Every kind of antibody reclaims to sample pool by adding 50 μ l LMB, and simple vortex is then at 1000x g circle round (back-spinning) 10 minutes.Protein concentration utilizes UV Spec to analyze at 280nm and utilizes optical extinction coefficient 1.48 mensuration.Utilize LMB that every kind of antibody is regulated down to 10.5mg/ml subsequently.
Described antibody utilizes 1.0M boric acid (pH 8.6, that Chelex handles and 0.2 μ m is filtering) to be adjusted to～pH 8.6.CHx-DTPA (being dissolved in 1.0M boric acid) is subsequently with 3 moles of inner complexs: the mol ratio of 1 mole of antibody adds.The amount of the boric acid that adds is 1/10th of an antibody volume.This mixture is subsequently by vortex and room temperature insulation 16-18 hour.Go into to form metal 5mM sodium acetate by mixture being added Centricon 30 and diafiltration new, pre-rinse, 150mM sodium-chlor, pH 5 stops as each saturating filter in advance.The concentration of every kind of antibody transfers to 3mg/mL.
Female nude mouse the inboard s.c. inoculation of right lateral thigh be suspended in HBSS (Biowhittaker, in Cat#10-547F) the 174T cell.The tumour size was measured in the day before yesterday on-test.Gross tumor volume calculates by half [L * ((W2)/2)] that length multiply by width square.Mice group makes mean tumour volume be～200mm 3
In 0 time, 42 nude mouses are injected 111The antibody of the CH2 structural domain disappearance of In-mark.The distribution of antibody 7 time point processes followed the trail of in described research, and each time point comprises 6 mouse.By on (tared weight) paper that mouse is placed on the weight taring and push bladder and come to collect urine from every mouse.Blood obtains via " eye bloodletting " (approximately every mouse of 200ul).For every mouse independently, collect any ight soil in blood sample collection and the urine sample process.Behind the blood collecting, put to death mouse by disconnected neck.Once put to death in six mouse each, and collect other sample via dissecting.Every kind of sample (removing outer skin) blots on paper handkerchief, and weighs with the washing of 3% formalin.All samples utilizes the paper of weight taring to weigh.
After the sample collecting, sample is placed borosilicate (borosilicate) check pipe and count on the gamma calculating instrument, the decay contrast of utilization is the antibody of the mark of dilution in 1: 10.The per-cent radioactivity relevant with each organ or tissue calculates with respect to decay contrast (dosage of % injection/g tissue or organ), and these values are presented among Figure 22 (CC49 V2 G1/G3/Pro243Ala244Pro245+[Gly/Ser]) and 23 (HuCC49[gly/ser]).Embodiment shows that the gathering of antibody molecule in kidney that comprises this new connection peptides reduces, and the gathering in blood has minimizing slightly, and the gathering in tumour obviously reduces.This collection of illustrative plates comprises these stable in vivo increases and effectiveness and security enhanced molecule.
Embodiment 11. comprises the susceptibility reduction of the antibody of connection peptides to reductive agent
Embodiment confirms the CC49 G1/G3/Pro243Ala244Pro245+[Gly/Ser of structural domain disappearance] (SEQ ID NO:9) more stable to Triptide (GSH) reduction, as parent CC49, is higher than the CC49 of the structural domain disappearance with Gly-Ser knuckle joint.
In brief, 50ug ddCC49 (Gly-Ser), ddCC49G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) or parent CC49 be 0,1,5 or 10mM GSH in, room temperature insulation 1 hour.Used reaction buffer comprises 100mM PBS, pH7.2 or 100mM sodium acetate, and 100mM NaCl, pH 4.5.The antibody that GSH-handles heats with SDS and is splined on 4-20% gradient SDS-PAGE, and is non--the reduction gel.Allow the sample of application of sample to pass through gel with 120 Volts, migration in 90 fens in room temperature.The albumen Coomassie blue stain, desiccant gel.Gel is shown in Figure 24.
As shown in figure 24, the CC49 G1/G3/Pro243Ala244Pro245+[Gly/Ser of structural domain disappearance] (SEQ ID NO:9) more stable to Triptide (GSH) reduction, as parent CC49, is higher than the CC49 of the structural domain disappearance with Gly-Ser knuckle joint.In addition, compare with 100mM PBS pH 7.2,100mM sodium acetate pH 4.5 is the CC49G1/G3/Pro243Ala244Pro245+[Gly/Ser of protection structural domains disappearance further] (SEQ ID NO:9) do not reduced by GSH.The susceptibility of reductive agent is reduced this unexpected observation prompting G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) hinge design makes and can adopt the chemical process of utilizing reductive agent, be connected in the technology of antibody such as those (for example SPDP joints) of being used to prepare drug conjugates or with emitting isotope (for example 99MTc), keep the physical integrity of antibody simultaneously.As if this advantage of reductive agent susceptibility do not changed the pharmacokinetics advantage (seeing the mouse bio distribution data among the embodiment 10) of the construct of CH2-structural domain disappearance.Reductive agent susceptibility is reduced also and can increase prediction from the body internal stability.
