CN100408097C - Pharmaceutical compositions directed to erb-b1 receptors - Google Patents

Abstract

The invention relates to novel bispecific antibodies and their use in tumor therapy. The novel antibodies have the ability to bind to ErbB receptors, preferably ErbB1 receptors, which are overexpressed on many cancer tissues. Since the different specificities of the antigen-binding sites are directed to different epitopes within the binding domain of same or different ErbB receptors, these antibodies are more effective with respect to inhibition and down-regulation of the ErbB receptor and the corresponding signaling cascade.

Description

Anti-ERB-B antibody of bispecific and the purposes in oncotherapy thereof

Invention field

The present invention relates to novel bi-specific antibody and the purposes in oncotherapy thereof.This novel antibodies can with the ErbB receptor, particularly ErbB1 receptors bind of on multiple cancerous tissue, cross expressing.Because the not homospecificity of antigen binding site at identical or different ErbB receptor in conjunction with the different epi-positions in the territory, these antibody are suppressing and downward modulation ErbB receptor and more effective aspect the signal cascade accordingly.The invention still further relates to pharmaceutical composition, its comprise described bi-specific antibody or its fragment and other pharmaceutically efficacious agents such as monospecific antibody, immunoconjugates and/or cytotoxic agent.

Background of invention

Surpassing in the time in 20 years, known to the multiple receptor on the tumor cell surface and other antigenic biomolecule, such as monoclonal antibody (MAb) or other protein/polypeptide, and little chemical compound is applicable to oncotherapy.About antibody method, these MAb great majority are by chimericization or humanized to improve the toleration of human immunity system.MAb or above-mentioned chemical body with its on the tumor cell and the target structure specificity that in most cases also is present on the normal structure combine, and can cause depending on the different effect of its epitope specificity and/or concrete antigenic functional characteristic.Expected at the MAb of orphan receptor (orphan receptor) or other non-functional cell surface molecule and at the MAb of the part-binding site external structure of functional activity receptor (growth factor receptors that for example has kinase activity) and can be induced the immunological effect subfunction (cytotoxicity (ADCC) of antibody-dependant cell mediation, complement dependent cytotoxicity (CDC)) that is primarily aimed at target cell.In addition, according to the characteristic of antigen and Mab, the combination of antibody can cause the crosslinked of receptor.The receptor-antibody complex internalization that causes thus can cause the downward modulation over a long time of Rd on the cell surface.

With in part-binding site or its direct neighborhood in the bonded Mab of epi-position and native ligand competition and its receptors bind, reduce thus or suppressed the part combination fully, and replaceable with the part of its receptors bind.The blocking-up of this receptor has suppressed ligand dependent receptor activation and downstream signal conduction.For example, the ErbB receptor, (EGFR) can be caused the various kinds of cell effect by the monoclonal antibody blocking-up such as EGF-R ELISA, comprises suppressing the synthetic and propagation of DNA, inducing cell cycle arrest and apoptosis and metastasis and blood vessel formation against function.

In the 1980s, the ErbB receptor is the related typical receptor tyrosine kinase of cancer.Tyrosine kinase is a class of enzymes, and the tyrosine residue of the terminal phosphate of its catalysis adenosine triphosphate in protein substrate shifts.It is believed that tyrosine kinase passes through substrate phosphorylation, in the signal conduction of many cell functions, play a crucial role.Really the cutter reason is still unclear though signal conducts, and tyrosine kinase is at cell proliferation, and demonstrating in carcinogenesis and the cell differentiation is the important factor that works.

Tyrosine kinase can be divided into receptor type and non-receptor type.Two kinds of tyrosine kinase of receptor type and non-receptor type all participate in the cell signal approach, and these approach can cause many pathological conditions, comprise cancer, psoriasis and hyperimmune response.Many tyrosine kinase participate in cell growth and angiogenesis.

The non-receptor type tyrosine kinase also is made up of many subfamilies, comprises Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack and LIMK.Each of these subfamilies also can be divided into different receptors again.For example, the Src subfamily is one of maximum subfamily, and it comprises Src, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.Enzyme in the Src subfamily forms relevant with tumor.To the more detailed discussion of non-receptor type tyrosine kinase, referring to BolenOncogene, 8:2025-2031 (1993).

Receptor type tyrosine kinase has the extracellular, strides film and intracellular portion, but not receptor type tyrosine kinase is intracellular fully.The tyrosine kinase that receptor connects is a transmembrane protein, and it comprises extracellular ligand in conjunction with the territory, strides the film sequence, and the cytoplasmic tyrosine kinase territory.Receptor type tyrosine kinase is formed by having different bioactive transmembrane receptors in a large number.

Identified the receptor type tyrosine kinase of different subfamilies.The tyrosine kinase that relates to comprises fibroblast growth factor (FGF) receptor, epidermal growth factor (EGF) receptor in the ErbB major families type and platelet derived growth factor (PDGF) receptor.Also relate to nerve growth factor (NGF) receptor, brain derived neurotrophic factor (BDNF) receptor, and neurotrophin-3 (NT-3) receptor and neurotrophin-4 (NT-4) receptor.

A receptor type tyrosine kinase subfamily is named as HER or ErbB subfamily, by EGFR (ErbB1), and HER2 (ErbB2 or p185neu), HER3 (ErbB3) and HER4 (ErbB4 or tyr02) form.The part of this subfamily receptor comprises epidermal growth factor (EGF), TGF-a, amphiregulin (amphiregulin), HB-EGF, beta cell regulin (betacellulin), heregulin and neuregulin.The PDGF subfamily comprises the FLK family that is made up of kinases insertion domain receptor (KDR).

EGFR, by the ErbB1 gene code, itself and human malignancies causal correlation.Particularly, at mammary gland, bladder, lung, head, the expression of having observed EGFR in neck and stomach cancer and the glioblastoma strengthens.The enhancing of EGFR expression of receptor usually is attended by EGFR part in the same tumor cell---and the generation of transforming growth factor (TGF-α) increases, thereby cause receptor activation (Baselga and Mendelsohn, Pharmac.Ther.64:127-154 (1994)) through the autocrine stimulation approach.

The EGF receptor is that a kind of molecular weight is 170,000 transmembrane glycoprotein, and finds that it is present on many epithelial cell types.It can be by at least three kinds of parts---EGF, and TGF-α (transforming growth factor) and amphiregulin activate.Verified epidermal growth factor (EGF) and transforming growth factor (TGF-α) can both and then cause cell proliferation and tumor growth with the EGF receptors bind.These somatomedin can not combine (Ulrich and SCH1esinger, 1990, Cell 61,203) with HER2.With induce several growth factor families (as PDGF) of receptor dimerizationization different by self dimerization character, the monomers grow factor such as EGF have 2 receptor binding sites, therefore, it can make the crosslinked (Lemmon etc. of EGF receptor of two vicinities, 1997, EMBO J.16,281).Receptor dimerizationization is essential to the inherent catalytic activity of activation growth factor receptors and to the autophosphorylation of growth factor receptors on tyrosine residue.The latter can be used as the docking site of multiple adapter protein or enzyme, and these protein or enzyme can start many signal cascades simultaneously.In higher eucaryotic cells, it is abundant interactive multitiered network that simple linear path develops, and wherein the combination of component is expressed and activated and makes and can react in the sight of the formation from start to finish specific biological of growing.The ErbB network can not only be integrated the input signal of himself, can also integrate the allos signal, comprises hormone, lymphokine, neurotransmitter and stress-induced thing.

Should be noted that receptor protein tyrosine kinase (PTK) can carry out same dimerization, also can carry out different dimerization, wherein the mitogenesis of homodimer receptor combination and transformation (not starting or the conduction of weak enabling signal) are lower than corresponding heterodimer combination.The heterodimer that comprises ErbB2 be the most effective complex (referring to following summary: Yarden and Sliwkowski, 2001, NatureReviews, Molecular cell Biology, volume 2,127-137; Tzahar and Yarden, 1998, BBA 1377, M25-M37).

The antibody of verified anti-EGF receptor demonstrates tumor cell proliferation inhibition in blocking-up EGF and TGF-α and receptors bind.Consider these discoveries, developed a lot of mices and rat monoclonal anti EGF receptor antibody, and in vivo with vitro detection its ability that growth of tumour cell is suppressed (Modjtahedi and Dean, 1994, J.Oncology 4,277).Humanized monoclonal antibodies 425 (hMAb 425, and US 5,558, and 864; EP 0531472) and resistant chimeric monoclonal antibody 225 (cMAb 225, and US 4,943,533 and EP 0359282) all at the EGF receptor, and in clinical trial, all show effectively.Proved that C225 antibody (Catuximab) can be at the growth of tumour cell of vitro inhibition by EGF mediation, and the formation that in nude mouse, suppresses people's tumor.And, the most important thing is that it can (be to eradicate people's tumor in amycin (doxorubicin, adriamycin), paclitaxel and cisplatin) the synergism body with some chemotherapeutics that this antibody also demonstrates in the xenotransplantation mouse model.Ye etc. (1999, Oncogene 18,731) report can successfully be treated Proliferation of Human Ovarian Cell by use in conjunction mosaic type MAb225 with at the humanization MAb 4D5 of HER2 receptor.

Second member HER2 (ErbB2 or p185neu) of ErbB family, it is accredited as the transformed gene product of the neuroblastoma of the chemically treated rat of hanging oneself at first.The activation form of neu proto-oncogene is caused by the point mutation (valine becomes glutamic acid) of striding in the film district of this encoding proteins.Can in mammary gland and ovary cancer, observe the amplification of neu people's homologue, its relevant (Slamon etc., Science, 235:177-182 (1987) with prognosis mala; Slamon etc., Science, 244:707-712 (1989); US 4,968, and 603).The molecular weight of ErbB2 (HER2) is about 185,000, though so far the ligands specific of HER2 is determined clearly that as yet ErbB2 (HER2) has quite high homology with EGF receptor (HER1).

Further find to make the cytotoxic effect sensitivity (US 5,677,171) of the breast tumor cell line of expression ErbB2 to TNF α at the antibody 4D5 of HER2 receptor.Humanization recombinant (huMAb4D5-8, rhuMAb HER2 or the HERCEPTIN of mouse-anti-ErbB2 antibody 4D5

US 5,821,337) cross among the patient who expresses metastatic breast cancer and have clinical activity (Baselga etc., J.Clin.Oncol.14:737-744 (1996)) at the ErbB2-that suffers from that carried out the significant antitumor previous tretament.HERCEPTIN

Be used for the treatment of the suffer from metastatic breast cancer patient of (its tumor is crossed expression ErbB2 albumen) in 1988 by official approval listing.

