CN102884084B - Anti-HER 2 and compositions - Google Patents

Anti-HER 2 and compositions Download PDF

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CN102884084B
CN102884084B CN201180022307.6A CN201180022307A CN102884084B CN 102884084 B CN102884084 B CN 102884084B CN 201180022307 A CN201180022307 A CN 201180022307A CN 102884084 B CN102884084 B CN 102884084B
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antibody
sequence
heavy chain
light
chain
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CN102884084A (en
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M·W·佩德森
A·詹森
P-J·梅杰
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Symphogen AS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell

Abstract

The present invention relates to novel HER2 (ErbB2) targeted therapeutic antibody and anti-HER2 recombinant polyclonal antibody compositions, and this antibody and antibody compositions are used for treating the purposes of cancer.Described antibody compositions comprises the recombinant antibodies that at least three kinds of epi-positions different from HER2 combine.The HER2 of two of which antibodies cell surface, causes them to produce the antibody receptor grid of crosslinking at cell surface, and therefore causes HER2 receptor internalisation.The third antibodies HER2 in compositions, therefore it block the different dimerization between HER2 and HER3.

Description

Anti-HER 2 and compositions
Invention field
The present invention relates to (comprise 2 for the novel HER2 receptor target recombinant antibodies treating human cancer and combinations thereof thing Plant or this antibody-like multiple).
Background of invention
Epidermal growth factor (EGFR) family (i.e. ErbB family) is a subtribe of tyrosine kinase (RTK) receptor, its Comprise 4 members: EGFR/ErbB, HER2/ErbB2, HER3/ErbB3 and HER4/ErbB4.EGFR family member adjusts with strand Joint glycoprotein is closely related, and the latter has the outer ligand binding domain of born of the same parents, single cross-film district and intracellular kinase and (sees summary Ferguson (2008) Annu Rev Biophys.37:353-373).Under normal physiological status, ErbB family regulates also Coordinate cell growth, breed and migrate in critical event (Citri et al. (2006) Nat Rev Mol Cell Biol.7:505-516).It is presently believed that EGFR, HER2 and HER3 are in Normocellular vicious transformation and the lasting life of cancerous cell Length plays pivotal role.Have now been found that the situation (Slamon that there is EGFR and HER2 process LAN in many epithelial cancers Et al. (1987) Science, 235:177-182；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 process LAN and the progression of disease in several people's epithelial cancer, survival rate reduce, chemotherapy response Difference and generation relevant (Slamon et al. (1987) supra of drug resistance；Baselga et al.(2002)Oncologist 7 Suppl 4:2-8).
Human epidermal growth factor receptor 2 (HER2, also referred to as ErbB2 or Neu；UniProtKB/Swiss-Prot No.P04626) being made up of 1233 aminoacid, its structure is similar with EGFR, has the born of the same parents comprising 4 subdomain I-IV Foreign lands: the other territory of membrane-spanning domain, film, intracellular endochylema tyrosine kinase and modulability C-terminal territory (Yamamoto et al. (1986) Nature 319:230-234).
HER2 is member (Klapper et al. (1999) Proc not uniquely being combined with known ligand in ErbB family Natl AcadSci USA 96:4995-5000).HER2 is by forming different dimeric complexes and quilt with other ErbB family member Activating, therefore it is indirectly regulated by EGFR and HER3 part and (sees summary Yarden et al. (2001) NatRev Mol Cell Biol.2:127-137).HER2 is the first-selected partner (Graus-of other 3 kinds of ErbB receptor generation Heterodimerization Porta et al. (1997) EMBO J 16:1647-1655；Tzahar et al. (1996) Mol Cell Biol. 16: 5276-5287), it is by reducing the speed of dissociating of ligand-receptor complex, thus strengthens the parent of other ErbB receptor and part And power, such HER2 can strengthen and extend signal transduction (Pedersen et al. (2009) Mol CancerRes.7:275- 284).Another ligand binding receptor generation Heterodimerization of HER2 Yu ErbB family, can induce cross phosphorylation, make C-terminal Aminoacid phosphorylation.So in reverse as the support (King et al. (1988) EMBOJ7:1647-1651) of signaling molecule. The highest HER2 heterodimer of activity is HER2-HER3 complex (Pinkas-Kramarski et al. (1996) EMBOJ 15:2452-2467), wherein HER2 passes through active kinase (Guy et al. (1994) Proc complementary with kinase defective HER3 Natl Acad Sci USA 91:8132-8136.).Contrary with EGFR, HER2 has internalization resistance (Hommelgaard et Al. (2004) MolBiol Cell 15:1557-1567), it can escape lysosomal degradation, thus is retained in serous coat.
HER2 Main Function in the normal tissue seemingly regulates the signal transduction of the ErbB receptor that part combines.With EGFR is similar to, and HER2 mainly expresses in epithelial cell and (sees summary Freudenberg et al. (2009) Exp Mol Pathol.87:1-11), and have now been found that it plays growth regulation, differentiation, apoptosis and reconstruction in normal mammary gland development Deng non-carcinogenic effect (Troyer et al. (2001) J Mammary Gland Biol Neoplasia 6:7-21).With EGFR Situation is similar to, and cell surface HER2 too much can cause epithelial cell to be converted into Various Tissues (Freudenberg et al. (2009)supra).At present it has been reported that there is HER2 copy in a large amount of human tumors to increase the situation with process LAN, including The aggressive duct carcinoma of 20%-30%, and it is by the predictive factors as Clinical Outcome poor (overall survival reduction) (Slamon et al.(1987)supra；Ravdin et al. (1995) Gene 159:19-27).Refer in early days occurring converting The human breast tissue levied but convert the most completely can detect that HER2 high expressed, in this prompting HER2 vicious transformation in early days Play a significant role (Freudenberg et al. (2009) supra).The most also at other epithelial cancer, such as colorectal cancer, Ovarian cancer, cancer of pancreas, pulmonary carcinoma and bladder transitional cell carcinoma, find HER2 high expressed (Freudenberg et al. (2009) supra). HER2 activate can induced cell proliferation out of control, protect cells from apoptosis, and affect normal epithelial tissue (Muthuswamy Et al. (2001) Nat Cell Biol.3:785-792).And the HER2 that metastasis cancer cell is expressed may be at cancer cell migration In play a role (De Potter (1994) Hum Pathol.25:1264-1268).
EGFR and HER2 is the cancer target through checking, and monoclonal antibody and the little molecule of present these receptors of targeting press down Preparation has been approved by the treatment for kinds cancer.But, initially often resistance to owing to producing to the patient of these treatment responses Medicine causes disease relapse (Pao et al. (2005) PLoSMed2:e73).Herceptin (trade name Trastuzumab) can targeting HER2, for treating the breast carcinoma of HER2 receptor process LAN.In January, 2010, European Union approval Trastuzumab combination chemotherapy HER2 Positive stomach metastatic carcinoma patient.The monoclonal antibody handkerchief trastuzumab of another kind of part targeting HER2 receptor currently carries out many Item clinical trial.Herceptin is by combining HER2 and then blocking HER2 function and onset, and handkerchief trastuzumab is HER dimerization Change inhibitor, HER2 Yu HER3 and other EGFR receptor generation dimerization can be suppressed.
Owing to handkerchief trastuzumab is at present still at clinical experimental stage, its final Clinical efficacy is still unaware of.For song Trastuzumab, although Herceptin combined chemotherapy more single medicine chemotherapy can obtain the clinical income of life span extension, but most of HER2 breast cancer patients with positive produces response to this medicine, and (total response rate of Trastuzumab combined chemotherapy is 45%, and single medicine Chemotherapy is 29%；Trastuzumab prescription information, Genentech, in March, 2009).Slamon etc. report similar result, deliver In N Engl J Med (2001), 344 (11): 783-92, it points out simultaneously, compared with single medicine chemotherapy, Trastuzumab combined chemotherapy 1 term mortality rate (22% and 33%, P=0.008) can be reduced, extend median survival interval (25.1 and 20.3 months, P= 0.046).Therefore, although the monoclonal antibody of targeting HER2 can improve therapeutic effect, such as at the transitivity breast of HER2 process LAN In the treatment of adenocarcinoma, but its to improve space the biggest.
Summary of the invention
The present invention relates to (comprise 2 for the novel HER2 receptor target recombinant antibodies treating human cancer and combinations thereof thing Plant or this antibody-like multiple), such as breast carcinoma, ovarian cancer, gastric cancer and the cancer of other process LAN HER2.With current this type of The Therapeutic Method of cancer is compared, and including the targeting monoclonal antibody of other receptor of HER2 or EGFR family, the antibody of the present invention can Alone, or preferably 2 kinds or this type of antibody compositions multiple combination, in the case of optional and other therapies is combined (such as chemotherapy) Remarkable clinical efficacy is provided.
On the one hand, the present invention relates to novel anti-HER2 recombinant antibodies, based on antibody 4380/4381 described in this patent, 4382, 4383,4384,4385,4386,4387,4517,4518 and 4519 and its humanized antibody.In one embodiment, the present invention Relating in one aspect to a kind of anti-HER2 recombinant antibody molecule, it comprises antibody 4380/4381 described in this patent, and 4382,4383, The heavy chain CDR3 sequence of arbitrary antibody in 4384,4385,4386,4387,4517,4518 and 4519.
Another embodiment of this aspect of the present invention: a kind of anti-HER2 recombinant antibody molecule, it comprises antibody 4380/4381, The heavy chain CDR3 sequence of arbitrary antibody and light chain in 4382,4383,4384,4385,4386,4387,4517,4518 and 4519 CDR3 sequence；A kind of anti-HER2 recombinant antibody molecule, it comprises heavy chain CDR1, CDR2 and the CDR3 of arbitrary antibody in these antibody Sequence and light chain CDR1, CDR2 and CDR3 sequence；A kind of anti-HER2 recombinant antibodies, it comprises the weight of arbitrary antibody in these antibody Chain variable region sequence and light-chain variable sequence, or comprise and have at least 80%, the heavy chain of 85%, 90% or 95% identical sequence Variable region sequences and light-chain variable 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 is including at least the weight of the first and second anti-HER2 Group antibody, the combinations of both epi-positions different from HER2, and also one of which or two kinds are selected from above-mentioned antibody group.
Another aspect of the present invention relates to a kind of anti-HER2 recombinant polyclonal antibody compositions, and it is not including at least with HER2 The first, second, and third anti-HER2 recombinant antibodies composition combined with epi-position, wherein the first and second antibody can be combined with HER2 And cause HER2 receptor internalisation, and the 3rd antibody is combined the HER3 phosphorylation that part can be suppressed to induce with HER2.
Another aspect of the present invention relates to a kind of immune conjugate, and it comprises the anti-HER2 recombinant antibodies of a kind of present invention, Can be with a kind of cancer therapy drug coupling.The related fields of the present invention relate to antibody compositions, and it is including at least anti-HER2 in the present invention First and second antibody of recombinant antibodies, in described compositions, at least a kind of Anti-HER 2 is immune conjugate.
Another aspect of the present invention relates to nucleic acid molecules, and it has the nucleotides sequence of Anti-HER 2 in code book invention Row, and comprise expression vector and the host cell of this expression vector of transfection of polynucleotide.
Another aspect of the present invention relates to producing antibody of the present invention and the method for polyclonal antibody compositions.
Another aspect of the present invention relates to treating the mankind or the method for animal subjects disease, particularly human cancer, logical Cross and give described experimenter's one Anti-HER 2 or the present composition and realize.Another related fields are for using in the present invention The preparation of one or more Anti-HER 2s for treating the medicine of the mankind or Animal diseases, particularly treat human cancer.
Another aspect of the present invention relates to a kind of method inducing process LAN HER2 cell surface HER2 internalization, the method bag Include and make anti-HER2 recombinant antibody composition in a kind of anti-HER2 recombinant antibodies of cells contacting, immune conjugate or a kind of present invention.
The other side of the present invention and some embodiment should be readily appreciated that in following description and example.
Picture describes
Fig. 1 shows that the representative epi-position of Anti-HER 2 of the present invention combines result.
Fig. 2 A, 2B, 2C and 2D show through containing 2,3, N87 and SKBR3 of 4 kind of Anti-HER 2 mixture of the present invention induction swell Relative ADCC percent under two kinds of different antibodies concentration in oncocyte system.
Fig. 3 A, 3B, 3C and 3D show that N87 and the SKBR3 tumor through the induction of 2 or 3 kind Anti-HER 2 of the present invention mixture is thin The ADCC measured value of born of the same parents.
Fig. 4 shows through 2,3, the CDC measured value of the N87 cell of 4 kind of Anti-HER 2 mixture of the present invention induction.
Fig. 5 shows the CDC measured value of the N87 cell through the induction of 2 or 3 kind Anti-HER 2 of the present invention mixture.
Fig. 6 shows the titration results that N87 gastric carcinoma cell lines metabolic activity is suppressed by 4 kinds of mixtures of antibodies of the present invention.
Fig. 7 shows the titration results that HCC202 breast cancer cell line metabolic activity is suppressed by 4 kinds of mixtures of antibodies of the present invention.
Fig. 8 shows the titration results that BT474 breast cancer cell line metabolic activity is suppressed by 4 kinds of mixtures of antibodies of the present invention.
Fig. 9 shows the titration results that ZR-75-30 breast cancer cell line metabolic activity is suppressed by 4 kinds of mixtures of antibodies of the present invention.
Figure 10 shows the Anti-HER 2 mixture of the present invention interior curative effect in NCI-N87 Gastric Cancer Xenograft of Nude Mice model.
Figure 11 shows the Anti-HER 2 mixture of the present invention interior curative effect in OE19 Gastric Cancer Xenograft of Nude Mice model.
Figure 12 shows that NCI-N87 cell processes 15 points with tune albumen β after described antibody or mixtures of antibodies overnight process again The Western blot analysis of intracellular EGFR, pEGFR, HER2, pHER2, HER3 and pHER3 expression after clock.
Figure 13 A shows in human cancer HER2 homodimer and HER2/HER3 heterodimer in oncogene cell transduction Different but relevant effect.Figure 13 B shows that 3 kinds of Anti-HER 2 mixture of the present invention block the guess mechanism of two paths, wherein Two kinds of antibody can induce HER2 internalization and degraded, and the 3rd antibody can block compensatory by HER2/HER3 heterodimer and believe Number transduction.
Figure 14 shows that ZR-75-30, NCI-N87, BT474 and HCC202 cancerous cell line is through above-mentioned antibody and mixtures of antibodies mistake The Western blot analysis of HER2 expression after processing night.
Figure 15 show HCC202 and NCI-N87 cancerous cell line after above-mentioned antibody and mixtures of antibodies overnight process HER2 and The Western blot analysis of HER3 expression.
Figure 16 show OE19, N87, ZR-75-30, BT474 and HCC202 cell line through above-mentioned 10 μ g/ml monoclonal antibodies or Mixtures of antibodies processes the Western blot analysis of HER2 expression after 48 hours.
Figure 17-19 shows that different antibodies mixture is to 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 suppression titration results.
Detailed Description Of The Invention
Definition
" antibody " or " antibody molecule " refers to functional serum component, is often referred to Molecule Set (antibody or immunoglobulin) or Plant molecule (antibody molecule or immunoglobulin molecules).Antibody can combine with special antigenic determinant (antigen or epitope) Or reaction, immunological effect mechanism can be induced in turn.It has been generally acknowledged that single antibody is narrow spectrum, antibody compositions can be single Clone (being i.e. made up of identical antibody molecule) or polyclone (i.e. identical or different by with same antigen or not even synantigen 2 kinds or multiple different antibodies of epi-position reaction) antibody.Each antibody all has can be specific binding with its corresponding antigens Unique texture, all natural antibodies have identical basic structure, i.e. 2 identical light chains and 2 identical heavy chains.Resist simultaneously Body is also generically and collectively referred to as immunoglobulin.
" antibody " in this patent also includes chimeric and single-chain antibody and the binding fragment of antibody, such as Fab, Fv fragment Or scFv (scFv) fragment, and multimeric forms, such as dimer IgA molecule or pentavalent IgM.Antibody can be people source Or inhuman source, such as mice or other rodent sources antibody, or chimeric, humanization of based on mouse antibodies or mould again Shape antibody.
Each heavy chain of usual a kind of antibody comprises a variable region of heavy chain (VH) and a CH light chain constant District generally comprises 3 domains, i.e. CH1, CH2 and CH3.Each light chain of usual a kind of antibody comprises a variable region of light chain (VL) and one constant region of light chain constant region of light chain generally comprises single structure territory, i.e. CL.VH and VL district can be subdivided into high change District (" hypervariable region ", it is variable that its sequence and/or structure define ring camber).The most also referring to complementarity-determining region (CDR), it is dispersed in It is distributed in the most conservative skeleton district (FR).Each VH and VL generally includes 3 CDR and 4 FR, presses to c-terminus from aminoterminal Following sequence arranges: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.In variable region, amino acid residue generally uses standard to compile Number method is numbered (Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, MD, USA).
In the sequence table of annex, light chain (LC) DNA and aminoacid sequence comprise variable region of light chain (VL) sequence and people κ is permanent Determine region sequence.As described in example 1, people κ constant region starts with aminoacid-TVAAP-(Thr Val Ala Ala Pro), with C end Aminoacid-NRGEC (Asn Arg Gly Glu Cys) terminates.Therefore, in this patent, " light-chain variable sequence " or " VL " can It is understood the N end portion (i.e. aminoacid TVAAP) of sequence of light chain in the sequence table before starting for people κ constant region.
Whole antibody numbering used in this patent, such as " antibody 4517 ", the definition in instruction example and in annex sequence table Specific antibody.Such as, in antibody 4517, heavy chain comprises variable region of heavy chain SEQ ID NO:2 and IGHG1 CH SEQ IDNO:44, sequence of light chain is SEQ ID NO:4, as it has been described above, sequence of light chain comprises variable region of light chain (in SEQ ID NO:4 Residue 1-110) and people's κ constant-region sequences (the residue 111-216 in SEQIDNO:4).
In this patent describe a kind of antibody for " deriving from " or " based on " a kind of specific antibodies time, it is meant that " source " is anti- It is a kind of below that body comprises (according to specific background): the heavy chain CDR3 sequence of described specific antibodies；The heavy chain of described specific antibodies CDR3 sequence and light chain CDR3 sequence；Heavy chain CDR1, CDR2 and the CDR3 sequence of described specific antibodies and light chain CDR1, CDR2 and CDR3 sequence；Or the variable region of heavy chain of described specific antibodies and light-chain variable sequence, or described weight chain variabl area sequence and/or Light-chain variable sequence humanized antibody, or heavy chain and/or light-chain variable sequence at least 80%, 85%, 90% or 95% sequence Arranging identical with corresponding heavy chain and/or light-chain variable sequence, for example, at least 96%, 97%, 98% or 99% sequence is identical.This Described in patent, source or a kind of antibody based on a certain specific antibodies generally combine the HER2 table identical with described specific antibodies Position, and preferably essentially identical with described specific antibodies activity antibody.
The specificity that antibody interacts with target antigen depends primarily on the amino being positioned in heavy chain and 6 CDR of light chain Acid residue.Therefore between antibody, the variation of the aminoacid sequence in CDR is much more compared with the outer sequence of CDR region.Because CDR sequence is with big Most antibody antigens interact relevant, can express a certain recombinant antibodies naturally occurring antibody characteristic of simulation, or given The specific antibodies of aminoacid sequence, by construction of expression vector, is transplanted to the skeleton district of another antibody by specific antibodies CDR sequence Sequence.As such, it is possible to by one non-human source antibodies " humanization ", retain the binding specificity of original antibodies and affine the most in a large number Property.Humanization will be carried out more detail discussion below.
" chimeric antibody " referring broadly to comprising the one or more regions from a kind of antibody, and from one or more The antibody in one or more regions of other antibody.In this patent, " chimeric antibody " typically groups of people source and part is inhuman The antibody in source, i.e. a portion from inhuman animal, such as mice or other Rodents, or birds, such as chicken.Chimeric Antibody is better than non-human source antibodies, and this is that the response risk in order to reduce mankind's antagonist, i.e. mouse antibodies can make human body generation anti- Murine antibody.Such as, in typical chimeric antibody, variable region sequences is from the Mus source sequence after mouse immune, and behaviour source, constant region Sequence.For chimeric antibody, the part in inhuman source, the most usually the skeleton district of variable region sequences, change the most further, thus Antagonist carries out humanization.