Embodiment 12. comprises the anti-CD 20 antibodies of connection peptides
Hinge area connection peptides G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) as being imported into the C2B8 antibody of CH2 structural domain-disappearance as described in the embodiment 3.C2B8 is chimeric anti-CD 20 monoclonal antibody, is made up of the mouse weight and the light chain variable structural domain that are blended in people's heavy chain and constant region of light chain respectively.Correct modification to hinge area is determined by DNA analysis.Plasmid DNA is used to transform the CHODG44 cell with instantaneous preparation antibody protein.
Supernatant is collected from producing and is contained G1/G3/Pro243Ala244Pro245+[Gly/Ser] cell of the C2B8 antibody of the CH2 structural domain-disappearance of connection peptides, the antibody concentration in the culture supernatant is measured by immunoassay.To comparing from the total antibody protein of about 3ng of instantaneous (transient) cell cultures and the huCC49 Mab of CH2 structural domain-disappearance, describedly relatively undertaken by the Western trace then, wherein utilize Anti-Human IgG HRP link coupled antibody to detect the huCC49 A type and the Type B isotype of CH2 structural domain disappearance by non--reduction SDS-PAGE electrophoresis.Under these conditions, the A type moves as single 120kDa homodimer, and Type B moves as 60kDa binary (doublet).Be shown in the ratio of mixing the A type that is found basic increase generation of the connection peptides of SEQ ID NO:9.Example results is shown in Figure 27.This result shows G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge (SEQ ID NO:9) causes producing the C2B8 antibody of all A type CH2 structural domain-disappearances basically and Type B seldom maybe can not detect, and has confirmed that the availability that this hinge can prepare A type isotype can generally be used for each species specific antibody.
Embodiment 13. comprises the anti--CD23 antibody of connection peptides
Hinge area connection peptides G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) be used for substantially making up 5E8 (the 5E8 Δ CH2) antibody of CH2 structural domain-disappearance as described in embodiment 3.5E8 is inosculating antibody-CD23 monoclonal antibody, by being blended in the heavy and light chain constant domain of people respectively, the heavy and light chain variable structural domain composition of primates.Correct modification to hinge area is determined by DNA analysis.The nucleic acid of 5E8 light chain and heavy chain and aminoacid sequence are shown in Figure 28 and 29 respectively.Plasmid DNA is used to transform CHO DG44 cell with instantaneous preparation antibody protein.
Containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser that imports 5E8 Δ CH2 antibody sequence] clone (1A7) of (SEQ ID NO:9) connection peptides is used for Antibody Preparation.Utilize embodiment 4 described methods, preparation and antibody purification.The 5E8 Δ CH2G1/G3/Pro243Ala244Pro245 antibody that only utilizes the Protein G column purification need not further HIC purifying as single peak purity 〉=97% wash-out basically.The protein sample of reductive and non--reductive purifying is by the SDS-PAGE electrophoretic analysis.Under the non-reduced condition, expection A type moves as single 120kDa heterodimer, and Type B moves as the 60kDa binary.Be shown in the ratio of mixing the A type that is found basic increase generation of the connection peptides of SEQ ID NO:9.Example results is shown in Figure 30.This result shows G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge (SEQ ID NO:9) causes producing basically all A type 5E8 Δ CH2 antibody and Type B seldom maybe can not detect, ÷ (seeing swimming lane 2) has confirmed that the availability that this hinge can prepare A type isotype can be used for each species specific antibody usually.5E8 Δ CH2G1/G3/Pro243Ala244Pro245 antibody shows not obviously gathering or the decomposition of antibody product by the size exclusion chromatography check and as single peak wash-out.The solvable CD23 of 5E8 Δ CH2G1/G3/Pro243Ala244Pro245 antibody further detection Cy5-mark in FRET (FRET (fluorescence resonance energy transfer) (fluorescenceresonance energy transfer)) competition binding assay combines with the 5E8 IgG's of Eu-mark, described detection utilize Delphia fluorimeter (Wallac1420 Multilabel Counter Victor V, PerkinElmer).The competition binding curve is shown in Figure 31.5E8 Δ CH2 G1/G3/Pro243Ala244Pro245 (containing the connection peptides that is shown in SEQ ID NO:9) and contrast parent 5E8 IgG antibody are by wash-out.The relative active undistinguishable of relative combination of the antibody that hinge is transformed in conjunction with activity and contrast parent 5E8 IgG antibody.From these results obviously as seen, will cause the preferred synthetic of A isotype in hinge area (containing the connection peptides that is shown in SEQ ID NO:9) the importing 5E8 Δ CH2 antibody, maintenance simultaneously combination fully is active, supports the generally useful property of the hinge of transformation.
Embodiment 14. comprises connection peptides chB3F6 antibody
Hinge area connection peptides G1/G3/Pro243Ala244Pro245+[Gly/Ser] (SEQ ID NO:9) be used for substantially making up chimeric B3F6 (the chB3F6 Δ CH2) antibody of CH2 structural domain-disappearance as described in embodiment 3.ChB3F6 is inosculating antibody-CRIPTO monoclonal antibody, and people mouse heavy and the light chain constant domain weighs and the light chain variable structural domain is formed by being blended in respectively.The correct modification of hinge area is determined by DNA analysis.The nucleic acid of chB3F6 light chain and heavy chain and aminoacid sequence are shown in Figure 32 and 33 respectively.Plasmid DNA is used to transform CHO DG44 cell with instantaneous preparation antibody protein.
