CN104093742A

CN104093742A - Antibodies for epidermal growth factor receptor 3 (HER3) directed to domain III and domain IV of her3

Info

Publication number: CN104093742A
Application number: CN201280068966.8A
Authority: CN
Inventors: W·埃利斯; S·埃滕伯格; A·P·加纳; N·豪布斯特; H·于埃; C·C·S·昆茨; E·A·赖辛格斯普拉格; Q·盛
Original assignee: Novartis AG
Current assignee: Novartis AG
Priority date: 2011-12-05
Filing date: 2012-12-04
Publication date: 2014-10-08
Also published as: JP2015500830A; EP2788381A2; AU2012349739A1; EA201491120A1; UY34488A; IL232950A0; AR089085A1; BR112014013495A2; CA2857939A1; TW201331225A; WO2013084151A3; IN2014CN04374A; KR20140099315A; WO2013084151A2; MX2014006731A; BR112014013495A8; SG11201402784WA

Abstract

The present invention relates to antibodies or fragments thereof that bind to a non-linear epitope within domain 3 of the HER3 receptor and inhibit both ligand-dependent and ligand-independent signal transduction. The invention also relates antibodies or fragments thereof that bind to amino acid residues within domains 3-4 of HER3 and inhibit both ligand-dependent and ligand-independent signal transduction; and compositions and methods of use of the antibodies or the fragments.

Description

EGF-R ELISA 3 (HER3) antibody of the Domain III of anti-HER3 and structural domain IV

Related application

The application requires the right of priority of the U.S. Provisional Application submitted on December 5th, 2011 number 61/566,912, and its content is incorporated herein by reference with its integral body at this.

Technical field

The present invention relates to non-linear epi-position in the structural domain 3 with HER3 acceptor is combined and suppresses the two antibody or its fragment of ligand dependent and non-ligand dependent signal transduction.The invention still further relates to amino-acid residue in the structural domain 3-4 with HER3 is combined and suppresses the two antibody or its fragment of ligand dependent and non-ligand dependent signal transduction; And the method for the composition of this antibody-like or its fragment and this antibody-like of use or its fragment.

Background technology

Human epidermal growth factor receptor 3 (ErbB3, also claim HER3) be receptor protein tyrosine kinase the EGF-R ELISA that belongs to receptor protein tyrosine kinase (EGFR) subfamily, this subfamily also comprises EGFR (HER1, ErbB1), HER2 (ErbB2, Neu) and HER4 (ErbB4) (people such as Plowman, (1990) Proc.Natl.Acad.Sci.U.S.A.87:4905-4909; The people such as Kraus, (1989) Proc.Natl.Acad.Sci.U.S.A.86:9193-9197; With the people such as Kraus, (1993) Proc.Natl.Acad.Sci.U.S.A.90:2900-2904).The same with prototype EGF-R ELISA, transmembrane receptor HER3 is comprised of the dimerization structural domain in the outer ligand binding domains (ECD) of born of the same parents, ECD, membrane spaning domain, intracellular protein Tyrosylprotein kinase spline structure territory (TKD) and C-end phosphorylation domain.Different from other HER family members, the kinase domain of HER3 shows extremely low inherent kinase activity.

Part neuregulin 1 (NRG) or neuregulin 2 be in conjunction with the ectodomain of HER3, and by promoting to come with the dimerization of other dimerization mating partners (as HER2) signal transduction path of activated receptor mediation.Allos dimerization causes activation and the transphosphorylation of HER3 born of the same parents' intracellular domain, and this is not only the diversified means of signal, and is the means that signal amplifies.In addition, HER3 allos dimerization also can occur in the situation that lacking activation part, and this is commonly referred to non-ligand dependent HER3 and activates.For example, at HER2, for example, due to gene amplification (in mammary cancer, lung cancer, ovarian cancer or cancer of the stomach), during with high level expression, can form spontaneous HER2/HER3 dimer.In this case, think that HER2/HER3 is that the highest active ErbB signal transmits dimer, and be therefore high-degree of conversion.

In some cancer types, found the HER3 increasing, for example, in mammary cancer, lung cancer, gastrointestinal cancer and carcinoma of the pancreas, found.What is interesting is, shown the expression of HER2/HER3 and advanced to dependency (people such as Alimandi, (1995) Oncogene10:1813-1821 between stage of invasion from the non-intruding phase; The people such as DeFazio, (2000) Cancer 87:487-498; The people such as Naidu, (1988) Br.J.Cancer 78:1385-1390).Therefore, need to disturb the medicine of the signal granting of HER3 mediation.

Summary of the invention

The present invention is based in conjunction with the non-linear epi-position of the amino-acid residue in the structural domain that comprises HER3 3 of HER3 acceptor block ligand dependency (for example neuregulin) and non-ligand dependent HER3 signal transduction path the two antibody or the surprising discovery of its fragment.The present invention also the amino-acid residue in the structural domain 3-4 based in conjunction with HER3 block ligand dependency (for example neuregulin) and non-ligand dependent HER3 signal transduction path the two antibody or the discovery of its fragment.

Therefore, on the one hand, the present invention relates to identify separated antibody or its fragment of the non-linear epi-position of HER3 acceptor, amino-acid residue in the structural domain 3 that wherein this non-linear epi-position comprises HER3 acceptor, wherein this antibody or its fragment are combined with mating surface B, and wherein this antibody or its fragment block ligand dependency and non-ligand dependent signal transduction the two.

In one embodiment, mating surface B comprises the amino-acid residue that at least one is selected from amino-acid residue 335-342,398,400,424-428,431,433-434 and 455.In another embodiment, this antibody or its fragment are further combined with mating surface A.In one embodiment, mating surface A comprises the amino-acid residue that at least one is selected from amino-acid residue 362-376.

On the other hand, the present invention relates to identify separated antibody or its fragment of the non-linear epi-position of HER3 acceptor, amino-acid residue in the structural domain 3 that wherein this non-linear epi-position comprises HER3 acceptor, wherein this antibody or its fragment are combined with the mating surface that comprises at least one amino-acid residue that is selected from mating surface A and at least one and be selected from the amino-acid residue of mating surface B, and wherein this antibody or its fragment block ligand dependency and non-ligand dependent signal transduction the two.

In one embodiment, this antibody or the combination on HER3 acceptor of its fragment blocking-up HER3 part.In one embodiment, this HER3 part is selected from neuregulin 1 (NRG), neuregulin 2, β tunicin, Heparin-binding Urogastron and epiregulin (epiregulin).In one embodiment, this antibody or its fragment have any that is selected from following feature: with the HER3 receptors bind of inactive state, because the sterically hindered HER3 that stops between this antibody or its fragment and the structural domain of HER3 adopts activity conformation, by the flexible degree reducing in structural domain 3, stop HER3 to adopt activity conformation, in the residue 371-377 of structural domain 3, induction stops HER3 to adopt the conformational change of activity conformation, destabilization HER3 makes it be subject to degraded, accelerate the downward of cell surface HER3, and produce be subject to proteolysis degraded or can not with the non-natural HER3 dimer of other receptor tyrosine kinase dimerizations.In one embodiment, mating surface A comprises amino-acid residue 362-376.In one embodiment, mating surface B comprises amino-acid residue 335-342,398,400,424-428,431,433-434 and 455.

In one embodiment, this non-linear epi-position comprises amino-acid residue 335-342,362-376,398,400,424-428,431,433-434 and 455 (in structural domain 3), or its subset.In one embodiment, at least one in the following HER3 residue of the VH of this antibody or its fragment combination: Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400 and Lys434.In one embodiment, at least one in the following HER3 residue of the VL of this antibody or its fragment combination: Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455.In one embodiment, this antibody or its fragment have reduced the non-ligand dependent formation of HER2-HER3 albumen composition in the cell of expressing HER2 and HER3 in the situation that lacking HER3 part with the combination of HER3 acceptor.In one embodiment, as assessed by the non-ligand dependent phosphorylation assay of HER3, this antibody or its fragment suppress the phosphorylation of HER3.In one embodiment, the non-ligand dependent phosphorylation assay of this HER3 is used the cell of HER2 amplification, and wherein the cell of this HER2 amplification is SK-Br-3 cell and BT-474.In one embodiment, this antibody or its fragment have reduced the ligand dependent formation of HER2-HER3 albumen composition in the cell of expressing HER2 and HER3 under the existence of HER3 part with the combination of HER3 acceptor.In one embodiment, as assessed by HER3 ligand dependent phosphorylation assay, this antibody or its fragment suppress the phosphorylation of HER3.In one embodiment, this HER3 ligand dependent phosphorylation assay is used the MCF7 cell through stimulating under the existence of neuregulin (NRG).In one embodiment, this antibody is selected from monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody and synthetic antibody.

On the other hand, the present invention relates to identify separated antibody or its fragment of the epi-position of HER3 acceptor, amino-acid residue in the structural domain 3-4 that wherein this epi-position comprises HER3 acceptor, and wherein this antibody or its fragment block ligand dependency and non-ligand dependent signal transduction the two.

In one embodiment, the amino-acid residue that this epi-position comprises at least one amino-acid residue 329-498 that is selected from SEQ ID NO:1 (structural domain 3), and at least one is selected from the amino-acid residue of the amino-acid residue 499-642 (structural domain 4) of SEQ ID NO:1.In one embodiment, this epi-position that comprises the amino-acid residue in structural domain 3-4 is selected from linear epitope, non-linear epi-position and conformational epitope.In one embodiment, this antibody or its fragment have reduced the non-ligand dependent formation of HER2-HER3 albumen composition in the cell of expressing HER2 and HER3 in the situation that lacking HER3 part with the combination of HER3 acceptor.In one embodiment, as assessed by the non-ligand dependent phosphorylation assay of HER3, this antibody or its fragment suppress the phosphorylation of HER3.In one embodiment, the non-ligand dependent phosphorylation assay of this HER3 is used the cell of HER2 amplification, and wherein the cell of this HER2 amplification is SK-Br-3 cell and BT-474.In one embodiment, this antibody or its fragment have reduced the ligand dependent formation of HER2-HER3 albumen composition in the cell of expressing HER2 and HER3 under the existence of HER3 part with the combination of HER3 acceptor.In one embodiment, as assessed by HER3 ligand dependent phosphorylation assay, this antibody or its fragment suppress the phosphorylation of HER3.In one embodiment, this HER3 ligand dependent phosphorylation assay is used the MCF7 cell through stimulating under the existence of neuregulin (NRG).

On the other hand, the present invention relates to separated antibody or its fragment of anti-HER3 acceptor, there is at least 1x 10 ⁷m ^-1, 10 ⁸m ^-1, 10 ⁹m ^-1, 10 ¹⁰m ^-1, 10 ¹¹m ^-1, 10 ¹²m ^-1, 10 ¹³m ^-1(the K that dissociates _d), wherein this antibody or its fragment block ligand dependency and non-ligand dependent signal transduction the two.In one embodiment, as measured measurement by being selected from the phosphorylation in vitro of phosphoric acid-HER3 and phosphoric acid-Akt, this antibody or its fragment suppress the phosphorylation of HER3.

On the other hand, the present invention relates to separated antibody or its fragment, it is in conjunction with the non-linear epi-position in the structural domain 3 of the HER3 identical with the antibody described in table 1.

On the other hand, the present invention relates to separated antibody or its fragment, it is in conjunction with the amino-acid residue in the structural domain 3-4 of the HER3 identical with the antibody described in table 2.

On the other hand, the present invention relates to the antibody fragment in conjunction with HER3, it is selected from Fab, F (ab ₂) ', F (ab) ₂', scFv, VHH, VH, VL, dAb, wherein this antibody fragment block ligand dependency and non-ligand dependent signal transduction the two.

On the other hand, the present invention relates to the pharmaceutical composition that comprises antibody or its fragment and pharmaceutically acceptable carrier.In one embodiment, this pharmaceutical composition further comprises additional treatment agent.In one embodiment, this additional treatment agent is selected from HER1 inhibitor, HER2 inhibitor, HER3 inhibitor, HER4 inhibitor, mTOR inhibitors and PI3 kinase inhibitor.In one embodiment, this additional treatment agent be selected from horse trastuzumab (Matuzumab) (EMD72000), cetuximab (Cetuximab), victibix (Panitumumab), mAb 806, Buddhist nun not pearl monoclonal antibody (Nimotuzumab), gefitinib (Gefitinib), CI-1033 (PD183805), lapatinibditosylate (Lapatinib) (GW-572016), xylene monosulfonic acid lapatinibditosylate (Lapatinib Ditosylate), erlotinib hydrochloride (Erlotinib HCL) (OSI-774), PKI-166 and hER1 inhibitor; Be selected from handkerchief trastuzumab (Pertuzumab), Herceptin (Trastuzumab), MM-111, HKI-272 (neratinib), lapatinibditosylate or xylene monosulfonic acid lapatinibditosylate hER2 inhibitor; Be selected from the micromolecular HER3 inhibitor of MM-121, MM-111, IB4C3,2DID12 (U3Pharma AG), AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech), MOR10703 (Novartis) and inhibition HER3; With HER4 inhibitor.In one embodiment, this additional treatment agent is HER3 inhibitor, and wherein this HER3 inhibitor is MOR10703.In one embodiment, this additional treatment agent is to be selected from CCI-779 (Temsirolimus) ridaforolimus/ rapamycin (Deforolimus), AP23573, MK8669, everolimus (everolimus) mTOR inhibitors.In one embodiment, this additional treatment agent is the PI3 kinase inhibitor that is selected from GDC 0941, BEZ235, BKM120 and BYL719.

On the other hand, the present invention relates to treat the method for cancer, it comprises the individuality of selecting to suffer from the cancer of expressing HER3, this individuality is used to the composition that comprises disclosed antibody in table 1 or table 2 or its fragment of significant quantity.In one embodiment, this individuality is people, and this cancer is selected from mammary cancer, colorectal carcinoma, lung cancer, multiple myeloma, ovarian cancer, liver cancer, cancer of the stomach, acute myeloid leukaemia, chronic myelogenous leukemia, osteosarcoma, squamous cell carcinoma, Peripheral Nerve Sheath Tumors, schwannoma, head and neck cancer, bladder cancer, the esophageal carcinoma, the Barretts esophageal carcinoma, glioblastoma, research of clear cell sarcoma of soft tissue, malignant mesothe, neurofibromatosis, kidney, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia and endometriosis.In one embodiment, this cancer is mammary cancer.

On the one hand, the present invention relates to this antibody or its fragment as the purposes of medicine.On the one hand, the present invention relates to this antibody or its fragment and be used for the treatment of the purposes by the cancer of HER3 ligand dependent signal transduction pathway or the mediation of non-ligand dependent signal transduction pathway.On the one hand, the present invention relates to this antibody or its fragment in the purposes in the medicine of the cancer of HER3 ligand dependent signal transduction pathway or the mediation of non-ligand dependent signal transduction pathway for the preparation for the treatment of, this cancer is selected from mammary cancer, colorectal carcinoma, lung cancer, multiple myeloma, ovarian cancer, liver cancer, cancer of the stomach, carcinoma of the pancreas, acute myeloid leukaemia, chronic myelogenous leukemia, osteosarcoma, squamous cell carcinoma, Peripheral Nerve Sheath Tumors, schwannoma, head and neck cancer, bladder cancer, the esophageal carcinoma, the Barretts esophageal carcinoma, glioblastoma, research of clear cell sarcoma of soft tissue, malignant mesothe, neurofibromatosis, kidney, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia (gynacomastica) and endometriosis.

Accompanying drawing summary

Fig. 1. the representative MOR12615SET curve that employment HER3 obtains;

Fig. 2. the SK-Br-3 Cell binding being undertaken by FACS titration is measured;

Fig. 3 .HER3 structural domain is in conjunction with ELISA;

Fig. 4. (A) surface of HER3/MOR12604x ray crystal structure represents.In the conformation of HER3 (by structural domain D2, D3 and D4 mark) in sealing, MOR12604 and structural domain 3 combinations.(B) HER3 from HER3/MOR12064 structure comparing by structural domain 3 (Dark grey) and unconjugated HER3 structure (light gray; The people such as Cho, (2003) Nature421:756-760) C α stack.(C) view of structural domain 3 epi-positions of 12604 identifications.The HER3 residue highlighting in x ray crystal structure in MOR12604's within.(D) the interactional view of structural domain 3/MOR12604.With Dark grey, highlight MOR12604 mating surface, indicate surface A (solid line) and surperficial B (dotted line);

(A) HER3 and (B) inhibition of Akt phosphorylation of part induction in Fig. 5 .MCF7 cell;

Ligand dependent (A) HER3 and (B) inhibition of Akt phosphorylation in the SKBr3 cell of Fig. 6 .HER2 amplification;

Non-ligand dependent (A) HER3 and (B) inhibition of Akt phosphorylation in the BT474 cell of Fig. 7 .HER2 amplification;

The inhibition of the propagation of the ligand stimulation of Fig. 8 .MCF7 cell;

The inhibition of the non-ligand dependent propagation of Fig. 9 .SKBr3 cell;

The inhibition of the non-ligand dependent propagation of Figure 10 .BT474 cell;

Figure 11. show the data that suppress the tumor growth in vivo in BxPC3 (A) and BT474 (B) with MOR12606 and MOR13655; With

Figure 12. show the data of improving the inhibition of tumor growth in vivo in BxPC3 with the combination of MOR12606 (DIII bonding agent) and MOR10703 (DII+IV bonding agent).

Detailed Description Of The Invention

Definition

In order to make the present invention be easier to understand, first define some term.Detailed Description Of The Invention in the whole text shown in other definition.

Phrase used herein " signal transduction " or " signal is provided active " refer to generally pass through the initial biochemical cause-effect relationship of protein-protein interaction (as the combination of somatomedin and acceptor), cause signal from a part for cell, to be delivered to another part of cell.For HER3, this transmission relates to the specificity phosphorylation of one or more tyrosine, Serine or threonine residues on one or more protein in causing the serial reaction of signal transduction.Penultimate process generally comprises nuclear event, causes the change of genetic expression.

Term used herein " HER3 " or " HER3 acceptor " claim again " ErbB3 ", refer to Mammals HER3 albumen, and " her3 " or " erbB3 " refers to Mammals her3 gene.Preferred HER3 albumen is the people HER3 albumen being present in the cytolemma of cell.People her3 gene is at U.S. Patent number 5,480, and 968 and the people such as Plowman, (1990) Proc.Natl.Acad.Sci.USA, describes in 87:4905-4909.

In searching number NP_001973 (people), the people HER3 of definition, hereinafter represents with SEQ ID NO:1.All names represent total length, immature HER3 (amino acid/11-1342).Between position 19 and 20, cut immature HER3, produce ripe HER3 albumen (20-1342 amino acid).

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Term used herein " HER3 part " refers in conjunction with and activates the polypeptide of HER3.The example of HER3 part includes but not limited to neuregulin 1 (NRG) and neuregulin 2, Heparin-binding Urogastron and epiregulin.This term comprises bioactive fragment and/or the variant of naturally occurring polypeptide.

" HER2-HER3 albumen composition " is the oligomer of the non-covalent combination that contains HER2 acceptor and HER3 acceptor.When the cell of expressing these two kinds of acceptors is exposed to HER3 part (as NRG), or when HER2 has the expression of activity/mistake, can form this mixture.

Phrase used herein " HER3 is active " or " HER3 activation " refer to the increase that the downstream signal of oligomerization (for example comprising the increase of the mixture of HER3), HER3 phosphorylation, allosteric rearrangement (those of for example being induced by part) and HER3 mediation is provided.

The term using in the background of HER3 " stabilization " or " stabilization " refer to directly to maintain (locking, constraint, keep, preferential in conjunction with, be beneficial to) the inactivation state of HER3 or inactivation conformation and non-block ligand be in conjunction with HER3, makes ligand binding no longer can activate antibody or its fragment of HER3.

Term used herein " granting of ligand dependent signal " refers to activate by the HER3 of part.For example, by allos dimerization and/or the HER3 phosphorylation proof HER3 of the increase of downstream signal pathway (PI3K) activation are activated.When measuring by the mensuration described in embodiment, with respect to untreated (contrast) cell, in the cell of irriate that is exposed to antibody or its fragment, antibody or its fragment can be added up the amount that reduces significantly phosphorylation HER3.The cell of expressing HER3 can be naturally occurring clone (for example MCF7), can be maybe the cell producing by introduce the nucleic acid restructuring of coding HER3 albumen in host cell.Can add and activate HER3 part or by endogenous expression, activate part and carry out cytositimulation by external source.

Antibody or its fragment of " reducing the HER3 being induced by neuregulin in cell activates " are such antibody or its fragments, when measuring by the mensuration described in embodiment, with respect to untreated (contrast) cell, in this antibody or its fragment statistics, reduce significantly HER3 tyrosine phosphorylation.This can measure by the HER3 phosphorylation level based on after HER3 being exposed to NRG and object antibody.The cell of expressing HER3 albumen can be naturally occurring cell or clone (for example MCF7), can be maybe cell or clone that restructuring produces.

Term used herein " non-ligand dependent signal is provided " refers to not need the cell HER3 active (for example phosphorylation) of ligand binding.For example, non-ligand dependent HER3 activation can be the result that HER2 crosses expression or the activated mutant in HER3 heterodimer mating partner (as EGFR and HER2).With respect to untreated (contrast) cell, in being exposed to the cell of antibody or its fragment, antibody or its fragment can be added up the amount that reduces significantly phosphorylation HER3.The cell of expressing HER3 can be naturally occurring clone (for example SK-Br-3), can be maybe the cell producing by introduce the nucleic acid restructuring of coding HER3 albumen in host cell.

Term used herein " blocking-up " refers to stop or stops interact or process, for example, stops the grantings of ligand dependent or non-ligand dependent signal.

Term used herein " identification " refers to find epi-position in the two of its structural domain 3 at the structural domain 3 of HER3, the structural domain 4 of HER3 or HER3 and structural domain 4 and antibody or its fragment of interact with it (for example in conjunction with).This epi-position can be linear epitope, non-linear epi-position or conformational epitope.For example, amino-acid residue 335-342, the 362-376,398,400 of antibody or its fragment and HER3,424-428,431,433-434 and 455 (in structural domain 3) or its subset interact.In another example, antibody or its fragment and at least one are selected from amino-acid residue 329-498 (structural domain 3) amino-acid residue or its subset interact.In another example, antibody or its fragment and at least one are selected from amino-acid residue 499-642 (structural domain 4) amino-acid residue or its subset interact.In another example, antibody or its fragment and at least one are selected from the amino-acid residue of structural domain 3 (the amino-acid residue 329-498 of SEQ ID NO:1) of HER3 and amino-acid residue or its subset that at least one is selected from structural domain 4 (the amino-acid residue 499-642 of SEQ ID NO:1) interacts.

Phrase used herein " in conjunction with " simultaneously refer to can be together with HER3 antibody or its fragment in conjunction with the HER3 part of the ligand binding site on HER3 acceptor.This mean antibody and part the two all can together with in conjunction with HER3 acceptor.Only for illustrative purposes, HER3 part NRG can be in conjunction with HER3 acceptor together with HER3 antibody.Measurement part and antibody being simultaneously determined in embodiment chapters and sections of combination are described.

Antibody or its fragment of particular event " failed " to refer to not carry out in term used herein.For example, the antibody of " fail activation signal transduction " or its fragment are antibody or its fragments of not triggering signal transduction.

The whole antibody that term used herein " antibody " refers to HER3 epi-position interacts (for example,, by combination, steric hindrance, stabilization/stabilization removal, spatial distribution) and Inhibitory signal is transduceed.Naturally occurring " antibody " is the glycoprotein comprising by interconnective at least 2 weights (H) chain of disulfide linkage and 2 light (L) chains.Every heavy chain is comprised of variable region of heavy chain (being called for short VH herein) and CH.CH is comprised of three domain C H1, CH2 and CH3.Every light chain is comprised of variable region of light chain (being called for short VL herein) and constant region of light chain.Constant region of light chain is comprised of a domain C L.VHHe VL district can be further subdivided into the hypervariable region that is known as complementary determining region (CDR), wherein scatters the more conservative region that is called framework region (FR).Each VH and VL are comprised of three CDR and 4 FR, arrange in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from aminoterminal to carboxyl terminal.The variable region of heavy chain and light chain comprises the binding domains with AI.The constant region of antibody can mediated immunity sphaeroprotein and the combination of host tissue or the factor (first component (Clq) that comprises immune various kinds of cell (for example effector cell) and classical complement system).Term " antibody " comprises Fv (sdFv), Fab fragment, F (ab') fragment and antiidiotype (anti-Id) antibody that monoclonal antibody for example, people's antibody, humanized antibody, camel (camelised) antibody, chimeric antibody, scFv (scFv), disulphide connects (for example comprising the anti-Id antibody for antibody of the present invention) and any epi-position binding fragment above.Antibody can belong to any isotype (for example IgG, IgE, IgM, IgD, IgA and IgY), kind (for example IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

Light chain and heavy chain are all divided into the region with structure and function homology.Term " constant " and " variable " are used in function.Put at this point, should be appreciated that the variable domains of light (VL) chain and heavy (VH) chain portion all determines antigen recognition and specificity.On the contrary, the constant domain of light chain (CL) and heavy chain (CH1, CH2 or CH3) is given important biological characteristics, as secretion, transplacental migration, Fc receptors bind, complement combination, etc.By convention, constant region structural domain become from the antigen-binding site of antibody or aminoterminal more and more away from time, it numbers increase.N-end is variable region, and C-end is constant region; CH3 and CL structural domain comprise in fact respectively the carboxyl terminal of heavy chain and light chain.

Phrase used herein " antibody fragment " refers to retain and HER3 epitope specificity interacts (for example,, by combination, steric hindrance, stabilization/stabilization removal, spatial distribution) and one or more parts of the antibody of the ability of Inhibitory signal transduction.The example of binding fragment includes but not limited to Fab fragment, the unit price fragment being comprised of VL, VH, CL and CH1 structural domain; F (ab) ₂fragment, is included in the divalence fragment of two Fab fragments that hinge area connects by disulfide linkage; The Fd fragment being formed by VH and CH1 structural domain; The Fv fragment being formed by VL and the VH structural domain of antibody wall scroll arm; DAb fragment (Ward etc., (1989) Nature 341:544-546), it is comprised of VH structural domain; With separated complementary determining region (CDR).

In addition, although two structural domain VL of Fv fragment and VH are by the genes encoding separating, but available recombination method, by synthetic linker, they are connected, this synthetic linker makes them can be prepared into wall scroll protein chain, and wherein VLHe VH district pairing formation monovalent molecule (is called scFv (scFv); Consult such as Bird etc., (1988) Science 242:423-426; With Huston etc., (1988) Proc.Natl.Acad.Sci.85:5879-5883).Within this type of single-chain antibody is also intended to be covered in term " antibody fragment ".With routine techniques well known by persons skilled in the art, obtain these antibody fragments, and screen this fragment, in the mode identical with complete antibody, use this fragment.

Antibody fragment also can be incorporated in single domain antibody, huge antibody (maxibody), miniantibody (minibody), intracellular antibody, double antibody, three antibody, four antibody, v-NAR and bis-scFv and (consult for example Hollinger and Hudson, (2005) Nature Biotechnology 23:1126-1136).Antibody fragment portable to based on polypeptide as (consult U.S. Patent number 6,703,199, it has described fibronectin polypeptide monoclonal antibody body) in the support of III type fibronectin (Fn3).

Antibody fragment can be incorporated in the single chain molecule that comprises pair of series Fv section (VH-CH1-VH-CH1), this series connection Fv section forms a pair of antigen binding domain (Zapata etc., (1995) Protein Eng.8:1057-1062 together with complementary light chain polypeptide; With U.S. Patent number 5,641,870).

Term " epi-position " comprise can specific binding immunoglobulin (Ig) or otherwise with the protein determinant of interaction of molecules.Epi-position determinant generally, by the chemically reactive surface group of molecule, as amino acid or carbohydrate or sugared side chain composition, and can have special Three Dimensions Structure, and special charge characteristic.Epi-position can be " linearity ", " non-linear " or " conformation " epi-position.

Term " linear epitope " refers to such epi-position, and all interaction points between its protein and interacting molecule (as antibody) exist (continuous) along the one-level aminoacid sequence linearity of protein.Once determine the expectation epi-position on antigen, likely produce the antibody for this epi-position, for example use the technology described in the present invention.Alternatively, in discovery procedure, the generation of antibody and sign can be illustrated the information of relevant expectation epi-position.By this information, then likely screen competitively the antibody in conjunction with identical epi-position.Implementation method is to carry out cross competition research, to find the antibody of competitive binding each other, for example, competes the antibody of conjugated antigen.The high throughput method for " vanning (binning) " antibody of the cross competition based on antibody has been described in international patent application no WO 2003/48731.It will be appreciated by those skilled in the art that all epi-positions of arbitrary substance that antibody in fact can specific binding.Epi-position can comprise those residues of antibodies.

Term " non-linear epi-position " refers to have and forms in ad hoc structure territory the discontinuous amino acid whose epi-position of the three-dimensional structure of (for example, in structural domain 1, in structural domain 2, in structural domain 3 or structural domain 4 interior).In one embodiment, this non-linear epi-position is in structural domain 2.This non-linear epi-position for example can also be present in, between two or more structural domains (interface between structural domain 3-4).Non-linear epi-position also refers to the discontinuous amino acid as the three-dimensional structure in ad hoc structure territory.

Term " conformational epitope " refers to such epi-position, and wherein discontinuous amino acid flocks together in 3-d modelling.In conformational epitope, interaction point appears on the amino-acid residue being separated from each other in sequence on protein.It will be appreciated by those skilled in the art that by residue or the occupied space of side chain of creating shape of molecule and contribute to determine what epi-position is.

In one embodiment, this epi-position is in the structural domain 3 of HER3.In one embodiment, this epi-position is the non-linear epi-position of the amino-acid residue in the structural domain 3 that comprises HER3.In one embodiment, amino-acid residue 335-342, the 362-376,398,400 that this non-linear epi-position comprises SEQ ID NO:1,424-428,431,433-434 and 455 (in structural domain 3), or its subset.

In another embodiment, this epi-position is in the structural domain 4 of HER3.In one embodiment, this epi-position comprises at least one amino-acid residue that is selected from structural domain 4 (the amino-acid residue 499-642 of SEQ ID NO:1), or its subset.In one embodiment, this epi-position is the linear epitope in the structural domain 4 of HER3.In one embodiment, this epi-position is the non-linear epi-position in the structural domain 4 of HER3.In another embodiment, this epi-position is the conformational epitope in the structural domain 4 of HER3.

In another embodiment, this epi-position is in the structural domain 3-4 of HER3.In one embodiment, this epi-position comprises at least one amino-acid residue that is selected from structural domain 3 (the amino-acid residue 329-498 of SEQ ID NO:1), or its subset.In one embodiment, this epi-position comprises at least one amino-acid residue that is selected from structural domain 4 (the amino-acid residue 499-642 of SEQ ID NO:1), or its subset.In one embodiment, this epi-position comprises at least one amino-acid residue that is selected from structural domain 3 (the amino-acid residue 329-498 of SEQ ID NO:1) and at least one and is selected from amino-acid residue or its subset of structural domain 4 (the amino-acid residue 499-642 of SEQ ID NO:1).In one embodiment, this epi-position is linear epitope.In one embodiment, this epi-position is non-linear epitope.In another embodiment, this epi-position is conformational epitope.

Generally speaking, to the antibody of particular target antigen-specific by the epi-position on the target antigen in preferential identification of protein and/or macromolecular complex mixture.

Can use the epitope mapping technical evaluation well known in the art of arbitrary number to comprise the region of the given polypeptide of epi-position.See for example Methods in Molecular Biology, 66 volumes (Glenn E.Morris compiles 1996) Humana Press, Totowa, the Epitope Mapping Protocols in New Jersey.For example, can be by for example synthesize a large amount of peptides on solid support simultaneously, this peptide, corresponding to the part of protein molecule, makes peptide and antibody response measure linear epitope when peptide is still attached to upholder.This class technology is known in the art, and at for example U.S. Patent number 4,708,871; The people such as Geysen, (1984) Proc.Natl.Acad.Sci.USA 8:3998-4002; The people such as Geysen, (1985) Proc.Natl.Acad.Sci.USA 82:78-182; The people such as Geysen, describe in (1986) Mol.Immunol.23:709-715.Similarly, for example, for example pass through, hydrogen/deuterium exchange, X-ray crystallography and two dimensional NMR, easily identify non-linear epi-position and conformational epitope by measuring amino acid whose space conformation.See for example Epitope Mapping Protocols, above.Also the antigenicity region of available standards antigenicity and hydropathic profile (standard antigen and the hydropathic profiles that for example calculate with Omiga version 1.0 software programs that for example obtain from Oxford Molecular Group) identification of protein.This computer program utilizes the Hopp/Woods method (people such as Hopp, (1981) Proc.Natl.Acad.Sci USA 78:3824-3828) measure antigenicity spectrum, utilize Kyte-Doolittle technology people such as (, (1982) J.MoI.Biol.157:105-132) Kyte to measure hydropathic profile.

Term used herein " mating surface " refers to a plurality of continuous or discrete surface in the 3D configuration on HER3, for example the structural domain 3 of HER3.These surfaces form the part of epi-position, and interact with antibody or its fragment.For example, mating surface can comprise at least two surfaces (for example surface A and surperficial B, see Fig. 4 D), at least three surfaces (surface A for example, surface B and surface C), at least four surfaces (surface A for example, surface B, surface C and surperficial D), at least five surfaces (surface A for example, surface B, surface C, surface D and surperficial E), at least six surfaces (surface A for example, surface B, surface C, surface D, surface E and surperficial F), at least seven surfaces (surface A for example, surface B, surface C, surface D, surface E, surface F and surperficial G), at least eight surfaces (surface A for example, surface B, surface C, surface D, surface E, surface F, surface G and surperficial H), at least nine surfaces (surface A for example, surface B, surface C, surface D, surface E, surface F, surface G, surface H and surperficial I) or at least ten surfaces (surface A for example, surface B, surface C, surface D, surface E, surface F, surface G, surface H, surface I and surperficial J).

Term used herein " mating surface A " refers to the surface on the structural domain 3 of HER3, and it comprises at least one amino-acid residue that is selected from amino-acid residue 362-376.

Term used herein " mating surface B " refers to the surface on the structural domain 3 of HER3, and it comprises at least one amino-acid residue that is selected from amino-acid residue 335-342,398,400,424-428,431,433-434 and 455.

The polypeptide that phrase used herein " monoclonal antibody " or " monoclonal antibody combination " refer to have essentially identical aminoacid sequence or derive from identical genetic origin, comprises antibody, antibody fragment, bi-specific antibody etc.This term also comprises the preparation of the antibody molecule of unit molecule composition.Monoclonal antibody combination is shown single binding specificity and the avidity to defined epitope.

Phrase used herein " people's antibody " comprises the antibody with following variable region, the sequence that wherein HeCDR district in framework region all originates from people.In addition, if antibody comprises constant region, this constant region is also from such human sequence, people's germline sequence for example, or the mutant form of people's germline sequence, or contain such as from people such as Knappik (2000) J Mol Biol 296:57-86) described in the antibody of the total frame sequence analyzed of people's Frame sequence.The structure of immunoglobulin variable structural domain (for example CDR) and position can define by the numbering plan of knowing, for example, the combination of Kabat numbering plan, Chothia numbering plan or Kabat and Chothia (is for example shown in, Sequences of Proteins of Immunological Interest, U.S.Department of Health and Human Services (1991), the people such as Kabat compile; The people such as Lazikani, (1997) J.Mol.Bio.273:927-948); The people such as Kabat, (1991) Sequences of Proteins of Immunological Interest, the 5th edition, NIH publication number 91-3242U.S.Department of Health and Human Services; The people such as Chothia, (1987) J.Mol.Biol.196:901-917; The people such as Chothia, (1989) Nature 342:877-883; With the people such as Al-Lazikani, (1997) J.Mol.Biol.273:927-948.

