CN102884084A - Anti-her2 antibodies and compositions - Google Patents

Anti-her2 antibodies and compositions Download PDF

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CN102884084A
CN102884084A CN2011800223076A CN201180022307A CN102884084A CN 102884084 A CN102884084 A CN 102884084A CN 2011800223076 A CN2011800223076 A CN 2011800223076A CN 201180022307 A CN201180022307 A CN 201180022307A CN 102884084 A CN102884084 A CN 102884084A
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her2
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M·W·佩德森
A·詹森
P-J·梅杰
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Abstract

The present invention relates to novel therapeutic antibodies directed against HER2 (ErbB2), as well as recombinant polyclonal anti-HER2 antibody compositions, and use of the antibodies and antibody composition for treatment of cancers. The antibody composition comprises at least three recombinant antibodies that bind distinct epitopes of HER2. Two of the antibodies bind to HER2 on the surface of a cell such that they generate a cross-linked antibody-receptor lattice on the cell surface and thereby result in HER2 receptor internalization. The third antibody in the composition binds HER2 such that it blocks heterodimerization between HER2 and HER3.

Description

Anti-HER 2 and composition
Invention field
The present invention relates to be used for the treatment of novel HER2 receptor target recombinant antibodies and the composition (comprising 2 kinds or multiple this antibody-like) thereof of human cancer.
Background of invention
Urogastron (EGFR) family (being ErbB family) is a subtribe of Tyrosylprotein kinase (RTK) acceptor, and it comprises 4 member: EGFR/ErbB, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4.It is closely related that EGFR family member and strand are regulated glycoprotein, and the latter has the outer ligand binding domain of born of the same parents, single cross-film district and intracellular kinase (referring to summary Ferguson (2008) Annu Rev Biophys.37:353-373).Under normal physiological status, ErbB family regulates and is coordinating critical event (Citri et al. (2006) Nat Rev Mol Cell Biol.7:505-516) in Growth of Cells, propagation and the migration.Think that now EGFR, HER2 and HER3 bring into play keying action in the lasting growth of Normocellular vicious transformation and cancer cells.At present found in many epithelial cancers, to exist EGFR and HER2 crosses situation (Slamon et al. (1987) Science, the 235:177-182 of expression; Arteaga (2002) Oncologist 7 Suppl 4:31-39; Bodey et al. (1997) Anticancer Res.17:1319-1330; Rajkumar et al. (1996) J Pathol.179:381-385).And EGFR and HER2 cross express with several people's epithelium cancers in progression of disease, survival rate reduce, chemotherapy is replied poor and relevant (Slamon et al. (1987) supra of generation resistance; Baselga et al. (2002) Oncologist 7 Suppl 4:2-8).
(HER2 is also referred to as ErbB2 or Neu to the human epidermal growth factor receptor 2; UniProtKB/Swiss-Prot No.P04626) formed by 1233 amino acid, its structure and EGFR are similar, have an extracellular domain that comprises 4 subdomain I-IV: the other territory of membrane-spanning domain, film, born of the same parents' endoplasm Tyrosylprotein kinase and modulability C-terminal territory (Yamamoto et al. (1986) Nature 319:230-234).
HER2 is the member of not being combined with known ligand (Klapper et al. (1999) Proc Natl AcadSci USA 96:4995-5000) unique in the ErbB family.HER2 is activated by forming different aggressiveness mixture with other ErbB family member, so it is subjected to EGFR and HER3 part indirect regulation (referring to summary Yarden et al. (2001) NatRev Mol Cell Biol.2:127-137).HER2 is first-selected partner (Graus-Porta et al. (1997) the EMBO J 16:1647-1655 of other 3 kinds of ErbB acceptor generation allos dimerizations; Tzahar et al. (1996) Mol Cell Biol. 16:5276-5287), it is by reducing the speed of dissociating of ligand-receptor mixture, thereby strengthen the avidity of other ErbB acceptor and part, HER2 can strengthen and prolong signal transduction (Pedersen et al. (2009) Mol CancerRes.7:275-284) like this.Another ligand binding acceptor generation allos dimerization of HER2 and ErbB family can be induced cross phosphorylation, makes C-terminal amino acid phosphorylation.So conversely as the support (King et al. (1988) EMBOJ7:1647-1651) of signaling molecule.The highest active HER2 heterodimer is HER2-HER3 mixture (Pinkas-Kramarski et al. (1996) EMBOJ 15:2452-2467), and wherein HER2 is by active kinase and kinase deficiency type HER3 complementary (Guy et al. (1994) Proc Natl Acad Sci USA 91:8132-8136.).Opposite with EGFR, HER2 has internalization resistance (Hommelgaard et al. (2004) MolBiol Cell 15:1557-1567), and it can escape the lysosome degraded, thereby is retained in the serous coat.
The Main Function of HER2 in healthy tissues seemingly regulated the signal transduction of the ErbB acceptor of ligand binding.Similar with EGFR, HER2 mainly expresses (referring to summary Freudenberg et al. (2009) Exp Mol Pathol.87:1-11) in epithelial cell, and finds that at present it is bringing into play the non-carcinogenic effects (Troyer et al. (2001) J Mammary Gland Biol Neoplasia 6:7-21) such as growth regulation, differentiation, apoptosis and reconstruction in normal breast is grown.Similar with the EGFR situation, cell surface HER2 too much can cause epithelial cell to be converted into Various Tissues (Freudenberg et al. (2009) supra).Report at present, in a large amount of human tumors, exist the HER2 copy to increase and cross the situation of expression, the aggressive duct carcinoma that comprises 20%-30%, and it has been used as predictive factors (Slamon et al. (1987) supra of Clinical Outcome poor (overall survival reduction); Ravdin et al. (1995) Gene 159:19-27).Can detect the HER2 high expression level in the human mammary tissue that occur to transform Symptoms at Primary Stage but transform not yet fully, this points out HER2 play a significant role in the vicious transformation in early days (Freudenberg et al. (2009) supra).Also in other epithelial cancer, for example colorectal cancer, ovarian cancer, carcinoma of the pancreas, lung cancer and bladder transitional cell carcinoma are found HER2 high expression level (Freudenberg et al. (2009) supra) simultaneously.But HER2 activates the inducing cell proliferation out of control, and Cell protection is avoided apoptosis, and affects normal epithelium (Muthuswamy et al. (2001) Nat Cell Biol.3:785-792).And the HER2 that expresses of metastasis cancer cell may in the cancer cells migration, play a role (De Potter (1994) Hum Pathol.25:1264-1268).
EGFR and HER2 are the cancer targets through checking, and the monoclonal antibody of these acceptors of target and micromolecular inhibitor have been ratified the treatment for kinds cancer now.But the patient who at first these treatments is replied often can the feelings recurrences (Pao et al. (2005) PLoSMed2:e73) because the generation resistance is caused a disease.(commodity are called Trastuzumab to Herceptin
Figure BDA00002350442700021
But) target HER2, be used for the treatment of the mammary cancer that the HER2 acceptor is crossed expression.In January, 2010, the stomach metastatic carcinoma patient of the approval Trastuzumab combination chemotherapy HER2 of the European Union positive.The monoclonal antibody handkerchief trastuzumab of another kind of part target HER2 acceptor is carrying out multinomial clinical trial at present.Herceptin passes through in conjunction with HER2 and then blocks the HER2 function and onset, and the handkerchief trastuzumab is the HER dimerisation inhibitor, can suppress HER2 and HER3 and other EGFR acceptor generation dimerization.
Because the handkerchief trastuzumab is at present still at clinical experimental stage, its final Clinical efficacy is not still known.For Herceptin, although the more single medicine chemotherapy of Herceptin combined chemotherapy can obtain the clinical income that prolong lifetime, (total response rate of Trastuzumab combined chemotherapy is 45%, and single medicine chemotherapy is 29% but most of HER2 breast cancer patients with positives are not replied this medicine generation; The Trastuzumab prescription information, Genentech, in March, 2009).Slamon etc. have reported similar result, be published in N Engl J Med (2001), 344 (11): 783-92, it is pointed out simultaneously, compare with single medicine chemotherapy, the Trastuzumab combined chemotherapy can reduce by 1 term mortality ratio (22% and 33%, P=0.008), prolong median survival interval (25.1 and 20.3 months, P=0.046).Therefore, although the monoclonal antibody of target HER2 can improve result for the treatment of, for example cross in the treatment of metastatic breast cancer of expression at HER2, it is still very large that it improves the space.
Summary of the invention
The present invention relates to be used for the treatment of novel HER2 receptor target recombinant antibodies and the composition (comprising 2 kinds or multiple this antibody-like) thereof of human cancer, for example be used for mammary cancer, ovarian cancer, cancer of the stomach and other crosses the cancer of expressing HER2.Compare with the methods for the treatment of of current this type of cancer, the target monoclonal antibody that comprises HER2 or other acceptor of EGFR family, antibody of the present invention can be alone, or preferred 2 kinds or multiple this type of antibody compositions coupling, remarkable clinical efficacy is provided in the situation of optional and other therapies coupling (for example chemotherapy).
On the one hand, the present invention relates to novel anti-HER2 recombinant antibodies, based on the described antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 of this patent with and humanized antibody.In one embodiment, one aspect of the present invention relates to a kind of anti-HER2 recombinant antibody molecule, and it comprises the heavy chain CDR3 sequence of arbitrary antibody in the described antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 of this patent.
Another embodiment of this aspect of the present invention: a kind of anti-HER2 recombinant antibody molecule, it comprises antibody 4380/4381,4382, heavy chain CDR3 sequence and the light chain CDR3 sequence of arbitrary antibody in 4383,4384,4385,4386,4387,4517,4518 and 4519; A kind of anti-HER2 recombinant antibody molecule, it comprises the heavy chain CDR1 of arbitrary antibody in these antibody, CDR2 and CDR3 sequence and light chain CDR1, CDR2 and CDR3 sequence; A kind of anti-HER2 recombinant antibodies, it comprises weight chain variabl area sequence and the light chain variable region sequence of arbitrary antibody in these antibody, or comprise and have at least 80%, 85%, the weight chain variabl area sequence of 90% or 95% identical sequence and light chain variable region sequence, for example at least 96%, 97%, 98% or 99% sequence is identical.
Another aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the recombinant antibodies of the first and second anti-HER2 at least, both and the different epi-position combinations of HER2, and also wherein one or both can be selected from above-mentioned antibody group.
Another aspect of the present invention relates to a kind of anti-HER2 recombinant polyclonal antibody composition, its first, second, and third anti-HER2 recombinant antibodies that comprises at least different epi-position combinations from HER2 forms, wherein the first and second antibody can be combined with HER2 and be caused HER2 acceptor internalization, and the 3rd antibody is combined with HER2 and can be suppressed the HER3 phosphorylation that part is induced.
Another aspect of the present invention relates to a kind of immune conjugate, and it comprises a kind of anti-HER2 recombinant antibodies of the present invention, can with a kind of cancer therapy drug coupling.Related fields of the present invention relate to antibody compositions, and it comprises the first and second antibody of anti-HER2 recombinant antibodies among the present invention at least, and having a kind of Anti-HER 2 in the described composition at least is immune conjugate.
Another aspect of the present invention relates to nucleic acid molecule, and it has the nucleotide sequence of Anti-HER 2 in the code book invention, and comprises the expression vector of polynucleotide and the host cell of this expression vector of transfection.
Another aspect of the present invention relates to the method for producing antibody of the present invention and polyclonal antibody composition.
Another aspect of the present invention relates to the method, particularly human cancer for the treatment of the mankind or animal subjects disease, realizes by giving a kind of Anti-HER 2 of described experimenter or the present composition.Another related fields are particularly treated human cancer for the medicine of one or more Anti-HER 2s among use the present invention for the preparation of the treatment mankind or Animal diseases.
Another aspect of the present invention relates to a kind of method of expressing HER2 cell surface HER2 internalization of inducing, and the method comprises makes anti-HER2 recombinant antibody composition among a kind of anti-HER2 recombinant antibodies of cells contacting, immune conjugate or a kind of the present invention.
Other side of the present invention and some embodiment below description and example in easy to understand.
Picture is described
Fig. 1 shows that the representative epi-position of Anti-HER 2 of the present invention is in conjunction with the result.
Fig. 2 A, 2B, 2C and 2D show through contain 2,3, relative ADCC percentage ratio in N87 that 4 kind of Anti-HER 2 mixture of the present invention induced and the SKBR3 tumor cell line under two kinds of different antibodies concentration.
Fig. 3 A, 3B, 3C and 3D show through 2 or the N87 that induces of 3 kind of Anti-HER 2 mixture of the present invention and the ADCC observed value of SKBR3 tumour cell.
The CDC observed value of the N87 cell that Fig. 4 shows through 2,3,4 kind of Anti-HER 2 mixture of the present invention induced.
Fig. 5 shows through 2 or the CDC observed value of 3 kind of Anti-HER 2 mixture of the present invention N87 cell of inducing.
Fig. 6 shows the titration results that 4 kinds of mixtures of antibodies of the present invention suppress N87 gastric carcinoma cell lines metabolic activity.
Fig. 7 shows the titration results that 4 kinds of mixtures of antibodies of the present invention suppress HCC202 breast cancer cell line metabolic activity.
Fig. 8 shows the titration results that 4 kinds of mixtures of antibodies of the present invention suppress BT474 breast cancer cell line metabolic activity.
Fig. 9 shows the titration results that 4 kinds of mixtures of antibodies of the present invention suppress ZR-75-30 breast cancer cell line metabolic activity.
Figure 10 shows the interior curative effect of Anti-HER 2 mixture of the present invention in NCI-N87 Gastric Cancer Xenograft of Nude Mice model.
Figure 11 shows the interior curative effect of Anti-HER 2 mixture of the present invention in OE19 Gastric Cancer Xenograft of Nude Mice model.
Figure 12 show the NCI-N87 cell through described antibody or mixtures of antibodies spend the night process after again with cell after transferring albumen β to process 15 minutes in EGFR, pEGFR, HER2, pHER2, the protein immunoblotting analysis of HER3 and pHER3 expression level.
Figure 13 A shows in the human cancer the different but relevant effect of HER2 homodimer and HER2/HER3 heterodimer in the oncogene cell transduction.Figure 13 B shows the guess mechanism of two paths of 3 kinds of Anti-HER 2 mixture blocking-up of the present invention, and wherein two kinds of antibody can be induced HER2 internalization and degraded, and the 3rd antibody can be by HER2/HER3 heterodimer blocking-up compensatory signal transduction.
Figure 14 shows ZR-75-30, NCI-N87, BT474 and HCC202 cancerous cell line through above-mentioned antibody and mixtures of antibodies spend the night process after the protein immunoblot analysis of HER2 expression level.
Figure 15 show HCC202 and NCI-N87 cancerous cell line through above-mentioned antibody and mixtures of antibodies spend the night process after the protein immunoblot analysis of HER2 and HER3 expression level.
Figure 16 shows OE19, N87, ZR-75-30, the protein immunoblot analysis of BT474 and the HCC202 clone HER2 expression level after 48 hours through above-mentioned 10 μ g/ml monoclonal antibodies or mixtures of antibodies processing.
Figure 17-19 shows the titration results that the different antibodies mixture suppresses NCI-N87 gastric carcinoma cell lines (Figure 17), BT474 breast cancer cell line (Figure 18) and HCC202 breast cancer cell line (Figure 19) metabolic activity.
Detailed Description Of The Invention
Definition
" antibody " or " antibody molecule " refers to functional serum component, is often referred to Molecule Set (antibody or immunoglobulin (Ig)) or a kind of molecule (antibody molecule or immunoglobulin molecules).Antibody can with special antigenic determinant (antigen or epitope) in conjunction with or reaction, but induction of immunity effect mechanism conversely.It has been generally acknowledged that single antibody is narrow spectrum, antibody compositions can be mono-clonal (namely being comprised of identical antibody molecule) or polyclone (namely by with 2 kinds or multiple different antibodies of same antigen or the not even identical or different epi-position reaction of synantigen) antibody.Each antibody all have can with the unique texture of its corresponding antigens specific binding, all natural antibodies have identical basic structure, i.e. 2 identical light chains and 2 identical heavy chains.Antibody also is generically and collectively referred to as immunoglobulin (Ig) simultaneously.
" antibody " in this patent also comprises binding fragment chimeric and single-chain antibody and antibody, for example Fab, Fv fragment or scFv (scFv) fragment, and polymer form, for example dimer IgA molecule or pentavalent IgM.Antibody can be the source people source or inhuman, mouse or other rodent source antibody for example, or based on chimeric, the humanization of mouse antibodies or moulding antibody again.
Each heavy chain of common a kind of antibody comprises a variable region of heavy chain (VH) and a CH CH comprises 3 structural domains, i.e. CH1, CH2 and CH3 usually.Each light chain of common a kind of antibody comprises a variable region of light chain (VL) and a constant region of light chain constant region of light chain comprises single structure territory, i.e. CL usually.VH and VL district can be subdivided into hypervariable region (it is variable that " hypervariable region ", its sequence and/or structure define the ring camber).Also refer to simultaneously complementarity-determining region (CDR), it is dispersed in and is distributed in more conservative skeleton district (FR).Each VH and VL generally include 3 CDR and 4 FR, arrange in the following order from aminoterminal to carboxyl terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.Amino-acid residue adopts the standard number method to be numbered (Kabat et al. (1991) Sequences of Proteins of Immunological Interest usually in the variable region, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD, USA).
In the sequence table of appendix, light chain (LC) DNA and aminoacid sequence comprise variable region of light chain (VL) sequence and people κ constant region sequence.Described in example 1, people κ constant region finishes with C terminal amino acid-NRGEC (Asn Arg Gly Glu Cys) with amino acid-TVAAP-(Thr Val Ala Ala Pro) beginning.Therefore, " light chain variable region sequence " or " VL " can be understood the N end parts (being amino acid TVAAP) that begins sequence of light chain in the front sequence table for people κ constant region in this patent.
Whole antibody used in this patent is numbered, and for example " antibody 4517 " indicates in the example and the specific antibody of the definition in the appendix sequence table.For example, heavy chain comprises variable region of heavy chain SEQ ID NO:2 and IGHG1 CH SEQ IDNO:44 in the antibody 4517, sequence of light chain is SEQ ID NO:4, as mentioned above, sequence of light chain comprises variable region of light chain (the residue 1-110 among the SEQ ID NO:4) and people κ constant region sequence (the residue 111-216 among the SEQIDNO:4).
In this patent describe a kind of antibody for " deriving from " or " based on " during a kind of specific antibodies, it is a kind of to mean that " source " antibody comprises below (according to specific background): the heavy chain CDR3 sequence of described specific antibodies; The heavy chain CDR3 sequence of described specific antibodies and light chain CDR3 sequence; The heavy chain CDR1 of described specific antibodies, CDR2 and CDR3 sequence and light chain CDR1, CDR2 and CDR3 sequence; Or the variable region of heavy chain of described specific antibodies and light chain variable region sequence, or described weight chain variabl area sequence and/or light chain variable region sequence humanized antibody, or heavy chain and/or light chain variable region sequence at least 80%, 85%, 90% or 95% sequence is identical with corresponding heavy chain and/or light chain variable region sequence, for example at least 96%, 97%, 98% or 99% sequence is identical.Source described in this patent or common in conjunction with the HER2 epi-position identical with described specific antibodies based on a kind of antibody of a certain specific antibodies, and the active essentially identical antibody of preferred and described specific antibodies.
The interactional specificity of antibody and target antigen depends primarily on the amino-acid residue that is arranged in heavy chain and 6 CDR of light chain.Therefore the variation of the aminoacid sequence in the CDR is much more than sequence outside the CDR district between the antibody.Because the CDR sequence interacts relevant with most of antibody antigens, can express a certain natural recombinant antibodies that has the antibody characteristic of simulation, perhaps the specific antibodies of given aminoacid sequence by construction of expression vector, is transplanted to specific antibodies CDR sequence the skeleton region sequence of another antibody.Like this, can with a kind of non-human antibody " humanization ", still keep in a large number binding specificity and the affinities of original antibody.The below will discuss in more detail to humanization.