Except that anti-ErbB antibody, known multiple little chemical molecular also is effective inhibitor of ErbB acceptor molecule, it can block the binding site (referring to detailed description) of native ligand, or the tyrosine residue of the binding site of blocking-up receptor kinase, stops phosphorylation and further cascade signal thus.

Demonstrating a dynamical representative in clinical trial is Iressa ^TM(ZD-1839), it can be used for NSCLC indication (nonsmall-cell lung cancer).

Though more existing be in the medicine and the method that are hopeful to be used for the treatment of tumor in research and development and the listing, but still need have improved characteristic and other medicament and the pharmaceutical composition and the combination of the effect that increases.

Summary of the invention

The present invention is based on the inventor's following discovery: some is for example crossed receptor tyrosine kinase such as the ErbB acceptor molecule of expressing on the tumor cell surface and has the specificity epitope site at native ligand in conjunction with the territory in diseased cells, different antibodies or generally speaking, homospecificity can not combine with these sites simultaneously, and insignificant mutual obstruction can not occur or only exist.Obviously, these antibody or specificity bonded epi-position aspect 3-d modelling, compare relative less with the full size in conjunction with the territory of acceptor molecule.It can induce increase to the active downward modulation of signal path, the preferred blocking-up that increases, and the blocking-up that increases thus to whole signal cascade to the ErbB receptor.

The present invention has described the new ideas of oncotherapy first, promptly individuality is used effective fragment on bi-specific antibody or its function, the first specific antigen binding site by described bi-specific antibody combines with the second different epi-positions of identical or different receptor with the first epi-position combination and the second specific antigen binding site, and blocking-up or inhibition ErbB receptor, preferred EGF receptor (EGFR).

Can find, this bi-specific antibody can be by two different antigen binding site and simultaneously with the native ligand of same receptor molecule (for example EGFR or Her-2) or different acceptor molecule (for example EGFR and Her-2) in conjunction with the different epi-position combinations in the territory, and hindering significantly mutually between the different antigen binding sites of antibody can not occur, thereby make and higher antibody density occurs on the receptor and cause (by reducing the binding ability with natural (excitability) part such as EGF or TGF a) much better than inhibition of signal cascade the corresponding acceptor molecule of monomer or dimer unit form.This should cause the stronger inhibition of tumor growth and/or cause solid tumor or the apoptosis of neoplasm metastasis increases.Preferred antibody especially above and anti-EGFR and the anti-Her2 antibody hereinafter described, and fragment, preferred bispecific F (ab ') 2 fragments, this is because it has less size.In embodiment preferred of the present invention, described by first antigen binding site that comes from humanization, chimeric or Mus type MAb 425 and come from bi-specific antibody (fragment) (BAb＜425 that humanization, second antigen binding site chimeric or Mus type MAb 225 are formed, 225 〉, F (ab '＜425 〉, ab '＜225 〉)).In other embodiments of the present invention, described by first antigen binding site that comes from humanization, chimeric or Mus type MAb 425 and come from bi-specific antibody (fragment) (BAb＜425 that humanization, second antigen binding site chimeric or Mus type MAb 4D5 are formed, 4D5 〉, F (ab '＜425 〉, ab '＜4D5 〉)).In other embodiments of the present invention, described by first antigen binding site that comes from humanization, chimeric or Mus type MAb 225 and come from bi-specific antibody (fragment) (BAb＜225 that humanization, second antigen binding site chimeric or Mus type MAb4D5 are formed, 4D5 〉, F (ab '＜425 〉, ab '＜4D5 〉)).In the above-described embodiment, humanization MAb 425, chimeric MAb225 (CETUXIMAB

) and humanization 4D5 (HERCEPT1N

) preferably as source antibody.The present invention also comprises hybrid antibody (heteroantibody) or its fragment in principle.The hybrid antibody of synthetic preparation even can partly form (for example＜425,225,4D5 〉) by coming from three kinds of different anti-egfr antibodies or its segmental three different antigen binding sites.

Find, compare with corresponding monospecific antibody, can strengthen the crosslinked/dimerization of similar and different ErbB receptor according to bi-specific antibody of the present invention, strengthen blocking-up/inhibitions, strengthen inducing ErbB receptor-specific signals path adjusting to the ErbB receptor.Interesting is, this crosslinked action can further involved described bi-specific antibody (fragment) and the mixture of the anti-ErbB antibody of monospecific (fragment) strengthen, wherein said monospecific antibody (fragment) preferably has the described first or second identical antigen binding site of antigen binding site with bi-specific antibody (fragment).In other words: for example, (i) MAb 425 or MAb 225 or MAb 4D5 and BAb＜425,225 〉; Or (ii) MAb 425 or MAb 225 or MAb 4D5 and BAb＜425,4D5 〉; Or (iii) MAb 425 or MAb 225 or MAb 4D5 and BAb＜4D5,225〉mixture compare with MAb or BAb are used with same concentrations as medicament separately, the former causes the inhibition and the downward modulation to the ErbB receptor of increase.

Though above-mentioned discovery only to the ErbB receptor is made as target receptor molecule, be should be noted that disclose by the inventor and as above-mentioned and following the principles of science also be applicable to other biological acceptor outside the ErbB.

Randomly, compositions of the present invention also comprises the therapeutical active compound that can support and strengthen the effect of above-mentioned molecule.

Described medicament can be a cytotoxic agent, and antagonism molecule preferably is such as tyrosine kinase antagonist, other ErbB antagonist, hormone receptor antagonists, protein kinase antagonist or anti-angiogenic agent.To specifically describe in more detail below and can be used for described molecule of the present invention.

Therapeutic activity agent of the present invention also can provide with the form of the pharmaceutical kit that comprises following packing, described packing be included in the container separately or one or more the described antagonisies in single container.Utilize the Therapeutic Method of this associating can randomly comprise radiation treatment.On the whole go up, this medicament administration can be followed with radiotherapy and carry out, wherein radiotherapy can with medicament administration basically simultaneously or before or after carry out.Can carry out simultaneously basically or carry out in succession according to using also of different medicaments in the conjoint therapy of the present invention.There is the tumor of the receptor that participates in tumor vessel formation successfully to treat on the cell surface with conjoint therapy of the present invention.

The growth of known cancer and growth have many interchangeable routes.If one route is blocked, then they often can be by expressing and using other receptors and signal pathway to be transformed on another route.Therefore, owing to of the present inventionly medication combinedly can block several so possible tumors and grow tactfully, thereby have a plurality of advantages.Combination according to the present invention can be used for treating and prevents those to be present in associated hormone receptor on the tumor cell surface and growth and growing tumors, tumor sample disease and neoplasia disease and neoplasm metastasis by activation.

Different pharmacy optimizations in the present invention's associating promptly, are lower than the dosage that conventional clinical condition uses down with the low dosage use in conjunction.When using chemical compound of the present invention, compositions, medicament and treatment to individuality, the advantage that reduces dosage comprises the incidence rate that has reduced the relevant side reaction of high dose.For example, by reducing as mentioned and the dosage of medicament hereinafter described, compare with observed effect under the high dose condition, the frequency and the order of severity of nausea and vomiting all are reduced.Expection reduces the quality of life that rate of side effects can be improved the cancer patient.The advantage that reduces rate of side effects also comprises the compliance that improves the patient, reduce because of side reaction need hospitalization number of times, reduce the use of required analgesic when curing the pain that side reaction brings.Therapeutic effect maximization when in addition, method of the present invention and associating can also make high dose.

Combinations thereof demonstrates amazing synergism.Can observe this medication combined tumor that makes of use in the clinical research and occur dwindling really and disintegrating, not detect tangible medicine side reaction simultaneously.

The present invention relates generally to:

Bi-specific antibody, or effective fragment on its function, its comprise with bonded first antigen binding site of first epi-position of an ErbB receptor and with the second epi-position bonded second different antigen binding sites of the 2nd ErbB receptor.

Corresponding bi-specific antibody or its fragment, wherein said first and/or described second epi-position be positioned at described receptor native ligand in conjunction with the territory.

Corresponding bi-specific antibody or its fragment are compared with corresponding monospecific antibody, and it strengthens blocking-up and/or inhibition to the ErbB receptor, and strengthen inducing ErbB receptor-specific signals path downward modulation.

Corresponding bi-specific antibody or its fragment, it strengthens inducing the crosslinked and/or dimerization with identical or different specific acceptor molecule.

Corresponding bi-specific antibody or its fragment, first epi-position of a wherein said ErbB receptor is different with second epi-position of the 2nd ErbB receptor.

The bi-specific antibody of claim 5 or its fragment, a wherein said ErbB receptor is different with described the 2nd ErbB receptor.

Corresponding bi-specific antibody or its fragment, the wherein said first and second ErbB receptors are identical.

Corresponding bi-specific antibody or its fragment, a wherein said ErbB receptor is EGF receptor (EGFR).

Corresponding bi-specific antibody or its fragment, wherein said the 2nd ErbB receptor is ErbB-2 (Her-2).

Corresponding bi-specific antibody or its fragment, the wherein said first and second ErbB receptors are EGF receptor (EGFR).

Corresponding bi-specific antibody or its fragment, wherein said first antigen binding site comes from MAb 425 humanized, chimeric or Mus.

Corresponding bi-specific antibody or its fragment, wherein said first antigen binding site comes from MAb 225 humanized, chimeric or Mus.

Corresponding bi-specific antibody or its fragment, wherein said first antigen binding site comes from MAb 425 humanized, chimeric or Mus, and described second antigen binding site comes from MAb 225 humanized, chimeric or Mus, and the native ligand of each antigen binding site and identical EGF acceptor molecule is in conjunction with the different epi-position combinations in the territory.

Corresponding bi-specific antibody or its fragment, a wherein said ErbB receptor are EGF receptors (EGFR) and described the 2nd ErbB receptor is ErbB-2 (Her-2).

Corresponding bi-specific antibody or its fragment, wherein said first antigen binding site comes from MAb 425 humanized, chimeric or Mus or 225, and described second antigen binding site comes from MAb 4D5 (Herceptin

).

Corresponding bi-specific antibody or its fragment, wherein fragment is F (ab ') 2.

Pharmaceutical composition, it comprises as effective fragment and pharmaceutically acceptable alternatively carrier, diluent or excipient on each described bi-specific antibody of above-mentioned claim or its function.

Corresponding pharmaceutical compositions, it also comprises effective fragment on the anti-ErbB antibody of monospecific or its function.