People's antibody of the present invention can comprise can't help the amino-acid residue (for example,, by the somatic mutation in external random or site-directed mutagenesis or body or conservatively replace the sudden change of introducing, to promote stability or production) of human sequence coding.

Phrase used herein " human monoclonal antibodies " refers to show the antibody of single binding specificity, and it has following variable region, and wherein HeCDR district in framework region is all from human sequence.In one embodiment, by hybridoma, produce human monoclonal antibodies, this hybridoma comprise have comprise people's heavy chain transgenosis and light chain genetically modified genomic, from transgenic nonhuman animal transgenic mice B cell that obtain, that merge with immortality cell for example.

Phrase used herein " recombinant human antibody " comprises by recombinant means to be prepared, express, produce or separated everyone antibody, for example, for example, from the antibody of human immunoglobulin gene transgenosis or trans-chromosome animal (mouse) or prepare from it hybridoma separation, from transforming for expressing the separated antibody of host cell (for example, from transfectoma) of people's antibody, the antibody of combination people antibody library separation from restructuring, with by relating to human immunoglobulin gene, the all or part of montage of sequence is to any other means preparations of other DNA sequence dnas, express, produce or separated antibody.Such recombinant human antibody has following variable region, and wherein HeCDR district in framework region is from people's germline immunoglobulin sequences.But, in certain embodiments, can carry out vitro mutagenesis (or, carry out body endosome cell mutation when the transgenic animal of end user Ig sequence) to such recombinant human antibody, thus the V of recombinant antibodies _hand V _lthe aminoacid sequence in district is such sequence, although this sequence is from people's germline V _hand V _lsequence is also associated, but may not naturally be present in the people's antibody germline storehouse in body.

Specific binding between two entities represents with at least 10 ²m ^-1, 5x10 at least ²m ^-1, at least 10 ³m ^-1, 5x10 at least ³m ^-1, at least 10 ⁴m ^-1, 5x10 at least ⁴m ^-1, at least 10 ⁵m ^-1, 5x10 at least ⁵m ^-1, at least 10 ⁶m ^-1, 5x10 at least ⁶m ^-1, at least 10 ⁷m ^-1, 5x10 at least ⁷m ^-1, at least 10 ⁸m ^-1, 5x10 at least ⁸m ^-1, at least 10 ⁹m ^-1, 5x10 at least ⁹m ^-1, at least 10 ¹⁰m ^-1, 5x10 at least ¹⁰m ^-1, at least 10 ¹¹m ^-1, 5x10 at least ¹¹m ^-1, at least 10 ¹²m ^-1, 5x10 at least ¹²m ^-1, at least 10 ¹³m ^-1, 5x10 at least ¹³m ^-1, at least 10 ¹⁴m ^-1, 5x10 at least ¹⁴m ^-1, at least 10 ¹⁵m ^-1, or 5x10 at least ¹⁵m ^-1the equilibrium constant (K _a) (k _on/ k _off) combination.

Phrase " specificity (or selectivity) combination " refers to HER3 binding antibody and the association reaction of HER3 acceptor in the heterogeneous population of protein and other biological product.Except the equilibrium constant (K above pointing out _a) outside, HER3 binding antibody of the present invention also has lower than 5x10 conventionally ^-2m, lower than 10 ^-2m, lower than 5x10 ^-3m, lower than 10 ^-3m, lower than 5x10 ^-4m, lower than 10 ^-4m, lower than 5x10 ^-5m, lower than 10 ^-5m, lower than 5x10 ^-6m, lower than 10 ^-6m, lower than 5x10 ^-7m, lower than 10 ^-7m, lower than 5x10 ^-8m, lower than 10 ^-8m, lower than 5x10 ^-9m, lower than 10 ^-9m, lower than 5x10 ^-10m, lower than 10 ^-10m, lower than 5x10 ^-11m, lower than 10 ^-11m, lower than 5x10 ^-12m, lower than 10 ^-12m, lower than 5x10 ^-13m, lower than 10 ^-13m, lower than 5x10 ^-14m, lower than 10 ^-14m, lower than 5x10 ^-15m or lower than 10 ^-15m or lower dissociation rate constant (K _d) (k _off/ k _on), and with than its for example, avidity in conjunction with at least 2 times of the avidity height of heterogenetic antigen (HSA) in conjunction with HER3.

In one embodiment, antibody or its fragment have lower than 3000pM, lower than 2500pM, lower than 2000pM, lower than 1500pM, lower than 1000pM, lower than 750pM, lower than 500pM, lower than 250pM, lower than 200pM, lower than 150pM, lower than 100pM, lower than 75pM, lower than 10pM, lower than the dissociation constant (K of 1pM _d), this dissociation constant for example, with describing or well known to a person skilled in the art method assessment (, BIAcore mensuration, ELISA, FACS, SET) (Biacore International AB, Uppsala, Sweden) herein.

Term " K used herein _assoc" or " K _a" refer to the association rate of specific antibodies-AI, and term " K used herein _dis" or " K _d" refer to the dissociation rate of specific antibodies-AI.Term " K used herein _d" referring to dissociation constant, it is from K _dwith K _aratio (be K _d/ K _a) obtain and be expressed as volumetric molar concentration (M).Can use method well known in the art to measure the K of antibody _dvalue.Be used for measuring antibody K _dmethod be by using surperficial plasmon to resonate, or use bio-sensor system as system.

Term used herein " avidity " refers to antibody and antigen interactional intensity on single antigen position.In each antigen position, variable region and the antigen of antibody " arm " interact by weak noncovalent force on many sites; Interact more, avidity is stronger.

Term used herein " avidity " refers to that the general stability of Antibody-antigen complex or the informationization of intensity measure.It is controlled by three principal elements: antibody epitope avidity; Tiring of antigen and antibody; And the structural arrangement of the part that interacts.Finally, these factors have determined the specificity of antibody, and specific antibodies is in conjunction with the possibility of accurate epitope.

Term used herein " is tired " and is referred to the number of potential target combining site in polypeptide.Specificity position (being epi-position) on target molecule of each target combining site specific binding or target molecule.When polypeptide comprises more than one target combining site, each target combining site can the identical or different molecule (for example can be in conjunction with differing molecular, for example different antigen, or the different epi-positions on same molecular) of specific binding.

Phrase used herein " inhibition antibody " refers in conjunction with HER3 and suppresses the antibody of the biological activity that HER3 signal provides (for example, for example reducing, reduces and/or suppresses the signal granting activity of HER3 induction in phosphoric acid-HER3 or phosphoric acid-Akt measure).The example of measuring is described in more detail in following examples.Therefore, be to be understood that, (for example biochemistry, immunochemistry, cell, physiology or other biological are active for " inhibition " one or more these HER3 functional performances of measuring according to method known in this field and as herein described, etc.) antibody relate to respect to when there is no antibody (for example, maybe when there is irrelevant specific control antibodies) viewed given activity, the statistically evident minimizing of given activity.The antibody that suppresses HER3 activity causes so statistically evident minimizing, it is at least 10% of measuring parameter, at least 50%, 80% or 90% minimizing, in certain embodiments, antibody of the present invention can suppress to surpass 95%, 98% or 99% HER3 functionally active, and this reduction by cell HER3 phosphorylation level proves.

Phrase " separated antibody " refers to antibody, and it is substantially not for example, containing other antibody (, the separated antibody of specific binding HER3 does not contain the antibody of the antigen of specific binding except HER3 substantially) with different antigen-specifiies.Yet the separated antibody of specific binding HER3 can have cross reactivity to other antigen.In addition, separated antibody can be substantially containing other cellular materials and/or chemical.

Phrase " the conservative variant of modifying " is applicable to amino acid and nucleotide sequence.For specific nucleic acid sequence, the conservative variant of modifying refer to encode those nucleic acid of identical or basic identical aminoacid sequence, or not during encoding amino acid sequence, refer to essentially identical sequence at this nucleic acid.Since the degeneracy of genetic code, a large amount of identical any given protein of nucleic acid encoding of function.For example, the equal coded amino acid L-Ala of codon GCA, GCC, GCG and GCU.Therefore,, on each position (wherein codon is determined L-Ala), codon can be changed over to the codon of described any correspondence, and not change coded polypeptide.This type of nucleic acid variation is " silent variant ", and it is a kind of for conservative modification variation.Each nucleotide sequence of coded polypeptide has also been described each possible silent variant of nucleic acid herein.Those skilled in the art will recognize that each codon (except AUG, it is unique password of methionine(Met) normally, and TGG, and it is unique password of tryptophane normally) in can modification of nucleic acids, to produce molecule identical in function.Therefore each silent variant that, has implied the nucleic acid of coded polypeptide described in each in sequence.

For peptide sequence, " variant of conservative modification " comprises each replacement, the disappearance of peptide sequence or adds, and it causes with chemically similar amino acid, amino acid being replaced.It is known in the art that similar amino acid whose conservative substitution table in function is provided.This type of is conservative modify variant except and do not repel polymorphism variant of the present invention, plant between homologue and allelotrope.8 groups contain each other for guarding the amino acid of replacing below: 1) L-Ala (A), glycine (G); 2) aspartic acid (D), L-glutamic acid (E); 3) l-asparagine (N), glutamine (Q); 4) arginine (R), Methionin (K); 5) Isoleucine (I), leucine (L), methionine(Met) (M), α-amino-isovaleric acid (V); 6) phenylalanine (F), tyrosine (Y), tryptophane (W); 7) Serine (S), Threonine (T); With 8) halfcystine (C), methionine(Met) (M) (for example consulting Creighton, Proteins (1984)).In some embodiments, term " conserved sequence modification " is used in reference to combination feature amino acid modified of the antibody that not remarkably influenced or change contain aminoacid sequence.

Term " intersects-competition " and " intersect-competition " is used interchangeably herein, means the ability that antibody or its fragment disturb other antibody or its fragment to be combined with HER3 in the competitive binding assay of standard.

Useful Standard Competition binding assay is measured ability or the degree that antibody or its fragment can disturb another antibody or its fragment to be combined with HER3, thereby determines whether it to be called to cross competition of the present invention.Suitable assay method relates to uses Biacore technology (for example, by using BIAcore 3000 instruments (Biacore, Uppsala, Sweden)), and it can measure interactional degree with surperficial plasmon resonance technique.For measuring another assay method of cross competition, use the method based on ELISA.

Term used herein " optimization " refers to codon, change nucleotide sequence, with encoding amino acid sequence, this codon is to produce cell or biology, generally eukaryotic cell, for example preferred in Pichia (Pichia) cell, Trichoderma (Trichoderma) cell, Chinese hamster ovary cell (CHO) or people's cell.By the nucleotide sequence transformation of optimizing, to retain the aminoacid sequence of the initial coding of nuclei originis nucleotide sequence (it is also referred to as " parent " sequence) completely or as much as possible.

For evaluating antibody, to the standard test of the binding ability of the HER3 of a plurality of species, be well known in the art, comprise for example ELISA, Western trace and RIA.Suitable mensuration is described in detail in an embodiment.Also can for example, by the binding kinetics (binding affinity) of standard test assessment antibody well known in the art, for example by Biacore, analyze, or FACS relative affinity (Scatchard).Also describe in detail in an embodiment for for example evaluating antibody, to the mensuration of the effect of the functional performance of HER3 (, receptors bind is measured, and regulates HER3 signal path).

In the background of two or more nucleic acid or peptide sequence, phrase " per-cent is identical " or " per-cent identity " refer to identical two or more sequences or subsequence.Maximum in for comparison window or in designated area is corresponding and compare when comparing and comparing with visual inspection mensuration with following sequence comparison algorithm or by craft, if (two sequences has particular percentile, in specific region, or when not specifying, 60% identity in full length sequence, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity optionally) same amino acid residue or Nucleotide, so two sequences " basic identical ".Optionally, identity is present in length at least about the region of 50 Nucleotide (or 10 amino acid), or in length, is more preferably 100 to 500 or 1000 or the region of polynucleotide (or 20,50,200 or more amino acids) more.

For sequence comparison, a common sequence is as with reference to sequence, and cycle tests compares with it.When using sequence comparison algorithm, will test and reference sequences input computer, if needed, specify subsequence coordinate, and specified sequence algorithm routine parameter.Default program parameter can be used, maybe alternative parameter can be specified.Then sequence comparison algorithm calculates cycle tests with respect to the per-cent sequence identity of reference sequences based on program parameter.

" comparison window " used herein comprises with reference to being selected from 20 to 600, conventionally approximately 50 to approximately 200, more generally the section of the consecutive position of any some amount of approximately 100 to approximately 150, wherein can carry out after best comparison, the reference sequences of sequence and equal amts consecutive position being compared at two sequences.For sequence alignment method relatively, be known in the art.For example, by local homology's algorithm of Smith and Waterman (1970) Adv.Appl.Math.2:482c, by Needleman and Wunsch, (1970) the sequence analysis algorithm of J.Mol.Biol.48:443, by search Pearson and Lipman, (1988) similarity method of Proc.Nat ' l.Acad.Sci.USA 85:2444, computerize by these algorithms realizes (Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, GAP in WI, BESTFIT, FASTA and TFASTA), or compare and visual inspection (is consulted by craft, such as Brent etc., (2003) Current Protocols in Molecular Biology) carry out the best comparison of sequence for comparing.

Two examples that are suitable for measuring the algorithm of per-cent sequence identity and sequence similarity are BLAST and BLAST 2.0 algorithms, and it is described in respectively Altschul etc., (1977) Nuc.Acids Res.25:3389-3402; With Altschul etc., in (1990) J.Mol.Biol.215:403-410.For carrying out the software of BLAST analysis, can openly obtain by National Center for Biotechnology Information.This algorithm relates to and is first tested and appraised short word that in search sequence, length is W and identifies that high sub-sequence is to (HSP), when with database sequence in the word of equal length while comparing, this short word coupling or meet a certain on the occasion of threshold value score T.T be called neighborhood word score threshold value (Altschul etc., above).These initial neighborhood word are hit the seed as initial search, with the longer HSP that finds to contain them.As long as can increase accumulation comparison score, the both direction extension word along each sequence hits.For nucleotide sequence, by parameter M (the award score to a pair of coupling residue; Always be greater than 0) and the N (point penalty to mispairing residue; Always be less than 0) calculate and accumulate score.For aminoacid sequence, with score matrix, calculate accumulation score.In accumulation comparison score, from its value of being up to, fall X amount, accumulation comparison score because the accumulation of one or more negative score residues comparisons arrives zero or below or while arriving the end of arbitrary sequence, stop word and hit the extension making progress each party.BLAST algorithm parameter W, T and X have determined sensitivity and the speed of comparison.BLASTN program (for nucleotide sequence) is used the comparison of acquiescence word length (W) 11, expected value (E) 10, M=5, N=-4 and two chains.For aminoacid sequence, BLASTP program is used the comparison of acquiescence word length 3, expected value (E) 10 and BLOSUM62 score matrix (consulting Henikoff and Henikoff, (1989) Proc.Natl.Acad.Sci.USA89:10915) comparison (B) 50, expected value (E) 10, M=5, N=-4 and two chains.

BLAST algorithm also carries out the statistical analysis (for example consulting Karlin and Altschul, (1993) Proc.Natl.Acad.Sci.USA 90:5873-5787) of similarity between two sequences.A kind of similarity measurement that BLAST algorithm provides is minimum summation probability (P (N)), and it provides the indication that the probability of coupling accidentally occurs between two Nucleotide or aminoacid sequence.For example, if test nucleic acid and reference nucleic acid relatively in minimum summation probability lower than approximately 0.2, more preferably less than approximately 0.01, and most preferably lower than approximately 0.001, think that so nucleic acid is similar to reference sequences.

Also can use E.Meyers and the W.Miller of the ALIGN program that has been integrated into (version 2 .0), (1988) Comput.Appl.Biosci., algorithm 4:11-17), the per-cent identity of using PAM120 weighting residue table, room length point penalty 12 and gap penalty 4 to measure between two aminoacid sequences.In addition, can use the Needleman and Wunsch (1970) J.Mol that have been integrated into the GAP program in GCG software package (can obtain) on www.gcg.com, Biol.48:444-453) algorithm, use Blossom 62 matrixes or PAM250 matrix, and room weighting 16,14,12,10,8,6 or 4 and length weighting 1,2,3,4,5 or 6 measure two per-cent identity between aminoacid sequence.

Except the per-cent of the sequence identity above pointed out, the antibody mediated immunity cross reaction that the polypeptide that two nucleotide sequences or essentially identical another indication of polypeptide are article one nucleic acid encodings and the polypeptide for second nucleic acid encoding produce, as described below.Therefore, polypeptide is common and second polypeptide is basic identical, and for example, wherein two peptides are only because of the conservative difference of replacing.Article two, essentially identical another indication of nucleotide sequence is two molecules or the hybridization each other under stringent condition of its complementary sequence, as described below.Article two, the essentially identical another indication of nucleotide sequence is this sequence that can increase with identical primer.

Phrase " nucleic acid " is used interchangeably with term " polynucleotide " herein, and refers to deoxyribonucleotide or ribonucleotide and the polymkeric substance thereof of strand or double chain form.The framework residue that contains known nucleotide analog or modification or the nucleic acid of key contained in term, it is for that synthesize, naturally occurring and non-natural existence, it has similar binding property to reference nucleic acid, and it carries out metabolism in the mode similar to reference nucleotide.The example of this type of analogue includes but not limited to thiophosphatephosphorothioate, phosphoramidate, methylphosphonate, chirality-methylphosphonate, 2-O-methyl ribonucleotides, peptide nucleic acid(PNA) (PNA).

Unless otherwise noted, specific nucleic acid sequence is also contained its conservative variant (for example degenerate codon replacement) and complementary sequence of modifying in the dark, and the sequence explicitly pointing out.Especially, as described in detail, can complete degenerate codon and replace by producing such sequence, wherein with mixed base and/or Hypoxanthine deoxyriboside residue, replace the 3rd position (Batzer etc., (1991) Nucleic Acid Res.19:5081 of one or more selected (or all) codons; Ohtsuka etc., (1985) J.Biol.Chem.260:2605-2608; With Rossolini etc., (1994) Mol.Cell.Probes 8:91-98).

Phrase " effectively connect " refers to the functional relationship of two and more polynucleotide (for example DNA) section.Conventionally, it refers to the functional relationship between transcriptional regulatory sequences and transcription sequence.For example, if promotor or enhancer sequence stimulate or regulate encoding sequence transcribing in suitable host cell or other expression systems, this promotor or enhancer sequence are effectively connected to encoding sequence so.Usually, the promoter transcription that is effectively connected to transcription sequence regulates sequence and transcription sequence contiguous in physical space, and they are cis actings.Yet some transcriptional regulatory sequences, as enhanser does not need with encoding sequence (enhanser strengthens it and transcribes) contiguous or be positioned near it in physical space.

Term " polypeptide " and " protein " are used interchangeably herein to refer to the polymkeric substance of amino-acid residue.Term is applicable to aminoacid polymers, one of them or more amino acids residue be corresponding naturally occurring amino acid whose artificial chemistry stand-in, be also applicable to the aminoacid polymers that naturally occurring aminoacid polymers and non-natural exist.Unless otherwise noted, specific peptide sequence is also contained its conservative variant of modifying in the dark.

Term used herein " individuality " comprises people and non-human animal.Non-human animal comprises all vertebratess, and for example Mammals and nonmammalian, as non-human primates, sheep, dog, milk cow, chicken, Amphibians and Reptilia.When pointing out, term " patient " or " individuality " are used interchangeably herein.

Term used herein " carcinostatic agent " refers to can be used for treating any medicine of cell proliferation disorders (as cancer), comprises cytotoxic agent, chemotherapeutics, radiotherapy and radiotherapy dose, target anticancer agent and immunotherapeutic agent.

Term used herein " tumour " refers to no matter be pernicious or optimum neoplastic cell growth and propagation, and all cancerations are front and cell and the tissue of canceration.

Term used herein " anti-tumor activity " refers to the reduction of tumor cell proliferation speed, vigor or transfer activity.The possible mode that shows anti-tumor activity is to be presented at the abnormal cell growth speed that occurs during treatment or decline or the reduction of tumor size stability.This activity can be used generally acknowledged external or in-vivo tumour model evaluation, includes but not limited to xenograft models, allograft model, MMTV model and other known models for Effect of Anti tumor promotion well known in the art.

Term used herein " malignant tumour " refers to non-benign tumour or cancer.

Term used herein " cancer " refers to be characterized by the malignant tumour of imbalance or Growth of Cells out of control.Exemplary cancer comprises: cancer, sarcoma, leukemia and lymphatic cancer.Term " cancer " comprises that primary malignant tumor (for example, cell wherein does not move to the malignant tumour at the position beyond the original tumor locus in individual body) and second malignant neoplasm is (for example, the malignant tumour producing by transfer, tumor cell migration is to the secondary position different from original tumor locus).

Following chapters and sections and segmentation describe in more detail in chapters and sections of the present invention multiple aspect.

The structure of HER acceptor and activation mechanism

The structural domain that all 4 kinds of HER acceptors all have the outer ligand binding domains of born of the same parents, single membrane spaning domain and contain tenuigenin Tyrosylprotein kinase.Tyrosine kinase domain high conservative in the born of the same parents of HER acceptor, although the replacement that the kinase domain of HER3 contains key amino acid and therefore lack kinase activity (people such as Guy, (1994): PNAS 91,8132-8136).The dimerization inducible kinase of the HER acceptor of part induction activates, the acceptor transphosphorylation on the tyrosine residues in C-end afterbody and intracellular signal are subsequently provided effector raising and activating (Yarden and Sliwkowski, (2001) Nature Rev 2,127-137; The people such as Jorissen, (2003) Exp Cell Res 284,31-53.

The crystalline structure of the ectodomain of HER provide the receptor activation process of some parts inductions information (Schlessinger, (2002) Cell 110,669-672).The ectodomain of every kind of HER acceptor is comprised of 4 subdomains: subdomain I and III cooperation form ligand binding site, and subdomain II (perhaps also having subdomain IV) participates in receptor dimerization by direct acceptor-acceptor interaction.In the HER1 of binding partner structure, the dimerization ring of the beta hairpin in subdomain II (being called dimerization ring) and mating partner acceptor interacts, mediation receptor dimerization (people such as Garrett, (2002) Cell 110,763-773; The people such as Ogiso, (2002) Cell 110,775-787).On the contrary, in HER1, the HER3 of inactivation and the structure of HER4, dimerization ring participates in and the intramolecular interaction of subdomain IV, and this is at the receptor dimerization (Cho and the Leahy that have stoped there is no part in the situation that, (2002) Science 297,1330-1333; The people such as Ferguson, (2003) Mol Cell 12,541-552; The people such as Bouyan, (2005) PNAS102,15024-15029).The structure of HER2 is unique in HER.In the situation that there is no part, HER2 have with the part activated state of HER1 similarly, there is the conformation of outstanding dimerization ring, can with other HER acceptor interactions (people such as Cho, (2003) Nature 421,756-760; The people such as Garrett, (2003) Mol Cell 11,495-505).This can explain the allos dimerization ability that HER2 strengthens.

Although homology and allos dimerization that HER acceptor crystalline structure is HER acceptor provide model, but the background (people such as Franklin that some HER homologies and heterodimer are more general than other dimers, (2004) Cancer Cell 5,317-328), and the effect of each structural domain in receptor dimerizationization and self the suppress (people such as Burgess, (2003) Mol Cell 12,541-552; The people such as Mattoon, (2004) PNAS101,923-928) still not clear.

HER3 structure and epi-position

Have been described in PCT/EP2011/064407 and USSN:61/375 before the conformational epitope of anti-HER3 antibodies, in 408, the two is all submitted on August 22nd, 2011, at this, with its integral body, is incorporated herein by reference.Show the structural domain 2 that comprises HER3 and the conformational epitope of structural domain 4 with the three-dimensional structure of the HER3 of the compound clipped form of HER3 antibody fragment.

The invention provides another kind of antibody or its fragment, it is in conjunction with the non-linear epi-position in the structural domain 3 of HER3.These antibody or its fragment in conjunction with HER3 suppress ligand dependent and non-ligand dependent signal transduction the two.

The present invention also provides an antibody-like or its fragment, its in the structural domain 3-4 of HER3 in conjunction with suppress ligand dependent and non-ligand dependent signal transduction the two.In one embodiment, this antibody-like or its fragment in conjunction with the structural domain 3 of HER3 or structural domain 4 suppress ligand dependent and non-ligand dependent signal transduction the two.In another embodiment, this antibody-like or its fragment in conjunction with the structural domain 3 of HER3 and structural domain 4 suppress ligand dependent and non-ligand dependent signal transduction the two.

The present embodiment is presented on the crystalline structure of the HER3 that Fab fragment resolving power determination and MOR12604 is combined.

The three-dimensional structure that has shown the clipped form (residue 20-640) of the ectodomain of the HER3 compound with antibody. resolving power determination HER3-MOR12604Fab mixture, and be presented in Fig. 4.

Although unnecessary, provide theoretical, a kind of possible mechanism of action model is that HER3 exists with inactivation (sealing, constraint) or active (opening) state conventionally.Ligand binding has been induced conformational change, makes HER3 existing in conjunction with activity (opening) state of heterodimer mating partner, the activation that causes downstream signal to be provided.Such as inactivation (constraint) state of the antibodies HER3 of MOR12604, and obviously sealed ligand binding site.

The combination of MOR12604 in structural domain 3 show, MOR12604 can play a role by being selected from following mechanism: the required HER3 residue of sealing ligand binding; Because stoping HER3, the steric hindrance between antibody and HER3 structural domain 3 adopts activity conformation; By the flexible degree reducing in HER3 hinge area (structural domain 3), stop HER3 to adopt activity conformation; In structural domain 3 ring 371-377, induce conformational change, this conformational change prevents that HER3 from changing open conformation into; Stabilization removal HER3, easily degrades it; As partial agonist, play a role to accelerate the downward of HER3; And every arm by MOR12604 is in conjunction with a part HER3, makes antibody produce non-natural HER3 dimer, this dimer be easy to proteolysis degraded or can not with other receptor tyrosine kinase dimerizations.

In order to check in conjunction with structural domain 3 antibody of HER3 or the crystalline structure of its fragment, by express the nucleotide sequence of coding HER3 or its variant in suitable host cell, then under the existence of the Fab of relevant HER3 target, make the crystallization of protein of purifying, the crystal of preparation HER3.Preferably, this HER3 polypeptide contains ectodomain (amino acid 20 to 640 or its clipped form of human polypeptides (SEQ ID NO:1), preferably comprise amino acid 20-640), but lacks membrane spaning domain and born of the same parents' intracellular domain.

Also HER3 polypeptide can be produced as to fusion rotein, for example, be convenient to extract and purifying.The example of fusion rotein mating partner comprises glutathione-S-transferase (GST), Histidine (HIS), hexahistine (6HIS), GAL4 (DNA combination and/or transcriptional activation domains) and beta-galactosidase enzymes.Also can between fusion rotein mating partner and target protein matter sequence, add proteolysis cleavage site easily, to allow to remove fusion rotein sequence.

After expression, can purifying and/or proteins concentrate, for example, by immobilized metal affinity chromatography, ion exchange chromatography and/or gel-filtration.

Available technology crystallization of protein described herein.Conventionally, in crystallisation process, the drop that contains protein soln is mixed with crystallization damping fluid, and allow balance in the container of sealing.Can by known technology for example " hanging drop " or " sit drip " method realize balance.In these methods, drop is suspended on much more top, crystallization damping fluid storehouse or is seated its side, by vapor diffusion, reaches balance.Alternatively, can reach balance by additive method, for example, below oil, by semipermeable partition, or spread (for example seeing the people such as Chayen, (2008) Nature Methods 5,147 – 153) by free interface.

Once obtain crystal, can be by known X-ray diffraction technology analytic structure.The crystal of many utilization chemically modifieds, as derived the crystal of modifying mutually for approximate by heavy atom.In fact, for example, in the solution that contains heavy metal atom salt or organometallic compound (lead chloride, mercaptosuccinic acid gold, Thiomersalate or uranyl acetate), soak crystal, this heavy metal atom salt or organometallic compound can diffuse through crystal and be combined with protein surface.Then can be by the X-ray diffraction analysis of the crystal soaking being measured to the position of the heavy metal atom of combination.Can resolve the spectrum that crystal atoms (scattering center) diffraction X ray homogeneous beam obtains by math equation, to obtain mathematical coordinates.With diffraction data, calculate the electron density map of crystal repeating unit.The another kind of method that obtains phase information is to use the technology that molecule is replaced that is called.In this method, to the retrieval model application rotation and the translation algorithm that obtain from dependency structure, obtain the approximate location (seeing Rossmann, (1990) Acta Crystals A46,73-82) of target protein matter.With electron density map, determine the position (people such as Blundel, (1976) Protein Crystallography, Academic Press) of each atom in the structure cell of crystal.

The disclosure has been described the three-dimensional structure of the Fab of HER3 and anti-HER3 antibody.The border, rough structure territory of HER3 ectodomain is as follows: structural domain 1: amino acid 20-207; Structural domain 2: amino acid 208-328; Structural domain 3: amino acid 329-498; With structural domain 4: amino acid 499-642.The three-dimensional structure of HER3 and antibody allows to identify the target combining site of potential HER3 instrumentality.Preferred target combining site relates to the target combining site that HER3 activates.In one embodiment, target combining site is positioned at the structural domain 3 of HER3.Therefore, can be for example by change structure territory, with respect to the relative position of himself or other HER3 structural domains, regulate HER3 to activate with the antibody of structural domain 3 combinations or its fragment.Therefore, the combination of the amino-acid residue in antibody or its fragment and structural domain 3 can cause protein to adopt and stop the conformation activating.

In some embodiments, antibody or its fragment are in conjunction with the mating surface of HER3.A plurality of continuous or discrete surface that this mating surface comprises the part of formation and antibody or the interactional epi-position of its fragment in 3D configuration.For example, mating surface can comprise at least two surfaces (for example surface A and surperficial B, see Fig. 4 D), at least three surfaces (surface A for example, surface B and surface C), at least four surfaces (surface A for example, surface B, surface C and surperficial D), at least five surfaces (surface A for example, surface B, surface C, surface D and surperficial E), at least six surfaces (surface A for example, surface B, surface C, surface D, surface E and surperficial F), at least seven surfaces (surface A for example, surface B, surface C, surface D, surface E, surface F and surperficial G), at least eight surfaces (surface A for example, surface B, surface C, surface D, surface E, surface F, surface G and surperficial H), at least nine surfaces (surface A for example, surface B, surface C, surface D, surface E, surface F, surface G, surface H and surperficial I) or at least ten surfaces (surface A for example, surface B, surface C, surface D, surface E, surface F, surface G, surface H, surface I and surperficial J).

In one embodiment, antibody or its fragment are combined with mating surface A.In one embodiment, antibody or its fragment are combined with mating surface B.In another embodiment, antibody or its fragment and mating surface A and the two combination of mating surface B.In one embodiment, mating surface A comprises the amino-acid residue that at least one is selected from amino-acid residue 362-376.In one embodiment, mating surface B comprises the amino-acid residue that at least one is selected from amino-acid residue 335-342,398,400,424-428,431,433-434 and 455.In another embodiment, antibody or its fragment with below in conjunction with surface bonding: mating surface A, wherein at least one amino-acid residue is selected from amino-acid residue 362-376; Mating surface B, wherein at least one amino-acid residue is selected from amino-acid residue 335-342,398,400,424-428,431,433-434 and 455.

In some embodiments, antibody or its fragment and the people HER3 protein binding with the non-linear epi-position of the HER3 amino-acid residue 335-342,362-376,398,400,424-428,431,433-434 and 455 (in structural domain 3) or its subset that comprise SEQ ID NO:1.In some embodiments, within amino-acid residue 335-342,362-376,398,400,424-428,431,433-434 and 455 (in structural domain 3) or its subset of antibody or its fragment and SEQ ID NO:1 or with it overlapping amino acid is combined.In some embodiments, the amino acid within amino acid 335-342, the 362-376,398,400 of antibody or its fragment and SEQ ID NO:1,424-428,431,433-434 and 455 (in structural domain 3) or its subset (and/or consisting of aminoacid sequence) combination.

In some embodiments, antibody or its fragment and the people HER3 protein binding with the epi-position (linear, non-linear, conformation) of the HER3 amino-acid residue 499-642 (structural domain 4) that comprises SEQ ID NO:1 or its subset.In some embodiments, antibody or its fragment within the amino-acid residue 499-642 of SEQ ID NO:1 (structural domain 4) or its subset or with it overlapping amino acid be combined.In some embodiments, the amino-acid residue 499-642 (structural domain 4) of antibody or its fragment and SEQ ID NO:1 or the amino acid within its subset (and/or consisting of aminoacid sequence) combination.

In some embodiments, antibody or its fragment and people HER3 protein binding, this HER3 albumen has the non-linear epi-position of the HER3 amino-acid residue 335-342,362-376,398,400,424-428,431,433-434 and 455 (in structural domain 3) or its subset that comprise SEQ ID NO:1, and the epi-position (linear, non-linear, conformation) of the HER3 amino-acid residue 499-642 (structural domain 4) that comprises SEQ ID NO:1 or its subset.In some embodiments, within amino-acid residue 335-342, the 362-376,398,400 of antibody or its fragment and SEQ ID NO:1,424-428,431,433-434 and 455 (in structural domain 3) or with it overlapping amino acid and epi-position (linear, non-linear, the conformation) combination of the HER3 amino-acid residue 499-642 (structural domain 4) that comprises SEQ ID NO:1 or its subset.In some embodiments, the amino acid within amino acid 335-342, the 362-376,398,400 of antibody or its fragment and SEQ ID NO:1,424-428,431,433-434 and 455 (in structural domain 3) or its subset (and/or consisting of aminoacid sequence) and the amino-acid residue 499-642 (structural domain 4) that comprises SEQ ID NO:1 or epi-position (linear, non-linear, the conformation) combination of its subset.

In some embodiments, antibody or its fragment and people HER3 protein binding, the HER3 amino-acid residue in the structural domain 4 that this people HER3 albumen has the epi-position (linear, non-linear, conformation) of HER3 amino-acid residue in the structural domain 3 that comprises SEQ ID NO:1 or its subset and comprises SEQ ID NO:1 or the epi-position (linear, non-linear, conformation) of its subset.In some embodiments, antibody or its fragment within the amino-acid residue of the structural domain 3 of SEQ ID NO:1 and the amino acid of structural domain 4 or its subset or with it overlapping amino acid be combined.

In some embodiments, antibody or its fragment are combined with the inactivation state of HER3 acceptor, thereby stop HER3 to adopt activity conformation.In some embodiments, antibody or its fragment stop HER3 to adopt activity conformation due to the steric hindrance between antibody or its fragment and the structural domain of HER3 (for example,, for MOR12604, with the spatial interference of the structural domain 1 of HER3).In some embodiments, antibody or its fragment stop HER3 to adopt activity conformation by the flexible degree reducing in structural domain 3.In some embodiments, the conformational change in the structural domain 3 ring 371-377 of antibody or its fragment induction SEQ ID NO:1, this conformational change stops HER3 to adopt activity conformation.In some embodiments, antibody or its fragment stabilization removal HER3, make it be easy to degraded.In some embodiments, antibody or its fragment are accelerated the downward of cell surface HER3.In some embodiments, antibody or its fragment produce non-natural HER3 dimer, this dimer be easy to proteolysis degraded or can not with other receptor tyrosine kinase dimerizations.