" humanization " refers to antibody and is all or part of and obtains behind inhuman source, such as application target mice immunized with antigen Antibody or chimeric antibody based on Mus source antibody, can replace some aminoacid, particularly heavy chain and light chain framework district and constant The aminoacid in district, so it can be avoided that or reduce Human immune responses.Now it is known that, all antibody can excite people Body creates antagonism the reaction of antibody, and this is relevant to " humanization " degree of antibody.Exempt from although we can not calculate to a nicety Epidemic focus, thus predict the human body opposing reaction to a certain antibody, but non-human source antibodies is more higher than human antibody immunogenicity. External source (usually rodent origin) constant region of chimeric antibody is substituted by people's source sequence, with the antibody phase of complete exogenous array Ratio, its immunogenicity is the most relatively low, and therapeutic antibodies is generally more likely to use humanization or complete human antibody.Therefore, right In chimeric antibody or other non-human source antibodies, preferably through humanization to reduce the antagonism response risk of human body antagonist.For Chimeric antibody, humanization often designs the skeleton district amendment of variable region sequences.The partial amino-acid of complementarity-determining region (CDR) Residue generally will not be changed in humanization, although needs to change an other cdr amino acid residue in some cases, such as Remove glycosylation site, desamidization site or unwanted cysteine residues.
The most existing humanized method of numerous antibody sequences；See summary Almagro & Fransson (2008) Front Biosci.13:1619-1633.Most common method is that CDR transplants, such as, and the chimeric antibody ginseng in a mice source The Human germ cells gene similar to Mus source variable region gene to discriminating, implants Mus source CDR sequence in this skeleton.Can basis Kabat CDR defines and carries out CDR transplanting, but the article delivered recently (Magdelaine-Beuzelin etal. (2007) CritRev.Oncol Hematol.64:210-225) point out IMGT definition (www.imgt.org) that humanization result can be improved. Owing to CDR transplants binding specificity and the affinity of the non-human source antibodies that can reduce implantation CDR, thus reduce biological activity, can Introduce inverse transition in the selected location transplanting CDR antibody, thus retain binding specificity and the affinity of female antibody.Can use Available information in document neutralizing antibody data base carries out position qualification to possible inverse transition.Candidate's amino of inverse transition Acid residue is usually located at antibody molecule surface, and hiding or that surface degrees of exposure is low residue generally will not be changed.Except CDR Transplanting and outside inverse transition, another substituting humanization technologies, for reinventing antibody, retains the residual of those non-surfaces, inhuman source exposure Base, and be people source residue by surface residue change.
As it was previously stated, the present invention comprises humanized antibody, the most humanized antibody.Antibody of the present invention can also be referred to " humanization hypotype ".Especially, this patent middle finger acts as " weight chain variabl area sequence " of what specific amino acid sequence and " light chain can Become region sequence " comprise any humanization hypotype of this particular sequence and this particular sequence simultaneously.
In this patent, compared with a certain specific heavy chain or light-chain variable sequence, there is the unison minimum levels such as sequence Weight chain variabl area sequence or light-chain variable sequence, i.e. have at least 80%, sequence of 85%, 90% or 95% etc. is unison, The sequences of for example, at least 96%, 97%, 98% or 99% etc. are unison, include but not limited to that the humanization of this type of reference sequences is sub- Type.
" recombinant antibodies " refers to cell or transfection expression contains this antibody coding sequence carrier (or multiple expression carries Body, usually 2 kinds of expression vectors) a kind of antibody of expression of cell lines, wherein coded sequence is the most unrelated with this cell.
" carrier " is a kind of nucleic acid molecules, can insert a nucleotide sequence wherein, for the transport of different genes environment And/or the expression in host cell.The carrier carrying nucleotide sequence transcriptional regulatory element is " expression vector "." plasmid " and " load Body " it is used interchangeably.The expression vector used in the present invention can be any applicable carrier known in the art, such as plasmid Or viral vector.
" polyclonal antibody " refers to combine from same or not synantigen different antigenic determinants or 2 kinds of reaction Or Multiple Antibodies molecular composition.In the present invention, the single antibody of polyclonal antibody can determine with the not synantigen of HER2 Bunch combine.Preferably in the present invention, the single antibody of polyclonal antibody epi-position different from HER2 combines, more preferably with the most overlapping Different epi-positions combine.It has been generally acknowledged that the diversity of polyclonal antibody is positioned at the variable region of antibody molecule." anti-HER2 recombinates many grams Grand antibody compositions " it is a kind of 2 kinds or the compositions of multiple Monoclonal Antibody Mixture comprising and being combined with HER2.
Prior art is established, and antibody also exists different homotype, such as mankind's homotype IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, or mice homotype IgG1, IgG2a, IgG2b, IgG3 and IgA.The antibody of the present invention can be any homotype.Though So the single antibody of polyclonal antibody compositions of the present invention can comprise the antibody of multiple homotype, but the most same homotype.
The recombinant antibody composition that one comprises " first and second recombinant antibodies of the most anti-HER2 " will comprise at least 2 kinds Specify antibody, but Anti-HER 2 of more than two kinds in this patent may be comprised.In some cases, this kind of recombinant antibodies combination Thing may comprise considerable single Anti-HER 2, and such as 10 or more, 15-20 is individual, but generally comprises less than 10 not Same HER2 antibody, i.e. 2,3,4,5,6,7,8 or 9 antibody.More commonly, the recombinant antibody composition of the present invention comprises The Anti-HER 2 different less than 6, in most of the cases, the Anti-HER 2 different less than 4.The most real Executing in example, the recombinant antibody composition of the present invention will comprise 2,3 or 4 kind of different Anti-HER 2, and usually 2 or 3 kind of difference Anti-HER 2.
" CDR " or " complementarity-determining region " refers to " high change " district in antibody variable region, and it mainly determines the knot of antibody Close specificity.See CDR to define: 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 region, i.e. CDR1, CDR2 and CDR3, wherein CDR3 difference is maximum.
" epi-position " has antigen active or immunogenic relatively macromole (such as antigen and antigen active position in referring to animal body Point) a part.There is the part that immunogenic epi-position is the relatively macromole that animal's antibody can be excited to react.There is antigen The epi-position of activity is that the antibody that determines through existing known method can the part of relatively macromole of immunity specific bond.Epitope Need not have immunogenicity.Antigen be antibody or antibody fragment can the material of immunity specific bond, such as toxin, virus, antibacterial, Albumen or DNA.Antigen or antigen site usual more than one epi-position, unless it is the least, and generally can immune response stimulating. Epi-position can be linear epitope or comformational epitope.Linear epitope is typically by about 6-10 aminoacid neighbouring on a protein molecular Composition, this protein molecular can be by antibody recognition.On the contrary, the aminoacid that comformational epitope is arranged by non-sequence forms, but antibody A recognizable specific three dimensional structure.But when a protein molecular is folded into three dimensional structure, the aminoacid of composition epi-position is arranged side by side, So antibody can recognize that comformational epitope.In the protein of degeneration, only linear epitope can be identified.According to definition, comformational epitope Must be positioned at the outside of folded protein.
" different epi-position " refers to, when in the present invention, 2 kinds of different antibodies combine from different epi-positions, antigen combines competition and is less than 100%, preferred antigens combines competition and is less than 80%, and more preferably antigen combines and competes less than 50%, and most preferably competes and to the greatest extent may be used Can be low, such as antigen combines competition less than 25%.The antibody competing with one another for combining same antigen can be in conjunction with identical or overlapping Epi-position, or there is binding site in next-door neighbour district mutually, so competition is mainly caused by sterically hindered.Prior art can be used Perception method carries out antibody and analyzes " different epi-position ", such as, by the Binding experiment under the conditions of saturated antibody, FACS can be used (glimmering Photoactivation cell sorting) or other flow cytometer and fluorescent-labeled antibody the cell expressing HER2 is analyzed, or pass through Applications of surface plasmon resonance (SPR), uses HER2 antigen capture or is bound to flow cell surface.Use described in example SPR determines the method for competition between antibody.
Different epi-positions preferably " non-overlapped " epi-position, the antigen of 2 kinds of different Anti-HER 2s in the present composition combines Competing of a sufficiently low, 2 kinds of antibody can the most corresponding epi-position combine.Know those skilled in the art's understanding can exist not With the overlap of degree, although and the overlap of some degree can be there is, different epi-positions can consider " the most overlapping " epi-position, if phase Antibody is answered can substantially to combine its epi-position.When between two kinds of antibody, antigen combines competition below about 50%, generally it is contemplated that be suitable for This situation.
It is different that the antibody that epi-positions different from same antigen combine combines activity influence to antigen, and this depends on the position of epi-position Put.The antibody being combined with antigen active site epi-position can block antigen function completely, but the antibody being combined with another epi-position may Individually can be less to antigen activity influence, or not impact.But this antibody-like still can activating complement, thus cause antigen to disappear Remove, when one or more antibody of epi-positions different from combining same antigen is combined, may result in synergism.In the present invention In, epi-position preferred HER2 ectodomain.The antigen preferred HER2 ectodomain of the present invention, polypeptide or with antibody or antibody sheet The fragment of section immunity specific bond.HER2 related antigen can also be HER2 ectodomain, polypeptide or with antibody or antibody sheet The analog of the fragment of section immunity specific bond or derivant.
" immunoglobulin " usually blood or general name of Serum Antibody mixture, but can also be used for referring to other source Mixtures of antibodies.
" homology VHAnd VLEncode to " refer to the V of original pair that same antibody-producting cell is contained within or originatesHAnd VLCoding Sequence.Therefore, homology VHAnd VLEncode the V referring to that derived cell donor itself existsHAnd VLRight.“VHAnd VLEncode expressing anti- Body " refer to by a carrier, plasmid or other contain VHAnd VLA kind of antibody of the polynucleotide generation of coded sequence or antibody sheet Section.Expressing homology VHAnd VLDuring coding pair, either complete antibody still stablizes fragment, and it all maintains derived cell itself Express binding affinity and the specificity of antibody.Homology also refers to the set of homology pair to storehouse, can individually preserve.
" albumen " or " polypeptide " refers to any length or the amino acid chain of rear translation modification.Albumen can as monomer or Polymer exists, and comprises 2 or the polypeptide chain of multiple assembling, protein fragments, polypeptide, oligopeptide and peptide.
" head to head promoter " refers to a pair promoter pair the most adjacent, by the 2 of promoters driven genetic fragments Transcribe in the opposite direction.Head to head promoter is also referred to as bidirectional promoter.
" transfection " in this patent is by the broad terms of exogenous DNA into cells.It also comprises other functional equivalent By the method for exogenous DNA into cells, such as, convert, infect, transduce or the fusion of donorcells nuclear receptor cell.
As described in " background of invention ", " HER2 " (also referred to as HER2/neu and ErbB-2) refers to " people's table somatomedin Receptor 2 ".This patent middle finger includes the variant of HER2, hypotype and homologous genes.Preferably, a kind of antibody in the present invention with HER2 combines suppression and expresses cell (such as the be usually tumor cell) growth of HER2, this suppression by suppression HER2 and other The formation of heterodimer between ErbB family member and realize, such as with EGFR or HER3 Heterodimerization.
" Developing restraint " in this patent (such as, cell growth) comprises propagation (cell quantity increase) or metabolism generation is any The minimizing that can measure, when contacting with a kind of Anti-HER 2, with same cell growth phase when there is not Anti-HER 2 Ratio, for example, at least suppression growth of cell culture about 10%, and the preferably higher person of suppression ratio, the most about 20% or 30%, More preferably suppression ratio at least about 40% or 50%, the most about 60%, 70%, 80%, 90%, 99% or even 100%.
In this patent, " suppression dimerization " or " suppression dimer is formed " refers to and dimerization bodily form when there is not Anti-HER 2 One-tenth situation is compared, and owing to it is combined with a kind of Anti-HER 2, HER2 and such as EGFR, HER3 or HER4 form dimeric energy There is any measurable reduction in power.
" treatment " in this patent refers to enough Anti-HER 2s giving a kind of present invention or antibody compositions to subtract Gently, slow down or eliminate (healing) morbid state.Give 2 kinds in the present invention or more Anti-HER 2 generally by simultaneously to Give antibody, preferably comprise all Anti-HER 2 compositionss for treating.But also can give 2 kinds or more in the present invention respectively Anti-HER 2.Here the recombinant antibody composition giving to be made up of at least 2 kinds of Anti-HER 2s is appreciated that not It is given only the compositions comprising these type of at least 2 kinds of antibody compositions, also comprises and give antibody respectively.Therefore can simultaneously, sequential or point Do not give 2 kinds or the combination of multiple Anti-HER 2 of the present invention.
The same percentage of two sequences, such as variable region sequences, refer to the quantity of same position in sequence and (be calculated as Same position quantity/total number of positions * 100), it is considered to must be introduced into for the optimally aligned interval of two sequences.Comparative sequences With determine that the mutually unison percent that two sequences are shown in is to be completed by ready-made software.Suitably software program may be from various Resource, can use online or download use, for comparison albumen and nucleotide sequence.A kind of suitably program is ClustalW (Thompson et al.(1994)Nucleic Acids Res 11；22 (22): 4673-80), Ke Yicong Www.clustal.org or European Union's biometrics institute (www.ebi.ac.uk) download, its provide all kinds of other albumen and Nucleotide statistical tool.
Particular embodiment
An aspect of of the present present invention relates to various new Anti-HER 2.In one embodiment, the present invention relates to a kind of resisting HER2 recombinant antibodies, it comprises antibody 4380/4381 described in this patent, and 4382,4383,4384,4385,4386,4387, The heavy chain CDR3 sequence of arbitrary antibody in 4517,4518 and 4519.
In another embodiment, the present invention relates to a kind of anti-HER2 recombinant antibodies, it comprises antibody 4380/4381, and 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, and 4382, In 4383,4384,4385,4386,4387,4517,4518 and 4519 arbitrary antibody heavy chain CDR1, CDR2 and CDR3 sequence and Light chain CDR1, CDR2 and CDR3 sequence.
In another embodiment, the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises antibody 4380/4381, The weight chain variabl area sequence of arbitrary antibody and light chain in 4382,4383,4384,4385,4386,4387,4517,4518 and 4519 Variable region sequences；Or comprise the humanized sequence of described heavy chain and/or light-chain variable sequence, or comprise weight chain variabl area sequence And light-chain variable sequence, it has at least 80% with described heavy chain and light-chain variable sequence respectively, and 85%, 90% or 95% Identical sequence, such as with described sequence at least 96%, the sequence of 97%, 98% or 99% is identical.
In another embodiment, the present invention relates to a kind of anti-HER2 recombinant antibodies, it can be identical with any of the above-described antibody Epi-position combination therewith competition binding, and the antibody compositions being made up of one or more these antibody-likes, preferably at least comprise 2 Plant 2 kinds or 3 kinds of these antibody-likes described in other parts in this antibody-like, such as this patent.
Such as annex sequence table, table 1 below shows antibody 4380/4381,4382,4383,4384,4385,4386, The variable region of heavy chain (VH) of 4387,4517,4518 and 4519 antibody and variable region of light chain (LC) DNA and the sequence of aminoacid sequence No. ID.As explained on, in sequence table, light chain DNA and aminoacid sequence include light-chain variable sequence (VL) and people κ constant region sequence Row.
Table 1: selected Anti-HER 2 variable region of heavy chain and the DNA of light chain and the serial ID number of aminoacid sequence
One specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the heavy chain CDR3 of antibody 4517 Sequence, preferably comprise the heavy chain of antibody 4517 and the heavy chain of light chain CDR3 sequence, such as antibody 4517 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4517 weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its people Source sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with the heavy chain of antibody 4517 At least 80% is had, the identical sequence of 85%, 90% or 95% with light-chain variable sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the heavy chain CDR3 of antibody 4518 Sequence, preferably comprise the heavy chain of antibody 4518 and the heavy chain of light chain CDR3 sequence, such as antibody 4518 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4518 weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its people Source sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with the heavy chain of antibody 4518 At least 80% is had, the identical sequence of 85%, 90% or 95% with light-chain variable sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the weight of antibody 4380/4381 Chain CDR3 sequence, preferably comprises heavy chain and the light chain CDR3 sequence, the such as heavy chain of antibody 4380/4381 of antibody 4380/4381 With light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4380 weight chain variabl area sequence or its humanized sequence, and light chain Variable region sequences or its humanized sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, it is respectively At least 80% is had, the identical sequence of 85%, 90% or 95% with heavy chain and the light-chain variable sequence of antibody 4380/4381. In the present embodiment, preferably 4380.
Antibody " 4380/4381 " refer to VH and LC aminoacid sequence be respectively SEQ ID NO:10 and 12 one resist Body.As shown in SEQ IDNO:12,40 amino acids residues can be tyrosine or threonine.Antibody 4380 and 4381 is direct Unique difference is in antibody 4380, and LC sequence 40 amino acids residue is tyrosine, and in antibody 4381,40 bit aminos Acid residue is threonine.In original mouse antibody, this position is free cysteine.In both antibody, it is usually preferred to 4380。
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the heavy chain CDR3 of antibody 4382 Sequence, preferably comprise the heavy chain of antibody 4382 and the heavy chain of light chain CDR3 sequence, such as antibody 4382 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4382 weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its people Source sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with the heavy chain of antibody 4382 At least 80% is had, the identical sequence of 85%, 90% or 95% with light-chain variable sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the heavy chain CDR3 of antibody 4383 Sequence, preferably comprise the heavy chain of antibody 4383 and the heavy chain of light chain CDR3 sequence, such as antibody 4383 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4383 weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its people Source sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with the heavy chain of antibody 4383 At least 80% is had, the identical sequence of 85%, 90% or 95% with light-chain variable sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the heavy chain CDR3 of antibody 4384 Sequence, preferably comprise the heavy chain of antibody 4384 and the heavy chain of light chain CDR3 sequence, such as antibody 4384 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4384 weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its people Source sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with the heavy chain of antibody 4384 At least 80% is had, the identical sequence of 85%, 90% or 95% with light-chain variable sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the heavy chain CDR3 of antibody 4385 Sequence, preferably comprise the heavy chain of antibody 4385 and the heavy chain of light chain CDR3 sequence, such as antibody 4385 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4385 weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its people Source sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with the heavy chain of antibody 4385 At least 80% is had, the identical sequence of 85%, 90% or 95% with light-chain variable sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the heavy chain CDR3 of antibody 4386 Sequence, preferably comprise the heavy chain of antibody 4386 and the heavy chain of light chain CDR3 sequence, such as antibody 4386 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4386 weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its people Source sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with the heavy chain of antibody 4386 At least 80% is had, the identical sequence of 85%, 90% or 95% with light-chain variable sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the heavy chain CDR3 of antibody 4387 Sequence, preferably comprise the heavy chain of antibody 4387 and the heavy chain of light chain CDR3 sequence, such as antibody 4387 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4387 weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its people Source sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with the heavy chain of antibody 4387 At least 80% is had, the identical sequence of 85%, 90% or 95% with light-chain variable sequence.
Another specific embodiments of the present invention relates to a kind of anti-HER2 recombinant antibodies, and it comprises the heavy chain CDR3 of antibody 4519 Sequence, preferably comprise the heavy chain of antibody 4519 and the heavy chain of light chain CDR3 sequence, such as antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence, or comprise antibody 4519 weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its people Source sequence, or comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with the heavy chain of antibody 4519 At least 80% is had, the identical sequence of 85%, 90% or 95% with light-chain variable sequence.
Another aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least the first and second of anti-HER2 Body, the first and second antibody epi-position different from HER2 combine, and one of which or both be selected from above-mentioned antibody group.This One embodiment of bright this aspect relates to a kind of recombinant antibody composition, and it is including at least first and second antibody of anti-HER2, First and second antibody epi-position different from HER2 combines, and wherein the first and second antibody comprise and are selected from antibody 4380/ A kind of heavy chain CDR3 sequence of the antibody of 4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519.