Containing the G1/G3/Pro243Ala244Pro245+[Gly/Ser that imports chB3F6 Δ CH2 antibody sequence] clone (3C7) of (SEQ ID NO:9) connection peptides is used for Antibody Preparation.Utilize embodiment 4 described methods, preparation and antibody purification.The 5E8 Δ CH2G1/G3/Pro243Ala244Pro245 antibody that only utilizes the Protein G column purification need not further HIC purifying as single peak purity 〉=97% wash-out basically.The protein sample of reductive and non--reductive purifying is by the SDS-PAGE electrophoretic analysis.Under the non-reduced condition, expection A type moves as single 120kDa heterodimer, and Type B moves as the 60kDa binary.Be shown in the ratio of mixing the A type that basic increase produces of the connection peptides of SEQ ID NO:9.Example results is shown in Figure 34.This result shows G1/G3/Pro243Ala244Pro245+[Gly/Ser] hinge (SEQ ID NO:9) causes producing basically all A type 5E8 Δ CH2 antibody and Type B seldom maybe can not detect, and has confirmed that the availability that this hinge can prepare A type isotype can generally be used for each species specific antibody.5E8 Δ CH2 G1/G3/Pro243Ala244Pro245 antibody is by size exclusion chromatography check and as the single peak wash-out of 93-98%, shows that the antibody product obviously do not assemble or decompose.ChB3F6 CH2 G1/G3/Pro243Ala244Pro245 antibody further detects in flow cytometry competition binding assay, and described detection utilization is incorporated into the B3F6 IgG of FITC-mark of GEO tumour cell as the antigenic source of CRIPTO.The competition binding curve is shown in Figure 35.ChB3F6 Δ CH2 G1/G3/Pro243Ala244Pro245 (containing the connection peptides that is shown in SEQ ID NO:9) and contrast parent 5E8 IgG antibody are by wash-out.The relative active undistinguishable of relative combination of the antibody that hinge is transformed in conjunction with activity and contrast parent chB3F6 IgG antibody.From these results obviously as seen, in chB3F6 Δ CH2 antibody, import hinge area (contain be shown in SEQ ID NO:9 connection peptides) and cause the preferred synthetic of A isotype, keep simultaneously fully, further support the generally useful property of the hinge transformed in conjunction with active.
Equivalent
Those skilled in the art's utilization is no more than routine test and will recognizes, maybe can determine many equivalents of specific embodiments of the present invention.Described equivalent intention is included in the following claim.
Sequence table
Sequence table
SEQ ID NO：1
GGGSSGGGSG
SEQ ID NO：2
EPKSCDKTHT
SEQ ID NO：3
CPPCP
SEQ ID NO：4
APELLGGP
SEQ ID NO：5
ELKTPLGDTTHT
SEQ ID NO：6
CPRCPEPKSCDTPPPCPRCP
SEQ ID NO：7
EPKSCDKTHTCPPCGGGSSGGGSG
SEQ ID NO：8
EPKSCDKTHTCPPCPEPKSCDTPPPCPRCPGGGSSGGGSG
SEQ ID NO：9
EPKSCDKTHTCPPCPEPKSCDTPPPCPRCPAPGGGSSGGGSG
SEQ ID NO：10
EPKSCDKTHTSPPCPGGGSSGGGSG
SEQ ID NO：11
EPKSCDKTHTSPPCPAPGGGSSGGGSG
SEQ ID NO：12
EPKSCDKTHTCPPSPGGGSSGGGSG
SEQ ID NO：13
EPKSCDKTHTCPPSPAPGGGSSGGGSG
SEQ ID NO：14
EPKSCDKTHTCPPCPAPGGGSSGGGSG
SEO ID NO：15
EPKSCDKTHTCPPCPGGGSSGGGSG
SEQ ID NO：16
CAGGTCCAGCTGGTGCAGTCCGGCGCTGAGGTGGTGAAACCTGGGGCTTCCGTGAAGATTTCCTGCAAG
GCAAGCGGCTACACCTTCACTGATCACGCAATCCACTGGGTGAAACAGAATCCTGGACAGCGCCTGGAG
TGGATTGGATATTTCTCTCCCGGAAACGATGATTTTAAGTACAATGAGAGGTTCAAGGGCAAGGCCACA
CTGACTGCAGACACATCTGCCAGCACTGCCTACGTGGAGCTCTCCAGCCTGAGATCCGAGGATACTGCA
GTGTACTTCTGCACAAGATCCCTGAATATGGCCTACTGGGGACAGGGAACCCTGGTCACCGTCTCCAGC
GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCT
GGAGGTGGCTCGAGTGGAGGCGGATCCGGAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCA
TCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC
ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGAC
TCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTC
TTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG
GGTAAATGA
SEQ ID NO：17
GACATCGTGATGAGCCAGTCTCCAGACTCCCTGGCCGTGTCCCTGGGCGAGAGGGTGACTCTGAATTGC
AAGTCCAGCCAGTCCCTGCTCTATAGCGGAAATCAGAAGAACTATCTCGCCTGGTATCAGCAGAAACCA
GGGCAGAGCCCTAAACTGCTGATTTACTGGGCATCCGCTAGGGAATCCGGCGTGCCTGATCGCTTCAGC
GGCAGCGGATCTGGGACAGACTTCACTCTGACAATCAGCAGCGTGCAGGCAGAAGACGTGGCAGTCTAT
TATTGTCAGCAGTATTATAGCTATCCCCTCACATTCGGCGCTGGCACCAAGCTGGAACTGAAACGTACG
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT
GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAA
TCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC
CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG
AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA
SEQ ID NO：18
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYNERFKGKAT
LTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD
IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP
GK