" chimeric antibody " broadly refers to comprise the one or more zones from a kind of antibody, and from the antibody in one or more zones of one or more other antibody.In this patent, " chimeric antibody " be the antibody in groups of people source and the inhuman source of part normally, namely wherein a part from inhuman animal, for example mouse or other rodents, or bird, for example chicken.Chimeric antibody is better than non-human antibody, and this is in order to reduce the risk of replying of human antagonist, and namely mouse antibodies can make human body produce anti-mouse antibody.For example, the mouse source sequence of variable region sequences after from mouse immune in the typical chimeric antibody, and constant region behaviour source sequence.For chimeric antibody, the part in inhuman source namely is generally the skeleton district of variable region sequences, further change easily, thereby antagonist carries out humanization.
" humanization " refers to antibody and is all or part of inhuman source, the antibody that for example obtains behind the application target antigen immune mouse or based on the chimeric antibody of mouse source antibody, can replace some amino acid, particularly Human immune responses can be avoided or reduce to the amino acid of heavy chain and light chain skeleton district and constant region like this.We know now, can both exciting human the create antagonism reaction of antibody of all antibody, and this " humanization " degree with antibody is relevant.The immunogenicity although we can not calculate to a nicety, thus human body predicted to the opposing reaction of a certain antibody, and non-human antibody is stronger than human antibody immunogenicity.The external source of chimeric antibody (being generally the rodents source) constant region is substituted by people's source sequence, compares with the antibody of complete exogenous array, and its immunogenicity is usually lower, and therapeutic antibodies is more prone to use humanization or complete human antibody usually.Therefore, for chimeric antibody or other non-human antibody, preferably reply risk through humanization with the antagonism that reduces the human body antagonist.For chimeric antibody, revise in the skeleton district that humanization often can design variable region sequences.The partial amino-acid residue of complementarity-determining region (CDR) can not be changed in humanization usually, although need in some cases to change other cdr amino acid residue, for example remove glycosylation site, desamidization site or unwanted cysteine residues.
The at present existing humanized method of numerous antibody sequences; Referring to summary Almagro ﹠amp; Fransson (2008) Front Biosci.13:1619-1633.The most frequently used method is that CDR transplants, and for example, the chimeric antibody in a mouse source participates in differentiating the Human germ cells gene similar to mouse source variable region gene, mouse source CDR sequence is implanted in this skeleton.Can carry out CDR according to Kabat CDR definition and transplant, but the article of delivering recently (Magdelaine-Beuzelin etal. (2007) CritRev.Oncol Hematol.64:210-225) prompting IMGT definition (www.imgt.org) can improve the humanization result.Because CDR transplants binding specificity and the affinity that can reduce the non-human antibody of implanting CDR, thereby reduce biological activity, can introduce inverse transition in the selected location of transplanting CDR antibody, thereby keep binding specificity and the affinity of female antibody.Can use the available information in the document neutralizing antibody database that possible inverse transition is carried out the position evaluation.The candidate amino acid residue of inverse transition is usually located at the antibody molecule surface, and the residue of hiding or surperficial degrees of exposure is low can not be changed usually.Except CDR transplanting and inverse transition, another substituting humanization technology keeps the residue that non-surface, those inhuman sources exposes, and the surface residue change is people source residue for reinventing antibody.
As previously mentioned, the present invention comprises humanized antibody, i.e. humanized antibody." the humanization hypotype " that also can refer to antibody of the present invention.Especially, this patent middle finger " weight chain variabl area sequence " and " light chain variable region sequence " of acting as what specific amino acid sequence comprises any humanization hypotype of this particular sequence and this particular sequence simultaneously.
In this patent, compare with a certain specific heavy chain or light chain variable region sequence, weight chain variabl area sequence or light chain variable region sequence with unison minimum levels such as sequences, it is unison namely to have sequence of at least 80%, 85%, 90% or 95% etc., for example at least 96%, 97%, 98% or 99% sequences etc. are unison, include but not limited to the humanization hypotype of this type of reference sequences.
" recombinant antibodies " refers to a kind of antibody of the expression of cell lines of the carrier that cell or transfection expression contain this antibody coding sequence (or a plurality of expression vector, be generally 2 kinds of expression vectors), and wherein encoding sequence has nothing to do with this cell usually.
" carrier " is a kind of nucleic acid molecule, can to wherein inserting a nucleotide sequence, be used for the transportation of different genes environment and/or the expression in the host cell.The carrier that carries the nucleotide sequence transcriptional regulatory element is " expression vector "." plasmid " and " carrier " is used interchangeably.The expression vector that uses among the present invention can be the known suitable carrier of any prior art, for example plasmid or virus vector.
" polyclonal antibody " refer to can from different antigenic determinant same or not synantigen in conjunction with or 2 kinds or Multiple Antibodies molecular composition of reaction.In the present invention, the single antibody of polyclonal antibody can from the different antigenic determinant combinations of HER2.The different epi-position combinations from HER2 of single antibody of polyclonal antibody among preferred the present invention are more preferably from substantially nonoverlapping different epi-position combinations.It has been generally acknowledged that the otherness of polyclonal antibody is positioned at the variable region of antibody molecule." anti-HER2 recombinant polyclonal antibody composition " is a kind of 2 kinds or composition of multiple monoclonal antibody mixture of being combined with HER2 that comprise.
Prior art is established, and antibody exists different homotypes, human homotype IgG1 for example, IgG2, IgG3, IgG4, IgA1 and IgA2, or mouse homotype IgG1, IgG2a, IgG2b, IgG3 and IgA.Antibody of the present invention can be any homotype.Although the single antibody of polyclonal antibody composition of the present invention can comprise the antibody of multiple homotype, be preferably same homotype.
The recombinant antibody composition of a kind of comprising " the first and second recombinant antibodies of anti-at least HER2 " will comprise at least 2 kinds and specify antibody, but may comprise the Anti-HER 2 more than 2 kinds in this patent.In some cases, this class recombinant antibody composition may comprise considerable single Anti-HER 2, for example 10 or more, 15-20, is less than 10 different HER2 antibody but usually comprise, i.e. 2,3,4,5,6,7,8 or 9 antibody.More commonly, recombinant antibody composition of the present invention comprises and is no more than 6 different Anti-HER 2s, in most of the cases, is no more than 4 different Anti-HER 2s.In a preferred embodiment, recombinant antibody composition of the present invention will comprise 2,3 or 4 kind of different Anti-HER 2, be generally 2 or 3 kind of different Anti-HER 2.
" CDR " or " complementarity-determining region " refers to " hypermutation " district in the antibody variable region, the binding specificity of its major decision antibody.Define referring to CDR: Lefranc et al (2003), IMGT unique numbering for immunoglobulin and T cellreceptor variable domains and Ig superfamily V-like domains, Dev.Comp Immunol.27,55-77.Each heavy chain of antibody and light chain all comprise 3 CDR districts, i.e. CDR1, and CDR2 and CDR3, wherein CDR3 difference is maximum.
" epi-position " refers to have in the animal body antigenic activity or an immunogenic part than macromole (for example antigen and antigenic activity site).Having immunogenic epi-position is the more macromolecular part that can excite the animal's antibody reaction.Epi-position with antigenic activity is the more macromolecular part that the antibody determined through existing currently known methods can immune specific combination.Epitope need not to have immunogenicity.Antigen is the material that antibody or antibody fragment can immune specific combination, for example toxin, virus, bacterium, albumen or DNA.Antigen or antigen site be not only epi-position usually, unless it is very little, but and common immune response stimulating.Epi-position can be linear epitope or conformational epitope.Linear epitope generally is comprised of about 6-10 contiguous on protein molecular amino acid, and this protein molecular can be by antibody recognition.On the contrary, conformational epitope is comprised of the amino acid of non-series arrangement, but antibody can be identified a specific three-dimensional structure.But when a protein molecular was folded into three-dimensional structure, the amino acid that forms epi-position was arranged side by side, and antibody can be identified conformational epitope like this.In the protein of sex change, only linear epitope can be identified.According to definition, conformational epitope must be positioned at the outside of folded protein.
" different epi-position " refer to, when 2 kinds of different antibodies among the present invention and different epi-positions in conjunction with the time, antigen is lower than 100% in conjunction with competition, preferred antigens is lower than 80% in conjunction with competition, more preferably antigen is lower than 50% in conjunction with competition, and most preferably competes alaply, and for example antigen is lower than 25% in conjunction with competition.Mutually competition can be in conjunction with identical or overlapping epi-position in conjunction with the antibody of same antigen, or has binding site in mutual next-door neighbour district, and competition mainly is to be caused by sterically hindered like this.Can adopt the prior art currently known methods to carry out antibody analyzes " different epi-position ", for example by the combination experiment under the saturated antibody condition, can adopt FACS (fluorescence-activated cell sorting) or other flow cytometer and fluorescent-labeled antibody that the cell of expressing HER2 is analyzed, or by Applications of surface plasmon resonance (SPR), adopt the HER2 antigen capture or be bonded to the wandering cells surface.Describe in the example and adopt SPR to determine the method for competing between antibody.
Preferred " non-overlapped " epi-position of different epi-positions, the antigen of 2 kinds of different Anti-HER 2s in the present composition is enough low in conjunction with competition, 2 kinds of simultaneously corresponding epi-position combinations with it of antibody.Know those skilled in the art and understand and can have in various degree overlapping, although and can have the overlapping of some degree, different epi-positions can be considered " not overlapping " epi-position, as long as corresponding antibodies can be substantially in conjunction with its epi-position.When antigen is lower than about 50% in conjunction with competition between two kinds of antibody, usually can consider applicable this situation.
The antibody of different epi-position combinations from same antigen is different in conjunction with activity influence to antigen, and this depends on the position of epi-position.The antibody of being combined with antigenic activity site epi-position can be blocked antigen function fully, but the antibody of being combined with another epi-position may separately can be less to the antigen activity influence, or not impact.But but this antibody-like activating complement still, thereby cause antigen to be eliminated, from conjunction with one or more antibody couplings of the different epi-positions of same antigen the time, may cause synergy.In the present invention, the preferred HER2 ectodomain of epi-position.The preferred HER2 ectodomain of antigen of the present invention, polypeptide or with the fragment of antibody or antibody fragment immunity specific combination.The HER2 related antigen also can be HER2 ectodomain, polypeptide or with analogue or the derivative of the fragment of antibody or antibody fragment immunity specific combination.
" immunoglobulin (Ig) " is generally the general name of blood or Serum Antibody mixture, but also can be used for referring to other source mixtures of antibodies.
" homology V HAnd V LCoding to " refer to that same antibody-producting cell contains or the V of the original pairing of originating HAnd V LEncoding sequence.Therefore, homology V HAnd V LThe V of coding to referring to that derived cell donor itself exists HAnd V LRight." V HAnd V LCoding is to expressing antibody " refer to contain V by a carrier, plasmid or other HAnd V LA kind of antibody or antibody fragment that the polynucleotide of encoding sequence generate.Expressing homology V HAnd V LCoding to the time, no matter be complete antibody or stable fragment, it is all keeping binding affinity and specificity that derived cell itself is expressed antibody.Homology also refers to the set that homology is right to the storehouse, can preserve separately.
" albumen " or " polypeptide " refers to the amino acid chain of any length or rear translation modification.Albumen can be used as monomer or polymer and exists, and comprises polypeptide chain, protein fragments, polypeptide, oligopeptides and the peptide of 2 or a plurality of assemblings.
" head to head promotor " refers to a pair of closely adjacent promotor pair, transcribed in the opposite direction by 2 gene fragments of promoters driven.Head to head promotor is also referred to as bidirectional promoter.
" transfection " in this patent is the broad terms with the foreign DNA transfered cell.The method with the foreign DNA transfered cell that it also comprises other functional equivalent for example transforms, infects, transduction or donorcells nuclear receptor cytogamy.
Described in " background of invention ", " HER2 " (being also referred to as HER2/neu and ErbB-2) refers to " people shows growth factor acceptor 2 ".This patent middle finger comprises varient, hypotype and the homologous gene of HER2.Preferably, a kind of antibody among the present invention is combined cell (for example being generally tumour cell) growth that suppress to express HER2 with HER2, this inhibition realizes by the formation that suppresses heterodimer between HER2 and other ErbB family member, for example with EGFR or HER3 allos dimerization.
(for example " suppress growth " in this patent, Growth of Cells) comprises propagation (cell quantity increase) or metabolism any minimizing that measures occurs, when contacting with a kind of Anti-HER 2, same cell growth phase ratio when not having Anti-HER 2, for example suppress at least growth of cell culture about 10%, and the preferred higher person of inhibiting rate, for example at least about 20% or 30%, more preferably inhibiting rate is at least about 40% or 50%, for example be at least about 60%, 70%, 80%, 90%, 99% or even 100%.
" inhibition dimerization " or " suppress dimer form " refers to that the dimer formational situation is compared when not having Anti-HER 2 in this patent, because it is combined with a kind of Anti-HER 2, HER2 and for example EGFR, HER3 or HER4 form dimeric ability any measurable reduction occur.
" treatment " in this patent refers to capacity and gives a kind of Anti-HER 2 of the present invention or antibody compositions to alleviate, to slow down or to eliminate (healing) morbid state.Give among the present invention 2 kinds or more Anti-HER 2 usually by giving simultaneously antibody, preferably contain all Anti-HER 2 compositions and be used for the treatment of.But also can give respectively 2 kinds or more Anti-HER 2 among the present invention.Here give to be appreciated that by the recombinant antibody composition that at least 2 kinds of Anti-HER 2s form not only comprises the composition that these type of at least 2 kinds of antibody form, and also comprises to give respectively antibody.Therefore can be simultaneously, sequential or give respectively 2 kinds or the combination of multiple Anti-HER 2 of the present invention.
The same percentage of two sequences, variable region sequences for example refers to the quantity (being calculated as same position quantity/total number of positions * 100) of same position in the sequence, considers to introduce to be used for the best interval of aiming at of two sequences.Comparative sequences is to finish by ready-made software with the mutually unison percentage ratio that definite two sequences are seen.Suitable software program can come from various resources, can use online or download use, is used for comparison albumen and nucleotide sequence.A kind of suitable program is ClustalW (Thompson et al. (1994) Nucleic Acids Res 11; 22 (22): 4673-80), can download from www.clustal.org or biometrics institute of European Union (www.ebi.ac.uk), it provides all kinds of other albumen and Nucleotide statistical tools.
Special embodiment
An aspect of of the present present invention relates to the various new Anti-HER 2.In one embodiment, the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of arbitrary antibody in the described antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 of this patent.
In another embodiment, the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises antibody 4380/4381,4382, the heavy chain CDR3 sequence of arbitrary antibody in 4383,4384,4385,4386,4387,4517,4518 and 4519.
In another embodiment, the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises antibody 4380/4381,4382,4383,4384,4385,4386, heavy chain CDR1, the CDR2 of arbitrary antibody and CDR3 sequence and light chain CDR1, CDR2 and CDR3 sequence in 4387,4517,4518 and 4519.
In another embodiment, of the present inventionly relate to a kind of anti-HER2 recombinant antibodies, it comprises antibody 4380/4381,4382, weight chain variabl area sequence and the light chain variable region sequence of arbitrary antibody in 4383,4384,4385,4386,4387,4517,4518 and 4519; Or comprise the humanization sequence of described heavy chain and/or light chain variable region sequence, or comprise weight chain variabl area sequence and light chain variable region sequence, it has at least 80% with described heavy chain and light chain variable region sequence respectively, 85%, 90% or 95% identical sequence, for example the sequence with described sequence at least 96%, 97%, 98% or 99% is identical.
In another embodiment, the present invention relates to a kind of anti-HER2 recombinant antibodies, it can also compete with it combination with the identical epi-position combination of above-mentioned arbitrary antibody, and the antibody compositions that is formed by one or more these antibody-likes, preferably comprise at least 2 kinds of these antibody-likes, for example described 2 kinds or 3 kinds of these antibody-likes of other parts in this patent.
As the appendix sequence table, following table 1 shows antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and the serial ID of the variable region of heavy chain (VH) of 4519 antibody and variable region of light chain (LC) DNA and aminoacid sequence number.As above release, light chain DNA and aminoacid sequence comprise light chain variable region sequence (VL) and people κ constant region sequence in the sequence table.
Table 1: the DNA of selected Anti-HER 2 variable region of heavy chain and light chain and the serial ID of aminoacid sequence number
Figure BDA00002350442700121
A specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4517, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4517, the for example heavy chain of antibody 4517 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4517 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4517 respectively, 85%, 90% or 95% identical sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4518, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4518, the for example heavy chain of antibody 4518 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4518 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4518 respectively, 85%, 90% or 95% identical sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4380/4381, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4380/4381, the for example heavy chain of antibody 4380/4381 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4380 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380/4381 respectively, 85%, 90% or 95% identical sequence.In the present embodiment, preferred 4380.
Antibody " 4380/4381 " refers to a kind of antibody that VH and LC aminoacid sequence are respectively SEQ ID NO:10 and 12.Shown in SEQ IDNO:12,40 amino acids residues can be tyrosine or Threonine.Antibody 4380 and 4381 direct unique differences are in antibody 4380, and LC sequence 40 amino acids residues are tyrosine, and in antibody 4381,40 amino acids residues are Threonine.In the original mouse antibodies, this position is free halfcystine.In these two kinds of antibody, common preferred 4380.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4382, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4382, the for example heavy chain of antibody 4382 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4382 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4382 respectively, 85%, 90% or 95% identical sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4383, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4383, the for example heavy chain of antibody 4383 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4383 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4383 respectively, 85%, 90% or 95% identical sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4384, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4384, the for example heavy chain of antibody 4384 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4384 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4384 respectively, 85%, 90% or 95% identical sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4385, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4385, the for example heavy chain of antibody 4385 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4385 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4385 respectively, 85%, 90% or 95% identical sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4386, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4386, the for example heavy chain of antibody 4386 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4386 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4386 respectively, 85%, 90% or 95% identical sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4387, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4387, the for example heavy chain of antibody 4387 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4387 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4387 respectively, 85%, 90% or 95% identical sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises the heavy chain CDR3 sequence of antibody 4519, the heavy chain and the light chain CDR3 sequence that preferably comprise antibody 4519, the for example heavy chain of antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4519 weight chain variabl area sequences or its humanization sequence, and light chain variable region sequence or its humanization sequence, or comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4519 respectively, 85%, 90% or 95% identical sequence.
Another aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody of anti-HER2 at least, the different epi-position combinations of the first and second antibody and HER2, and wherein a kind of or both can be selected from above-mentioned antibody group.An embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, it comprises the first and second antibody of anti-HER2 at least, the different epi-position combinations of the first and second antibody and HER2, and also wherein the first and second antibody comprise and can be selected from antibody 4380/4381,4382,4383,4384,4385,4386, the heavy chain CDR3 sequence of a kind of antibody of 4387,4517,4518 and 4519.
An embodiment of this aspect of the present invention relates to a kind of recombinant antibodies component, it comprises the first and second antibody of anti-HER2 at least, the wherein different epi-position combinations of the first and second antibody and HER2, and also wherein the first and second antibody comprise and can be selected from antibody 4380/4381,4382,4383,4384,4385,4386, the heavy chain of a kind of antibody of 4387,4517,4518 and 4519 and light chain CDR3 sequence.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody of anti-HER2 at least, wherein the different epi-position combinations of the first and second antibody and HER2, and wherein the first and second antibody comprise and can be selected from antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517, the heavy chain of a kind of antibody of 4518 and 4519 and light chain CDR1, CDR2 and CDR3 sequence.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody of anti-HER2 at least, wherein the different epi-position combinations of the first and second antibody and HER2, and wherein the first and second antibody comprise and can be selected from antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517, the weight chain variabl area sequence of a kind of antibody of 4518 and 4519 or its humanization sequence, and light chain variable region sequence or its humanization sequence; Wherein the first and second antibody comprise a weight chain variabl area sequence and a light chain variable region sequence, its respectively with can be selected from antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517, the weight chain variabl area sequence of a kind of antibody of 4518 and 4519 and light chain variable region sequence have at least 80%, 85%, 90% or 95% identical sequence.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, it comprises the first and second antibody of anti-HER2 at least, the wherein different epi-position combinations of the first and second antibody and HER2, and also wherein the first and second antibody comprise and can be selected from antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 or its humanized antibody.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, it comprises the first and second antibody of anti-HER2 at least, the wherein different epi-position combinations of the first and second antibody and HER2, and also wherein the first and second antibody comprise and can be selected from and antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 antibody in conjunction with same epi-position and competition combination.