One embodiment of this aspect of the present invention relates to a kind of recombinant antibodies component, and it is including at least first He of anti-HER2 Second antibody, wherein the first and second antibody epi-position different from HER2 combines, and wherein the first and second antibody comprise can Weight selected from a kind of antibody of antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 Chain and light chain CDR3 sequence.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it is including at least the first of anti-HER2 And second antibody, wherein the first and second antibody epi-position different from HER2 combines, and wherein the first and second antibody comprise It is selected from a kind of antibody of antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 Heavy chain 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 is including at least the first of anti-HER2 And second antibody, wherein the first and second antibody epi-position different from HER2 combines, and wherein the first and second antibody comprise It is selected from a kind of antibody of antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 Weight chain variabl area sequence or its humanized sequence, and light-chain variable sequence or its humanized sequence；Wherein first and second Antibody comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively be selected from antibody 4380/4381, The weight chain variabl area sequence of a kind of antibody of 4382,4383,4384,4385,4386,4387,4517,4518 and 4519 and light chain Variable region sequences has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it is including at least the first of anti-HER2 And second antibody, wherein the first and second antibody epi-position different from HER2 combines, and wherein the first and second antibody comprise It is selected from antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 or its humanization resists Body.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it is including at least the first of anti-HER2 And second antibody, wherein the first and second antibody epi-position different from HER2 combines, and wherein the first and second antibody comprise It is selected from combining same table with antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 and 4519 Position and the antibody of competition binding.
Another embodiment of the present invention is a kind of antibody compositions, the anti-HER2 combined including at least epi-positions different from HER2 First and second antibody of recombinant antibodies, antibody described at least one of which is selected from following compositions:
A () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:56) and the light chain CDR3 sequence of antibody 4382 (SEQ ID NO:82)；
B () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:59) and the light chain CDR3 sequence of antibody 4383 (SEQ ID NO:84)；
C () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:62) and the light chain CDR3 sequence of antibody 4384 (SEQ ID NO:86)；
D () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:65) and the light chain CDR3 sequence of antibody 4385 (SEQ ID NO:88)；
E () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:68) and the light chain CDR3 sequence of antibody 4386 (SEQ ID NO:90)；
F () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:71) and the light chain CDR3 sequence of antibody 4387 (SEQ ID NO:92)；
G () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:47) and the light chain CDR3 sequence of antibody 4517 (SEQ ID NO:76)；
H () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:50) and the light chain CDR3 sequence of antibody 4518 (SEQ ID NO:78)；
I () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:74) and the light chain CDR3 sequence of antibody 4519 (SEQ IDNO:93)；And
J () a kind of antibody, comprises heavy chain CDR3 sequence (SEQ ID NO:53) and the light chain CDR3 sequence of antibody 4380 (SEQ ID NO:80).
First and second antibody of the most described Anti-HER 2 are all selected from antibody (a)-(j).Antibody compositions also may be used Comprise the 3rd antibody of anti-HER2 recombinant antibodies, be preferably selected from a kind of antibody of above-mentioned antibody (a)-(j).In another embodiment In, antibody compositions can comprise the first and second antibody of the anti-HER2 recombinant antibodies that at least different from HER2 epi-position combines, Wherein said first and second antibody all can epi-position identical with any of the above-described antibody (a)-(j) combine and competition binding therewith.
Another embodiment of the present invention is a kind of antibody compositions, the anti-HER2 combined including at least epi-positions different from HER2 First and second antibody of recombinant antibodies, antibody described at least one of which is selected from following compositions:
(A) a kind of antibody, comprise antibody 4382 variable region of heavy chain (SEQ ID NO:14) CDR1, CDR2 and CDR3 and CDR1, CDR2 and CDR3 of variable region of light chain (SEQ ID NO:16)；
(B) a kind of antibody, comprise antibody 4383 variable region of heavy chain (SEQ ID NO:18) CDR1, CDR2 and CDR3 and CDR1, CDR2 and CDR3 of variable region of light chain (SEQ ID NO:20)；
(C) a kind of antibody, comprise antibody 4384 variable region of heavy chain (SEQ ID NO:22) CDR1, CDR2 and CDR3 and CDR1, CDR2 and CDR3 of variable region of light chain (SEQ ID NO:24)；
(D) a kind of antibody, comprise 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；
(E) a kind of antibody, comprise antibody 4386 variable region of heavy chain (SEQ ID NO:30) CDR1, CDR2 and CDR3 and CDR1, CDR2 and CDR3 of variable region of light chain (SEQ ID NO:32)；
(F) a kind of antibody, comprise antibody 4387 variable region of heavy chain (SEQ ID NO:34) CDR1, CDR2 and CDR3 and CDR1, CDR2 and CDR3 of variable region of light chain (SEQ ID NO:36)；
(G) a kind of antibody, comprise 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；
(H) a kind of antibody, comprise antibody 4518 variable region of heavy chain (SEQ ID NO:6) CDR1, CDR2 and CDR3 and CDR1, CDR2 and CDR3 of variable region of light chain (SEQ ID NO:8)；
(I) a kind of antibody, comprise antibody 4519 variable region of heavy chain (SEQ ID NO:38) CDR1, CDR2 and CDR3 and CDR1, CDR2 and CDR3 of variable region of light chain (SEQ ID NO:40)；And
(J) a kind of antibody, comprise antibody 4380 variable region of heavy chain (SEQ ID NO:10) CDR1, CDR2 and CDR3 and CDR1, CDR2 and CDR3 of variable region of light chain (SEQ ID NO:12).
In the present embodiment, the first and second antibody of the most described Anti-HER 2 are selected from above-mentioned antibody (A)-(J).Anti- Body compositions may further include at least the 3rd antibody of anti-HER2 recombinant antibodies, is preferably selected from the one of above-mentioned antibody (A)-(J) Antibody.In another embodiment, antibody compositions can comprise the anti-HER2 recombinant antibodies that at least different from HER2 epi-position combines The first and second antibody, the first and second antibody described in each of which all can be with the identical table of any of the above-described antibody (A)-(J) Position combination therewith competition binding.
One particular embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it is including at least first and the Two antibody, 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 of antibody 4382 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises antibody 4382 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380, and a kind of antibody, bag Heavy chain containing antibody 4382 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380, and a kind of antibody, comprises antibody 4382 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4382 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of compositions based on antibody 4380 and 4382, and it includes at least Other antibody a kind of, has especially selected from antibody based on antibody 4385,4517 and 4518.This type of embodiment of one of which relates to one Planting recombinant antibody composition, comprise first, second, and third recombinant antibodies, 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 of antibody 4382 Sequence；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 the weight of antibody 4382 Chain and light chain CDR3 sequence；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 light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380；A kind of antibody, comprises The heavy chain of antibody 4382 and light chain CDR1, CDR2 and CDR3 sequence；With a kind of antibody, comprise heavy chain and the light chain of antibody 4385 CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380；A kind of antibody, comprises antibody 4382 Heavy chain and light-chain variable sequence；With a kind of antibody, comprise heavy chain and the light-chain variable sequence of antibody 4385；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4382 Row have at least 80%, the identical sequence of 85%, 90% or 95%；With a kind of antibody, comprise a weight chain variabl area sequence and One light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4385 have at least 80%, 85%, The identical sequence of 90% or 95%.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises first, second, and third and resists Body, 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 of antibody 4382 Sequence；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 the weight of antibody 4382 Chain and light chain CDR3 sequence；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 light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380；A kind of antibody, comprises The heavy chain of antibody 4382 and light chain CDR1, CDR2 and CDR3 sequence；With a kind of antibody, comprise heavy chain and the light chain of antibody 4517 CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380；A kind of antibody, comprises antibody 4382 Heavy chain and light-chain variable sequence；With a kind of antibody, comprise heavy chain and the light-chain variable sequence of antibody 4517；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4382 Row have at least 80%, the identical sequence of 85%, 90% or 95%；With a kind of antibody, comprise a weight chain variabl area sequence and One light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4517 have at least 80%, 85%, The identical sequence of 90% or 95%.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises first, second, and third and resists Body, Qi Zhong, second and the 3rd antibody be:
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 of antibody 4382 Sequence；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 the weight of antibody 4382 Chain and light chain CDR3 sequence；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 light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380；A kind of antibody, comprises The heavy chain of antibody 4382 and light chain CDR1, CDR2 and CDR3 sequence；With a kind of antibody, comprise heavy chain and the light chain of antibody 4518 CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380；A kind of antibody, comprises antibody 4382 Heavy chain and light-chain variable sequence；With a kind of antibody, comprise heavy chain and the light-chain variable sequence of antibody 4518；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4382 Row have at least 80%, the identical sequence of 85%, 90% or 95%；With a kind of antibody, comprise a weight chain variabl area sequence and One light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4518 have at least 80%, 85%, The identical sequence of 90% or 95%.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises first, second, third and the Four antibody, wherein first, second, third and fourth 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 of antibody 4382 Sequence；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 the weight of antibody 4382 Chain and light chain CDR3 sequence；A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385；A kind of antibody, comprises antibody The heavy chain of 4518 and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380；A kind of antibody, comprises The heavy chain of antibody 4382 and light chain CDR1, CDR2 and CDR3 sequence；A kind of antibody, comprises heavy chain and the light chain of antibody 4385 CDR1, CDR2 and CDR3 sequence；A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4518；Or
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380；A kind of antibody, comprises antibody 4382 Heavy chain and light-chain variable sequence；A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4385；A kind of antibody, bag Heavy chain and light-chain variable sequence containing antibody 4518；
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4382 Row have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, comprises a weight chain variabl area sequence and Individual light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4385 have at least 80%, 85%, 90% Or the identical sequence of 95%；A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with Heavy chain and the light-chain variable sequence of antibody 4518 have at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4383 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises antibody 4383 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380, and a kind of antibody, bag Heavy chain containing antibody 4383 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380, and a kind of antibody, comprises antibody 4383 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4383 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of compositions based on antibody 4380 and 4383, and it includes at least Other antibody a kind of.This type of embodiment of one of which relates to a kind of recombinant antibody composition, and it comprises first, second, and third and resists Body, 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 of antibody 4383 Sequence；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 the weight of antibody 4383 Chain and light chain CDR3 sequence；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 light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380；A kind of antibody, comprises The heavy chain of antibody 4383 and light chain CDR1, CDR2 and CDR3 sequence；With a kind of antibody, comprise heavy chain and the light chain of antibody 4384 CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380；A kind of antibody, comprises antibody 4383 Heavy chain and light-chain variable sequence；With a kind of antibody, comprise heavy chain and the light-chain variable sequence of antibody 4384；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4383 Row have at least 80%, the identical sequence of 85%, 90% or 95%；With a kind of antibody, comprise a weight chain variabl area sequence and One light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4384 have at least 80%, 85%, The identical sequence of 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4384 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises antibody 4384 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380, and a kind of antibody, bag Heavy chain containing antibody 4384 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380, and a kind of antibody, comprises antibody 4384 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4384 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of compositions based on antibody 4380 and 4384, and it includes at least Other antibody a kind of, has especially selected from antibody based on antibody 4385,4517,4518 and 4519.This type of embodiment of one of which Relating to a kind of recombinant antibody composition, comprise first, second, and third recombinant antibodies, 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 of antibody 4380 Sequence；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 the weight of antibody 4384 Chain and light chain CDR3 sequence；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 light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380；A kind of antibody, comprises The heavy chain of antibody 4384 and light chain CDR1, CDR2 and CDR3 sequence；With a kind of antibody, comprise heavy chain and the light chain of antibody 4517 CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380；A kind of antibody, comprises antibody 4384 Heavy chain and light-chain variable sequence；With a kind of antibody, comprise heavy chain and the light-chain variable sequence of antibody 4517；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4384 Row have at least 80%, the identical sequence of 85%, 90% or 95%；With a kind of antibody, comprise a weight chain variabl area sequence and One light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4517 have at least 80%, 85%, The identical sequence of 90% or 95%.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises first, second, and third and resists Body, 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 of antibody 4380 Sequence；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 the weight of antibody 4384 Chain and light chain CDR3 sequence；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 light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380；A kind of antibody, comprises The heavy chain of antibody 4384 and light chain CDR1, CDR2 and CDR3 sequence；With a kind of antibody, comprise heavy chain and the light chain of antibody 4518 CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380；A kind of antibody, comprises antibody 4384 Heavy chain and light-chain variable sequence；With a kind of antibody, comprise heavy chain and the light-chain variable sequence of antibody 4518；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4384 Row have at least 80%, the identical sequence of 85%, 90% or 95%；With a kind of antibody, comprise a weight chain variabl area sequence and One light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4518 have at least 80%, 85%, The identical sequence of 90% or 95%.
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, 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 of antibody 4380 Sequence；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 the weight of antibody 4384 Chain and light chain CDR3 sequence；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 light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380；A kind of antibody, comprises The heavy chain of antibody 4384 and light chain CDR1, CDR2 and CDR3 sequence；With a kind of antibody, comprise heavy chain and the light chain of antibody 4519 CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380；A kind of antibody, comprises antibody 4384 Heavy chain and light-chain variable sequence；With a kind of antibody, comprise heavy chain and the light-chain variable sequence of antibody 4519；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4384 Row have at least 80%, the identical sequence of 85%, 90% or 95%；With a kind of antibody, comprise a weight chain variabl area sequence and One light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4519 have at least 80%, 85%, The identical sequence of 90% or 95%.
Another this type of embodiment of the present invention relates to a kind of recombinant antibody composition, and it comprises first, second, third and the Four antibody, wherein first, second, third and fourth 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 of antibody 4384 Sequence；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 the weight of antibody 4384 Chain and light chain CDR3 sequence；A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385；A kind of antibody, comprises antibody The heavy chain of 4518 and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380；A kind of antibody, comprises The heavy chain of antibody 4384 and light chain CDR1, CDR2 and CDR3 sequence；A kind of antibody, comprises heavy chain and the light chain of antibody 4385 CDR1, CDR2 and CDR3 sequence；A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4518；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380；A kind of antibody, comprises antibody 4384 Heavy chain and light-chain variable sequence；A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4385；A kind of antibody, bag Heavy chain and light-chain variable sequence containing antibody 4518；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4384 Row have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, comprises a weight chain variabl area sequence and Individual light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4385 have at least 80%, 85%, 90% Or the identical sequence of 95%；A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with Heavy chain and the light-chain variable sequence of antibody 4518 have at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4385 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises antibody 4385 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380, and a kind of antibody, bag Heavy chain containing antibody 4385 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380, and a kind of antibody, comprises antibody 4385 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4385 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4386 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises antibody 4386 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380, and a kind of antibody, bag Heavy chain containing antibody 4386 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380, and a kind of antibody, comprises antibody 4386 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4386 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4387 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises antibody 4387 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380, and a kind of antibody, bag Heavy chain containing antibody 4387 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380, and a kind of antibody, comprises antibody 4387 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4387 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4517 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises antibody 4517 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380, and a kind of antibody, bag Heavy chain containing antibody 4517 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380, and a kind of antibody, comprises antibody 4517 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4517 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4518 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises antibody 4518 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380, and a kind of antibody, bag Heavy chain containing antibody 4518 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380, and a kind of antibody, comprises antibody 4518 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4518 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4519 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4380, and a kind of antibody, comprises antibody 4519 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4380, and a kind of antibody, bag Heavy chain containing antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4380, and a kind of antibody, comprises antibody 4519 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4380 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4519 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4384 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises antibody 4384 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4382, and a kind of antibody, bag Heavy chain containing antibody 4384 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4382, and a kind of antibody, comprises antibody 4384 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4382 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4384 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4385 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises antibody 4385 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4382, and a kind of antibody, bag Heavy chain containing antibody 4385 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4382, and a kind of antibody, comprises antibody 4385 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4382 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4385 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of compositions based on antibody 4382 and 4385, and it includes at least Other antibody a kind of.This type of embodiment of one of which relates to a kind of recombinant antibody composition, and it comprises first, second, and third and resists Body, 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 of antibody 4385 Sequence；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 the weight of antibody 4385 Chain and light chain CDR3 sequence；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 light chain CDR1, CDR2 and the CDR3 sequence of antibody 4382；A kind of antibody, comprises The heavy chain of antibody 4385 and light chain CDR1, CDR2 and CDR3 sequence；With a kind of antibody, comprise heavy chain and the light chain of antibody 4518 CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4382；A kind of antibody, comprises antibody 4385 Heavy chain and light-chain variable sequence；With a kind of antibody, comprise heavy chain and the light-chain variable sequence of antibody 4518；
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4382 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4385 Row have at least 80%, the identical sequence of 85%, 90% or 95%；With a kind of antibody, comprise a weight chain variabl area sequence and One light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4518 have at least 80%, 85%, The identical sequence of 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4386 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises antibody 4386 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4382, and a kind of antibody, bag Heavy chain containing antibody 4386 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4382, and a kind of antibody, comprises antibody 4386 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4382 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4386 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4387 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises antibody 4387 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4382, and a kind of antibody, bag Heavy chain containing antibody 4387 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4382, and a kind of antibody, comprises antibody 4387 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4382 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4387 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of compositions based on antibody 4382 and 4387, and it includes at least Other antibody a kind of.This type of embodiment of one of which relates to a kind of recombinant antibody composition, and it comprises first, second, and third and resists Body, 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 of antibody 4387 Sequence；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 the weight of antibody 4387 Chain and light chain CDR3 sequence；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 light chain CDR1, CDR2 and the CDR3 sequence of antibody 4382；A kind of antibody, comprises The heavy chain of antibody 4387 and light chain CDR1, CDR2 and CDR3 sequence；With a kind of antibody, comprise heavy chain and the light chain of antibody 4517 CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4382；A kind of antibody, comprises antibody 4387 Heavy chain and light-chain variable sequence；With a kind of antibody, comprise heavy chain and the light-chain variable sequence of antibody 4517；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4382 have at least 80%, the identical sequence of 85%, 90% or 95%；A kind of antibody, bag Containing a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain sequence of antibody 4387 Row have at least 80%, the identical sequence of 85%, 90% or 95%；With a kind of antibody, comprise a weight chain variabl area sequence and One light-chain variable sequence, its respectively heavy chain and light-chain variable sequence with antibody 4517 have at least 80%, 85%, The identical sequence of 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4517 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises antibody 4517 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4382, and a kind of antibody, bag Heavy chain containing antibody 4517 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4382, and a kind of antibody, comprises antibody 4517 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4382 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4517 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4518 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4382, and a kind of antibody, comprises antibody 4518 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4382, and a kind of antibody, bag Heavy chain containing antibody 4518 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4382, and a kind of antibody, comprises antibody 4518 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4382 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4518 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4384 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises antibody 4384 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4383, and a kind of antibody, bag Heavy chain containing antibody 4384 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4383, and a kind of antibody, comprises antibody 4384 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4383 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4384 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4385 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises antibody 4385 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4383, and a kind of antibody, bag Heavy chain containing antibody 4385 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4383, and a kind of antibody, comprises antibody 4385 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4383 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4385 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4386 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises antibody 4386 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4383, and a kind of antibody, bag Heavy chain containing antibody 4386 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4383, and a kind of antibody, comprises antibody 4386 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4383 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4386 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4517 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises antibody 4517 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4383, and a kind of antibody, bag Heavy chain containing antibody 4517 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4383, and a kind of antibody, comprises antibody 4517 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4383 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4517 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4518 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises antibody 4518 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4383, and a kind of antibody, bag Heavy chain containing antibody 4518 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4383, and a kind of antibody, comprises antibody 4518 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4383 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4518 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4519 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4383, and a kind of antibody, comprises antibody 4519 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4383, and a kind of antibody, bag Heavy chain containing antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4383, and a kind of antibody, comprises antibody 4519 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4383 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4519 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4385 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises antibody 4385 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4384, and a kind of antibody, bag Heavy chain containing antibody 4385 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4384, and a kind of antibody, comprises antibody 4385 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4384 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4385 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4387 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises antibody 4387 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4384, and a kind of antibody, bag Heavy chain containing antibody 4387 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4384, and a kind of antibody, comprises antibody 4387 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4384 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4387 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4517 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises antibody 4517 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4384, and a kind of antibody, bag Heavy chain containing antibody 4517 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4384, and a kind of antibody, comprises antibody 4517 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4384 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4517 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4519 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4384, and a kind of antibody, comprises antibody 4519 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4384, and a kind of antibody, bag Heavy chain containing antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4384, and a kind of antibody, comprises antibody 4519 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4384 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4519 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4386 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises antibody 4386 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4385, and a kind of antibody, bag Heavy chain containing antibody 4386 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4385, and a kind of antibody, comprises antibody 4386 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4385 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4386 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4517 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises antibody 4517 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4385, and a kind of antibody, bag Heavy chain containing antibody 4517 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4385, and a kind of antibody, comprises antibody 4517 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4385 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4517 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4518 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises antibody 4518 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4385, and a kind of antibody, bag Heavy chain containing antibody 4518 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4385, and a kind of antibody, comprises antibody 4518 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4385 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4518 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4519 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4385, and a kind of antibody, comprises antibody 4519 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4385, and a kind of antibody, bag Heavy chain containing antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4385, and a kind of antibody, comprises antibody 4519 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4385 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4519 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4387 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises antibody 4387 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4386, and a kind of antibody, bag Heavy chain containing antibody 4387 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4386, and a kind of antibody, comprises antibody 4387 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4386 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4387 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4517 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises antibody 4517 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4386, and a kind of antibody, bag Heavy chain containing antibody 4517 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4386, and a kind of antibody, comprises antibody 4517 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4386 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4517 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4518 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises antibody 4518 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4386, and a kind of antibody, bag Heavy chain containing antibody 4518 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4386, and a kind of antibody, comprises antibody 4518 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4386 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4518 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4519 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4386, and a kind of antibody, comprises antibody 4519 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4386, and a kind of antibody, bag Heavy chain containing antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4386, and a kind of antibody, comprises antibody 4519 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4386 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4519 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4517 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4387, and a kind of antibody, comprises antibody 4517 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4387, and a kind of antibody, bag Heavy chain containing antibody 4517 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4387, and a kind of antibody, comprises antibody 4517 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4387 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4517 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4518 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4387, and a kind of antibody, comprises antibody 4518 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4387, and a kind of antibody, bag Heavy chain containing antibody 4518 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4387, and a kind of antibody, comprises antibody 4518 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4387 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4518 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4519 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4387, and a kind of antibody, comprises antibody 4519 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4387, and a kind of antibody, bag Heavy chain containing antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4387, and a kind of antibody, comprises antibody 4519 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4387 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4519 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4519 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4517, and a kind of antibody, comprises antibody 4519 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4517, and a kind of antibody, bag Heavy chain containing antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4517, and a kind of antibody, comprises antibody 4519 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4517 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4519 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
Another embodiment of this aspect of the present invention relates to a kind of recombinant antibody composition, and it resists including at least first and second Body, 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 of antibody 4519 CDR3 sequence；
A kind of antibody, comprises heavy chain and the light chain CDR3 sequence of antibody 4518, and a kind of antibody, comprises antibody 4519 Heavy chain and light chain CDR3 sequence；
A kind of antibody, comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of antibody 4518, and a kind of antibody, bag Heavy chain containing antibody 4519 and light chain CDR1, CDR2 and CDR3 sequence；
A kind of antibody, comprises heavy chain and the light-chain variable sequence of antibody 4518, and a kind of antibody, comprises antibody 4519 Heavy chain and light-chain variable sequence；Or
A kind of antibody, comprises a weight chain variabl area sequence and a light-chain variable sequence, its respectively with antibody Heavy chain and the light-chain variable sequence of 4518 have at least 80%, the identical sequence of 85%, 90% or 95%, and a kind of antibody, Comprise a weight chain variabl area sequence and a light-chain variable sequence, its respectively with heavy chain and the variable region of light chain of antibody 4519 Sequence has at least 80%, the identical sequence of 85%, 90% or 95%.