SEQ ID NO：19
DIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASARESGVPDRFS
GSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
SEQ ID NO：20
CAGGTCCAGCTGGTGCAGTCCGGCGCTGAGGTGGTGAAACCTGGGGCTTCCGTGAAGATTTCCTGCAAG
GCAAGCGGCTACACCTTCACTGATCACGCAATCCACTGGGTGAAACAGAATCCTGGACAGCGCCTGGAG
TGGATTGGATATTTCTCTCCCGGAAACGATGATTTTAAGTACAATGAGAGGTTCAAGGGCAAGGCCACA
CTGACTGCAGACACATCTGCCAGCACTGCCTACGTGGAGCTCTCCAGCCTGAGATCCGAGGATACTGCA
GTGTACTTCTGCACAAGATCCCTGAATATGGCCTACTGGGGACAGGGAACCCTGGTCACCGTCTCCAGC
GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGAGCCC
AAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGGAGGTGGCTCGAGTGGAGGCGGATCCGGA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTC
AGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG
CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAG
CTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTG
CACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO：21
CAGGTCCAGCTGGTGCAGTCCGGCGCTGAGGTGGTGAAACCTGGGGCTTCCGTGAAGATTTCCTGCAAG
GCAAGCGGCTACACCTTCACTGATCACGCAATCCACTGGGTGAAACAGAATCCTGGACAGCGCCTGGAG
TGGATTGGATATTTCTCTCCCGGAAACGATGATTTTAAGTACAATGAGAGGTTCAAGGGCAAGGCCACA
CTGACTGCAGACACATCTGCCAGCACTGCCTACGTGGAGCTCTCCAGCCTGAGATCCGAGGATACTGCA
GTGTACTTCTGCACAAGATCCCTGAATATGGCCTACTGGGGACAGGGAACCCTGGTCACCGTCTCCAGC
GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGAGCCC
AAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGCACCTGGAGGTGGCTCGAGTGGAGGCGGA
TCCGGAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC
AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAG
GCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO：22
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYNERFKGKAT
LTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPEPKSCDTPPPCPRCPGGGSSGGGSGGQPREPQVYTLPPSRDELTKNQV
SLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGK
SEQ ID NO：23
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYNERFKGKAT
LTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPEPKSCDTPPPCPRCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK
SEQ ID NO：24
CAGGTCCAGCTGGTGCAGTCCGGCGCTGAGGTGGTGAAACCTGGGGCTTCCGTGAAGATTTCCTGCAAG
GCAAGCGGCTACACCTTCACTGATCACGCAATCCACTGGGTGAAACAGAATCCTGGACAGCGCCTGGAG
TGGATTGGATATTTCTCTCCCGGAAACGATGATTTTAAGTACAATGAGAGGTTCAAGGGCAAGGCCACA
ATCACTGCAGACACATCTGCCAGCACTGCCTACGTGGAGCTCTCCAGCCTGAGATCCGAGGATACTGCA
GTGTACTTCTGCGCCAGATCCCTGAATATGGCCTACTGGGGACAGGGAACCCTGGTCACCGTCTCCAGC
GCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCG
GCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTG
ACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTG
ACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACC
AAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGAGCCC
AAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGCACCTGGAGGTGGCTCGAGTGGAGGCGGA
TCCGGAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC
CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAAT
GGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTAC
AGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAG
GCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO：25
GACATCGTGATGAGCCAGTCTCCAGACTCCCTGGCCGTGTCCCTGGGCGAGAGGGTGACTCTGAATTGC
AAGTCCAGCCAGTCCCTGCTCTATAGCGGAAATCAGAAGAACTATCTCGCCTGGTATCAGCAGAAACCA
GGGCAGCCCCCTAAACTGCTGATTTACTGGGCATCCGCTAGGGAATCCGGCGTGCCTGATCGCTTCAGC
GGCAGCGGATCTGGGACAGACTTCACTCTGACAATCAGCAGCGTGCAGGCAGAAGACGTGGCAGTCTAT
TATTGTCAGCAGTATTATAGCTATCCCCTCACATTCGGCGCTGGCACCAAGCTGGAACTGAAACGTACG
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTT
GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAA
TCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACC
CTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTG
AGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA
SEQ ID NO：26
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYNERFKGKAT
ITADTSASTAYVELSSLRSEDTAVYFCARSLNMAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTA
ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT
KVDKKVEPKSCDKTHTCPPCPEPKSCDTPPPCPRCPAPGGGSSGGGSGGQPREPQVYTLPPSRDELIKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGK
SEQ ID NO：27
DIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQPPKLLIYWASARESGVPDRFS
GSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASV
VCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGL
SSPVTKSFNRGEC
SEQ ID NO：28
DIVMSQSPSSLPVSVGEKVTLSCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASARESGVPDRFT
GSGSGTDFTLSISSVKTEDLAVYYCQQYYSYPLTFGAGTKLVLK
SEQ ID NO：29
DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNSKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFS
GSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPYSFGQGTKLEIK
SEQ ID NO：30
DIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQSPKLLIYWASARESGVPDRFS
GSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLTFGAGTKLELK
SEQ ID NO：31
DIVMSQSPDSLAVSLGERVTLNCKSSQSLLYSGNQKNYLAWYQQKPGQPPKLLIYWASARESGVPDRFS
GSGSGTDFTLTISSVQAEDVAVYYCQQYYSYPLTFGAGTKLELK
SEQ ID NO：32
QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQNPEQGLEWIGYFSPGNDDFKYNERFKG
KATLTADKSSSTAYVQLNSLTSEDSAVYFCTRSLNMAYWGQGTSVTVSS
SEQ ID NO：33
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYAMHWVRQAPGQRLEWMG
WINAGNGNTKYSQKFQGRVTITRDTSASTAYMELSSLRSEDTAVYYCAR
GGYYGSGSNYWGQGTLVTVSS
SEQ ID NO：34
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYNERFKGKAT
LTADTSASTAYVELSSLRSEDTAVYFCTRSLNMAYWGQGTLVTVSS
SEQ ID NO：35
QVQLVQSGAEVVKPGASVKISCKASGYTFTDHAIHWVKQNPGQRLEWIGYFSPGNDDFKYNERFKGKAT
ITADTSASTAYVELSSLRSEDTAVYFCARSLNMA YWGQGTLVTVSS
SEQ ID NO：36
IGKTISKKAK
SEQ ID NO：37
CPEPKSCDTPPPCPR
SEQ ID NO：38
GAGGTGCAGCTGGTGGAGTCTGGGGGCGGCTTGGCAAAGCCTGGGGGGTCCCTGAGACTCTCCTGCGCAGCCTCCGGGTTCAGGTT
CACCTTCAATAACTACTACATGGACTGGGTCCGCCAGGCTCCAGGGCAGGGGCTGGAGTGGGTCTCACGTATTAGTAGTAGTGGTG
ATCCCACATGGTACGCAGACTCCGTGAAGGGCAGATTCACCATCTCCAGAGAGAACGCCAAGAACACACTGTTTCTTCAAATGAAC
AGCCTGAGAGCTGAGGACACGGCTGTCTATTACTGTGCGAGCTTGACTACAGGGTCTGACTCCTGGGGCCAGGGAGTCCTGGTCAC
CGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGG
GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCG
GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTG
CAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGT
GCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGCACCTGGAGGTGGCTCGAGTGGAGGCGGTTCCGGA
GGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGT
CAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGC
TGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCC
GTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
SEQ ID NO：39
GACATCCAGATGACCCAGTCTCCATCTTCCCTGTCTGCATCTGTAGGGGACAGAGTCACCATCACTTGCAGGGCAAGTCAGGACAT
TAGGTATTATTTAAATTGGTATCAGCAGAAACCAGGAAAAGCTCCTAAGCTCCTGATCTATGTTGCATCCAGTTTGCAAAGTGGGG
TCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGAGTTCACTCTCACCGTCAGCAGCCTGCAGCCTGAAGATTTTGCGACTTAT
TACTGTCTACAGGTTTATAGTACCCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGTACGGTGGCTGCACCATCTGT
CTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG
CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACC
TACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCT
GAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT
SEQ ID NO：40
EVQLVESGGGLAKPGGSLRLSCAASGFRFTFNNYYMDWVRQAPGQGLEWVSRISSSGDPTWYADSVKGRFTISRENAKNTLFLQMN
SLRAEDTAVYYCASLTTGSDSWGQGVLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPEPKSCDTPPPCPRCPAPGGGSSGGGSG
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VMHEALHNHYTQKSLSLSPGK
SEQ ID NO：41
DIQMTQSPSSLSASVGDRVTITCRASQDIRYYLNWYQQKPGKAPKLLIYVASSLQSGVPSRFSGSGSGTEFTLTVSSLQPEDFATY
YCLQVYSTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDST
YSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO：42
CAGGTCCAACTGCAGCAGGTTGGGGCTGAACTGGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTT
CACCAGCTACTGGATACACTGGGTGAAGCAGAGGCCTGGACAGGGCCTTGAGTGGATTGGAGAGAATGATCCTAGCAACGGTCGTA
CTAACTACAATGAGAAGTTCAAGAACAAGGCCACACTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCATCTCAGCAGCCTG
ACATCTGAGGACTCTGCGGTCTATTACTGTTCAAGGGGCCCTAATTACTTCTATTCTATGGACTACTGGGGTCAAGGAACCTCAGT
CACCGTCTCCTCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCC
TGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC
CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACAT
CTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCAC
CGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGCACCTGGAGGTGGCTCGAGTGGAGGCGGTTCC
GGAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCT
GGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCG
TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGC
TCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGT
SEQ ID NO：43
GATTTTTTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATeAAGTCAGAGCAT
TGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTCATCTACAAAGTTTCCA
ACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAG
GATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAGCGTACGGT
GGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT
TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGAC
AGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGT
CACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT
SEQ ID NO：44
QVQLQQVGAELVKPGASVKLSCKASGYTFTSYWIHWVKQRPGQGLEWIGENDPSNGRTNYNEKFKNKATLTVDKSSSTAYMHLSSL
TSEDSAVYYCSRGPNYFYSMDYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTF
PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPEPKSCDTPPPCPRCPAPGGGSSGGGS
GGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SVMHEALHNHYTQKSLSLSPG
SEQ ID NO：45
DFLMTQTPLSLPVSLGDQASISCRSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAE
DLGVYYCFQGSHVPLTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD
SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
SEQ ID NO：46
CAGGTACAACTGCAGCAGCCTGGGGCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACATT
TACCAGTTACAATATGCACTGGGTAAAACAGACACCTGGTCGGGGCCTGGAATGGATTGGAGCTATTTATCCCGGAAATGGTGATA
CTTCCTACAATCAGAAGTTCAAAGGCAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTG
ACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATCGACTTACTACGGCGGTGACTGGTACTTCAATGTCTGGGGCGCAGGGAC
CACGGTCACCGTCTCTGCAGCTAGCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAG
CGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCAC
ACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGAC
CTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACAT
GCCCACCGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGCACCTGGAGGTGGCTCGAGTGGAGGC
GGTTCCGGAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGAC
CTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGC
CTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTC
TCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
SEQ ID NO：47
QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSL
TSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVH
TFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPEPKSCDTPPPCPRCPAPGGGSSGG
GSGGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSLSPGK*
SEQ ID NO：48
CAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGT
AAGTTACATCCACTGGTTCCAGCAGAAGCCAGGATCGTCCCCCAAACCCTGGATTTATGCCACATCCAACCTGGCTTCTGGAGTCC
CTGTTCGCTTCAGTGGCAGTGGGTCTGGGACTTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTAC
TGCCAGCAGTGGACTAGTAACCCACCCACGTTCGGAGGGGGGACCAAGCTGGAAATCAAACGTACGGTGGCTGCACCATCTGTCTT
CATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCA
AAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC
AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAG
CTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTGA
SEQ ID NO：49
QIVLSQSPAILSASPGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYY
CQQWTSNPPTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC*
SEQ ID NO：50
ESKYGPP
SEQ ID NO：51
CPSCP
SEQ ID NO：52
APEFLGGP
SEQ ID NO：53
ESKYGPPCPSCPEPKSCDTPPPCPRCPAP

Claims (58)

1. composition, it comprises the polypeptide dimer with at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain part and synthetic connection peptides, wherein surpass 50% described polypeptide dimer and comprise polypeptide chain via at least one interchain disulfide bond connection.
2. the composition of claim 1 wherein surpasses 90% polypeptide dimer and connects via at least one interchain disulfide bond.
3. the composition of claim 1, wherein said polypeptide chain comprises via connection peptides and VL, the CH3 structural domain of VH or CH1 gene fusion.
4. the composition of claim 1, wherein said polypeptide chain lack all or part CH2 structural domain.
5. the composition of claim 1, wherein said polypeptide dimer connects via two or more interchain disulfide bonds.
6. the composition of claim 1, wherein said heavy chain partly is derived from and is selected from IgG1, IgG2, the antibody isotype of the group that IgG3 and IgG4 form.
7. the composition of claim 6, wherein said heavy chain partly comprise to be derived from and are selected from γ 1 hinge, γ 2 hinges, the aminoacid sequence of the hinge area of the group that γ 3 hinges or γ 4 hinges are formed.
8. the composition of claim 7, wherein said heavy chain partly comprises chimeric hinge.
9. the composition of claim 1, wherein said synthetic connection peptides comprises to small part IgG1 hinge arrangement territory, to small part IgG3 hinge arrangement territory.
10. the composition of claim 1, wherein said binding site independently is selected from: antigen binding site, the ligand binding moiety of acceptor, and the group formed of the receptor binding moiety of part.
11. the composition of claim 1, wherein said polypeptide dimer comprise four polypeptide chains, wherein two polypeptide chains comprise at least one heavy chain part and synthetic connection peptides.
12. composition, it comprises the polypeptide dimer with at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain part and synthetic connection peptides, wherein surpass about 50% polypeptide dimer and comprise the polypeptide chain that connects via at least one interchain disulfide bond, wherein said connection peptides comprises the proline residue that is positioned at Kabat numbering system position 243.