Another embodiment of the present invention is a kind of antibody compositions, comprises at least the first and second antibody of the anti-HER2 recombinant antibodies of different epi-position combinations from HER2, and wherein at least a described antibody is selected from following composition:
(a) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:56) and the light chain CDR3 sequence (SEQ ID NO:82) of antibody 4382;
(b) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:59) and the light chain CDR3 sequence (SEQ ID NO:84) of antibody 4383;
(c) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:62) and the light chain CDR3 sequence (SEQ ID NO:86) of antibody 4384;
(d) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:65) and the light chain CDR3 sequence (SEQ ID NO:88) of antibody 4385;
(e) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:68) and the light chain CDR3 sequence (SEQ ID NO:90) of antibody 4386;
(f) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:71) and the light chain CDR3 sequence (SEQ ID NO:92) of antibody 4387;
(g) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:47) and the light chain CDR3 sequence (SEQ ID NO:76) of antibody 4517;
(h) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:50) and the light chain CDR3 sequence (SEQ ID NO:78) of antibody 4518;
(i) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:74) and the light chain CDR3 sequence (SEQ IDNO:93) of antibody 4519; And
(j) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:53) and the light chain CDR3 sequence (SEQ ID NO:80) of antibody 4380.
The first and second antibody of preferred described Anti-HER 2 all can be selected from antibody (a)-(j).Antibody compositions also can comprise the 3rd antibody of anti-HER2 recombinant antibodies, is preferably a kind of antibody that is selected from above-mentioned antibody (a)-(j).In another embodiment, antibody compositions can comprise the first and second antibody of the anti-HER2 recombinant antibodies of different from HER2 at least epi-position combinations, and wherein said the first and second antibody all can also be competed with it combination with the identical epi-position combination of above-mentioned arbitrary antibody (a)-(j).
Another embodiment of the present invention is a kind of antibody compositions, comprises at least the first and second antibody of the anti-HER2 recombinant antibodies of different epi-position combinations from HER2, and wherein at least a described antibody is selected from following composition:
(A) a kind of antibody comprises CDR1, the CDR2 of antibody 4382 variable region of heavy chain (SEQ ID NO:14) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:16);
(B) a kind of antibody comprises CDR1, the CDR2 of antibody 4383 variable region of heavy chain (SEQ ID NO:18) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:20);
(C) a kind of antibody comprises CDR1, the CDR2 of antibody 4384 variable region of heavy chain (SEQ ID NO:22) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:24);
(D) a kind of antibody comprises CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:28) CDR1, CDR2 and the CDR3 of antibody 4385 variable region of heavy chain (SEQ ID NO:26);
(E) a kind of antibody comprises CDR1, the CDR2 of antibody 4386 variable region of heavy chain (SEQ ID NO:30) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:32);
(F) a kind of antibody comprises CDR1, the CDR2 of antibody 4387 variable region of heavy chain (SEQ ID NO:34) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:36);
(G) a kind of antibody comprises CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:4) CDR1, CDR2 and the CDR3 of antibody 4517 variable region of heavy chain (SEQ ID NO:2);
(H) a kind of antibody comprises CDR1, the CDR2 of antibody 4518 variable region of heavy chain (SEQ ID NO:6) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:8);
(I) a kind of antibody comprises CDR1, the CDR2 of antibody 4519 variable region of heavy chain (SEQ ID NO:38) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:40); And
(J) a kind of antibody comprises CDR1, the CDR2 of antibody 4380 variable region of heavy chain (SEQ ID NO:10) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:12).
In the present embodiment, the first and second antibody of preferred described Anti-HER 2 are selected from above-mentioned antibody (A)-(J).Antibody compositions also can comprise the 3rd antibody of anti-HER2 recombinant antibodies at least, is preferably a kind of antibody that is selected from above-mentioned antibody (A)-(J).In another embodiment, antibody compositions can comprise the first and second antibody of the anti-HER2 recombinant antibodies of different from HER2 at least epi-position combinations, and wherein each described first and second antibody all can also be competed with it combination with the identical epi-position combination of above-mentioned arbitrary antibody (A)-(J).
A special embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4380 and 4382 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4382;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4382;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4382, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4382; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4382 respectively.
Another embodiment of this aspect of the present invention relates to a kind of composition based on antibody 4380 and 4382, and it comprises a kind of other antibody at least, is selected from especially the antibody based on antibody 4385,4517 and 4518.Wherein this type of embodiment relates to a kind of recombinant antibody composition, comprises first, second, and third recombinant antibodies, and wherein first, second, and third antibody is:
Antibody 4380,4382 and 4385 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382; With a kind of antibody, comprise the heavy chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382; With a kind of antibody, comprise heavy chain and the light chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, CDR2 and CDR3 sequence; With a kind of antibody, comprise heavy chain and the light chain CDR1 of antibody 4385, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382; With a kind of antibody, comprise heavy chain and the light chain variable region sequence of antibody 4385; Or
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4380 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4382 respectively; With a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4385 respectively.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises first, second, and third antibody, and wherein first, second, and third antibody is:
Antibody 4380,4382 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382; With a kind of antibody, comprise the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382; With a kind of antibody, comprise heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, CDR2 and CDR3 sequence; With a kind of antibody, comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382; With a kind of antibody, comprise heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4380 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4382 respectively; With a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises first, second, and third antibody, wherein, the second and the 3rd antibody is:
Antibody 4380,4382 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382; With a kind of antibody, comprise the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382; With a kind of antibody, comprise heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, CDR2 and CDR3 sequence; With a kind of antibody, comprise heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382; With a kind of antibody, comprise heavy chain and the light chain variable region sequence of antibody 4518; Or
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4380 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4382 respectively; With a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises the first, second, third and the 4th antibody, and wherein the first, second, third and the 4th antibody is:
Antibody 4380,4382,4385 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4385; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4385; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4385, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence; Or
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4385; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4518;
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4380 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4382 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4385 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4380 and 4383 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4383;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4383;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4383, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4383; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4383 respectively.
Another embodiment of this aspect of the present invention relates to a kind of composition based on antibody 4380 and 4383, and it comprises a kind of other antibody at least.Wherein this type of embodiment relates to a kind of recombinant antibody composition, and it comprises first, second, and third antibody, and wherein first, second, and third antibody is:
Antibody 4380,4383 and 4384 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4383; With a kind of antibody, comprise the heavy chain CDR3 sequence of antibody 4384;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4383; With a kind of antibody, comprise heavy chain and the light chain CDR3 sequence of antibody 4384;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4383, CDR2 and CDR3 sequence; With a kind of antibody, comprise heavy chain and the light chain CDR1 of antibody 4384, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4383; With a kind of antibody, comprise heavy chain and the light chain variable region sequence of antibody 4384; Or
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4380 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4383 respectively; With a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4384 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4380 and 4384 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4384;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4384;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4384, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4384; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4384 respectively.
Another embodiment of this aspect of the present invention relates to a kind of composition based on antibody 4380 and 4384, and it comprises a kind of other antibody at least, has especially to be selected from based on antibody 4385,4517,4518 and 4519 antibody.Wherein this type of embodiment relates to a kind of recombinant antibody composition, comprises first, second, and third recombinant antibodies, and wherein first, second, and third antibody is:
Antibody 4380,4384 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; With a kind of antibody, comprise the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4384; With a kind of antibody, comprise heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4384, CDR2 and CDR3 sequence; With a kind of antibody, comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4384; With a kind of antibody, comprise heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4380 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4384 respectively; With a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises first, second, and third antibody, and wherein first, second, and third antibody is:
Antibody 4380,4384 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; With a kind of antibody, comprise the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4384; With a kind of antibody, comprise heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4384, CDR2 and CDR3 sequence; With a kind of antibody, comprise heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4384; With a kind of antibody, comprise heavy chain and the light chain variable region sequence of antibody 4518; Or
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4380 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4384 respectively; With a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises, the second and the 3rd antibody, and wherein first, second, and third antibody is:
Antibody 4380,4384 and 4519 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; With a kind of antibody, comprise the heavy chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4384; With a kind of antibody, comprise heavy chain and the light chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4384, CDR2 and CDR3 sequence; With a kind of antibody, comprise heavy chain and the light chain CDR1 of antibody 4519, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4384; With a kind of antibody, comprise heavy chain and the light chain variable region sequence of antibody 4519; Or
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4380 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4384 respectively; With a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4519 respectively.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises the first, second, third and the 4th antibody, and wherein the first, second, third and the 4th antibody is:
Antibody 4380,4384,4385 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4384; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4385; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4384; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4385; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4384, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4385, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4384; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4385; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4518; Or
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4380 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4384 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4385 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4380 and 4385 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4385, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4385; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4385 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4380 and 4386 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4386;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4386;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4386, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4386; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4386 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4380 and 4387 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4387;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4387;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4387, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4387; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4387 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4380 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4380 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4518; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4380 and 4519 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4380, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4519, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4380, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4519; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4380 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4519 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4382 and 4384 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4384;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4384;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4384, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4384; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4382 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4384 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4382 and 4385 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4385, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4385; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4382 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4385 respectively.
Another embodiment of this aspect of the present invention relates to a kind of composition based on antibody 4382 and 4385, and it comprises a kind of other antibody at least.Wherein this type of embodiment relates to a kind of recombinant antibody composition, and it comprises first, second, and third antibody, and wherein first, second, and third antibody is:
Antibody 4382,4385 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4385; With a kind of antibody, comprise the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4385; With a kind of antibody, comprise heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4385, CDR2 and CDR3 sequence; With a kind of antibody, comprise heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4385; With a kind of antibody, comprise heavy chain and the light chain variable region sequence of antibody 4518;
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4382 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4385 respectively; With a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4382 and 4386 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4386;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4386;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4386, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4386; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4382 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4386 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4382 and 4387 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4387;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4387;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4387, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4387; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4382 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4387 respectively.
Another embodiment of this aspect of the present invention relates to a kind of composition based on antibody 4382 and 4387, and it comprises a kind of other antibody at least.Wherein this type of embodiment relates to a kind of recombinant antibody composition, and it comprises first, second, and third antibody, and wherein first, second, and third antibody is:
Antibody 4382,4387 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382; A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4387; With a kind of antibody, comprise the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382; A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4387; With a kind of antibody, comprise heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, CDR2 and CDR3 sequence; A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4387, CDR2 and CDR3 sequence; With a kind of antibody, comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382; A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4387; With a kind of antibody, comprise heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4382 respectively; A kind of antibody comprises a weight chain variabl area sequence and a light chain variable region sequence, and it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4387 respectively; With a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4382 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4382 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4382 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4382, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4382, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4518; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4382 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4383 and 4384 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4384;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4384;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4383, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4384, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4384; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4383 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4384 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4383 and 4385 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4383, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4385, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4385; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4383 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4385 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4383 and 4386 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4386;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4386;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4383, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4386, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4386; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4383 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4386 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4383 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4383, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4383 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4383 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4383, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4518; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4383 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4383 and 4519 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4383, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4519, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4383, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4519; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4383 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4519 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4384 and 4385 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4384, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4385, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4384, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4385; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4384 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4385 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4384 and 4387 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4387;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4387;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4384, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4387, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4384, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4387; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4384 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4387 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4384 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4384, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4384, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4384 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4384 and 4519 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4384, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4519, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4384, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4519; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4384 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4519 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4385 and 4386 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4386;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4386;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4385, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4386, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4385, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4386; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4385 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4386 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4385 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4385, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4385, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4385 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4385 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4385, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4385, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4518; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4385 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4385 and 4519 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4385, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4519, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4385, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4519; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4385 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4519 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4386 and 4387 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4387;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4387;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4386, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4387, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4386, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4387; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4386 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4387 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4386 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4386, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4386, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4386 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4386 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4386, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4386, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4518; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4386 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4386 and 4519 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4386, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4519, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4386, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4519; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4386 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4519 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4387 and 4517 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4387, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4387, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4517;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4387, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4517, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4387, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4517; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4387 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4517 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4387 and 4518 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4387, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4387, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4518;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4387, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4518, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4387, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4518; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4387 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4518 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4387 and 4519 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4387, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4387, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4387, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4519, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4387, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4519; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4387 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4519 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4517 and 4519 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4517, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4517, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4517, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4519, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4517, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4519; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4517 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4519 respectively.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it comprises the first and second antibody at least, and wherein the first and second antibody are:
Antibody 4518 and 4519 or its humanized antibody;
A kind of antibody comprises the heavy chain CDR3 sequence of antibody 4518, and a kind of antibody, comprises the heavy chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR3 sequence of antibody 4518, and a kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4519;
A kind of antibody comprises heavy chain and the light chain CDR1 of antibody 4518, and CDR2 and CDR3 sequence, and a kind of antibody comprise heavy chain and the light chain CDR1 of antibody 4519, CDR2 and CDR3 sequence;
A kind of antibody comprises heavy chain and the light chain variable region sequence of antibody 4518, and a kind of antibody, comprises heavy chain and the light chain variable region sequence of antibody 4519; Or
A kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80% with heavy chain and the light chain variable region sequence of antibody 4518 respectively, 85%, 90% or 95% identical sequence, and a kind of antibody, comprise a weight chain variabl area sequence and a light chain variable region sequence, it has at least 80%, 85%, 90% or 95% identical sequence with heavy chain and the light chain variable region sequence of antibody 4519 respectively.
Therefore, preferred recombinant polyclonal Anti-HER 2 composition of the present invention, the first and second antibody that wherein comprise are respectively following antibody:
4380 and 4382,
4380 and 4383,
4380 and 4384,
4380 and 4385,
4380 and 4386,
4380 and 4387,
4380 and 4517,
4380 and 4518,
4380 and 4519,
4382 and 4384,
4382 and 4385,
4382 and 4386,
4382 and 4387,
4382 and 4517,
4382 and 4518,
4383 and 4384,
4383 and 4385,
4383 and 4386,
4383 and 4517,
4383 and 4518,
4383 and 4519,
4384 and 4385,
4384 and 4387,
4384 and 4517,
4384 and 4519,
4385 and 4386,
4385 and 4517,
4385 and 4518,
4385 and 4519,
4386 and 4387,
4386 and 4517,
4386 and 4518,
4386 and 4519,
4387 and 4517,
4387 and 4518,
4387 and 4519,
4517 and 4519,
4518 and 4519;
Or its humanized antibody; Or antibody sources is in the single antibody of every antagonist centering of listing, for example, the first and second antibody wherein comprise the CDR3 sequence of described heavy chain of antibody, or the first and second antibody wherein comprise the CDR3 sequence of described heavy chain of antibody and light chain, or the first and second antibody wherein comprise the CDR1 of described heavy chain of antibody and light chain, CDR2 and CDR3 sequence, for example described heavy chain of antibody and variable region of light chain and humanized antibody thereof.
More preferably recombinant polyclonal Anti-HER 2 composition of the present invention wherein comprises 2,3 or 4 kind of antibody of being selected from following combination:
4380 and 4382,
4380 and 4384,
4380 and 4518,
4382 and 4385,
4382 and 4518,
4383 and 4518,
4384 and 4385,
4384 and 4517,
4385 and 4518,
4380,4382 and 4385,
4380,4382 and 4517,
4380,4382 and 4518,
4380,4383 and 4384,
4380,4384 and 4517,
4380,4384 and 4518,
4380,4384 and 4519,
4382,4385 and 4518,
4382,4387 and 4517,
4380,4382,4385 and 4518,
4380,4384,4385 and 4518;
Or its humanized antibody; Or the single antibody of antibody sources in each antibody combination of listing, for example, wherein single antibody comprises the CDR3 sequence of described heavy chain of antibody, or wherein single antibody comprises the CDR3 sequence of described heavy chain of antibody and light chain, or wherein single antibody comprises the CDR1 of described heavy chain of antibody and light chain, CDR2 and CDR3 sequence, for example described heavy chain of antibody and variable region of light chain and humanized antibody thereof.
Still more preferably recombinant polyclonal Anti-HER 2 composition of the present invention wherein comprises 2 or 3 kind of antibody of being selected from following combination:
4382 and 4518,
4384 and 4517,
4382,4385 and 4518,
4382,4387 and 4517;
Or its humanized antibody; Or the single antibody of antibody sources in each antibody combination of listing, for example, wherein single antibody comprises the CDR3 sequence of described heavy chain of antibody, or wherein single antibody comprises the CDR3 sequence of described heavy chain of antibody and light chain, or wherein single antibody comprises the CDR1 of described heavy chain of antibody and light chain, CDR2 and CDR3 sequence, for example described heavy chain of antibody and variable region of light chain and humanized antibody thereof.
Another embodiment relates to a kind of recombinant antibody composition, it comprises the first and second antibody of anti-HER2 at least, the wherein different epi-position combinations of the first and second antibody and HER2, and the same epi-position of single antibodies of the first and second antibody and above-mentioned each composition of listing.
One preferred aspect in, a kind of anti-HER2 recombinant polyclonal antibody composition of the present invention, it is at least by 3 kinds of antibody that can different epi-position combinations from HER2, more preferably the first and second antibody are combined with HER2 and can be caused HER2 acceptor internalization, and the 3rd antibody is combined with HER2 and can be suppressed the HER3 phosphorylation that part is induced.It is generally acknowledged, this antibody-like composition can be by following machine-processed onset: wherein 2 kinds of antibody are in case be combined with the HER2 of cell surface, can generate a kind of antibody of coupling-be subjected to volume mesh at cell surface, this is with rising HER2 acceptor internalization level, and the 3rd antibody is combined with HER2, the allos dimerization of HER2 capable of blocking like this and HER3, thus the HER3 phosphorylation suppressed.
Being exemplified as of this type of recombinant polyclonal antibody composition, the first antibody of Anti-HER 2 are 4517 or 4518, and the second antibody of Anti-HER 2 is 4380,4385 or 4387, and the 3rd antibody of Anti-HER 2 is 4382,4383 or 4519;
Or its humanized antibody; Or antibody sources is in the single antibody of every antagonist centering of listing, for example, first, second, and third antibody wherein comprises the CDR3 sequence of described heavy chain of antibody, or first, second, and third antibody wherein comprises the CDR3 sequence of described heavy chain of antibody and light chain, or first, second, and third antibody wherein comprises the CDR1 of described heavy chain of antibody and light chain, CDR2 and CDR3 sequence, for example described heavy chain of antibody and variable region of light chain and humanized antibody thereof.
This type of preferred recombinant polyclonal antibody examples of compositions comprises following composition, and wherein the first and second antibody of Anti-HER 2 are 4518+4385 or 4517+4387, and the 3rd antibody of Anti-HER 2 is 4382.
Or its humanized antibody; Or antibody sources is in the single antibody of every antagonist centering of listing, for example, first, second, and third antibody wherein comprises the CDR3 sequence of described heavy chain of antibody, or first, second, and third antibody wherein comprises the CDR3 sequence of described heavy chain of antibody and light chain, or first, second, and third antibody wherein comprises the CDR1 of described heavy chain of antibody and light chain, CDR2 and CDR3 sequence, for example described heavy chain of antibody and variable region of light chain and humanized antibody thereof.
In a certain embodiment, the present invention relates to a kind of antibody compositions, it comprises first, second, and third antibody of Anti-HER 2 that can different epi-position combinations from HER2, wherein:
(a) first antibody of Anti-HER 2 comprises:
The heavy chain CDR3 sequence of antibody 4517 (SEQ ID NO:47) and light chain CDR3 sequence (SEQ IDNO:76), perhaps
The heavy chain CDR3 sequence of antibody 4518 (SEQ ID NO:50) and light chain CDR3 sequence (SEQ IDNO:78);
(b) second antibody of Anti-HER 2 comprises:
The heavy chain CDR3 sequence of antibody 4380 (SEQ ID NO:53) and light chain CDR3 sequence (SEQ IDNO:80),
The heavy chain CDR3 sequence of antibody 4385 (SEQ ID NO:65) and light chain CDR3 sequence (SEQ IDNO:88), perhaps
The heavy chain CDR3 sequence of antibody 4387 (SEQ ID NO:71) and light chain CDR3 sequence (SEQ IDNO:92); With
(c) the 3rd antibody of Anti-HER 2 comprises:
The heavy chain CDR3 sequence of antibody 4382 (SEQ ID NO:56) and light chain CDR3 sequence (SEQ IDNO:82),
The heavy chain CDR3 sequence of antibody 4383 (SEQ ID NO:59) and light chain CDR3 sequence (SEQ IDNO:84), perhaps
The heavy chain CDR3 sequence of antibody 4519 (SEQ ID NO:74) and light chain CDR3 sequence (SEQ IDNO:93).