It is therefore preferable that the recombinant polyclonal Anti-HER 2 compositions of the present invention, the first and second antibody wherein comprised 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 the every antagonist centering listed, such as, therein One and second antibody comprise the CDR3 sequence of described heavy chain of antibody, or the first and second antibody therein comprise described heavy chain of antibody With the CDR3 sequence of light chain, or the first and second antibody therein comprise described heavy chain of antibody and light chain CDR1, CDR2 and CDR3 sequence, the most described heavy chain of antibody and variable region of light chain and humanized antibody thereof.
More preferably the recombinant polyclonal Anti-HER 2 compositions of the present invention, wherein comprises 2 selected from following combination, 3 or 4 Kind antibody:
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 that antibody sources is in each Antibody Combination listed, such as, Qi Zhongdan Individual antibody comprises the CDR3 sequence of described heavy chain of antibody, or the most single antibody comprises the CDR3 sequence of described heavy chain of antibody and light chain Arrange, or the most single antibody comprises CDR1, CDR2 and the CDR3 sequence of described heavy chain of antibody and light chain, the most described heavy chain of antibody With variable region of light chain and humanized antibody thereof.
The most more preferably the recombinant polyclonal Anti-HER 2 compositions of the present invention, wherein comprises 2 selected from following combination Or 3 kinds of antibody:
4382 and 4518,
4384 and 4517,
4382,4385 and 4518,
4382,4387 and 4517；
Or its humanized antibody；Or the single antibody that antibody sources is in each Antibody Combination listed, such as, Qi Zhongdan Individual antibody comprises the CDR3 sequence of described heavy chain of antibody, or the most single antibody comprises the CDR3 sequence of described heavy chain of antibody and light chain Arrange, or the most single antibody comprises CDR1, CDR2 and the CDR3 sequence of described heavy chain of antibody and light chain, the most described heavy chain of antibody With variable region of light chain and humanized antibody thereof.
Another embodiment relates to a kind of recombinant antibody composition, and it is including at least first and second antibody of anti-HER2, Wherein the first and second antibody epi-position different from HER2 combines, and the first and second antibody and the above-mentioned each combination listed The same epi-position of single antibodies of thing.
In a preferred aspect, the one anti-HER2 recombinant polyclonal antibody compositions of the present invention, it is at least by can be with 3 kinds of antibody that HER2 difference epi-position combines, the more preferably first and second antibody are combined with HER2 and may result in HER2 receptor internalisation, and 3rd antibody is combined the HER3 phosphorylation that part can be suppressed to induce with HER2.It is generally believed that this kind of antibody compositions can by with Lower mechanism onset: wherein 2 kinds of antibody are once combined with the HER2 of cell surface, it is possible to generate the anti-of a kind of coupling at cell surface Body-by volume mesh, this will raise HER2 receptor internalisation level, and the 3rd antibody is combined with HER2, so can block HER2 with The Heterodimerization of HER3, thus suppress HER3 phosphorylation.
The example of this type of recombinant polyclonal antibody compositions is, the first antibody of Anti-HER 2 is 4517 or 4518, anti- The second antibody of HER2 antibody 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 the every antagonist centering listed, such as, therein One, second and the 3rd antibody comprise the CDR3 sequence of described heavy chain of antibody, or first, second, and third antibody therein comprises institute State the CDR3 sequence of heavy chain of antibody and light chain, or first, second, and third antibody therein comprises described heavy chain of antibody and light chain CDR1, CDR2 and CDR3 sequence, the most 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 includes following compositions, wherein the first He of Anti-HER 2 Second antibody is 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 the every antagonist centering listed, such as, therein One, second and the 3rd antibody comprise the CDR3 sequence of described heavy chain of antibody, or first, second, and third antibody therein comprises institute State the CDR3 sequence of heavy chain of antibody and light chain, or first, second, and third antibody therein comprises described heavy chain of antibody and light chain CDR1, CDR2 and CDR3 sequence, the most 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 and can epi-positions different from HER2 combine First, second, and third antibody of Anti-HER 2, wherein:
A the first antibody of () Anti-HER 2 comprises:
The heavy chain CDR3 sequence (SEQ ID NO:47) of antibody 4517 and light chain CDR3 sequence (SEQ IDNO:76), or
The heavy chain CDR3 sequence (SEQ ID NO:50) of antibody 4518 and light chain CDR3 sequence (SEQ IDNO:78)；
B the second antibody of () Anti-HER 2 comprises:
The heavy chain CDR3 sequence (SEQ ID NO:53) of antibody 4380 and light chain CDR3 sequence (SEQ IDNO:80),
The heavy chain CDR3 sequence (SEQ ID NO:65) of antibody 4385 and light chain CDR3 sequence (SEQ IDNO:88), or
The heavy chain CDR3 sequence (SEQ ID NO:71) of antibody 4387 and light chain CDR3 sequence (SEQ IDNO:92)；With
C 3rd antibody of () Anti-HER 2 comprises:
The heavy chain CDR3 sequence (SEQ ID NO:56) of antibody 4382 and light chain CDR3 sequence (SEQ IDNO:82),
The heavy chain CDR3 sequence (SEQ ID NO:59) of antibody 4383 and light chain CDR3 sequence (SEQ IDNO:84), or
The heavy chain CDR3 sequence (SEQ ID NO:74) of antibody 4519 and light chain CDR3 sequence (SEQ IDNO:93).
In certain preferred embodiments, the present invention relates to a kind of antibody compositions, it comprises can epi-positions different from HER2 knot First, second, and third antibody of the Anti-HER 2 closed, wherein:
A () this anti-HER2 first antibody comprises:
CDR1, CDR2 and CDR3 of antibody 4517 variable region of heavy chain (SEQ ID NO:2) and variable region of light chain (SEQ IDNO:4) CDR1, CDR2 and CDR3, or
CDR1, CDR2 and CDR3 of antibody 4518 variable region of heavy chain (SEQ IDNO:6) and variable region of light chain (SEQ IDNO:8) CDR1, CDR2 and CDR3；
B () this anti-HER2 second antibody comprises:
CDR1, CDR2 and CDR3 of antibody 4380 variable region of heavy chain (SEQ ID NO:10) and variable region of light chain (SEQIDNO:12) CDR1, CDR2 and CDR3,
CDR1, CDR2 and CDR3 of antibody 4385 variable region of heavy chain (SEQ ID NO:26) and variable region of light chain (SEQ ID NO:28) CDR1, CDR2 and CDR3, or
CDR1, CDR2 and CDR3 of antibody 4387 variable region of heavy chain (SEQ ID NO:34) and variable region of light chain (SEQ ID NO:36) CDR1, CDR2 and CDR3；And
C () this anti-HER2 the 3rd antibody comprises:
CDR1, CDR2 and CDR3 of antibody 4382 variable region of heavy chain (SEQ ID NO:14) and variable region of light chain (SEQIDNO:16) CDR1, CDR2 and CDR3,
CDR1, CDR2 and CDR3 of antibody 4383 variable region of heavy chain (SEQ ID NO:18) and variable region of light chain (SEQIDNO:20) CDR1, CDR2 and CDR3, or
CDR1, CDR2 and CDR3 of antibody 4519 variable region of heavy chain (SEQ ID NO:38) and variable region of light chain (SEQ ID NO:40) CDR1, CDR2 and CDR3.
In a certain particularly preferred 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).At this In the case of Zhong, Anti-HER 2 compositions may is that
The first antibody of (a) Anti-HER 2 comprise 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 comprise 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；Or
The first antibody of (b) Anti-HER 2 comprise 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 comprise 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 compositions of the present invention may is that
The first antibody of Anti-HER 2 comprise 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 comprise 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；Or
3rd antibody of Anti-HER 2 comprise 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 HER2 domain II, and handkerchief trastuzumab is also kindred circumstances, and as described in example 9, anti- Body 4382 and handkerchief trastuzumab analog all can block the HER3 phosphorylation of part induction.(the Cancer Cell such as Franklin 2004,5 (4): 317-28) disclosing, handkerchief trastuzumab can be combined with HER2 at domain II immediate vicinity, structure blocks HER2- Binding pocket needed for HER3 Heterodimerization and signal transduction.Therefore, in addition to such as antibody 4382 and handkerchief trastuzumab, The blocking-up HER2-HER3 Heterodimerization that other Anti-HER 2s being combined with dimerization interface in a similar manner will have like Effect, three that therefore this antibody-like is suitable as the present invention in terms of this respect resist.
Although related to the present invention it was found that, 2 kinds or multiple Anti-HER 2 energy described in combination this patent Enough effectively cause HER2 internalization and degraded, but HER2 may be raised the generation of HER3 by the cell of antibody target.It is now recognized that Anti-HER 2 monoclonal antibody (such as Herceptin) drug resistance may raise HER3 to it relevant, this may be by following machine System produces, i.e. HER2-HER3 heterodimer constitutes a complete receptor, it is possible in the situation without HER2 homodimer signal Under still mediated cancer signal transduction.Following example 9 describes the antibody compositions of the present invention being made up of 3 kinds of Anti-HER 2s, Wherein 2 kinds of antibody can be combined with HER2, so can generate coupled antibody by volume mesh, thus cause HER2 internalization and fall Solve, and three resist and can so block HER2-HER3 Heterodimerization in conjunction with HER2, thus avoid HER2 phosphorylation and HER3 to be situated between The signal transduction led.Assuming that these with similar Anti-HER 2 compositions in terms of blocking HER family phosphorylation and signal transduction Having the strongest advantage, i.e. this antibody compositions and comprise the mixture of at least Anti-HER 2, wherein 2 kinds of antibody may result in HER2 Internalization and degraded, the 3rd antibody blocks HER2-HER3 Heterodimerization.Especially, it is assumed that this kind of antibody compositions can show Write and reduce the growth of the tumor cell to Anti-HER 2 drug resistance.Therefore, this kind of Anti-HER 2 compositions to preventing and may subtract Light this kind of drug resistance of Anti-HER 2 is the most valuable, and to treatment to certain anti-HER 2 monoclonal antibody (such as toltrazuril list Anti-) treatment drug-resistant tumor is the most valuable.
Another aspect of the present invention and suppression are raw to the tumor cell of a kind of Anti-HER 2 treatment drug resistance or part drug resistance Long method is correlated with, and the method comprises a kind of for cells contacting anti-HER2 recombinant polyclonal antibody compositions as above, its Comprise 2 kinds can cell surface produce coupled antibody by volume mesh Anti-HER 2 (causing HER2 internalization) and a kind of with The blocked HER2 and the three of HER3 Heterodimerization that HER2 combines is anti-(the HER3 phosphorylation of suppression part induction).Before such as Once Herceptin treatment tumor cell was used.
Table 2 below and 3 show the heavy chain (table 2) of all kinds of Anti-HER 2s according to the present invention and the CDR1 of light chain (table 3), CDR2 and CDR3 sequence.The sequence table of annex is listed these antibody heavy chain variable regions and the ammonia of light chain (including variable region of light chain) Base acid sequence, and DNA sequences encoding (optimizing for expressing cho cell).The SEQID numbering seeing these sequences in table 1 is converged Always.
Table 2: selected Anti-HER 2 heavy chain CDR1, CDR2 and CDR3 sequence
Table 3: selected Anti-HER 2 light chain CDR1, CDR2 and CDR3 sequence
Another aspect of the present invention relates to nucleic acid molecules, and it comprises the nucleotides sequence of a kind of antibody in a code book invention Row, i.e. a kind of antibody is selected from antibody 4380/4381,4382,4383,4384,4385,4386,4387,4517,4518 Hes 4519, or its humanization variant；Or the heavy chain of encoding such antibody and/or light-chain variable sequence, or a kind of heavy chain and/or Sequence of light chain, it is with this type of heavy chain and/or light-chain variable sequence has at least 80%, 85%, 90% or 95% identical sequence.
In an embodiment of this aspect of the present invention, nucleic acid molecules comprises a nucleotide sequence, is 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 coding is arbitrary with described The nucleotide sequence of the aminoacid sequence that nucleotide sequence is identical.
Another aspect of the present invention relates to a kind of expression vector, and it comprises above-mentioned nucleic acid molecules.As it has been described above, in the present invention Middle expression carrier used thereof can be any suitable type known in the art, such as plasmid or viral vector.
Another aspect of the present invention relates to a kind of host cell, and it comprises above-mentioned nucleic acid molecules, and described host cell can Express the Anti-HER 2 of described nucleic acid molecule encoding.
On the other hand, the binding specificity of the antibody disclosed in any 2 kinds of this patents can be combined with a kind of bispecific point Sub-portfolio.The most this kind of bi-specific binding molecule comprises heavy chain and light chain CDR1, CDR2 and the CDR3 sequence of selected 2 kinds of antibody Row.Bi-specific binding molecule can be bidirectional variable domain antibodies, and wherein antibody two-arm comprises 2 different variable regions, or Can be antibody fragment, such as bi-specific Fab-fragment or bispecific scFv.
Prepare Anti-HER 2 and antibody compositions
Another aspect of the present invention relates to preparing Anti-HER 2 of the present invention or the method for anti-HER2 polyclonal antibody.This A bright embodiment relates to a kind of method preparing described Anti-HER 2, comprises and can express anti-HER2 described in offer one The host cell of antibody, cultivates described host cell, and the antibody of isolated under conditions of this antibody of applicable expression.
In another embodiment, a kind of method that the present invention relates to prepare anti-HER2 polyclone recombinant antibody composition, should Compositions includes at least the first and second host cells, and wherein can to express anti-HER2 restructuring anti-for the first and second host cells Body, cultivates the first and second host cells, and the first He of isolated under conditions of applicable expression the first and second antibody Second antibody
A kind of antibody or antibody compositions in the present invention can produce recombinant monoclonal or monoclonal anti by prior art Prepared by body method.Therefore, when producing the single antibody of the present invention, any side of existing production recombinant monoclonal antibodies can be used Method.When producing a kind of antibody compositions being made up of in the present invention 2 kinds or multiple Anti-HER 2, can produce single anti-respectively Body, i.e. prepares single antibody in independent bioreactor respectively, or to produce single in a bioreactor anti-simultaneously Body.When the different antibodies quantity in a kind of antibody compositions exceedes, such as 2 kinds or 3 kinds, then it is generally in cost-efficient examining Consider, in a bioreactor, preferably produce antibody simultaneously.On the other hand, a small amount of different antibodies is only comprised when said composition Time, such as 2 kinds, 3 kinds or 4 kinds of different antibodies, then need to determine as the case may be to be respectively in different bioreactors Produce, or produce in a bioreactor simultaneously.If producing antibody compositions in multiple bioreactors, then may be used By obtaining supernatant from each bioreactor purification respectively, then collect the antibody in supernatant, finally obtain the anti-of purification HER2 antibody compositions.All kinds of methods producing polyclonal antibody in multiple bioreactors see WO 2009/129814 (incorporated by reference), is wherein described in upstream later time point or before Downstream processing or period merges cell line or antibody and prepares Thing.
2 kinds or multiple single antibody is produced in single biomass generator, can be by WO 2004/061104 or WO2008/ (incorporated by reference in this patent) is carried out described in 145133.Method described in WO2004/061104 is special based on antibody coding sequence site Specific integration enters in host cell, it is ensured that VHAnd VLMaintain its original pair in process of production.Furthermore, site-specific integration Position effect can be minimized, it is therefore contemplated that in multi-clone cell line, the growth of individual cells can be similar with expression characterization.Logical Often, the method relates to: i) a kind of host cell with one or more recombinase recognition sites；Ii) compatible with this host cell The expression vector including at least a recombinase recognition site；Iii) by by selected VHAnd VLCoding is to being transferred to a table Reach in carrier, form expression vector collection, thus can from this vector expression full length antibody or antibody fragment, if screening vector and table Reach carrier identical, then without this transfer；Iv) can be with host cell gene in middle restructuring with expression vector collection and a coding The carrier transfection host cell that enzyme recognition site combines；V) host cell from transfection obtains/generates multi-clone cell line；With Vi) express from this multi-clone cell line and collect antibody compositions.
WO 2008/145133 describes a kind of 2 kinds or another kind side of Multiple Antibodies of production in single biomass generator Method.The method relates to generating a kind of multi-clone cell line, and this cell line can express a kind of polyclonal antibody or other comprises 2 kinds Or the polyclonal protein of multiple heterogeneity, method is a) to provide a set of expression vector, wherein said carrier to comprise at least one Unique nucleotide copies, a kind of unique member of its coding polyclonal protein, is avoiding expression vector site-specific integration Enter cytogene under the conditions, respectively by expression vector transfection host cell, thus obtain 2 kinds or various kinds of cell compositions, Each compositions expresses a kind of unique component of this polyclonal protein；And c) at least 2 kinds of cell compositions are mixed to get more than one Cloned cell line.The method advantage of WO 2004/061104 and WO 2008/145133 is: can give birth in single bioreactor Produce all compositionss of recombinant polyclonal antibody, and by single program purification, thus avoid producing respectively with purification each Antibody, meanwhile can unify to produce different antibodies.WO 2008/145133 method also has the advantage improving yield, because often A kind of multiple copies of the polynucleotide producing the cell portability a certain antibody of coding.