In some embodiments, antibody or its fragment can be in conjunction with activated state or the inactivation states of HER3.In some embodiments, antibody or its fragment stabilization, in the HER3 of inactivation state acceptor, make HER3 acceptor fail to form acceptor-receptor complex with coreceptor dimerization.The failure that forms acceptor-receptor complex has stoped the two the activation of ligand dependent and non-ligand dependent signal transduction.

In some embodiments, the dimerization of antibody or its fragment induction HER3 and HER3, to form the acceptor-receptor complex of inactivation.The formation of the acceptor-receptor complex of inactivation has stoped the activation of the signal transduction of HER3 mediation, because HER3 is hidden in the acceptor-receptor complex of inactivation.

Shown in structure also allow to identify the specificity HER3 amino-acid residue at antibody or its fragment (for example MOR12604) and the interaction interface of HER3.This is defined as MOR12604 albumen VH chain interior residue.This residue is as follows: Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400 and Lys434.Shown in structure also allow to identify the specificity HER3 amino-acid residue at antibody or its fragment (for example MOR12604) and the interaction interface of HER3.This is defined as MOR12604 albumen VL chain interior residue.This residue is as follows: Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455.As visible in table 5 and 6 (MOR12604) respectively, light chain and heavy chain all relate to the combination of the amino-acid residue in the structural domain 3 of antigen-binding proteins and epi-position.

Similarly, consider current instruction, measurable which residue and the region that changes antigen-binding proteins of those skilled in the art, and not excessive interference antigen-binding proteins in conjunction with the structural domain 3 of HER3 and 4 ability.

Measured core interaction interface amino acid, there is distance H ER3 mating partner albumen and be less than or equal to all amino-acid residues of at least one atom.Select as core area cut-off distance, to allow atom within a van der Waals radius adds the hydrogen bond of possible water mediation.

In some embodiments, in conjunction with, shelter or stop MOR012604 and the interactional any antigen-binding proteins of any above-mentioned residue can be used in conjunction with or in and HER3.In some embodiments, antibody or its fragment in conjunction with at least one following HER3 residue (SEQ ID NO:1) or with its interaction: Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400 and Lys434.In some embodiments, antibody and fragment thereof in conjunction with at least one following HER3 residue (SEQ ID NO:1) or with its interaction: Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455.In some embodiments, antibody and fragment thereof in conjunction with at least one following HER3 residue (SEQ ID NO:1) or with its interaction: Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400, Lys434, Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455.In some embodiments, antibody and fragment thereof in conjunction with at least one following HER3 residue (SEQ ID NO:1) or with its interaction: Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400, Lys434, Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455.In some embodiments, antibody or its fragment in conjunction with all following HER3 residues (SEQ ID NO:1) or with its interaction: Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400, Lys434, Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455.In some embodiments, antibody or its fragment are in 5 dusts of one or more above-mentioned residues.In some embodiments, antibody or its fragment are in 5 to 8 dust distances of one or more above-mentioned residues.In some embodiments, antibody or its fragment and 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,20,25,30,35,40,45, or 50 above-mentioned residues interact, seal this residue or in 8 dusts of this residue.

For example, the acquisition of the 3D structure of HER3 and HER3:MOR12604 mixture provides framework for exploring in more detail other HER3 antibody.The 3D structure of HER3 allows the epi-position of monoclonal antibody map and infer its binding mode, because some cell growth inhibiting, some stimulate cell growth, and the not impact of other cell growth.The non-linear epi-position of MOR12604 has been located in the structural domain 3 of HER3.The acquisition of the 3D structure of this acceptor is measured the accurate mechanism of action of these inhibitor and designs new method and disturb HER3 function of receptors being convenient to.In one embodiment, the antibodies of the present invention non-linear epi-position identical with MOR12604.

The non-linear epi-position of any antibody institute combination of listing in table 1 in some embodiments, is particularly useful.In certain embodiments, the non-linear epi-position of HER3 can be used for antibody or its fragment of separation and combination HER3.In certain embodiments, the non-linear epi-position of HER3 can be used for producing antibody or its fragment in conjunction with HER3.In certain embodiments, the non-linear epi-position of HER3 can be used as original antibody or its fragment producing in conjunction with the non-linear epi-position of HER3 of immunity.In certain embodiments, can use the non-linear epi-position of HER3 to animal, can from animal, obtain the antibody in conjunction with HER3 subsequently.

The present invention also provides an antibody-like, and it is in conjunction with the epi-position in the structural domain 3-4 of HER3 (linear, non-linear or conformation).In table 2, show this type of antibody of combination or example of its fragment in structural domain 3-4.Can with aforesaid method and below the method described in embodiment chapters and sections produce the structural domain 3-4 antibody compound with HER3 or its fragment.

In some embodiments, can for example, by the specific residue in sudden change HER3 (wild-type antigen) and measure antibody or whether its fragment can, in conjunction with the HER3 albumen of sudden change or variant HER3 albumen or the variation of measuring the relative wild-type of avidity, identify structural domain/region of containing the residue that contacts with antibody or hidden by antibody.By producing many single mutation, can identifying, in combination, play direct acting residue, thereby or enough closely make sudden change can affect the residue of the combination between antibody and antigen apart from antibody.By these amino acid whose information, can illustrate antigen (HER3) structural domain/region of containing the residue that contacts with antibody or sheltered by antibody.The mutagenesis of the known technology of use such as Alanine-scanning can help epi-position relevant on defined function.Also can apply the mutagenesis of use arginine/L-glutamic acid sweeping scheme (for example sees, the people such as Nanevicz, (1995), J.Biol.Chem.270 (37): the people such as 21619-21625 and Zupnick, (2006), J.Biol.Chem.281 (29): 20464-20473).Generally speaking, arginine and L-glutamic acid are replaced the amino acid in (generally individually) wild type peptide, because these amino acid be charged and volume large, therefore there are the potentiality of destroying the combination between antigen-binding proteins and antigen in the antigen region of introducing sudden change.The arginine existing in wild-type antigen replaces with L-glutamic acid.Can obtain multiple such single mutant, and which residue is the combination result of analyze collecting measure and affect combination.Can produce a series of mutant HER3 antigen, each mutant antigen has single mutation.Can measure the combination of each mutant HER3 antigen and multiple HER3 antibody or its fragment and be combined the ability comparison of wild-type HER3 (SEQ ID NO:1) with selected antibody or its fragment.

The change of the combination between antibody used herein or its fragment and mutant or variant HER3 (for example reduce or increase) means binding affinity (for example, detecting or below the mensuration based on ball described in embodiment is measured as Biacore by currently known methods), EC ₅₀variation, and/or the total binding ability of antigen-binding proteins (for example, by antigen-binding proteins concentration to the B in the figure of antigen concentration _maxminimizing prove) variation (for example reducing).In conjunction with the residue of remarkable change indication sudden change relate to the combination with antibody or its fragment.

In some embodiments, in conjunction with remarkable minimizing for example mean, with respect to the combination between antibody or its fragment and wild-type HER3 (, SEQ ID NO:1), binding affinity, EC between antibody or its fragment and mutant HER3 antigen ₅₀and/or ability reduce to surpass 10%, surpasses 20%, surpasses 40%, surpasses 50%, surpasses 55%, surpasses 60%, surpasses 65%, surpasses 70%, surpasses 75%, surpasses 80%, surpasses 85%, surpasses 90% or surpass 95%.

In some embodiments, with wild-type HER3 albumen (for example, SEQ ID NO:1) compare, the combination of the mutant HER3 albumen of antibody or its fragment one or more to having (for example, 1,2,3,4,5,6,7,8,9,10 or more) sudden change significantly reduces or increases.

Although variant form is with reference to the wild-type sequence shown in SEQ ID NO:1, should be understood that amino acid can be different in the allele variant of HER3 or splice variant.Also consider such antibody or its fragment, its demonstration significantly changes (for example lower or higher combination) to the combination of the allelic form of this type of HER3.

Except the general structure aspect of antibody, by structural approach, can study the more special interaction between paratope and epi-position.In one embodiment, the structure of CDR is facilitated paratope, by this paratope antibody, can be combined with epi-position.Available various ways is measured the shape of this type of paratope.Can use traditional structure inspection method, as NMR or X-ray crystallography.These methods can be studied the shape of independent paratope, or at paratope the shape when epi-position is combined.Alternatively, can generate on computers molecular model.Can be by for example, carrying out generating structure with the auxiliary homology modeling of commercial packages (from Accelrys (San Diego, Calif.) InsightII modeling software bag).In brief, the antibody sequence of available examine is retrieved for the database (as Protein Data Bank) with the protein of known structure.After identifying the homologous protein with known structure, with these homologous proteins as modeling template.Can compare each possible template, thereby produce the sequence alignment based on structure between template.Then antibody sequence and these templates with unknown structure can be compared, to generate the molecular model of the antibody with unknown structure.It will be appreciated by those skilled in the art that to exist manyly for generating on computers the alternative approach of this class formation, can use wherein any.For example, can use the method described in the people such as similar Hardman, it is with U.S. Patent number 5,958,708 issues, application QUANTA (Polygen Corp., Waltham, Mass.) and CHARM (people such as Brooks, (1983), J.Comp.Chem.4:187) (are incorporated herein by reference with its integral body at this).

Not only the shape of paratope is to determining that whether possible paratope is in connection with epi-position and how well very important in conjunction with having, and the interaction between epi-position and paratope is from providing bulk information source as design variable antibody.It will be appreciated by those skilled in the art that various ways can study this interaction.A kind of mode is to use the structural models (perhaps as above-mentioned) generating, then use for example InsightII (Accelrys, San Diego, Calif.) program, this program has connection module, particularly can in the conformation between paratope and its epi-position and directional space, carry out Monte Carlo retrieval.Result is can estimate epi-position and the interactional position of paratope and how to interact.In one embodiment, only with fragment or the variant of epi-position, help determine relevant interaction.In one embodiment, in the interactional modeling between paratope and epi-position, use whole epi-position.

Which by using these model structures, can predict in the interaction of residue between epi-position and paratope most important.Therefore, in one embodiment, can easily select to change which residue to change the combination feature of antibody.For example, apparent from docking model, in paratope, the side chain of some residue can spatially hinder the combination of epi-position, and it can be favourable therefore these residues being changed into the residue with smaller side chain.Can determine this point by many modes.For example, can observe simply 2 models and estimate to interact based on functional group and proximity.Alternatively, can be as the above-mentioned pairing that repeats of carrying out epi-position and paratope, to obtain more favourable energy interaction.Also can measure the interaction to Multiple Antibodies variant, to determine that antibody can be in which kind of alternative mode in conjunction with epi-position.Also multiple model capable of being combined determines should how to change antibody structure to obtain having the antibody of the special characteristic of expectation.

Can test definite model above by multiple technologies.For example, available program determination interaction energy discussed above, to determine which variant of further inspection.In addition, with coulomb and Van der Waals, interact to measure the interaction energy of epi-position and variant paratope.Also with site-directed mutagenesis, come the change of the antibody structure of observation post's prediction whether really to cause the change of the combination feature of expectation.Alternatively, can change to epi-position that to carry out verification model be correct, or measure between paratope and epi-position, can exist generally in conjunction with theme.

Although it will be appreciated by those skilled in the art that these models will provide antibody and the necessary guidance of variant thereof of preparation the present embodiment, but still wish computer model to carry out conventionally test, perhaps by vitro study.In addition, it will be apparent to those skilled in the art ground, modify arbitrarily the also activity of possibility antagonist and there is extra side effect.For example, although prediction causes any change of stronger combination can induce stronger combination, it also can cause other structural modifications, and this structural modification may reduce or change antibody activity.Determine whether it is so the routine of this area, and available various ways reaches.For example, can test activity by ELISA.Alternatively, can be by testing sample with surperficial plasmon resonance equipment.

HER3 antibody

The invention provides the antibody-like shown in table 1, the non-linear epi-position in the structural domain 3 of its identification HER3, and suppress ligand dependent and non-ligand dependent HER3 signal transduction pathway the two.The present invention also provides the antibody-like shown in table 2, the epi-position (linear, non-linear, conformation) in the structural domain 3-4 of its identification HER3, and suppress ligand dependent and non-ligand dependent HER3 signal transduction pathway the two.

Table 1: in conjunction with the example of the HER3 antibody of the non-linear epi-position in the structural domain 3 of HER3.

Table 2: in conjunction with the example of the amino acid whose HER3 antibody in the structural domain 3-4 of HER3.

On the one hand, (for example the invention provides specific binding HER3 albumen, people and/or cynomolgus monkey HER3) the antibody of structural domain 3, this antibody comprise there is SEQ ID NO:14, the VH structural domain of 34,54,74,94,114,134,154 and 174 aminoacid sequence.On the other hand, (for example the invention provides specific binding HER3 albumen, people and/or cynomolgus monkey HER3) the antibody of structural domain 3, this antibody comprise there is SEQ ID NO:15, the VL structural domain of 35,55,75,95,115,135,155 and 175 aminoacid sequence.

On the one hand, (for example the invention provides specific binding HER3 albumen, people and/or cynomolgus monkey HER3) the antibody of structural domain 3-4, this antibody comprise there is SEQ ID NO:194, the VH structural domain of 214,234,254,274,294,314,334,354,374,394,414,434,454,474,494,514,534,554,574,594,614,634,654,674 and 694 aminoacid sequence.On the other hand, (for example the invention provides specific binding HER3 albumen, people and/or cynomolgus monkey HER3) the antibody of structural domain 3, this antibody comprise there is SEQ ID NO:195, the VL structural domain of 215,235,255,275,295,315,335,355,375,395,415,435,455,475,495,515,535,555,575,595,615,635,655,675 and 695 aminoacid sequence.

Because each in these antibody or its fragment can, in conjunction with HER3, can " be mixed and mate " VH, VL, full-length light chains and total length sequence of heavy chain (nucleotide sequence of aminoacid sequence and encoding amino acid sequence) and produce other HER3 antibody of the present invention.Available combination well known in the art measure (for example ELISA and in embodiment chapters and sections, describe other measure) test the HER3 antibody that this type of " mixes and coupling ".When mixing and mating these chains, should replace the VH sequence from concrete VH/VL pairing by the VH sequence of structural similitude.Similarly, should replace the total length sequence of heavy chain from the pairing of concrete total length heavy chain/full-length light chains with the total length sequence of heavy chain of structural similitude.Similarly, should replace the VL sequence from concrete VH/VL pairing by the VL sequence of structural similitude.Similarly, should replace the full-length light chains sequence from the pairing of concrete total length heavy chain/full-length light chains by the full-length light chains sequence of structural similitude.

Therefore, on the one hand, the invention provides separated monoclonal antibody or its fragment, it has: comprise the variable region of heavy chain that is selected from SEQ ID NO:14,34,54,74,94,114,134,154 and 174 aminoacid sequence; With comprise the variable region of light chain that is selected from SEQ ID NOs:15,35,55,75,95,115,135,155 and 175 aminoacid sequence; Wherein this antibodies specific for example, in conjunction with the structural domain 3 of HER3 (, people and/or cynomolgus monkey).

Therefore, on the one hand, the invention provides separated monoclonal antibody or its fragment, it has: comprise the variable region of heavy chain that is selected from SEQ ID NO:194,214,234,254,274,294,314,334,354,374,394,414,434,454,474,494,514,534,554,574,594,614,634,654,674 and 694 aminoacid sequence; With comprise the variable region of light chain that is selected from SEQ ID NO:195,215,235,255,275,295,315,335,355,375,395,415,435,455,475,495,515,535,555,575,595,615,635,655,675 and 695 aminoacid sequence; Wherein this antibodies specific for example, in conjunction with the structural domain 3 of HER3 (, people and/or cynomolgus monkey).

In specific embodiments, the VH that comprises SEQ ID NO:14 in conjunction with the antibody of the structural domain 3 of HER3 and the VL of SEQ ID NO:15.In specific embodiments, the VH that comprises SEQ ID NO:34 in conjunction with the antibody of the structural domain 3 of HER3 and the VL of SEQ ID NO:35.In specific embodiments, the VH that comprises SEQ ID NO:54 in conjunction with the antibody of the structural domain 3 of HER3 and the VL of SEQ ID NO:55.In specific embodiments, the VH that comprises SEQ ID NO:74 in conjunction with the antibody of the structural domain 3 of HER3 and the VL of SEQ ID NO:75.In specific embodiments, the VH that comprises SEQ ID NO:94 in conjunction with the antibody of the structural domain 3 of HER3 and the VL of SEQ ID NO:95.In specific embodiments, the VH that comprises SEQ ID NO:114 in conjunction with the antibody of the structural domain 3 of HER3 and the VL of SEQ ID NO:115.In specific embodiments, the VH that comprises SEQ ID NO:134 in conjunction with the antibody of the structural domain 3 of HER3 and the VL of SEQ ID NO:135.In specific embodiments, the VH that comprises SEQ ID NO:154 in conjunction with the antibody of the structural domain 3 of HER3 and the VL of SEQ ID NO:155.In specific embodiments, the VH that comprises SEQ ID NO:174 in conjunction with the antibody of the structural domain 3 of HER3 and the VL of SEQ ID NO:175.

In specific embodiments, the VH that comprises SEQ ID NO:194 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:195.In specific embodiments, the VH that comprises SEQ ID NO:214 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:215.In specific embodiments, the VH that comprises SEQ ID NO:234 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:235.In specific embodiments, the VH that comprises SEQ ID NO:254 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:255.

In specific embodiments, the VH that comprises SEQ ID NO:274 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:275.In specific embodiments, the VH that comprises SEQ ID NO:294 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:295.In specific embodiments, the VH that comprises SEQ ID NO:314 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:315.In specific embodiments, the VH that comprises SEQ ID NO:334 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:335.In specific embodiments, the VH that comprises SEQ ID NO:354 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:355.In specific embodiments, the VH that comprises SEQ ID NO:374 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:375.In specific embodiments, the VH that comprises SEQ ID NO:394 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:395.In specific embodiments, the VH that comprises SEQ ID NO:414 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:415.In specific embodiments, the VH that comprises SEQ ID NO:434 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:435.In specific embodiments, the VH that comprises SEQ ID NO:454 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:255.In specific embodiments, the VH that comprises SEQ ID NO:474 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:475.In specific embodiments, the VH that comprises SEQ ID NO:494 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:495.In specific embodiments, the VH that comprises SEQ ID NO:514 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:515.In specific embodiments, the VH that comprises SEQ ID NO:534 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:535.In specific embodiments, the VH that comprises SEQ ID NO:554 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:555.In specific embodiments, the VH that comprises SEQ ID NO:574 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:575.In specific embodiments, the VH that comprises SEQ ID NO:594 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:595.In specific embodiments, the VH that comprises SEQ ID NO:614 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:615.In specific embodiments, the VH that comprises SEQ ID NO:634 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:635.In specific embodiments, the VH that comprises SEQ ID NO:654 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:655.In specific embodiments, the VH that comprises SEQ ID NO:674 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:675.In specific embodiments, the VH that comprises SEQ ID NO:694 in conjunction with the antibody of the structural domain 3-4 of HER3 and the VL of SEQ ID NO:695.

On the other hand, the invention provides the HER3 antibody of binding domains 3, it comprises heavy chain and light chain CDR1, CDR2 and CDR3 described in table 1, or its combination.The aminoacid sequence of the VH CDR1 of antibody shows in SEQ ID NO:2,22,42,62,82,102,122,142 and 162.The aminoacid sequence of the VH CDR2 of antibody shows in SEQ ID NO:3,23,43,63,83,103,123,143 and 163.The aminoacid sequence of the VH CDR3 of antibody shows in SEQ ID NO:4,24,44,64,84,104,124,144 and 164.The aminoacid sequence of the VL CDR1 of antibody shows in SEQ ID NO:8,28,48,68,88,108,128,148 and 168.The aminoacid sequence of the VL CDR2 of antibody shows in SEQ ID NO:9,29,49,69,89,109,129,149 and 169.The aminoacid sequence of the VL CDR3 of antibody shows in SEQ ID NO:10,30,50,70,90,110,130,150 and 170.With Kabat system (people such as Kabat, (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH publication number 91-3242; The people such as Chothia, (1987) J.Mol.Biol.196:901-917; The people such as Chothia, (1989) Nature 342:877-883; With the people such as Al-Lazikani, (1997) J.Mol.Biol.273,927-948) CDR district described.

In specific embodiments, in conjunction with the antibody of the structural domain 3 of HER3, comprise: the variable region of heavy chain CDR1 of SEQ ID NO:142; The variable region of heavy chain CDR2 of SEQ ID NO:143; The variable region of heavy chain CDR3 of SEQ ID NO:144; The variable region of light chain CDR1 of SEQ ID NO:148; The variable region of light chain CDR2 of SEQ ID NO:149; Variable region of light chain CDR3 with SEQ ID NO:150.

In specific embodiments, in conjunction with the antibody of the structural domain 3 of HER3, comprise: the variable region of heavy chain CDR1 of SEQ ID NO:162; The variable region of heavy chain CDR2 of SEQ ID NO:163; The variable region of heavy chain CDR3 of SEQ ID NO:164; The variable region of light chain CDR1 of SEQ ID NO:168; The variable region of light chain CDR2 of SEQ ID NO:169; Variable region of light chain CDR3 with SEQ ID NO:170.

On the other hand, the invention provides the HER3 antibody of binding domains 3-4, it comprises heavy chain and light chain CDR1, CDR2 and CDR3 described in table 2, or its combination.The aminoacid sequence of the VH CDR1 of antibody shows in SEQ ID NO:182,202,222,242,262,282,302,322,342,362,382,402,422,442,462,482,502,522,542,562,582,602,622,642,662 and 682.The aminoacid sequence of the VH CDR2 of antibody shows in SEQ ID NO:183,203,223,243,263,283,303,323,343,363,383,403,423,443,463,483,503,523,543,563,583,603,623,643,663 and 683.The aminoacid sequence of the VH CDR3 of antibody shows in SEQ ID NO:184,204,224,244,264,284,304,324,344,364,384,404,424,444,464,484,504,524,544,564,584,604,624,644,664 and 684.The aminoacid sequence of the VL CDR1 of antibody shows in SEQ ID NO:188,208,228,248,268,288,308,328,348,368,388,408,428,448,468,488,508,528,548,568,588,608,628,648,668 and 688.The aminoacid sequence of the VL CDR2 of antibody shows in SEQ ID NO:189,209,229,249,269,289,309,329,349,369,389,409,429,449,469,489,509,529,549,569,589,609,629,649,669 and 689.The aminoacid sequence of the VL CDR3 of antibody shows in SEQ ID NOs:190,210,230,250,270,290,310,330,350,370,390,410,430,450,470,490,510,530,550,570,590,610,630,650,670 and 690.With Kabat system (people such as Kabat, (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH publication number 91-3242; The people such as Chothia, (1987) J.Mol.Biol.196:901-917; The people such as Chothia, (1989) Nature 342:877-883; With the people such as Al-Lazikani, (1997) J.Mol.Biol.273,927-948) CDR district described.

In specific embodiments, in conjunction with the antibody of the structural domain 3-4 of HER3, comprise: the variable region of heavy chain CDR1 of SEQ ID NO:662; The variable region of heavy chain CDR2 of SEQ ID NO:663; The variable region of heavy chain CDR3 of SEQ ID NO:664; The variable region of light chain CDR1 of SEQ ID NO:668; The variable region of light chain CDR2 of SEQ ID NO:669; Variable region of light chain CDR3 with SEQ ID NO:670.

In specific embodiments, in conjunction with the antibody of the structural domain 3-4 of HER3, comprise: the variable region of heavy chain CDR1 of SEQ ID NO:682; The variable region of heavy chain CDR2 of SEQ ID NO:683; The variable region of heavy chain CDR3 of SEQ ID NO:684; The variable region of light chain CDR1 of SEQ ID NO:688; The variable region of light chain CDR2 of SEQ ID NO:689; Variable region of light chain CDR3 with SEQ ID NO:690.

Other antibody of the present invention comprise the amino acid having suddenlyd change, and in Dan CDR district, have at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with CDR district shown in sequence described in table 1 or table 2.In some embodiments, it comprises the aminoacid sequence of sudden change, wherein, when comparing with CDR district shown in sequence described in table 1 or table 2, in CDR district, suddenlyd change and be no more than 1,2,3,4 or 5 amino acid, but still maintained its specificity to original antibody epitope.

Other antibody of the present invention comprise the amino acid having suddenlyd change, but have at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the framework region shown in sequence described in table 1 or table 2 in framework region.In some embodiments, it comprises the aminoacid sequence of sudden change, wherein, when comparing with the framework region shown in sequence described in table 1 or table 2, in framework region, suddenlyd change and be no more than 1,2,3,4,5,6 or 7 amino acid, but still maintained its specificity to original antibody epitope.The present invention also provides nucleotide sequence, VH, VL, total length heavy chain and the full-length light chains of the antibody of its coding specific binding HER3 albumen (for example, people and/or cynomolgus monkey HER3).

Other antibody of the present invention comprise following antibody, wherein the nucleic acid of amino acid or coded amino acid suddenlys change, but has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with sequence described in table 1 or table 2.In some embodiments, it comprises the aminoacid sequence of sudden change, wherein when comparing with the variable region shown in sequence described in table 1 or table 2, and no more than 1,2,3,4 or 5 amino acid that suddenlyd change in variable region, but retain essentially identical therapeutic activity.

People's antibody used herein comprise " produce from " or " derived from " heavy chain or variable region of light chain or total length heavy chain or the light chain of specific germline sequence, if the variable region of antibody or total length chain are available from the system of end user's racial immunity globulin gene.This type systematic comprises with the transgenic mice of object antigen immune carrier immunoglobulin gene or the human immunoglobulin gene library of showing on phage with object antigen selection.For example can be by the relatively aminoacid sequence and the aminoacid sequence of people's germline immunoglobulin (Ig) of people's antibody, and people's germline immunoglobulin sequences of the sequence of selection and people's antibody the most close in sequence (i.e. the highest % identity) identify " generation oneself " or " derived from " people's antibody of people's germline immunoglobulin sequences." produce from " or " derived from " people's antibody of particular person racial immunity sphaeroprotein sequence can contain the amino acid difference than germline sequence, for example, by naturally occurring somatic mutation or deliberately introduce the amino acid difference that rite-directed mutagenesis causes.Yet, in VH or VL framework region, the antibody of the choosing aminoacid sequence of conventionally encoding with people's germline immunoglobulin gene on aminoacid sequence there is at least 90% identity, and contain such amino-acid residue, at the racial immunity sphaeroprotein aminoacid sequence with other species (for example, while mouse germline sequence) comparing, this amino-acid residue is behaved people's Identification of the antibodies.In some cases, people's antibody has at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the aminoacid sequence of racial immunity globulin gene coding on aminoacid sequence.Conventionally, the aminoacid sequence of recombinant human antibody and people's germline immunoglobulin gene coding has no more than 10 amino acid whose differences in VH or VL framework region.In some cases, the aminoacid sequence of people's antibody and racial immunity globulin gene coding has no more than 5, or not even more than 4,3,2 or 1 amino acid whose differences.

Antibody disclosed herein can be the derivative of single-chain antibody, double antibody, domain antibodies, nano antibody and monoclonal antibody body (unibody)." single-chain antibody " (scFv) is comprised of the wall scroll polypeptide chain that comprises the VL structural domain being connected with VH structural domain, and wherein VL structural domain and the pairing of VH structural domain form monovalent molecule.Can prepare single-chain antibody (for example seeing the people such as Bird, the people such as (1988) Science 242:423-426 and Huston, (1988) Proc.Natl.Acad.Sci.USA85:5879-5883) according to method well known in the art." double antibody " is comprised of two chains, every chain is included in variable region of heavy chain and the variable region of light chain connecting by small peptide joint on same polypeptide chain, wherein two regions on same chain are not paired with each other, but form bispecific molecule with the complementary structure territory pairing on another chain.The method of preparing double antibody is (for example seeing the people such as Holliger, the people such as (1993) Proc.Natl.Acad.Sci.USA 90:6444-6448 and Poljak, (1994) Structure 2:1121-1123) well known in the art.Domain antibodies (dAb) is little functional bonding unit, the heavy chain of corresponding antibody or the variable region of light chain of antibody.Domain antibodies is good representation in bacterium, yeast and mammalian cell system.The further details of domain antibodies and production method thereof is well known in the artly (for example to see U.S. Patent number 6,291,158; 6,582,915; 6,593,081; 6,172,197; 6,696,245; European patent 0368684 & 0616640; WO05/035572, WO04/101790, WO04/081026, WO04/058821, WO04/003019 and WO03/002609.Nano antibody is derived from the heavy chain of antibody.Nano antibody comprises single variable domains and 2 constant domain (CH2 and CH3) conventionally, and retains the antigen binding capacity of original antibody.Can prepare by means commonly known in the art nano antibody (for example see, U.S. Patent number 6,765,087, U.S. Patent number 6,838,254, WO 06/079372).Monoclonal antibody body is comprised of a light chain and a heavy chain of IgG4 antibody.Can prepare monoclonal antibody body by removing the hinge area of IgG4 antibody.The visible WO2007/059782 of further details of monoclonal antibody body and preparation method thereof.

Homologous antibody

In another embodiment, the invention provides the antibody or its fragment that comprise with the aminoacid sequence of the sequence homology described in table 1 or table 2, and this antibodies HER3 albumen (for example, people and/or cynomolgus monkey HER3), and the functional performance of the expectation of those antibody described in reservation table 1 or table 2.

For example, the invention provides the separated monoclonal antibody (or its function fragment) that comprises variable region of heavy chain and variable region of light chain, wherein this variable region of heavy chain comprises and is selected from SEQ ID NO:14,34,54,74,94,114,134,154 and 174 aminoacid sequence at least 80%, 90%, 95%, 96%, 97%, 98% or 99% same aminoacid sequence; This variable region of light chain comprises and is selected from SEQ ID NO:15,35,55,75,95,115,135,155 and 175 aminoacid sequence at least 80%, 90%, 95%, 96%, 97%, 98% or 99% same aminoacid sequence; Wherein this antibodies HER3 (for example, people and/or cynomolgus monkey HER3) structural domain 3, and the signal that suppresses HER3 is provided active, this activity can for example, be measured in other measuring methods (phosphoric acid of, describing in embodiment-HER3 mensuration, phosphoric acid-Akt mensuration, cell proliferation and part blocking-up are measured) of phosphorylation assay or the granting of HER signal.Also comprise within the scope of the invention the parent nucleotide sequence of variable heavy chain and light chain; And for express total length heavy chain and the sequence of light chain of optimizing in mammalian cell.Other antibody of the present invention comprise amino acid or the nucleic acid having suddenlyd change, but have at least 60,70,80,90,95,98 or 99% per-cent identity with above-mentioned sequence.In some embodiments, it comprises the aminoacid sequence of sudden change, wherein when comparing with the variable region shown in above-mentioned sequence, by aminoacid deletion, insertion or Substitution in variable region, be no more than 1,2,3,4 or 5 amino acid.

For example, the invention provides the separated monoclonal antibody (or its function fragment) that comprises variable region of heavy chain and variable region of light chain, wherein this variable region of heavy chain comprises and is selected from ID NO:194,214,234,254,274,294,314,334,354,374,394,414,434,454,474,494,514,534,554,574,594,614,634,654,674 and 694 aminoacid sequence at least 80%, 90%, 95%, 96%, 97%, 98% or 99% same aminoacid sequence; This variable region of light chain comprises and is selected from SEQ ID NO:195,215,235,255,275,295,315,335,355,375,395,415,435,455,475,495,515,535,555,575,595,615,635,655,675 and 695 aminoacid sequence at least 80%, 90%, 95%, 96%, 97%, 98% or 99% same aminoacid sequence; Wherein this antibodies HER3 (for example, people and/or cynomolgus monkey HER3) structural domain 3-4, and the signal that suppresses HER3 is provided active, this activity can for example, be measured in other measuring methods (phosphoric acid of, describing in embodiment-HER3 mensuration, phosphoric acid-Akt mensuration, cell proliferation and part blocking-up are measured) of phosphorylation assay or the granting of HER signal.Also comprise within the scope of the invention the parent nucleotide sequence of variable heavy chain and light chain; And for express total length heavy chain and the sequence of light chain of optimizing in mammalian cell.Other antibody of the present invention comprise amino acid or the nucleic acid having suddenlyd change, but have at least 60,70,80,90,95,98 or 99% per-cent identity with above-mentioned sequence.In some embodiments, it comprises the aminoacid sequence of sudden change, wherein when comparing with the variable region shown in above-mentioned sequence, by aminoacid deletion, insertion or Substitution in variable region, be no more than 1,2,3,4 or 5 amino acid.

In other embodiments, VH and/or VL aminoacid sequence can have with the sequence shown in table 1 or table 2 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity.In other embodiments, VH and/or VL aminoacid sequence can be identical, just have amino acid substitution being no more than 1,2,3,4 or 5 amino acid position place.Can for example, by mutagenesis (site-directed mutagenesis or PCR mediation mutagenesis), obtain such antibody, this antibody has the VHHe VL district that VHHe VL district with antibody described in table 1 or table 2 has height (80% or higher) identity, then by the function of the reservation of the coded antibody through changing of functional examination method test as herein described.

In other embodiments, heavy chain and/or light chain variable region nucleotide sequence have 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% identity with the sequence above.

" per-cent identity " between two sequences used herein is the function (being that % identity equals same position number/total number of positions x 100) of the total same position number of this sequence, wherein consider the length in room number and each room, need to introduce this room for the best comparison of two sequences.Described in below non-limiting example, available mathematical algorithm is realized the comparison of sequence between two sequences and determining of per-cent identity.

In addition or alternatively, protein sequence of the present invention can further be used as " search sequence " and search for for public database, for example, to identify correlated series.For example, available Altschul etc., the blast program of (1990) J.Mol.Biol.215:403-10 (version 2 .0) carries out this type of search.

There is the conservative antibody of modifying

In certain embodiments, antibody of the present invention has the variable region of heavy chain that comprises CDR1, CDR2 and CDR3 sequence and comprises CDR1, CDR2 and the variable region of light chain of CDR3 sequence, one of them or more these CDR sequences have the aminoacid sequence that indicates based on antibody described herein or its conservative modification, and wherein this antibody retains the functional performance of the expectation of HER3 antibody of the present invention.

Therefore, the invention provides separated HER3 monoclonal antibody or its fragment in conjunction with the structural domain 3 of HER3, it is by the variable region of heavy chain that comprises CDR1, CDR2 and CDR3 sequence and comprise CDR1, CDR2 and the variable region of light chain of CDR3 sequence forms, wherein: variable region of heavy chain CDR1 aminoacid sequence is selected from SEQ ID NO:2,22,42,62,82,102,122,142 and 162, and conservative modification; Variable region of heavy chain CDR2 aminoacid sequence is selected from SEQ ID NO:3,23,43,63,83,103,123,143 and 163, and conservative modification; Variable region of heavy chain CDR3 aminoacid sequence is selected from SEQ ID NO:4,24,44,64,84,104,124,144 and 164, and conservative modification; Variable region of light chain CDR1 aminoacid sequence is selected from SEQ ID NO:8,28,48,68,88,108,128,148 and 168, and conservative modification; Variable region of light chain CDR2 aminoacid sequence is selected from SEQ ID NO:9,29,49,69,89,109,129,149 and 169, and conservative modification; Variable region of light chain CDR3 aminoacid sequence is selected from SEQ ID NO:10,30,50,70,90,110,130,150 and 170, and conservative modification; This antibody or its fragments specific are in conjunction with HER3, and by suppressing the conduction of HER3 signal by way of suppressing HER3 activity, this activity can for example, be measured in other measuring methods (, the phosphoric acid described in embodiment-HER3 mensuration, phosphoric acid-Akt mensuration, cell proliferation and part blocking-up are measured) of phosphorylation assay or the granting of HER signal.