In a certain preferred embodiment, the present invention relates to a kind of antibody compositions, it comprises first, second, and third antibody of Anti-HER 2 that can different epi-position combinations from HER2, wherein:
(a) this anti-HER2 first antibody comprises:
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4517 variable region of heavy chain (SEQ ID NO:2) and CDR3 and variable region of light chain (SEQ IDNO:4), perhaps
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4518 variable region of heavy chain (SEQ IDNO:6) and CDR3 and variable region of light chain (SEQ IDNO:8);
(b) this anti-HER2 second antibody comprises:
CDR1, the CDR2 of antibody 4380 variable region of heavy chain (SEQ ID NO:10) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQIDNO:12),
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4385 variable region of heavy chain (SEQ ID NO:26) and CDR3 and variable region of light chain (SEQ ID NO:28), perhaps
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4387 variable region of heavy chain (SEQ ID NO:34) and CDR3 and variable region of light chain (SEQ ID NO:36); And
(c) this anti-HER2 the 3rd antibody comprises:
CDR1, the CDR2 of antibody 4382 variable region of heavy chain (SEQ ID NO:14) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQIDNO:16),
CDR1, the CDR2 of antibody 4383 variable region of heavy chain (SEQ ID NO:18) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQIDNO:20), perhaps
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4519 variable region of heavy chain (SEQ ID NO:38) and CDR3 and variable region of light chain (SEQ ID NO:40).
In a certain particularly preferably embodiment, the 3rd antibody of Anti-HER 2 comprises: antibody 4382 variable region of heavy chain CDR1, CDR2 and CDR3 (SEQ ID NO:14) and variable region of light chain CDR1, CDR2 and CDR3 (SEQ ID NO:16).In this case, the Anti-HER 2 composition can be:
(a) first antibody of Anti-HER 2 comprises antibody 4518 variable region of heavy chain (SEQIDNO:6) CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:8) CDR1, CDR2 and CDR3,
The second antibody of Anti-HER 2 comprises antibody 4385 variable region of heavy chain (SEQ ID NO:26) CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:28) CDR1, CDR2 and CDR3; Perhaps
(b) first antibody of Anti-HER 2 comprises antibody 4517 variable region of heavy chain (SEQ ID NO:2) CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:4) CDR1, CDR2 and CDR3,
The second antibody of Anti-HER 2 comprises antibody 4387 variable region of heavy chain (SEQ ID NO:34) CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:36) CDR1, CDR2 and CDR3.
Especially, Anti-HER 2 composition of the present invention can be:
The first antibody of Anti-HER 2 comprises antibody 4518 variable region of heavy chain (SEQ ID NO:6) CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:8) CDR1, CDR2 and CDR3,
The second antibody of Anti-HER 2 comprises antibody 4385 variable region of heavy chain (SEQ ID NO:26) CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:28) CDR1, CDR2 and CDR3; Perhaps
The 3rd antibody of Anti-HER 2 comprises antibody 4382 variable region of heavy chain 3 (SEQ ID NO:14) CDR1, CDR2 and CDR and variable region of light chain (SEQ ID NO:16) CDR1, CDR2 and CDR3.
Antibody 4382 is combined with the HER2 domain II, and the handkerchief trastuzumab also is kindred circumstances, and described in example 9, the HER3 phosphorylation that the equal energy of antibody 4382 and handkerchief trastuzumab analogue block ligand is induced.Franklin etc. (Cancer Cell 2004,5 (4): 317-28) disclose, can be combined with HER2 near the domain II center, structure blocking-up HER2-HER3 allos dimerization and the required binding pocket of signal transduction by the handkerchief trastuzumab.Therefore, except for example antibody 4382 and handkerchief trastuzumab, other Anti-HER 2s of being combined with the dimerization interface in a similar manner will have the effect of similar blocking-up HER2-HER3 allos dimerization, and it is anti-therefore to see that from this respect this antibody-like is suitable as three the present invention.
Find that in experiment related to the present invention although 2 kinds or multiple Anti-HER 2 described in the coupling this patent can effectively cause HER2 internalization and degraded, HER2 may be raised the generation of HER3 by the cell of antibody target.Thinking at present may be relevant with its rise HER3 to anti-HER 2 monoclonal antibody (for example Herceptin) resistance, this may be to produce by following mechanism, be that the HER2-HER3 heterodimer consists of a complete acceptor, can in without the situation of HER2 homodimer signal, still mediate the cancer signal transduction.Following example 9 has been described the antibody compositions of the present invention that is comprised of 3 kinds of Anti-HER 2s, wherein 2 kinds of antibody can be combined with HER2, can generate like this coupling antibody receptor grid, thereby cause HER2 internalization and degraded, and three anti-can be in conjunction with HER2, block like this HER2-HER3 allos dimerization, thereby avoided the signal transduction of HER2 phosphorylation and HER3 mediation.Suppose that these and similar Anti-HER 2 composition have very strong advantage aspect blocking-up HER family's phosphorylation and the signal transduction, namely this antibody compositions comprises at least mixture of Anti-HER 2, wherein 2 kinds of antibody can cause HER2 internalization and degraded, the 3rd antibody HER2-HER3 allos capable of blocking dimerization.Especially, suppose that this antibody-like composition can significantly reduce the tumour cell growth to the Anti-HER 2 resistance.Therefore, this class Anti-HER 2 composition may to prevention and to alleviate this class resistance of Anti-HER 2 very valuable, and very valuable to certain anti-HER 2 monoclonal antibody (for example Herceptin) treatment drug-resistant tumor to treatment.
Another aspect of the present invention is relevant to the method for the growth of tumour cell of a kind of Anti-HER 2 treatment resistance or part resistance with inhibition, the method comprises a kind of aforesaid anti-HER2 recombinant polyclonal antibody composition of cells contacting, and it comprises 2 kinds of three anti-(suppressing the HER3 phosphorylation that part is induced) that can produce at cell surface Anti-HER 2 (causing the HER2 internalization) and a kind of HER2 capable of blocking of being combined with HER2 and the HER3 allos dimerization of coupling antibody receptor grid.Once adopted Herceptin treatment tumour cell for example.
Following table 2 and 3 shows CDR1, CDR2 and the CDR3 sequence according to heavy chain (table 2) and the light chain (table 3) of all kinds of Anti-HER 2s of the present invention.List these antibody heavy chain variable regions and the light chain aminoacid sequence of (comprising variable region of light chain) in the sequence table of appendix, and DNA sequences encoding (optimize and be used for expressing cho cell).SEQID numbering referring to these sequences in the table 1 gathers.
Table 2: selected Anti-HER 2 heavy chain CDR1, CDR2 and CDR3 sequence
Figure BDA00002350442700551
Table 3: selected Anti-HER 2 light chain CDR1, CDR2 and CDR3 sequence
Figure BDA00002350442700561
Another aspect of the present invention relates to nucleic acid molecule, and it comprises the nucleotide sequence of a kind of antibody in the code book invention, and namely a kind of antibody can be selected from antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519, or its humanization varient; Or the heavy chain of this antibody-like of encoding and/or light chain variable region sequence, or a kind of heavy chain and/or sequence of light chain, itself and this type of heavy chain and/or light chain variable region sequence have at least 80%, 85%, 90% or 95% identical sequence.
In the embodiment of the present invention aspect this, nucleic acid molecule comprises a nucleotide sequence, can be selected from SEQ ID NOS 1,3,5,7,9,11,13,15,17,19,21,23,25,27,29,31,33,35,37 and 39, or the nucleotide sequence of the coding aminoacid sequence identical with described arbitrary nucleotide sequence.
Another aspect of the present invention relates to a kind of expression vector, and it comprises above-mentioned nucleic acid molecule.As mentioned above, expression carrier used thereof can be the known any adequate types of prior art, for example plasmid or virus vector in the present invention.
Another aspect of the present invention relates to a kind of host cell, and it comprises above-mentioned nucleic acid molecule, and described host cell can be expressed the Anti-HER 2 of described nucleic acid molecule encoding.
The binding specificity of the antibody that discloses in any 2 kinds of this patents on the other hand, can make up with a kind of dual specific binding molecule.Preferred this class dual specific binding molecule comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of selected 2 kinds of antibody.The dual specific binding molecule can be the bidirectional variable domain antibodies, and namely wherein antibody two arms comprise 2 different variable regions, maybe can be antibody fragment, for example dual specific Fab fragment or dual specific scFv.
Preparation Anti-HER 2 and antibody compositions
Another aspect of the present invention relates to the method for preparing Anti-HER 2 of the present invention or anti-HER2 polyclonal antibody.One embodiment of the present invention relates to a kind of method for preparing described Anti-HER 2, and comprising provides a kind of described host cell that can express Anti-HER 2, cultivates described host cell under the condition that is fit to this antibody of expression, and separates the antibody that obtains.
In another embodiment, the present invention relates to prepare a kind of method of anti-HER2 polyclone recombinant antibody composition, said composition comprises the first and second host cells at least, wherein the first and second host cells can be expressed anti-HER2 recombinant antibodies, under the condition that is fit to expression the first and second antibody, cultivate the first and second host cells, and separate the first and second antibody that obtain
A kind of antibody among the present invention or antibody compositions can be by prior art Restruction mono-clonal or the preparations of monoclonal anti body method.Therefore, when producing single antibody of the present invention, can adopt any method of existing Restruction monoclonal antibody.During a kind of antibody compositions that production is comprised of 2 kinds or multiple Anti-HER 2 among the present invention, can produce respectively single antibody, namely preparation is single respectively in bio-reactor independently plants antibody, or in a bio-reactor manufacture order kind antibody simultaneously.Different antibodies quantity in a kind of antibody compositions surpasses, and for example 2 kinds or 3 kinds, then usually be in cost-efficient consideration, preferably in a bio-reactor, produce simultaneously antibody.On the other hand, when said composition only comprised a small amount of different antibodies, for example 2 kinds, 3 kinds or 4 kinds of different antibodies then needed to determine as the case may be to be to produce respectively in different bio-reactors, or produced simultaneously in a bio-reactor.If in a plurality of bio-reactors, produce antibody compositions, then can by from each bio-reactor respectively purifying obtain supernatant liquor, gather again the antibody in the supernatant liquor, obtain at last the Anti-HER 2 composition of purifying.In a plurality of bio-reactors, produce each class methods of polyclonal antibody referring to WO 2009/129814 (incorporated by reference), wherein be described in the upstream a little later time point or before the Downstream processing or during merge clone or antibody preparations.
In single biomass generator, produce 2 kinds or multiple single antibody, can by WO 2004/061104 or WO2008/145133 is described carry out (incorporated by reference in this patent).The described method of WO2004/061104 enters in the host cell based on the antibody coding sequence site-specific integration, guarantees V HAnd V LKeep in process of production its original pairing.Therefore moreover site-specific integration can minimize the position effect, expects that growth and the expression characterization of individual cells can be very similar in the multi-clone cell line.Usually, the method relates to: i) a kind of host cell with one or more recombinase recognition sites; Ii) with the expression vector that comprises at least a recombinase recognition site of this host cell compatibility; Iii) pass through selected V HAnd V LCoding forms the expression vector collection, thereby can from this vector expression full length antibody or antibody fragment, if screening vector is identical with expression vector, then need not this transfer being transferred in the expression vector; Iv) the carrier transfection host cell that can be combined at middle recombinase recognition site with host cell gene with expression vector collection and coding; V) host cell from transfection obtains/generates multi-clone cell line; And vi) expresses and collects antibody compositions from this multi-clone cell line.
WO 2008/145133 has described a kind of another kind of method of producing 2 kinds or Multiple Antibodies in single biomass generator.The method relates to a kind of multi-clone cell line of generation, this clone can be expressed the polyclone albumen that a kind of polyclonal antibody or other comprise 2 kinds or multiple heterogeneity, method is that a cover expression vector a) is provided, wherein said carrier comprises the Nucleotide copy of at least a uniqueness, a kind of unique member of its coding polyclone albumen, avoid the expression vector site-specific integration enter cytogene condition under, respectively with the expression vector transfection host cell, thereby obtain 2 kinds or various kinds of cell composition, each composition is expressed a kind of unique component of this polyclone albumen; And c) at least 2 kinds of cell compositions is mixed to get a multi-clone cell line.The method advantage of WO 2004/061104 and WO 2008/145133 is: can be in single bio-reactor all compositions of Restruction polyclonal antibody, and by single program purifying, thereby avoid producing respectively and each antibody of purifying, meanwhile can unify the production different antibodies.WO 2008/145133 method also has the advantage that improves output, a plurality of copies of the polynucleotide of a certain antibody because each production cell portability is encoded.
Can in various types of cells, produce antibody of the present invention, comprise eucaryon or the prokaryotic cell prokaryocyte of mammalian cell and nonmammalian, such as vegetable cell, insect cell, yeast cell, fungi, intestinal bacteria etc.But preferably in mammalian cell, produce antibody, for example Chinese hamster ovary celI, COS cell, bhk cell, myeloma cell are (for example, Sp2/0 or NS0 cell), inoblast, NIH 3T3 for example, or immortalization human cell, for example HeLa cell, HEK 293 cells or PER.C6 cell.
At present those skilled in the art know with nucleotide sequence be transfected into host cell method (referring to, Sambrook et al. for example, Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Edition, 2001).Site-specific integration described in the WO 2004/061104 comprises one or more recombinase recognition sites in the suitable host cell gene group.In this case, suitable expression vector comprises the restructuring recognition site of a coupling host cell recombinase.WO2004/061104 describes more heterogeneous pass information in detail, for example adopts the method for site-specific integration to shift selected V from a screening vector HAnd V LIt is right to encode.
When in single biomass generator, generating a kind of antibody compositions of the present invention that comprises 2 or a plurality of Anti-HER 2s, can select to have similar rate of propagation and preferred class and generate a multi-clone cell line like the clone of antibody expression level.Then obtain multi-clone cell line by mix individual cells system by predetermined proportion.Relevant generation is a kind of to be expressed the multi-clone cell line of polyclonal antibody and uses this type of clone to produce polyclonal antibody, understand more detailed information and example such as need, see also WO 2009/129814, WO 2004/061104 and WO 2008/145133 (this patent incorporated by reference).
An embodiment among the present invention is a kind of multi-clone cell line, and it can express 2 kinds or multiple Anti-HER 2 among the present invention.Another embodiment is a kind of multi-clone cell line, and wherein single cell can be expressed single V HAnd V LRight, and multi-clone cell line integral body can be expressed V HAnd V LPair set, wherein each V HAnd V LTo a kind of Anti-HER 2 of encoding.
Can in single bio-reactor, produce a kind of recombinant antibody composition of the present invention, method is as follows: in suitable medium, to cultivate for some time from an ampoule in polyclone working cardial cell storehouse (pWCB), to reach enough antibody expression levels, keep simultaneously the basic homogeneous of relative expression's level that multi-clone cell line is expressed different antibodies.Usually the production time approximately had been advisable in 15-50 days.Can adopt existing culture technique, for example fed-batch type is cultivated or perfusion culture.Medium optimization serum free medium, more preferably serum-free and protein culture medium, for example chemically defined substratum.This class substratum is generally used for the cell cultures of certain production type, and present commercially available a large amount of suitable medium preparations that have.
Obtain recombinant antibody composition from substratum, and adopt traditional purification technique to carry out purifying.These technology comprise, for example, affinity chromatography coupling subsequent purification step, for example ion exchange chromatography, hydrophobic chromatography and gel-filtration, these purification techniques are through being usually used in the recombinant antibodies purifying.In single bio-reactor, produce 2 kinds or Multiple Antibodies by a multi-clone cell line, usually behind purifying, assess all single composition, for example ion exchange chromatographies in the polyclonal antibody composition.For example, can adopt method described in WO2006/007853 and the WO 2009/065414 (this patent incorporated by reference) to the qualitative analysis of a kind of polyclonal antibody composition.
Therapeutic composition
Another aspect of the invention is a kind of pharmaceutical composition, comprise a kind of activeconstituents, can be a kind of Anti-HER 2 conduct among at least the present invention, or a kind of anti-HER2 recombinant Fab or another kind of anti-HER2 recombinant antibody fragment composition.The activeconstituents of preferred this type of pharmaceutical composition is the aforesaid anti-HER2 recombinant antibody composition that comprises 2 kinds or multiple Anti-HER 2.This composition can be used for improving, preventing and/or treating cancer.Pharmaceutical composition can be used for the mankind, domestic animal or pet administration, but normally human administration.
Ratio in a kind of therapeutic composition of the present invention between different antibodies, or at the same time, sequential or give respectively in the situation of different antibodies of the present invention, the ratio between different antibodies often is equivalent, but also inequality.Therefore, the antibody compositions that contains 2 kinds of Anti-HER 2s among the present invention often adopts 1: 1 ratio, and comprises the frequent ratio that adopts 1: 1: 1 of antibody compositions of 3 kinds of Anti-HER 2s.But, according to the characteristic of different antibodies, may need the inequality proportioning.The proper ratio of different Anti-HER 2s can be by WO2010/040356 described definite (this patent incorporated by reference) in the antibody compositions of the present invention, definite and the system of selection of optimum chemical metered proportions between chemical substance in the medicinal composition has wherein been described, a kind of polyclonal antibody composition for example is to obtain to have best effectiveness and the medicinal composition of curative effect.
Except at least a antibody of the present invention or fragment, pharmaceutical composition also comprises at least a medicinal thinner, carrier or the vehicle accepted.These can comprise, for example sanitas, stablizer, tensio-active agent/wetting agent, emulsifying agent, solubilizing agent and be used for regulating salt and/or the damping fluid of osmotic pressure.Solution or suspension also can comprise thickening material, for example Xylo-Mucine, carboxymethyl cellulose, dextran, polyvinylpyrrolidone or gelatin.The suitable pH value of pharmaceutical composition usually in the 5.5-8.5 scope, about 6-8, for example 7, can use damping fluid to keep suitable pH value.
Can adopt traditional pharmaceutical methods to provide, for example cancer patients's administration, dosage forms or composition.Administration is generally used for therapeutic purpose, this means administration after making a definite diagnosis cancer.Can adopt any suitable route of administration, for example in parenteral, intravenously, intra-arterial, subcutaneous, intramuscular, intraperitoneal, the nose, aerosol, suppository or oral administration.Common pharmaceutical composition of the present invention is with liquor or suspensions administration, and more frequent with the aqueous solution or suspension, some situation is with isotonic solution or suspension.
Pharmaceutical composition of the present invention prepares with currently known methods, for example by traditional dissolving, freeze-drying, mixing, granulation or moulding process.Can (for example, consult Remington:The Science and Practice ofPharmacy (21st edition), ed.A.R.Gennaro, 2005, Lippincott Williams ﹠amp according to traditional pharmaceutical technology pharmaceutical compositions; Wilkins, Philadelphia, PA, USA; And Encyclopedia of Pharmaceutical Technology, ed.J.Swarbrick, 3 RdEdition, 2006, InformaHealthcare, New York, NY, USA).
As the alternative formulation of liquid dosage form, composition of the present invention can be made into lyophilized powder, comprises at least a antibody or with carrier, for example N.F,USP MANNITOL, before use available liquid dissolved composition, for example sterilized water.
Pharmaceutical composition comprises about 1% to about 95% activeconstituents, and preferred about 20% to about 90%.According to the present invention, pharmaceutical composition can adopt for example single dose form, for example ampoule, bottle, suppository, tablet or capsule.Can give treatment or the prevention significant quantity (for example, preventing, eliminate or alleviate the dosage of pathological condition) of these formulations of human body, thus treatment cancer or other diseases.The preferred dosage of medicine may depend on following variable, for example cancer severity, certain patient's holistic health state, compound vehicle prescription and route of administration.