Corresponding pharmaceutical compositions, effective fragment is selected from MAb 425, MAb 225 on the anti-ErbB antibody of wherein said monospecific or its function, or MAb4D5 (Herceptin

).

Corresponding pharmaceutical compositions, it also comprises cytotoxic agent.

Corresponding pharmaceutical compositions, wherein said cytotoxic agent is a chemotherapeutics.

Corresponding pharmaceutical compositions, wherein said chemotherapeutics is selected from: cisplatin, amycin, gemcitabine, docetaxel, paclitaxel, bleomycin.

Corresponding pharmaceutical compositions, wherein said cytotoxic agent are ErbB acceptor inhibitor, tyrosine kinase inhibitor, protein kinase A inhibitor, or anti-angiogenic agent.

A kind of pharmaceutical kit, it comprises

(i) first packing, its comprise at least on above-mentioned bi-specific antibody or its function effectively fragment and

(ii) second pack, it comprises effective fragment on the anti-ErbB antibody of monospecific or its function at least.

Corresponding pharmaceutical kit, it comprise comprise BAb＜h425, c225 or segmental first packing of its F (ab ') 2 and comprise effectively segmental second packing on humanization MAb 425 (h425), chimeric MAb 225 (c225) or humanization Mab 4D5 or its function.

Corresponding pharmaceutical kit, it also comprises the 3rd packing, and this packing comprises other medicament.

Corresponding pharmaceutical kit, wherein said other medicament is the cytotoxicity medicine.

Corresponding pharmaceutical kit, wherein said cytotoxicity medicine is selected from: cisplatin, amycin, gemcitabine, docetaxel, paclitaxel, bleomycin, ErbB acceptor inhibitor, tyrosine kinase inhibitor, protein kinase A inhibitor and anti-angiogenic agent.

Above-mentioned bi-specific antibody or pharmaceutical composition/test kit were used for the treatment of purposes in the medicine of the tumor of expressing the ErbB receptor and neoplasm metastasis in preparation.

A kind of tumor of expressing the ErbB receptor and method of neoplasm metastasis for the treatment of in individuality, it comprises effectively fragment or pharmaceutical composition/test kit on the bi-specific antibody described in the above-mentioned and claim of effective dose on the described individual administering therapeutic or its function.

A kind of method that strengthens ErbB receptor-specific signals path downward modulation in crossing the tumor express the ErbB receptor, this method is by to effectively fragment or pharmaceutical composition/test kit are implemented on the above-mentioned bi-specific antibody of effective dose on the individual administering therapeutic or its function.

Corresponding method also comprises the cytotoxic agent of the patient being used effective dose.

Corresponding method, wherein said cytotoxic agent is a chemotherapeutics, and is selected from: cisplatin, amycin, gemcitabine, docetaxel, paclitaxel, bleomycin.

Corresponding method, wherein said cytotoxic agent is ErbB acceptor inhibitor, tyrosine kinase inhibitor, protein kinase A inhibitor, or anti-angiogenic agent.

In a preferred embodiment of the invention, bi-specific antibody of the present invention bonded ErbB acceptor type of antigen binding site is ErbB1 receptor (EGFR).

Therefore, the present invention relates more specifically to:

Bi-specific antibody or its fragment, its can with the different epi-position combinations on being positioned at identical or different ErbB acceptor molecule type, described antibody comprise with bonded first antigen binding site of the epi-position of the first acceptor type ErbB1 and with the different epi-positions bonded second different antigen binding sites of the 2nd ErbB acceptor molecule type.

Bi-specific antibody, wherein said the 2nd ErbB acceptor molecule type is ErbB1 (EGFR).

Bi-specific antibody, wherein said the 2nd ErbB acceptor molecule type is ErbB2 (Her-2).

Bi-specific antibody, wherein at least one described epi-position is positioned at receptor binding domains.

Bi-specific antibody, wherein said receptor binding domains are that the native ligand of described receptor is in conjunction with the territory.

Bi-specific antibody, wherein first or second antigen binding site combines the epi-position combination in the territory with the native ligand of described ErbB acceptor molecule type.

Bi-specific antibody, wherein first and second antigen binding sites combine the epi-position combination in the territory with the native ligand of described ErbB acceptor molecule type.

Bi-specific antibody, wherein antigen binding site be positioned at identical ErbB acceptor molecule type on different epi-position combinations.

Bi-specific antibody, wherein antigen binding site be positioned at different ErbB acceptor molecule types on different epi-position combinations.

Bi-specific antibody, wherein first and second antigen binding sites separately with the native ligand of described ErbB receptor in conjunction with the different epi-position combinations in the territory, block thus and/or suppress this receptor, compare with corresponding monospecific antibody whereby, strengthen blocking-up and/or inhibition and enhancing inducing ErbB receptor-specific signals path downward modulation to the ErbB receptor.

Bi-specific antibody is wherein compared with the combination of epi-position on bi-specific antibody and the identical ErbB acceptor molecule type, has strengthened inducing crosslinked and/or dimerization with identical or different specific different ErbB acceptor molecules.

Bi-specific antibody, wherein said first antigen binding site comes from MAb 425 humanized, chimeric or Mus.

According to each bi-specific antibody of claim 1-11, wherein said first antigen binding site comes from MAb 225. humanized, chimeric or Mus

Bi-specific antibody, it is named as " BAb＜h425; c225〉", wherein said first antigen binding site comes from MAb 425 humanized, chimeric or Mus, and described second antigen binding site comes from MAb 225 humanized, chimeric or Mus, the different epi-position combinations on each antigen binding site and ErbB1 receptor (EGFR) molecule.

Bi-specific antibody, wherein said different epi-positions be positioned at native ligand in conjunction with the territory.

Bi-specific antibody, wherein second antigen binding site combines with ErbB 2 acceptor molecules (Her-2) or vegf receptor molecule.

The bi-specific antibody of claim 16, wherein said second antigen binding site comes from MAb4D5 (Herceptin

).

Come from above-mentioned and bispecific antibody fragment each described bi-specific antibody of claim, wherein fragment is F (ab ') 2.

Pharmaceutical composition, it comprises one or more bi-specific antibodys or its fragment described in above-mentioned and the claim, and randomly pharmaceutically acceptable carrier, diluent or excipient.

Pharmaceutical composition, it also comprises effective fragment on the anti-ErbB antibody of monospecific or its function.

Pharmaceutical composition, effective fragment is selected from MAb 425, MAb 225 on the anti-ErbB antibody of wherein said monospecific or its function, or MAb 4D5 (Herceptin

).

Pharmaceutical composition, it also comprises cytotoxic agent.

Pharmaceutical composition, wherein said cytotoxic agent is a chemotherapeutics.

Pharmaceutical composition, wherein said chemotherapeutics is selected from: cisplatin, amycin, gemcitabine, docetaxel, paclitaxel, bleomycin.

Pharmaceutical composition, wherein said cytotoxic agent are ErbB acceptor inhibitor, vegf receptor inhibitor, tyrosine kinase inhibitor, protein kinase A inhibitor, anti-angiogenic agent, antihormone agent, or cytokine.

Immunoconjugates, it comprises above-mentioned bi-specific antibody, and this antibody directly or by linkers merges at C-terminal and biologically effective peptide, polypeptide or protein, and wherein preferred described protein or polypeptide are cytokines.

Pharmaceutical kit, it comprises

(i) first packing, its comprise the bi-specific antibody described in the above-mentioned at least and claim or immunoconjugates and

(ii) second packing, it comprises at least on the anti-ErbB antibody of monospecific or its function effectively fragment.

Pharmaceutical kit, it comprises first packing and second packing, this first comprises bi-specific antibody " BAb＜h425; c225〉" or its F (ab ') 2 fragments or its immunoconjugates, and described second comprises on humanization MAb 425 (h425), chimeric MAb 225 (c225) or humanization Mab 4D5 or its function effectively antibody fragment or its immunoconjugates.

Pharmaceutical kit, it also comprises the 3rd packing, and this packing comprises the cytotoxicity medicine.

Pharmaceutical kit, wherein said cytotoxicity medicine is selected from: cisplatin, amycin, gemcitabine, docetaxel, paclitaxel, bleomycin, ErbB acceptor inhibitor, vegf receptor inhibitor, tyrosine kinase inhibitor, protein kinase A inhibitor, antihormone agent and anti-angiogenic agent.

Above-mentioned bi-specific antibody or pharmaceutical composition/test kit were used for the treatment of purposes in the medicine of the tumor of expressing the ErbB receptor and neoplasm metastasis and relevant disease in preparation.

Detailed Description Of The Invention

The present invention is based on following observed result, specific two or more MAb that have at different immunogenic structures combine with its epi-position with can simultaneously and unhinderedly or only having inapparent obstruction, described epi-position can be positioned at same receptor, or even in the identical receptor domain, for example in the ligand binding domain.Therefore, have specific bispecific antibody at the different epi-positions of same receptor and can combine with two specificity epitope and form the bivalence receptor-antibody complex thus, when using monospecific Mab, in most cases on single receptor, cannot see this complex.

Perhaps, the antigen binding site of described bi-specific antibody can interact with other contiguous same receptor, makes up complex thus between these receptors.In addition, the bi-specific antibody at the antigenic structure on the not isoacceptor of identical or different receptor family also can be used to make up complex between these receptors.

With only compare with a kind of therapeutic effect of monospecific antibody, use and can greatly improve therapeutic effect at one or more bi-specific antibodys of identical or different receptor or the associating of monospecific and bi-specific antibody:

Each antigen binding site of bi-specific antibody combines with its specificity epitope on target receptor (for example EGFR) independently.

Two antigen binding sites of bi-specific antibody can interact with its specificity epitope, and described epi-position can be positioned on the same receptor and may be positioned at the same receptor territory or be positioned at not on the isoacceptor.

Bi-specific antibody can be independently in conjunction with two kinds of different epi-positions on the same receptor.With the affinity of monospecific antibody in most of the cases be subject to the unit price of this receptor in conjunction with comparing, this has increased the total affinity of bi-specific antibody to single receptor.

Since to single receptor than high affinity, MAb compares with monospecific, effectively blocks the bi-specific antibody that receptor will need low concentration.

Because bi-specific antibody is more effective than monospecific MAb aspect receptor blocking, it can be induced receptor activation and downstream signal are conducted more significant inhibitory effect.

Similarly, have in the ligand binding domain or the mixture of specific one or more bi-specific antibodys of contiguous different epi-positions in conjunction with the territory or monospecific and bi-specific antibody can increase the effect of receptor blocking.