The antibody of the present invention can be produced, including mammalian cell and the eucaryon of nonmammalian or former in various types of cells Nucleus, such as plant cell, insect cell, yeast cells, fungus, escherichia coli etc..But preferably in mammalian cell Produce antibody, such as Chinese hamster ovary celI, COS cell, bhk cell, myeloma cell (such as, Sp2/0 or NS0 cell), one-tenth fiber Cell, such as NIH 3T3, or immortalised human cells, such as HeLa cell, HEK 293 cell or PER.C6 cell.
The method that nucleotide sequence is transfected into known to those skilled in the art host cell at present (see, e.g. Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd Edition, 2001).Site-specific integration described in WO 2004/061104, applicable One or more recombinase recognition sites are comprised in host cell gene group.In this case, suitable expression vector comprises one The restructuring recognition site of individual coupling host cell recombinase.WO2004/061104 describes more relevant informations in detail, for example with site The method of specific integration is from a selected V of screening vector transferHAnd VLIt is right to encode.
A kind of antibody compositions of the present invention comprising 2 or multiple Anti-HER 2 is generated in single biomass generator Time, can be chosen with similar growth rate and generate a multi-clone cell line with the cell line of similar antibody expression. Then by mixing individual cells system acquisition multi-clone cell line by predetermined ratio.Relevant generate a kind of polyclonal antibody of expressing Multi-clone cell line and use this type of cell line produce polyclonal antibody, as more detailed information and example need to be understood, please join Read WO 2009/129814, WO 2004/061104 and WO 2008/145133 (this patent incorporated by reference).
An embodiment in the present invention is a kind of multi-clone cell line, and it can express in the present invention 2 kinds or multiple anti- HER2 antibody.Another embodiment is a kind of multi-clone cell line, and wherein single cell can express single VHAnd VLRight, and many grams Grand cell line entirety can express VHAnd VLTo set, each of which VHAnd VLAnti-HER 2 a kind of to coding.
Can produce a kind of recombinant antibody composition of the present invention in single bioreactor, method is as follows: suitably In culture medium, a period of time will be cultivated from an ampoule in polyclone working cell storehouse (pWCB), to reach enough antibody Expression, maintains the relative expression levels of multi-clone cell line expression different antibodies substantially uniform simultaneously.The generally production time It was advisable at about 15-50 days.Existing culture technique, such as fed-batch type can be used to cultivate or perfusion cultures.Culture medium is preferred Serum-free medium, more preferably serum-free and protein culture medium, the most chemically defined culture medium.This kind of culture medium is generally used for certain The cell of kind of production type is cultivated, and presently commercially available has a large amount of suitably culture medium preparation.
Obtain recombinant antibody composition from culture medium, and use conventional purification techniques to be purified.These technology include, example As, affinity chromatograph coupling following purification steps, such as ion-exchange chromatography, hydrophobic chromatography and gel filtration, these purification techniques It is frequently used for recombinant antibodies purification.2 kinds or Multiple Antibodies is produced by a multi-clone cell line in single bioreactor, logical All single compositions in assessment polyclonal antibody compositions, such as ion-exchange chromatography the most after purification.Such as, can use Method described in WO2006/007853 and WO 2009/065414 (this patent incorporated by reference) is to a kind of polyclonal antibody compositions Qualitative analysis.
Therapeutic composition
Another aspect of the invention is a kind of pharmaceutical composition, comprise a kind of active component, can be at least in the present invention A kind of Anti-HER 2 conduct, or a kind of anti-HER2 recombinant Fab or another kind of anti-HER2 recombinant antibody fragment compositions.Preferably The active component of this type of pharmaceutical composition is the anti-HER2 recombinant antibodies group comprising 2 kinds or multiple Anti-HER 2 as above Compound.This compositions can be used for improving, preventing and/or treat cancer.Pharmaceutical composition can be used for the mankind, domestic animal or house pet to Medicine, but typically the mankind are administered.
Ratio between different antibodies in a kind of therapeutic composition of the present invention, or at the same time, sequential or give this respectively In the case of invention different antibodies, the ratio between different antibodies, often equivalent, it is possible to inequality.Therefore, the present invention contains The antibody compositions of 2 kinds of Anti-HER 2s through frequently with 1: 1 ratio, and comprise the antibody compositions warp of 3 kinds of Anti-HER 2s Frequently with the ratio of 1: 1: 1.But, according to the characteristic of different antibodies, it may be necessary to inequality proportioning.Antibody compositions of the present invention The proper ratio of middle different Anti-HER 2 can determine (this patent incorporated by reference) as described in WO2010/040356, described in it The determination of optimum chemical metered proportions and system of selection, such as a kind of polyclonal antibody combination between chemical substance in composition of medicine Thing, to obtain the composition of medicine with optimal effect and curative effect.
In addition to the antibody or fragment of at least one present invention, pharmaceutical composition also includes that at least one is medicinal acceptable dilute Release agent, carrier or excipient.These can include, such as preservative, stabilizer, surfactant/wetting agent, emulsifying agent, solubilising Agent and for regulating salt and/or the buffer of osmotic pressure.Solution or suspension also can comprise thickening agent, such as carboxymethyl cellulose Sodium, carboxymethyl cellulose, glucosan, polyvinylpyrrolidone or gelatin.The suitable ph of pharmaceutical composition is generally at 5.5-8.5 In the range of, about 6-8, such as 7, buffer can be used to maintain suitable pH value.
Traditional pharmaceutical methods can be used to provide, and such as cancer patient is administered, dosage forms or compositions.It is administered and generally uses In therapeutic purposes, it means that be administered after making a definite diagnosis cancer.Any suitable route of administration, such as parenteral, vein can be used In, intra-arterial, subcutaneous, intramuscular, intraperitoneal, intranasal, aerosol, suppository or oral administration.The generally pharmaceutical composition of the present invention Being administered with liquid solution or suspensions, more often with aqueous solution or suspension, some situation is with isosmotic solution or suspension.
The pharmaceutical composition of the present invention is prepared in a known manner, such as by traditional dissolving, lyophilizing, mixing, pelletize or Moulding process.Pharmaceutical composition can be prepared (such as, refering to Remington:The Science and according to conventional pharmaceutical technology Practice ofPharmacy (21st edition), ed.A.R.Gennaro, 2005, Lippincott Williams & Wilkins, Philadelphia, PA, USA；And Encyclopedia of Pharmaceutical Technology, Ed.J.Swarbrick, 3rdEdition, 2006, InformaHealthcare, New York, NY, USA).
As the replacement dosage form of liquid dosage form, the compositions of the present invention can be made into lyophilized powder, comprise at least one antibody or Together with carrier, such as mannitol, liquid dissolved composition, such as sterilized water can be used before use.
Pharmaceutical composition comprises the active component of about 1% to about 95%, and preferably from about 20% to about 90%.According to the present invention, Pharmaceutical composition can use such as single dose form, such as ampoule, bottle, suppository, tablet or capsule.Human body can be given The treatment of these dosage forms or prevention effective dose (such as, prevent, eliminate or alleviate the dosage of pathological condition), thus treat cancer or Other diseases.The preferred dosage of medicine is likely to be dependent on following variable, and such as cancer severity, certain patient is whole Body health status, compound vehicle formula and route of administration.
Antibody and the therapeutic use of compositions in the present invention
Anti-HER 2 prepared in accordance with the present invention and pharmaceutical composition can be used for treating or improving mammalian diseases, special It it not treatment human cancer.One embodiment of the present of invention is one prevention, treats or improve the mankind or other mammal cancer The method of one or more symptoms that disease is relevant, comprises and gives the present invention anti-HER2 recombinant antibody composition to described mammal Effective dose.
One of them embodiment suffers from the disease method being characterized with HER2 process LAN, particularly cancer, the party with treatment Method comprises the Anti-HER 2 given defined in a kind of this patent of described patient, or preferably one comprises at least 2 kinds of this patents The anti-HER2 recombinant antibody composition of definition.
Another embodiment relates to allos between a kind of reduction intracellular HER2 of process LAN HER2 and other ErbB family receptors The method that dimer is formed, the method comprises the anti-HER2 recombinant antibodies defined in a kind of for described cells contacting this patent, or A kind of anti-HER2 recombinant antibody composition comprised defined at least 2 kinds of this patents.
Another embodiment of the present invention is to apply the one anti-HER2 recombinant antibodies of the present invention or antibody compositions to prepare one Plant compositions, one or more diseases that said composition is correlated with for treating, improve or prevent the mankind or other mammalian cancer Shape, such as one patient for the treatment of, its illnesses is characterized as HER2 process LAN.
Based on some factors, including HER2 expression, following cancer types is particularly suitable for using the one of the present invention to resist Body compositions treat: breast carcinoma, ovarian cancer, gastric cancer, colon and rectum carcinoma, carcinoma of prostate, bladder cancer, cancer of pancreas, head and neck cancer and Nonsmall-cell lung cancer.The antibody compositions of the present invention is particularly suitable for treating the cancer of process LAN HER2, such as some epithelium Cell carcinoma, such as breast carcinoma, ovarian cancer and gastric cancer.
2 Major Clinical paths that these indications are relevant are 1) auxiliary therapy, at least one additional procedures is treated； Or 2) single therapy.The two discussed briefly below selects.
1) auxiliary therapy: in auxiliary therapy, also becomes therapeutic alliance, it will use antibody in the present invention with extremely Few a kind of other therapeutic treatment associating, a kind of chemotherapy or cancer therapy drug and/or radiotherapy.Or or extraly, it is possible to Anti-HER 2 in the present invention is combined with a kind of different anticancrin from compositions, such as a kind of targeting EGFR or VEGF Antibody.Therefore, in addition to standard one line and two gamma therapies, can be treated by the antibody or compositions that give a kind of present invention State major cancers target.Conceptual design will alleviate and reduce the dose assessment curative effect of conventional criteria chemotherapy by tumor quality.This Decrease in dose can additionally increase by reducing the dose limiting toxicity of chemotherapeutics and/or extend quality.
Induce cancerous cell terminal differentiation medication combined by antibody compositions of the present invention with known, treatment can be improved further Effect.This compounds is selected from, such as, comprise tretinoin, trans retinoic acid, cis-retinoic acid, PB, nerve growth The factor, dimethyl sulfoxide, vitamin D3 activity form, peroxisome proliferators activated receptor γ, 12-O-myristoyl Buddhist ripple Alcohol-13-acetas, Vitro By Hexamethylene Bisacetamide, transforming growth factor-β, butanoic acid, cyclic adenosine monophosphate and the base of vesnarinone Group.Preferred compound is selected from comprising tretinoin, PB, all-trans retinoic acid and the group of vitamin D activities form.
The medicine of a kind of antibody compositions comprising the present invention and at least one chemotherapy or anticancer compound can be used for cancer The while of in treatment, separate or the therapeutic alliance of successive administration.Chemotherapy compound can be by any conjunction being suitable to treat a certain cancer Suitable chemotherapeutic, a kind of selected from the medicine containing alkylating agent, such as platinum analog derivative, such as cisplatin, carboplatin or oxaliplatin； Plant alkaloid, such as paclitaxel, Docetaxel or irinotecan；Antitumor antibiotics, such as doxorubicin (amycin)； Topoisomerase enzyme inhibitor, such as hycamtin；And antimetabolite, such as fluorouracil or other fluoropyrimidines.
Estimate that antibody of the present invention can be used as auxiliary therapy simultaneously, be combined with tyrosinase inhibitor (TKI).These are to close Becoming, usually quinazoline derivant, low-molecular-weight molecule, it can be with receptor intracellular tyrosine collection U.S. domain generation phase interaction With, by competition intracellular Mg-ATP binding site, the receptor phosphorylation of suppression part induction.Currently clinical development some can Block the kinase whose tyrosinase inhibitor of HER2.Some of them can be with targeting EGFR or other EGFR family receptors.Relevant this The summary of a little TKI, refering to Spector et al. (2007) Breast Cancer Res.9 (2): 205.Therefore by the present invention's The medicine of the TKI composition of a kind of antibody compositions and at least one targeting HER2 can be additionally used in treatment of cancer simultaneously, separately or The drug combination of successive administration.
In other embodiments, the antibody compositions of the present invention can be with other antibody drug use in conjunction.These include target To EGFR (Or) or VEGFMedicine.In other embodiments, the antibody compositions of the present invention The immune-mediated present invention can be strengthened with a kind of drug combination being known to stimulating immune system cell, this kind of drug combination The curative effect of antibody compositions.This kind of immunostimulation medicine includes recombinant interleukin (such as IL-21 and IL-2).
2) single therapy: apply antibody in tumor single therapy according to the present invention, can when difference treatment with chemotherapy or anti- Antibody, i.e. monotherapy is given in the case of cancer drug.
Immune conjugate
In the present invention, another of the therapeutical effect of antibody and compositions is chosen as immune conjugate, will antibody and a kind or Multiple cancer therapy drug coupling.Especially, when compositions comprises 2 kinds or the multiple different antibodies of the present invention, and they can be from different HER2 Epi-position combines, it is contemplated that it can generate coupled antibody by volume mesh at cell surface, compared with using single monoclonal antibody, so Receptor internalisation level may be raised.Therefore can be special with one or more anticarcinogen couplings by one or more antibody of this compositions Strange land, effectively coupling anticarcinogen is delivered to inside tumor cell, thus strengthens the effect of Anti-HER 2 of the present invention, improve The activity of killing tumor cell.
The antibody of the present invention can with all kinds of cancer therapy drug couplings, including cytotoxic drug (bag classic chemotherapy medicine and other Small molecule anti-cancer drug), cytokine (wherein conjugate is referred to as " immune cell factor "), (wherein conjugate is referred to as toxin " immunotoxin ") and radionuclide, and the immune conjugate of some approved clinical practices.Including(even Connection90The mice anti-CD 20 antibodies of Y),(coupling131The mice anti-CD 20 antibodies of I) and(coupling calicheamycin Mice anti-CD 33 antibody). other immune conjugate at clinical trial testing includes being coupled to, such as amycin or maytansine The antibody of compound.The immunotoxin of clinical trial testing includes some Pseudomonas aeruginosa Exotoxin A being coupled to block Antibody.The most also tested the immune cell factor comprising humanization EpCAM antibody.
With the antibody of cytotoxic drug coupling in the present invention, may belong to, any the one of such as chemotherapeutic primary categories Kind, including alkylating agent (such as, carboplatin, cisplatin, oxaliplatin), antimetabolite (such as, methotrexate, capecitabine, Ji Xi His shore), anthracycline antibiotics (such as bleomycin, amycin, Mitomycin-C) and plant alkaloid (such as, as docetaxel, Paclitaxel, taxanes and vinca alkaloids, such as vincaleucoblastine, vincristine, vinorelbine).Owing to immune conjugate can Specificity by inside targets neoplastic cells after anticarcinogen target tumor, particularly internalization, Anti-HER 2 based on the present invention Immune conjugate is based on high cell toxicity agent, such as calicheamycin or maytansine derivant, or based on toxin, such as bacteriotoxin (such as Pseudomonas aeruginosa Exotoxin A, diphtheria toxin, diphtherotoxin) or phytotoxin (such as Ricin), relatively have superiority.
In immune conjugate, the anticarcinogen of coupling is generally connected with antibody by a unstable joint, and this joint is at blood In Qing metastable, but when immune conjugate is entered after targeted cells by internalization, and it can by drug release out.Suitably connect Head includes, such as, chemical linker, stable under serum pH neutral, but after internalization easily in lysosome under solutions of weak acidity Acid hydrolysis occurs, and disulfide bond joint can be cracked by intracellular mercaptan, and peptide linker is stable in serum, but at intracellular easily quilt Enzymolysis.
Various coupling can be designed in the compositions containing 2 kinds or multiple antibody of the present invention.Such as, if 2 kinds of antibody, can With by antibody and 2 kinds or multiple different anticarcinogen coupling, or by one antibody and a kind of prodrug coupling, this prodrug can by with another The substance activating (such as enzyme) of antibody coupling.Antibdy directed enzyme-the prodrug therapy (ADEPT) of monoclonal antibody the most general Read discussed, wherein prodrug by monoclonal antibody-enzyme conjugates by the enzyme activition of a kind of target tumor, but the present invention provide A kind of adjustable the method is for the method for some situation.Therefore, it can specificity and increase tumor cell killing potential, avoid simultaneously Or alleviate the damage of normal tissue.
About the more information of antitumor immune conjugate, see 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
Give the antibody of the present invention of effective dose and compositions for treating disease, i.e. reach the dosage of results needed And the time.Treatment effective dose changes with many factors, the disease specific such as treated, age, sex, weight in patients and anti- HER2 Antybody therapy be whether single therapy still with one or more other anti-cancer therapies therapeutic alliances.
The effective dose of oncotherapy can be in progress and/or improve the ability of patients symptomatic and measure by its stable disease, Preferably reverse disease progression, such as by reducing tumor size.The ability of a kind of antibody compositions suppression tumor of the present invention can Assessed by vitro tests, as described in example, it is possible to by its animal model to human tumor curative effect the most measurable It is estimated.Optional suitable dosage thus provide optimal treatment response for each situation, such as, can single defeated Note or continuous infusion, and carry out dose titration according to the emergency of each patient.
Although not yet determining at present the antibody administration dosage of the present invention, but by with the most granted similar medicine for treatment Product (a kind of anti-HER 2 monoclonal antibody) compare to determine a certain dosage.Therefore, a kind of antibody compositions of the present invention Rational Dosage will be in anti-HER 2 monoclonal antibody Herceptin (Trastuzumab) recommended dose be similar to.As the case may be, may be used Giving Trastuzumab (infusion administration) and be used for treating breast carcinoma, initial dose is 4mg/kg, and then every weekly dose is 2mg/kg, or rises Beginning dosage is 8mg/kg, and the most every 3 weekly doses are 6mg/kg.
The suitable dose of antibody compositions of the present invention in the range of 0.1-100mg/kg, e.g., from about 0.5-50mg/kg, such as About 1-20mg/kg.Such as, the dosage of antibody compositions is at least 0.25mg/kg, for example, at least 0.5mg/kg, the most extremely Few 1mg/kg, for example, at least 1.5mg/kg, for example, at least 2mg/kg, for example, at least 3mg/kg, for example, at least 4mg/kg, the most extremely Few 5mg/kg, and such as up to most 50mg/kg, the most most 30mg/kg, the most most 20mg/kg are the highest Reach most 15mg/kg.Repeat administration the most at suitable intervals, the most once in a week, once every 2 weeks, every 3 weeks are once, or every 4 weeks Once, feeling properly as long as being responsible for doctor, doctor can increase and decrease dosage on demand.
Antibody of the present invention can use 3 kinds of different administering modes.Traditional intravenous be administered be most of tumor standard to Medicine technology.But belly cavity tumor, such as ovarian cancer, cancer of biliary duct, other pipeline cancer and similar cancer, verified, intraperitoneal administration is to obtaining Obtain tumor locus high concentration antibody favourable, cleaning antibody can be made to be down to minimum.Similarly, some entity tumor has applicable local The blood vessel structure of perfusion.Regional perfusion can obtain high concentration antibody at tumor locus, and is down to minimum by the removing of antibody short-term.
With any albumen or antibody infusion medicine for treatment condition seemingly, safety issue specifically includes that (i) release of cytokines Syndrome, i.e. hypotension, generate heat, tremble, shiver with cold；(ii) to this albumen generation immunogenic response, (i.e. patient is to recombinant antibodies Medicine produces antibody), and the Normocellular toxicity that (iii) is to expression HER2 receptor, such as: many epithelial cells.Can use Standard testing and down-stream monitor this type of safety issue any.
It is hereby incorporated by reference in the application cited all patents and non-patent literature.
In following example (non-limiting example), present invention will be further described.
Example
Example 1: the clone of Anti-HER 2
Immunity
Inject purified different albumen and HER2 overexpressing cell to female BAl BIc/c (strain A) or C57B16 mice (8-10 week old) carries out immunity.