The therepic use of antibody and composition among the present invention
Anti-HER 2 prepared in accordance with the present invention and pharmaceutical composition can be used for treatment or improve mammalian diseases, particularly treat human cancer.One embodiment of the present of invention are the methods of one or more relevant symptoms of a kind of prevention, treatment or the improvement mankind or other mammalian cancer, comprise to described Mammals to give the present invention the effective dose of anti-HER2 recombinant antibody composition.
One of them embodiment suffers from HER2 and crosses the disease method that is expressed as feature with treatment, cancer particularly, the method comprises and gives the Anti-HER 2 that defines in a kind of this patent of described patient, or preferred a kind of anti-HER2 recombinant antibody composition that defines at least 2 kinds of this patents that comprises.
Another embodiment relates to a kind of method that heterodimer forms between the interior HER2 of HER2 cell and other ErbB family receptors of expressing that reduced, the method comprises the anti-HER2 recombinant antibodies that will define in a kind of this patent of described cells contacting, or preferred a kind of anti-HER2 recombinant antibody composition that defines at least 2 kinds of this patents that comprises.
Another embodiment of the present invention prepares a kind of composition for using a kind of anti-HER2 recombinant antibodies of the present invention or antibody compositions, one or more symptoms that said composition is used for the treatment of, improves or prevents the mankind or other mammalian cancer to be correlated with, for example treat a patient, its disease that takes a disease is characterized as HER2 and crosses expression.
Based on some factors, comprise the HER2 expression level, following type of cancer is particularly suitable for adopting a kind of antibody compositions treatment of the present invention: mammary cancer, ovarian cancer, cancer of the stomach, colon and rectum carcinoma, prostate cancer, bladder cancer, carcinoma of the pancreas, head and neck cancer and nonsmall-cell lung cancer.Antibody compositions of the present invention is particularly suitable for treating the cancer of expressing HER2, for example some cell carcinoma, for example mammary cancer, ovarian cancer and cancer of the stomach.
2 main clinical paths that these indications are relevant are 1) auxiliary therapy, at least a additional procedures treatment; Or 2) single therapy.Below concise and to the point these two selections are discussed.
1) auxiliary therapy: in auxiliary therapy, also become combination therapy, will use antibody and at least a other therapeutic treatment associating among the present invention, be generally a kind of chemotherapy or cancer therapy drug and/or radiotherapy.Perhaps or extraly, also can be with the Anti-HER 2 among the present invention and composition and the associating of a kind of different anticancrin, for example antibody of a kind of targeting EGFR or VEGF.Therefore, except standard one line and two roentgenism ies, can be by giving a kind of antibody of the present invention or combination treatment above-mentioned main cancer target.Conceptual design will alleviate and reduce by tumor quality the dosage assessment curative effect of conventional criteria chemotherapy.Thereby reducing, this dosage can additionally increase and/or prolong quality by the dosage that reduces chemotherapeutics is xicity related.
But by antibody compositions of the present invention and the eventually last differentiation medicament associating of known inducing cancer cell, can further improve curative effect.This compounds can be selected from, and for example comprises the group of vitamin A acid, trans retinoic acid, cis-retinoic acid, PB, nerve growth factor, methyl-sulphoxide, Vitamin D3 500,000 I.U/GM activity form, peroxisome proliferation-activated receptors γ, 12-O-TPA, Vitro By Hexamethylene Bisacetamide, transforming growth factor-beta, butyric acid, cyclic monophosphate and vesnarinone.Preferred compound is selected from the group that comprises vitamin A acid, PB, all-trans retinoic acid and vitamins D activity form.
The medicine that comprises a kind of antibody compositions of the present invention and at least a chemotherapy or anticancer compound can be used in the cancer therapy simultaneously, separately or the combination therapy of successive administration.Chemotherapy compound can be by any suitable chemotherapeutic that is suitable for treating a certain cancer, for example a kind of medicine that contains alkylating agent that is selected from, for example platinum analog derivative, for example cis-platinum, carboplatin or oxaliplatin; Plant alkaloid, for example taxol, Docetaxel or irinotecan; Antitumor antibiotics, for example Dx (Zorubicin); Topoisomerase enzyme inhibitor, for example Hycamtin; And metabolic antagonist, for example Fluracil or other 5-FUs.
Estimate that simultaneously antibody of the present invention can be used as auxiliary therapy, with tyrosinase inhibitor (TKI) coupling.These synthesize, and are generally quinazoline derivant, low-molecular-weight molecule, and it can interact with the U.S. structural domain of tyrosine collection in the acceptor born of the same parents, by Mg-ATP binding site in the competition born of the same parents, suppresses the receptor phosphorylation that part is induced.At present just at the kinase whose tyrosinase inhibitor of some HER2 capable of blocking of clinical development.Some of them can also targeting EGFR or other EGFR family receptors.The summary of relevant these TKI is consulted Spector et al. (2007) Breast Cancer Res.9 (2): 205.Therefore the medicine that is comprised of the TKI of a kind of antibody compositions of the present invention and at least a target HER2 also can be used in the cancer therapy simultaneously, separates or the drug combination of successive administration.
In other embodiments, antibody compositions of the present invention can with other antibody drug combined utilization.These comprise targeting EGFR (
Figure BDA00002350442700621
Or
Figure BDA00002350442700622
) or VEGF
Figure BDA00002350442700623
Medicine.In other embodiments, antibody compositions of the present invention can with a kind of known drug combination that can the stimulating immune system cell, this class drug combination can strengthen the curative effect of immune-mediated antibody compositions of the present invention.This para-immunity stimulating drug comprises recombinant interleukin (for example IL-21 and IL-2).
2) single therapy: in the tumour single therapy, use antibody according to the present invention, can not give antibody in the situation for the treatment of with chemotherapy or cancer therapy drug simultaneously, i.e. monotherapy.
Immune conjugate
Another of the therapeutic action of antibody and composition is chosen as immune conjugate among the present invention, is about to antibody and one or more cancer therapy drug couplings.Especially, when composition comprises 2 kinds or multiple different antibodies of the present invention, its can from the combination of different HER2 epi-position, estimate that it can generate coupling antibody receptor grid at cell surface, compare the acceptor internalization that to raise like this level with the single monoclonal antibody of use.Therefore with one or more antibody of this composition and one or more anticarcinogen couplings can be specifically, effectively the coupling anticarcinogen is delivered to the tumour cell inboard, thereby strengthen the effect of Anti-HER 2 of the present invention, the activity of raising killing tumor cell.
Antibody of the present invention can with all kinds of cancer therapy drug couplings, comprise cytotoxic drug (wrapping traditional chemotherapeutic and other small molecules anticarcinogen), cytokine (wherein conjugate is called as " immune cell factor "), toxin (wherein conjugate is called as " immunotoxin ") and radionuclide, and the immune conjugate of some approved clinical applications.Comprising
Figure BDA00002350442700624
(coupling 90The mouse anti-CD20 antibodies of Y),
Figure BDA00002350442700625
(coupling 131The mouse anti-CD20 antibodies of I) and
Figure BDA00002350442700626
(the mouse anti-CD 33 antibody of coupling calicheamycin). comprise at other immune conjugate of clinical trial testing being coupled to, for example the antibody of Zorubicin or maytenin compound.The immunotoxin of clinical trial testing comprises that some are coupled to the antibody of the Pseudomonas Exotoxin A that blocks.Also tested simultaneously the immune cell factor that comprises humanization EpCAM antibody.
Among the present invention with the antibody of cytotoxic drug coupling, can belong to, for example the chemotherapeutic primary categories is any, comprise that alkylating agent (for example, carboplatin, cis-platinum, oxaliplatin), metabolic antagonist (for example, methotrexate, capecitabine, gemcitabine), anthracycline antibiotics (such as bleomycin, Zorubicin, Mitomycin-C) and plant alkaloid (for example, such as docetaxel, taxol, taxanes and vinca alkaloids, such as vincaleucoblastine, vincristine(VCR), vinorelbine).Because but the immune conjugate specificity is with the anticarcinogen target tumor, target tumor cell interior after the internalization particularly, based on the immune conjugate of Anti-HER 2 of the present invention based on the high cell toxicity agent, for example calicheamycin or maytansine derivative, or based on toxin, such as bacteriotoxin (such as Pseudomonas Exotoxin A, diphtheria toxin) or plant poison (such as Ricin), have superiority.
The anticarcinogen of coupling is connected with antibody by a unsettled joint usually in the immune conjugate, and this joint is metastable in serum, but after immune conjugate was entered targeted cells by internalization, it can be with drug release out.Suitable joint comprises, for example, and chemical joint, stable under the serum pH neutral, but after the internalization under the solutions of weak acidity acid hydrolysis occurs easily in lysosome, the disulfide linkage joint can be by intracellular mercaptan cracking, peptide linker is stable in serum, but in born of the same parents easily by enzymolysis.
Can the various couplings of design within containing the composition of 2 kinds or multiple antibody of the present invention.For example, 2 kinds of antibody can be with antibody and 2 kinds or multiple different anticarcinogen coupling, or with a kind of antibody and a kind of prodrug coupling, this prodrug can by with the substance activating (for example enzyme) of another antibody coupling.The universal of the antibdy directed enzyme-prodrug therapy of monoclonal antibody (ADEPT) is discussed, wherein prodrug is activated by a kind of enzyme of target tumor by monoclonal antibody-enzyme conjugates, but the invention provides the method that a kind of capable of regulating the method is used for some situation.Therefore, can specificity increase tumor cytotoxicity power, the damage of avoiding simultaneously or alleviating normal tissue.
The more information of relevant antitumor immune conjugate is referring to Wu et al. (2005) Nature Biotechnology23 (9): 1137-1146; Schrama et al. (2006) Nature Reviews/Drug Discovery 5:147-159; AndRohrer (2009) chimica oggi/Chemistry Today 27 (5): 56-60.
Dosage and approach
The antibody of the present invention and the composition that give effective dose are used for the treatment of disease, namely reach dosage and the time of results needed.The treatment effective dose changes with many factors, for example the treatment of the disease specific, age, sex, weight in patients for the treatment of and Anti-HER 2 whether be single therapy or with one or more other anti-cancer therapies combination therapys.
The effective dose of oncotherapy can be measured by its stable disease progress and/or the ability of improving patient's symptom, preferably reverses progression of disease, for example by reducing tumor size.The ability that a kind of antibody compositions of the present invention suppresses tumour can be assessed by in vitro tests, described in example, also can assess by suitable measurable its animal model to the human tumor curative effect.Thereby can select suitable dosage to reply for each situation provides best treatment, for example, but single infusion or continuous infusion, and carry out dose titration according to each patient's emergency situation.
Although not yet determine at present antibody administration dosage of the present invention, can by relatively coming to determine a certain dosage with the similar granted medicine that is used for the treatment of (a kind of anti-HER 2 monoclonal antibody).Therefore, the Rational Dosage of a kind of antibody compositions of the present invention will be in anti-HER 2 monoclonal antibody Herceptin (Trastuzumab
Figure BDA00002350442700641
) recommended dose similar.As the case may be, can give Trastuzumab (transfusion administration) and be used for the treatment of mammary cancer, initial dose is 4mg/kg, and then every weekly dose is 2mg/kg, or initial dose is 8mg/kg, and then per 3 weekly doses are 6mg/kg.
The appropriate dose of antibody compositions of the present invention in the 0.1-100mg/kg scope, for example about 0.5-50mg/kg, for example about 1-20mg/kg.For example, the dosage of antibody compositions is at least 0.25mg/kg, for example 0.5mg/kg at least, 1mg/kg at least for example, 1.5mg/kg at least for example, 2mg/kg at least for example, for example 3mg/kg at least, 4mg/kg at least for example, 5mg/kg at least for example, and for example up to maximum 50mg/kg, for example up to maximum 30mg/kg, for example up to maximum 20mg/kg, for example up to maximum 15mg/kg.Usually with the appropriate interval repeat administration, for example weekly, per 2 weeks, once per 3 weeks, once or per 4 weeks once, feels suitable as long as be responsible for the doctor, and the doctor can increase and decrease dosage on demand.
Antibody of the present invention can adopt 3 kinds of different administering modes.The traditional intravenous administration is the standard medicine-feeding technology of most of tumours.But belly cavity tumor, for example ovarian cancer, cholangiocarcinoma, other pipeline cancer and similar cancer are verified, and intraperitoneal administration is favourable to obtaining the tumor locus high concentration antibody, and it is minimum that cleaning antibody is down to.Similarly, some noumenal tumour has the blood vessel structure of suitable regional perfusion.Regional perfusion can obtain high concentration antibody at tumor locus, and is down to the removing of antibody short-term minimum.
Similar with medicine to any albumen or antibody infusion of therapeutic, safety issue mainly comprises: (i) cytokines release syndrome was, namely ypotension, generate heat, tremble, shiver with cold; (ii) to this albumen generation immunogenic response (being that the patient produces antibody to the restructuring antibody drug), and (iii) to expressing the Normocellular toxicity of HER2 acceptor, for example: many epithelial cells.Can adopt standard testing and down-stream to monitor any this type of safety issue.
All patents and the non-patent literature in this incorporated by reference the application, quoted.
Present invention will be further described in the example below (non-limiting example).
Example
Example 1: the clone of Anti-HER 2
Immunity
Inject purified different albumen and HER2 overexpressing cell female BALB/c (strain A) or C57B16 mouse (8-10 age in week) are carried out immunity.
Adopt commercially available HER2 albumen (R﹠amp; D Systems cat.#1129-ER) is used for immunity.MCF-7 AU565 (ATCC, CRL-2351) is used for cellular immunization.Culturing cell in the RPMI-1640 substratum that adds 10% foetal calf serum (FBS) and 1% mycillin (P/S).Before each immunity, use the PBS washed cell, with TrypLE digestion, then resuspended in growth medium.Then use PBS washed cell suspension 2 times, under the 250g centrifugal 5 minutes, then take out and resuspended in the aseptic PBS of 15ml.
Then diluting cells or antigen in PBS mix with freund's adjuvant at 1: 1.Freund's adjuvant can strengthen and regulate immune response.For the first time immunity is used complete Freund's adjuvant (CFA), but use incomplete Freund's adjuvant (IFA) in follow-up immunity.IFA is a kind of oil-in-water mineral oil emulsion, and CFA makes for the mycobacterium tuberculosis that adds the heat-killed drying to IFA.Two kinds of adjuvants all have the deposit effect.CFA can produce the long-term immune response that continues, and can be used for first immunisation with the enhancing immune response, and IFA is used for follow-up immunization.Can be by one after another drop of surface at one glass of water be tested.When this still remains one, then emulsion is stable, can be used for injection.Can only be to the stable emulsion of injected in mice.
According to schedule (referring to table 4), per injection 25-100 μ g antigen or 10 7Individual cell.Inject altogether mouse 4 times.All mouse are injected 300 μ l or 200 μ l emulsions.According to schedule, can subcutaneous (s.c.), abdominal cavity (i.p.) or vein (i.v.) injection.
At last, disconnected neck is put to death mouse, takes out spleen, is transferred in the 74 μ m cell filter screens (Corning#136350-3479).Cell is emanated by filter screen, resuspended with the ice-cold RPMI-1640 substratum that contains 10% foetal calf serum (FBS), and under 300g centrifugal 5 minutes.With the RPMI-1640 substratum re-suspended cell group that contains 1% foetal calf serum (FBS), filter again centrifugal collection by 50 μ m injection filters (BD#340603).With frozen behind the foetal calf serum that contains 10%DMSO (FCS) re-suspended cell, freeze-stored cell is stored in-80 ℃ down until the FACS sorting.
Mouse plasmocyte FACS sorting
Frozen splenocyte bottle is thawed under 37 ℃, then in the situation that still has ice to exist, be transferred in the 15ml pipe.Xiang Guanzhong dropwise adds the ice-cold RPMI of 10ml, 10%FBS, and shake test tube.With 10ml FACS PBS washing once, cell is crossed 50 μ m Filcon and filtered the front 5ml of adding FCS PBS.Then sedimentation cell and resuspended with 1ml PBS+2%FBS (final volume) is the anti-CD43-FITC of 5 μ g/ml and anti-CD138-PE antibody staining with being diluted to final concentration then.Hatched 20 minutes at 4 ℃ under the dark condition.Then, use 2mlFACS damping fluid washed cell 2 times.Add at most 15mlFACSPBS.Add propidium iodide (PI) in 1: 100 ratio, 1 part PI to 100 portion FACS PBS damping fluid, then cell is divided to the 96 hole PCR plates (seeing below) that contain the PCR reaction buffer, then at 400g centrifugal 2 minutes, then that cell plate are frozen under-80 ℃.The plasmocyte reject gate is that the CD43-positive/CD-138 is positive.
Homology V HAnd V LTo chain
Carry out V to minute electing plasmacytic cell as HAnd V LEncoding sequence carries out chain, is beneficial to V HAnd V LEncoding sequence autosyndetic pairing.This program is used two step method PCR, i.e. the multiple overlapping extension RT-PCR of single stage method, and then carry out nest-type PRC.The used primer mixture κ light chain that only increases in this example.But, can be to the primer of interpolation amplification lambda light chain in many primer mixtures and the nest-type PRC primer mixture.If add the λ primer, can revise process of separation, the λ positive cell can not be excluded like this.Homology V HAnd V LThe chain principle of sequence sees WO 2005/042774 and Meijer et al. (2006) J Mol Biol.358 (3) for details: described in the 764-72.
The 96 hole PCR plates that thaw, sorting cells are used for the template of multiple overlapping extension RT-PCR.Contain reaction buffer (OneStep RT-PCR Buffer at unicellular sorting forward direction; Qiagen), add the sorting damping fluid in each hole of RT-PCR primer (referring to table 5) and RNase inhibitor (RNasin, Promega).With OneStep RT-PCR5Enzyme Mix (25 ' dilution; Qiagen) and dNTP mixture (every hole 200 μ M) complement to the appointment final concentration under the 20 μ l reaction volumes.Under 55 ℃, hatched 30 minutes, and made every hole carry out the reverse transcription of RNA (RT).Behind RT, the PCR plate is carried out following PCR circulation: 94 ℃ lower 10 minutes, 35 ' (94 ℃ lower 40 seconds, 60 ℃ lower 40 seconds, 72 ℃ are lower 5 minutes), 72 ℃ lower 10 minutes.
The PCR that carries out 24 96 orifice plates (ABgene) in the H20BIT thermal cycler reacts to obtain high-throughput.After finishing circulation the PCR plate is stored under-20 ℃.
In the nest-type PRC, prepare 96 hole PCR plates, following mixture (20 μ l reaction) is contained in every hole, to obtain the final concentration of appointment: 1 ' FastStart damping fluid (Roche), dNTP mixture (each 200 μ M), nested primer mixture (referring to table 6), Phusion archaeal dna polymerase (0.08U; Finnzymes) and FastStart high-fidelity enzyme mixture (0.8U; Roche).As the template of nest-type PRC, 1 μ l is transferred to multiple overlapping extension RT-PCR reaction.The nest-type PRC plate carries out following thermal cycling: 35 ' (95 ℃ lower 30 seconds, 60 ℃ lower 30 seconds, 72 ℃ are lower 90 seconds), 72 ℃ lower 10 minutes.Then there is the overlapping extension fragment of about 890 base pairs (bp) in the selective reaction thing with checking with 1% agarose electrophoretic analysis at random.The PCR plate is stored under-20 ℃, until the PCR fragment is further processed.
Collect the chain V that nest-type PRC obtains HAnd V LIt is right to encode, and the coding that does not mix different donor sources is right, then prepares 1% agarose gel electrophoresis purifying.It is described to press WO 2008/104183, and the constant light chain encoding sequence of overlapping extension people κ is to chain V HAnd V LCoding is to the V of PCR product LEncoding sequence.From containing the constant light chain of plasmid amplification people κ with the encoding sequence of the human antibodies of κ light chain, reaction system comprises: Phusion enzyme (2U; Finnzymes), 1x Phusion damping fluid, dNTP mixture (each 200 μ M), hKCforw-v2 primer and Kappa3 ' primer (table 5) and plasmid template pLL138 (10ng/ μ l), cumulative volume 50 μ l.Then carry out following thermal cycling: 25 ' (95 ℃ lower 30 seconds, 55 ℃ lower 30 seconds, 72 ℃ are lower 45 seconds), 72 ℃ lower 10 minutes.Then prepare the PCR fragment that 1% agarose gel electrophoresis purifying obtains.