Because it is more effective than the receptor blocking that only causes by single a kind of monospecific or bi-specific antibody by the receptor blocking that causes at uniting of two or more monospecifics of same receptor domain and/or bi-specific antibody, so can obtain part-, thereby cause more effective receptor inactivation in conjunction with more effective inhibitory action.

This more effective receptor inactivation causes the downstream receptor signal is conducted more effective inhibition, and causes thus the influence of part-dependent cell function is strengthened.

Because this more effective receptor blocking can not lose effect so can reduce the dosage (or concentration) of employed each monospecific and/or bi-specific antibody.When use was lower than the suitableeest therapeutic dose and still demonstrates the therapeutic antibodies of dose-limiting toxicity or side effect, this may be very significant.

To at first form the receptor-antibody complex of dimerization with the bi-specific antibody and the monospecific antibody of different receptors bind on the same cell.But because its different antigenic specificity, bi-specific antibody can form such receptor-antibody complex, and this complex is not limited to the dimer of same receptor.Thus, the receptor clustering thing that is formed by bi-specific antibody can comprise receptor big, theory unlimited quantity.

These big receptor-antibody complex have improved the internalization of receptor, and thus may be more effective for removing receptor from cell surface and reducing receptor-dependent cell function subsequently.

The formation of big receptor-antibody complex can be further strengthened by uniting two or more monospecifics and/or bi-specific antibody, the internalization of receptor can be further strengthened thus and the downward modulation of receptor-dependent cell function.

Bi-specific antibody and uniting of two or more monospecifics and/or bi-specific antibody at identical or different receptor can be used for treating the tumor that has suitable receptor.The EGFR positive tumor is a typical example, but Therapeutic Principle's of the present invention application is not limited to this indication.Thus, adopt identical principle can treat the miscellaneous tumor that has other receptor, receptor family or other antigenicity structure.

Utilize the treatment that bi-specific antibody carries out or utilize the therapeutic alliance of carrying out at different antigenic two or more monospecifics on the identical or different receptor and/or bi-specific antibody, can also combined chemotherapy medicine and/or radiation enforcement therapeutic alliance.

Utilize the treatment that bi-specific antibody carries out or utilize the therapeutic alliance of carrying out at different antigenic two or more monospecifics on the identical or different receptor and/or bi-specific antibody, also can unite use with other Therapeutic Principle, described other Therapeutic Principle includes but not limited to hormone antagonist or hormone agonist, angiogenesis inhibitor therapy and other therapies.

Be example with treatment herein, described and used the Therapeutic Principle who has at the associating of the specific bi-specific antibody of the different antigenic structures on the identical or different receptor or monospecific and bi-specific antibody the EGFR positive tumor.But this principle is not limited to EGFR, and it is applicable to the application of any other target antigen.

If there is not other explanation, term and word that the present invention uses have implication given below and definition.And these definition and implication have been described the present invention in more detail, comprise preferred embodiment.

" receptor " or " acceptor molecule " is meant soluble or film combination/relevant albumen or glycoprotein, and it contains one or more can the combination with part to form the territories of receptor-ligand complex.By combining with the part that may be agonist or antagonist, receptor can be activated or inactivation, can start thus or the disabling signal path.

Term " acceptor molecule type " or " ErbB (ErbB1) acceptor molecule type " are meant concrete acceptor type such as ErbB1, ErbB2 etc., but not the concrete unimolecule of this receptor type.In other words: bi-specific antibody of the present invention can combine with first epi-position of ErbB1 acceptor molecule individuality by its first antigen binding site, and the second different epi-position combinations of second antigen binding site of this antibody ErbB1 acceptor molecule individuality identical with this.Also may be the second different epi-position combinations of second antigen binding site with the acceptor molecule individuality of another same type (ErbB1) of this antibody.In addition, also may be the second different epi-position combinations of acceptor molecule individuality of the different ErbB acceptor molecule of second antigen binding site type (for example ErbB2) of this antibody with another.

" part " or " receptors ligand " is meant so natural or synthetic chemical compound, its can with acceptor molecule in conjunction with forming the receptor-ligand complex.The term part comprises agonist, antagonist and the chemical compound with partial agonist/antagonist activities.

" agonist " or " receptor stimulating agent " is meant so natural or synthetic chemical compound, and itself and receptors bind form receptor-agonist complex, activate described receptor or receptor-agonist complex and enabling signal path and further biological process.

" antagonist " or " receptor antagonist " is meant the natural or synthetic chemical compound with biological effect opposite with agonist.Antagonist and receptors bind are by competing the effect that receptor is blocked receptor stimulating agent with agonist.Antagonist is that the ability of effect according to its blocking-up agonist defines.Receptor antagonist also can be an antibody or it has the fragment of immune therapeutic activity.To enumerate and discuss the preferred antagonist of the present invention below.

" ErbB receptor " is meant the receptor protein tyrosine kinase that belongs to (as above-mentioned) ErbB receptor family, comprises EGFR (ErbB1), and ErbB2 awaits in ErbB3 and ErbB4 receptor and this family other members that differentiate in the future.The ErbB receptor generally contain one can with the bonded cell of ErbB part foreign lands; A lipophilic membrane-spanning domain; A conservative intracellular tyrosine kinases territory; And one contain some can be by the c-terminus signal domain of the tyrosine residue of phosphorylation.The ErbB receptor can be " native sequences " ErbB receptor or its " variant amino acid sequence body ".Preferred ErbB receptor is the people ErbB receptor of native sequences.ErbB1 is meant the gene of coding EGFR protein product.Most preferably the EGF receptor (EGFR, HER1)." ErbB1 " and " HER 1 " and " EGFR " are expressed in that this paper is used interchangeably and all refer to people HER 1 albumen." ErbB2 " and " HER 2 " is used interchangeably and all refers to people HER 2 albumen at this paper.The preferred ErbB1 receptor of the present invention (EGFR).

" ErbB part " is the polypeptide of combination and/or activation ErbB receptor.Comprise for example EGF with the bonded ErbB part of EGFR, TGF-α, amphiregulin, β cell regulin (betacellulin), HB-EGF and epiregulin (epiregulin), preferred EGF and TGF-α.

In the context of the present invention, " ErbB receptor binding domains " is the regional area (binding sequence/ring/bag) of ErbB receptor, and this zone and native ligand or antagonism or excitability medicine combine.This zone not only can comprise a kind of specific binding site or epi-position, can also comprise two or more epi-positions or binding site.Species specificity associative list position in this territory and a class antagonism or excitability medicine or part combine.Yet different medicaments usually can by inhibition or activation cause unique and unique signal path in conjunction with different epi-positions in the territory or that be close in conjunction with the territory in conjunction with being considered to the same receptor type, and described signal path is distinctive to described antibody.In addition, should be understood that employed phrase " in the territory " also comprises the true position in conjunction with the territory that is close to corresponding native ligand in this description and claims.

" ErbB is in conjunction with epi-position or binding site " be meant the ErbB receptor in conjunction with in the territory or with the amino acid whose conformation and/or the configuration that combine territory next-door neighbour, describedly combine with part or receptor antagonist/agonist in conjunction with the territory.

" identical ErbB/ErbB1 acceptor molecule " not necessarily means same acceptor molecule, but also comprises other acceptor molecule of same type.Preferably, mean same acceptor molecule.

Term " ErbB receptor antagonist/inhibitor " is meant can be in conjunction with the biologically effective molecule of also blocking or suppress the ErbB receptor.Therefore, by the blocking-up receptor, antagonist has stoped the combination of ErbB part (agonist) and the activation of agonist/ligand receptor complex.The ErbB antagonist can at HER 1 (ErbB1, EGFR), HER2 (ErbB2) and ErbB3 and ErbB4.

The preferred antagonist of the present invention at the EGF receptor (EGFR, HER1).The ErbB receptor antagonist can be the immune therapeutic activity fragment or the non-immunobiology molecule of antibody or antibody, such as peptide, polypeptide protein.In chemical molecular is also included within, but the preferred antagonist of the present invention is anti-egfr antibodies and Anti-HER 2.

The preferred antibody of the present invention is anti-HER1 and anti-Her2 antibody, more preferably anti-HER1 antibody.Preferred anti-HER1 antibody is MAb425, and preferred humanized MAb425 (hMAb425, US 5,558, and 864; EP 0,531 472) and chimeric MAb 225 (CETUXIMAB

).Monoclonal antibody h425 most preferably, it has demonstrated the untoward reaction and the side reaction of high curative effect and reduction in single Drug therapy.Most preferred anti-Her2 antibody is the HERCEPTIN of Genentech/Roche listing

Effective EGF receptor antagonist of the present invention can also be natural or synthetic chemical compound.Some examples of this type of preferred molecule include the salt of organic compounds, organo-metallic compound, organic compound and organo-metallic compound.The example of chemistry HER2 receptor antagonist has: the heteroaryl compound (US 5,656,655) that styryl replaces; Two monocycles and/or bicyclic aryl heteroaryl, carbocyclic ring and assorted carbocyclic compound (US 5,646,153); Trinucleated pyrimidine compound (US 5,679,683); Have receptor tyrosine kinase and suppress active quinazoline derivant (US 5,616,582); Heteroaryl ethylene two bases or heteroaryl ethylene two basic aryl compounds (US 5,196,446); Name is called 6-(2,6-dichloro-phenyl)-2-(4-(2-diethyl-amino ethoxy) phenyl amino)-8-methyl-8H-pyrido (2,3)-chemical compound (Panek of 5-pyrimidin-7-ones, Deng, 1997, J.Pharmacol.Exp.Therap.283,1433), it can suppress EGFR, PDGFR and FGFR receptor family.

According to the present invention, term " tyrosine kinase antagonist/inhibitor " is meant and naturally or synthetic can suppresses or block the tyrosine kinase medicament of (comprising receptor tyrosine kinase).Therefore, this term comprises above-mentioned ErbB receptor antagonist/inhibitor in fact.Except above and the hereinafter mentioned anti-ErbB receptor antibody, this definition preferred tyrosine kinase antagonist down is to show in single Drug therapy breast carcinoma and carcinoma of prostate effective chemical chemical compound.Suitable indolocarbazole type tyrosine kinase inhibitor can utilize as United States Patent (USP) 5,516,771; 5,654,427; 5,461,146; The information acquisition of 5,650,407 documents such as grade.United States Patent (USP) 5,475,110; 5,591,855; 5,594,009 and WO96/11933 pyrrolocarbazole type tyrosine kinase inhibitor and carcinoma of prostate are disclosed.Most promising a kind of anticarcinogen is gefitinib (IRESSA in the literary composition

Astra Zeneca), its be in the news to suffer from nonsmall-cell lung cancer (NSCLC) and late period head and neck cancer the patient have notable therapeutic effect and good tolerability.