Use commercially available HER2 albumen (R&D Systems cat.#1129-ER) for immunity.MCF-7 AU565 (ATCC, CRL-2351) is used for cellular immunization.Adding 10% hyclone (FBS) and 1% mycillin (P/S) RPMI-1640 culture medium is cultivated cell.Before each immunity, use PBS washed cell, digest with TrypLE, then giving birth to In long culture medium resuspended.Then use PBS washed cell suspension 2 times, be centrifuged 5 minutes under 250g, then take out and aseptic at 15ml In PBS resuspended.
Diluting cells or antigen in PBS, then mix with Freund adjuvant 1: 1.Freund adjuvant can strengthen and regulate immunity Reaction.Immunity for the first time, uses complete Freund's adjuvant (CFA), but uses incomplete Freund's adjuvant in follow-up immunity (IFA).IFA is a kind of oil-in-water mineral oil emulsion, and CFA is to add heat-killed dry mycobacterium tuberculosis system to IFA Become.Two kinds of adjuvants are respectively provided with deposit effect.CFA can produce the most lasting immunoreation, can be used for first immunisation and exempts to strengthen Epidemic disease is reacted, and IFA is for follow-up immunization.Can be by the one after another drop of surface at one glass of water be tested.When this is maintained as one Drip, then stable emulsion, can be used for injecting.Can only be to the stable Emulsion of injected in mice.
According to schedule (seeing table 4), per injection 25-100 μ g antigen or 107Individual cell.Injection mice 4 times altogether.Institute Mouse is had to inject 300 μ l or 200 μ l Emulsions.According to schedule, can subcutaneous (s.c.), abdominal cavity (i.p.) or vein (i.v.) note Penetrate.
Finally, disconnected neck puts to death mice, takes out spleen, is transferred to (Corning#136350-in 74 μm cell strainer 3479).Cell is isolated by filter screen, resuspended by the ice-cold RPMI-1640 culture medium containing 10% hyclone (FBS), It is centrifuged 5 minutes under 300g.With the RPMI-1640 culture medium re-suspended cell group containing 1% hyclone (FBS), noted by 50 μm Penetrate filter (BD#340603) to filter, more centrifugal collection.With frozen after hyclone (FCS) re-suspended cell containing 10%DMSO, freeze Deposit storage of cells at-80 DEG C until FACS sorts.
Mice plasma cell FACS sorts
Frozen splenocyte bottle is thawed at 37 DEG C, in the presence of still having ice, is then transferred to 15ml pipe In.Xiang Guanzhong is added dropwise over RPMI, the 10%FBS that 10ml is ice-cold, and shakes test tube.Washed once with 10ml FACS PBS, will Cell is crossed before 50 μm Filcon filter and is added 5ml FCS PBS.Then sedimentation cell with 1ml PBS+2%FBS (final volume) Resuspended, then with being diluted to the final concentration of 5 anti-CD43-FITC of μ g/ml and anti-CD138-PE antibody staining.4 under dark condition DEG C hatch 20 minutes.Then, with 2mlFACS buffer solution cell 2 times.At most add 15mlFACSPBS.By the ratio of 1: 100 Example adds propidium iodide (PI), 1 part of PI to 100 part of FACS PBS, is then divided by cell to containing PCR reaction buffer 96 hole PCR plate (seeing below), be then centrifuged 2 minutes at 400g, then by frozen for cell plates at-80 DEG C.Plasma cell sorts Door is that the CD43-positive/CD-138 is positive.
Homology VHAnd VLTo chain
Sorting is carried out V for plasmacytic cellHAnd VLCoded sequence carries out chain, beneficially VHAnd VLCoded sequence homology Pairing.This program uses two step method PCR, i.e. one-step method multiple overlap extension RT-PCR, carries out nest-type PRC the most again.This example Primer mixture used by only expands κ light chain.But, can add in many primer mixtures and nest-type PRC primer mixture The primer of amplification lambda light chain.If adding λ primer, can revise process of separation, such λ positive cell will not be excluded.Homology VHWith VLThe chain principle of sequence refers to WO 2005/042774 and Meijer et al. (2006) J Mol Biol.358 (3): 764-72 Described in.
Thawing 96 hole PCR plate, sorting cells is for the template of multiple overlap extension RT-PCR.Contain at unicellular sorting forward direction Respond buffer (OneStep RT-PCR Buffer；Qiagen), RT-PCR primer (seeing table 5) and RNase inhibitor Each hole of (RNasin, Promega) is added sorting buffer.With OneStep RT-PCR5Enzyme Mix (25 ' dilution；And dNTP mixture (200 μMs of every hole) complements to the appointment final concentration under 20 μ l reaction volumes Qiagen).55 Hatch at DEG C 30 minutes, make every hole carry out the reverse transcription (RT) of RNA.After RT, PCR plate is carried out following PCR cycle: at 94 DEG C 10 minutes, 35 ' (at 94 DEG C 40 seconds, at 60 DEG C 40 seconds, at 72 DEG C 5 minutes), at 72 DEG C 10 minutes.
The PCR carrying out 24 piece of 96 orifice plate (ABgene) in H20BIT thermal cycler reacts to obtain high flux.Complete to follow After ring, PCR plate is stored at-20 DEG C.
In nest-type PRC, preparing 96 hole PCR plate, following mixture (20 μ l reaction) is contained in every hole, dense to obtain the end specified Degree: 1 ' FastStart buffer (Roche), dNTP mixture (each 200 μMs), nested primer mixture (seeing table 6), Phusion archaeal dna polymerase (0.08U；And FastStart high-fidelity enzymatic mixture (0.8U Finnzymes)；Roche).As The template of nest-type PRC, is transferred to multiple overlap extension RT-PCR reaction by 1 μ l.Nest-type PRC plate carries out following thermal cycle: 35 ' (at 95 DEG C 30 seconds, at 60 DEG C 30 seconds, at 72 DEG C 90 seconds), at 72 DEG C 10 minutes.Then reactant 1% agarose is randomly choosed The overlap extension fragment of about 890 base pairs (bp) is there is in electrophoretic analysis with checking.At PCR plate is stored in-20 DEG C, until right PCR fragment is further processed.
Collect the chain V that nest-type PRC obtainsHAnd VLIt is right to encode, and the coding not mixing different donor source is right, then prepares 1% agarose gel electrophoresis purification.As described in WO 2008/104183, overlap extension people's κ constant light coded sequence is to chain VHAnd VLThe coding V to PCR primerLCoded sequence.Plasmid amplification from the coded sequence containing the human antibodies with κ light chain People's κ constant light, reaction system comprises: Phusion enzyme (2U；Finnzymes), 1x Phusion buffer, dNTP mixture (each 200 μMs), hKCforw-v2 primer and Kappa3 ' primer (table 5) and plasmid template pLL138 (10ng/ μ l), cumulative volume 50μl.Then carry out following thermal cycle: 25 ' (at 95 DEG C 30 seconds, at 55 DEG C 30 seconds, at 72 DEG C 45 seconds), at 72 DEG C 10 minutes.So The PCR fragment that rear preparation 1% agarose gel electrophoresis purification obtains.
Spliced by following Overlap extension PCR, the PCR fragment of every part of purification is spliced the pure man κ constant light coding region and expands Increase and the PCR fragment (SEQ ID NO:41) of purification: 50 μ l reaction systems comprise people κ constant light coding domain segment (1.4ng/ μ L), the PCR fragment (1.4ng/ μ l) of purification, Phusion archaeal dna polymerase (0.5U；Finnzymes), FastStart high-fidelity Enzymatic mixture (0.2U；Roche), 1xFastStart buffer (Roche), dNTP mixture (each 200 μMs), mhKCrev draw Thing and mJH primer (table 7).Then following thermal cycle is carried out: at 95 DEG C 2 minutes, 25 ' (at 95 DEG C 30 seconds, at 55 DEG C 30 seconds, 72 At DEG C 1 minute), at 72 DEG C 10 minutes.Then the PCR fragment (about 4518bp) that 1% agarose gel electrophoresis purification obtains is prepared.
By homology VHAnd VLEncode inserting in screening vector
In order to determine and the antibody of HER2 specific bond, it is thus achieved that VHAnd VLCoded sequence is expressed as full length antibody.This relates to By VHAnd VLEncode inserting expression vector and transfection host cell.
Two step method cloning process is used to generate containing chain VHAnd VLCoding is to expression vector.Statistically, if expressed Carrier comprises 10 times of VHAnd VLHomology, to PCR primer quantity, is used for generating screening storehouse, and all unique gene can to existence 99% Can property.Therefore, if obtaining 400 overlap extension V genetic fragments, can generate at least 4000 clone banks with carry out screening all solely There is 99% probability in special gene pairs.
In simple terms, chain VHAnd VLEncode the PCR primer purified to storehouse, splice the pure man κ constant light coding region, Shear in the recognition site introducing PCR primer end with XhoI and NotI DNA restriction endonuclease.By standard linker, will cut Cut the fragment with purification and be connected to the mammal IgG expression vector OO-VP-002 of XhoI/NotI enzyme action (see WO2008/ Described in 104183).Connection mixture electricity is proceeded to escherichia coli, then adds to the 2 ' YT plates containing suitable antibiotic, hatch Overnight.Use the carrier storehouse that the cell purification recovered in plate is expanded by standard DNA purification method (Qiagen).With AscI and NheI enzyme Cut plasmid, be used for inserting promoter leader fragment.The restriction endonuclease sites of these enzymes is positioned at VHAnd VLEncoding gene is to it Between.After plasmid purification, by standard linker, the two-way mammalian promoter leader fragment of AscI-NheI enzyme action is inserted AscI and NheI restriction endonuclease sites.The plasmid connected expands in escherichia coli, then uses standard method purification matter Grain.By traditional program, the screening plasmid storehouse generated is converted escherichia coli.Obtain is cloned in 384 orifice plates storage.
Screening combines HER2 overexpressing cell
First, use in Laser Scanning Confocal Microscope screening antibodies storehouse and combine with HER2 process LAN breast cancer cell line (SKBR-3) Antibody.5000 SKBR-3 cells are seeded to the every of 384 porocyte culture plates (Perkin Elmer, cat.#6007439) Kong Zhong, cell pellet overnight is adherent.10 μ l antibody supernatant are transferred in every hole, then culture plate are hatched 2 hours, abandon every afterwards Culture medium in hole, then add the 30 new culture medium of μ l to every hole, comprise 2 μ g/mlAlexa-488 labellings goat anti-human igg (H+L, Invitrogen cat.#A11013), 2 μ g/ml CellMask Blue (Invitrogen cat.#H34558) and 1 μM Hoechst33342 (Invitrogen cat.#H3570), hatches 30 minutes the most again.Again abandon culture medium, washed cell, use 2% formalin solution (Aldrich cat.#533998) is fixed.Then OPERA high flux Laser Scanning Confocal Microscope is used to measure Fluorescence level (Perkin Elmer).
Copolymerization Jiao's garbled data identifies 266 positive targets, corresponding to the 3.46% of total clone.
Sequence analysis and Immune Clone Selection
The clone with SKBR-3 Cell binding identified dates back 384 original orifice plates, and is transferred to new culture plate In.From clone, extract DNA, then carry out the DNA sequencing of V gene.Aligned sequences, selects all Unique clones.Obtain sequence Multiple ratio to show each clone uniqueness, carry out the discriminating of unique antibodies.266 clones are being carried out sequence analysis After, identify the antibody sequence bunch unique more than 70 genes.The somatic hypermutation cloned by common precursor obtains every One correlated series.In a word, 1-2 the clone selecting every bunch carries out sequence and specificity verification.Selected antibody variable sequences ginseng See the sequence table of annex.As explained on, the sequence of light chain in sequence table comprises identical people's κ constant light district, its initial amino Acid is TVAAP, and terminating aminoacid is C-end NRGEC.
Sequence and specificity verification
In order to verify that antibody coding is cloned, prepare DNA plasmid, and transfect FreeStyle CHO-S cell with 2ml (Invitrogen) express.Transfect latter 96 hours and collect supernatant.Expression, its specificity is estimated by standard anti-igg ELISA The antibody being combined cell by HER2 specific ELISA and OPERA analysis determines specificity.
In simple terms, for ELISA, it is coated with 1 μ g/ml HER2 albumen (R&D Systems cat.#1129-ER) NuncMaxisorb plate (cat.#464718), dilutes with PBS, and 4 DEG C overnight.Before 50 μ l 2%Milk-PBS-T close, use PBS+0.05%Tween20 (PBS-T) washs No. 1 plank.Wash No. 1 plank with PBS-T and 20 μ l2%milk-PBS-T, add Enter 5 μ l FreeStyle CHO-S transfection thing supernatant (seeing below), hatch at room temperature 1.5 hours, then wash with PBS-T No. 1 plank, every hole 20 μ l.Add the 2%milk-PBS-T bis-that dilution factor is 1: 25000 anti-(HRP-goat-anti human kappa light chain, Serotec, cat.#STAR 100P), the antibody being combined with hole with detection, and at room temperature hatch 1 hour.Adding at the bottom of 25 μ l Wash No. 1 plank with PBS-T before thing (Kem-En-Tec Diagnostics, cat.#4518), hatch 5 minutes.After incubation Add 25 μ l 1M sulphuric acid and terminate reaction.Nonspecific signal is detected at 450 nm by ELISA microplate reader.HER2ELISA also sends out Existing 266 clones there are 178 clones and SKBR-3 Cell binding.
Table 4. is for generating the immunisation schedule table of anti-HER2 clone's initial substance
Table 5.RT-PCR multiple overlap extension primer mixture
W=A/T, R=A/G, S=G/C, Y=C/T, K=G/T M=A/C, H=ACT, B=GCT；Conc.-final concentration.
Table 6. nested primer collection
K=G/T, M=A/C, D=AGT；Conc.-final concentration.
Table 7. κ constant light splicing primer collection
Example 2: selected Anti-HER 2 function is qualitative
Employing vigor is tested, and selects 41 unique antibodies for Function detection.Cell injury inevitably leads to cell Maintain and metabolizing cells function and the ability of growth energy are provided.Carry out metabolic activity detection based on this premise, generally measure Mitochondria activity.Cell proliferation reagent WST-1 (Roche Cat.No.11 644 807 001) is a kind of end that can be used directly Thing, for measuring the metabolic activity in living cells.Assume that metabolic activity is relevant to living cells quantity.In this example, WST-1 test For measuring the quantity of metabolic active cells, these cancerous cell process 96 hours through 2 μ g/ml difference Anti-HER 2s.
By 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 the concentration of 1000 cells in every hole 96 orifice plates, its culture medium comprises 2 μ g/ml Anti-HER 2s.By cell plates in incubator 37 DEG C cultivate 4 days.Then every hole adds 20 μ l WST-1 reagent, hatch 1 hour in 37 DEG C.Cell plates are transferred to orbital shaker, shake 1 hour.In ELISA microplate reader On under 450nm and 620nm (reference wavelength) measure absorbance.Metabolic active cells (MAC) level difference is calculated as follows For compareing the percent of supernatant:
Selected antibody analysis result sees table 8, wherein lists single cancerous cell line and average and standard deviation data.From this A little results can be regarded as, has identified the HER2 antibody with functional activity, and the antibody exhibits in antibody library goes out all or big The inhibitory action of the cancerous cell line of most tests.
Metabolic active cells (MAC) percent in the presence of table 8. Anti-HER 2
Antibody is numbered 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
* with the antibody 4380/4381 of cysteine on light chain 40
Example 3: determine overlapping epitope
In order to select containing the Anti-HER 2 compound playing cooperative effect after associating, the antibody in each mixture should When being combined with non-overlapping epitopes.Therefore, in order to study degree overlapping between Anti-HER 2, surface plasma resonance technology is used (SPR) technology carries out epi-position merging.Can be at ProteOnTMXPR 36 protein-interacting array system (Biorad Laboratories) SPR analysis is carried out.This system two-way (being defined as L and A) can measure 6 kinds of interactions, generates altogether simultaneously 36 kinds may interact.
Arrange
ProteOn GLC sensor chip (BioRad) coupling can inject the 3600-3620 of fluidic cell L1 to L6 altogether The anti-Fc antibody (Biacore, GE Healthcare) of unit of shaking (RU), can be illustrated to use ProteOn amino coupled by manufacturer Test kit (Biorad).Use flow velocity 25 μ l/min, inject Fc coupling HER2 (HER2-Fc) that 125 μ l concentration are 50nM, and Fluidic cell L1-L5 captures, and is injected into by running buffer (relevant compositions sees following reagent portion) similarly simultaneously In fluidic cell L6.In order to close free anti-Fc site, by the envelope of 125 μ l 0.33mg/ml before injection Anti-HER 2 Close monoclonal antibody (mAb)Running buffer, to fluidic cell L1 to L5, is injected in L6 by (Abbott), and flow velocity is equal It is 25 μ l/min.Blocking monoclonal antibodies should be made at least 300 seconds to dissociate before injection 100 μ l 50nM Anti-HER 2s, flow velocity is 50 μ l/min, the most at least dissociate 10 seconds.Conversion multi-channel module is to A direction afterwards, then the 2nd injection Anti-HER 2 (use and press the identical flow conditions of L direction injection Anti-HER 2 and concentration with the 1st time).In circulating at totally 8, test is all The overlapping degree the most in the two directions of HER2 antibody, and mutual overlapping degree is to determine oneself's overlapping (comparison generation Between two antibody of table, 100% is overlapping).Direct in each circulation, available 25 μ l 3M MgCl2(flow velocity is 50 μ l/min) regeneration is anti- Body-EGFR complex.
Reagent
GLC chip (Biorad, Cat.No.176-5012)
·ProteOnTMAmino 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 MgCl2Regeneration buffer, Biacore cat.No 344-ER-050
Antigen: HER2-Fc, R&D systems cat.No 1129-ER, is redeveloped into 100 μ g/ml with PBS.
Blocking monoclonal antibodies:(Abbott).It is dissolved as storing solution 5mg/ml with distilled water, slow running Rush and liquid is diluted to 0.33mg/ml
Anti-HER 2: 4517,4518,4519,4382,4383,4384,4385,4386,4387+ TrastuzumabsAnd handkerchief (wherein handkerchief trastuzumab analog has and drapes over one's shoulders in WO2006/033700 and US 2006/0121044A1 trastuzumab analog The heavy chain of the handkerchief trastuzumab of dew and light chain amino acid)
Result
With obtaining RU maximum after running buffer reference substance and baseline correction normalization, and by every before and after having compared The RU value of individual Anti-HER 2 determines the suppression degree of the Anti-HER 2 of detection, by immediate record before and after each sample of injection Reporting point realize.Typical XPR ProteOn circulates as shown in Figure 1.The overlap of the first and second antibody/combine suppression hundred The calculating of mark is different based on RU value difference before and after the first and second antibody, such first and second antibody identical representatives of RU value 100% overlapping (i.e. 100% suppression).At least carry out 2 experiments and confirm that 2 kinds of antibody exist suppression on 2 directions.Will suppression Value 50-100% (at least calculating average from 2 independent experiments) combines the epi-position of overlapping epitope or close antigen as prompting The mark of significance competition between antibody pair, inhibiting value points out antibody not to be adjacent especially to the epi-position identified less than 50% simultaneously Closely, so can reduce sterically hindered.Inhibiting value will not be included into the analysis of overlapping epitope, because judging that it does not represents less than 25% Non-limiting suppresses.List the antibody pair of 50-100% and 25-50% degree of suppression separately below.
The antibody of 50-100% degree of suppression is to combination:
4517 and 4518
4517 and Trastuzumab
4384 and 4518
4382 and 4519
4382 and 4383
4382 and handkerchief trastuzumab analog
4383 and 4387
4383 and handkerchief trastuzumab analog
4384 and 4386
4385 and 4387
The antibody of 25-50% degree of suppression is to combination:
4387 and 4519
4519 and handkerchief trastuzumab analog
4382 and 4387
4383 and 4387
4387 and handkerchief trastuzumab analog
Fig. 1 shows the representative epi-position amalgamation result of Anti-HER 2 of the present invention." HER2 " refers to that Fc-coupling HER2 combines the phase. " close " the combination phase referring to blocking antibody Synagis to close free anti-Fc site.Block site is by " HER2 " end and " envelope Closing " RU value difference value represents (in both cases, determining, i.e. 600 and 1500 seconds) in the free end of term between end, refer to " The free anti-Fc site closed "." mAb1 → " refer to the first binding time to Anti-HER 2." ROTATE CHIP " points to Second to skew." mAb2 ↓ " refers to the second binding time to Anti-HER 2.Suppression percent is calculated as an anti-binding (being referred to as " mAb1 combination ") RU part, two anti-(being referred to as " mAb2 combination ") obtain after combining.In this example, at sample A1 2 running buffers of middle injection, inject same antibody in sample A2, and A2 antibody is with 4 kinds in sample A3, A4, A5 and A6 not Synantibody is together injected.The oneself's overlap observing sample A2 (significantly changes at mAb2 ↓ period RU；Referred to by lower right cursor Show), but between A2 antibody and other antibody, do not observe notable overlap.