By following overlapping extension PCR splicing, with the PCR fragment assembly of the every part of purifying PCR fragment (SEQ ID NO:41) to the amplification of the constant light chain of people κ coding region and purifying: 50 μ l reaction systems comprise the constant light chain of people κ coding region fragment (1.4ng/ μ l), the PCR fragment of purifying (1.4ng/ μ l), Phusion archaeal dna polymerase (0.5U; Finnzymes), FastStart high-fidelity enzyme mixture (0.2U; Roche), 1xFastStart damping fluid (Roche), dNTP mixture (each 200 μ M), mhKCrev primer and mJH primer (table 7).Then carry out following thermal cycling: 95 ℃ lower 2 minutes, 25 ' (95 ℃ lower 30 seconds, 55 ℃ lower 30 seconds, 72 ℃ are lower 1 minute), 72 ℃ lower 10 minutes.Then prepare the PCR fragment (about 4518bp) that 1% agarose gel electrophoresis purifying obtains.
With homology V HAnd V LCoding is to inserting in the screening vector
For antibody definite and the HER2 specific combination, the V of acquisition HAnd V LEncoding sequence is expressed as full length antibody.This relates to V HAnd V LCoding is to inserting expression vector and transfection host cell.
Adopt two step method clone step to generate and contain chain V HAnd V LCoding is to expression vector.Statistically, if expression vector comprises 10 times to V HAnd V LHomology is used for generating the screening storehouse to PCR product quantity, and there is 99% possibility in all unique gene pairss.Therefore, if obtain 400 overlapping extension V gene fragments, can generate at least 4000 clone banks and have 99% possibility to screen all unique gene pairss.
In simple terms, chain V HAnd V LThe PCR product that coding is purified to the storehouse splices to the constant light chain of people κ coding region, shears at the recognition site of introducing PCR product end with XhoI and NotI DNA restriction endonuclease.By the standard linker, will shear and the fragment of purifying is connected to the Mammals IgG expression vector OO-VP-002 (seeing that WO2008/104183 is described) that the XhoI/NotI enzyme is cut.To connect the mixture electricity and change intestinal bacteria over to, and be added into again and contain in suitable antibiotic 2 ' YT plate overnight incubation.The carrier storehouse of adopting standard DNA method of purification (Qiagen) that the cell purification of recovering in the plate is increased.With AscI and NheI digested plasmid, be used for inserting the leading fragment of promotor.The restriction endonuclease sites of these enzymes is positioned at V HAnd V LEncoding gene between.Behind plasmid purification, insert AscI and NheI restriction endonuclease sites by the leading fragment of two-way mammalian promoter that the standard linker is cut the AscI-NheI enzyme.The plasmid that connects increases in intestinal bacteria, then adopts the standard method plasmid purification.Press the screening plasmid storehouse conversion intestinal bacteria that traditional program will generate.Being cloned in 384 orifice plates of obtaining stored.
Screening is in conjunction with the HER2 overexpressing cell
At first, cross the antibody of expression breast cancer cell line (SKBR-3) combination with HER2 in the employing Laser Scanning Confocal Microscope screening antibodies storehouse.5000 SKBR-3 cells are seeded in every hole of 384 porocyte culture plates (Perkin Elmer, cat.#6007439), cell spends the night adherent.10 μ l antibody supernatant liquors are transferred in every hole, then culture plate is hatched 2 hours, abandon afterwards the substratum in every hole, add again the new substratum of 30 μ l to every hole, goat anti-human igg (the H+L that comprises 2 μ g/mlAlexa-488 marks, Invitrogen cat.#A11013), 2 μ g/ml CellMask Blue (Invitrogen cat.#H34558) and 1 μ M Hoechst33342 (Invitrogen cat.#H3570), and then hatched 30 minutes.Again abandon substratum, washed cell, fixing with 2% formalin solution (Aldrich cat.#533998).Then use OPERA high-throughput Laser Scanning Confocal Microscope to measure fluorescence level (Perkin Elmer).
The burnt garbled data of copolymerization identifies 266 positive targets, corresponding to total clone's 3.46%.
Sequential analysis and clonal selection
The clone with the SKBR-3 Cell binding who identifies dates back 384 original orifice plates, and is transferred in the new culture plate.From the clone, extract DNA, then carry out the dna sequencing of V gene.Aligned sequences is selected all unique clones.Obtain the multiple ratio of sequence to showing each clone's uniqueness, carry out the discriminating of unique antibody.After 266 clones are carried out sequential analysis, identify the antibody sequence bunch that surpasses 70 gene uniquenesses.Somatocyte high frequency sudden change by the common precursor clone obtains each correlated series.In a word, select every bunch 1-2 clone to carry out sequence and specificity checking.Selected antibody variable region sequence is referring to the sequence table of appendix.As above release, the sequence of light chain in the sequence table comprises the identical constant light chain of people κ district, and its initial amino acid is TVAAP, and finishing amino acid is C-end NRGEC.
Sequence and specificity checking
In order to verify the antibody coding clone, prepare the DNA plasmid, and express with 2ml transfection FreeStyle CHO-S cell (Invitrogen).96 hours collection supernatants after the transfection.Anti-IgG ELISA estimates expression level with standard, and its specificity is analyzed by HER2 specific ELISA and OPERA and determined specificity in conjunction with the antibody of cell.
In simple terms, for ELISA, with 1 μ g/ml HER2 albumen (R﹠amp; D Systems cat.#1129-ER) coated NuncMaxisorb plate (cat.#464718), with the PBS dilution, 4 ℃ are spent the night.Before 50 μ l 2%Milk-PBS-T sealing, with No. 1 plank of PBS+0.05%Tween20 (PBS-T) washing.With PBS-T and No. 1 plank of 20 μ l2%milk-PBS-T washing, add 5 μ l FreeStyle CHO-S transfection thing supernatant liquors (seeing below), under room temperature, hatched 1.5 hours, then with No. 1 plank of PBS-T washing, every hole 20 μ l.Add extent of dilution and be 1: 25000 2%milk-PBS-T two anti-(HRP-goat-anti human kappa light chain, Serotec, cat.#STAR 100P), detecting the antibody of being combined with the hole, and at room temperature hatched 1 hour.Adding 25 μ l substrates (Kem-En-Tec Diagnostics, cat.#4518) before with No. 1 plank of PBS-T washing, hatched 5 minutes.After hatching, add 25 μ l 1M sulfuric acid termination reactions.Under 450nm, detect special signal with the ELISA microplate reader.Also finding in HER2ELISA has 178 clones and SKBR-3 Cell binding among 266 clones.
Table 4. is used for generating the immune schedule of anti-HER2 clone initial substance
Figure BDA00002350442700691
Figure BDA00002350442700701
The multiple overlapping extension primer mixture of table 5.RT-PCR
W=A/T, R=A/G, S=G/C, Y=C/T, K=G/T M=A/C, H=ACT, B=GCT; The Conc.-final concentration.
Table 6. nested primer collection
Figure BDA00002350442700703
K=G/T, M=A/C, D=AGT; The Conc.-final concentration.
The constant light chain splicing of table 7. κ primer collection
Figure BDA00002350442700711
Example 2: selected Anti-HER 2 function is qualitative
The test of employing vigor selects 41 unique antibody to be used for Function detection.Cell injury causes cell to keep and provide the ability of metabolism cell function and growth energy inevitably.Carry out metabolic activity based on this prerequisite and detect, usually measure mitochondria activity.Cell proliferation reagent WST-1 (Roche Cat.No.11 644 807 001) is a kind of substrate that can directly use, and is used for measuring the metabolic activity in the viable cell.Suppose that metabolic activity is relevant with viable cell quantity.In this example, the WST-1 test is used for measuring the quantity of metabolic activity cell, and these cancer cells were processed 96 hours through the different Anti-HER 2s of 2 μ g/ml.
Cancerous cell line SKBR-3 (ATCC cat.#HTB-30), BT-474 (ATCC cat.#HTB-20), NCI-N87 (ATCC cat.#CRL-5822) and MDA-453 (ATCC cat.#HTB-130) are seeded to 96 orifice plates with the concentration of 1000 cells in every hole, and its substratum comprises 2 μ g/ml Anti-HER 2s.With cell plate in incubator 37 ℃ cultivated 4 days.Then every hole adds 20 μ l WST-1 reagent, hatches 1 hour in 37 ℃.Cell plate are transferred to the track shaking table, jolting 1 hour.On the ELISA microplate reader, under 450nm and 620nm (reference wavelength), measure absorbance.Metabolic activity cell (MAC) level difference by following be calculated as the contrast supernatant percentage ratio:
Figure BDA00002350442700712
Selected antibody analysis result wherein lists single cancerous cell line and average and standard deviation data referring to table 8.Can regard as from these results, identify the HER2 antibody with functionally active, and the antibody in the antibody library shows the restraining effect to the cancerous cell lines of all or great majority test.
There is lower metabolic activity cell (MAC) percentage ratio in table 8. Anti-HER 2
The antibody numbering SKBR-3 BT-474 N87 MDA-453 Average Standard deviation
3165* 83 78 83 108 88 14
4382 94 68 88 90 85 12
4383 91 98 64 115 92 21
4384 88 66 61 78 73 12
4385 95 95 82 84 89 7
4386 87 89 47 98 80 23
4387 82 82 65 97 81 13
4517 94 83 69 81 82 10
4518 99 79 81 102 90 12
4519 98 79 105 96 95 11
* upper antibody 4380/4381 with halfcystine of 40 of light chains
Example 3: determine overlapping epi-position
In order to select to contain the Anti-HER 2 compound of the rear performance of associating synergistic effect, the antibody in each mixture should be combined with non-overlapped epi-position.Therefore, in order to study degree overlapping between Anti-HER 2, adopt surface plasma resonance technology (SPR) technology to carry out epi-position and merge.Can be at ProteOn TMXPR 36 protein-interacting array systems (Biorad Laboratories) carry out SPR and analyze.6 kinds of interactions can two-way (being defined as L and A) be measured by this system, and generating simultaneously totally 36 kinds may interact.
Arrange
ProteOn GLC sensor chip (BioRad) coupling can inject the anti-Fc antibody (Biacore of the 3600-3620 resonance units (RU) of fluidic cell L1 to L6, GE Healthcare), can use ProteOn amino coupled test kit (Biorad) by manufacturers's explanation.Adopt flow velocity 25 μ l/min, inject the Fc coupling HER2 (HER2-Fc) that 125 μ l concentration are 50nM, and catch at fluidic cell L1-L5, similarly running buffer (relevant composition is referring to following reagent part) is injected among the fluidic cell L6 simultaneously.For the free anti-Fc site of sealing before the injection Anti-HER 2, with the sealing monoclonal antibody (mAb) of 125 μ l 0.33mg/ml (Abbott) to fluidic cell L1 to L5, running buffer is injected L6, flow velocity is 25 μ l/min.The sealing monoclonal antibody was dissociated 300 seconds at least, and flow velocity is 50 μ l/min, then dissociates at least 10 seconds.Change afterwards multi-channel module to the A direction, then the 2nd injection Anti-HER 2 (adopt with the 1st time and press L direction injection Anti-HER 2 identical flow conditions and concentration).In totally 8 circulations, test the each other overlapping degree on both direction of all HER2 antibody, and mutual overlapping degree is to determine oneself's overlapping (contrast represent between two antibody 100% overlapping).In each circulation directly, available 25 μ l 3M MgCl 2(flow velocity is 50 μ l/min) regeneration antibody-EGFR mixture.
Reagent
GLC chip (Biorad, Cat.No.176-5012)
ProteOn TMAmino coupled test kit (Biorad) EDC-NHS (cat#176-2410)
Anti-Fc antibody (Biacore kit, cat#BR-1008-39)
Running buffer: PBS, 0.005%Tween-20 (PBS-T)
3M MgCl 2The regeneration damping fluid, Biacore cat.No 344-ER-050
Antigen: HER2-Fc, R﹠amp; D systems cat.No 1129-ER is redeveloped into 100 μ g/ml with PBS.
The sealing monoclonal antibody:
Figure BDA00002350442700731
(Abbott).Be dissolved as storing solution 5mg/ml with distilled water, in running buffer, be diluted to 0.33mg/ml
Anti-HER 2: 4517,4518,4519,4382,4383,4384,4385,4386,4387+ Trastuzumab With handkerchief trastuzumab analogue (wherein handkerchief trastuzumab analogue has heavy chain and the light chain amino acid of the handkerchief trastuzumab that discloses in WO2006/033700 and US 2006/0121044A1)
The result
With obtaining the RU maximum value after running buffer reference substance and the baseline correction normalization method, and determine by the RU value of each Anti-HER 2 before and after relatively finishing to realize the inhibition degree of the Anti-HER 2 that detects by the reporting point of injecting each sample front and back immediate record.Typical XPR ProteOn circulation as shown in Figure 1.The first and second antibody overlapping/be based on the first and second antibody in conjunction with the calculating that suppresses percentage ratio before and after the RU value difference different, the identical representatives 100% of such the first and second antibody RU values overlapping (i.e. 100% inhibition).At least carry out 2 experiments and confirm that there is inhibition in 2 kinds of antibody in 2 directions.With inhibiting value 50-100% (at least computation of mean values from 2 independent experiments) as prompting in conjunction with overlapping epi-position or near the antibody of the epi-position of antigen to a sign of significance competition, it is not to be close to especially to the epi-position of identifying that the while inhibiting value is lower than 50% prompting antibody, can reduce so sterically hindered.Inhibiting value is lower than 25% will not be included into the analysis of overlapping epi-position, not represent non-significance and will suppress because judge it.The below lists respectively the antibody pair of 50-100% and 25-50% inhibition degree.
The antibody of 50-100% inhibition degree is to making up:
4517 and 4518
4517 and Trastuzumab
4384 and 4518
4382 and 4519
4382 and 4383
4382 and handkerchief trastuzumab analogue
4383 and 4387
4383 and handkerchief trastuzumab analogue
4384 and 4386
4385 and 4387
The antibody of 25-50% inhibition degree is to making up:
4387 and 4519
4519 and handkerchief trastuzumab analogue
4382 and 4387
4383 and 4387
4387 and handkerchief trastuzumab analogue
Fig. 1 shows the representative epi-position amalgamation result of Anti-HER 2 of the present invention." HER2 " refers to that Fc-coupling HER2 is in conjunction with the phase." sealing " refer to blocking antibody Synagis with seal free anti-Fc site in conjunction with the phase.Block site refers to " the free anti-Fc site of having sealed " by RU value difference value representative between " HER2 " end and " sealing " end (in both cases, determining namely 600 and 1500 seconds in the free end of term)." mAb1 → " refer to the first binding time to Anti-HER 2." ROTATE CHIP " point to second to skew." mAb2 ↓ " refers to the second binding time to Anti-HER 2.Suppress percentage ratio and be calculated as primary antibodie in conjunction with (being referred to as " mAb1 combination ") RU part, two anti-(being referred to as " mAb2 combination ") are in conjunction with rear acquisition.In this example, 2 running buffers of injection in sample A1, the same antibody of injection in sample A2, A2 antibody and sample A3, A4,4 kinds of different antibodies among A5 and the A6 are together injected.Observe sample A2 self-overlapping (mAb2 ↓ during RU significantly change; By the indication of lower right cursor), but between A2 antibody and other antibody, do not observe significantly overlapping.
The function of example 4:2 kind or 3 kinds of Anti-HER 2 mixtures is qualitative
This example has been described the vitro detection of all 2 kinds or the 3 kinds mixtures of antibodies in the Anti-HER 2 among the present invention that 10 kinds of selecting have been determined at people HER2 combination.The assessment mixtures of antibodies suppresses the growth of 3 kinds of different carcinoma clones: N87 (cancer of the stomach), SKBR-3 (mammary cancer) and BT474 (mammary cancer).
Method
Detect antibody 4380,4381,4382,4383,4384,4385,4387,4517,4518 and 4519 in all mixtures of 2 kinds or 3 kinds antibody, each antibody is determined at people HER2 receptors bind, to determine to have the antibody combination of best validity.For example, the method for the preparation of different antibodies combination in 384 orifice plates sees WO 2010/040356 for details.Details are described below.
Plant mixtures of antibodies
10 kinds of antibody are diluted to 25 μ g/ml with 1xPBS, add in 384 orifice plates 100 μ l antibody-solutions for the preparation of for detection of 2 kinds of mixtures of antibodies.
For 3 kinds of clones that detect, each uses 2 384 orifice plates, and 46 μ l are contained correlated measure cell (N87:3000 cell; SKBR-3:1000 cell; BT474:2000 cell) substratum is added in the hole.Adopt Biomek3000 laboratory work station (Beckman Coulter), from raise plate, take out 2 kinds of different antibodies of 2 μ l and add in 384 orifice bores that contain substratum and cell, comprise like this all combinations of 2 kinds of different antibodies.In addition, culture plate comprises substratum control wells (50 μ l 1xPBS substratum; Acellular), untreated control wells (50 μ l 1xPBS substratum+cells; Without antibody) and contain 4 μ l Trastuzumabs or handkerchief trastuzumab analogue and only contain one of 10 kinds of antibody of the present invention as the culture hole (except 46 μ l substratum+extracellulars) of the substratum of extra check as substratum or the 4 μ l of reference antibody.
Contain culture hole, cultivation datum hole, the untreated control hole of 2 kinds of mixtures of antibodies and contain Trastuzumab/handkerchief trastuzumab or a kind of antibody of the present invention, in 37 ℃ of incubators, hatched 4 days, then in all relevant hole of culture plate, add the cell proliferation reagent WST-1 that 5 μ l dilute with 1xPBS at 1: 1.Place again 37 ℃ to hatch 1 hour culture plate, then be transferred on the track shaking table, hatched again 1 hour.On the ELISA microplate reader, measure absorbance in 450nm and 620nm (reference wavelength).Metabolic activity cell (MAC) quantity is by the following percentage ratio that is calculated as untreated control:
Suppose that metabolic activity is relevant with viable cell quantity, %MAC numerical value hangs down the inhibition degree corresponding to the antibody cell growth of higher level.
3 kinds of mixtures of antibodies
The antibody of 10 kinds of identical empirical tests and people HER2 receptors bind is diluted to 16.67 μ g/ml with 1xPBS, add in 384 orifice plates 100 μ l antibody-solutions for the preparation of for detection of 3 kinds of mixtures of antibodies.
3 kinds of identical clones of vitro detection, i.e. N87, SKBR-3 and BT474.For each clone that detects, use 10 384 different orifice plates, the 44 μ l substratum that will contain the equal amts cell add in the hand-hole.Adopt Biomek3000 laboratory work station (Beckman Coulter), from raise plate, take out 3 kinds of different antibodies of 2 μ l and add in 384 orifice bores that contain substratum and cell, comprise like this all combinations of 3 kinds of different antibodies.In addition, culture plate comprises substratum control wells (50 μ l 1xPBS substratum; Acellular), untreated control wells (50 μ l 1xPBS substratum+cells; Without antibody) and contain 6 μ l Trastuzumabs or handkerchief trastuzumab analogue and only contain one of 10 kinds of antibody of the present invention as the culture hole (except 44 μ l substratum+extracellulars) of the substratum of extra check as substratum or the 6 μ l of reference antibody.
Contain culture hole, cultivation datum hole, the untreated control hole of 3 kinds of mixtures of antibodies and contain Trastuzumab/handkerchief trastuzumab or a kind of antibody of the present invention, in 37 ℃ of incubators, hatched 4 days, then in all relevant hole of culture plate, add the cell proliferation reagent WST-1 that 5 μ l dilute with 1xPBS at 1: 1.Place again 37 ℃ to hatch 1 hour culture plate, then be transferred on the track shaking table, hatched again 1 hour.Measure absorbance in 450nm and 620nm (reference wavelength), and by above-mentioned calculating %MAC.