Therefore, the invention provides separated HER3 monoclonal antibody or its fragment in conjunction with the structural domain 3-4 of HER3, its by the variable region of heavy chain that comprises CDR1, CDR2 and CDR3 sequence and comprise CDR1, CDR2 and the variable region of light chain of CDR3 sequence forms, wherein: variable region of heavy chain CDR1 aminoacid sequence is selected from SEQ ID NO:182,202,222,242,262,282,302,322,342,362,382,402,422,442,462,482,502,522,542,562,582,602,622,642,662 and 682, and conservative modification; Variable region of heavy chain CDR2 aminoacid sequence is selected from SEQ ID NO:183,203,223,243,263,283,303,323,343,363,383,403,423,443,463,483,503,523,543,563,583,603,623,643,663 and 683, and conservative modification; Variable region of heavy chain CDR3 aminoacid sequence is selected from SEQID NO:184,204,224,244,264,284,304,324,344,364,384,404,424,444,464,484,504,524,544,564,584,604,624,644,664 and 684, and conservative modification; Variable region of light chain CDR1 aminoacid sequence is selected from SEQ ID NO:188,208,228,248,268,288,308,328,348,368,388,408,428,448,468,488,508,528,548,568,588,608,628,648,668 and 688, and conservative modification; Variable region of light chain CDR2 aminoacid sequence is selected from SEQ ID NO:189,209,229,249,269,289,309,329,349,369,389,409,429,449,469,489,509,529,549,569,589,609,629,649,669 and 689, and conservative modification; Variable region of light chain CDR3 aminoacid sequence is selected from SEQ ID NO:190,210,230,250,270,290,310,330,350,370,390,410,430,450,470,490,510,530,550,570,590,610,630,650,670 and 690, and conservative modification; This antibody or its fragments specific are in conjunction with HER3, and by suppressing the conduction of HER3 signal by way of suppressing HER3 activity, this activity can for example, be measured in other measuring methods (, the phosphoric acid described in embodiment-HER3 mensuration, phosphoric acid-Akt mensuration, cell proliferation and part blocking-up are measured) of phosphorylation assay or the granting of HER signal.

Antibody in conjunction with identical epi-position

The invention provides with epi-position the antibody of interact (for example, by combination, steric hindrance, stabilization/stabilization removal, spatial distribution), this epi-position is identical with the interactional epi-position of HER3 antibody institute with described in table 1 or table 2.Therefore can be based on them at HER3 in conjunction with for example, identifying other antibody with the ability of other antibody cross competitions of the present invention (with statistically significant mode competitive inhibition be combined) in measuring.The ability that test antibody suppresses antibody of the present invention and HER3 albumen (for example, people and/or cynomolgus monkey HER3) combination proves that this test antibody can be combined with this antibody competition HER3; According to non-limiting theory, this antibody can with the epi-position of identical or relevant (for example, approaching on similar in structure or space) on antibodies HER3 albumen with its competition.In certain embodiment, be human monoclonal antibodies with the antibody of the upper identical epi-position of antibodies HER3 of the present invention.Can be by preparation described herein separated this type of human monoclonal antibodies.

In one embodiment, this antibody or its fragment be in conjunction with the structural domain 3 of HER3, and suppress ligand dependent and non-ligand dependent HER3 signal transduction the two.In one embodiment, this antibody or its fragment be in conjunction with the structural domain 3-4 of HER3, and suppress ligand dependent and non-ligand dependent HER3 signal transduction the two.

Ligand dependent and dependent/non-dependent that antibody of the present invention or its fragment suppress HER3 activate the two and do not stop ligand binding.Based on following reason, think that this is favourable:

(i) compare the antibody of target list kind HER3 activation mechanism (being ligand dependent or non-ligand dependent), therapeutic antibodies will have clinical application in broad-spectrum tumor, because different tumor types is subject to mechanism drives separately.

(ii) therapeutic antibodies will relate in the tumor type of two kinds of HER3 activation mechanisms effectively at the same time.The antibody of target list kind HER3 activation mechanism (being ligand dependent or non-ligand dependent) will be shown low effect or inefficacious in these tumor types.

(iii) ligand dependent that suppresses HER3 activates and does not stop the dysgenic possibility of the ligand concentration that the effect of the antibody of ligand binding increased lower.This will be converted into the effect of increase in the tumor type being driven by high concentration HER3 part, or the drug resistance reducing during by the rise mediation of HER3 part in resistance is inclined to.

(iv) by stablizing inactivation form, suppress antibody that HER3 activates and will be difficult for producing the drug resistance that the alternate mechanism that activated by HER3 drives.

Therefore, antibody of the present invention can be used for treating the clinical invalid illness of existing therapeutic antibodies.

Transformation and the antibody of modifying

Also can as parent material, prepare antibody of the present invention with the antibody with VH shown in one or more this paper and/or VL sequence, to transform the antibody of modification, the antibody of this modification can have the character changing from initial antibody.Can be by modifying in one or two variable region (being VH and/or VL), for example in one or more CDR district and/or the one or more residues in one or more framework region carry out engineered antibody.In addition or alternatively, can carry out engineered antibody by the residue of modifying in constant region, for example, change the effector function of this antibody.

The variable region transformation of one type that can carry out is that CDR transplants.Antibody mainly interacts by amino-acid residue and the target antigen being positioned in six heavy chains and light chain complementary determining region (CDR).For this reason, the aminoacid sequence in CDR is more diversified than the sequence outside CDR between each antibody.Because CDR sequence is responsible for most antibody-AIs, so expressing the recombinant antibodies of the character of the specific naturally occurring antibody of simulation by construction of expression vector is possible, this expression vector comprises the CDR sequence of being transplanted on the frame sequence with different antibodies of different nature, this CDR sequence (is for example consulted from specific naturally occurring antibody, Riechmann etc., (1998) Nature332:323-327; Jones etc., (1986) Nature 321:522-525; Queen etc., (1989) Proc.Natl.Acad., U.S.A.86:10029-10033; The U.S. Patent number 5,225,539 of Winter; U.S. Patent number 5,530,101 with Queen etc.; 5,585,089; 5,693,762 and 6,180,370).

Therefore, another embodiment of the invention relates to separated HER3 monoclonal antibody or its fragment in conjunction with the structural domain 3 of HER3, it comprises: variable region of heavy chain, and it contains respectively and has the CDR1 sequence that is selected from SEQ ID NO:2,22,42,62,82,102,122,142 and 162 aminoacid sequence; There is the CDR2 sequence that is selected from SEQ ID NO:33,23,43,63,83,103,123,143 and 163 aminoacid sequence; The CDR3 sequence with SEQ ID NO:4,24,44,64,84,104,124,144 and 164 aminoacid sequence; And variable region of light chain, it contains respectively and has the CDR1 sequence that is selected from SEQ ID NO:8,28,48,68,88,108,128,148 and 168 aminoacid sequence; There is the CDR2 sequence that is selected from SEQ ID NO:9,29,49,69,89,109,129,149 and 169 aminoacid sequence; And there is the CDR3 sequence that is selected from SEQ ID NO:10,30,50,70,90,110,130,150 and 170 aminoacid sequence.

Therefore, another embodiment of the invention relates to separated HER3 monoclonal antibody or its fragment in conjunction with the structural domain 3-4 of HER3, it comprises: variable region of heavy chain, and it contains respectively and has the CDR1 sequence that is selected from SEQ ID NO:182,202,222,242,262,282,302,322,342,362,382,402,422,442,462,482,502,522,542,562,582,602,622,642,662 and 682 aminoacid sequence; There is the CDR2 sequence that is selected from SEQ ID NO:183,203,223,243,263,283,303,323,343,363,383,403,423,443,463,483,503,523,543,563,583,603,623,643,663 and 683 aminoacid sequence; The CDR3 sequence with SEQ ID NO:184,204,224,244,264,284,304,324,344,364,384,404,424,444,464,484,504,524,544,564,584,604,624,644,664 and 684 aminoacid sequence; And variable region of light chain, it contains respectively and has the CDR1 sequence that is selected from SEQ ID NO:188,208,228,248,268,288,308,328,348,368,388,408,428,448,468,488,508,528,548,568,588,608,628,648,668 and 688 aminoacid sequence; There is the CDR2 sequence that is selected from SEQ ID NO:189,209,229,249,269,289,309,329,349,369,389,409,429,449,469,489,509,529,549,569,589,609,629,649,669 and 689 aminoacid sequence; And there is the CDR3 sequence that is selected from SEQ ID NO:190,210,230,250,270,290,310,330,350,370,390,410,430,450,470,490,510,530,550,570,590,610,630,650,670 and 690 aminoacid sequence.

The VH that this antibody-like contains monoclonal antibody and VL CDR sequence, but can also contain the different frame sequences from these antibody.Can or comprise the reference of publication of germline antibody gene sequence from public DNA database and obtain this type of frame sequence.For example, the germline DNA sequence dna of people's heavy chain and chain variable region gene is found in " Vbase " people germline sequence library (can obtain on internet www.mrc-cpe.cam.ac.uk/vbase), and the people such as Kabat, (1991) Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH publication number 91-3242; The people such as Chothia, (1987) J.Mol.Biol.196:901-917; The people such as Chothia, (1989) Nature 342:877-883; With the people such as Al-Lazikani, (1997) J.Mol.Biol.273:927-948; The people such as Tomlinson, (1992) J.fol.Biol.227:776-798; With the people such as Cox, (1994) Eur.J Immunol.24:827-836; Content separately is clearly incorporated herein by reference at this.

For the example of the frame sequence of antibody of the present invention, be structurally similar those frame sequences of the frame sequence (consensus sequence and/or frame sequence that for example monoclonal antibody of the present invention is used) that uses to the selected antibody of the present invention.VH CDR1,2 and 3 sequences and VL CDR1,2 and 3 sequence portables to see frame sequence derived from racial immunity globulin gene in sequence have on the framework region of identical sequence, or this CDR sequence portable is to comparing on the framework region of containing one or more sudden changes with germline sequence.For example, have been found that residue in the framework region that suddenlys change in some cases maintains or the antigen binding capacity that strengthens antibody is favourablely (for example to consult the U.S. Patent number 5,530,101 of Queen etc.; 5,585,089; 5,693,762 and 6,180,370).

It is the amino-acid residue in sudden change VH and/or VL CDR1, CDR2 and/or CDR3 district that the variable region of another type is modified, and improves thus one or more binding properties (for example, avidity) of object antibody, is called " affinity maturation ".Can carry out the mutagenesis of site-directed mutagenesis or PCR mediation and introduce sudden change, and the impact of antagonist combination or other object functional propertys is provided in the external or in vivoassay providing in as described herein and embodiment.Can introduce conservative modification (as discussed) above.This sudden change can be amino acid substitution, interpolation or disappearance.In addition, conventionally change 1,2,3,4 or 5 residue that is no more than in CDR district.

Therefore, in another embodiment, the invention provides separated HER3 monoclonal antibody or its fragment in conjunction with the structural domain 3 of HER3, it is comprised of variable region of heavy chain, this variable region of heavy chain has: by being selected from SEQ ID NO:22,22,42,62,82,102,122,142 and 162 aminoacid sequence, or compare with 162 and have the VH CDR1 district that the aminoacid sequence of 1,2,3,4 or 5 amino acid substitution, disappearance or interpolation forms with SEQ ID NO:22,22,42,62,82,102,122,142; Have and be selected from SEQ ID NO:3,23,43,63,83,103,123,143 and 163 aminoacid sequence, or compare the VH CDR2 district of the aminoacid sequence with 1,2,3,4 or 5 amino acid substitution, disappearance or interpolation with 163 with SEQ ID NO:3,23,43,63,83,103,123,143; Have and be selected from SEQ ID NO:4,24,44,64,84,104,124,144 and 164 aminoacid sequence, or compare the VH CDR3 district of the aminoacid sequence with 1,2,3,4 or 5 amino acid substitution, disappearance or interpolation with 370 with SEQ ID NO:4,24,44,64,84,104,124,144,164; Have and be selected from SEQ ID NO:8,28,48,68,88,108,128,148 and 168 aminoacid sequence, or compare the VL CDR1 district of the aminoacid sequence with 1,2,3,4 or 5 amino acid substitution, disappearance or interpolation with 168 with SEQ ID NO:8,28,48,68,88,108,128,148; Have and be selected from SEQ ID NO:9,29,49,69,89,109,129,149 and 169 aminoacid sequence, or compare the VL CDR2 district of the aminoacid sequence with 1,2,3,4 or 5 amino acid substitution, disappearance or interpolation with 169 with SEQ ID NO:9,29,49,69,89,109,129,149; And have and be selected from SEQ ID NO:10,30,50,70,90,110,130,150,170 and 373 aminoacid sequence, or compare the VL CDR3 district of the aminoacid sequence with 1,2,3,4 or 5 amino acid substitution, disappearance or interpolation with 170 with SEQ ID NO:10,30,50,70,90,110,130,150.

Therefore, in another embodiment, the invention provides separated HER3 monoclonal antibody or its fragment in conjunction with the structural domain 3-4 of HER3, it is comprised of variable region of heavy chain, this variable region of heavy chain has: by being selected from SEQ ID NO:182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, 522, 542, 562, 582, 602, 622, 642, 662 and 682 aminoacid sequence, or with SEQ ID NO:182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, 522, 542, 562, 582, 602, 622, 642, 662 compare and have 1 with 682, 2, 3, 4 or 5 amino acid substitutions, the VH CDR1 district that disappearance or the aminoacid sequence adding form, there is the SEQ of being selected from ID NO:183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, 523, 543, 563, 583, 603, 623, 643, 663 and 683 aminoacid sequence, or with SEQ ID NO:183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, 523, 543, 563, 583, 603, 623, 643, 663 compare and have 1 with 683, 2, 3, 4 or 5 amino acid substitutions, the VH CDR2 district of the aminoacid sequence of disappearance or interpolation, there is the SEQ of being selected from ID NO:184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, 524, 544, 564, 584, 604, 624, 644, 664 and 684 aminoacid sequence, or with SEQ ID NO:184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, 524, 544, 564, 584, 604, 624, 644, 664 compare and have 1 with 684, 2, 3, 4 or 5 amino acid substitutions, the VH CDR3 district of the aminoacid sequence of disappearance or interpolation, there is the SEQ of being selected from ID NO:188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, 528, 548, 568, 588, 608, 628, 648, 668 and 688 aminoacid sequence, or with SEQ ID NO:188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, 528, 548, 568, 588, 608, 628, 648, 668 compare and have 1 with 688, 2, 3, 4 or 5 amino acid substitutions, the VL CDR1 district of the aminoacid sequence of disappearance or interpolation, there is the SEQ of being selected from ID NO:189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, 529, 549, 569, 589, 609, 629, 649, 669 and 689 aminoacid sequence, or with SEQ ID NO:189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, 529, 549, 569, 589, 609, 629, 649, 669 compare and have 1 with 689, 2, 3, 4 or 5 amino acid substitutions, the VL CDR2 district of the aminoacid sequence of disappearance or interpolation, and there is the SEQ of being selected from ID NO:190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, 530, 550, 570, 590, 610, 630, 650, 670 and 690 aminoacid sequence, or with SEQ ID NO:190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, 530, 550, 570, 590, 610, 630, 650, 670 compare and have 1 with 690, 2, 3, 4 or 5 amino acid substitutions, the VL CDR3 district of the aminoacid sequence of disappearance or interpolation.

Antibody fragment is implanted into alternative framework or support

Can use Multiple Antibodies/immunoglobulin frameworks or support, as long as resulting polypeptide comprises the land of at least one specific binding HER3.This type of framework or support comprise 5 main idiotypes or its fragment of human normal immunoglobulin, and comprise the immunoglobulin (Ig) of other animal species, preferably have the immunoglobulin (Ig) of humanization aspect.Those skilled in the art constantly find and develop new framework, support and fragment.

On the one hand, the present invention relates to produce the antibody based on NIg with the NIg support of portable CDR of the present invention on it.Can utilize known or unknown NIg framework and support, as long as they for example comprise, to the special land of target HER3 protein (, people and/or cynomolgus monkey HER3).Known NIg framework or support comprise, but be not limited to fibronectin (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd., Cambridge, MA, and Ablynx nv, Zwijnaarde, Belgium), NGAL (Pieris Proteolab AG, Freising, Germany), little module immune drug (Trubion Pharmaceuticals Inc., Seattle, WA), huge antibody (Avidia, Inc., Mountain View, CA), albumin A (Affibody AG, Sweden) and affilin (γ-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany).

Fibronectin support based on III type fibronectin structural domain (for example, the tenth of III type fibronectin the module ( ¹⁰fn3 structural domain)).III type fibronectin structural domain has 7 or 8 β chains that are distributed between two β-pleated sheet structures, and these two β-pleated sheet structures self are packed the core that forms protein each other, and also contains and β chain is connected to each other and is exposed to the ring (similar with CDR) in solvent.At each edge of β-pleated sheet structure interlayer, have at least three such rings, wherein this edge is the protein border (consult US 6,818,418) vertical with β chain direction.These supports based on fibronectin are not immunoglobulin (Ig)s, although the minimum function antibody fragment (variable region of heavy chain that comprises whole antigen recognition unit) in overall folded and camel and vigone IgG is very close.Due to this structure, NIg antibody analogue antigen binding property, the antigen-binding matter of this antigen-binding matter and antibody in character with similar in avidity.These supports can be used for external ring randomization and reorganization strategy, the affinity maturation similar process of this strategy and internal antibody.These molecules based on fibronectin can be used as support, wherein use standard clone technology to replace molecule Huan district with CDR of the present invention.

Ankyrin technology is based on using the protein of the replicated blocks with ankyrin source as the support that carries variable region, and this variable region can be used in conjunction with different targets.33 amino acid whose polypeptide that these ankyrin replicated blocks are comprised of two antiparallel α spirals and β-bend.By using ribosomal display to carry out largest optimization to the combination of variable region.

Avimers is derived from the protein that contains natural A structural domain, as HER3.These structural domains are natively for protein-protein interaction, and in people, surpass 250 protein structurally based on A structural domain.Avimers is comprised of a large amount of different " A structural domain " monomer (2-10) connecting by amino acid joint.Method described in available for example U.S. Patent Application Publication No. 20040175756,20050053973,20050048512 and 20060008844 produces can be in conjunction with the Avimers of target antigen.

Affibody affinity ligand is the little simple protein in conjunction with the triple helical Shu Zucheng of the support in territory by an IgG based on albumin A.Albumin A is the surface protein from bacterium streptococcus aureus (Staphylococcus aureus).This supporting structure territory is comprised of 58 amino acid, randomization wherein 13 produce the affibody library (for example consulting US5,831,012) with a large amount of ligand variants.Affibody molecular simulation antibody, compares with the molecular weight of antibody 150kDa, and they have the molecular weight of 6kDa.Although its size is little, the combining site of affibody molecule and the combining site of antibody are similar.

Anticalins is the product of Pieris ProteoLab AG company exploitation.They are derived from NGAL, and NGAL is the little and firm protein of a large class, are usually directed to physiology transportation or the storage of chemical-sensitive or undissolved compound.Some natural NGAL exist in people's tissue or body fluid.Protein structure shows it is on stiff frame, to have hypermutation ring immunoglobulin (Ig).Yet, to compare with antibody or its recombinant fragment, NGAL is comprised of the wall scroll polypeptide chain with 160 to 180 amino-acid residues, only slightly large than single immunoglobulin domains.The Fourth Ring group that forms binding pocket shows obvious structure plasticity-and tolerates multiple side chain.Therefore combining site can be reinvented in proprietary method, to identify the target molecule with high-affinity and specific difform regulation.The bilin (bilin) of a protein of NGAL family---Pieris brassicae (Pieris Brassicae) in conjunction with albumen (BBP) for developing anticalins by mutagenic treatment Fourth Ring group.An example of the patent application of description anticalins is in PCT publication number WO 199916873.

Affilin molecule is for the little NIg protein to protein and the design of micromolecular specificity avidity.Can from two libraries, select very rapidly new affilin molecule, the scaffolding protein from people of each of this library based on different.Affilin molecule and immunoglobulin (Ig) protein do not show any structural homology.At present, utilize two kinds of affilin supports, wherein a kind of is γ crystallin---people's structure crystallins, another kind is " ubiquitin " superfamily protein.Two kinds of people's supports are all very little, show high temperature stability, and pH change and denaturing agent are almost had to resistance.This high stability is mainly the β-pleated sheet structure structure due to protein expansion.The example of the protein in γ crystallin source is described in WO200104144, and the example of " ubiquitin sample " protein is described in WO2004106368.

Protein epitope stand-in (PEM) are median size, annular, the peptide sample molecules (MW 1-2kDa) of simulated albumin matter beta hairpin secondary structure, and protein beta hairpin secondary structure relates to the main secondary structure of protein-protein interaction.

In some embodiments, by changing Fc district, change Fab into reticent IgG1 form.For example, can change the antibody in table 1 or table 2 into IgG form.

People's antibody or humanized antibody

The invention provides the human antibody of specific binding HER3 protein (for example, people and/or cynomolgus monkey/mouse/rat HER3).Compare with chimeric antibody or humanized antibody, when individual human is used, people HER3 antibody or its fragment have the antigenicity of further reduction.

Available methods known in the art produce people's HER3 antibody or its fragment.For example, by mankind's engineering, non-human antibody is transformed into people's antibody of transformation.U.S. Patent Publication No. 20050008625 has been described the interior method of body of replacing non-human antibody variable region with the people variable region in antibody, and the method remains identical simultaneously or provides than non-human antibody's combination feature better in conjunction with feature.The method depends on the replacement to the epi-position guiding of inhuman reference antibody variable region with human antibody.Income earner's antibody is generally structurally with uncorrelated with reference to non-human antibody, but with this reference antibody in conjunction with the identical epi-position in same antigen.In brief, under the reporting system of response test antibodies antigen exists, by set up the competition in conjunction with limited amount antigen, the complementary replacement method that makes it possible to carry out continuous epitope guiding between the library of the multiple heterocomplex of " rival " and reference antibody (" test antibody ") in cell.This rival can be reference antibody or derivatives thereof, as Single-Chain Fv Fragment of Murine.This rival can be also natural or artificial antigen part, its with reference antibody in conjunction with identical epi-position.The only requirement of this rival is it in conjunction with the epi-position identical with reference antibody, and it competes conjugated antigen with reference antibody.Test antibody has from one of inhuman reference antibody common antigen in conjunction with V district, and from multiple source (as the repertoire library of people's antibody) random other V districts of selecting.From the common V district of reference antibody, as guiding, this test antibody is positioned in the identical epi-position on antigen, and in same direction, thereby makes to select to be partial to the highest antigen for reference antibody in conjunction with fidelity of reproduction.

Permitted eurypalynous reporting system and be can be used for detecting the interaction of expecting between test antibody and antigen.For example, the sub-fragment of complementation reporter can connect respectively antigen and test antibody, and report only occurring when test antibody conjugated antigen by fragment complementation is activated.When test antibody and antigen-report section fusions and rival's coexpression, report activates to become and depends on the ability of this test antibody and rival's competition, and this ability and this test antibody are proportional to the avidity of antigen.Spendable other reporting systems comprise as U.S. Patent Application Serial 10/208, the reactivation of the disclosed sub-reactivation system of report (RAIR) from suppressing in 730 (publication numbers 20030198971), or in U.S. Patent Application Serial 10/076,845 (publication number 20030157579) disclosed competition activation system.

Utilize the complementary replacement system of continuous epitope guiding, select the cell of identifying that the single test antibody of expression and rival, antigen and report subgroup are divided.In these cells, each test antibody is competed in conjunction with limited amount antigen with rival one to one.Report that sub activity is proportional with the amount in conjunction with the antigen of test antibody, this antigen amount then proportional to the stability of the avidity of antigen and test antibody with test antibody.When being expressed as test antibody, starting take its activity with respect to reference antibody and select test antibody as basis.The result that the first round is selected is " heterozygosis " antibody group, and wherein each antibody comprises from the identical inhuman V district of reference antibody with from the people V district in library, and wherein each antibody and identical epi-position on reference antibody conjugated antigen.The one or more hybrid antibodies of selecting in the first round to the avidity of antigen by equal or higher to the avidity of antigen with reference antibody.

In second V district replacement step, be used in the people V district selected in first step as the guidance of selecting people to replace, this people replaces and uses various library in same clansman V district to replace residue inhuman reference antibody V district.The hybrid antibody of selecting in the first round also can be used as the second rival who takes turns selection.The second result of taking turns selection is human antibody group, and it is structurally different from reference antibody, but competes in conjunction with identical antigen with reference antibody.Some the antibody of choosing with reference antibody in conjunction with the identical epi-position in same antigen.At these, choose in antibody, one or more antibody with the avidity with reference antibody quite or than its high avidity in conjunction with identical epi-position.

By a kind of mouse mentioned above or chimeric HER3 antibody or its fragment, as with reference to antibody, can easily utilize the method to produce the people's antibody in conjunction with people HER3 with identical binding specificity and identical or better binding affinity.In addition, also can be from the company of common production people antibody, KaloBios for example, Inc. (Mountain View, CA) obtains this type of people HER3 antibody or its fragment by commercial sources.

Camel (camelid) antibody

From the member of camel and dromedary camel (Bactrian camel (Camelus bactrianus) and Calelus dromaderius) family, comprise that New World member is if the antibody protein obtaining in yamma species (Lama paccos, llama (Lama glama) and thin camel (Lama vicugna)) is about size, structural complexity with characterize aspect the antigenicity of individual human.Some IgG antibody from this Mammals family that occurring in nature is found lacks light chain, and is therefore structurally different from the four chain quaternary structure of the typical case with two heavy chains and two light chains from the antibody of other animals.Consult PCT/EP93/02214 (disclosed WO 94/04678 on March 3rd, 1994).

Can obtain by genetic modification the region of the camel antibody of the little single variable domains that is accredited as VHH, to produce small protein matter target to high-affinity, obtain low-molecular-weight antibody derived protein, be called " camel nano antibody ".Consult the U.S. Patent number 5,759,808 of authorizing on June 2nd, 1998; Also consult the people such as Stijlemans, (2004) J Biol Chem 279:1256-1261; The people such as Dumoulin, (2003) Nature 424:783-788; The people such as Pleschberger, (2003) Bioconjugate Chem 14:440-448; The people such as Cortez-Retamozo, (2002) Int J Cancer 89:456-62; And the people such as Lauwereys, (1998) EMBO J 17:3512-3520.For example, from Ablynx, Ghent, the transformation library of camel antibody and antibody fragment can be buied by Belgium.(for example, US20060115470; Domantis (US20070065440, US20090148434).As other antibody in inhuman source, the aminoacid sequence that changes camel antibody of can recombinating obtain with human sequence more as sequence, nano antibody can carry out " humanization ".Therefore, the natural low antigenicity of camel antibody on human can further reduce.

Camel nano antibody has general 1/10th molecular weight of the molecular weight of human IgG molecule, and this protein physical diameter several nanometers only.Undersized a kind of result is that the combination of camel nano antibody is for larger antibody protein sightless antigen position in function, be the detection reagent that camel nano antibody can be used as using the antigen that classical immunological technique can't detect, and may be used as therapeutical agent.Therefore therefore, undersized another result is that camel nano antibody can suppress the combination with the ditch of target protein or the different position of characteristic in narrow slit, and thereby can there is such ability, its than classical antibody more as the function of classical low-molecular-weight drug.

Lower molecular weight and closely size also cause camel nano antibody extremely thermally-stabilised, stable to extreme pH and proteolysis digestion, and a little less than antigenicity.Another result is that camel nano antibody is easily transferred to tissue from the recycle system, even through hemato encephalic barrier, can treat the obstacle of the tissue that affects the nerves.Nano antibody can be further convenient to medicament transport and stride across hemato encephalic barrier.Consult disclosed U.S. Patent application 20040161738 on August 19th, 2004.These features are indicated huge treatment potential with the low antigenicity combination to people.In addition, these molecules can, at prokaryotic cell prokaryocyte, as given full expression in intestinal bacteria (E.coli), and be fusion rotein with phage expression, and have function.

Therefore, feature of the present invention is HER3 to be had to camel antibody or the nano antibody of high-affinity.In some embodiment herein, the natural generation in camel animal of this camel antibody or nano antibody, is used this paper for the technology described in other antibody, with HER3 or its peptide fragment immunity camel, produces.Alternatively, transformation is in conjunction with the camel nano antibody of HER3, as described in embodiment herein with HER3 the elutriation method as target, by for example selecting to produce the camel nano antibody in conjunction with HER3 from the phage display library of the camel nano antibody protein of suitable mutagenic treatment.Can pass through the nano antibody of the further customized development of genetic modification, to have from the transformation period of 45 minutes to two weeks in acceptor individuality.In specific embodiments, described in for example PCT/EP93/02214, by the CDR sequence of the heavy chain of people's antibody of the present invention or light chain being implanted into nano antibody or single domain antibody frame sequence obtains camel antibody or nano antibody.In one embodiment, camel antibody or the nano antibody amino-acid residue in the structural domain 3 of at least one HER3 that is selected from following amino-acid residue is combined: 335-342,362-376,398,400,424-428,431,433-434 and 455 (in structural domain 3), or its subset.In one embodiment, camel antibody or the nano antibody residue in the structural domain 3 of at least one HER3 that is selected from following amino-acid residue is combined: 571,582-584,596-597,600-602,609-615 (structural domain 4), or its subset.

Bispecific molecule and multivalent antibody

On the other hand, the present invention relates to two paratopes, dual specific or polyspecific molecule, it comprises in conjunction with the antibody of the non-linear epi-position in the structural domain 3 of HER3 or conformational epitope or its fragment.On the other hand, this pair of paratope, dual specific or polyspecific molecule comprise antibody or its fragment in conjunction with the epi-position in the structural domain 4 of HER3.This antibody or its fragment can derive for or be connected to another functional molecular (for example another peptide or protein (for example, the part of another antibody or acceptor)), to produce the bispecific molecule in conjunction with at least two different combining sites or target molecule.In fact this antibody or its fragment can derive for or be connected to over a kind of other functional moleculars, to produce in conjunction with two paratopes or polyspecific molecule more than two different combining sites and/or target molecule; This type of pair of paratope or polyspecific molecule.In order to produce bispecific molecule, antibody or its fragment can (for example connect in function, by chemical coupling, heredity fusion, non-covalent combination or other modes) one or more other binding molecules, as another antibody, antibody fragment, peptide or in conjunction with stand-in, make to produce bispecific molecule.

By providing other clinical benefit (people such as Coloma, (1997) at 2 of anti-Binding in vivos or a plurality of antigen; The people such as Merchant, (1998); The people such as Alt, (1999); The people such as Zuo, (2000); The people such as Lu, (2004); The people such as Lu, (2005); The people such as Marvin, (2005); The people such as Marvin, (2006); The people such as Shen, (2007); The people such as Wu, (2007); The people such as Dimasi, (2009); The people such as Michaelson, (2009)). (people such as Morrison, (1997) Nature Biotech.15:159-163; The people such as Alt (1999) FEBS Letters 454:90-94; The people such as Zuo, (2000) Protein Engineering 13:361-367; The people such as Lu, (2004) JBC 279:2856-2865; The people such as Lu, (2005) JBC 280:19665-19672; The people such as Marvin, (2005) Acta Pharmacologica Sinica26:649-658; The people such as Marvin, (2006) Curr Opin Drug Disc Develop 9:184-193; The people such as Shen, (2007) J Immun Methods 218:65-74; The people such as Wu, (2007) Nat Biotechnol.11:1290-1297; The people such as Dimasi, (2009) J Mol Biol.393:672-692; With the people such as Michaelson, (2009) mAbs 1:128-141.

Can use methods known in the art, by puting together composition binding specificity, prepare bispecific molecule.For example, can separately produce each binding specificity of bispecific molecule, then mutually put together, for example, can carry out covalency with multiple coupling or linking agent and put together.The example of linking agent comprises albumin A, carbodiimide, N-succinimido-S-acetyl-thioacetate (SATA), 5,5'-dithio two (2-nitrobenzoic acids) (DTNB), o-phenylene dimaleimide (oPDM), N-succinimido-3-(2-pyridyl dithio) propionic ester (SPDP) and sulfosuccinimide base 4-(N-maleimide ylmethyl) hexanaphthene-l-carboxylicesters (sulfo group-SMCC) (be for example shown in, the people such as Karpovsky, (1984) J.Exp.Med.160:1686; The people such as Liu, (1985) Proc.Natl.Acad.Sci.USA 82:8648).Additive method is included in Paulus (1985) Behring Ins.Mitt.No.78:118-132; The people such as Brennan, (1985) Science 229:81-83), and the people such as Glennie, those that describe (1987) J.Immunol.139:2367-2375).Puting together agent is SATA and sulfo group-SMCC, and both all can obtain from Pierce Chemical Co. (Rockford, IL).

With regard to antibody, they can hold the sulfydryl of hinge area to become key to put together by the C of two heavy chains.In specific embodiments, before puting together, modify hinge area to contain odd number sulfhydryl residue, for example 1.

Alternatively, two kinds of binding specificities can be encoded in same vehicle, and in identical host cell, express and assembling.At bispecific molecule, be mAb x mAb, mAb x Fab, Fab x F (ab') ₂or during part x Fab fusion rotein, the method is particularly useful.Bispecific molecule of the present invention can be to comprise a single-chain antibody and in conjunction with the single chain molecule of determinant, or comprises two in conjunction with the strand bispecific molecule of determinant.Bispecific molecule can comprise at least two single chain molecules.For example, in U.S. Patent number 5,260,203; U.S. Patent number 5,455,030; U.S. Patent number 4,881,175; U.S. Patent number 5,132,405; U.S. Patent number 5,091,513; U.S. Patent number 5,476,786; U.S. Patent number 5,013,653; U.S. Patent number 5,258,498; With U.S. Patent number 5,482, the method for the preparation of bispecific molecule has been described in 858.

Can for example, by for example enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), facs analysis, biological assay (growth-inhibiting) or Western trace, measure to verify that bispecific molecule is combined with its specific target target.Each in these assay methods is generally for example, by utilizing the labelled reagent (antibody) special to object mixture to detect the existence of the protein-antibody complex of particularly important.

On the other hand, the invention provides multivalent compounds, it comprises in conjunction with at least two of the antibody of HER3 identical or different fragments.This antibody fragment can by protein merge or covalently or non-covalently key link together.For example, by by the antibody of antibody of the present invention and for example, the antibody linked tetravalence compound that obtains in conjunction with the constant region (Fc or hinge area) of antibody of the present invention.For example in Borean patent EP 1012280B1, trimerizing structural domain has been described.Five dimerization modules have for example been described in PCT/EP97/05897.