The preferred dose of chemical tyrosine kinase inhibitor defined above be every day the 1pg/kg body weight to the 1g/kg body weight.More preferably, the dosage of tyrosine kinase inhibitor be every day the 0.01mg/kg body weight to the 100mg/kg body weight.

According to biomolecule of the present invention preferably any variant of antibody or its fragment or antibody such as immunoconjugates.

In this article, term " antibody " or " immunoglobulin " have the most generalized implication, the multi-specificity antibody (for example bi-specific antibody) and the antibody fragment that constitute particularly including complete monoclonal antibody, polyclonal antibody, by at least 2 complete antibodies are as long as it demonstrates and has required biologic activity.This term generally comprises by 2 or a plurality ofly has the antibody of different binding specificities or a hybrid antibody that antibody fragment links together and constitutes.

According to the aminoacid sequence of antibody constant region, complete antibody can be divided into different " antibody (immunoglobulin) type ".Complete antibody has 5 main type: IgA, IgD, and IgE, IgG and IgM, wherein some can further be divided into " subclass " (isotype), as IgG1, IgG2, IgG3, IgG4, IgA and IgA2.CH corresponding to the different antibodies type is called α, δ, ε, γ and μ.The main type of the preferred antibody of the present invention is IgG, and IgG1 and IgG2 more specifically say so.

Antibody usually is the glycoprotein of molecular weight about 150,000, is made up of 2 same light (L) chain and 2 same weights (H) chains.Every light chain all passes through a covalent disulfide bonds and links to each other with heavy chain, and disulfide bond number difference between the heavy chain of different immunoglobulin isotypes.Every heavy chain and light chain be the intrachain disulfide bond at regular interval also.Every heavy chain all at one end has a variable region (VH), is thereafter a plurality of constant regions.Every light chain all has a variable region (VL) at one end, and a constant region is arranged on the other end.First constant region of the constant region of light chain and heavy chain is arranged side by side, and the variable region of light chain and the variable region of heavy chain are arranged side by side.It is believed that particular amino acid residue constitutes the interface between light chain and the variable region of heavy chain.The antibody " light chain " of invertebrate species 2 kinds of clear and definite different types be can be divided into based on the aminoacid sequence of its constant region, handkerchief (κ) and lambda (λ) promptly blocked.

Term used herein " monoclonal antibody " is meant the antibody that obtains from the antibody population of homogenizing basically, that is, except the sudden change (these sudden changes may exist in a small amount) of possible natural generation, each strain antibody in the antibody population all is identical.Monoclonal antibody has the specificity of height, and it is at single antigen site.And different with the polyclonal antibody goods, polyclonal antibody comprises the different antibodies at different determinants (epi-position), and each monoclonal antibody is all only at a determinant on the antigen.Except their specificity, the superior part of monoclonal antibody also is to synthesize does not have the monoclonal antibody that other antibody pollute.The MONOCLONAL ANTIBODIES SPECIFIC FOR method comprises Kohler and Milstein (1975, Nature 256,495) and " monoclonal antibody technique; the preparation of Rodents and human hybridoma and sign " (1985, Burdon etc., Eds, Laboratory Techniquesin Biochemistry and Molecular Biology, Volume 13, Elsevier SciencePublishers, the hybridoma method of describing in Amsterdam) can be prepared perhaps that (example can be referring to US 4 with well-known recombinant DNA method, 816,567).For example also can adopt Clackson etc., Nature, 352:624-628 (1991) and Marks etc., J.Mol.Biol., the technology of describing among the 222:58,1-597 (1991) is separated monoclonal antibody from phage antibody library.

Term " chimeric antibody " is meant such antibody, the part of its heavy chain and/or light chain with from the antibody of specific species or belong to the identical or homology of corresponding sequence in the antibody of specific antibodies type or subclass, yet the remainder of chain then with from the antibody of another species or belong to the identical or homology of corresponding sequence in the antibody of another antibody type or subclass, this term also refers to the fragment of this kind antibody, (for example: US 4 as long as it has required biologic activity, 816,567; Morrison etc., Proc.Nat.Acad.Sci.USA, 81:6851-6855 (1984)).The method of producing mosaic type and humanized antibody is well-known to those skilled in the art.For example, the method for preparing chimeric antibody comprise Boss (Celltech) and Cabilly (Genentech) (US 4,816,397; US 4,816,567) method of describing in the patent.

" humanized antibody " is the antibody of inhuman (as Rodents) chimeric antibody form, and it contains the sequence that comes from non-human immunoglobulin of minimum flow.With regard to most of, humanized antibody is human normal immunoglobulin's (receptor antibody), and wherein the residue of the hypervariable region of receptor (CDR) is replaced by inhuman species hypervariable region residue (as mice, rat, rabbit or non-human primates) (donor antibody), that have required specificity, affinity and performance.Sometimes, human normal immunoglobulin's framework region (FR) residue is replaced by corresponding inhuman residue.In addition, humanized antibody can contain the residue that does not all have on receptor antibody and the donor antibody.Can further the become more meticulous performance of antibody of these modifications.Usually, humanized antibody contains all basically variable region (at least one, two variable regions typically), wherein all or all basically hypermutation ring be all corresponding to the appropriate section of non-human immunoglobulin, and all or all basically FR have human normal immunoglobulin's sequence.Humanized antibody also can randomly contain at least a portion constant region for immunoglobulin (Fc), is typically human normal immunoglobulin's constant region.The preparation method of humanized antibody is described in for example Winter, and US 5,225,539 and Boss (Celltech, US 4,816,397) in.

" antibody fragment " comprises the part of complete antibody, preferably comprises the antigen binding domain or the variable region of antibody.The example of antibody fragment comprises Fab, Fab ', F (ab ') 2, Fv and Fc fragment, double antibody (diabody), linear antibody, single-chain antibody molecule; And the multi-specificity antibody that constitutes by antibody fragment." complete " antibody is meant the variable region that comprises conjugated antigen and the antibody of constant region of light chain (CL) and CH (CH1, CH2 and CH3).

Preferably, complete antibody has one or more effector functions.Papain digestion antibody produces " Fc " fragment (its title has reflected that it is easy to crystalline ability) of 2 same antigen binding fragments (be referred to as " Fab " fragment, each fragment contains an antigen binding site and a CL district and a CH1 district) and remnants.

CH2, the hinge region of CH3 and IgG1 or the main type of IgG2 antibody are generally contained in " Fc " district of antibody.Hinge region is the group with about 15 amino acid residues, and its CH1 district and CH2-CH3 district combine.

Pepsin produces " F (ab ') a 2 " fragment, and it contains 2 antigen binding sites and still has crosslinked antigenic ability.

" Fv " is minimum antibody fragment, and it contains a complete antigen recognition and antigen binding site.This zone is made up of by the non-covalent dimer that is connected to form closely a variable region of heavy chain and variable region of light chain.3 of each variable region hypervariable regions (CDR) interact and determine an antigen binding site on VH-VL dimer surfaces in this configuration.Generally speaking, the antigen-binding specificity of antibody has been given in 6 hypervariable regions.But, even only variable region (or only contain half Fv of 3 the antigenic specificity hypervariable regions) also has the ability of identification and conjugated antigen, though its affinity than complete binding site is low.

" Fab " fragment also comprises first constant region (CH1) of the constant region of light chain and heavy chain and only has an antigen binding site.The segmental difference of " Fab ' " fragment and Fab has been more than the c-terminus in heavy chain CH1 district several residues, comprises one or more cysteine from antibody hinge region.

F (ab ') 2 antibody fragments produce with the right form of Fab ' fragment at first, between these two Fab ' fragments the cysteine hinge are arranged.Other chemical coupling of antibody fragment also be known (for example referring to Hermanson, Bioconjugate Techniques, Academic Press, 1996; US4,342,566).

" strand Fv ' " or " scFv " antibody fragment comprise the V of antibody, and V, the district, and wherein these domains are present on the polypeptide chain.Preferably, the Fv polypeptide also is included in the peptide linker between VH and the VL domain, and it can make scFv form antigen in conjunction with required structure.Strand FV antibody also can be from for example Pl ü ckthun (Rosenburg and Moore compile, Springer-Verlag, NewYork, pp.269-315 (1994) for The Pharmacology Of Monoconal Antibodies, Vol.113), WO93/16185; US 5,571, and 894; US 5,587, and 458; Huston etc. (1988, Proc.Natl.Acad.Sci.85,5879) or Skerra and Plueckthun (1988, Science 240,1038) are known.

Term " variable " or " FR " are meant the following fact, and promptly some part in the variable region has very different sequences between antibody and the antibody, and these parts are used for combination and the specificity of each specific antibodies to its specific antigen.Yet this transmutability is not the whole variable region that is evenly distributed in antibody.Its three of concentrating on light chain and variable region of heavy chain are called in the section of hypervariable region.The higher conservative part of variable region is called framework region (FR).Each all comprises four FR (FR1-FR4) variable region of natural heavy chain and light chain, and it mainly adopts the beta sheet configuration, is connected by three hypervariable regions, and the hypervariable region forms ring and connects the beta sheet structure, and forms the part of beta sheet structure sometimes.The hypervariable region of every chain closely keeps together by FR, and facilitate the formation of the antigen binding site of antibody (referring to Kabat etc. with the hypervariable region of another chain, Sequences Of Proteins Of ImmunologicalInterest, 5th Ed.Public Health Service, National Institutes Of Health, Bethesda, MD. (1991).Though constant region is not participated in antibody and antigenic combination directly, it demonstrates multiple effector function, as participating in the antibody dependent cellular cytotoxicity (ADCC) of antibody.

Term used herein " hypervariable region " or " CDR " are meant the amino acid residue of being responsible for the bonded antibody of antigen.The amino acid residue that the hypervariable region generally comprises " complementary determining region " or " CDR " (for example, the 24-34 position (L1) of variable region of light chain, 50-56 position (L2) and 89-97 position (L3) residue, the 31-35 position (H1) of variable region of heavy chain, 50-65 position (H2) and 95-102 position (H3) residue); And/or the amino acid residue of " hypermutation ring " (for example, the 26-32 position (L1) of variable region of light chain, 50-52 position (L2) and 91-96 position (L3) residue, the 26-32 position (H1) of variable region of heavy chain, 53-55 position (H2) and 96-101 position (H3) residue; Chothia and Lesk J.Mol.Biol.196:901-917 (1987))." framework region " or " FR " residue is meant those variable region residues except that the hypervariable region residue of this place definition.