The result
The people HER2 of all 2 kinds or 3 kinds of mixtures Anti-HER 2 of being combined with to(for) selected 10 kinds of conclusive evidences, the assessment phase is suppressed the ability of 3 kinds of cancerous cell line N87 (cancer of the stomach), SKBR-3 (mammary cancer) and BT474 (mammary cancer).Calculate the %MAC of 10 kinds of monoclonal antibodies and 2 kinds or 3 kinds mixtures.Then sort according to the effect of mixture cell growth, table 8 has been listed have the highest average curative effect 55 kinds of mixtures of (the highest average %MAC is based on these 3 kinds of clone results' average).
The result shows that the effect difference that various mixture cell growth suppress between the different clones is very large, although the difference of average %MAC is so not large.Although selected 55 kinds of mixtures of antibodies listing in the table 8 based on average %MAC, but the result according to any one or more clone can with single mixtures of antibodies as research object, only have high inhibition level (low %MAC) can be understood in a kind of body for the very effective antibody combination of certain class cancer at the single cell cording.
The level that 55 kinds of mixtures the most effective that table 8 is comprised of 2 kinds or 3 kinds of Anti-HER 2s suppress growth of cancer cells in 3 kinds of cancerous cell lines (N87, BT474 and SKBR-3).Suppress water-glass and be shown metabolic activity percentage of cells (%MAC.)
Figure BDA00002350442700771
Example 5:ADCC and CDC measure
In order to study HER2 specific antibody mixture of the present invention in the ability of external evoked antibody dependent cellular cytotoxicity (ADCC) and CDC (CDC), adopt N87 and/or SKBR3 as target cell to by 2,3 or 20 kinds of different Anti-HER 2 mixtures forming of 4 kind of antibody test.
Method
External ADCC
Adopt Lymphoprep TMIsolation medium is separating periphery blood monocytic cell (PBMC) from the blood of Healthy Volunteers, and with its action effect cell.To express the N87 of HER2 and SKBR3 cell as targeted cells.At 37 ℃ lower per 10 7Individual cell with 334 μ Ci Sodium chromates ( 51Cr, 334) mark is 1 hour.After the washing, with targeted cells (5x10 3/ hole) with Anti-HER 2 under 37 ℃ in the RPMI-1640+10%FCS+1% mycillin preincubate 30 minutes, then according to the effector cell: targeted cells (E/T) ratio adds the effector cell at 50: 1.Cell was additionally hatched under 37 ℃ 4 hours, then use TopCount microwell plate scintillometer by detecting in the substratum 51The content of Cr detects lysis.By to containing 51Add 2%Triton-X100 in the hole of Cr labeled cell and obtain maximum cracking.From containing effector cell and targeted cells but do not contain and measure synchronous cracking situation the hole of antibody.
Calculate by the following method specific cytotoxic:
Compare with positive control antibody Trastuzumab, press the relative cytotoxicity of following calculating:
The relative cytotoxicity of %=(antibody X cell toxicity %/Trastuzumab cytotoxicity %) * 100
External CDC
Use separates the serum that obtains from fresh extraction centrifugal blood and originates as complement.To express the N87 cell of HER2 as targeted cells.At 37 ℃ lower per 10 7Individual cell with 334 μ Ci Sodium chromates ( 51Cr, 334) mark is 1 hour.After the washing, with targeted cells (5x10 3/ hole, 100 μ l) add in the Anti-HER 2 with RPMI-1640+10%FCS+1% mycillin (50 μ l) dilution, then add fresh serum (50 μ l).In under 37 ℃ cell being hatched in serum and antibody 3 hours, then use TopCount microwell plate scintillometer by detecting in the substratum 51The content of Cr detects lysis.By to containing 51Add 2%Triton-X100 in the hole of Cr labeled cell and obtain maximum cracking.Measure synchronous cracking situation from containing targeted cells but the hole of serum-free.
Press following calculating cytotoxicity percentage ratio:
Figure BDA00002350442700801
The result
20 kinds of antibody combination of test all can abduction delivering HER2 N87 and SKBR3 clone ADCC occurs.When antibody concentration was 0.1 μ g/ml, in the 16-24% scope, this compared with Trastuzumab to the maximum special toxicity of N87 clone for Anti-HER 2 combination, relatively cracking percentage ratio 46-69% quite (referring to Fig. 2 A and 2B and Fig. 3 A and 3B).In the SKBR3 cell, when antibody concentration was 0.1 μ g/ml, the specificity ADCC percentage ratio of inducing was in the 20-34% scope, with the relative cytotoxicity 48-83% (referring to Fig. 2 C and 2D and Fig. 3 C and 3D) corresponding to Trastuzumab.
Opposite with the ADCC test-results, Trastuzumab is induced targeted cells generation cracking, but Trastuzumab can't be induced CDC (Figure 4 and 5).On the contrary, contain among the present invention 2,3 or 16 kinds of Anti-HER 2 mixtures of 4 kind of antibody can induce CDC, 5%-31% is special to walk alone, average is 12% (Figure 4 and 5).Do not observe CDC when using the SKBR3 cell.Onrelevant between antibody quantity in the mixture and the maximum cell toxicity percentage ratio of inducing, contain 2,3 or the mixture of 4 kind of antibody be front 4 kinds of mixtures of antibodies (Figure 4 and 5) of inducing CDC.
This illustration is bright, and all Anti-HER 2 mixtures all show very high ADCC and CDC level among the present invention.In the test, the mechanism of action except ADCC and CDC may play a role in the effect of mixtures of antibodies in vivo.Especially, mixtures of antibodies of the present invention can be combined with non-overlapping HER2 epi-position, and this can cause high-caliber acceptor internalization, can improve antitumous effect like this.Therefore result shown in this example can unite with example 4 and show that mixtures of antibodies of the present invention shows high-caliber growth of cancer cells usually to be suppressed in one or more test cell systems, simultaneously in the example 4 institute's growth-inhibiting of releasing not necessarily with, ADCC Horizontal correlation for example.
Example 6: mixtures of antibodies is to the titration of different carcinoma clone
In this example, adopt the quantity of WST-1 experimental measurement metabolism active cells after the different antibodies mixture process of different concns described in example 2 and the example 4.Test antibody mixture in a plurality of cancerous cell lines is listed the result of 4 kinds of clones: N87 (cancer of the stomach), HCC202 (mammary cancer), BT474 (mammary cancer) and ZR-75-30 (mammary cancer) in this example.
Method
Before carrying out WST-1 test, with individual cells be suitable medium (having replenished 2%FBS and 1%P/S) with mixtures of antibodies of the present invention and control antibodies (
Figure BDA00002350442700811
Negative control antibodies, the positive control antibodies of handkerchief trastuzumab analogue) being diluted to total antibody final concentration is 100 μ g/ml, and the antibody final concentration is 50 μ g/ml in the hole that antibody concentration is the highest like this.Antibody is carried out 3 times of serial dilutions.With correlated measure cell (N87:3000 cell; HCC202, a BT474 and ZR-75-30:2000 cell) be added in the hole of 384 orifice plates, then plank is placed incubator under 37 ℃, to hatch 4 days.Then in all relevant hole of culture plate, add reagent WST-1.Place again 37 ℃ to hatch 1 hour culture plate, then be transferred on the track shaking table, hatched again 1 hour.Measure absorbance in 450nm and 620nm (reference wavelength), and calculate as stated above the metabolic activity cell and account for untreated control percentage ratio (%MAC).
The result
4 kinds of mixtures of antibodies are to clone N87, HCC202, and the titration results of BT474 and ZR-75-30 is shown in accompanying drawing 6-9.In addition, table 9 shows the IC of 4 kinds of mixtures of antibodies and handkerchief trastuzumab analogue 50Value (the maximum inhibition concentration of half) and maximum inhibition level.(only it should be noted that and below the situation that corresponding maximum inhibition level is more or less the same each other, can compare IC 50Value).Can find out that from accompanying drawing and table 9 mixture shows inhibition and the effect of different levels in the different clones of test.But these 4 kinds of mixtures are suppressing aspect 4 kinds of cell line growths all owing to the handkerchief trastuzumab.
The IC that table 9.4 kind of Anti-HER 2 compound and inhibition handkerchief trastuzumab analogue suppress 4 kinds of clones 50Value and curative effect
Figure BDA00002350442700812
Figure BDA00002350442700821
Example 7: the Anti-HER 2 mixture is the curative effect in the N87 model in nude mice in vivo
In nude mice NCI-N87 transplantation model, studied Anti-HER 2 mixture 4384+4517,4382+4518, the interior curative effect of 4382+4385+4518 and 4382+4387+4518.This model is widely used in effectiveness and the curative effect of Effect of Anti carcinoma monoclonal antibody, comprises Anti-HER 2.The nude mice hypoimmunity lacks the T cell, and the human cell can grow in mouse like this.
Method
With 10 7Individual NCI-N87 cell is inoculated in the left side subcutaneous area of female nude mouse in 8 ages in week.Use the kind of calliper tumor size 2 times weekly, and calculate gross tumor volume (mm according to following formula 3): (width) 2X length x 0.5.It is 300mm that tumour is estimated size 3, mouse is carried out randomization distributes and begin treatment.Inject 2 50mg/kg4384+4517 to mouse peritoneal weekly, 4382+4318,4382+4385+4518 or 4382+4387+4517 in totally 5 weeks (injecting altogether 10 times), then observed for 3 weeks.This experiment also comprises anti-HER 2 monoclonal antibody Herceptin (Trastuzumab
Figure BDA00002350442700822
), its dosage and schedule are consistent with the Anti-HER 2 mixture.
The result
This experimental result is referring to Figure 10.The 59th day the time, the tumour mean size is 300mm behind tumor inoculation 3, the mouse random assignment is entered in 6 groups, every group of 10 animals begin to give 50mg/kg 4382+4518,4384+4517,4382+4385+4518,4382+4387+4517, Herceptin and vehicle (control group) treatment.Originally, the tumour in all treatment groups is grown always, until (the 66th day) begins to occur atrophy after treating for 1 week.In the mouse that gives the treatment of 4384+4517 or Herceptin, tumour is only compared with the big or small 300mm that begins 3Smaller, behind inoculated tumour, began in the 83rd day and the 73rd day respectively afterwards to increase.Afterwards, the growth kinetics of animal tumor and vehicle control group are similar in the treatment group.On the contrary, through 4382+4518, the mouse of 4382+4385+4518 or 4382+4387+4517 treatment with compare through Herceptin, show tumor suppression and prolong and significantly improve.When the treatment phase finishes (the 91st day), the tumor growth of 3 groups suppresses all to reduce, and tumour is stopping to begin slow growth after the treatment.
In a word, compare with Herceptin, accept anti-HER2 mixture 4382+4518, the animal of 4382+4385+4518 and 4382+4387+4517 treatment shows N87 tumour transplatation growth-inhibiting and significantly improves.
Example 8: the interior curative effect of anti-HER2 mixture in the OE19 model in nude mice
In nude mice OE19 cancer of the stomach transplantation model, studied Anti-HER 2 mixture 4384+4517,4382+4518, the interior curative effect of 4382+4385+4518 and 4382+4387+4518.
Method
With 5x10 6Individual OE19 cell is inoculated in the left side subcutaneous area of female nude mouse in 8 ages in week.Use the kind of calliper tumor size 2 times weekly, and calculate gross tumor volume (mm according to following formula 3): (width) 2X length x 0.5.It is 110mm that tumour is estimated size 3, mouse is carried out randomization distributes and begin treatment.Inject 2 50mg/kg4384+4517 to mouse peritoneal weekly, 4382+4318,4382+4385+4518 or 4382+4387+4517 in totally 5 weeks (injecting altogether 10 times), then observed for 3 weeks.This experiment also comprises anti-HER 2 monoclonal antibody Herceptin (Trastuzumab
Figure BDA00002350442700831
), its dosage and schedule are consistent with the Anti-HER 2 mixture.
The result
This experimental result is referring to Figure 11.After the treatment, namely in 4382+4385+4518 and 4382+4387+4517 treatment group, observe the tumor growth restraining effect of comparing with control group first.In the time of the 23rd day, the equal atrophy of the mouse tumor of 4382+4385+4518 treatment group, and significantly be better than Herceptin; This improvement continues during the whole research.Behind inoculated tumour the 37th day, namely finish after 7 times in 10 treatments, in 8 mouse 6 the complete atrophy of tumour is arranged in this group, and when the research end fully without tumour.In the 4382+4387+4517 treatment group, except 1 mouse, the equal atrophy of the tumour of all the other mouse.From the 32nd day, the mixture of 3 kinds of antibody was evident in efficacy than Herceptin.In the time of the 37th day, there are 4 not have tumour fully in 7 animals in this group, and continue to the research end.There is 1 mouse that treatment is not produced and replys, because gross tumor volume is excessive, in the time of the 53rd day, implement euthanasia.Mouse through the 4384+4517 treatment is also replied well treatment, shows than the remarkable better curative effect of Herceptin in the 28th day behind tumor inoculation.In the time of the 43rd day, 3 fully not treatments are arranged in 8 mouse of this group.Through the mouse of 4382+4518 treatment from the 16th day (begin treatment) to the 28th day, tumor growth all is under the state of a control, compares with Herceptin with vehicle group afterwards, Antybody therapy still suppresses tumor growth, although tumor size slowly increases.However, compare with Herceptin, in the time of the 44th day, until research finishes (the 67th day), 4382+4518 suppressed the evident in efficacy better of tumor growth from the 53rd day.
In a word, all Anti-HER 2 mixtures of the present invention are compared with Herceptin in the OE19 model in nude mice, and the effect of its inhibition and control tumor growth is significantly better.
Example 9
This illustration is bright, and HER2 degraded and the HER3 phosphorylation of blocking simultaneously the HER2 mediation can farthest suppress HER2 dependency cancerous cell line.Obtain best double blocking effect in the mixture of 3 kinds of Anti-HER 2s, 2 kinds of antibody in the mixture can effectively be induced the HER2 degraded, and the compensatory signal transduction of the 3rd kind of antibody HER2/HER3 heterodimer capable of blocking.It is generally acknowledged that compensatory raises the compensatory signal transduction (referring to example 11) that HER3 can partly accelerate the HER2/HER3 heterodimer.
Method
For the inherent mechanism of Effect of Anti HER2 mixtures of antibodies growth-inhibiting effect, in gastric carcinoma cell lines NCI-N87, study EGFR, the phosphorylation level of HER2 and HER3.The NCI-N87 cell adopts 10nM HER3 part to transfer albumen-β to stimulate 15 minutes after antibody spends the night pre-treatment, then its full cell lysate is carried out the protein immunoblotting analysis.Cell is inoculated in 6 orifice plates, when cell reaches 80% degrees of fusion, removes substratum, use the 1xPBS washed cell, and process with 10 μ g/ml antibody (containing the substratum dilution of 0.5%FBS with 2ml).After the processing of spending the night, then transfer albumen-β to stimulate 15 minutes with 10nM HER3 part.With 2ml 1xPBS washed cell, then add 200 μ l1xNuPAGELDS sample buffers.10-15 μ l sample is carried out the protein immunoblotting analysis, adopt anti-EGFR, pEGFR (Tyr1068), HER2, pHER2 (Tyr1221), the primary antibodie of HER3 or pHER3 (Tyr1289) detects.
The result
Transfer protein induced HER2 for antibody-mediated inhibition, the analytical results of HER3 and EGFR phosphorylation shows (Figure 12), and Anti-HER 2 and antibody compound are to EGFR, and HER2 and HER3 phosphorylation state and HER2 level have not same-action.But transfer protein induced HER3 on EGFR and HER2 phosphorylation level without impact.Only monoclonal antibody 4382 and the HER3 phosphorylation of inducing with reference to monoclonal antibody handkerchief trastuzumab analogue part capable of blocking.These results show that 4382 can block HER2 and HER3 allos dimerization.All mixtures of antibodies all can reduce EGFR and HER2 phosphorylation level and HER2 level.But only containing 4382 mixture can induce simultaneously the HER2 degraded and suppress EGFR and HER2 phosphorylation and HER3 phosphorylation.These results also show, contain the mixture (4382+4385+4518 and 4382+4387+4517) of 3 kinds of antibody at the mixture (4382+4518 and the Herceptin+handkerchief trastuzumab analogue), particularly HER2 that are better than 2 kinds of antibody aspect 3 kinds of receptor phosphorylations of blocking-up.It is generally acknowledged, contain the mixture of 3 kinds of antibody because it has the HER2 phosphorylation (by antibody 4382) of inducing simultaneously HER2 degraded (by the antibody in the corresponding mixture to 4385+4518 or 4387+4517) and block ligand mediation.These results and our pharmacology data have good dependency; pharmacology data shows; in vivo with vitro inhibition cell and tumor growth aspect; the mixture of 3 kinds of Anti-HER 2s is better than single anti-HER 2 monoclonal antibody, and usually also is better than the mixture (referring to example 4 and 6) of 2 kinds of Anti-HER 2s.
Figure 13 A and B show respectively the hypothesis mechanism of HER2/HER3 signal transduction system synoptic diagram and 3 kinds of Anti-HER 2 mixture superiority.Figure 13 A left side shows the carcinogenic signal path of HER2 homodimer mediation, but this path hyperamization pipe generates, cell migration and proliferation.Figure 13 A right side shows after HER3 part and the HER3 receptors bind by the signal path of HER2/HER3 heterodimer mediation (usually owing to raise HER3 after a kind of Anti-HER 2 is processed cause).This can be by impact mechanism, and for example albumen is synthetic, propagation and carbohydrate metabolism cause carcinogenic signal transduction.Figure 13 B shows the hypothesis mechanism of 3 kinds of Anti-HER 2 mixture blocking-up HER2 homodimers of the present invention and the carcinogenic signal path of HER2/HER3 heterodimer.2 kinds of Anti-HER 2s of being combined with the non-overlapped epi-position of HER2 of left diagram can form a kind of cross-coupling acceptor-antibody grid structure, and this can cause HER2 acceptor internalization and degraded.Figure 13 B right side shows the 3rd kind of mechanism that Anti-HER 2 blocking-up HER2/HER3 heterodimer compensatory forms can being combined with HER2 dimer arm, it also blocks the signal path of HER3 mediation simultaneously, and antagonism HER2 Antybody therapy produced resistance due to obstruction HER3 raised.
Example 10
This illustration is bright, can effectively induce the HER2 degraded in conjunction with the Anti-HER 2 compound of non-overlapped epi-position in multiple human cancer cell line.
Method
The acceptor palliating degradation degree of inducing in order to study the Anti-HER 2 compound, to through 48 hours ZR-75-30 of 10 μ g/ml antibody treatment, NCI-N87, the full cell lysate of BT474 and HCC202 carries out the protein immunoblotting analysis.Cell cultures in the T-75 culturing bottle, is removed substratum when cell reaches 80% degrees of fusion, use the 1xPBS washed cell, and process with 20 μ g/ml antibody (containing the substratum dilution of 0.5%FBS with 5ml).After the processing of spending the night, adopt standard RIPA damping fluid to prepare full cell lysate.Determine the total protein content in every duplicate samples, then 1-5 μ g sample is carried out the protein immunoblotting analysis, adopt the primary antibodie of anti-HER2 to detect.
The result
The result of study (Figure 14) of target degraded proves that the mixtures of antibodies of being combined with non-overlapped epi-position (4384+4517,4382+4385+4518 and 4382+4387+4517) can be induced HER2 degraded in all cells system.Mixture 4382+4518 and Herceptin+handkerchief trastuzumab analogue is renderd a service slightly low, and this points out the epi-position of these antibody is not to be optimum for inducing the antibody degraded.3 kinds of mixtures of antibodies are all stronger than the mixture effectiveness of single anti-HER 2 monoclonal antibody and 2 kinds of Anti-HER 2s, and the mixture that particularly contains antibody 4382+4385+4518 is renderd a service the strongest.Herceptin or handkerchief trastuzumab analogue all can not be induced the HER2 degraded, and Herceptin is induced in ZR-75-30 clone except the HER2 degraded.These results show can induce its degraded for the mixtures of antibodies of the non-overlapped epi-position of HER2, but mixture to have different effectiveness.
Example 11
This illustration is bright, thereby some cancerous cell line can be replied rise HER3 level to keep the carcinogenic signal path of HER2/HER3 mediation to target HER2.