In one embodiment, this pair of paratope/bi-specific antibody is in conjunction with the amino-acid residue in the structural domain 3 of HER3 at least, it is selected from amino-acid residue: 335-342,362-376,398,400,424-428,431,433-434 and 455 (in structural domain 3), or its subset.In one embodiment, this pair of paratope/bi-specific antibody is in conjunction with the amino-acid residue in the structural domain 3 of HER3 at least, and it is selected from amino-acid residue: 571,582-584,596-597,600-602,609-615 (structural domain 4), or its subset.

In another embodiment, the present invention relates to bifunctional antibody, wherein modify single monoclonal antibody, make antigen-binding site in conjunction with more than one antigen, as for example, in conjunction with the two bifunctional antibody of HER3 and another antigen (, HER1, HER2 and HER4).In another embodiment, the present invention relates to the bifunctional antibody of the antigen (antigen for example with the HER3 in " sealing " or " inactivation " state with phase isomorphic map) that target has phase isomorphic map.The example with the HER3 of " sealing " or " inactivation " state with the antigen of phase isomorphic map includes but not limited to HER1 and HER4.Therefore, bifunctional antibody can in conjunction with HER3 and HER1 the two; HER3 and HER4 the two; Or HER1 and HER4 the two.The dual combination specificity of bifunctional antibody can be further converted to double activity, or double activity suppresses.(for example see the people such as Jenny Bostrom, (2009) Science:323; 1610-1614).

The antibody with the transformation period of prolongation

The invention provides antibody or its fragment of the transformation period in vivo with prolongation of non-linear epi-position in the structural domain 3 of specific binding HER3 or conformational epitope.The present invention also provides the antibody of the transformation period in vivo with prolongation of the epi-position in the structural domain 4 of specific binding HER3.

Many factors can affect the protein transformation period in vivo.For example, metabolism in, kidney filtration, liver, the degraded of passing through proteolysis enzyme (proteolytic enzyme) and immunogenic response (for example,, by the protein neutralization of antibody and by the picked-up of scavenger cell and dendritic cell).Multiple strategy can be used for extending the transformation period of antibody of the present invention.For example, by chemistry, connect polyoxyethylene glycol (PEG), reCODEPEG, antibody support, Polysialic acid (PSA), hydroxyethylamyle (HES), albumin bound part and carbohydrate protective layer; By the protein heredity with in conjunction with serum protein (as albumin, IgG, FcRn), merge and shift; By coupling (in heredity or chemically), to other bound fractions, this bound fraction is in conjunction with serum protein, as nano antibody, Fab, DARPin, avimer, affibody and anticalin; By heredity, merge rPEG, albumin, albuminous structural domain, albumin bound protein and Fc; Or by being incorporated in nano-carrier, sustained release preparation or medical facilities.

In order to extend antibody serum circulation in vivo, can use or not use multifunction conjunction, by PEG locus specificity being conjugated to antibody N end or C end or by the ε amino existing on lysine residue, inertia polymerizable molecular (as high molecular weight PEGs) being attached in antibody or its fragment.For Pegylation antibody, antibody or its fragment conventionally with polyoxyethylene glycol (PEG) (as the active ester of PEG or aldehyde derivatives) therein one or more PEG groups become to be attached under the condition of antibody or antibody fragment and react.Available reactive PEG molecule (or similar reaction water-soluble polymkeric substance) carries out Pegylation by acylation reaction or alkylation reaction.Term used herein " polyoxyethylene glycol " is intended to comprise any form of PEG, and it is for other protein of derivatize, as list (C1-C10) alkoxyl group or aryloxy-polyoxyethylene glycol or polyoxyethylene glycol-maleimide.In certain embodiments, the antibody for the treatment of Pegylation is deglycosylated antibody.Use causes linearity or the branched polymer derivatize of biological activity loss reduction.Can be by SDS-PAGE and the mass spectrum degree that monitoring is puted together closely, to guarantee correctly puting together of PEG molecule and antibody.Can be by size exclusion chromatography or by ion exchange chromatography separated unreacted PEG from antibody-PEG conjugate.Can use method well known to those skilled in the art, for example, by immunoassay described herein, test effect in the combination activity of the derivative antibody of PEG and body.The method of pegylated protein is known in the art, and can be applicable to antibody of the present invention.For example consult the EP 0 401384 of the EP 0 154 316 of Nishimura etc. and Ishikawa etc.

The Pegylation technology of other modifications comprises the renovation technique (ReCODE PEG) that reconstruct chemistry quadrature instructs, and it is incorporated into the side chain of chemically appointment in biosynthesizing protein by comprising the reconfiguration system of tRNA synthetic enzyme and tRNA.This technology can will be incorporated in biosynthesizing protein more than 30 amino acids in intestinal bacteria, yeast and mammalian cell.This tRNA is incorporated into alpha-non-natural amino acid any position of amber codon location, makes amber codon change into from terminator codon the codon that signalling makes the amino acid incorporation of chemically appointment.

Restructuring Pegylation technology (rPEG) also can be used for serum half-life and extends.This technology relates to 300-600 amino acid whose non-structure protein tail heredity is fused in existing pharmaceutical protein.Because about 15 times than its actual molecular weight of the apparent molecular weights of this non-structure protein chain, so greatly increased the serum half-life of this protein.Chemically conjugated from needs different with traditional Pegylation repurity, this preparation method extremely simplifies and product is homogeneous.

Many sialylated be another kind of technology, the stability that it extends the activity phase and improve therapeutic peptides and proteins with natural polymer Polysialic acid (PSA).PSA is sialic polymkeric substance (sugar).When for protein and therapeutic peptide drug delivery, Polysialic acid provides protectiveness microenvironment for puting together.This has increased the active phase of therapeutic protein in circulation and has prevented that it is by immune system recognition.PSA polymkeric substance is natural to be seen in human body.It is adopted by some bacterium, and this bacterium has been evolved and surpassed millions of years, with the wall of this PSA polymer coating self.By molecular mimicry, then these natural Polysialic acid bacteriums can defeat the system of defense of health.PSA is natural final stealth technique, can be easily from then on bacterioid produce in a large number and there is predetermined physical features.Even when with protein coupling, bacterium PSA is also complete non-immunogenic because it with the PSA in human body chemically identical.

Another technology comprises uses hydroxyethylamyle (" the HES ") derivative that connects antibody.HES is the modified natural polymer derived from waxy corn starch, and can carry out metabolism by the enzyme of health.Generally use the rheological property that HES solution is replaced not enough blood volume and improved blood.The hydroxyethylamyle of antibody makes it possible to by putting forward high molecular stability, and extends circulating half-life by reducing renal clearance, causes the biologic activity improving.By changing different parameters, as the molecular weight of HES, the customizable antibody conjugates of HES widely.

Also can be by introduce the antibody that one or more amino acid modified (replace, insert or lack) produce the Half-life in vivo with increase in IgG constant domain or its FcRn binding fragment (preferably Fc or hinge Fc structural domain fragment).For example consult international publication number WO 98/23289; International publication number WO 97/34631; With U.S. Patent number 6,277,375.

In addition, antibody can be conjugated in albumin, so that antibody or antibody fragment are more stable or have in vivo a longer transformation period in vivo.Technology is known in the art, consults for example international publication number WO 93/15199, WO 93/15200 and WO 01/77137; With european patent number EP 413,622.

HER3 antibody or its fragment also can with one or more human serum albumin (HSA) polypeptide or its meromixis.The HSA of mature form is 585 amino acid whose protein, is responsible for the major part of serum osmotic pressure, also serves as endogenous and carrier exogenous ligand.Albumin is as the carrier of polypeptide and the desirable properties of transporter in body as function and the inert nature thereof of carrier molecule.In WO 93/15199, WO 93/15200 and EP 413 622, propose albumin and as the carrier of multiple proteins, be used as the purposes of the component of albumin fusion protein.Also proposed to merge (EP 399 666) with N-end fragment and the polypeptide of HSA.Therefore, by antibody or its fragment and albumin heredity or chemistry are merged or puted together, can stablize or extend shelf life, and/or within the period extending, keep molecular activity in solution, in external and/or body.

Can by genetic manipulation, realize the fusion of albumin and another kind of protein, DNA or its fragment of the HSA that makes to encode are connected with the DNA of coded protein.Then with the nucleotide sequence merging, transform or the suitable host of transfection, the nucleotide sequence of this fusion is arranged on suitable plasmid to express fusion polypeptide.Can carry out vivoexpression from for example protokaryon or eukaryotic cell, or carry out expression in vivo from genetically modified organism.Relate to additive method that HSA merges and be for example found in WO 2001077137 and WO200306007, be hereby incorporated by.In specific embodiments, at mammal cell line, for example, in Chinese hamster ovary celI system, carry out the expression of fusion rotein.Also consider that within the scope of the invention antibody is combined with the difference that acceptor changes under low or high pH.For example, by the CDR at antibody, comprise that additional amino acid (as Histidine) carrys out modified antibodies, avidity that can antagonist is modified, make it for example, in the lower maintenance of low pH (the low pH in lysosome), (for example see people (2010) the Nature Biotechnology such as Tomoyuki Igawa with its receptors bind; 28,1203-1207).

Antibody conjugates

The invention provides antibody or its fragment of specific binding HER3, this antibody or its fragment restructuring fusion or chemically conjugated (comprising covalency and non-covalent puting together) are to heterologous protein or polypeptide (or its fragment, preferred at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acid whose polypeptide), to produce fusion rotein.Particularly, the invention provides the fusion rotein that comprises antibody fragment described herein (for example, Fab fragment, Fd fragment, Fv fragment, F (ab) 2 fragments, VH structural domain, VH CDR, VL structural domain or VL CDR) and heterologous protein, polypeptide or peptide.Known in the art for method protein, polypeptide or peptide being merged or be conjugated on antibody or antibody fragment.Consult for example U.S. Patent number 5,336,603,5,622,929,5,359,046,5,349,053,5,447,851 and 5,112,946; European patent number EP307,434 and EP 367,166; International publication number WO 96/04388 and WO 91/06570; Ashkenazi etc., (1991), Proc.Natl.Acad.Sci.USA 88:10535-10539; Zheng etc., (1995), J.Immunol.154:5590-5600; With Vil etc., (1992), Proc.Natl.Acad.Sci.USA 89:11337-11341.

Can produce other fusion roteins by the technology of gene shuffling, motif reorganization, exon reorganization and/or codon reorganization (being jointly called " DNA reorganization ").Can utilize DNA to reorganize to change the activity (for example, thering is more antibody or its fragment of high-affinity and lower dissociation rate) of antibody of the present invention or its fragment.Generally consult U.S. Patent number 5,605,793,5,811,238,5,830,721,5,834,252 and 5,837,458; Patten etc., (1997), Curr.Opinion Biotechnol.8:724-33; Harayama, (1998), Trends Biotechnol.16 (2): 76-82; Hansson etc., (1999), J.Mol.Biol.287:265-76; And Lorenzo and Blasco, (1998), Biotechniques24 (2): 308-313 (each in these patents and publication is incorporated herein by reference with its integral body).Can before restructuring, by fallibility PCR, random nucleotide insertion or additive method, carry out random mutagenesis and change antibody or its fragment, or antibody or its fragment of coding.The antibody of coding specific binding HER3 protein or the polynucleotide of its fragment can be with one or more components of one or more heterologous molecule, motif, sections, partly, the restructuring such as structural domain, fragment.

In addition, can be by antibody or its segment composition to flag sequence (as peptide) so that purifying.In preferred embodiments, marker amino acid sequence is six Histidine peptides, and as label providing in pQE carrier (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311) etc., wherein many is commercially available.As Gentz etc., (1989), described in Proc.Natl.Acad.Sci.USA 86:821-824, for example, six Histidines are that purified fusion protein is provided convenience.Other peptide tags that can be used for purifying include, but are not limited to hemagglutinin (" HA ") label and " flag " label corresponding to the epi-position derived from influenza hemagglutinin matter (Wilson etc., (1984), Cell 37:767).

In other embodiments, antibody of the present invention or its fragment are puted together with diagnostic reagent or detection agent.This antibody-like can be used as a part (as measured the effect of specific therapy) for clinical trial program, for to disease or obstacle initial, develop, carry out and/or seriousness is monitored or prognosis.Can be by antibody and detectable substance coupling be realized to this type of diagnosis and detection, this detectable substance includes but not limited to plurality of enzymes, as but be not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes or acetylcholinesterase; Prothetic group, as but be not limited to streptavidin vitamin H and avidin/biotin; Fluorescent substance, as but be not limited to Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine base amine fluorescein, dansyl chloride or phycoerythrin; Luminophore, as but be not limited to luminol,3-aminophthalic acid cyclic hydrazide; Noclilucence material, as but be not limited to luciferase, luciferin and aequorin; Radioactive substance, as but be not limited to iodine ( ¹³¹i, ¹²⁵i, ¹²³i and ¹²¹i), carbon ( ¹⁴c), sulphur ( ³⁵s), tritium ( ³h), indium ( ¹¹⁵in, ¹¹³in, ¹¹²in and ¹¹¹in), technetium ( ⁹⁹tc), thallium ( ²⁰¹ti), gallium ( ⁶⁸ga, ⁶⁷ga), palladium ( ¹⁰³pd), molybdenum ( ⁹⁹mo), xenon ( ¹³³xe), fluorine ( ¹⁸f), ¹⁵³sm, ¹⁷⁷lu, ¹⁵⁹gd, ¹⁴⁹pm, ¹⁴⁰la, ¹⁷⁵yb, ¹⁶⁶ho, ⁹⁰y, 47Sc, ¹⁸⁶re, ¹⁸⁸re, ¹⁴²pr, ¹⁰⁵rh, ⁹⁷ru, ⁶⁸ge, ⁵⁷co, ⁶⁵zn, ⁸⁵sr, ³²p, ¹⁵³gd, ¹⁶⁹yb, ⁵¹cr, ⁵⁴mn, ⁷⁵se, ¹¹³sn and ¹¹⁷tin; And use the positron emitting metal of multiple positron emission fault art and on-radiation paramagnetic metal ion.

The present invention also comprises the antibody partly puted together with therapeutic or the purposes of its fragment.Antibody or its fragment can be conjugated to therapeutic part, for example, for example, as cytotoxin (cytostatic agent or cell killing agent), therapeutical agent or radioactive metal ion, alpha-particle radiator.Cytotoxin or cytotoxic agent comprise the arbitrary substance harmful to cell.

In addition, antibody or its fragment can be conjugated to therapeutic part or the drug moiety of modifying given biological answer-reply.Therapeutic part or drug moiety are not interpreted as and are limited to classical chemotherapeutic.For example, drug moiety can be protein, peptide or the polypeptide with the biologic activity of wanting.This proteinoid for example can comprise, toxin, as toxalbumin, ricin A, Pseudomonas exotoxin, Toxins,exo-, cholera or diphtheria toxin; Protein, as tumour necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, Thr6 PDGF BB, tissue plasminogen activator, apoptosis agent, anti-angiogenic agent; Or biological response modifying factor, for example lymphokine.In one embodiment, HER3 antibody or its fragment and therapeutic part (for example cytotoxin, medicine (for example immunosuppressor) or radiotoxin) are puted together.This class conjugate is referred to herein as " immunoconjugates ".Comprise that one or more cytotoxic immunoconjugates are called " immunotoxin ".Cytotoxin or cytotoxic agent comprise the arbitrary substance that is harmful to (for example killing) cell.Example comprises taxon, cytochalasin B, Gramicidin D, ethidium bromide, ipecamine, mitomycin, Etoposide, teniposide, vincristine(VCR), vinealeucoblastine(VLB), colchicine, Zorubicin, daunorubicin, dihydroxy anthracin diketone, mitoxantrone, mithramycin, dactinomycin, 1-boldenone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte and tetracycline and analogue or homologue.Therapeutical agent also comprises, for example, metabolic antagonist (for example, methotrexate, 6-MP, 6-Tioguanine, cytarabine, 5 FU 5 fluorouracil dacarbazine), cauterant (ablating agent) (for example, mustargen, phosphinothioylidynetrisaziridine Chlorambucil, melphalan, carmustine (BSNU) and chlorethyl cyclohexyl nitrosourea (CCNU), endoxan, busulfan, mitobronitol, U-9889, ametycin and cis-dichloro diamines platinum (II) is cis-platinum (DDP), anthracycline antibiotics (for example, daunorubicin (being called in the past promise mycin) and Zorubicin), microbiotic (for example, more raw rhzomorph (being called in the past actinomycin), bleomycin, mithramycin and anthramycin (AMC)) and antimitotic agent is (for example, vincristine(VCR) and vinealeucoblastine(VLB)).(for example seeing Seattle Genetics US20090304721).

Other examples of the therapeutic cells toxin that can put together with antibody of the present invention or its fragment comprise duocarmycin, calicheamycin, maytenin (maytansine) and auristatin, and derivative.The example of calicheamycin antibody conjugates can be buied (MylotargTm; Wyeth-Ayerst).

The existing joint technique in available this area is puted together cytotoxin and antibody of the present invention or its fragment.For the example of joint categories that cytotoxin and antibody are puted together, hydrazone, thioether, ester class, disulphide have been included but not limited to and containing peptide linker.Can select such joint, it is for example easy to cut by the low pH in lysosome compartment, or is easy to by proteolytic enzyme (as preferential proteolytic enzyme of expressing in tumor tissues, for example, as kethepsin (, cathepsin B, C, D)) cutting.

The type of cells involved toxin, joint and for the people such as the further discussion of method that therapeutical agent and antibody are puted together also visible Saito, (2003) Adv.Drug Deliv.Rev.55:199-215; The people such as Trail, (2003) Cancer Immunol.Immunother.52:328-337; Payne, (2003) Cancer Cell 3:207-212; Allen, (2002) Nat.Rev.Cancer 2:750-763; Pastan and Kreitman, (2002) Curr.Opin.Investig.Drugs 3:1089-1091; Senter and Springer, (2001) Adv.Drug Deliv.Rev.53:247-264.

Antibody of the present invention or its fragment also can be puted together with radio isotope, to produce radioactive cytotoxic drugs, claim again radioimmunoassay conjugate.Can put together for the radioisotopic example of diagnosing or treat and include but not limited to antibody, iodine ^l31, indium ¹¹¹, yttrium ⁹⁰and lutetium ¹⁷⁷.Method for the preparation of radioimmunoassay conjugate is known in this field.The example of radioimmunoassay conjugate can be buied, and comprises Zevalin ^tM(DEC Pharmaceuticals) and Bexxar ^tM(Corixa Pharmaceuticals), and can use antibody of the present invention to prepare radioimmunoassay conjugate by similar method.In certain embodiments, macrocyclic chelants is Isosorbide-5-Nitrae, 7,10-tetraazacyclododecanand-N, N ', N ", N " '-tetraacethyl (DOTA), it can be attached on antibody by linkers.This type of linkers is known in this field, and is described in Denardo etc., (1998), Clin Cancer Res.4 (10): 2483-90; Peterson etc., (1999), Bioconjug.Chem.10 (4): 553-7; With Zimmerman etc., (1999), Nucl.Med.Biol.26 (8): in 943-50, each reference is incorporated herein by reference with its integral body.

The technology that is used for therapeutic to be partly conjugated on antibody is well-known, consult such as Arnon etc., " Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy ", Monoclonal Antibodies And Cancer Therapy, Reisfeld etc. (editor), 243-56 page (Alan R.Liss, Inc.1985); Hellstrom etc., " Antibodies For Drug Delivery ", Controlled Drug Delivery (the 2nd edition Robinson etc. (editor), 623-53 page (Marcel Dekker, Inc.1987); Thorpe, " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:A Review ", Monoclonal Antibodies84:Biological And Clinical Applications, Pinchera etc. (editor), 475-506 page (1985); " Analysis; Results; And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy ", Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin etc. (editor), 303-16 page (Academic Press 1985); With Thorpe etc., (1982), Immunol.Rev.62:119-58.

Antibody also can be attached on solid support, and this solid support is particularly useful for immunoassay or the purifying of target antigen.This type of solid support includes but not limited to glass, Mierocrystalline cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.

Antibody combination

On the other hand, the present invention relates to HER3 antibody of the present invention or its fragment uses together with another kind of antibody, micromolecular inhibitor, mTOR inhibitors or PI3 kinase inhibitor with other treatment agent.Example includes but not limited to following:

HER1 inhibitor: HER3 antibody or its fragment can be used together with HER1 inhibitor, this HER1 inhibitor comprises but is not limited to, horse trastuzumab (EMD72000), cetuximab (Imclone), victibix (Amgen), mAb 806 and Buddhist nun's trastuzumab (TheraCIM), gefitinib (Astrazeneca); CI-1033 (PD183805) (Pfizer), lapatinibditosylate (GW-572016) (GlaxoSmithKline), xylene monosulfonic acid lapatinibditosylate (SmithKlineBeecham), erlotinib hydrochloride (OSI-774) (OSI Pharma), PKI-166 (Novartis) and the chloro-4-fluorophenyl of N-[4-[(3-) amino]-7-[[(3 " S ")-tetrahydrochysene-3-furyl] oxygen]-6-quinazolyl]-4 (dimethylamino)-2-butylene acid amides are (by Boehringer Ingelheim with trade name sell).

HER2 inhibitor: HER3 antibody or its fragment can be used together with HER2 inhibitor, this HER2 inhibitor comprises but is not limited to, handkerchief trastuzumab is (by Genentech with trade mark sell), Herceptin is (by Genentech/Roche with trade mark sale), MM-111, HKI-272 (claim again HKI-272, (2E) the chloro-4-[(pyridine-2-of-N-[4-[[3-yl) methoxyl group] phenyl] amino]-3-cyano group-7-ethoxyquinoline-6-yl]-4-(dimethylamino) but-2-enamides, in PCT publication number WO 05/028443, describe), lapatinibditosylate or xylene monosulfonic acid lapatinibditosylate be (by GlaxoSmithKline with trade mark sell).

HER3 inhibitor: HER3 antibody or its fragment can be used together with HER3 inhibitor, this HER3 inhibitor comprises but is not limited to, the small molecules of MM-121, MM-111, IB4C3,2DID12 (U3Pharma AG), AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech) and inhibition HER3.

HER4 inhibitor: HER3 antibody or its fragment can be used together with HER4 inhibitor.

PI3K inhibitor: HER3 antibody or its fragment can be used together with PI3 kinase inhibitor, this PI3 kinase inhibitor includes but not limited to, 4-[2-(1H-indoles-4-yl)-6-[[4-(methylsulfonyl) piperazine-1-yl] methyl] thieno-[3, 2-d] pyrimidine-4-yl] morpholine (claims again GDC 0941, in PCT publication number WO09/036082 and WO 09/055730, describe), 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinoline-3-yl)-2, 3-glyoxalidine also [4, 5-c] quinoline-1-yl] phenyl] propionitrile (claims again BEZ 235 or NVP-BEZ235, in PCT publication number WO 06/122806, describe), BKM120 and BYL719.

MTOR inhibitors: HER3 antibody or its fragment can be used together with mTOR inhibitors, this mTOR inhibitors includes but not limited to, CCI-779 is (by Pfizer with trade name sell), ridaforolimus (was called deferolimus in the past, (1R, 2R, 4S)-4-[(2R)-2[(1R, 9S, 12S, 15R, 16E, 18R, 19R, 21R, 23S, 24E, 26E, 28Z, 30S, 32S, 35R)-1, 18-sodium catchol disulfonate 9, 30-dimethoxy-15, 17, 21, 23, 29, 35-hexamethyl-2, 3, 10, 14, 20-five oxo-11, 36-dioxa-4-aza-tricycle [30.3.1.04, 9] hexatriacontane-16, 24, 26, 28-tetraene-12-yl] propyl group]-2-methoxyl group cyclohexyl dimethyl phosphine acid esters, claim again rapamycin, AP23573 and MK8669 (Ariad Pharm.), in PCT publication number WO 03/064383, describe), everolimus (RAD001) is (by Novartis with trade name sell).One or more therapeutical agents can be used with HER3 antibody of the present invention or its fragment simultaneously, or use before or after using HER3 antibody of the present invention or its fragment.

Produce the method for antibody of the present invention

(i) nucleic acid of encoding antibody

The invention provides the nucleic acid molecule of basic purifying, its encoded packets is containing the section of HER3 antibody chain or the polypeptide of structural domain mentioned above.The nucleotide sequence that nucleic acid more of the present invention comprise the HER3 antibody heavy chain variable region of encoding, and/or the nucleotide sequence of encoded light chain variable region.In specific embodiments, nucleic acid molecule is those nucleic acid molecule of identifying in table 1 or table 2.Other nucleic acid molecule more of the present invention comprise with table 1 or table 2 in the nucleotide sequence of the nucleotide sequence of those nucleic acid molecule of identifying basic identical (for example at least 80%, 90%, 95%, 96%, 97%, 98% or 99%).When expression vector from suitable is expressed, by the polypeptide of these polynucleotide encodings, can show HER3 antigen binding capacity.

The present invention also provides such polynucleotide, and this polynucleotide encoding is from antibody or the heavy chain of its fragment or at least one CDR district of light chain and the common all San Ge CDR district shown in above.The heavy chain of the antibody of some other polynucleotide encodings shown in above or its fragment and/or light chain all or substantially all variable region sequences.Due to the degeneracy of codon, multiple nucleic acids sequence each immunoglobulin amino acid sequence of encoding.

The variable region of nucleic acid molecule codified antibody of the present invention and constant region the two.The Nucleotide that nucleotide sequences more of the present invention comprise encoding mature weight chain variabl area sequence, the ripe weight chain variabl area sequence basic identical (for example, at least 80%, 90%, 95%, 96%, 97%, 98% or 99%) of the HER3 antibody shown in this maturation weight chain variabl area sequence and table 1 or table 2.The Nucleotide that some other nucleotide sequences comprise encoding mature light chain variable region sequence, the ripe light chain variable region sequence basic identical (for example, at least 80%, 90%, 95%, 96%, 97%, 98% or 99%) of the HER3 antibody shown in this maturation light chain variable region sequence and table 1 or table 2.

PCR mutagenesis that is can be by solid phase DNA from the beginning synthetic or the existing sequence by encoding antibody or its fragment produces polynucleotide sequence.Can realize by methods known in the art the direct chemosynthesis of nucleic acid, as Narang etc., (1979), the phosphotriester method of Meth.Enzymol.68:90; Brown etc., the phosphodiester method of (1979) Meth.Enzymol.68:109; Beaucage etc., (1981) Tetra.Lett., the diethyl phosphoramidite method of 22:1859; With U.S. Patent number 4,458,066 solid support method.Can be by PCR Technology:Principles and Applications for DNA Amplification, H.A.Erlich (editor), Freeman Press, NY, NY, 1992; PCR Protocols:A Guide to Methods and Applications, Innis etc. (editor), Academic Press, San Diego, CA, 1990; Mattila etc., (1991) Nucleic Acids Res.19:967; With Eckert etc., described in (1991) PCR Methods and Applications 1:17, carry out in polynucleotide sequence, introducing sudden change by PCR.

The present invention is also provided for producing expression vector and the host cell of antibody or its fragment.Can express with multiple expression vector the polynucleotide of coding HER3 antibody chain or its fragment.Expression vector based on viral and non-viral expression vector are all used in and in mammalian host cell, produce antibody.Non-virus carrier and system comprise plasmid, episomal vector (conventionally having the expression cassette for marking protein or RNA) and artificial chromosome (for example consulting Harrington etc., (1997) Nat Genet 15:345).For example, for for example, non-virus carrier at Mammals (people) cells HER3 polynucleotide and polypeptide, comprise pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV carrier and many other carriers for expression of other proteins matter known in the art.Useful virus vector comprises the carrier based on retrovirus, adenovirus, adeno associated virus, simplexvirus, the carrier based on SV40, papilloma virus, HBP Epstein-Barr virus, vaccinia virus vector and Semliki Forest virus (SFV).Consult, Brent etc., (1995) above; Smith, Annu.Rev.Microbiol.49:807; With Rosenfeld etc., (1992) Cell68:143.

The selective dependency of expression vector is in expressing therein the expection host cell of this carrier.The promotor of the polynucleotide that conventionally, this expression vector contains effective connection encoding antibody chain or its fragment and other adjusting sequences (for example enhanser).In some embodiments, with inducible promoter, prevent the expression of insertion sequence except under inductive condition.Inducible promoter comprises, for example pectinose, lacZ, metallothionein promoter or heat-inducible promoter.Can under non-inductive condition, expand the cultivation of inverting biological, and not be partial to the colony of encoding sequence, the expression product of this encoding sequence can be tolerated by host cell better.Except promotor, also can require or wish by other regulatory elements the effective expression for antibody chain or its fragment.These elements generally include ATG initiator codon and contiguous ribosome bind site or other sequences.In addition, the enhanser that can be suitable for by comprising used cell system strengthens the efficiency of expression and (for example consults Scharf etc., (1994) Results Probl.Cell Differ.20:125; With Bittner etc., (1987) Meth.Enzymol., 153:516).For example, SV40 enhanser or cmv enhancer can be used for improving the expression in mammalian host cell.

Expression vector also can provide secretory signal sequence position, with the polypeptide formation fusion rotein of the antibody with being inserted or fragment coding.More frequently, before comprising and entering carrier, the antibody of this insertion or fragment sequence are connected with signal sequence.The carrier that is ready to use in the sequence of accepting encoding antibody or fragment light chain and weight chain variable structural domain sometimes also encode constant region or its part.Examples of such carriers permission variable region is expressed as the fusion rotein with constant region, causes thus the generation of complete antibody or its fragment.Conventionally, this type of constant region is people's constant region.

For the host cell comprising and express antibody or fragment chain, can be prokaryotic cell prokaryocyte or eukaryotic cell.Intestinal bacteria are for cloning and express a kind of prokaryotic hosts of polynucleotide of the present invention.Other microorganism host that are suitable for using comprise genus bacillus, as subtilis (Bacillus subtilis), with other enterobacteriaceaes (enterobacteriaceae), as salmonella (Salmonella), serratia (Serratia) and multiple Rhodopseudomonas (Pseudomonas) species.In these prokaryotic hosts, also can prepare expression vector, it contains the expression control sequenc compatible with this host cell (for example replication orgin) conventionally.In addition, will there is the multiple well-known promotor of arbitrary number, as Lac operon system, tryptophane (trp) promoter systems, β-lactamase promoter systems, or from the promoter systems of lambda particles phage.This promotor is controlled expression conventionally alternatively together with operon sequence, and has ribosome bind site sequence etc., for initial and complete and transcribe and translate.Other microorganisms, as yeast also can be used for expressing antibody or its fragment.Also insect cell and baculovirus vector can be used in combination.

In some preferred embodiments, mammalian host cell is for expressing and produce antibody or its fragment.For example, they can be to express the hybridoma cell line of endogenous immunoglobulin gene or the mammal cell line that comprises heterogenous expression carrier.These comprise zooblast or people's cell of the normal or improper immortality of any natural death.For example, develop a large amount of suitable host cell system that can secrete complete immunoglobulin (Ig), comprised Chinese hamster ovary celI system, multiple Cos clone, HeLa cell, myeloma cell line, B cell and hybridoma through transforming.Generally at for example Winnacker, FROM GENES TO CLONES, VCH Publishers, N.Y., N.Y., has discussed the purposes of mammalian tissues cell culture in express polypeptide in 1987.Expression vector for mammalian host cell can comprise expression control sequenc, as replication orgin, promotor and enhanser (are for example consulted, Queen etc., (1986) Immunol.Rev.89:49-68), and necessary machining information site, as ribosome bind site, RNA splice site, polyadenylation site and Transcription Termination subsequence.These expression vectors generally contain derived from mammalian genes or from the promotor of mammalian virus.Suitable promotor can be composing type, cell type Idiotype, stage Idiotype and/or adjustable or adjustable.Useful promotor includes but not limited to metallothionein promoter, composing type adenovirus major late promoter, induced by dexamethasone type MMTV promotor, SV40 promotor, MRP polIII promotor, composing type MPSV promotor, tsiklomitsin induction type CMV promotor (as people's early stage CMV promotor immediately), composing type CMV promotor and promoter-enhancer known in the art combination.

For the method for introducing the expression vector that contains polynucleotide of interest sequence according to the type of cell host and difference.For example, calcium chloride transfection is generally used for prokaryotic cell prokaryocyte, and calcium phosphate is processed or electroporation can be used for other cell hosts.(generally consult Sambrook, etc., above).Additive method comprises, for example electroporation, calcium phosphate processing, liposome-mediated conversion, injection and microinjection, biological projectile method, virion, immunoliposome, polycation: nucleic acid conjugate, naked DNA, artificial viral body, with the fusions (Elliot and O'Hare, (1997) Cell88:223) of simplexvirus structural protein VP22, the promoting agent of DNA strengthens picked-up and transduction in vitro.Long-term, high yield for recombinant protein produce, and conventionally will wish stably express.For example, can prepare with the expression vector of the present invention that contains virus replication starting point or endogenous Expression element and selectable marker gene the clone of stably express antibody chain or fragment.Introduce after carrier, can make the cell 1-2 days that grows in enrichment medium, be then transformed into selective medium.The object of selective marker is the resistance of giving selecting, and it exists and allows the cell of the sequence that successful expression introduces to grow in selective medium.Can there is by the tissue culture technique propagation that is suitable for this cell type the cell of the stable transfection of resistance.

(ii) generation of monoclonal antibody of the present invention

Can produce monoclonal antibody (mAb) by multiple technologies, comprise conventional monoclonal anti body method, for example Kohler and Milstein, the standard body hybridoma technique of (1975) Nature 256:495.Can utilize the many technology that produce monoclonal antibody, for example, the virus Transformation of bone-marrow-derived lymphocyte or carinogenicity transform.

Animal system for the preparation of hybridoma is mouse system.In mouse, the generation of hybridoma is perfect method.Separated known in the art for the immunization protocol and the technology that merge through immune splenocyte.Fusion partner (for example rat bone marrow tumour cell) and fusion steps are also known.

Can on by the sequence basis of the mouse monoclonal antibody of preparation mentioned above, prepare chimeric antibody of the present invention or humanized antibody.Available standards Protocols in Molecular Biology obtains the DNA of encoding heavy chain and light chain immunoglobulin (Ig) from object murine hybridoma, and transforms to contain non-mouse (for example people) immunoglobulin sequences.For example, in order to produce chimeric antibody, available methods known in the art are connected to human constant region (for example consulting the U.S. Patent number 4,816,567 of Cabilly etc.) by mouse variable region.In order to produce humanized antibody, available methods known in the art Jiang Shu CDR district inserts in people's framework.For example consult the U.S. Patent number 5530101,5585089,5693762 and 6180370 of the U.S. Patent number 5225539 of Winter and Queen etc.

In certain embodiment, antibody of the present invention is human monoclonal antibodies.The transgenosis of the immune part of available carrier but not mouse system or transchromosomic mice produce this type of human monoclonal antibodies for HER3.These transgenosiss and transchromosomic mice comprise the mouse that is called HuMAb mouse and KM mouse herein, and are referred to as herein " people Ig mouse ".