The function of example 4:2 kind or 3 kinds of Anti-HER 2 mixture is qualitative
This example describes 10 kinds of all 2 kinds had determined that in Anti-HER 2 in the present invention that people HER2 combines of selection Or the vitro detection of 3 kinds of mixtures of antibodies.The growth of assessment mixtures of antibodies 3 kinds of different carcinoma cell lines of suppression: N87 (gastric cancer), SKBR-3 (breast carcinoma) and BT474 (breast carcinoma).
Method
Detection antibody 4380 in all mixture of 2 kinds or 3 kinds antibody, 4381,4382,4383,4384,4385, 4387,4517,4518 and 4519, each antibody is determined at people's HER2 receptor and combines, and has the anti-of optimal effectiveness to determine Body combines.Such as, in preparation 384 orifice plates, the method for different antibodies combination refers to WO 2010/040356.Describe below in detail Thin information.
Plant mixtures of antibodies
10 kinds of antibody 1xPBS are diluted to 25 μ g/ml, in 384 orifice plates, add 100 μ l antibody-solutions for preparing use 2 kinds of mixtures of antibodies in detection.
For detection 3 kinds of cell lines, 2 piece of 384 orifice plate of each use, 46 μ l are contained correlated measure cell (N87: 3000 cells；SKBR-3:1000 cell；BT474:2000 cell) culture medium add in hole.Use Biomek3000 laboratory stations (Beckman Coulter), from raise plate take out 2 kinds of different antibodies of 2 μ l add to containing In 384 orifice bores of culture medium and cell, so comprise all combinations of 2 kinds of different antibodies.Additionally, culture plate comprises culture medium Control wells (50 μ l 1xPBS culture medium；Acellular), untreated control wells (50 μ l 1xPBS culture medium+cell；Without antibody) With containing 4 μ l Trastuzumabs or handkerchief trastuzumab analog as the culture medium of reference antibody or 4 μ l only 10 kinds of antibody Han the present invention One of as the culture hole (except 46 μ l culture medium+extracellular) of culture medium of extra check.
Culture hole containing 2 kinds of mixtures of antibodies, cultivate datum hole, untreated control hole and single containing the appropriate pearl of Trastuzumab/handkerchief Resist or a kind of antibody of the present invention, hatch in 37 DEG C of incubators 4 days, in all relevant hole of culture plate, then add 5 μ l Cell proliferation reagent WST-1 with 1xPBS 1: 1 dilution.Culture plate is placed in 37 DEG C again hatch 1 hour, is then transferred to track On shaking table, then hatch 1 hour.ELISA microplate reader measures absorbance in 450nm and 620nm (reference wavelength).Metabolism is lived Sexual cell (MAC) quantity is calculated as follows the percent into untreated control:
Assuming that metabolic activity is relevant to living cells quantity, the low antibody on cell corresponding to higher level of %MAC numerical value is raw Long suppression degree.
3 kinds of mixtures of antibodies
The antibody 1xPBS that 10 kinds of identical empirical tests are combined with people's HER2 receptor is diluted to 16.67 μ g/ml, to 384 Orifice plate adds 100 μ l antibody-solutions for preparation for the 3 kinds of mixtures of antibodies detected.
Vitro detection 3 kinds of identical cell lines, i.e. N87, SKBR-3 and BT474.For each of detection cell line, make With 10 pieces of 384 different orifice plates, the 44 μ l culture medium containing equal number cell are added in hand-hole.Use Biomek3000 experiment Room work station (Beckman Coulter), takes out 3 kinds of different antibodies of 2 μ l add to containing culture medium and cell from raising plate In 384 orifice bores, so comprise all combinations of 3 kinds of different antibodies.Additionally, culture plate comprises culture medium control wells (50 μ l 1xPBS culture medium；Acellular), untreated control wells (50 μ l 1xPBS culture medium+cell；Without antibody) and containing 6 μ l He Sai Spit of fland or handkerchief trastuzumab analog are the most right as culture medium or the 6 μ l only conduct containing one of 10 kinds of antibody of the present invention of reference antibody According to the culture hole (except 44 μ l culture medium+extracellular) of culture medium.
Culture hole containing 3 kinds of mixtures of antibodies, cultivate datum hole, untreated control hole and single containing the appropriate pearl of Trastuzumab/handkerchief Resist or a kind of antibody of the present invention, hatch in 37 DEG C of incubators 4 days, in all relevant hole of culture plate, then add 5 μ l Cell proliferation reagent WST-1 with 1xPBS 1: 1 dilution.Culture plate is placed in 37 DEG C again hatch 1 hour, is then transferred to track On shaking table, then hatch 1 hour.Absorbance is measured in 450nm and 620nm (reference wavelength), and by above-mentioned calculating %MAC.
Result
For all 2 kinds or 3 kinds of mixture of the Anti-HER 2 that 10 kinds of selected confirmations are combined with people HER2, assess the phase Suppress 3 kinds of cancerous cell line N87 (gastric cancer), SKBR-3 (breast carcinoma) and the ability of BT474 (breast carcinoma).Calculate 10 kinds of monoclonal antis Body and the %MAC of 2 kinds or 3 kinds mixture.Then according to the effect of mixture cell growth is ranked up, table 8 lists tool There are 55 kinds of mixture of the highest average curative effect (the highest average %MAC, averages based on these 3 kinds of cell line results).
Result shows, between different cell lines, the effect difference of various mixture cell growth suppression is very big, although flat All difference of %MAC are the biggest.Although have selected the 55 kinds of mixtures of antibodies listed in table 8 based on average %MAC, but root Only height can be had at single cell cording using single mixtures of antibodies as object of study according to the result of any one or more cell line Suppression level (low %MAC) is understood to be a kind of internal for certain very effective Antibody Combination of class cancer.
55 kinds of maximally effective mixture that table 8 is made up of 2 kinds or 3 kinds of Anti-HER 2s 3 kinds of cancerous cell lines (N87, BT474 and SKBR-3) in growth of cancer cells suppression level.Suppression water-glass is shown as metabolic active cells percent (% MAC。)
Example 5:ADCC and CDC measure
In order to study HER2 specific antibody mixture induction of antibodies dependent cellular cytotoxicity (ADCC) in vitro of the present invention With the ability of CDC (CDC), use N87 and/or SKBR3 as target cell to by 2,3 or 4 kind of antibody group The 20 kinds of different Anti-HER 2 mixture become are tested.
Method
External ADCC
Use LymphoprepTMIsolation medium is separating periphery blood monocytic cell from the blood of Healthy Volunteers (PBMC), and as effector lymphocyte.N87 and the SKBR3 cell of HER2 will be expressed as targeted cells.At 37 DEG C often 107Individual cell with 334 μ Ci sodium chromates (51Cr, 334) labelling 1 hour.After washing, by targeted cells (5x103/ hole) use anti-HER2 Antibody at 37 DEG C in RPMI-1640+10%FCS+1% mycillin preincubate 30 minutes, then according to effector lymphocyte: target Effector lymphocyte is added to cell (E/T) ratio 50: 1.Cell is additionally hatched 4 hours at 37 DEG C, then uses TopCount Microplate scintillation counter is by detection culture medium51The content of Cr detects cell cracking.By to containing51Cr labelling is thin The hole of born of the same parents adds 2%Triton-X100 and obtains maximum cracking.From containing effector lymphocyte and targeted cells but without the hole of antibody Middle measurement synchronizes cracking situation.
Calculate specific cytotoxic by the following method:
Compare with positive control antibodies Trastuzumab, be calculated as follows relative cytotoxicity:
% relative cytotoxicity=(antibody X cell toxicity %/Trastuzumab cytotoxicity %) * 100
External CDC
Use from the serum of fresh extraction centrifugal blood isolated as Complement source.By thin for the N87 of expression HER2 Born of the same parents are as targeted cells.At 37 DEG C every 107Individual cell with 334 μ Ci sodium chromates (51Cr, 334) labelling 1 hour.After washing, will Targeted cells (5x103/ hole, 100 μ l) the anti-HER2 that dilutes of addition RPMI-1640+10%FCS+1% mycillin (50 μ l) In antibody, it is subsequently adding fresh serum (50 μ l).At 37 DEG C, cell is hatched 3 hours in serum and antibody, then use TopCount Microplate scintillation counter is by detection culture medium51The content of Cr detects cell cracking.By to containing51The hole of Cr labeled cell adds 2%Triton-X100 and obtains maximum cracking.From the hole containing targeted cells but serum-free Measure and synchronize cracking situation.
It is calculated as follows percent cytotoxicity:
Result
Test 20 kinds of Antibody Combination all can abduction delivering HER2 N87 and SKBR3 cell line occur ADCC.Antibody is dense When degree is 0.1 μ g/ml, Anti-HER 2 combination is to the maximum specific toxicity of N87 cell line in the range of 16-24%, and this is with conspicuous Sai Ting compares, and cracking percent 46-69% is quite (seeing Fig. 2 A and 2B and Fig. 3 A and 3B) relatively.In SKBR3 cell, anti- When bulk concentration is 0.1 μ g/ml, the specificity ADCC percent of induction is in the range of 20-34%, relative with corresponding to Trastuzumab Cytotoxicity 48-83% (sees Fig. 2 C and 2D and Fig. 3 C and 3D).
Contrary with ADCC result of the test, Trastuzumab induction targeted cells cracks, but Trastuzumab cannot induce CDC (Fig. 4 With 5).On the contrary, the present invention can induce CDC, 5%-31% special containing 2,3 or 16 kinds of Anti-HER 2 mixture of 4 kind of antibody Different walking alone, average is 12% (Figure 4 and 5).CDC is not observed when using SKBR3 cell.Antibody levels in mixture and induction Maximum cell toxicity percent between onrelevant, the mixture containing 2,3 or 4 kind of antibody be induction CDC front 4 kinds of antibody mix Compound (Figure 4 and 5).
This illustration is bright, and in the present invention, all Anti-HER 2 mixture all show the highest ADCC and CDC level.At body In interior test, the mechanism of action in addition to ADCC and CDC may play a role in the effect of mixtures of antibodies.Especially, The mixtures of antibodies of the present invention can be combined with non-overlapping HER2 epi-position, and this may result in high-caliber receptor internalisation, so can carry High antitumaous effect.Therefore result shown in this example can be combined with example 4 and shown, the mixtures of antibodies of the present invention is generally at a kind or many Kind of test cell system shows the suppression of high-caliber growth of cancer cells, simultaneously in example 4 released growth inhibited not necessarily with, such as ADCC level is correlated with.
Example 6: the mixtures of antibodies titration to different carcinoma cell line
In this example, WST-1 test described in example 2 and example 4 is used to measure at the different antibodies mixture of variable concentrations The quantity of metabolic active cells after reason.Test antibody mixture in multiple cancerous cell lines, lists 4 kinds of cell lines in this example Result: N87 (gastric cancer), HCC202 (breast carcinoma), BT474 (breast carcinoma) and ZR-75-30 (breast carcinoma).
Method
Before carrying out WST-1 test, incite somebody to action this with the suitable culture medium of individual cells system (supplementing 2%FBS and 1%P/S) Invention mixtures of antibodies and control antibodies (For negative control antibody, handkerchief trastuzumab analog is that positive control resists Body) it is diluted to the final concentration of 100 μ g/ml of total antibody, the final concentration of 50 μ g/ml of antibody in the hole that such antibody concentration is the highest.Will Antibody carries out 3 times of serial dilutions.By correlated measure cell (N87:3000 cell；HCC202, BT474 and ZR-75-30:2000 Individual cell) add to the hole of 384 orifice plates, then plank is placed in incubator and hatches 4 days at 37 DEG C.Then to culture plate All relevant hole add reagent WST-1.Culture plate is placed in 37 DEG C again hatch 1 hour, is then transferred on orbital shaker, then Hatch 1 hour.Measure absorbance in 450nm and 620nm (reference wavelength), and calculating metabolic active cells accounts for as stated above Untreated control percent (%MAC).
Result
4 kinds of mixtures of antibodies titration results such as accompanying drawing 6-9 institute to cell line N87, HCC202, BT474 and ZR-75-30 Show.Additionally, table 9 shows 4 kinds of mixtures of antibodies and the IC of handkerchief trastuzumab analog50Value (half maximum inhibition concentration) and maximum Suppression level.(it should be noted that and only can compare IC in the case of each corresponding maximum suppression level is more or less the same50 Value).From accompanying drawing and table 9 it can be seen that mixture shows suppression and the effect of varying level the different cell lines of test. But, these 4 kinds of mixture suppression 4 kinds of cell line growths in terms of all due to handkerchief trastuzumab.
The IC that 4 kinds of cell lines are suppressed by 9.4 kinds of Anti-HER 2 compounds of table and suppression handkerchief trastuzumab analog50Value and Curative effect
Example 7: the curative effect in Anti-HER 2 mixture N87 model in nude mice in vivo
Anti-HER 2 mixture 4384+4517,4382+4518,4382 is have studied in nude mice NCI-N87 transplantation model The interior curative effect of+4385+4518 and 4382+4387+4518.This model be widely used in Effect of Anti carcinoma monoclonal antibody effect and Curative effect, including Anti-HER 2.Nude mice hypoimmunity, lacks T cell, and such human cell can grow in mice.
Method
By 107Individual NCI-N87 cell is inoculated in the left side subcutaneous area of 8 week old female athymic nude mice.2 times a week with card Tumor size measured by chi, and calculates gross tumor volume (mm according to below equation3): (width)2X length x 0.5.Tumor assessment size For 300mm3, mice is carried out randomization distribution and starts treatment.2 50mg/kg4384+ are injected weekly to mouse peritoneal 4517,4382+4318,4382+4385+4518 or 4382+4387+4517, totally 5 weeks (injection 10 times altogether), then observe 3 weeks. This experiment also includes anti-HER 2 monoclonal antibody Herceptin (Trastuzumab), its dosage and schedule and anti-HER2 Mixtures of antibodies is consistent.
Result
This experimental result sees Figure 10.After tumor inoculation when the 59th day, tumor mean size is 300mm3, by mice with Machine is dispensed in 6 groups, and often 10 animals of group, start to give 50mg/kg 4382+4518,4384+4517,4382+4385+ 4518,4382+4387+4517, Herceptin and excipient (matched group) treatment.Originally, the tumor one in all treatment groups Straight growth, until (the 66th day) starts atrophy occur after treating 1 week.Give 4384+4517 or Herceptin treatment little In Mus, tumor is only compared with beginning size 300mm3Smaller, within the 83rd day and the 73rd day after inoculated tumour, start the most respectively to increase.It After, in treatment group, the growth kinetics of animal tumor is similar with excipient control group.On the contrary, through 4382+4518,4382+ The mice of 4385+4518 or 4382+4387+4517 treatment, with compared with Herceptin, shows tumor suppression and extends and aobvious Write and improve.At the end of the treatment phase (the 91st day), the Tumor growth inhibition of 3 groups all reduces, and tumor starts after stopping the treatment Slowly increase.
In a word, compared with Herceptin, accept anti-HER2 mixture 4382+4518,4382+4385+4518 and 4382+ The animal of 4387+4517 treatment shows N87 tumour transplatation growth inhibited and significantly improves.
Example 8: anti-HER2 mixture interior curative effect in OE19 model in nude mice
Anti-HER 2 mixture 4384+4517,4382+4518 is have studied in nude mice OE19 gastric cancer transplantation model, The interior curative effect of 4382+4385+4518 and 4382+4387+4518.
Method
By 5x106Individual OE19 cell is inoculated in the left side subcutaneous area of 8 week old female athymic nude mice.2 times a week with card Tumor size measured by chi, and calculates gross tumor volume (mm according to below equation3): (width)2X length x 0.5.Tumor assessment size For 110mm3, mice is carried out randomization distribution and starts treatment.2 50mg/kg4384+ are injected weekly to mouse peritoneal 4517,4382+4318,4382+4385+4518 or 4382+4387+4517, totally 5 weeks (injection 10 times altogether), then observe 3 weeks. This experiment also includes anti-HER 2 monoclonal antibody Herceptin (Trastuzumab), its dosage and schedule and anti-HER2 Mixtures of antibodies is consistent.
Result
This experimental result sees Figure 11.First after treatment, i.e. treat at 4382+4385+4518 and 4382+4387+4517 Group is observed the Tumor growth inhibition effect compared with matched group.As far back as the 23rd day time, 4382+4385+4518 treatment group The equal atrophy of mouse tumor, and it is significantly better than Herceptin；During this improvement continues whole research.After inoculated tumour the 37th My god, after i.e. completing 7 times in 10 treatments, this group has in 8 mices the complete atrophy of tumor of 6, and terminates in research Time entirely without tumor.In 4382+4387+4517 treatment group, in addition to 1 mice, the equal atrophy of tumor of remaining mice.From 32 days start, and the mixture of 3 kinds of antibody is evident in efficacy compared with Herceptin more relatively.When the 37th day, this group has in 7 animals 4 entirely without tumor, and continue to research terminate.There is 1 mice that treatment is not produced response, owing to gross tumor volume is excessive, Euthanasia is implemented in the 53rd day time.Also good to treatment response through the mice of 4384+4517 treatment, after tumor inoculation the 28th day Show the notable more preferably curative effect than Herceptin.When the 43rd day, 8 mices of this group there are 3 entirely without treatment. From the 16th day (starting treatment), to the 28th day, tumor growth was under controlled state the mice treated through 4382+4518, it Afterwards compared with excipient group and Herceptin, Antybody therapy still suppresses tumor growth, although tumor size is slowly increased.Although So, compared with Herceptin, when the 44th day, from the 53rd day until research terminates (the 67th day), 4382+4518 suppression is swollen Tumor growth evident in efficacy more preferably.
In a word, all Anti-HER 2 mixture of the present invention are in OE19 model in nude mice, with Herceptin phase Ratio, the effect of its suppression and control tumor growth is notable the most more preferably.
Example 9
This illustration is bright, and the HER3 phosphorylation of HER2 degraded and blocking-up HER2 mediation simultaneously can farthest suppress HER2 to depend on Rely property cancerous cell line.Obtaining optimal double blocking effect in the mixture of 3 kinds of Anti-HER 2s, 2 kinds in mixture resist Body can effectively induce HER2 to degrade, and the compensatory signal transduction of the 3rd kind of antibody blocks HER2/HER3 heterodimer.Typically Thinking, compensatory raises HER3 can partly accelerate the compensatory signal transduction (seeing example 11) of HER2/HER3 heterodimer.
Method
For the inherent mechanism of Effect of Anti HER2 mixtures of antibodies growth inhibited effect, in gastric carcinoma cell lines NCI-N87 The phosphorylation level of research EGFR, HER2 and HER3.NCI-N87 cell, after antisera overnight pretreatment, uses 10nM HER3 to join Body adjusts albumen-β to stimulate 15 minutes, then its full cell lysate is carried out Western blot analysis.Cell is inoculated in 6 In orifice plate, remove culture medium when cell reaches 80% degrees of fusion, use 1xPBS washed cell, and (use 2ml with 10 μ g/ml antibody Culture medium dilution containing 0.5%FBS) process.After overnight processing, then albumen-β is adjusted to stimulate 15 points with 10nM HER3 part Clock.With 2ml 1xPBS washed cell, it is subsequently adding 200 μ l1xNuPAGELDS sample buffers.10-15 μ l sample is carried out egg White matter immunoblotting assay, uses anti-EGFR, pEGFR (Tyr1068), HER2, pHER2 (Tyr1221), HER3 or pHER3 (Tyr1289) one is anti-to be detected.