Method
Reply impact on the HER3 level in order to study target HER2, NCI-N87 (cancer of the stomach) and HCC202 (mammary cancer) cell are through 20 μ g/ml antibody (Herceptin, handkerchief trastuzumab analogue, Herceptin+handkerchief trastuzumab analogue, 4384+4517,4382+4518,4382+4385+4518 or 4382+4387+4517)) processing of spending the night.Cell cultures in the T-75 culturing bottle, is removed substratum when cell reaches 80% degrees of fusion, use the 1xPBS washed cell, and process with 20 μ g/ml antibody or mixtures of antibodies (containing the substratum dilution of 0.5%FBS with 5ml).Cell adopts standard RIPA damping fluid to prepare full cell lysate after spending the night.Determine the total protein content in every duplicate samples, then 1 or 10 μ g samples are carried out the protein immunoblotting analysis, adopt the primary antibodie of anti-HER2 and HER3 to detect.
The result
The horizontal result of study of HER3 (Figure 15) proves that target HER2 can cause HER3 rise in 2 kinds of different clones.This result shows that simultaneously cell most probable after the mixtures of antibodies of inducing the HER2 degraded is processed raises HER3.
Example 12: the degraded of HER2 in the cancerous cell line of antibody treatment
This illustration is bright, can induce the HER2 degraded in conjunction with the Anti-HER 2 mixture of non-overlapped epi-position, the combination of 3 kinds of antibody 4382+4385+4518 is renderd a service byer force than the induced degradation aspect that is combined in of 2 kinds of antibody, and antibody 4385+4518 is the main drive that drives the HER2 internalization in the 4382+4385+4518 mixture.
Method
The HER2 acceptor Degradation Level of inducing in order to study mixtures of antibodies, OE19 (cancer of the stomach), NCI-N87 (cancer of the stomach), ZR-75-30 (mammary cancer), HCC202 (mammary cancer) and BT474 (mammary cancer) cell carry out the protein immunoblotting analysis to its full cell lysate after 10 μ g/ml monoclonal antibodies or the mixtures of antibodies that contains 2 kinds or 3 kinds antibody are processed 48 hours.Cell cultures in the T-75 culturing bottle, is removed substratum when cell reaches 80% degrees of fusion, use the 1xPBS washed cell, and process with 10 μ g/ml antibody or mixtures of antibodies (containing the substratum dilution of 0.5%FBS with 5ml).After hatching 48 hours, adopt standard RIPA damping fluid to prepare full cell lysate.Determine the total protein content in every duplicate samples, then 1-5 μ g sample is carried out the protein immunoblotting analysis, adopt the primary antibodie of anti-HER2 and actin to detect.Negative control antibody (anti-RSV (respiratory syncytial virus) antibody of debond HER2), Herceptin, handkerchief trastuzumab analogue, handkerchief trastuzumab+Herceptin analogue, antibody 4382,4385 and 4518 and the mixture of 2 kinds or 3 kinds are wherein adopted in antibody test.
The result
The result shows (Figure 16), and in the monoclonal antibody and mixtures of antibodies of test, mixtures of antibodies 4382+4385+4518 induces the degraded of HER2 target to render a service the strongest in the clone of all tests.In addition, 4385+4518 induces the HER2 degraded to render a service the strongest in the mixture that contains 2 kinds of antibody.Antibody change in other 2 kinds of 4382+4385+4518 mixtures of antibodies, i.e. 4382+4385 and 4382+4518, induce render a service aspect the target internalization a little less than.This explanation, antibody 4385 and 4518 are the main drives that drive the HER2 internalization in the 4382+4385+4518 mixtures of antibodies.
Example 13: the growth-inhibiting effect of different components in a kind of mixtures of antibodies
In this example, adopt vigor detection (WST-1) described in the example 2,4 and 6 to determine the antiproliferative effect of different compositions in the 4382+4385+4518 mixtures of antibodies.In addition, substitute antibody 4382 with handkerchief trastuzumab analogue and form new mixtures of antibodies, also it is assessed similarly to determine whether the antibody with similar binding specificity and biology department's function can substitute each other.Described in example 3, antibody 4382 and handkerchief trastuzumab analogue can be gone up contiguous epi-position in conjunction with overlapping epi-position or HER2, and 2 kinds of HER3 phosphorylations (example 9) that antibody part capable of blocking is induced.In multiple cancerous cell line, test mixtures of antibodies, comprised NCI-N87 (cancer of the stomach), OE19 (cancer of the stomach), HCC202 (mammary cancer), BT474 (mammary cancer) and SKBR3.
The result
Different antibodies mixture burette test result shows, all 2 kinds of antibody combinations in the 4382+4385+4518 mixtures of antibodies, i.e. and 4382+4385,4382+4518 and 4385+4518 all can the anticancer growths.But the mixture 4382+4385+4518 of these 3 kinds of antibody has effectiveness in suppressing the growth of test cell system the strongest.3 kinds of clone NCI-N87, the burette test result of BT474 and HCC202 is shown in Figure 17-19.
Can find out that from Figure 17-19 when antibody 4382 was substituted by a kind of antibody (handkerchief trastuzumab) with HER3 phosphorylation ability that similar binding specificity and block ligand induce, its growth-inhibiting effect still kept.Compare with alone handkerchief trastuzumab analogue, the combination of handkerchief trastuzumab analogue and antibody 4385 or 4518, or both cell growth inhibitions of combination are stronger.But the combination that contains antibody 4382 is stronger than the respective combination that contains handkerchief trastuzumab analogue effect in all test cell systems.
Figure IDA00002350443300011
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Figure IDA00002350443300391

Claims (42)

1. antibody compositions, at least formed by first, second, and third anti--HER2 recombinant antibodies that can different epi-position combinations from HER2, wherein the combination of the first and second antibody and HER2 causes HER2 acceptor internalization, and the combination of the 3rd antibody and HER2 causes part to induce the inhibition of HER3 phosphorylation.
2. antibody compositions described in the claim 1, wherein the first and second antibody can be combined with the HER2 of cell surface, generate a kind of antibody of coupling-be subjected to volume mesh at cell surface by this way; And the 3rd antibody is combined with HER2, blocks by this way the allos dimerization between HER2 and the HER3.
3. the antibody compositions described in the claim 1 or 2, wherein:
(a) this anti--HER2 first antibody comprises:
The heavy chain CDR3 sequence of antibody 4517 (SEQ ID NO:47) and light chain CDR3 sequence (SEQ ID NO:76), perhaps
The heavy chain CDR3 sequence of antibody 4518 (SEQ ID NO:50) and light chain CDR3 sequence (SEQ ID NO:78);
(b) this anti--HER2 second antibody comprises:
The heavy chain CDR3 sequence of antibody 4380 (SEQ ID NO:53) and light chain CDR3 sequence (SEQ ID NO:80),
The heavy chain CDR3 sequence of antibody 4385 (SEQ ID NO:65) and light chain CDR3 sequence (SEQ ID NO:88), perhaps
The heavy chain CDR3 sequence of antibody 4387 (SEQ ID NO:71) and light chain CDR3 sequence (SEQ ID NO:92); With
(c) this anti--HER2 the 3rd antibody comprises:
The heavy chain CDR3 sequence of antibody 4382 (SEQ ID NO:56) and light chain CDR3 sequence (SEQ ID NO:82),
The heavy chain CDR3 sequence of antibody 4383 (SEQ ID NO:59) and light chain CDR3 sequence (SEQ ID NO:84), perhaps
The heavy chain CDR3 sequence of antibody 4519 (SEQ ID NO:74) and light chain CDR3 sequence (SEQ ID NO:93).
4. the antibody compositions described in the claim 1 or 2, wherein:
(a) this anti-HER2 first antibody comprises:
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4517 variable region of heavy chain (SEQ ID NO:2) and CDR3 and variable region of light chain (SEQ ID NO:4), perhaps
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4518 variable region of heavy chain (SEQ ID NO:6) and CDR3 and variable region of light chain (SEQ ID NO:8);
(b) this anti--HER2 second antibody comprises:
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4380 variable region of heavy chain (SEQ ID NO:10) and CDR3 and variable region of light chain (SEQ ID NO:12),
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4385 variable region of heavy chain (SEQ ID NO:26) and CDR3 and variable region of light chain (SEQ ID NO:28), perhaps
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4387 variable region of heavy chain (SEQ ID NO:34) and CDR3 and variable region of light chain (SEQ ID NO:36); And
(c) this anti--HER2 the 3rd antibody comprises:
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4382 variable region of heavy chain (SEQ ID NO:14) and CDR3 and variable region of light chain (SEQ ID NO:16),
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4383 variable region of heavy chain (SEQ ID NO:18) and CDR3 and variable region of light chain (SEQ ID NO:20), perhaps
CDR1, CDR2 and the CDR3 of CDR1, the CDR2 of antibody 4519 variable region of heavy chain (SEQ ID NO:38) and CDR3 and variable region of light chain (SEQ ID NO:40).
5. antibody compositions described in the claim 1 or 2 wherein should comprise CDR1, the CDR2 of antibody 4382 variable region of heavy chain (SEQ ID NO:14) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:16) by anti--HER2 the 3rd antibody.
6. the antibody compositions described in the claim 5, wherein:
(a) this anti--HER2 first antibody comprises CDR1, the CDR2 of antibody 4518 variable region of heavy chain (SEQ ID NO:6) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:8),
Should anti--HER2 second antibody comprise CDR1, the CDR2 of antibody 4385 variable region of heavy chain (SEQ ID NO:26) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:28); Perhaps
(b) this anti--HER2 first antibody comprises CDR1, the CDR2 of antibody 4517 variable region of heavy chain (SEQ ID NO:2) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:4), and
Should anti--HER2 second antibody comprise CDR1, the CDR2 of antibody 4387 variable region of heavy chain (SEQ ID NO:34) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:36).
7. antibody compositions, at least by can different epi-position combinations from HER2 anti--HER2 first, second, and third recombinant antibodies forms, wherein the first and second antibody are combined with HER2 and can be caused HER2 acceptor internalization, and the 3rd antibody is combined with HER2 and is caused part to induce the inhibition of HER2 phosphorylation, and wherein said each first, second, and third anti--HER2 antibody is all also competed with it combination with the identical epi-position combination of corresponding first, second, and third antibody described in the claim 4-6.
8. an antibody compositions comprises resisting-HER2 the first and second recombinant antibodies of different epi-position combinations from HER2 at least, and wherein at least a described antibody is selected from following:
(a) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:56) and the light chain CDR3 sequence (SEQID NO:82) of antibody 4382;
(b) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:59) and the light chain CDR3 sequence (SEQID NO:84) of antibody 4383;
(c) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:62) and the light chain CDR3 sequence (SEQID NO:86) of antibody 4384;
(d) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:65) and the light chain CDR3 sequence (SEQID NO:88) of antibody 4385;
(e) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:68) and the light chain CDR3 sequence (SEQID NO:90) of antibody 4386;
(f) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:71) and the light chain CDR3 sequence (SEQID NO:92) of antibody 4387;
(g) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:47) and the light chain CDR3 sequence (SEQID NO:76) of antibody 4517;
(h) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:50) and the light chain CDR3 sequence (SEQID NO:78) of antibody 4518;
(i) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:74) and the light chain CDR3 sequence (SEQID NO:93) of antibody 4519; And
(j) a kind of antibody comprises heavy chain CDR3 sequence (SEQ ID NO:53) and the light chain CDR3 sequence (SEQID NO:80) of antibody 4380.
9. the antibody compositions described in the claim 8, wherein said anti--HER2 the first and second recombinant antibodies both all be selected from antibody (a)-(j).
10. the antibody compositions described in the claim 9 comprises one and is selected from resisting-HER2 the 3rd restructuring antibody of antibody (a)-(j).
11. antibody compositions, at least the first and second anti--HER2 recombinant antibodies that comprise different epi-position combinations from HER2, the wherein identical epi-position combination of arbitrary antibody (a)-(j) of definition and be combined with its competition in each described first and second antibody and the claim 8.
12. an antibody compositions comprises the first and second anti-HER2 recombinant antibodies of different epi-position combinations from HER2 at least, wherein at least a described antibody is selected from following:
(A) a kind of antibody comprises CDR1, the CDR2 of antibody 4382 variable region of heavy chain (SEQ ID NO:14) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:16);
(B) a kind of antibody comprises CDR1, the CDR2 of antibody 4383 variable region of heavy chain (SEQ ID NO:18) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQIDNO:20);
(C) a kind of antibody comprises CDR1, the CDR2 of antibody 4384 variable region of heavy chain (SEQ ID NO:22) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:24);
(D) a kind of antibody comprises CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:28) CDR1, CDR2 and the CDR3 of antibody 4385 variable region of heavy chain (SEQ ID NO:26);
(E) a kind of antibody comprises CDR1, the CDR2 of antibody 4386 variable region of heavy chain (SEQ ID NO:30) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:32);
(F) a kind of antibody comprises CDR1, the CDR2 of antibody 4387 variable region of heavy chain (SEQ ID NO:34) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:36);
(G) a kind of antibody comprises CDR1, CDR2 and CDR3 and variable region of light chain (SEQ ID NO:4) CDR1, CDR2 and the CDR3 of antibody 4517 variable region of heavy chain (SEQ ID NO:2);
(H) a kind of antibody comprises CDR1, the CDR2 of antibody 4518 variable region of heavy chain (SEQ ID NO:6) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:8);
(I) a kind of antibody comprises CDR1, the CDR2 of antibody 4519 variable region of heavy chain (SEQ ID NO:38) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:40); And
(J) a kind of antibody comprises CDR1, the CDR2 of antibody 4380 variable region of heavy chain (SEQIDNO:10) and CDR1, CDR2 and the CDR3 of CDR3 and variable region of light chain (SEQ ID NO:12).
13. the antibody compositions described in the claim 12, both all are selected from antibody (A) – (J) wherein said first and second anti--HER2 recombinant antibodies.
14. the antibody compositions described in the claim 13 comprises one and is selected from antibody (the 3rd anti--HER2 recombinant antibodies of A) – (J).
15. antibody compositions, at least the first and second anti-HER2 recombinant antibodies that comprise different epi-position combinations from HER2, wherein in each described first and second antibody and the claim 12 the identical epi-position of arbitrary antibody (A)-(J) of definition in conjunction with and compete with it combination.
16. the described arbitrary recombinant antibody composition of claim 8-15, by 2,3,4,5 or 6 kind of anti--HER2 antibody form.
17. the recombinant antibody composition described in the claim 16 is comprised of 2 kinds of anti--HER2 antibody.
18. the restructuring described in the claim 17 resists-HER2 antibody, comprises the first and second recombinant antibodies, wherein this first and second antibody comprises heavy chain of antibody CDR3 and light chain CDR3 sequence:
4380 and 4382,
4380 and 4384,
4380 and 4518,
4382 and 4385,
4382 and 4518,
4383 and 4518,
4384 and 4385,
4384 and 4517, perhaps
4385 and 4518.
19. resisting-the HER2 recombinant antibody composition described in the claim 18, wherein this first and second antibody comprises heavy chain of antibody CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 sequence:
4380 and 4382,
4380 and 4384,
4380 and 4518,
4382 and 4385,
4382 and 4518,
4383 and 4518,
4384 and 4385,
4384 and 4517, perhaps
4385 and 4518.
20. the recombinant antibody composition described in the claim 16 is comprised of 3 kinds of anti--HER2 antibody.
21. resisting-the HER2 recombinant antibody composition described in the claim 20 comprises the one one, second and the 3rd restructuring antibody, wherein this first, second, and third antibody comprises heavy chain of antibody CDR3 and light chain CDR3 sequence:
4380,4382 and 4385,
4380,4382 and 4517,
4380,4382 and 4518,
4380,4383 and 4384,
4380,4384 and 4517,
4380,4384 and 4518,
4380,4384 and 4519,
4382,4385 and 4518, perhaps
4382,4387 and 4517.
22. resisting-the HER2 recombinant antibody composition described in the claim 21 comprises first, second, and third recombinant antibodies, wherein this first, second, and third antibody comprises heavy chain of antibody CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 sequence:
4380,4382 and 4385,
4380,4382 and 4517,
4380,4382 and 4518,
4380,4383 and 4384,
4380,4384 and 4517,
4380,4384 and 4518,
4380,4384 and 4519,
4382,4385 and 4518, perhaps
4382,4387 and 4517.
23. the recombinant antibody composition described in the claim 16 is comprised of 4 kinds of anti--HER2 antibody.
24. resisting-the HER2 recombinant antibody composition described in the claim 23 comprises restructuring the first, second, third and the 4th antibody, wherein this first, second, third and the 4th antibody comprises heavy chain of antibody CDR3 and light chain CDR3 sequence:
4380,4382,4385 and 4518, perhaps
4380,4384,4385 and 4518.
25. the anti-HER2 recombinant antibody composition described in the claim 24, comprise first, second, third and quadruple group antibody, wherein the first, second, third and the 4th antibody contains heavy chain of antibody CDR1, CDR2 and CDR3 and light chain CDR1, CDR2 and CDR3 sequence:
4380,4382,4385 and 4518, perhaps
4380,4384,4385 and 4518.
26. arbitrary recombinant antibody composition described in the claim 1-25, wherein in described composition, have at least a kind of anti--HER2 antibody is a kind of immune conjugate, it is comprised of the resisting of a kind of and a kind of cancer therapy drug coupling-HER2 recombinant antibodies.
27. the recombinant antibody composition described in the claim 26, wherein cancer therapy drug is selected from cell toxicity medicament, cytokine, toxin and radionuclide.
28. a dual specific binding molecule, but two kinds of antibody that define in arbitrary claim among its specific binding claim 1-25.
29. the dual specific binding molecule described in the claim 28 is comprised of the heavy chain of described two kinds of antibody and light chain CDR1, CDR2 and CDR3 sequence.
30. a nucleic acid molecule comprises the nucleotide sequence of defined any antibody of codified claim 1-25.
31. an expression vector comprises nucleic acid molecule according to claim 30.
32. a host cell comprises nucleic acid molecule according to claim 30 or expression vector according to claim 31, wherein said host cell can be expressed by a kind of of described nucleic acid molecule encoding and be resisted-HER2 antibody.
33. a method that generates anti--HER2 antibody comprises describedly according to claim 32 providing a kind of host cell, cultivates this host cell under the suitable condition of expressing this antibody, and separates the antibody that generates.
34. a method that generates anti--HER2 recombinant antibody composition, this antibody compositions comprise first and second anti--HER2 recombinant antibodies at least.This method comprises provides at least a the first host cell and the second host cell, and wherein the first and second host cells all can be expressed defined arbitrary resisting-the HER2 recombinant antibodies of claim 1-25; Under the condition of suitable this first and second antibody of expression, cultivate this first and second host cell, and separate at least the first and second antibody that generate.
35. method described in the claim 34, wherein at least the first and second host cells are to cultivate in the bio-reactor that separates.
36. method described in the claim 34, wherein at least the first and second host cells are cultivated in a biological reactor.
37. comprising, a method for the treatment of the mankind or other mammalian cancer, the method give described Mammals according to claim 1 a kind of-27 described arbitrary resisting-the HER2 recombinant antibody composition.
38. a treatment suffers from the method for crossing the Disease that is expressed as feature with HER2, the method comprise give described patient according to claim 1 a kind of-27 described arbitrary anti--the HER2 recombinant antibody composition.
39. one kind was reduced and expresses the method that HER2 and other ErbB family receptors heterodimer form in the HER2 cell, the method comprises makes according to claim 1 a kind of-27 described arbitrary the resisting-the HER2 recombinant antibody composition of described cells contacting.
40. induced the method for expressing HER2 cell surface HER2 internalization for one kind, the method comprise make described cells contacting according to claim 1 a kind of-27 described arbitrary anti--the HER2 recombinant antibody composition.
41. one kind suppress to resist-the HER2 Antybody therapy produces the method for the tumor cell proliferation of resistance or part resistance, the method comprise make described cells contacting according to claim 1 a kind of-7 described arbitrary anti--the HER2 recombinant antibody composition.
42. method described in the claim 41, wherein this tumour cell has previously been accepted the Herceptin treatment.
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CN106831987A (en) * 2015-12-04 2017-06-13 浙江大学 A kind of anti-human HER2 antibody and its encoding gene and application
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