HuMAb mouse (Medarex, Inc.) contain people's heavy chain (μ and γ) that coding do not reset and human immunoglobulin gene's minigene seat (miniloci) of κ light chain immunoglobulin sequences, and the sudden change of the target of the endogenous μ of inactivation and κ chain gene seat (is for example consulted, Lonberg, Deng, (1994) Nature368 (6474): 856-859).Therefore, this mouse shows and reduces Mouse IgM or the K expressing, and during response immunity, people's heavy chain of introducing and the kind conversion of light chain transgenosis experience and somatic mutation, with produce high-affinity human IgG κ monoclonal antibody (Lonberg, N. etc., (1994) above; Summarize in Lonberg (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg and Huszar, (1995) Intern.Rev.Immunol.13:65-93; And Harding and Lonberg, in (1995) Ann.N.Y.Acad.Sci.764:536-546).The preparation of HuMAb mouse and purposes and the entrained genomic modification of this mouse are further described in Taylor etc., (1992) Nucleic Acids Research 20:6287-6295; Chen etc., (1993) International Immunology 5:647-656; Tuaillon etc., (1993) Proc.Natl.Acad.Sci.USA94:3720-3724; Choi etc., (1993) Nature Genetics 4:117-123; Chen etc., (1993) EMBO is J.12:821-830; Tuaillon etc., (1994) J.Immunol.152:2912-2920; Taylor etc., (1994) International Immunology 579-591; And Fishwild etc., in (1996) Nature Biotechnology 14:845-851, the content of all reference is clearly incorporated herein by reference with its integral body at this.Further consult the U.S. Patent number 5,545,806,5,569,825,5,625,126,5,633,425,5,789,650,5,877,397,5,661,016,5,814,318,5,874,299 and 5,770,429 that is Lonberg and Kay; The U.S. Patent number 5,545,807 of Surani etc.; Be PCT publication number WO 92103918, WO 93/12227, WO94/25585, WO 97113852, WO 98/24884 and the WO 99/45962 of Lonberg and Kay; And the PCT publication number WO 01/14424 of Korman etc.

In another embodiment, can be used on the mouse of carrier's immunoglobulin sequences on transgenosis and transfection chromosome, as the mouse of carrier's heavy chain transgenosis and people's light chain transfection chromosome produces people's antibody of the present invention.In the open WO 02/43478 of PCT of Ishida etc., describe herein this type of mouse that is called " KM mouse " in detail.

In addition, the alternative transgenic animal system of expressing human immunoglobulin gene can obtain in the art, and can be used for preparing HER3 antibody of the present invention.For example, can use the alternative transgenosis system that is called Xenomouse (Abgenix, Inc.).Such as the U.S. Patent number 5,939,598,6,075,181,6,114,598,6,150,584 and 6,162 of Kucherlapati etc., this type of mouse has been described in 963.

In addition, the alternative trans-chromosome animal system of expressing human immunoglobulin gene can obtain in the art, and can be used for preparing HER3 antibody of the present invention.For example, can use and be called the two mouse of carrier's heavy chain transfection chromosome of " TC mouse " and people's light chain transfection chromosome; Tomizuka etc., have described this type of mouse in (2000) Proc.Natl.Acad.Sci.USA 97:722-727.In addition, the milk cow of carrier's heavy chain and light chain transfection chromosome is existing in the art describes (Kuroiwa etc., (2002) Nature Biotechnology 20:889-894), and can be used for preparing HER3 antibody of the present invention.

Also available for the phage display legal system of screening human immunoglobulin gene library for human monoclonal antibodies of the present invention.Set up in the art or below in embodiment, describing this type of phage display method for separating of people's antibody.Consult such as the U.S. Patent number 5,223,409,5,403,484 and 5,571,698 of Ladner etc.; The U.S. Patent number 5,427,908 and 5,580,717 of Dower etc.; The U.S. Patent number 5,969,108 and 6,172,197 of McCafferty etc.; And the U.S. Patent number 5,885,793,6,521,404,6,544,731,6,555,313,6,582,915 and 6,593 of Griffiths etc., 081.

Also available SCID mouse is prepared human monoclonal antibodies of the present invention, in this SCID mouse, reconstruct people's immunocyte, make after immunity, to produce people's antibody response.Such as the U.S. Patent number 5,476,996 and 5,698 of Wilson etc., this type of mouse has been described in 767.

(iii) framework or Fc transformation

Engineered antibody of the present invention comprises wherein to be modified the framework residue in VH and/or VL, for example, to improve those antibody of antibody character.Conventionally carry out this class framework and modify to reduce the immunogenicity of antibody.For example, a kind of method is that one or more framework residues " reverse mutation " are become to corresponding germline sequence.More specifically, the antibody that has experienced somatic mutation contain be different from antibody derived from the framework residue of germline sequence.Can by relatively antibody frame sequence and this antibody derived from germline sequence identify this type of residue.For framework region sequence being reverted to their germline configuration, can be for example by site-directed mutagenesis by somatic mutation " reverse mutation " in germline sequence.This type of " reverse mutation " antibody is also intended to be included in the present invention.

The framework of another type is modified the one or more residues that relate in sudden change framework region or even one or more CDR district, to remove t cell epitope, reduces thus the potential immunogenicity of antibody.The method is also referred to as " going immunization ", and is further described in detail in the U.S. Patent Publication No. 20030153043 of Carr etc.

Except modifying in framework HuoCDR district or as alternative, can transform antibody of the present invention to comprise the modification in Fc district, be commonly used to change one or more functional propertys of antibody, as serum half-life, complement fixation, Fc receptors bind and/or antigen dependent cellular cytotoxicity.In addition, can chemically modified (for example, one or more chemical part can be attached on antibody) or modify antibody of the present invention to change its glycosylation, same for changing one or more functional propertys of antibody.Each in these embodiments has hereinafter been described in further detail.In Fc district, the numbering of residue is the numbering of the EU index of Kabat.

In one embodiment, modify the hinge area of CH1, make to change, for example, increase or reduce the number of cysteine residues in hinge area.In the U.S. Patent number 5,677,425 of Bodmer etc., further described the method.In the hinge area of change CH1, the number of cysteine residues, for example, be used for being convenient to the assembling of light chain and heavy chain, or improve or reduce the stability of antibody.

In another embodiment, the Fc hinge area of sudden change antibody, to reduce the biological half-life of antibody.More specifically, to the CH2-CH3 structural domain interface region of Fc hinge fragment, introduce one or more amino acid mutations, make antibody there is impaired SP (SpA) combination with respect to natural Fc hinge arrangement territory SpA combination.In the U.S. Patent number 6,165,745 of Ward etc., the method has been described in further detail.

Also in other embodiments, by replacing at least one amino-acid residue with different aminoacids residue, change Fc district, to change the effector function of antibody.For example, available different aminoacids residue is replaced one or more amino acid, makes the sub-part of antibody pairing effect have the avidity of change, but retains the antigen binding capacity of parental antibody.Change for example can be to the effector part of its avidity, the C1 component of Fc acceptor or complement.In the U.S. Patent number 5,624,821 and 5,648,260 of Winter etc., the method has all been described in further detail.

In another embodiment, available different aminoacids residue is replaced the one or more amino acid that are selected from amino-acid residue, makes C1q that antibody has a change in conjunction with/or the CDC (CDC) that reduces or eliminate.In the U.S. Patent number 6,194,551 of Idusogie etc., the method has been described in further detail.

In another embodiment, change one or more amino-acid residues, change thus the ability of antibody complement-fixing.In the open WO 94/29351 of PCT of Bodmer etc., the method has been described in further detail.

Also in another embodiment, modify Fc district to improve the ability of antibody-mediated antibody dependent cellular cytotoxicity (ADCC) and/or to increase the avidity of antibody to Fc γ acceptor by modifying one or more amino acid.Further detailed description the method in the open WO 00/42072 of the PCT of Presta.In addition, locate the Fc γ Rl on human IgG1, Fc γ RII, Fc γ RIII and FcRn binding site, and described the variant (consulting Shields etc., (2001) J.Biol.Chen.276:6591-6604) of the combination with raising.

Also in another embodiment, the glycosylation of modified antibodies.For example, can prepare deglycosylated antibody (i.e. this antibody deficiency glycosylation).Can change glycosylation, for example, be used for increasing the avidity of antibody to " antigen ".Can realize this type of carbohydrate and modify by for example changing one or more glycosylation sites in antibody sequence.For example, one or more amino acid substitutions that can cause one or more variable regions framework glycosylation site to be eliminated, eliminate the glycosylation of this site thus.This de-glycosylation can increase the avidity of antibody to antigen.In the U.S. Patent number 5,714,350 and 6,350,861 of Co etc., the method has been described in further detail.

In addition or alternatively, can prepare and there is the glycosylated antibody that changes type, as have reduction fucosyl residues low fucosylation antibody or there is the antibody of the bisection GlcNac structure of increase.The glycosylation pattern of verified this type of change improves the ADCC ability of antibody.For example by express antibody in the host cell of glycosylation machine with change, realize this type of carbohydrate and modify.Described in the art the cell of the glycosylation machine with change, and this cell can be used as expressing therein the host cell of recombinant antibodies of the present invention, produce thus the glycosylated antibody with change.For example, the EP 1,176,195 of Hang etc. has described the clone with the FUT8 gene destroying in function, and this genes encoding fucosyltransferase makes the antibody of expressing in this clone show low fucosylation.It is Lecl3 cell that the open WO 03/035835 of PCT of Presta has described variant Chinese hamster ovary celI, its ability Fucose being attached on the carbohydrate of Asn (297)-connection reduces, also low fucosylated (also the consulting Shields etc., (2002) J.Biol.Chem.277:26733-26740) that causes the antibody of expressing in this host cell.The open WO 99/54342 of PCT of Umana etc. has described such clone, this clone through transformation with the glycosyltransferase of expressing modified glucoprotein (for example, β (1,4)-N acetylglucosaminyl transferase III (GnTIII)), make the antibody of expressing in the clone of transformation show the bisection GlcNac structure increasing, this causes the active raising of ADCC (also consulting Umana etc., (1999) Nat.Biotech.17:176-180) of antibody.

In another embodiment, modified antibodies is to increase its biological half-life.Can use several different methods.For example, as the U.S. Patent number 6,277 of Ward, described in 375, can introduce one or more following sudden changes: T252L, T254S, T256F.Alternatively, as the people's such as Presta U.S. Patent number 5,869,046 and 6,121, described in 022, in order to increase biological half-life, in KeCH1Huo CL district, change antibody to contain the relief receptor binding domain of 2 rings obtaining from the CH2 structural domain in IgG Fc district.

(iv) method of the antibody that transformation changes

Having herein the VH that shows and HER3 antibody of the present invention or its fragment of VL sequence or total length heavy chain and sequence of light chain can be used for producing new HER3 binding antibody by the constant region of modifying total length heavy chain and/or sequence of light chain, VH and/or VL sequence or be attached on it.Therefore, in another aspect of this invention, by the constitutional features of HER3 antibody or its fragment, produce HER3 antibody relevant in structure, this antibody has retained at least one functional property of antibody of the present invention, as in conjunction with people HER3, and one or more functional propertys that suppress HER3.For example, one or more CDR district of antibody of the present invention or its sudden change can be combined with known framework region and/or other CDR restructuring, to produce the HER3 antibody of other modified recombinants of the present invention discussed above.The modification of other types is included in those that describe in first fore portion.Parent material for remodeling method is one or more VH provided herein and/or VL sequence, or its one or more CDR district.In order to produce the antibody of transformation, needn't actual prepare (being expressed as protein) and there is one or more VH provided herein and/or VL sequence, or the antibody in its one or more CDR district.But, the information comprising in sequence is produced to " s-generation " sequence derived from initiation sequence as parent material, then preparation is somebody's turn to do " s-generation " sequence and is expressed as protein.

Therefore, in another embodiment, the invention provides the method for the preparation of the antibody of the structural domain 3 of the combination HER3 being formed by variable fragments of heavy chain sequence and variable region of light chain antibody sequence, this weight chain variable region amino acid sequence has the SEQ of being selected from ID NO:2, 22, 42, 62, 82, 102, 122, 142 and 162 CDR1 sequence, be selected from SEQ ID NO:3, 23, 43, 63, 83, 103, 123, 143 and 163 CDR2 sequence and/or be selected from SEQ ID NO:4, 24, 44, 64, 84, 104, 124, 144 and 164 CDR3 sequence, this variable region of light chain antibody sequence has the SEQ of being selected from ID NO:8, 28, 48, 68, 88, 108, 128, 148 and 168 CDR1 sequence, be selected from SEQ ID NO:9, 29, 49, 69, 89, 109, 129, 149 and 169 CDR2 sequence and/or be selected from SEQ ID NO:10, 30, 50, 70, 90, 110, 130, 150 and 170 CDR3 sequence, at least one amino-acid residue changing in this variable fragments of heavy chain sequence and/or variable region of light chain antibody sequence produces the antibody sequence of at least one change, and the antibody sequence of this change is expressed as to protein.Also can prepare by screening antibodies library the antibody sequence of this change, fixing CDR3 sequence or minimum that this antibody library has as described in US20050255552 must be in conjunction with the diversity in determinant and CDR1 and CDR2 sequence.Can screen according to any triage techniques (as display technique of bacteriophage) that is suitable for screening antibodies from antibody library.

Therefore, in another embodiment, the invention provides the method for the preparation of the antibody of the structural domain 3-4 of the combination HER3 being formed by variable fragments of heavy chain sequence and variable region of light chain antibody sequence, this weight chain variable region amino acid sequence has the SEQ of being selected from ID NO:182, 202, 222, 242, 262, 282, 302, 322, 342, 362, 382, 402, 422, 442, 462, 482, 502, 522, 542, 562, 582, 602, 622, 642, 662 and 682 CDR1 sequence, be selected from SEQ ID NO:183, 203, 223, 243, 263, 283, 303, 323, 343, 363, 383, 403, 423, 443, 463, 483, 503, 523, 543, 563, 583, 603, 623, 643, 663 and 683 CDR2 sequence and/or be selected from SEQ ID NO:184, 204, 224, 244, 264, 284, 304, 324, 344, 364, 384, 404, 424, 444, 464, 484, 504, 524, 544, 564, 584, 604, 624, 644, 664 and 684 CDR3 sequence, this variable region of light chain antibody sequence has the SEQ of being selected from ID NO:188, 208, 228, 248, 268, 288, 308, 328, 348, 368, 388, 408, 428, 448, 468, 488, 508, 528, 548, 568, 588, 608, 628, 648, 668 and 688 CDR1 sequence, be selected from SEQ ID NO:189, 209, 229, 249, 269, 289, 309, 329, 349, 369, 389, 409, 429, 449, 469, 489, 509, 529, 549, 569, 589, 609, 629, 649, 669 and 689 CDR2 sequence and/or be selected from SEQ ID NO:190, 210, 230, 250, 270, 290, 310, 330, 350, 370, 390, 410, 430, 450, 470, 490, 510, 530, 550, 570, 590, 610, 630, 650, 670 and 690 CDR3 sequence, at least one amino-acid residue changing in this variable fragments of heavy chain sequence and/or variable region of light chain antibody sequence produces the antibody sequence of at least one change, and the antibody sequence of this change is expressed as to protein.Also can prepare by screening antibodies library the antibody sequence of this change, fixing CDR3 sequence or minimum that this antibody library has described in US20050255552 must be in conjunction with the diversity in determinant and CDR1 and CDR2 sequence.Can screen according to any triage techniques (as display technique of bacteriophage) that is suitable for screening antibodies from antibody library.

Standard molecular biological technique can be used for preparation and expresses the antibody sequence changing.By the antibody of the antibody sequence coding of this change, be the antibody that retains a kind of, the some or all of functional propertys of antibody as herein described or its fragment, this functional property includes but not limited to: with people and/or cynomolgus monkey HER3 specific binding; Antibody suppresses HER3 biological activity in conjunction with HER3 and by suppressing HER signal granting activity in phosphoric acid-HER measures.

Available standard test and/or mensuration described herein in available this area, those as shown in embodiment (for example, ELISA) are assessed the functional property of the antibody through changing.

In some embodiment of the method for engineered antibody of the present invention, can introduce randomly or optionally sudden change along all or part of antibody or fragment coding sequence, and can be by combination activity and/or other functional propertys of the resulting modified HER3 antibody of screening described herein.Mutation method has been described in the art.For example, the open WO 02/092780 of the PCT of Short has described with saturation mutagenesis, synthetic be linked and packed or it combines the method producing and screening antibodies suddenlys change.Alternatively, the open WO 03/074679 of the PCT of Lazar etc. has described the method for optimizing antibody physico-chemical property with calculating screening method.

The sign of antibody of the present invention

Can measure and characterize antibody of the present invention by several functions.For example, can in phosphoric acid as herein described-HER is measured, by suppressing HER signal, provide to suppress bioactive ability by them, they for example, characterize in conjunction with, their abilities that the granting of HER3 downstream signal is blocked in the resistance of proteolysis and their with the avidity of HER3 albumen (, people and/or cynomolgus monkey HER3), epi-position.Available several different methods is measured the signal granting of HER3 mediation.For example, can by (i) measure phosphoric acid-HER3, (ii) measure HER3 or other downstream signals provide phosphorylation, (iii) part blocking-up as herein described mensurations of albumen (for example Akt), formations of (iv) heterodimer, (v) HER3 dependent gene expression characteristic, (vi) acceptor internalization and (vii) cell phenotype of HER3 driving (for example breeding) monitor HER signal transduction path.

Can detect by direct mark object antibody the ability of antibodies HER3, or traget antibody not, with multiple sandwich assay mode indirect detection known in the art combination.

In some embodiments, HER3 antibody blocking with reference to the combination of HER3 antibody and HER3 or with reference to HER3 antibody competition, be combined HER3.They can be total man HER3 antibody mentioned above.They can be also in conjunction with other mouse of identical epi-position, chimeric or humanization HER3 antibody with reference antibody.Blocking-up reference antibody in conjunction with or show tested HER3 antibodies and the same or analogous epi-position of the defined epi-position of reference antibody with the ability of reference antibody competition combination, or combination with reference to HER3 antibody in conjunction with the enough approaching epi-position of epi-position.This antibody-like especially may be shared the advantageous feature identifying for reference antibody.Can be by the ability that for example competition combination is measured blocking-up reference antibody or competed with reference antibody.Use competition in conjunction with measuring, the ability of the antibody suppression reference antibody that inspection is tested and the specific binding of common antigen (for example HER3 polypeptide or protein).If excessive test antibody suppresses the combination of reference antibody substantially, the specific binding of the competition of test antibody and reference antibody and antigen.Substantially suppressing known test antibody makes the specific binding of reference antibody reduce at least 10%, 25%, 50%, 75% or 90% conventionally.

Many known competitions can be used to assess HER3 antibody in conjunction with mensuration and are combined with the competition of HER3 with reference to HER3 antibody.This comprises, such as the direct or indirect radioimmunoassay of solid phase (RIA), the direct or indirect enzyme immunoassay of solid phase (EIA), sandwich competition assay (seeing the people such as Stahli, (1983) Methods in Enzymology 9:242-253); Direct vitamin H-the avidin of solid phase EIA (seeing the people such as Kirkland, (1986) J.Immunol.137:3614-3619); The direct marker determination of solid phase, the direct mark sandwich assay of solid phase (are shown in Harlow & Lane, above); Use the direct mark RIA of solid phase (seeing the people such as Morel, (1988) Molec.Immunol.25:7-15) of I-125 marker; Direct vitamin H-the avidin of solid phase EIA (people such as Cheung, (1990) Virology176:546-552); And direct mark RIA people such as (, (1990) Scand.J.Immunol.32:77-82) Moldenhauer.Conventionally, this mensuration relates to any in the reference antibody that uses the purifying antigen that is combined in solid phase surface or cell, this solid phase surface or cell to carry unlabelled test HER3 binding antibody and mark.Amount by the marker of mensuration and solid phase surface or Cell binding under the existence of test antibody is measured competitive inhibition.The common excessive existence of test antibody.Owing to there being steric hindrance, the antibody of identifying by competition assay (competition antibody) comprises the antibody of the epi-position combination identical with the epi-position of reference antibody combination and the antibody of the adjacent epi-position combination that the epi-position of being combined with reference antibody enough approaches.

In order whether to measure selected HER3 monoclonal antibody in conjunction with unique epi-position, available commercial reagent (for example, from Pierce, Rockford, the reagent of IL) carries out biotinylation to each antibody.The coated elisa plate of available HER3 polypeptide is used the competition research of unlabelled monoclonal antibody and biotinylated monoclonal antibody.The combination of the biotinylated MAb of available streptavidin-alkaline phosphatase probe in detecting.In order to measure the isotype of the HER3 binding antibody of purifying, can carry out isotype ELISA.For example, the anti-human IgG of available 1 μ g/ml is in the hole of 4 ℃ of coated microtiter plates that spend the night.With after 1%BSA sealing, make plate to impinging upon envrionment temperature, react 1-2 hour with the isotype of 1 μ g/ml or mono-clonal HER3 antibody still less or purifying.The probe reaction that then can make hole and human IgG l or people IgM specific alkaline phosphatase put together.Then plate developed the color and analyze, thereby can determine the isotype of the antibody of purifying.

For proof mono-clonal HER3 antibody and the combination of expressing the viable cell of HER3 polypeptide, can use flow cytometry.In brief, in the PBS that the clone (cultivating) of expressing HER3 can contained to 0.1%BSA and 10% foetal calf serum with the HER3 binding antibody of multiple concentration under standard growth conditions, mix, and hatch 1 hour at 4 ℃.After washing, dyeing under identical condition and making cell and fluorescein-labeled anti-human IgG antibody response with first antibody.Can pass through FACScan instrumental analysis sample, use scattering of light and the sorting of lateral scattering performance unicellular.Except Flow Cytometry Assay or replace Flow Cytometry Assay, can use the alternative mensuration of utilizing fluorescent microscopy.Completely by mentioned above, cell is dyeed and passes through fluorescence microscopy cell.This method allows the visual of individual cells, but depends on that antigen density can have the susceptibility of reduction.

Can further test the reactivity of antibody of the present invention or its fragment and HER3 polypeptide or antigen fragment by Western trace.In brief, can prepare HER3 polypeptide or the fusion rotein of purifying, or from the cell extract of expressing the cell of HER3, then carry out sodium dodecyl sulfate-polyacrylamide gel electrophoresis.After electrophoresis, the antigen separating is transferred to nitrocellulose filter, with 10% foetal calf serum sealing, and surveys by monoclonal antibody to be measured.Available anti-human IgG alkaline phosphatase detects the combination of human IgG, and develops the color with BCIP/NBT substrate tablet (Sigma Chem.Co., St.Louis, MO).

Available multiple playback mode is assessed effect and the specificity of HER3 antibody in the mensuration based on cell of the heterodimer formation of part induction.Can assess activity by following one or more:

(i) for example, in target cell system (MCF-7 breast cancer cell) inhibition of the allos dimerization of HER2 and other EGF family members' part induction.Available receptor specific antibody is from cell lysate immunoprecipitation HER2 mixture, after electrophoresis/Western shifts, by surveying with the antibody of other EGF acceptors, can analyze the shortage/existence of other EGF acceptors in mixture and biology associated ligands thereof.

(ii) inhibition of the signal transduction path that the heterodimer being activated by part activates.With the combination of HER3 seemingly other members of EGF family receptors after ligand binding, cause the key that maximum cell is replied.In the situation that the HER3 of kinase deficiency, HER2 provides functional tyrosine kinase domain, makes it possible to occur signal granting after somatomedin ligand binding.Therefore, can in the situation that lacking and having inhibitor, for example, with part (adjusting albumen), process the cell of coexpression HER2 and HER3, and monitor the impact on HER3 tyrosine phosphorylation by many modes, these modes comprise from treated cell lysate immunoprecipitation HER3, use subsequently the Western trace (details is shown in that Agus above) of antiphosphotyrosine antibody.Alternatively, can be by the HER3 from solubilising lysate being caught to the hole with 96 coated orifice plates of anti-HER3 receptor antibody, and use such as people such as Waddleton, the antiphosphotyrosine antibody of the europium mark that (2002) Anal.Biochem.309:150-157 implements is measured tyrosine phosphorylation level and is developed high throughput assay.

In the wider expansion of this method, the effector molecule that the known acceptor heterodimer downstream activating is activated (can carry out trace by the lysate immunoprecipitation from treated and with the antibody that detects the activation form of these protein as Mitogen-activated Protein Kinase Cascades (MAPK) with Akt), or directly analyze by analyzing the ability of these protein modification/activation specific substrates.

(iii) inhibition of the cell proliferation of part induction.Known various kinds of cell is the combination of coexpression ErbB acceptor, for example much mammary cancer and prostate cancer cell line.Can in 24/48/96-well format, measure, read increase (violet staining) based on relevant DNA synthetic (tritium-labeled thymidine mixes), cell count etc.

Available multiple playback mode is assessed effect and the specificity of HER3 antibody in the mensuration based on cell of non-ligand dependent homology and heterodimer formation.For example, because spontaneous dimer forms, HER2 crosses and expresses the non-ligand dependent activation that triggers kinase domain.Cross HER2 and other HER molecules (as HER1, HER3 and HER4) of expressing and produce homology or heterodimer.

The ability of the tumor growth of the tumor xenogeneic graft of antibody or its fragment blocking-up human tumor cell line, the tumour generation phenotype of known this clone depends at least partly the part that HER3 heterodimer cell signal provides and activates, such as BxPC3 pancreatic cancer cell etc.This ability can be in the mouse of immunocompromised host assessment separately, or with the suitable cytotoxic agent combined evaluation of clone for discussed.The example of functional examination has also been described in embodiment chapters and sections below.

Prevention and therepic use

The invention provides by the antibody of the present invention or its fragment that have the individuality needing to use significant quantity are treated to the disease relevant to HER3 signal transduction path or the method for obstacle.In specific embodiments, the invention provides by have the individuality needing to use the antibody of the present invention of significant quantity its fragment is treated or preventing cancer (for example, mammary cancer, colorectal carcinoma, lung cancer, multiple myeloma, ovarian cancer, liver cancer, cancer of the stomach, carcinoma of the pancreas, acute myeloid leukaemia, chronic myelogenous leukemia, osteosarcoma, squamous cell carcinoma, Peripheral Nerve Sheath Tumors, schwannoma, head and neck cancer, bladder cancer, the esophageal carcinoma, the Barretts esophageal carcinoma, glioblastoma, research of clear cell sarcoma of soft tissue, malignant mesothe, neurofibromatosis, kidney, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia and endometriosis) method.In some embodiments, the invention provides by the antibody of the present invention that has the individuality needing to use significant quantity being treated or the method for the cancer that prevention is relevant to HER3 signal transduction path.

In specific embodiments, the present invention is for the method for the treatment cancer relevant to HER3 signal transduction path, this cancer includes but not limited to mammary cancer, colorectal carcinoma, lung cancer, multiple myeloma, ovarian cancer, liver cancer, cancer of the stomach, carcinoma of the pancreas, acute myeloid leukaemia, chronic myelogenous leukemia, osteosarcoma, squamous cell carcinoma, Peripheral Nerve Sheath Tumors, schwannoma, head and neck cancer, bladder cancer, the esophageal carcinoma, the Barretts esophageal carcinoma, glioblastoma, research of clear cell sarcoma of soft tissue, malignant mesothe, neurofibromatosis, kidney, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia and endometriosis.

Also can treat or prevent the other diseases relevant to the HER3 signal granting of abnormal or defect by antibody of the present invention or its fragment, this disease includes but not limited to that respiratory tract disease, osteoporosis, osteoarthritis, POLYCYSTIC KIDNEY DISEASE, diabetes, schizophrenia, vascular disease, heart disease, non-carcinogenic proliferative disease, fibrosis and nerve degenerative diseases are as alzheimer's disease.

Be suitable for comprising the standard care agent known in the art that can regulate ErbB signal transduction path with the medicine of HER3 antibody combined therapy.The suitable example that is used for the standard care agent of HER2 includes but not limited to He Saiting (Herceptin) and lapatinibditosylate (Tykerb).For the suitable example of the standard care agent of EGFR, include but not limited to that above-mentioned Iressa (Iressa), Erlotinib (Tarceva), Erbitux (Erbitux) and dimension gram replace than (Vectibix).May be suitable for including but not limited to the other drug of HER3 antibody combined therapy those medicines that regulate receptor tyrosine kinase, g protein coupled receptor, growth/survival signaling transduction pathway, nuclear hormone receptor, apoptosis pathway, cell cycle and blood vessel to occur.

Diagnostic uses

On the one hand, the present invention includes in for example, background at biological sample (, blood, serum, cell, tissue) or suffer from the diagnostic assay of measuring HER3 and/or expression of nucleic acid and HER3 protein function in cancer or the individuality of risk in suffering from cancer.

Diagnostic assay (as competitive assay) depends on the analogue (" tracer ") of mark and the ability that test sample analytes is competed the combining site of limited quantity on common binding partners.Binding partners does not generally dissolve before competition or after competition, and the tracer and the analyte that are then attached on binding partners separate with unconjugated tracer and analyte.By pouring out (wherein binding partners does not dissolve in advance) or realizing this separately by centrifugal (wherein binding partners is at competing reaction postprecipitation).As the amount by mark substance is measured, the amount of the test amount of sample analytes and the tracer of combination is inversely proportional to.The dose response curve of the analyte of preparation known quantity, and compare with test result, the amount of the analyte existing in sample with quantitative assay, tested.When using enzyme as detectable label, these mensuration are called ELISA system.In the mensuration of this form, the competitive binding between antibody and HER3 antibody causes the HER3 of combination, and preferred HER3 epi-position of the present invention is measuring of antibody in serum sample, and the most especially the inhibition antibody in serum sample measures.

The significant advantage of measuring is directly to suppressing antibody (disturbing HER3 combination, especially those antibody of epi-position combination), to measure.This mensuration (particularly ELISA test form) has considerable application in clinical setting and conventional blood screening.

Another aspect of the present invention is provided for measuring HER3 expression of nucleic acid in individuality or the method for HER3 activity, is suitable therapeutical agent or the preventive (being called " pharmacogenomics " herein) of this individual selection thus.Pharmacogenomics allows the genotype based on individual to select promoting agent (for example medicine) for example, for individual therapeutic or preventative processing (, check individual genotype determine the ability that this individuality is replied particular active agent).

Another aspect of the present invention relates to monitors promoting agent (for example medicine) to the expression of HER3 or active impact in clinical trial.

Pharmaceutical composition

In order to prepare pharmaceutical composition or the aseptic composite that comprises antibody or its fragment, by antibody or its fragment and pharmaceutically acceptable carrier or mixed with excipients.Composition can contain be in addition suitable for treatment or preventing cancer (mammary cancer, colorectal carcinoma, lung cancer, multiple myeloma, ovarian cancer, liver cancer, cancer of the stomach, carcinoma of the pancreas, acute myeloid leukaemia, chronic myelogenous leukemia, osteosarcoma, squamous cell carcinoma, Peripheral Nerve Sheath Tumors, schwannoma, head and neck cancer, bladder cancer, the esophageal carcinoma, glioblastoma, research of clear cell sarcoma of soft tissue, malignant mesothe, neurofibromatosis, kidney, melanoma, the Barretts esophageal carcinoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia and endometriosis) one or more therapeutical agents.

Can be by mixing to prepare the preparation of therapeutical agent and diagnostic reagent with physiologically acceptable carrier, vehicle or stablizer, its can be in for example freeze-drying the form of powder, slurry, the aqueous solution, emulsion or suspension (for example see, the people such as Hardman, (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington:The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, waits people's (volume) (1993) Pharmaceutical Dosage Forms:Parenteral Medications, Marcel Dekker, NY; Lieberman, waits people's (volume) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, waits people's (volume) (1990) Pharmaceutical Dosage Forms:Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, N.Y.).

The selection of therapeutical agent application program depends on a number of factors, and comprises the serum of entity or organizes immunogenicity and the accessibility of target cell in bio-matrix of turnover rate, symptom level, entity.In certain embodiments, application program maximizes in the amount that meets the therapeutical agent that makes to be delivered to patient in the level of acceptable side effect.The amount of the biological agent of therefore, sending depends in part on concrete entity and sanatory seriousness.Antibody, cytokine and the micromolecular guidance of selecting appropriate dose is availablely (for example to see Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub.Ltd, Oxfordshire, UK; Kresina (volume) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, N.Y.; Bach (volume) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, N.Y.; The people such as Baert, (2003) New Engl.J.Med.348:601-608; The people such as Milgrom, (1999) New Engl.J.Med.341:1966-1973; The people such as Slamon, (2001) New Engl.J.Med.344:783-792; The people such as Beniaminovitz, (2000) New Engl.J.Med.342:613-619; The people such as Ghosh, (2003) New Engl.J.Med.348:24-32; The people such as Lipsky, (2000) New Engl.J.Med.343:1594-1602).

By clinician, determine suitable dosage, for example, use the parameter or the factor that affect treatment or expected impact treatment known in this field or that suspect.Dosage generally, from the amount a little less than optimal dose, slightly increases subsequently, until reach the desirable or best effect with respect to any negative side-effects.Important diagnostic measures comprises these symptoms, for example the diagnostic measures of the level of the inflammatory cytokine of inflammation or generation.

Can change the actual dose level of activeconstituents in pharmaceutical composition of the present invention, to obtain the amount of active ingredient, this amount is replied effectively reaching desirable treatment with regard to concrete patient, composition and method of application, and to patient's nontoxicity.Selected dosage level will depend on multi-medicament dynamic metabolism factor, other drug, compound and/or the material that comprises the activity, route of administration, time of application, the discharge rate of particular compound utilizing, the time length for the treatment of of concrete composition that the present invention utilizes or its ester, salt or acid amides, uses with the concrete combination of compositions of utilizing, patient's to be treated age, sex, body weight, illness, general health and passing medical history, and the known similar factor of medical field.

Can pass through continuous infusion, or by the interval by for example 1 day, 1 week or weekly 1-7 administration the composition that comprises antibody of the present invention or its fragment is provided.Can be by intravenously, subcutaneous, local, oral, nose, rectum, intramuscular, brain or provide dosage by suction.Concrete dosage relates to avoid the maximal dose of remarkable adverse side effect or the dosage of dose frequency.Weekly total dose can be at least 0.05 μ g/kg body weight, at least 0.2 μ g/kg, at least 0.5 μ g/kg, at least 1 μ g/kg, at least 10 μ g/kg, at least 100 μ g/kg, at least 0.2mg/kg, at least 1.0mg/kg, at least 2.0mg/kg, at least 10mg/kg, at least 25mg/kg or at least 50mg/kg (for example see, the people such as Yang, (2003) New Engl.J.Med.349:427-434; The people such as Herold, (2002) New Engl.J.Med.346:1692-1698; The people such as Liu, (1999) J.Neurol.Neurosurg.Psych.67:451-456; The people such as Portielji, (2003) Cancer Immunol.Immunother.52:133-144).The optimal dosage of antibody or its fragment and antibody or polypeptide roughly the same, mole/basis of kg body weight on.The desirable plasma concentration of antibody or its fragment greatly about mole/basis of kg body weight on.Dosage can be at least 15 μ g, at least 20 μ g, at least 25 μ g, at least 30 μ g, at least 35 μ g, at least 40 μ g, at least 45 μ g, at least 50 μ g, at least 55 μ g, at least 60 μ g, at least 65 μ g, at least 70 μ g, at least 75 μ g, at least 80 μ g, at least 85 μ g, at least 90 μ g, at least 95 μ g or at least 100 μ g.The dosage that individuality is used can be at least 1,2,3,4,5,6,7,8,9,10,11 or 12 time or more.

For antibody of the present invention or its fragment, the dosage that patient is used can be 0.0001mg/kg to 100mg/kg weight in patients.Dosage can 0.0001mg/kg to 20mg/kg, 0.0001mg/kg to 10mg/kg, 0.0001mg/kg to 5mg/kg, 0.0001 to 2mg/kg, 0.0001 to 1mg/kg, 0.0001mg/kg to 0.75mg/kg, 0.0001mg/kg to 0.5mg/kg, 0.0001mg/kg to 0.25mg/kg, 0.0001 to 0.15mg/kg, 0.0001 to 0.10mg/kg, 0.001 to 0.5mg/kg, 0.01 to 0.25mg/kg or 0.01 to 0.10mg/kg weight in patients between.