Term " monospecific " is meant that according to antibody of the present invention wherein two of antibody binding sites have identical specificity, thus, and the identical epi-position on can bind receptor.Preferably, according to the present invention, pharmaceutical composition comprises monospecific antibody.

" bi-specific antibody (BAb) " is antibody (or its immune therapeutic activity fragment) single, two valencys, and it contains 2 not homospecific antigen binding sites.According to the present invention, BAbs is characterised in that BAb＜MAb 1, and MAb 2 〉, wherein＜MAb 1〉and＜MAb 2〉name comes from the antigen binding site of MAb 1 and MAb 2.For example, first antigen binding site is at angiogenesis receptor (for example integrin or vegf receptor), and second antigen binding site is at ErbB receptor (for example EGFR or HER 2).Bi-specific antibody can use chemical method (for example referring to (1981) Proc.Natl.Acad.Sci.USA 78 such as Kranz, 5807) or " polydoma " technology (referring to US4,474,893) or recombinant DNA technology preparation, all these technology are known in fact technology.Additive method has description at WO 91/00360 among WO 92/05793 and the WO96/04305.The also available single-chain antibody of bi-specific antibody prepare (for example referring to (1988) Proc.Natl.Acad.Sci.85 such as Huston, 5879; Skerra and Plueckthun (1988) Science 240,1038).These are the antibody variable region analog that produce with wall scroll polypeptide chain form.In order to constitute bispecific binders, single-chain antibody can be coupled at together by chemical method or the genetic engineering method that those skilled in that art know.Bi-specific antibody of the present invention also can prepare with the leucine zipper sequence.Used sequence can come from transcription factor Fos and Jun the leucine zipper district (Landschulz etc., 1988, Science 240,1759; Summary is seen, Maniatis and Abel, and 1989, Nature 341,24).Leucine zipper is the long special acid sequence of about 20-40 residue, and typically per 7 residues just have a leucine.This slide fastener sequence forms amphipathic alpha-helix, leucine residue on hydrophobic side aligning so that form dimer.Preferentially form heterodimer (O ' Shea etc., 1989, Science 245,646) with Fos and the corresponding peptide of the proteic leucine zipper of Jun.Bi-specific antibody that contains slide fastener and preparation method thereof also has open in WO92/10209 and WO93/11162.

Term " fusion rotein " is meant the natural or synthetic molecules of being made up of above-mentioned one or more biomolecule, wherein has not homospecific two or more molecules based on peptide or protein (comprising glycoprotein) and randomly merges by chemistry or based on amino acid whose linkers.This connection can be merged or N-C fusion (with 5 ' → 3 ' direction) by C-N, and preferred C-N merges and realizes.

Yet preferred fusion protein matter is immunoconjugates described as follows according to the present invention.

Term " immunoconjugates " refers to fusion rotein, and its meaning is antibody or immunoglobulin or its immunologic competence fragment that merges by covalent bond and non-immunology effector molecule.This fusion partners (partner) is preferably can be by glycosylated peptide or protein.Described non-antibody molecule can be connected to the C-terminal of heavy chain of antibody constant region or be connected to light chain and/or the N-terminal of variable region of heavy chain.This fusion partners can connect by linkers, and general this linkers is the peptide that contains 3-15 amino acid residue.Immunoconjugates of the present invention is by the fragment of immunization therapy effect and cytokine are preferably arranged at the immunoglobulin of ErbB receptor or its, such as TNF α, IFN γ or IL-2, or the fusion rotein formed of other toxic agents.Preferably, these are continuous by the C-terminal (that is its Fc part) of its N-terminal and described immunoglobulin based on peptide or proteinic molecule.

" hybrid antibody " is the fusion rotein of being made up of two or more antibody that merge by chemical cross-linking agent routinely or antibodies fragment basically, and each all has different binding specificities wherein said antibody.Hybrid antibody can prepare by two or more antibody or antibody fragment are conjugated in together.

Preferred hybrid antibody is made up of crosslinked Fab/Fab ' fragment.A lot of couplings or cross-linking reagent can be used for puting together of antibody.Protein A for example, carbodiimide, N-succinimido-S-acetyl group-thiacetate (SATA) and N-succinimido-3-(2-pyridine radicals dithio) propionic ester (SPDP) (for example referring to (1984) J.EXP.Med.160 such as Karpovsky, 1686; Liu etc. (1985) Proc.Natl.Acad.Sci.USA 82,8648).Other method also has Paulus, BehringInst.Mitt., No.78,118 (1985); (1987) J.Immunol.139 such as Brennan etc. (1985) Science 30,81 or Glennie, 2367 methods of describing.Another kind method uses adjacent phenylene dimaleimide (oPDM) that three Fab ' fragments are coupled at together (WO91/03493).

Multi-specificity antibody also is suitable among the present invention, and can be for example be prepared according to the instruction of WO94/13804 and WO98/50431.The preferred hybrid antibody of the present invention is the fusion rotein (for example MAB 425-MAB 225) that comprises the two kinds of anti-egfr antibodies (each different epi-position at same receptor of each antibody) that link together as described.

Term " cytokine " " be to act on the proteinic common name of another cell as the iuntercellular medium to what discharge by cell mass.The example of this type cytokines has lymphokine, monokine and traditional polypeptide hormone.Cytokine comprises growth hormone such as human growth hormone, N-methionyl human growth hormone and bovine growth hormone; Parathyroid hormone; Thyroxine; Insulin; Proinsulin; Relaxin; Relaxation precipitinogen; Glycoprotein hormones such as follicle stimulating hormone (FSH), thyrotropin (TSH) and lutropin (LH); Liver growth factor; Fibroblast growth factor; Prolactin antagonist; Galactagogin; Mice promoting sexual gland hormone related peptides; Inhibin; Activin; VEGF (VEGF); Integrin; Thrombopoietin (TPO); Nerve growth factor such as NGF β; PDGF; Transforming growth factor (TGF) is as TGF α and TGF β; Erythropoietin (EPO); Interferon such as IFN α, IFN β and IFN γ; Colony stimulating factor such as M-CSF, GM-CSF and G-CSF; Interleukin such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; With TNF α or TNF β.The preferred cytokine of the present invention is interferon and TNF α and IL-2.

Antibody " effector function " is meant those biological activitys that antibody Fc district (native sequences Fc district or aminoacid sequence variant Fc district) causes.The example of antibody mediated effect subfunction comprises the cytotoxicity that complement relies on, Fc receptors bind, the cytotoxic effect (ADCC) of antibody dependent cellular mediation, phagocytosis; The downward modulation of cell surface receptor (as B-cell receptor) etc.

Term " ADCC " (cytotoxic effect of antibody dependent cellular mediation) is meant a kind of so cell-mediated reaction, the non-specific cell toxic cell of wherein expressing Fc receptor (FcR) is (as NK cell (NK) cell, neutrophil cell, and macrophage) discern the binding antibody on the target cell and cause the cracking of target cell subsequently.The mediation ADCC main cell---the NK cell is only expressed Fc γ R III, and mononuclear cell can be expressed Fc γ R I, Fc γ R II and Fc γ R III.Be the ADCC activity of estimation purpose molecule, for example can utilize prior art (US 5,500,362; US 5,821,337) the external ADCC that describes in tests and carries out.Useful effector lymphocyte comprises PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) and NK cell (NK) cell in this experiment.

Term " Fc receptor " or " FcR " are used for describing and the bonded receptor in antibody Fc district.Preferred FcR is native sequences people FcR.And preferred FcR is the receptor (γ receptor) in conjunction with IgG antibody and comprises Fc γ R I, and the receptor of Fc γ R II and Fc γ R III subclass comprises the allele variant and the alternative splicing form of these receptors.Can be referring to for example to the summary of FcR, Ravetch and Kinet, Annu.Rev.Immunol 9:457-92 (1991).

As a specific embodiment, Therapeutic Method of the present invention comprises that other treatment goes up using of efficacious agents, and this other medicament can need effect by auxiliary block post, and for example tumor toxicity or cell suppress effect, or reduces or prevent unwanted side effect.Therefore, the present invention includes described medicament with above and hereinafter described with the associating of the pharmaceutical composition of definition, wherein said medicament can be other ErbB receptor antagonist, vegf receptor antagonist, cytokine, cytokine immunoconjugates, anti-angiogenic agent, antihormone agent or cytotoxic agent usually.One object of the present invention also is to make compositions as herein described and radiotherapy associating according to known method.

Term used herein " cytotoxic agent " has definition very widely, it is meant such material, this material can suppress or stop cell function and/or cause cytoclasis (cell death) and/or produce neoplasia resisting/antiproliferative effect, for example, directly or indirectly stop progress, maturation or the diffusion of tumor tumor cell.This term expressivity ground also comprises such medicament, and it only causes cyto-inhibition and does not have pure cytotoxic effect.This term comprises the chemotherapeutics of describing in detail below, and other ErbB antagonist (such as anti-ErbB antibody), anti-angiogenic agent, tyrosine kinase inhibitor, the protein kinase A inhibitor, the cytokine family member, the enzyme activity toxin of radiosiotope and toxin such as antibacterial, fungus, plant or animal origin.

Term " chemotherapeutics " is the subclass of term " cytotoxic agent ", and it specifically refers to have antitumous effect, preferably directly acts on the tumor cell and less indirect chemical agent through working such as mechanism such as biological respinse modifications.The chemotherapeutics that the present invention suits is preferably natural or synthetic chemical compound.A large amount of existing be in commercial use, clinical evaluation and clinical before the neoplasia resisting chemotherapeutics of development all can be included in the present invention, be used for by the receptor antagonist of describing with the present invention incompatible treatment tumor/neoplasia that links.Should be understood that chemotherapeutics can be randomly and described ErbB receptor antagonist of the present invention, preferred described anti-egfr antibodies is used together.

The example of chemotherapeutics comprises alkylating agent, and as chlormethine, ethylene imine chemical compound, alkyl sulfonic ester and other have the chemical compound such as the nitroso ureas of alkylating, cisplatin and dacarbazine; Antimetabolite such as folic acid, purine or pyrimidine antagonist; Mitotic inhibitor such as vinca alkaloids and podophyllotoxin derivative; Cytotoxic antibiotics and camptothecin derivative.