13. the composition of claim 12 wherein surpasses 90% polypeptide dimer and connects via at least one interchain disulfide bond.
14. the composition of claim 12, wherein said synthetic connection peptides also comprise the position 239 that is positioned at the Kabat numbering system or 242 halfcystine.
15. the composition of claim 12, wherein at least one polypeptide chain comprises via connection peptides and is connected in VL, the CH3 structural domain of VH or CH1 structural domain.
16. the composition of claim 12, wherein said synthetic connection peptides also comprise the alanine residue of the position 244 that is positioned at the Kabat numbering system and the proline residue of position 245.
17. the experimenter's that treatment can benefit from the therapy of utilizing binding molecule method comprises that the composition with claim 1 or 11 gives described experimenter, so that described treatment takes place.
18. the method for claim 17, wherein said experimenter suffers from cancer.
19. the method for claim 17, wherein said experimenter suffers from lymphoma.
20. the method for claim 17, wherein said experimenter suffers from autoimmune disease or illness.
21. the method for claim 17, wherein said experimenter suffers from inflammatory diseases or illness.
22. the composition of claim 12, wherein said binding site independently is selected from: antigen binding site, the group that the ligand binding moiety of acceptor and the receptor binding moiety of part are formed.
23. nucleic acid molecule, it comprises the nucleotide sequence of the polypeptide chain of coding claim 1 or 12.
24. host cell, it comprises the nucleic acid molecule of claim 23.
25. the composition of claim 12, wherein said polypeptide dimer comprise four polypeptide chains, wherein two polypeptide chains comprise at least one heavy chain part and synthetic connection peptides.
26. connection peptides, it comprises the aminoacid sequence that is selected from SEQ ID NO:8-15 and 53 groups of forming.
27. connection peptides, it is made up of the aminoacid sequence that is selected from the group that SEQ ID NO:8-15 and 53 forms.
28. comprising, the nucleic acid molecule of coded polypeptide chain, wherein said nucleic acid molecule be selected from: SEQ IDNO:16, the nucleotide sequence of 20,21,38,42,46 and 47 groups of forming.
29. comprising, the nucleic acid molecule of coded polypeptide chain, wherein said nucleic acid molecule be selected from: the nucleotide sequence of SEQ ID NO:24 and 25 groups of forming.
30. the antibody molecule of structural domain disappearance, it comprises and is selected from: SEQ ID NO:18, the aminoacid sequence of 22,23,40 and 44 groups of forming.
31. the antibody molecule of structural domain disappearance, it comprises and is selected from: the aminoacid sequence of SEQ ID NO:26 and 27 groups of forming.
32. antibody molecule, it comprises aminoacid sequence SEQ ID NO:31.
33. antibody molecule, it comprises aminoacid sequence SEQ ID NO:35.
34. separate the method for first kind and the second peptide species dipolymer, the wherein said first peptide species dimer comprises at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise the heavy chain part, the wherein said first peptide species dimer comprises the polypeptide chain that connects via at least one disulfide linkage, wherein the second peptide species dimer comprises at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise the heavy chain part, wherein the second peptide species dimer comprises the polypeptide that does not connect via at least one disulfide linkage, said method comprising the steps of:
I) make and comprise first kind with the dimeric mixture of second peptide species and separate the medium contact of described polypeptide dimer based on hydrophobic reactant, described contact is carried out under the second peptide species dimer and the described medium bonded condition allowing; With
Ii) collect the first peptide species dimer
Separate first kind and the second peptide species dimer thus.
35. the method for claim 34, wherein said collection step comprise described medium is contacted with about solution neutral pH, the about 120mS/cm of electric conductivity.
36. the method for claim 34, wherein said electric conductivity are about 116mS/cm.
37. the method for claim 36 also is included in step I i) described medium contact with about solution neutral pH, the about 140mS/cm of electric conductivity, win kind and the second peptide species dimer are combined with described medium.
38. comprising via synthetic connection peptides, the method for claim 34, wherein said polypeptide chain be connected in VL, the CH3 structural domain of VH or CH1 structural domain.
39. the method for claim 34, wherein first kind and the dimeric heavy chain of second peptide species partly are derived from and are selected from: IgG1, IgG2, the antibody isotype of the group that IgG3 and IgG4 form.
40. the method for claim 34, wherein said heavy chain partly comprises the aminoacid sequence that is derived from hinge area, and described hinge area is selected from: γ 1 hinge, γ 2 hinges, the group that γ 3 hinges and γ 4 hinges are formed.
41. the method for claim 34, wherein said binding site independently is selected from: antigen binding site, the group that the ligand binding moiety of acceptor and the receptor binding moiety of part are formed.
42. the method for claim 41, wherein at least one binding site of polypeptide dimer comprises at least one CDR of antibody, and described antibody is selected from: 2B8, Lym1, Lym2, LL2, Her2, B1, MB1, BH3, B4, B72.3, the group that CC49 and 5E10 form.
43. the method for claim 41, wherein at least one binding site of polypeptide dimer comprises the receptor binding moiety of part.