Can be multiplied by the dosage that calculates antibody of the present invention or its fragment in the dosage to be administered of mg/kg in order to kilogram weight in patients of (kg) meter.The dosage of antibody of the present invention or its fragment can be 150 μ g/kg or lower, 125 μ g/kg or lower, 100 μ g/kg or lower, 95 μ g/kg or lower, 90 μ g/kg or lower, 85 μ g/kg or lower, 80 μ g/kg or lower, 75 μ g/kg or lower, 70 μ g/kg or lower, 65 μ g/kg or lower, 60 μ g/kg or lower, 55 μ g/kg or lower, 50 μ g/kg or lower, 45 μ g/kg or lower, 40 μ g/kg or lower, 35 μ g/kg or lower, 30 μ g/kg or lower, 25 μ g/kg or lower, 20 μ g/kg or lower, 15 μ g/kg or lower, 10 μ g/kg or lower, 5 μ g/kg or lower, 2.5 μ g/kg or lower, 2 μ g/kg or lower, 1.5 μ g/kg or lower, 1 μ g/kg or lower, 0.5 μ g/kg or lower, or 0.5 μ g/kg or lower weight in patients.

The unitary dose of antibody of the present invention or its fragment can be 0.1mg to 20mg, 0.1mg to 15mg, 0.1mg to 12mg, 0.1mg to 10mg, 0.1mg to 8mg, 0.1mg to 7mg, 0.1mg to 5mg, 0.1 to 2.5mg, 0.25mg to 20mg, 0.25 to 15mg, 0.25 to 12mg, 0.25 to 10mg, 0.25 to 8mg, 0.25mg to 7m g, 0.25mg to 5mg, 0.5mg to 2.5mg, 1mg to 20mg, 1mg to 15mg, 1mg to 12mg, 1mg to 10mg, 1mg to 8mg, 1mg to 7mg, 1mg to 5mg, or 1mg to 2.5mg.

The dosage of antibody of the present invention or its fragment can reach at least 0.1 μ g/ml in individuality, at least 0.5 μ g/ml, at least 1 μ g/ml, at least 2 μ g/ml, at least 5 μ g/ml, at least 6 μ g/ml, at least 10 μ g/ml, at least 15 μ g/ml, at least 20 μ g/ml, at least 25 μ g/ml, at least 50 μ g/ml, at least 100 μ g/ml, at least 125 μ g/ml, at least 150 μ g/ml, at least 175 μ g/ml, at least 200 μ g/ml, at least 225 μ g/ml, at least 250 μ g/ml, at least 275 μ g/ml, at least 300 μ g/ml, at least 325 μ g/ml, at least 350 μ g/ml, at least 375 μ g/ml, or the serum titer of at least 400 μ g/ml.Alternatively, the dosage of antibody of the present invention or its fragment can reach at least 0.1 μ g/ml in individuality, at least 0.5 μ g/ml, at least 1 μ g/ml, at least 2 μ g/ml, at least 5 μ g/ml, at least 6 μ g/ml, at least 10 μ g/ml, at least 15 μ g/ml, at least 20 μ g/ml, at least 25 μ g/ml, at least 50 μ g/ml, at least 100 μ g/ml, at least 125 μ g/ml, at least 150 μ g/ml, at least 175 μ g/ml, at least 200 μ g/ml, at least 225 μ g/ml, at least 250 μ g/ml, at least 275 μ g/ml, at least 300 μ g/ml, at least 325 μ g/ml, at least 350 μ g/ml, at least 375 μ g/ml, or the serum titer of at least 400 μ g/ml.

The dosage of antibody of the present invention or its fragment can repeat, and can within least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months or at least 6 months, use at interval.

The concrete patient factor and becoming that effectively amount can be depending on the seriousness of the general health such as treated illness, patient, the method and approach of using and dosage and side effect (is for example shown in, the people such as Maynard, (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch Publ., London, UK).

Route of administration can be by for example, part or dermal application, by in intravenously, intraperitoneal, brain, in intramuscular, intraocular, intra-arterial, myelencephalon, intralesional injection or infusion, or (for example see by sustained release system or implant, the people such as Sidman, (1983) Biopolymers 22:547-556; The people such as Langer, (1981) J.Biomed.Mater.Res.15:167-277; Langer (1982) Chem.Tech.12:98-105; The people such as Epstein, (1985) Proc.Natl.Acad.Sci.USA82:3688-3692; The people such as Hwang, (1980) Proc.Natl.Acad.Sci.USA 77:4030-4034; U.S. Patent number 6,350,466 and 6,316,024).If desired, composition can also comprise that solubilizing agent and local anesthetic (as lignocaine) alleviate the pain of injection site.In addition, also can utilize lung to use, for example, by using sucker or atomizer, and there is the preparation of smog agent.For example see U.S. Patent number 6,019,968,5,985,320,5,985,309,5,934,272,5,874,064,5,855,913,5,290,540 and 4,880,078, and PCT publication number WO 92/19244, WO 97/32572, WO97/44013, WO 98/31346 and WO 99/66903, it is incorporated herein by reference with its integral body separately.

Also can via one or more route of administration, use composition of the present invention with one or more in several different methods known in the art.Those of skill in the art will understand, and the approach of using and/or pattern will depend on desirable result and become.Select route of administration for antibody of the present invention or its fragment to comprise intravenously, intramuscular, intracutaneous, intraperitoneal, subcutaneous, backbone or other parenteral route of administration, for example, by injection or infusion.Parenteral is used the mode of administration that can represent except intestines and topical application, conventionally by injection, undertaken, and include but not limited in intravenously, intramuscular, intra-arterial, sheath, in capsule, in socket of the eye, in heart, intracutaneous, intraperitoneal, under tracheae, subcutaneous, epidermis, under intraarticular, capsule, under arachnoid membrane, in backbone, epidural and breastbone inner injection and infusion.Alternatively, composition of the present invention can be via non-parenteral approach, and for example local, epidermis or mucous membrane route of administration are used, and for example nose is interior, oral, vagina, rectum, hypogloeeis or topical application.In one embodiment, by infusion, use antibody of the present invention or its fragment.In another embodiment, subcutaneous administration Multispecific epitope binding proteins of the present invention.

If use antibody of the present invention or its fragment in controlled release system or sustained release system, available pump realizes controlled release or sustained release (is shown in Langer, above; Sefton, (1987) CRC Crit.Ref Biomed.Eng.14:20; The people such as Buchwald, (1980), Surgery 88:507; The people such as Saudek, (1989) N.Engl.J.Med.321:574).Useful polymeric material reaches controlled release or the sustained release of the present invention's treatment (for example to be seen, Medical Applications of Controlled Release, Langer and Wise (volume), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (volume), Wiley, New York (1984); Ranger and Peppas, (1983) J.Macromol.Sci.Rev.Macromol.Chem.23:61; Also see the people such as Levy, (1985) Science 228:190; The people such as During, (1989) Ann.Neurol.25:351; The people such as Howard, (1989) J.Neurosurg.71:105); U.S. Patent number 5,679,377; U.S. Patent number 5,916,597; U.S. Patent number 5,912,015; U.S. Patent number 5,989,463; U.S. Patent number 5,128,326; PCT publication number WO 99/15154; With PCT publication number WO 99/20253.The example of the polymkeric substance using in extended release preparation includes but not limited to, polymethyl acrylic acid 2-hydroxy methacrylate, polymethylmethacrylate, polyacrylic acid, ethylene-vinyl acetate copolymer, polymethyl acrylic acid, PGA (PLG), polyanhydride, PVP, polyvinyl alcohol, polyacrylamide, polyoxyethylene glycol, poly(lactic acid) (PLA), polylactic-co-glycolic acid (PLGA) and poe.In one embodiment, the polymkeric substance using in extended release preparation is inertia, stable, aseptic and biodegradable containing leachable impurity, while storing.Controlled release system or sustained release system can be placed in preventative or therapeutic target near, therefore only need a part for body dose (for example to see, Goodson, in Medical Applications of Controlled Release, above, the 2nd volume, 115-138 page (1984)).

At Langer, (1990), have discussed controlled release system in the summary of Science 249:1527-1533.Can produce the extended release preparation that comprises one or more antibody of the present invention or its fragment by any technology well known by persons skilled in the art.See, for example, U.S. Patent number 4,526,938; The open WO 91/05548 of PCT; The open WO 96/20698 of PCT; The people such as Ning, (1996), Radiotherapy & Oncology 39:179-189; The people such as Song, (1995) PDA Journal of Pharmaceutical Science & Technology 50:372-397; The people such as Cleek, (1997) Pro.Int'l.Symp.Control.Rel.Bioact.Mater.24:853-854; And the people such as Lam, (1997) Proc.Int'l.Symp.Control Rel.Bioact.Mater.24:759-760, it is incorporated herein by reference with its integral body separately.

If topical application antibody of the present invention or its fragment, can be formulated as them the form of ointment, frost, transdermal patch, emulsion, gel, shampoo, spraying, aerosol, solution, emulsion, or other forms well known to those skilled in the art.For example see Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, the 19th edition, Mack Pub.Co., Easton, Pa. (1995).To non-sprayable topical formulations, conventionally use stickiness to semisolid or solid form, this form comprises the carrier compatible with topical application or one or more vehicle, and has in some cases the dynamic viscosity that is greater than water.Suitable preparation includes but not limited to, solution, suspension, emulsion, frost, ointment, powder, liniment, ointment etc., if wished, by said preparation sterilizing or for example, with auxiliary agent (sanitas, stablizer, wetting agent, damping fluid or salt), mix to affect multiple performance, for example osmotic pressure.Other suitable topical formulations comprise sprayable aerosol, wherein activeconstituents (in some cases with the combination of solid or liquid inert support) is packaged in there is pressurization volatile matter (for example, gaseous propellant, for example freonll-11) mixture in or in squeeze bottle.If wish, also can add wetting Agent for Printing Inks or wetting agent in pharmaceutical composition and formulation.The example of this type of supplementary component is well known in the art.

If the composition that intranasal administration comprises antibody or its fragment, can be formulated as it aerosol form, spraying, mist or drops form.Especially, preventive used according to the invention or therapeutical agent can be easily sent with the arosol spray form of the pattern of pressurized package or spraying gun, use suitable propelling agent (for example, Refrigerant 12, trichlorofluoromethane, dichloro tetrafluoro ethane, carbonic acid gas or other suitable gas).The in the situation that of pressurised aerosol, can be by providing the valve of the amount of sending metering to determine dose unit.Can prepare capsule and box (being for example comprised of gelatin) for sucker or insufflator, this capsule and box contain compound and suitable powder base-material as the powder mixture of lactose or starch.

With the second therapeutical agent (for example, cytokine, steroid, chemotherapeutic, microbiotic or radiation) method of jointly using or treating is known in the artly (for example to see, the people such as Hardman, (volume) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, the 10th edition, McGraw-Hill, New York, N.Y.; Poole and Peterson (volume) (2001) Pharmacotherapeutics for Advanced Practice:A Practical Approach, Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (volume) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams & Wilkins, Phila., Pa.).The therapeutical agent of significant quantity can make symptom be reduced by least 10%, at least 20%, at least about 30%, at least 40% or at least 50%.

The adjunctive therapy (for example preventive or therapeutical agent) that can use with antibody of the present invention or its fragment combination can be separated by less than 5 minutes with antibody of the present invention or its fragment, be separated by less than 30 minutes, be separated by 1 hour, be separated by approximately 1 hour, be separated by approximately 1 to approximately 2 hour, be separated by approximately 2 hours to approximately 3 hours, be separated by approximately 3 hours to approximately 4 hours, be separated by approximately 4 hours to approximately 5 hours, be separated by approximately 5 hours to approximately 6 hours, be separated by approximately 6 hours to approximately 7 hours, be separated by approximately 7 hours to approximately 8 hours, be separated by approximately 8 hours to approximately 9 hours, be separated by approximately 9 hours to approximately 10 hours, be separated by approximately 10 hours to approximately 11 hours, be separated by approximately 11 hours to approximately 12 hours, be separated by approximately 12 hours to 18 hours, be separated by 18 hours to 24 hours, be separated by 24 hours to 36 hours, be separated by 36 hours to 48 hours, be separated by 48 hours to 52 hours, be separated by 52 hours to 60 hours, be separated by 60 hours to 72 hours, be separated by 72 hours to 84 hours, be separated by 84 hours to 96 hours or be separated by and use for 96 hours to 120 hours.Can in medical with a patient, use 2 kinds or multiple therapy.

Antibody of the present invention or its fragment and other therapies used capable of circulation.Circulation therapy relates to (for example uses the first therapy, the first preventive or therapeutical agent) for some time, then use the second therapy (for example, the second preventive or therapeutical agent) for some time, alternatively, then (for example use the 3rd therapy, preventive or therapeutical agent) for some time etc., and repeat this sequential application, circulate to reduce that one of therapy is produced to resistance, avoid or reduce the side effect of one of therapy, and/or improve the effect of therapy.

In certain embodiments, can prepare antibody of the present invention or its fragment to guarantee appropriate distribution in vivo.For example, hemato encephalic barrier (BBB) is got rid of many high-hydrophilic compounds.In order to ensure therapeutic compound of the present invention, stride across BBB (if wish), for example they can be formulated in liposome.The relevant method of preparing liposome, is shown in for example U.S. Patent number 4,522,811,5,374,548 and 5,399,331.Liposome can comprise one or more selective transports to the part of specific cells or organ, thereby strengthens targeted delivery of drugs (for example seeing Ranade, (1989) J.Clin.Pharmacol.29:685).Exemplary targeting moiety comprises folic acid or vitamin H (for example seeing the people's such as Low U.S. Patent number 5,416,016); Mannoside (people such as Umezawa, (1988) Biochem.Biophys.Res.Commun.153:1038); Antibody (people such as Bloeman, (1995) FEBS Lett.357:140; The people such as Owais, (1995) Antimicrob.Agents Chemother.39:180); Tensio-active agent albumin A acceptor (people such as Briscoe, (1995) Am.J.Physiol.1233:134); P 120 (people such as Schreier, (1994) J.Biol.Chem.269:9090); Also see K.Keinanen; M.L.Laukkanen (1994) FEBS Lett.346:123; J.J.Killion; I.J.Fidler (1994) Immunomethods 4:273.

The invention provides the scheme of the pharmaceutical composition to having the individuality needing to use separately or comprising antibody of the present invention or its fragment with other therapy combined administrations.Can be to individuality simultaneously or use in turn the therapy (for example, preventive or therapeutical agent) of combined therapy of the present invention.Also the therapy (for example, preventive or therapeutical agent) of using combined therapy of the present invention capable of circulation.Circulation therapy relates to (for example uses the first therapy, the first preventive or therapeutical agent) for some time, then (for example use the second therapy, the second preventive or therapeutical agent) for some time, and repeat this sequential application, circulate to reduce that one of therapy (for example medicine) is produced to resistance, avoid or reduce the side effect of one of therapy (for example medicine), and/or improve the effect of therapy.

Can use to individuality the therapy (for example, preventive or therapeutical agent) of combination treatment of the present invention simultaneously.Term " simultaneously " is not limited to (for example use therapy in the accurate same time, preventive or therapeutical agent), and refer in order and within the timed interval, individuality used to the pharmaceutical composition that comprises antibody of the present invention or its fragment, antibody of the present invention can be worked with other therapies one, to provide than otherwise using the benefit that they improve.For example, can with random order, to individuality, use each therapy in turn at one time or at different time points; Yet if do not used at one time, they should use within the enough approaching time, so that result for the treatment of or the preventive effect of hope to be provided.Can be with the form of any appropriate and the approach by any appropriate to each therapy of experimenter's separate administration.In multiple embodiments, with less than 15 minutes, less than 30 minutes, be separated by less than 1 hour, be separated by approximately 1 hour, be separated by approximately 1 hour to approximately 2 hours, be separated by approximately 2 hours to approximately 3 hours, be separated by approximately 3 hours to approximately 4 hours, be separated by approximately 4 hours to approximately 5 hours, be separated by approximately 5 hours to approximately 6 hours, be separated by approximately 6 hours to approximately 7 hours, be separated by approximately 7 hours to approximately 8 hours, be separated by approximately 8 hours to approximately 9 hours, be separated by approximately 9 hours to approximately 10 hours, be separated by approximately 10 hours to approximately 11 hours, be separated by approximately 11 hours to approximately 12 hours, be separated by 24 hours, be separated by 48 hours, be separated by 72 hours or be separated by and individuality (was for example used to therapy in 1 week, preventive or therapeutical agent).In other embodiments, in medical with a patient, use two or more therapies (for example, preventive or therapeutical agent).

Can in same pharmaceutical composition, to individuality, use preventive or the therapeutical agent of combination treatment.Alternatively, can in the pharmaceutical composition separating, to individuality, use preventive or the therapeutical agent of combination treatment simultaneously.Can to individuality, use preventive or therapeutical agent by identical or different route of administration.

Fully described now the present invention, by following examples and claim, further illustrated the present invention, embodiment and what is claimed is illustratively, is not intended to further restriction.

Embodiment

Embodiment 1: method, material and antibody screening

(i) clone

SK-Br-3, BT-474 and MCF-7 clone is purchased from ATCC, and in the substratum that supplements 10% foetal calf serum (FBS) cellar culture.

(ii) generation of recombinant human, cynomolgus monkey, Mouse and rat HER3 carrier

From mouse brain cDNA (Clontech) pcr amplification mouse HER3 ectodomain and by with Refseq NM_010153 comparatively validate sequence.From Rat-2 cell mRNA reverse transcription rat HER3ECD and by with NM_017218 comparatively validate sequence.Use the RNA (Zyagen Laboratories) from multiple cynomolgus monkey tissue to produce cynomolgus monkey HER3cDNA template, and RT-PCR product cloning is entered (Invitrogen), then carry out two strands order-checking.People HER3 derived from human fetal brain cDNA library (Source) and by with NM_001982 comparatively validate sequence.

In order to produce the recombinant protein of mark, with Pwo Taq polysaccharase (Roche Diagnostics) pcr amplification people, mouse, rat and cynomolgus monkey HER3.The PCR product of amplification is through gel-purified and clone into pDonR201 (Invitrogen) Gateway entry vector (entry vector), this carrier had previously been modified to and had comprised in-frame N-end CD33 leader sequence and C-end TAG, for example FLAG TAG.TAG allows by anti-TAG monoclonal antibody purifying monomeric protein.Target gene flank has AttB1 and AttB2, allows to use clone technology (Invitrogen) recombinate enter be applicable to Gateway proprietary object carrier (for example, pcDNA3.1).With Gateway LR reaction and the proprietary object carrier that contains CMV promotor, carry out recombining reaction, to produce TAG expression vector, although also can use any commercially available carrier.

Produce other restructuring HER3 albumen, it merges HER3 ECD in the protein that produces Fc mark in the upstream of C-end factor X cleavage site and human IgG hinge and Fc structural domain.For this reason, the multiple HER3 ECD of pcr amplification be cloned into carrier (for example, pcDNA3.1), this carrier modification is to contain to meet factor X site-hinge-hFc that the C-end of reading frame merges.The open reading-frame (ORF) flank producing has AttB1 and AttB2 site, for further using recombinant clone technology (Invitrogen) clone.With LR Gateway, react HER3-Fc is transferred to the object expression construct that contains CMV promotor.Use standard rite-directed mutagenesis scheme produces HER3 point mutation expression construct and verifies resulting carrier sequence.

The generation of table 3:HER3 expression vector.HER3 amino acid numbering is based on NP_001973 (people), NP_034283 (mouse) and NP_058914 (rat).

Title	Describe
		People HER3	CD33-[people HER3, residue 20-640]-TAG
Mouse HER3	CD33-[mouse HER3, residue 20-643]-TAG
		Rat HER3	CD33-[rat HER3, residue 20-643]-TAG
Cynomolgus monkey HER3	CD33-[cynomolgus monkey HER3, residue 20-643]-TAG
		HER3D1-2	CD33-[people HER3, residue 20-329]-TAG
HER3D2	CD33-[people HER3, residue 185-329]-TAG
		HER3D3-4	CD33-[people HER3, residue 330-643]-TAG
HER3D3	CD33-[people HER3, residue 330-495]-TAG
		HER3D4	CD33-[people HER3, residue 496-643]-TAG

People HER3-Fc	[people HER3, residue 1-643]-Fc
		Mouse HER3-Fc	[mouse HER3, residue 1-643]-Fc
Cynomolgus monkey HER3-Fc	[cynomolgus monkey HER3, residue 1-643]-Fc
		Rat HER3-Fc	[rat HER3, residue 1-643]-Fc
HER3D2-Fc	[people HER3 residue 207-329]-Fc

(iii) expression of restructuring HER3 albumen

Formerly preadaptation suspension culture and in the proprietary serum free medium of Novartis, grow derived from the clone of HEK293 in the HER3 recombinant protein of express wishing.In the instantaneous 6 orifice plate transfections based on lipofection are measured, carry out small-scale and express checking.By transient transfection, carry out the generation of large-scale protein matter, and at Wave ^tMin bioreactor system (Wave Biotech), with the scale of 10-20L, carry out.Using 1:3 (w:w) ratio with DNA polymine (Polysciences) as plasmid vector.7-10 days harvested cell culture supernatants after transfection, and concentrated by tangential flow filtration and diafiltration before purifying.

(iv) purifying of labelled protein

For example, by collecting cell culture supernatant and hold back filter (Fresenius) with 10 kDa and concentrate 10 times of HER3 albumen that carry out purification of Recombinant mark (, TAG-HER3) by tangential flow filtration.By the Sepharose 4B coupling of anti-TAG monoclonal antibody and CNBr activation being prepared to anti-TAG post by the final ratio of 10mg antibody/mL resin.By the flow velocity of 1-2 mL/ minute, concentrated supernatant is added to the anti-Tag post of 35ml.With PBS, carrying out after baseline washing, with material, neutralization the sterile filtration of 100 mM glycine (pH 2.7) elution of bound.By measuring in the absorbancy at 280 nm places also with the theoretical coefficient of the 0.66 AU/mg mensuration protein concn that is converted.Finally by the order-checking of SDS-PAGE, N-end and LC-MS, characterize the protein of purifying.

(v) Fc Tag purifying

By the flow velocity of 1 ml/ minute, concentrated cell culture supernatant is added to 50 ml a-protein Sepharose Fast Flow posts.With PBS, carrying out after baseline washing, with the 10mM NaH of 10 times of column volumes ₂pO ₄/ 30% (v/v) Virahol pH 7.3 washing columns, then wash with the PBS of 5 times of column volumes.Finally, use material, neutralization the sterile filtration of 50mM Citrate trianion/140mM NaCl (pH 2.7) elution of bound.

(vi) HuCAL or elutriation

For the antibody of selective recognition people HER3, utilized multiple elutriation strategy.With commercially available phage display library MorphoSys HuCAL or library is as the source of antibody variants albumen, and the clone by selection with high binding affinity produces the therapeutic antibodies for people HER3 albumen.Phagemid library based on concept people such as (, (2000) J Mol Biol 296:57-86) Knappik, and utilize technology is come at phage display Fab (WO01/05950 of Lohning).

For the anti-HER3 antibody of separation, by solid phase, solution, full cell and the full cell elutriation of difference method, carry out standard elutriation strategy and RapMAT elutriation strategy.

(vii) solid phase elutriation

In order to identify anti-HER3 antibody, with different restructuring HER3 albumen, carry out multiple solid phase elutriation strategy.In order to carry out each, take turns solid phase elutriation, with the coated Maxisorp plate (Nunc) of HER3 albumen.With the coated plate of the anti-Fc of previous use (goat or mouse anti human IgG, Jackson Immuno Research), anti-Tag antibody or carry out the protein of capture of labels by passive adsorption.With PBS, wash coated plate sealing.With PBS, wash coated flat board 2 times, then add HuCAL or phage-antibody, on shaking table, incubated at room is 2 hours.The phage of elution of bound, adds to intestinal bacteria TG-1 and hatches and carry out phage-infect.Separated infected bacterium being coated on agar plate subsequently.On slave plate, scrape bacterium colony, and rescue and amplification phage.Every kind of HER3 elutriation strategy comprises takes turns elutriation, and contains unique antigen, antigen concentration and washing stringency.

(viii) solution phase elutriation

With multiple biotinylated restructuring HER3 albumen, in the situation that existing or lack neuregulin 1-β 1 (R & D Systems), carry out each and take turns solution phase elutriation.With EZ-, connect sulfo group-NHS-LC biotinylation test kit (Pierce) according to the specification sheets biotinylated protein matter of manufacturers.With PBS, wash magnetic bead (Dynabeads, Dynal) that 800 μ l streptavidins connect once, and seal and spend the night with Chemiblocker (Chemicon).In reaction tubes, hatch HuCAL or phage-antibody and suitable biotinylation HER3.Add streptavidin magnetic bead 20 minutes, and collect magnetic bead with magnetic-particle separator (Dynal).By add the phage that carrys out elution of bound from Dynabeads containing the damping fluid of DTT to each pipe, and add to intestinal bacteria TG-1.By the mode that the mode with describing is identical, carry out phage-infect in solid phase elutriation.Every kind of HER3 elutriation strategy comprises takes turns elutriation, and contains unique antigen, antigen concentration and washing stringency.For the antibody of separated target defined epitope, carried out competition elutriation.In these elutriation strategies, with reference antibody, hatch and pre-sealing HER3, then add HuCAL or phage-antibody.As alternate strategies, with reference antibody, come specificity wash-out and the compound phage-antibody of HER3.

(ix) elutriation based on cell

For cell elutriation, by HuCAL or phage-antibody and about 10 ⁷cell incubated at room 2 hours on turner, then centrifugal.Isolated cell precipitation, wash-out bacteriophage from cell.Collect supernatant and add to intestinal bacteria TG-1 culture, then carrying out said process.Utilize two kinds of strategies based on cell to identify anti-HER3 antibody:

A) full cell elutriation: in this strategy, by multiple complete clone as antigen.

B) the full cell elutriation of difference: in this strategy, antigen is comprised of cell and restructuring HER3 albumen in succession.By the elutriation of carrying out based on cell mentioned above, and when using recombinant protein as antigen, utilize solid phase elutriation scheme.With PBS (2-3X) and PBST (2-3X), wash.

(x) RapMAT ^tMlibrary produces and elutriation

In order to improve antibody binding affinity, maintain the diversity in library simultaneously, make second of solution and solid phase elutriation take turns output and all enter RapMAT ^tMprocess, and the output of the third round of full cell and the full cell elutriation of difference strategy enters this process (people such as Prassler, (2009) Immunotherapy; 1:571-583).By the Fab coding Insert Fragment subclone of the phage of selecting via elutriation is entered to display carrier 25_bla_LHC produces RapMAT ^tMlibrary, and by further digesting and produce H-CDR2RapMAT with specificity restriction enzyme ^tMlibrary or and L-CDR3RapMAT ^tMlibrary.According to storehouse, form with the ripe box of TRIM people such as (, (1994) Nucleic Acids Research 22:5600-5607) Virnekas Insert Fragment is replaced with to H-CDR2 or L-CDR3.Library size is estimated at 8x10 ⁶-1x10 ⁸in individual clone's scope.Produce RapMAT antibody-phage and carry out again two-wheeled solution, solid phase or the elutriation based on cell with described before experimental technique.

Developed especially this elutriation strategy widely of the iterative refinement that relates to library design, with by directly comprising that in elutriation part blocking antibody makes screening depart from from pure Ligand Competition antibody.Secondly, revised the transition process of FAB to IgG, to maximize the recovery of candidate clone, and guaranteed that all selective binding agent are all analyzed in functional examination.From 44 initial elutriations, produce the specificity Her3 binding antibody of Liao Yue28Ge family, the two characteristic of the block ligand dependency of only having San Ge antibody family to show to wish and dependent/non-dependent signal transduction.In conjunction with the separated structural domain 1-2 of Her3 and 2 the A of family.In conjunction with separated structural domain 3-4 rather than the only B of family of structural domain 4 and the C of family of binding domains 3.

Embodiment 2: the transient expression of anti-HER3 IgG

In BioWave20, cultivate and adapt to the HEK293-6E cell suspending.With relevant aseptic DNA:PEI-MIX transient transfection cell further cultivation.After transfection, by using the tangential flow filtration of Fresenius filter to remove cell.With holding back filter (Fresenius), by tangential flow filtration, concentrate not celliferous material, by the aseptic Entkeimung of enriched material cup type (stericup) filter.Aseptic supernatant is stored in to 4 ℃.

Embodiment 3: the purifying of anti-HER3IgG

In cooling cabinet on 100explorer Air chromatographic system, carry out the purifying of IgG, use and there is 25mL from the XK16/20 post (being GE Healthcare) of loading MabSelect SuRe resin.Except loading, all flow velocitys are 3.5mL/ minute, and pressure limitation is 5bar.With the PBS balance columns of 3 times of column volumes, the filtered fermentation supernatant of loading then.Use PBS washing column.Using from Citrate trianion/NaCl (pH 4.5) and start and linearity drops to the pH gradient elution IgG of Citrate trianion/NaCl (pH 2.5), is then the constant step of identical pH 2.5 damping fluids.Merge the fraction that contains IgG, neutralization, and sterile filtration (Millipore Steriflip, 0.22um) immediately.Measure OD ₂₈₀, and calculate protein concn based on sequence data.Test respectively gathering (SEC-MALS) and the purity (SDS-PAGE and MS) of consolidated material (pool).

Expression and the purifying of embodiment 4:HuCAL-Fab antibody in intestinal bacteria

With the YT substratum that has supplemented paraxin, in diastatochromogenes, carry out the expression of the Fab fragment of coding in TG-1 cell.Wave and culture thing is until OD600nm reaches 0.5.By adding IPTG (isopropyl-β-D-thiogalactoside(IPTG)) to carry out abduction delivering.Use N,O-Diacetylmuramidase ruptured cell.By the Fab fragment of the separated His6-mark of IMAC (Bio-Rad).With PD10 post, by buffer exchange, be 1x Dulbecco ' s PBS (pH 7.2).Sterile filtration sample.By determined by ultraviolet spectrophotometry protein concn.Analytic sample purity in the 15%SDS-PAGE of sex change, reduction.By using the size exclusion chromatography (HP-SEC) of calibration criterion, measure the homogeneity of the Fab prepared product under native state.

Embodiment 5: by solution equilibria titration (SET), measure HER3 affinity of antibody (K _d)

Substantially by before described people such as (, (1985) J Immunol Methods 77:305-19) Friguet in solution, carry out avidity mensuration.In order to improve sensitivity and the accuracy of SET method, it is changed into the technology (people such as Haenel, (2005) Anal biochem339:182-84) based on ECL from classical ELISA.

With described before unlabelled HER3-Tag (people, rat, mouse or cynomolgus monkey), by SET, carry out avidity mensuration.

With XLfit software (ID Business Solutions), apply self-defined model of fit assessment data.K for every kind of IgG _dmeasure, use with drag (according to the people such as Piehler people such as (, (1997) J Immunol Methods 201:189-206 revises) Piehler.

y = \frac{{2 B}_{\max}}{[IgG]} (\frac{[IgG]}{2} - \frac{{(\frac{x + [IgG] + K_{D}}{2} - \sqrt{\frac{{(x + [kgG] + K_{D})}^{2}}{4} - x [IgG]})}^{2}}{2 [IgG]})

[IgG]: the total IgG concentration of application

X: total soluble antigen concentration (combining site) of application

B _max: the peak signal that there is no the IgG of antigen

K _d: avidity.

Embodiment 6: by FACS, measure antibody cell combination

By FACS, assess antibody and the endogenous human antigen's that expresses combination on human cancer cell.In order to measure antibody EC ₅₀value, gathers in the crops SK-Br-3 cell and be diluted to 1x10 in FACS damping fluid (PBS/3%FBS/0.2%NaN3) with accutase ⁶cell/mL.Each hole to 96 orifice plates (Nunc) adds 1x10 ⁵cells/well and with 210g 4 ℃ centrifugal 5 minutes, then remove supernatant.The serial dilutions of test antibody (diluting in the dilution step of 1:4 with FACS damping fluid) is added to the cell of precipitation, and on ice, hatch 1 hour.With 100 μ L FACS damping fluids washings sedimentation cell 3 times.The mountain goat anti-human igg (Jackson ImmunoResearch) that PE with 1/200 dilution of FACS damping fluid is puted together adds cell, and on ice, hatches 1 hour.With 100 μ LFACS damping fluids, carry out additional washing step 3 times, then carry out centrifugation step, 4 ℃ with 210g centrifugal 5 minutes.Finally, re-suspended cell in 200 μ L FACS damping fluids, measures fluorescent value with FACSArray (BD Biosciences).By measuring average channel fluorescence, assess the amount of the anti-HER3 antibody of cell surface combination.

The combination of embodiment 7:HER3 structural domain

By the suitable recombinant human protein's matter of 200ng (the irrelevant control protein of HER3-label, D1-2-label, D2-label, D3-4-label, D4-label and mark), spend the night and be coated with 96 hole Maxisorp plates (Nunc).Then porose with PBS/0.1%Tween-20 washing institute, with PBS/1%BSA/0.1%Tween-20 sealing, and wash with PBS/0.1%Tween-20.The final concentration that anti-HER3 antibody is added to associated orifices to 10 μ g/mL, and in incubated at room.Use PBS/0.1%Tween-20 wash plate, then add 1/10000 to be diluted in the detection antibody that the suitable peroxidase in PBS/1%BSA/0.1%Tween-20 connects.The detection antibody using is goat anti-mouse (Pierce, 31432), the anti-goat of rabbit (Pierce, 31402) and goat anti-human (Pierce, 31412).Plate, incubated at room 1 hour, then washs with PBS/0.1%Tween-20.Add institute porose 100 μ l TMB (3,3 ', 5,5 ' tetramethyl benzidine) substrate solution (BioFx), then use 50 μ l 2.5%H ₂sO ₄termination reaction.By measuring OD by SpectraMax microplate reader (Molecular Devices) ₄₅₀measure the combination degree of HER3 antibody and every kind of recombinant protein.In the time of suitably, with Graphpad Prism, analyze dose response curve.

Embodiment 8: the x-ray crystal structure of people HER3/MOR12604Fab mixture is measured

The present embodiment is presented on the crystalline structure of the HER3 that Fab fragment resolving power determination and MOR12604 is combined.In PBS (pH 7.3), on the HiLoad 26/60Superdex 200PrepGrade post (GE Healthcare) of balance, be further purified the people HER3 ectodomain of mark.By described at expression in escherichia coli MOR12604Fab purifying before.By by the mol ratio of 2:1 (concentration of estimating by LCUV method), excessive MOR12604Fab being mixed to prepare HER3/MOR12604-Fab mixture with the HER3 of mark, and Superdex 200 10/300 posts (GE Healthcare) of balance are gone up purifying in 25mM Tris (pH 7.5), 150mM NaCl.By SDS-PAGE and lcms analysis peak fraction.Merge containing the HER3 of the equimolar ratio of having an appointment and Fab the two fraction and be concentrated into 10mg/ml.By sit a vapor diffusion from the drop that contains 150nl HER3/MOR12604 mixture and 150nl pond liquid (200mM dipotassium hydrogen phosphate and 20%PEG 3350), come to cultivate HER3/MOR12604 crystal at 293K.Crystal is transferred to 200mM dipotassium hydrogen phosphate, 25%PEG 3350 and 15% glycerine, and quick-frozen in liquid nitrogen.