Result
The analysis result of protein induced HER2, HER3 and EGFR phosphorylation is adjusted to show (figure for antibody-mediated suppression 12), Anti-HER 2 and antibody complex have not same-action to EGFR, HER2 and HER3 phosphorylation state and HER2 level.But Adjust protein induced HER3 on EGFR and HER2 phosphorylation level without impact.Only monoclonal antibody 4382 and reference monoclonal antibody Handkerchief trastuzumab analog can block the HER3 phosphorylation of part induction.These results show, 4382 can block HER2 and HER3 Heterodimerization.All mixtures of antibodies all can reduce EGFR and HER2 phosphorylation level and HER2 level.The most only There is the mixture containing 4382 can induce HER2 degraded and suppression EGFR and HER2 phosphorylation and HER3 phosphorylation simultaneously.This A little results also show, the mixture (4382+4385+4518 and 4382+4387+4517) containing 3 kinds of antibody is blocking 3 kinds of receptors Phosphorylation aspect is better than the mixture (4382+4518 and Herceptin+handkerchief trastuzumab analog) of 2 kinds of antibody, particularly HER2.It is generally believed that the mixture containing 3 kinds of antibody has induction HER2 degraded simultaneously (by corresponding mixture due to it Antibody to 4385+4518 or 4387+4517) and block ligand-mediated HER2 phosphorylation (by antibody 4382).These Result and our pharmacology data have good dependency, and pharmacology data show, in vivo with vitro inhibition cell with Tumor growth aspect, the mixture of 3 kinds of Anti-HER 2s is better than single anti-HER 2 monoclonal antibody, and is generally also advantageous over 2 kinds The mixture (seeing example 4 and 6) of Anti-HER 2.
Figure 13 A and B shows HER2/HER3 signal transduction system schematic diagram and 3 kinds of Anti-HER 2 mixture superioritys respectively Hypothesis mechanism.Showing the oncogenic signals path that HER2 homodimer mediates on the left of Figure 13 A, this path can cause angiogenesis, cell Migrate and propagation.The signal that HER3 part is mediated by HER2/HER3 heterodimer after being combined is shown with HER3 receptor on the right side of Figure 13 A Path (is typically due to raise HER3 after a kind of Anti-HER 2 processes cause).This can be by influencing mechanism, and such as albumen closes One-tenth, propagation and carbohydrate metabolism cause oncogenic signals to be transduceed.Figure 13 B shows that 3 kinds of Anti-HER 2 mixture of the present invention block HER2 with two The hypothesis mechanism of aggressiveness and HER2/HER3 heterodimer oncogenic signals path.Left diagram 2 kinds is combined with HER2 non-overlapping epitopes Anti-HER 2 can form a kind of cross-coupling receptor-antibody network, this may result in HER2 receptor internalisation and degraded.Figure Show on the right side of 13B that the 3rd kind of Anti-HER 2 can being combined with HER2 dimer arm blocks HER2/HER3 heterodimer compensatory shape The mechanism become, it the most also blocks the signal path of HER3 mediation, hinders HER3 to raise caused antagonism HER2 Antybody therapy and produce Drug resistance.
Example 10
This illustration is bright, and the Anti-HER 2 compound in conjunction with non-overlapping epitopes effectively can lure in multiple human cancer cell line Lead HER2 degraded.
Method
For the receptor degradation degree of Effect of Anti HER2 antibody complex induction, to through 10 μ g/ml antibody treatment 48 hours The full cell lysate of ZR-75-30, NCI-N87, BT474 and HCC202 carry out Western blot analysis.Cell is cultivated In T-75 culture bottle, remove culture medium when cell reaches 80% degrees of fusion, use 1xPBS washed cell, and resist with 20 μ g/ml Body (containing the culture medium dilution of 0.5%FBS with 5ml) processes.After overnight processing, standard RIPA buffer is used to prepare full cell Lysate.Determine the total protein content in every part of sample, then 1-5 μ g sample is carried out Western blot analysis, use The one of anti-HER2 is anti-to be detected.
Result
The result of study (Figure 14) of target degraded proves, the mixtures of antibodies that is combined with non-overlapping epitopes (4384+4517, 4382+4385+4518 and 4382+4387+4517) HER2 degraded in all cells system can be induced.Mixture 4382+4518 and song Trastuzumab+handkerchief trastuzumab analog effect is slightly lower, and this points out the epi-position of these antibody for induction of antibodies degraded to be not Optimum.3 kinds of mixtures of antibodies are all stronger than the mixture effect of single anti-HER 2 monoclonal antibody and 2 kinds of Anti-HER 2s, Mixture effect especially with antibody 4382+4385+4518 is the strongest.No matter Herceptin or handkerchief trastuzumab analog HER2 all can not be induced to degrade, except Herceptin induces HER2 degraded in ZR-75-30 cell line.These results show, Mixtures of antibodies for HER2 non-overlapping epitopes can induce it to degrade, but mixture has different effect.
Example 11
This illustration is bright, some cancerous cell line can to targeting HER2 response thus raise HER3 level to maintain HER2/HER3 The oncogenic signals path of mediation.
Method
Thin in order to study the targeting HER2 response impact on HER3 level, NCI-N87 (gastric cancer) and HCC202 (breast carcinoma) Born of the same parents are through 20 μ g/ml antibody (Herceptin, handkerchief trastuzumab analog, Herceptin+handkerchief trastuzumab analog, 4384+ 4517,4382+4518,4382+4385+4518 or 4382+4387+4517)) overnight process.Cell is incubated at T-75 cultivate In Ping, remove culture medium when cell reaches 80% degrees of fusion, use 1xPBS washed cell, and mix with 20 μ g/ml antibody or antibody Compound (containing the culture medium dilution of 0.5%FBS with 5ml) processes.Cell, after overnight, uses the preparation of standard RIPA buffer complete thin Cellular lysate thing.Determine the total protein content in every part of sample, then 1 or 10 μ g samples carried out Western blot analysis, Use the one of anti-HER2 and HER3 to resist to detect.
Result
The horizontal result of study of HER3 (Figure 15) proves, targeting HER2 may result in HER3 in 2 kinds of different cell lines and raises.This knot Fruit shows simultaneously, and cell most probable after the mixtures of antibodies of induction HER2 degraded processes raises HER3.
Example 12: the degraded of HER2 in the cancerous cell line of antibody treatment
This illustration is bright, and the Anti-HER 2 mixture in conjunction with non-overlapping epitopes can induce HER2 to degrade, 3 kinds of antibody 4382 The combination of+4385+4518 is more higher than combination effect in terms of induced degradation of 2 kinds of antibody, and antibody 4385+4518 is 4382+ 4385+4518 mixture drives the main drive of HER2 internalization.
Method
In order to study the HER2 receptor degradation level of mixtures of antibodies induction, OE19 (gastric cancer), NCI-N87 (gastric cancer), ZR- 75-30 (breast carcinoma), HCC202 (breast carcinoma) and BT474 (breast carcinoma) cell are through 10 μ g/ml monoclonal antibodies or containing 2 kinds or 3 After the mixtures of antibodies of kind antibody processes 48 hours, its full cell lysate is carried out Western blot analysis.By cell It is incubated in T-75 culture bottle, removes culture medium when cell reaches 80% degrees of fusion, use 1xPBS washed cell, and with 10 μ g/ Ml antibody or mixtures of antibodies (containing the culture medium dilution of 0.5%FBS with 5ml) process.After hatching 48 hours, use standard RIPA Full cell lysate prepared by buffer.Determine the total protein content in every part of sample, then 1-5 μ g sample is carried out protein and exempt from Epidemic disease engram analysis, uses the one of anti-HER2 and actin to resist and detects.Antibody test uses negative control antibody (not combine Anti-RSV (respiratory syncytial virus) antibody of HER2), Herceptin, handkerchief trastuzumab analog, handkerchief trastuzumab+song Trastuzumab analog, antibody 4382,4385 and 4518 and wherein 2 kinds or the mixture of 3 kinds.
Result
Result shows (Figure 16), in the monoclonal antibody and mixtures of antibodies of test, and mixtures of antibodies 4382+4385+ 4518 induce HER2 target degraded effect the strongest in the cell line of all tests.Additionally, 4385+4518 is containing 2 kinds of antibody Mixture in induce HER2 degraded effect the strongest.Antibody change in other 2 kinds of 4382+4385+4518 mixtures of antibodies, i.e. 4382+4385 and 4382+4518, in terms of induction target internalization, effect is more weak.This explanation, antibody 4385 and 4518 is 4382+ 4385+4518 mixtures of antibodies drives the main drive of HER2 internalization.
Example 13: the growth inhibited effect of different component in a kind of mixtures of antibodies
In this example, viability examination (WST-1) described in example 2,4 and 6 is used to determine that 4382+4385+4518 antibody mixes The antiproliferative effect of different components in compound.Additionally, substitute antibody 4382 with handkerchief trastuzumab analog to form new antibody Mixture, also the assessment to it is similar to is the most permissible to determine the antibody with similar binding specificity and biology department's function Substitute each other.As described in example 3, antibody 4382 and handkerchief trastuzumab analog can be in conjunction with tables neighbouring on overlapping epitope or HER2 Position, and the HER3 phosphorylation (example 9) of 2 kinds of antibody blocks part inductions.Antibody mixing is tested in multiple cancerous cell line Thing, including NCI-N87 (gastric cancer), OE19 (gastric cancer), HCC202 (breast carcinoma), BT474 (breast carcinoma) and SKBR3.
Result
Different antibodies mixture burette test result shows, all 2 kinds of antibody in 4382+4385+4518 mixtures of antibodies Combination, i.e. 4382+4385,4382+4518 and 4385+4518, all can grow with anticancer.But, the mixing of these 3 kinds of antibody It is the strongest that thing 4382+4385+4518 has effect in the growth of suppression test cell system.3 kinds of cell lines NCI-N87, BT474 and The burette test result of HCC202 is as in figs. 17-19.
Can find out from Figure 17-19, when antibody 4382 is had similar binding specificity by a kind of and block part induction When the antibody (handkerchief trastuzumab) of HER3 phosphorylation abilities substitutes, its growth inhibited effect still retains.Pearl appropriate with alone handkerchief is single Anti-analog is compared, handkerchief trastuzumab analog and the combination of antibody 4385 or 4518, or the cell growth inhibition of both combinations Act on higher.But, the combination containing antibody 4382 is thinner in all tests than the respective combination containing handkerchief trastuzumab analog Born of the same parents system act on higher.

Claims (30)

1. an antibody compositions, its first restructuring Anti-HER 2 comprising epi-position different from HER2 combination or its humanization resist Body and the second restructuring Anti-HER 2 or its humanized antibody, wherein:
A () first restructuring Anti-HER 2 comprises heavy chain CDR1, CDR2, CDR3 sequence in SEQ ID NO:22 and SEQ ID Light chain CDR1, CDR2, CDR3 sequence in NO:24, the second restructuring Anti-HER 2 comprises the heavy chain in SEQ ID NO:2 Light chain CDR1, CDR2, CDR3 sequence in CDR1, CDR2, CDR3 sequence and SEQ ID NO:4；Or
B () first restructuring Anti-HER 2 comprises heavy chain CDR1, CDR2, CDR3 sequence in SEQ ID NO:14 and SEQ ID Light chain CDR1, CDR2, CDR3 sequence in NO:16, the second restructuring Anti-HER 2 comprises the heavy chain in SEQ ID NO:6 Light chain CDR1, CDR2, CDR3 sequence in CDR1, CDR2, CDR3 sequence and SEQ ID NO:8；Or
C () first restructuring Anti-HER 2 comprises heavy chain CDR1, CDR2, CDR3 sequence in SEQ ID NO:26 and SEQ ID Light chain CDR1, CDR2, CDR3 sequence in NO:28, the second restructuring Anti-HER 2 comprises the heavy chain in SEQ ID NO:6 Light chain CDR1, CDR2, CDR3 sequence in CDR1, CDR2, CDR3 sequence and SEQ ID NO:8.
2. the antibody compositions of claim 1, wherein said first restructuring Anti-HER 2 comprises the weight in SEQ ID NO:22 Light chain CDR1, CDR2, CDR3 sequence in chain CDR1, CDR2, CDR3 sequence and SEQ ID NO:24, described second restructuring is anti- HER2 antibody comprise heavy chain CDR1, CDR2, CDR3 sequence in SEQ ID NO:2 and the light chain CDR1 in SEQ ID NO:4, CDR2, CDR3 sequence.
3. the antibody compositions of claim 1, the weight chain variable domain amino acid sequence of wherein said first restructuring Anti-HER 2 It is in the weight chain variable domain amino acid sequence in SEQ ID NO:22 and SEQ ID NO:24 with light chain variable domain amino acid sequence Light chain variable domain amino acid sequence, and the weight chain variable domain amino acid sequence of wherein said second restructuring Anti-HER 2 and light Chain variable domain amino acid sequence is in the weight chain variable domain amino acid sequence in SEQ ID NO:2 and SEQ ID NO:4 respectively Light chain variable domain amino acid sequence.
4. the antibody compositions of claim 1, wherein said first restructuring Anti-HER 2 comprises the weight in SEQ ID NO:22 Light chain CDR1, CDR2, CDR3 sequence in chain CDR1, CDR2, CDR3 sequence and SEQ ID NO:24, described second restructuring is anti- HER2 antibody comprise heavy chain CDR1, CDR2, CDR3 sequence in SEQ ID NO:2 and the light chain CDR1 in SEQ ID NO:4, CDR2, CDR3 sequence, wherein changes the residue of one of described CDR to remove desamidization site.
5. the antibody compositions of claim 1, wherein said first restructuring Anti-HER 2 comprises the weight in SEQ ID NO:26 Light chain CDR1, CDR2 and CDR3 sequence in chain CDR1, CDR2 and CDR3 sequence and SEQ ID NO:28, and described second restructuring Anti-HER 2 comprises heavy chain CDR1, CDR2 and CDR3 sequence in SEQ ID NO:6 and the light chain in SEQ ID NO:8 CDR1, CDR2 and CDR3 sequence.
6. the antibody compositions of claim 1, the weight chain variable domain amino acid sequence of wherein said first restructuring Anti-HER 2 It is in the weight chain variable domain amino acid sequence in SEQ ID NO:26 and SEQ ID NO:28 with light chain variable domain amino acid sequence Light chain variable domain amino acid sequence, and the weight chain variable domain amino acid sequence of wherein said second restructuring Anti-HER 2 and light Chain variable domain amino acid sequence is the weight chain variable domain amino acid sequence in SEQ ID NO:6 and the light chain in SEQ ID NO:8 Variable domain amino acid sequence.
7. the antibody compositions of any one of claim 1-6, at least one Anti-HER 2 in wherein said compositions is people Source.
8. the antibody compositions of any one of claim 1-6, at least one Anti-HER 2 in wherein said compositions is IgG Isotype.
9. the antibody compositions of any one of claim 1-6, it also comprises the anticancrin that at least one is other.
10. the antibody compositions of any one of claim 1-6, a kind of anti-HER2 antibody It is in the immune conjugate comprising cancer therapy drug.
The antibody compositions of 11. claim 10, wherein said cancer therapy drug selected from cell toxicity medicament, cytokine, toxin and Radionuclide.
12. 1 kinds of bi-specific binding molecule, it comprises the first Anti-HER 2 any one of claim 1-6 or its people source Change the heavy chain of antibody and light chain CDR1, CDR2 and CDR3 sequence and the second Anti-HER 2 or the heavy chain of its humanized antibody and light Chain CDR1, CDR2 and CDR3 sequence.
The bi-specific binding molecule of 13. claim 12, it comprises:
Weight chain variable domain amino acid sequence in (a) SEQ ID NO:22 and the light chain variable domain amino acid in SEQ ID NO:24 Sequence；With
Weight chain variable domain amino acid sequence in (b) SEQ ID NO:2 and the light chain variable domain amino acid sequence in SEQ ID NO:4 Row.
The bi-specific binding molecule of 14. claim 12, it comprises:
Weight chain variable domain amino acid sequence in (a) SEQ ID NO:26 and the light chain variable domain amino acid in SEQ ID NO:28 Sequence；With
Weight chain variable domain amino acid sequence in (b) SEQ ID NO:6 and the light chain variable domain amino acid sequence in SEQ ID NO:8 Row.
15. 1 kinds of restructuring Anti-HER 2s, it comprises heavy chain CDR1, CDR2 and CDR3 sequence in one sequence, and light chain CDR1, CDR2 and CDR3 sequence:
A () is SEQ ID NO 14 and 16 respectively；
B () is SEQ ID NO 22 and 24 respectively；
C () is SEQ ID NO 2 and 4 respectively；
D () is SEQ ID NO 6 and 8 respectively；Or
E () is SEQ ID NO 26 and 28 respectively；
Or the humanized antibody of described antibody.
The restructuring Anti-HER 2 of 16. claim 15, heavy chain and the light chain variable domain amino acid sequence of wherein said antibody be:
Weight chain variable domain amino acid sequence in (a) SEQ ID NO 14 and the light chain variable domain amino acid in SEQ ID NO 16 Sequence；
Weight chain variable domain amino acid sequence in (b) SEQ ID NO 22 and the light chain variable domain amino acid in SEQ ID NO 24 Sequence；
Weight chain variable domain amino acid sequence in (c) SEQ ID NO 2 and the light chain variable domain amino acid sequence in SEQ ID NO 4 Row；
Weight chain variable domain amino acid sequence in (d) SEQ ID NO 6 and the light chain variable domain amino acid sequence in SEQ ID NO 8 Row；Or
Weight chain variable domain amino acid sequence in (e) SEQ ID NO 26 and the light chain variable domain amino acid in SEQ ID NO 28 Sequence.
The restructuring Anti-HER 2 of 17. claim 15 or 16, wherein said antibody is humanized.
18. the restructuring Anti-HER 2 of claim 15 or 16, wherein said antibody is IgG isotype.
19. 1 kinds of pharmaceutical compositions, it comprises the antibody compositions of any one of claim 1-11, and claim 12-14 is arbitrary The bi-specific binding molecule of item, or the recombinant antibodies of any one of claim 15-18, and medicinal acceptable excipient.
20. 1 kinds encode the heavy chain variable domain of claim 1-11 and any one of 15-18 defined restructuring Anti-HER 2, light Chain variable domain or both nucleotide sequences.
The nucleotide sequence of 21. claim 20, wherein said nucleotide sequence is SEQ ID NO:1,3,5,7,13,15,21, 23,25, or 27.
22. 1 kinds of expression vectors, it comprises the nucleotide sequence of claim 20 or 21.
23. 1 kinds of host cells, it comprises coding claim 1-11 and any one of 15-18 defined restructuring Anti-HER 2 The nucleotide sequence of heavy chain variable domain and coding claim 1-11 and any one of 15-18 defined restructuring Anti-HER 2 The nucleotide sequence of light-chain variable domain, wherein said host cell can express described Anti-HER 2.
24. 1 kinds of methods generating anti-HER2 antibody of recombinating, it host cell including providing claim 23, at suitable table Cultivate this host cell under conditions of reaching this antibody, and separate the antibody generated.
25. 1 kinds of methods generating antibody compositions, the method comprises following step: provide the first host cell and the second host Cell, wherein the first and second host cells can express the first and second restructuring defined in any one of claim 1-11 respectively Anti-HER2 antibody；This first and second host cell is cultivated under conditions of suitable this first and second recombinant antibodies of expression；And Separate the first and second recombinant antibodies generated, thus generate the antibody compositions comprising the first and second recombinant antibodies.
The method of 26. claim 25, wherein the first and second host cells are cultivated in separate bioreactor.
The method of 27. claim 25, wherein the first and second host cells are cultivated in a bioreactor.
28. the antibody compositions of any one of claim 1-11, the bi-specific binding molecule of any one of claim 12-14, Or the restructuring Anti-HER 2 of any one of claim 15-18 is used for treating the mankind or other mammal gastric cancer or breast in preparation Purposes in the medicine of adenocarcinoma.
29. the antibody compositions of any one of claim 1-11, the bi-specific binding molecule of any one of claim 12-14, Or the restructuring Anti-HER 2 of any one of claim 15-18 produces drug resistance for suppression to anti-HER2 Antybody therapy in preparation Or the purposes in the medicine of the gastric cancer of part drug resistance or breast cancer cell growth.
30. the purposes of claim 29, wherein said gastric cancer or breast cancer cell have previously accepted Herceptin treatment.
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