44. the method for claim 41, wherein at least one binding site of polypeptide dimer comprises the ligand binding moiety of acceptor.
45. composition, it comprises the first peptide species dimer by the method preparation of claim 34.
46. the experimenter's that treatment can benefit from the treatment that utilizes binding molecule method, the composition that comprises claim 45 gives the experimenter, so that treatment takes place.
47. the method for claim 34, wherein said polypeptide dimer comprise four polypeptide chains, wherein two described polypeptide chains comprise at least one heavy chain part.
48. separate the first kind of correct folding antibody molecule and the method for second kind of correct antibody molecule that folds, wherein said first kind and second kind of antibody molecule comprise four polypeptide chains respectively, wherein at least two chains comprise at least one heavy chain part, article at least two, chain comprises at least one light chain part, said method comprising the steps of:
I) make comprise first kind with the mixture of second kind of antibody molecule with can contact based on the medium of hydrophobic reactant separation antibody molecule;
The solution of the about 120mS/cm of described medium and electric conductivity and about neutral pH is contacted, make described first kind of described medium of antibody molecule debond and described second kind of antibody molecule in conjunction with described matrix, separate described first kind and second kind of antibody molecule thus.
49. composition comprises first peptide species by the method preparation of claim 48.
50. increase the method for the dimeric amount of first peptide species of cell generation with respect to the dimeric amount of second peptide species, wherein said first kind and the second peptide species dimer comprise at least two binding sites and at least two polypeptide chains, described at least two polypeptide chains comprise the heavy chain part, wherein said first kind of dimer comprises the polypeptide chain that connects via at least one disulfide linkage, described second kind of dimer comprises the polypeptide chain that does not connect via at least one disulfide linkage, said method comprising the steps of: transform described polypeptide chain to comprise synthetic connection peptides, the dimeric amount of first peptide species that makes cell produce increases for the dimeric amount of second peptide species.
51. pass through the composition of the method preparation of claim 50.
52. the composition of claim 50, wherein said polypeptide dimer comprise four polypeptide chains, wherein two polypeptide chains comprise at least one heavy chain part and synthetic connection peptides.
53. comprise the polypeptide of synthetic connection peptides, it comprises aminoacid sequence SEQ ID NO:37, wherein said polypeptide is not naturally occurring IgG3 molecule.
54. the polypeptide of claim 53, wherein said polypeptide are the IgG4 molecules.
55. the polypeptide of claim 54, wherein said polypeptide is in conjunction with VLA-4.
56. comprise the method for the dimeric amount of the polypeptide chain that connects via at least one disulfide linkage in the IgG4 molecule colony that the increase cell produces, may further comprise the steps: make cell expressing comprise the IgG4 molecule of synthetic connection peptides, make the dimeric amount that comprises the polypeptide chain that connects via at least one disulfide linkage in the IgG4 molecule colony increase.
57. composition, it comprises the polypeptide dimer with at least two binding sites and at least two polypeptide chains, wherein said at least two polypeptide chains comprise at least one heavy chain part and lack all or part CH2 structural domain, wherein surpass 50% polypeptide dimer and comprise the polypeptide chain that connects via at least one interchain disulfide bond.
58. the composition of claim 57, wherein said polypeptide dimer comprise four polypeptide chains, wherein two described polypeptide chains comprise at least one heavy chain part.
CNB2004800245766A 2003-06-27 2004-06-28 Use of hydrophobic-interaction-chromatography or hinge-region modifications for the production of homogeneous antibody-solutions Expired - Lifetime CN100497390C (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
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CN103328514A (en) * 2010-11-09 2013-09-25 阿尔蒂单抗治疗公司 Protein complexes for antigen binding and methods of use
CN111349166A (en) * 2018-12-20 2020-06-30 东莞市朋志生物科技有限公司 Recombinant antibody of anti-human CA72-4 glycoprotein
CN113416258A (en) * 2019-10-24 2021-09-21 高新 Multi-specificity antibody and preparation method and application thereof

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ES2667863T3 (en) * 2007-03-29 2018-05-14 Genmab A/S Bispecific antibodies and their production methods
CN110563845A (en) * 2019-09-12 2019-12-13 滨州医学院 anti-IGSF 9 antibody, pharmaceutical composition and application thereof

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103328514A (en) * 2010-11-09 2013-09-25 阿尔蒂单抗治疗公司 Protein complexes for antigen binding and methods of use
CN103328514B (en) * 2010-11-09 2015-12-02 阿尔蒂单抗治疗公司 The albumen composition combined for antigen and using method thereof
US9518132B2 (en) 2010-11-09 2016-12-13 Altimab Therapeutics, Inc. Protein complexes for antigen binding and methods of use
CN111349166A (en) * 2018-12-20 2020-06-30 东莞市朋志生物科技有限公司 Recombinant antibody of anti-human CA72-4 glycoprotein
CN113416258A (en) * 2019-10-24 2021-09-21 高新 Multi-specificity antibody and preparation method and application thereof
CN113416258B (en) * 2019-10-24 2023-08-29 北京免疫方舟医药科技有限公司 Multispecific antibody and preparation method and application thereof

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