Preferred chemotherapeutics is amifostine (ethyol), cisplatin, dacarbazine (DTIC), actinomycin D, chlormethine, streptozotocin, cyclophosphamide, carrnustine (BCNU), lomustine (CCNU), amycin, the little fat body of amycin (doxil), gemcitabine (gemzar), daunorubicin, the little fat body of daunorubicin (daunoxome), procarbazine, mitomycin, cytosine arabinoside, etoposide, methotrexate, 5-fluorouracil (5-FU), vincaleucoblastine, vincristine, bleomycin, paclitaxel (taxol), taxotere (taxotere), aldesleukin (aldesleukin), asparaginase, busulfan, carboplatin, cladibrine, camptothecine, CPT-11,10-hydroxyl-7-ethyl-camptothecine (SN38), gefitinib (Iressa), dacarbazine, floxuridine, fludarabine, hydroxyurea, ifosfamide, idarubicin, mesna, interferon-ALPHA, interferon beta, irinotecan (irinotecan), mitoxantrone, the holder pool is for bearing, leuprorelin, megestrol, chloramphenalan, mercaptopurine, plicamycin (plicamycin), mitotane, asparaginase (pegaspargase), pentostatin (pentostatin), Amedel, plicamycin, streptozotocin, tamoxifen, teniposide, testolactone, thioguanine, plug is for group, uracil mustard, Vinorelbine, chlorambucil and combination thereof.

The most preferred chemotherapeutics of the present invention is a cisplatin, gemcitabine, amycin, paclitaxel (taxol) and bleomycin.

Anti-angiogenic agent " refer to natural or synthetic chemical compound, it can block or disturb the generation of blood vessel to a certain extent.The angiogenesis inhibitor molecule can be, for example and angiogenic growth factor or growth factor receptors in conjunction with and with the biomolecule of its blocking-up.This locate preferred angiogenesis inhibitor molecule can with receptors bind, preferably combine with integrin receptor or with vegf receptor.This term also comprises the prodrug of described anti-angiogenic agent among the present invention.This term also comprises having described effect and also can be classified as cytotoxic agent, preferably the medicament of chemotherapeutics.

There are a lot of structures can cause angiogenesis inhibitor character with the different molecule in source.Suitable angiogenesis inhibitor or most of correlation types of blocker are among the present invention, for example:

(i) antimitotic agent, fluorouracil for example, ametycin, paclitaxel;

(ii) estrogen metabolism thing such as 2-methoxyestradiol;

Matrix metalloproteinase (MMP) inhibitor that (iii) suppresses the zinc metalloprotein enzyme (for example, betimastat, BB16, TIMPs, minocycline, GM6001, or those (materials) (Golub that in " inhibition of matrix metalloproteinase: treatment is used ", touches upon, Annals of the New YorkAcademy of Science, Vol.878a; Greenwald, Zucker (Eds.), 1999);

The (iv) multipurpose agent of angiogenesis inhibitor and the factor, as IFN α (US 4,530,901; US4,503,035; 5,231,176); Angiostatin and plasminogen fragment (kringle1-4 for example, kringle5, kringle 1-3 (O ' Reilly, M.S. etc., Cell (Cambridge, Mass.) 79 (2): 315-328,1994; Cao etc., J.Biol.Chem.271:29461-29467,1996; Cao etc., J.BiolChem 272:22924-22928,1997); Endostatin (endostatin) (Cell 88 (2) for O ' Reilly, M.S. etc., 277,1997 and WO 97/15666), thrombospondin (TSP-1; Frazier, 1991, Curr Opin Cell Biol 3 (5): 792); Platelet factor 4 (PF4);

(v) activator of plasminogen/urokinase inhibitors;

(vi) urokinase receptor antagonist;

(vii) heparinase;

(viii) Amebacilin analog such as TNP-470;

(ix) (a lot of ErbB receptor antagonists cited above and below (EGFR/Her 2 antagonisies) also are tyrosine kinase inhibitors for tyrosine kinase inhibitor such as SU 10, therefore it can demonstrate respectively that thereby anti-EGF receptor blocking is active to cause tumor growth to be suppressed, thereby and the activity that demonstrates angiogenesis inhibitor cause vascular development and endotheliocyte to be grown being suppressed);

(x) suramin and suramin analog;

(xi) opening property of tubulation (angiostatic) steroid;

(xii) VEGF and bFGF antagonist;

(xiii) vegf receptor antagonist such as anti-vegf receptor antibody (DC-101);

(xiv) flk-1 and flt-1 antagonist;

(xv) cyclo-oxygenase-II inhibitor such as COX-II;

(xvi) integrin antagonist and integrain receptor antagaonists such as α v antagonist and α v receptor antagonist, for example, anti-α v receptor antibody and RGD peptide.The preferred integrin of the present invention (receptor) antagonist.

Term " integrin antagonist/inhibitor " or " integrain receptor antagaonists/inhibitor " are meant natural or synthetic molecule, its blocking-up and inhibition of integrins receptor.Sometimes, this term comprises that part at described integrin receptor is (for example for α _vβ ₃: vitronectin, fibrin, Fibrinogen, Feng's von willebrand's factor, thrombospondin, laminin; For α _vβ ₅: vitronectin; For α _vβ ₁: fibronectin and vitronectin; For α _vβ ₆: antagonist fibronectin).

Preferred pin of the present invention is to the antagonist of integrin receptor.Integrin (receptor) antagonist can be natural or synthetic peptide, and non-peptide, peptide mimics (pepetidomimetica), immunoglobulin be the functional fragment of antibody or antibody for example, or immunoconjugates (fusion rotein).

The preferred integrin inhibitor of the present invention is at α _vIntegrin receptor (for example, α _vβ ₃, α _vβ ₅, α _vβ ₆And subclass) inhibitor.Preferred integrin inhibitor is α _vAntagonist, especially α _vβ ₃Antagonist.The preferred α of the present invention _vAntagonist is the RGD peptide, and peptide mimics (non-peptide) antagonist and anti-alpha 2 integrin receptor antibody are as blocking-up α _vThe antibody of receptor.Exemplary nonimmunologic α _vβ ₃Antagonist is at US 5,753,230 and US 5,766,591 in instruction is arranged.Preferred antagonist is linearity and the ring-like peptide that contains RGD.The common half-life more stable and in serum of cyclic peptide is longer.Yet the most preferred integrin antagonist of the present invention is ring (Arg-Gly-Asp-DPhe-NMeVal) (EMD121974, Cilengitide

Merck KgaA, Germany; EP 0770622), it can block integrin receptors alpha effectively _vβ ₃, α _vβ ₁, α _vβ ₆, α _vβ ₈, α _Nbβ ₃

α was all described in scientific and technical literature and the patent documentation _vβ ₃/ α _vβ ₅/ α _vβ ₆The suitable peptide agonist of integrin receptor and peptide simulation (non-peptide) antagonist.For example, can be referring to Hoekstra and Poulter, 1998, Curr.Med.Chem.5,195; WO 95/32710; WO 95/37655; WO97/01540; WO 97/37655; WO 97/45137; WO 97/41844; WO 98/08840; WO 98/18460; WO 98/18461; WO 98/25892; WO 98/31359; WO 98/30542; WO 99/15506; WO 99/15507; WO 99/31061; WO 00/06169; EP 0853084; EP 0854140; EP 0854145; US 5,780, and 426; With US 6,048,861.Disclose and also be applicable to benzo-aza azoles of the present invention and relevant benzodiazepine azoles and benzocyclohepta alkene α _vβ ₃The patent of integrain receptor antagaonists comprises WO 96/00574, and WO 96/00730, and WO 96/06087, WO96/26190, WO 97/24119, and WO 97/24122, WO 97/24124, and WO 98/15278, and WO 99/05107, WO 99/06049, and WO 99/15170, and WO 99/15178, WO 97/34865, WO 97/01540, and WO 98/30542, WO 99/11626 and WO 99/15508.At WO98/08840; WO 99/30709; WO 99/30713; WO 99/31099; WO 00/09503; US 5,919, and 792; US 5,925, and 655; US 5,981, and 546; With US 6,017, other integrain receptor antagaonists with main chain conformation ring binding characteristic have been described in 926.At US 6,048,861 and WO00/72801 in a series of n-nonanoic acid derivant is disclosed, they are effective α _vβ ₃Integrain receptor antagaonists.Other chemical micromolecule integrin antagonisies (most is vitronectin antagonists) are disclosed among the WO 00/38665.Other α _vβ ₃Receptor antagonist has been proved and can have suppressed angiogenesis effectively.For example, synthetic receptor antagonist is as (S)-10,11-dihydro-3-[3-(pyridine-2-base is amino)-1-propoxyl group]-[a, d1 cycloheptene-10-acetic acid (called after SB-265123) were testing in a lot of mammal model systems the 5H-dibenzo.(Keenan etc., 1998, Bioorg.Med.Chem.Lett.8 (22), 3171; Ward etc., 1999, Drug Metab.Dispos.27 (11), 1232).The selection experiment that is suitable for the integrin antagonist make antagonist is as Smith etc., and 1990, J.Bio1.Chem.265 has description in 12267 and in referring to Patent Document.