Under the light beam line 17-ID of Advanced Photon Source (Argonne National Laboratory), collect data.Process the data of HER3/MOR12604Fab mixture, and with autoPROC (Global Phasing, LTD) at spacer P2 ₁2 ₁2 ₁in be adjusted to unit cell dimension a=56.15, b=174.71, statistical is good.With Phaser (people such as McCoy, (2007) J.Appl.Cryst.40:658-674), by molecular replacement, resolve the structure of HER3/MOR12604Fab, with published HER3ECD structure 1mb6 and proprietary Fab as search model.In COOT (Emsley & Cowtan (2004) Acta Cryst.60:2126-2132), set up the final mask that each asymmetric unit contains 1 molecule HER3/MOR12604Fab mixture, and with BUSTER (Global Phasing, LTD) by R and R _freelybeing worth refine is respectively 19.9% and 23.3%, and the root-mean-square deviation of bond distance and bond angle is respectively with 1.19 °.PyMOL ( that LLC), identifies contains at the arbitrary atom of MOR12604 Fab hER3 residue with interior atom is listed in table 5 and 6.

Embodiment 9: phosphoric acid-HER3 cell in vitro is measured

In DMEM/F12,15mM HEPES, L-glutaminate, 10%FBS, routine maintains MCF-7 cell, in DMEM, 10%FBS, routine maintains BT474 cell, and in McCoy ' s5a, 10%FBS, 1.5mM L-glutaminate, routine maintains SK-Br-3 cell.Trysinization approaches the cell converging, and with PBS washing, and is diluted to 5x 10 ⁵cell/mL.Then each hole to 96 hole flat undersides (Nunc) adds 100 μ L cell suspensions, produces 5x10 ⁴the whole density of cells/well.Make MCF7 cell attachment approximately 3 hours, then substratum is replaced by the hungry substratum that contains 0.5%FBS.Then by all plates 37 ℃ of night incubation, with the HER3 antibody of suitable concn, at 37 ℃, process 80 minutes afterwards.Within last 20 minutes, with 50ng/mL NRG1, processing MCF7 cell stimulates HER3 and AKT phosphorylation, and BT474/SK-Br-3 cell is without additional stimulation.Suck lentamente all substratum, with containing 1mM CaCl ₂with 0.5mM MgCl ₂(Gibco) ice-cold PBS washed cell.By adding the lysis buffer that 50 μ L are ice-cold (20mM Tris (pH8.0)/137mM NaCl/10% glycerine/2mM EDTA/1%NP-40/1mM sodium vanadate/1x Phospho-Stop/1x Complete mini proteinase inhibitor (Roche)/0.1mM PMSF) to carry out lysing cell, oscillation incubation on ice 30 minutes.Then collect lysate, and with 1800g, within centrifugal 15 minutes, come to remove cell debris at 4 ℃.

With carbon plate (Mesoscale Discovery), prepare HER3 capture board, the 4 μ g/mL MAB3481 capture antibodies (R & D Systems) that this carbon plate dilutes in PBS with 20 μ L are 4 ℃ of coated spending the night, subsequently with 1x Tris damping fluid (Mesoscale the Discovery)/0.1%Tween-20 sealing containing 3% bovine serum albumin.By adding appropriate lysate and within 1 hour, catching HER3 at room temperature oscillation incubation flat board, then suck lysate, with 1x Tris damping fluid (Mesoscale Discovery)/0.1%Tween-20 washing hole.By room temperature, shaken and hatched 1 hour, the anti-pY1197 antibody (Cell Signaling) that is used in the 1:8000 preparing in 3% milk powder/1x Tris/0.1%Tween-20 detects the HER3 of phosphorylation.With 1x Tris/0.1%Tween-20 washing hole 4 times, and by the anti-rabbit Ab of goat (#R32AB) incubated at room of the S-Tag mark with diluting, within 1 hour, detect the protein of phosphorylation in 3% sealing damping fluid.Suck each hole, with 1x Tris/0.1%Tween-20 washing 4 times, then add 20 μ L containing the reading damping fluid T (Mesoscale Discovery) of tensio-active agent, with the quantitative signal of Mesoscale Sector Imager.

Embodiment 10: phosphoric acid-Akt (S473) cell in vitro is measured

The approaching MCF7 converging, SK-Br-3 and the BT-474 cell by accutase (PAA Laboratories) results, in perfect medium, cultivated, and in suitable substratum with 5x10 ⁵the final concentration of cell/mL is resuspended.Then each hole to 96 hole flat undersides (Nunc) adds 100 μ L cell suspensions, to produce 5x10 ⁴the final densities of cells/well.Make MCF7 cell attachment approximately 3 hours, then substratum is replaced by the hungry substratum that contains 0.5%FBS.Then by all plates 37 ℃ of overnight incubation, with the HER3 antibody of suitable concn, at 37 ℃, process 80 minutes afterwards.Within last 20 minutes, with 50ng/mL NRG1, processing MCF7 cell stimulates HER3 and AKT phosphorylation, and SK-Br-3 cell is without additional stimulation.Suck lentamente all substratum, with containing 1mM CaCl ₂with 0.5mM MgCl ₂(Gibco) ice-cold PBS washed cell.By adding the lysis buffer that 50 μ L are ice-cold (20mM Tris (pH8.0)/137mM NaCl/10% glycerine/2mM EDTA/1%NP-40/1mM sodium vanadate/Trypsin inhibitor,Trasylol (10 μ g/mL)/leupeptin (10 μ g/mL)) to carry out lysing cell, oscillation incubation on ice 30 minutes.Then collect lysate, and with 1800g, within centrifugal 15 minutes, remove cell debris at 4 ℃.20 μ L lysates are added to the multiple spot 384 hole phosphoric acid-Akt carbon plates (Mesoscale Discovery) that previously sealed with 3%BSA/1x Tris/0.1%Tween-20.Plate, room temperature oscillation incubation 2 hours, is then sucked to lysate, with 1x Tris damping fluid (Mesoscale Discovery)/0.1%Tween-20 washing hole 4 times.By room temperature oscillation incubation 2 hours, be used in and in 1%BSA/1x Tris/0.1%Tween-20, dilute the Akt that the 20 anti-phosphoric acid-Akt of μ L SULFO-TAG (S473) antibody (Mesoscale Discovery) of 50 times detect phosphorylations.With 1x Tris/0.1%Tween-20 washing hole 4 times, then add 20 μ L containing the reading damping fluid T (Mesoscale Discovery) of tensio-active agent, with the quantitative signal of Mesoscale Sector Imager.

Embodiment 11: cell line proliferation is measured

Cellar culture SK-Br-3 cell in McCoy ' the s 5A substratum of modification that supplements 10% foetal calf serum, and cultivate BT-474 cell in the DMEM that supplements 10%FBS.Trysinization approaches the cell converging, and with PBS washing, with growth medium dilution, is 5x 10 ⁴cell/mL, and inoculate in 96 hole clear bottom black plates (Costar 3904) with the density of 5000 cells/well.Cell, 37 ℃ of overnight incubation, then adds the HER3 antibody (final concentration is 10 or 1 μ g/mL conventionally) of suitable concn.Plate is put back to incubator, after 6 days, use CellTiter-Glo (Promega) assessment cell viability.To each hole, add 100 μ L CellTiter-Glo solution gentle oscillation incubation 10 minutes at room temperature.By SpectraMax microplate reader (Molecular Devices), measure luminous quantity.By by the luminous value obtaining with every kind of HER3 antibody with the luminous value that standard isotype control antibodies obtains, compare, the growth inhibiting degree of every kind of antibody acquisition for calculating.

For proliferation assay, cellar culture MCF-7 cell in the DMEM/F12 (1:1) that contains 4mM L-glutaminate/15mM HEPES/10%FBS.Trysinization approaches the cell converging, and with PBS washing, and is 1x 10 with DMEM/F12 (1:1) dilution that contains 4mM L-glutaminate/15mM HEPES/10 μ g/mL human transferrin/0.2%BSA ⁵cell/mL.Density with 5000 cells/well is inoculated cell in 96 hole clear bottom black plates (Costar).Then the HER3 antibody (final concentration is 10 or 1 μ g/mL conventionally) that adds suitable concn.Also to suitable hole, add 10ng/mL NRG1-β 1EGF structural domain (R & D Systems) to carry out stimulate cell growth.Plate is put back to incubator, after 6 days, use CellTiter-Glo (Promega) assessment cell viability.By subtracting background (impassivity adjusting albumen) luminous value and by the value obtaining with every kind of anti-HER3 antibody with by the value of standard isotype control antibodies acquisition, compare to calculate the growth inhibiting degree with every kind of antibody acquisition.

Embodiment 12: BxPC3 effect research in body

At the foetal calf serum that contains 10% heat inactivation and containing cultivating BxPC3 cell in antibiotic RPMI-1640 substratum until while implanting.

To the 10x10 in the subcutaneous mixture that is implanted in 50% phosphate buffered saline(PBS) and 50% matrigel of female athymia nu/nu Balb/C mouse (Harlan Laboratories) ⁶cell.The total volume injected that contains suspension cell is 200 μ L.Once tumour reaches about 200mm ³size, animal enters effect research.Generally speaking, every group totally 10 animals enter research.If animal shows uncommon tumor growth feature before entering research, animal is got rid of from research.

By side direction tail vein injection to animal intravenous administration.Animal adopts 20mg/kg, semiweekly scheme during studying.According to describing in detail and calculate gross tumor volume and T/C value for BT-474 institute.

Embodiment 13: BT474 effect research in body

At the foetal calf serum that contains 10% heat inactivation and containing cultivating BT-474 cell in antibiotic DMEM until while implanting.

At cell, inoculate first 1 day, to 17 beta estradiol particles of the subcutaneous implantation sustained release of female athymia nu/nu Balb/C mouse (Harlan Laboratories) (Innovative Research of America) to maintain serum estradiol level.At 17 beta estradiol particles, implant latter 1 day, by the 5x10 in suspension ⁶cell in-situ is injected into the 4th mammary fat pad, this suspension contain in Hank balanced salt solution 50% without phenol red matrigel (BD Biosciences).The total volume injected that contains suspension cell is 200 μ L.Cell is implanted latter 20 days, has about 200mm ³the animal of gross tumor volume enter effect research.Generally speaking, every group totally 10 animals enter effect research.

For single promoting agent research, by side direction tail vein injection, with contrasting IgG or MOR12606 or MOR13655, animal is carried out to intravenous administration.Animal adopts 20mg/kg, semiweekly dosage regimen during studying.For combination research, MOR10703 and MOR12606 press 20mg/kg twice pair of animals administer weekly.During studying, by measuring diameter, measure gross tumor volume twice weekly.With following formula, calculate per-cent treatment/contrast (T/C) value:

％T/C＝100×ΔT/ΔC if ΔT>0

Wherein:

The mean tumour volume of T=research medication therapy groups the last day;

The mean tumour volume of the average tumor Ti Ji – administration first day medication therapy groups of Δ T=research medication therapy groups the last day;

The mean tumour volume of C=research control group the last day; With

The mean tumour volume of the average tumor Ti Ji – administration first day control group of Δ C=treatment control group the last day.

Measure body weight twice weekly, according to body weight, adjust dosage.With (BW _current-BW _initially)/(BW _initially) x 100 % that calculates body weight changes.Data are expressed as the per-cent body weight change starting from the treatment first day.

All data are expressed as to mean value ± standard error of the mean (SEM).By Δ gross tumor volume and body weight, carry out statistical analysis.With single factor ANOVA and afterwards Tukey between organizing relatively.For all statistical evaluations, significance level is located to p<0.05.Reported the significance of comparing vehicle Control group.

Results and discussions

Jointly, these results show the antibody of the amino-acid residue in a class binding domains 3 or 4.The combination of these antibody suppresses ligand dependent and non-ligand dependent signal is provided.

(i) avidity is measured

By solution equilibria titration (SET) mentioned above, measure affinity of antibody.Result is summarised in table 3, and the example titration curve of MOR12615 and MOR12604 is included in Fig. 1.Data surface identifies the antibody of the many people of combining closely HER3.

Table 3: the K of the anti-HER3IgG measuring by solution equilibria titration (SET) _dvalue.Hu (people), Cy (cynomolgus monkey), Mu (mouse) and ra (rat), nd (undetermined).

(ii) SK-Br-3 cell EC ₅₀measure

By calculating the antibodies HER2 amplifying cells identifying, be the EC of SK-Br-3 ₅₀value is measured them in conjunction with the ability (seeing Fig. 2, table 4) of HER3 express cell.

Table 4: the FACS EC of anti-HER3IgG on cell ₅₀value.

MOR#	SK-Br-3FACS EC ₅₀(pM)
		14537	179
14538	279
		14533	42
14534	28

(iii) HER3 structural domain combination

In measuring, ELISA characterized anti-HER3 antibody subset in conjunction with the ability of the multiple ectodomain of people HER3.For this reason, the ectodomain of HER3 is divided into its 4 composing type structural domains, and by the multiple combination of these structural domains of clone mentioned above, expresses and purifying is protein independently.Use this strategy, following structural domain is successfully produced as to soluble protein: structural domain 1 and 2 (D1-2), structural domain 2 (D2), structural domain 3 and 4 (D3-4) and structural domain 4 (D4).With a series of inner antibody producing, as positive control, confirmed the integrity of every kind of separated structural domain before.

As shown in Figure 3, observe MOR12615 and MOR12604 success in conjunction with HER3 ectodomain and separated D3-4 protein.Do not observe and D1-2 or D2 protein bound.This shows in conjunction with data, and these antibody recognition are mainly included in the epi-position in structural domain 3 or 4.Enjoyably, MOR12604 can be in conjunction with separated D3 protein, show its epi-position further refining to the residue in structural domain 3.Because MOR12615 and MOR12604 are the representative members of two different families of anti-HER3 antibody, according to its hCDR3 sequence, antibody MOR12514, MOR12515, MOR12516, MOR12615, MOR12920, MOR12921, MOR12922, MOR13654, MOR13655, MOR13656, MOR13657, MOR13658, MOR13659, MOR13660, MOR13661, MOR13662, MOR13663, MOR13664, MOR13665, MOR13666, MOR13667, MOR13668, MOR13669, MOR13670, MOR14537 and MOR14538 can classify as D3-4 bonding agent.Antibody MOR12603, MOR12604, MOR12605, MOR12606, MOR14533 and MOR14534 can classify as D3 bonding agent.

(iv) HER3/MOR12604 crystalline structure

Resolved the MOR12604Fab fragment of being combined with HER3 ectodomain the x-ray crystal structure of resolving power, the HER3 epi-position (seeing Fig. 4) of being identified further to define the associated antibodies of this family.The stack of MOR12604/HER3 crystalline structure and disclosed HER3 crystalline structure shows, the HER3 of MOR12604 combination is in constraint (inactivation) conformation (seeing Fig. 4 B).The feature of this conformation is the significant interaction interface between the structural domain 2 and 4 that mediates of the β-hair clip dimerization ring in structural domain 2.People (the Cho & Leahy such as the HER3 conformation of observing and Cho, (2002), Science 297:1330-1333) previously described similar, they disclose the crystalline structure of HER3 ectodomain in the situation that lacking neuregulin.Because neuregulin can activate HER3, infer that the constraint conformation of HER3 is inactivation.In crystallization the relevant EGFR family member HER4 (people such as Bouyain, (2005) Proc.Natl.Acad.Sci.USA, 102:15024-15029) and the HER1 (people such as Ferguson, (2003), in the time of Molec.Cell 11:507-517), also observe similar constraint conformation.

Spatial relation in inactivation (constraint) state between the structural domain 1 to 4 of HER3 is significantly different from the spatial relation in stretching, extension (activation) state.This finds crystalline structure (people such as Cho, (2003) the Nature 421:756-760 of the HER1 based on relevant EGFR family member HER2 and ligand binding; The people such as Ogiso, (2002) Cell 110:775-787; The people such as Garrett, (2002) Cell 110:763-773), the two is all in stretching (activation) state.In stretching state, β-hair clip dimerization ring of structural domain 2 discharges from itself and the inhibition of structural domain 4 interact, so can be freely and its dimerization mating partner protein interaction.Therefore, β-hair clip dimerization ring of structural domain 2 maintain constraint (inactivation) state and mediation stretch state in the dimerization of EGF acceptor, thereby cause thering is critical function in the activation of intracellular kinase structural domain.

In crystalline structure, MOR12604Fab, HER3 structural domain 3, HER3 structural domain 4 and comprise that the electron density in the part-structure territory 2 (residue 261-278) of β-hair clip dimerization ring all obtains good definition.Observed the weak electron density of the HER3 residue 20-260 that is positioned in structural domain 1 and structural domain 2 and 279-303 or without electron density, shown when HER3 is incorporated into MOR12604, it has retained flexibility to a certain degree.This finds more consistent with other crystalline structure of independent HER3 and the HER3 of being combined with multiple Fab fragment, and this relatively shows the Light Difference that the relative structural domain in bound state is located.

Crystalline structure also discloses, and the HER3 epi-position of MOR12604 identification is the non-linear epi-position (seeing Fig. 4, table 5 and 6) comprising from the residue of structural domain 3.Therefore the HER3 epi-position that the antibody family of this height correlation identifies may be defined as:

Structural domain 3: residue 335-342,362-376,398,400,424-428,431,433-434 and 455.

MOR12604 mating surface can be further subdivided into two surfaces that are denoted as solid line circle and broken circle in Fig. 4 D:

Surface A: residue 362-376;

Surface B: residue 335-342,398,400,424-428,431,433-434 and 455.

Surface A or surperficial B are that roughly the same surface-area (surface is contributed at overall HER3/12604 interface surface ).

Enjoyably, the combination of MOR12604 and structural domain 3 causes by significant conformational change in the ring of HER3 residue 371-377 definition.This conformation of this ring is different from other disclosed HER3 structures, therefore shows that it is by MOR12604 zygotic induction.From the definite MOR12604 epi-position of crystalline structure, be combined data consistent with our ELISA structural domain, this ELISA structural domain in conjunction with data in, determined that MOR12604 is in conjunction with separated D3 structural domain.In addition the relatively demonstration high superposed of the EGFR residue that MOR12604 epi-position contacts with TGF α (Garrett etc., (2002) Cell110:763-773).Owing to thinking that neuregulin interacts to be similar to mode and the HER3 of TGF α/EGFR, MOR12604 probably will stop ligand binding, thereby block nerves regulates protein induced HER3 to activate.

The combination of MOR12604 in structural domain 3 can show, MOR12604 can play a role by any (or the combination) in following mechanism:

The required HER3 residue of-sealing ligand binding;

-because stoping HER3, the steric hindrance between antibody and HER3 structural domain adopts activity conformation;

-for example, by the flexible degree reducing in HER3 hinge area (structural domain 3), stop HER3 to adopt activity conformation;

-in the ring 371-377 of structural domain 3, induce conformational change, this conformational change prevents that HER3 from changing open conformation into;

-stabilization removal HER3, easily degrades it;

-as partial agonist, play a role to accelerate the downward of HER3;

The dimerization of-inhibition and binding partners;

-every arm by MOR12604 makes antibody produce non-natural HER3 dimer in conjunction with a part HER3, this dimer be easy to proteolysis degraded or can not with other receptor tyrosine kinase dimerizations.

Interaction between table 5:MOR12604Fab heavy chain and people HER3.The numbering of Fab VH residue is based on its linear aminoacid sequence (SEQ ID NO:1).The numbering of HER3 residue is based on NP_001973.Shown HER3 residue has at least one atom in MOR12604Fab within atom.

Interaction between table 6:MOR12604Fab light chain and people HER3.The numbering of Fab VL residue is based on its linear aminoacid sequence (SEQ ID NO:1).The numbering of HER3 residue is based on NP_001973.Shown HER3 residue has at least one atom in MOR12604Fab within atom.

(v) inhibition that cell signal is provided

In order to determine the impact of anti-HER3 antibody on ligand dependent HER3 activity, MCF7 cell and IgG are hatched, then with neuregulin, stimulate.Example suppresses curve and shows in Fig. 5, and is summarised in table 7.By HER2 amplifying cells, be also that SK-Br-3 and BT474 have studied the impact (Fig. 6, Fig. 7 and table 7) that anti-HER3 antibody activates the HER3 of HER2 mediation.

Table 7: the pHER3IC of anti-HER3IgG in MCF7, BT474 and SK-Br-3 cell ₅₀with inhibition degree value

In order to determine whether the inhibition of HER3 activity affects downstream cell signal granting, in the SK-Br-3/BT474 cell of the MCF7 cell with stimulating at NRG after anti-HER3 antibody treatment and HER2 amplification, measured Akt phosphorylation (seeing Fig. 5, Fig. 6 and table 8).

Table 8: the pAkt (S of anti-HER3IgG in SK-Br-3, BT-474 and MCF7 cell ⁴⁷³) IC ₅₀with inhibition degree value

In a word, MOR12514, MOR12515, MOR12516, MOR12615, MOR12920, MOR12921, MOR12922, MOR13654, MOR13655, MOR13656, MOR13657, MOR13658, MOR13659, MOR13660, MOR13661, MOR13662, MOR13663, MOR13664, MOR13665, MOR13666, MOR13667, MOR13668, MOR13669, MOR13670, MOR14537, MOR14538, MOR12603, MOR12604, MOR12605, MOR12606, MOR14533 and MOR14534 can both suppress cell HER3 activity and downstream signal granting with ligand dependent and non-ligand dependent mode separately.

(vi) inhibition of propagation

Due to MOR12514, MOR12515, MOR12516, MOR12615, MOR12920, MOR12921, MOR12922, MOR13654, MOR13655, MOR13656, MOR13657, MOR13658, MOR13659, MOR13660, MOR13661, MOR13662, MOR13663, MOR13664, MOR13665, MOR13666, MOR13667, MOR13668, MOR13669, MOR13670, MOR14537, MOR14538, MOR12603, MOR12604, MOR12605, MOR12606, MOR14533 and MOR14534 can suppress HER3 activity, (instance data is shown in Fig. 8 to have tested the ability of its subset block ligand dependency and dependent/non-dependent in-vitro cell growth, Fig. 9, in Figure 10, and be summarised in table 9).The anti-HER3 antibody of testing is all effective inhibition of cell proliferation, thereby has confirmed can suppress in conjunction with the antibody of D3 or D3-4 the phenotype that HER3 drives.

Table 9: suppress by the propagation after anti-HER3IgG treatment S K-Br-3, BT-474 and MCF7 cell

(vii) in the body of tumor growth, suppress

In order to measure the activity in vivo of described anti-HER3 antibody, at BxPC3 and BT-474 tumor model, tested the anti-tumor activity of MOR12606 and MOR13655 in the two.With MOR12606 or MOR13655, BxPC3 model repetitively administered being produced respectively to 25% disappears and 5%T/C (Figure 11 A).

With the BT474 model that MOR12606 or the mono-drug treating HER2 of MOR13655 drive, produce respectively 53% or 46%T/C (Figure 11 B).

We have also examined or check the effect of antibody of two kinds of target HER3 of two different non-overlapped epi-positions of built up section.With the combination of MOR10703 and MOR12606, BT474 model repetitively administered is produced to 7%T/C (Figure 12).

Introduce reference

All reference of quoting herein (comprising patent, patent application, paper, textbook etc.) and the reference (being also less than the degree of listing) of wherein quoting are incorporated herein by reference with its integral body at this.

Equivalent

We think that above-mentioned specification sheets is enough to make those skilled in the art to implement the present invention.Foregoing description and example have described some preferred embodiment of the present invention in detail and have described the optimal mode of contriver's imagination.Yet should be appreciated that and describe to such an extent that how detailedly have no matter above, the present invention can implement in many ways, should explain the present invention according to the claim of enclosing and any equivalents thereof.

Claims

1. identify separated antibody or its fragment of the non-linear epi-position of HER3 acceptor, amino-acid residue in the structural domain 3 that wherein non-linear epi-position comprises HER3 acceptor, wherein antibody or its fragment be in conjunction with comprising the mating surface that at least one is selected from the amino-acid residue of mating surface B, and wherein antibody or its fragment block ligand dependency and non-ligand dependent signal transduction the two.

2. the separated antibody of claim 1 or its fragment, wherein mating surface B comprises the amino-acid residue that at least one is selected from amino-acid residue 335-342,398,400,424-428,431,433-434 and 455.

3. separated antibody or the fragment of claim 1, wherein antibody or its fragment are further combined with mating surface A.

4. the separated antibody of claim 3 or its fragment, wherein mating surface A comprises the amino-acid residue that at least one is selected from amino-acid residue 362-376.

5. identify separated antibody or its fragment of the non-linear epi-position of HER3 acceptor, amino-acid residue in the structural domain 3 that wherein non-linear epi-position comprises HER3 acceptor, wherein antibody or its fragment be in conjunction with comprising at least one amino-acid residue that is selected from mating surface A and at least one is selected from the mating surface of the amino-acid residue of mating surface B, and wherein antibody or its fragment block ligand dependency and non-ligand dependent signal transduction the two.

6. the separated antibody of claim 5 or its fragment, wherein the HER3 ligand binding on antibody or its fragment blocking-up HER3 acceptor.

7. the separated antibody of claim 6 or its fragment, wherein HER3 part is selected from neuregulin 1 (NRG), neuregulin 2, β tunicin, Heparin-binding Urogastron and epiregulin.

8. the separated antibody of claim 5 or its fragment, wherein antibody or its fragment have and are selected from any one following feature: in conjunction with the inactivation state of HER3 acceptor; Because stoping HER3, the steric hindrance between antibody or its fragment and the structural domain of HER3 adopts activity conformation; By the flexible degree reducing in structural domain 3, stop HER3 to adopt activity conformation; In structural domain 3 residue 371-377, induction stops HER3 to adopt the conformational change of activity conformation; Stabilization removal HER3 makes it be easy to degraded; Accelerate the downward of cell surface HER3; And produce be easy to proteolysis degraded or can not with the non-natural HER3 dimer of other receptor tyrosine kinase dimerizations.

9. the separated antibody of claim 5 or its fragment, wherein mating surface A comprises amino-acid residue 362-376.

10. the separated antibody of claim 5 or its fragment, wherein mating surface B comprises amino-acid residue 335-342,398,400,424-428,431,433-434 and 455.

The separated antibody of 11. claims 5, wherein non-linear epi-position comprises amino-acid residue 335-342,362-376,398,400,424-428,431,433-434 and 455 (in structural domain 3), or its subset.

The separated antibody of 12. claims 5, wherein at least one in the following HER3 residue of the VH of antibody or its fragment combination: Ile365, Thr366, Asn369, Gly370, Asp371, Pro372, Trp373, His374, Lys375, Gln400 and Lys434.

The separated antibody of 13. claims 5, wherein at least one in the following HER3 residue of the VL of antibody or its fragment combination: Gly335, Ser336, Gly337, Ser338, Phe340, Gln341, Asp362, Leu364, Ile365, Thr366, His374, Ile376, Asn398, Gln400, Tyr424, Asn425, Arg426, Phe428, Leu431, Met433, Lys434, Tyr455.

The separated antibody of 14. claims 5 or its fragment, wherein antibody or its fragment have reduced the non-ligand dependent formation of HER2-HER3 protein complex in the cell of expressing HER2 and HER3 in the situation that lacking HER3 part with the combination of HER3 acceptor.

The separated antibody of 15. claims 5 or its fragment, wherein by the phosphorylation of the non-ligand dependent phosphorylation assay assessment antibody of HER3 or its fragment inhibition HER3.

The separated antibody of 16. claims 15 or its fragment, wherein the non-ligand dependent phosphorylation assay of HER3 is used the cell of HER2 amplification, and wherein the cell of HER2 amplification is SK-Br-3 cell and BT-474.

The separated antibody of 17. claims 5 or its fragment, wherein antibody or its fragment have reduced the ligand dependent formation of HER2-HER3 protein complex in the cell of expressing HER2 and HER3 under the existence of HER3 part with the combination of HER3 acceptor.

The separated antibody of 18. claims 5 or its fragment, wherein by the phosphorylation of HER3 ligand dependent phosphorylation assay assessment antibody or its fragment inhibition HER3.

The separated antibody of 19. claims 18 or its fragment, wherein HER3 ligand dependent phosphorylation assay is used the MCF7 cell stimulating under neuregulin (NRG) exists.

The separated antibody of 20. claims 5 or its fragment, wherein antibody is selected from monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody and synthetic antibody.

Separated antibody or its fragment of the epi-position of 21. identification HER3 acceptors, the amino-acid residue in the structural domain 3-4 that wherein epi-position comprises HER3 acceptor, and wherein antibody or its fragment block ligand dependency and non-ligand dependent signal transduction the two.

The separated antibody of 22. claims 21, the amino-acid residue that wherein epi-position comprises at least one amino-acid residue 329-498 that is selected from SEQ ID NO:1 (structural domain 3) is selected from the amino-acid residue of the amino-acid residue 499-642 (structural domain 4) of SEQ ID NO:1 with at least one.

The separated antibody of 23. claims 21, the epi-position that wherein comprises the amino-acid residue in structural domain 3-4 is selected from linear epitope, non-linear epi-position and conformational epitope.

The separated antibody of 24. claims 21, wherein antibody or its fragment have reduced the non-ligand dependent formation of HER2-HER3 protein complex in the cell of expressing HER2 and HER3 in the situation that lacking HER3 part with the combination of HER3 acceptor.

The separated antibody of 25. claims 21 or its fragment, wherein by the phosphorylation of the non-ligand dependent phosphorylation assay assessment antibody of HER3 or its fragment inhibition HER3.

The separated antibody of 26. claims 25 or its fragment, wherein the non-ligand dependent phosphorylation assay of HER3 is used the cell of HER2 amplification, and wherein the cell of HER2 amplification is SK-Br-3 cell and BT-474.

The separated antibody of 27. claims 21 or its fragment, wherein antibody or its fragment have reduced the ligand dependent formation of HER2-HER3 protein complex in the cell of expressing HER2 and HER3 under the existence of HER3 part with the combination of HER3 acceptor.

The separated antibody of 28. claims 21 or its fragment, wherein by the phosphorylation of HER3 ligand dependent phosphorylation assay assessment antibody or its fragment inhibition HER3.

The separated antibody of 29. claims 28 or its fragment, wherein HER3 ligand dependent phosphorylation assay is used the MCF7 cell stimulating under neuregulin (NRG) exists.

The separated antibody of 30. anti-HER3 acceptors or its fragment, it has at least 1x 10 ⁷m ^-1, 10 ⁸m ^-1, 10 ⁹m ^-1, 10 ¹⁰m ^-1, 10 ¹¹m ^-1, 10 ¹²m ^-1, 10 ¹³m ^-1(the K that dissociates _d), wherein antibody or its fragment block ligand dependency and non-ligand dependent signal transduction the two.

The separated antibody of 31. claims 30 or its fragment, wherein measure by being selected from the phosphorylation in vitro of phosphoric acid-HER3 and phosphoric acid-Akt the phosphorylation of measuring antibody or its fragment inhibition HER3.

The antibody of 32. separation or its fragment, it is in conjunction with the non-linear epi-position in the structural domain 3 of the HER3 identical with the antibody described in table 1.

The antibody of 33. separation or its fragment, it is in conjunction with the amino-acid residue in the structural domain 3-4 of the HER3 identical with the antibody described in table 2.

34. in conjunction with the antibody fragment of HER3, it is selected from Fab, F (ab ₂) ', F (ab) ₂', scFv, VHH, VH, VL, dAb, wherein antibody fragment block ligand dependency and non-ligand dependent signal transduction the two.

35. pharmaceutical composition, it comprises antibody or its fragment, and pharmaceutically acceptable carrier.

36. the pharmaceutical composition of claim 35, it further comprises additional treatment agent.

The pharmaceutical composition of 37. claims 36, wherein additional treatment agent is selected from HER1 inhibitor, HER2 inhibitor, HER3 inhibitor, HER4 inhibitor, mTOR inhibitors and PI3 kinase inhibitor.

The pharmaceutical composition of 38. claims 37, wherein additional treatment agent be selected from horse trastuzumab (EMD72000), cetuximab, victibix, mAb 806, Buddhist nun not pearl monoclonal antibody, gefitinib, CI-1033 (PD183805), lapatinibditosylate (GW-572016), xylene monosulfonic acid lapatinibditosylate, erlotinib hydrochloride (OSI-774), PKI-166 and hER1 inhibitor; Be selected from handkerchief trastuzumab, Herceptin, MM-111, HKI-272, lapatinibditosylate or xylene monosulfonic acid lapatinibditosylate hER2 inhibitor; Be selected from the micromolecular HER3 inhibitor of MM-121, MM-111, IB4C3,2DID12 (U3Pharma AG), AMG888 (Amgen), AV-203 (Aveo), MEHD7945A (Genentech), MOR10703 (Novartis) and inhibition HER3; And HER4 inhibitor.

The pharmaceutical composition of 39. claims 37, wherein additional treatment agent is HER3 inhibitor, wherein HER3 inhibitor is MOR10703.

The pharmaceutical composition of 40. claims 37, wherein additional treatment agent is to be selected from CCI-779 ridaforolimus/ rapamycin, AP23573, MK8669, everolimus mTOR inhibitors.

The pharmaceutical composition of 41. claims 37, wherein additional treatment agent is the PI3 kinase inhibitor that is selected from GDC 0941, BEZ235, BKM120 and BYL719.

The method of 42. treatment cancers, it comprises the individuality of selecting to suffer from the cancer of expressing HER3, described individuality is used to the composition that comprises disclosed antibody in table 1 or table 2 or its fragment of significant quantity.

The method of 43. claims 42, wherein individuality is people, cancer is selected from mammary cancer, colorectal carcinoma, lung cancer, multiple myeloma, ovarian cancer, liver cancer, cancer of the stomach, acute myeloid leukaemia, chronic myelogenous leukemia, osteosarcoma, squamous cell carcinoma, Peripheral Nerve Sheath Tumors, schwannoma, head and neck cancer, bladder cancer, the esophageal carcinoma, the Barretts esophageal carcinoma, glioblastoma, research of clear cell sarcoma of soft tissue, malignant mesothe, neurofibromatosis, kidney, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia and endometriosis.

The method of 44. claims 43, wherein cancer is mammary cancer.

The antibody of 45. claim 1-34 or its fragment, it is as medicine.

The antibody of 46. claim 1-34 or its fragment, it is used for the treatment of the cancer by HER3 ligand dependent signal transduction pathway or the mediation of non-ligand dependent signal transduction pathway.

47. antibody of claim 1-34 or the purposes of its fragment, for the preparation of the cancer being used for the treatment of by HER3 ligand dependent signal transduction pathway or the mediation of non-ligand dependent signal transduction pathway, described cancer is selected from mammary cancer, colorectal carcinoma, lung cancer, multiple myeloma, ovarian cancer, liver cancer, cancer of the stomach, carcinoma of the pancreas, acute myeloid leukaemia, chronic myelogenous leukemia, osteosarcoma, squamous cell carcinoma, Peripheral Nerve Sheath Tumors, schwannoma, head and neck cancer, bladder cancer, the esophageal carcinoma, the Barretts esophageal carcinoma, glioblastoma, research of clear cell sarcoma of soft tissue, malignant mesothe, neurofibromatosis, kidney, melanoma, prostate cancer, benign prostatic hyperplasia (BPH), gynecomastia and endometriosis.