The antibody of anti-alpha 2 integrin receptor also is well-known.Can be to suitable anti-alpha 2 integrin (as α _vβ ₃, α _vβ ₅, α _vβ ₆) monoclonal antibody modify, (comprise F (ab) to comprise its Fab ₂, the Fv of Fab and through engineering approaches or single-chain antibody).At integrin receptors alpha _vβ ₃A suitable and preferred monoclonal antibody of using be accredited as LM609 (Brooks etc., 1994, Cell 79,1157; ATCC HB 9537).An anti-α of strong specificity is disclosed in WO 97/45447 _vβ ₅Antibody, P1F6, it also is preferred for the present invention.The α that another is suitable _vβ ₆Antibodies selective is the α of selectivity at integrin receptor _vMab 14D9.F8 of chain (WO 99/37683, DSMACC2331, Merck KGaA, Germany) and MAb 17.E6 (EP 0719859, DSMACC2160, Merck KGaA).Another suitable anti-alpha 2 integrin antibodies is the Vitraxin that has gone on the market

As used herein, term " antihormone agent " comprises natural or synthetic organic or peptide compounds, its effect be regulate or inhibitory hormone to the effect of tumor.More specifically, " antihormone agent " (1) suppresses the generation of serum androgen, and (2) blocking-up serum androgen combines with androgen receptor, and perhaps (3) inhibition testosterone is converted into DHT, or the associating of two or more these compounds.Antihormone agent of the present invention generally comprises the steroid receptor antagonist, more particularly estrogen antagonist agent, as comprise tamoxifen, raloxifene, aromatase suppresses 4 (5)-imidazoles, 4-trans-Hydroxytamoxifen, Lilly 88571., keoxifene, LY117018, Ao Nasi ketone and toremifene (toremifene) are (Fareston); And the androgen antagonist agent, as flutamide (flutamide), Ni Luta amine (nilutamide), bicalutamide, leuprorelin (1euprolide) and Coserelin (goserelin); And above-mentioned any the acceptable salt of pharmacy, acid or derivant.This term also comprises glycoprotein hormones-as follicle stimulating hormone (FSH), agonist and/or the antagonist of thyrotropin (TSH) and lutropin (LH) and LHRH (luteotropic hormone releasing hormone).Can be used for LHRH agonist of the present invention is goserelin acetate, and its trade name is ZOLADEX

(Zeneca).Another example of the lhrh antagonist that is suitable for is cyproterone acetate (CPA) and megestrol acetate, and its commodity are MEGACE

(Bristol-Myers, Oncology).Steroid androgen antagonist agent prostate androgen receptor capable of blocking.It also can suppress the release of LH.Preferably the CPA dosage of using to human patients is 100mg/ days-250mg/ days.On-steroidal androgen antagonist agent blocking-up androgen receptor.They also can cause the increase of serum Lh level and level of serum testosterone.Preferred on-steroidal androgen antagonist agent be flutamide (2-methyl-N-[4-20 nitro-3-(trifluoromethyl) Phenylpropionamide], its commodity are called EULEXIN

(Schering Corp.).Flutamide performance be the androgen antagonist effect, it suppresses androgen picked-up, suppresses in the target tissue androgenic nuclear combination or both have both at the same time.Another on-steroidal androgen antagonist agent is a Ni Luta amine, and its chemical name is 5,5-dimethyl-3-[4-nitro-3-(trifluoromethyl-4 '-nitrobenzophenone)-4,4-dimethyl-imidazolidine-diketone.In some embodiments of the present invention, antihormone agent is the associating of LHRH agonist such as leuprorelin acetate and androgen antagonist agent such as flutamide or Ni Luta amine.As, can use leuprorelin acetate by subcutaneous injection, intramuscular injection or intravenous injection, and simultaneously can oral flutamide.Antihormone agent of the present invention comprises, as above-mentioned, and the antagonist of steroid/Thyroid Hormone Receptors, comprising the receptor of other non-permission (non-permissive)-as RAR, TR, the antagonist of VDR etc.Those skilled in the art can understand at an easy rate, and multiple synthetic and natural retinoic acid receptors (RAR) antagonist can be used according to the invention.

Can unite with other medicines according to bi-specific antibody of the present invention.These medicines are preferably selected from:

Tyrosine kinase inhibitor is such as Iressa

Anti-angiogenic agent, preferred integrin inhibitor, more preferably the RGD peptide comprises cyclic peptide, such as (the Cilengitide of ring-(Arg-Gly-Asp-DPhe-NMeVal)

Merck KGaA);

Anti-vegf receptor antibody, such as DC-101, or the VEGF antagonist;

The COX-II inhibitor;

Cytokine, such as TNF-α, IFN-α, IFN-β, IFN-γ, IL-2;

I type protein kinase A (PKAI) inhibitor, such as blended main chain antisense oligonucleotide, as HYB 165 (referring to, for example, Tortora etc., 1999, Clin.Cancer Res., 875-881);

Antihormone agent, such as Coserelin, boserelin, leuprorelin, tamoxifen.

Term " cancer " and " tumor " are meant or what describe is that typical characteristic is the mammal physiological decease of no regulating cell growth.Can treat tumor by using pharmaceutical composition of the present invention, such as mammary gland, heart, lung, small intestinal, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, the tumor of cervix uteri regulating liver-QI.More specifically, tumor is selected from: adenoma, angiosarcoma, astrocytoma, epithelial cancer, germinoma, glioblastoma, glioma, hamartoma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and teratoma.Specifically, tumor is selected from: acra mottle sample melanoma, actinic keratosis, adenocarcinoma, cystadenocarcinoma, adenoma, sarcoadenoma, adenosquamous carcinoma, astrocytoma, bartholin gland carcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary hemangioma, cancer, carcinosarcoma, spongy cancer of biliary duct, chondrosarcoma, venation silk papilloma/cancer, clear cell carcinoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, adenocarcinoma of endometrium, ependyma sarcoma, epithelioid sarcoma, Ewing sarcoma, fibrolamellar sample cancer, focal nodular hyperplasia, gastrinoma, germinoma, Glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, adenoma of liver, adenoma of liver disease, hepatocarcinoma, insulinoma, last Intradermal neoplasia, squamous cell carcinoma between epithelium, invasive squamous cell cancer, large cell carcinoma, leiomyosarcoma, pernicious lentigo type melanoma, malignant melanoma, malignant mesothe, medulloblastoma, medulloepithelioma, melanoma, meningeal tumor, mesothelium tumor, metastatic tumo(u)r, mucous epithelium cancer, neuroblastoma, neuroepithelium adenocarcinoma, NM, oat-cell carcinoma, oligodendroglioma, osteosarcoma, pancreas polypeptide, papillary serous adenocarcinoma, pinealocytoma, pituitary tumor, plasmocytoma, false sarcoma, pulmonary blastoma, renal cell carcinoma, retinoblast cancer, rhabdomyosarcoma, sarcoma, the serosity cancer, small cell carcinoma, soft tissue cancer, somatostatin secretory cell tumor, scale cancer, squamous cell carcinoma, subcutaneous, show the shallow type melanoma that spreads, undifferentiated cancer, the uvea melanoma, verrucous carcinoma, vasoactive intestinal polypeptide tumor, the fully cancer of differentiation, and nephroblastoma.

Preferred available tumor according to antibody molecule treatment of the present invention is a great expression ErbB receptor, the especially entity tumor or the neoplasm metastasis of ErbB 1 receptor, and such as breast carcinoma, carcinoma of prostate, head and neck cancer, SCLC, cancer of pancreas.

Term " biologically/function on effectively " or " in the treatment effective (amount) " are meant such medicine/molecule, it can cause in the body or the external biological function or the change of biological function, and, preferably in the mankind, can effectively treat disease or disease with specified quantitative mammal.For cancer, the treatment effective dose of medicine can reduce the quantity of cancerous cell; Reduce the size of tumor; Suppress (that is, slowing down to a certain extent and preferably termination) cancer cell infiltration in peripheral organs; Suppress the transfer of (that is, slowing down to a certain extent and preferably termination) tumor; Suppress growth of tumor to a certain extent; And/or alleviate one or more relevant symptoms of cancer to a certain extent.If medicine can stop had cancerous cell growth and/or kill already present cancerous cell, then this medicine may have cell inhibition and/or cytotoxicity.For treatment for cancer, curative effect can for example be determined by estimating that progression of disease time (TTP) and/or assaying reaction rate (RR) are next.

Term " effective in the immunization therapy " is meant the biomolecule that causes the mammalian immune reaction.More specifically, this term is meant the molecule that can discern with conjugated antigen.Typically, antibody, contain its antigen binding site (complementary determining region, antibody fragment CDRs) and antibody fusion protein be in the immunization therapy effectively.

" radiation therapy ": according to the present invention, tumor can also utilize lonizing radiation or radiopharmaceuticals to treat.Radioactive source both can be positioned at that the patient that will treat is external also can be positioned at body.If it is external that radioactive source is positioned at the patient, this Therapeutic Method is called extracorporeal irradiation radiotherapy (EBRT).If radioactive source can be positioned at patient's body, this treatment is called brachytherapy (BT).More already used typical radioactive atoms comprise radium, caesium-137, and iridium-192, americium-241 and gold-198, cobalt-57; Copper-67; Technetium-99; Iodo-123; Iodine-131; And indium-111.Also can use labelled with radioisotope medicament of the present invention.Current radiation therapy is the standard treatments of unresectable or inoperable tumor of control and/or neoplasm metastasis.Have been found that associating radiation therapy and chemotherapy can improve curative effect.The principle of radiation therapy foundation is the death that the high dose radiation that projects the target area will cause the proliferative cell in tumor and the normal structure.The radiation dose scheme is generally according to radiation absorbed dose (rad), and time and segmentation are determined, and must be undertaken carefully determining by the tumor expert.The amount of radiation that the patient accepts will depend on various factors, but two positions that most important factor is other important structure of the relative health of tumor or organ, and the degree of tumor diffusion.A preferred therapeutic scheme the patient being implemented radiation therapy is to continue 5-6 week course of treatment, uses for patient's segmentation according to the dosage of jede Woche 1 1.8-2.0Gy, 5 day every day the 50-60Gy accumulated dose.Gy is the abbreviation of gray(Gy), is meant 100rad dosage.If under the X-ray therapy background,, can be observed positive or even collaborative effect usually with anti-ErbB Antybody therapy tumor of the present invention.In other words, if described chemical compound and radiation and/or chemotherapeutics combination, then the inhibitory action to tumor growth will strengthen.Can randomly use radiotherapy according to the present invention.It is to recommend with preferred under the situation according to medicament of the present invention that can not use effective dose to the patient.

" Drug therapy ": with regard to step, method of the present invention comprises multiple form of implementation.Such as, medicament of the present invention can side by side one after the other or independently use.In addition, but medicament separate administration and administered twice at interval in about 3 weeks, promptly second kind of medicament begins immediately basically to be administered to after first kind of activating agent used and is no more than the time in about 3 weeks behind first kind of pharmacy application and begins to use.This method can be carried out after operation.Perhaps, operation can used first kind of active agents and use in second kind of interval between the active agents and carry out.The example of the method is with the inventive method and surgery ablation of tumors operation use in conjunction.Typically comprise with one or more administration period according to the treatment of the inventive method and to use this therapeutic combination.For example, marquis when using simultaneously, the therapeutic combination that contains 2 kinds of medicaments is held to continue in one-period and is used about 2 days to about 3 weeks.After this, treatment cycle can be carried out repetition on demand according to the judgement of practitioner.Similarly, if carry out sequential application, then every kind of therapeutic agent time of using is adjustable to and typically covers the same time.Interval between two cycles can not waited from about 0 to 2 months.

Medicament of the present invention can be by injection or infusion and through parenteral administration gradually in time.Tissue to be treated generally just can be treated with the method for systemic administration in the body, so the method for the most frequent use is that intravenous gives therapeutic combination, and still when destination organization may contain target molecule, its hetero-organization and application process also can be considered.Therefore, medicament of the present invention can be through ophthalmic, intravenous, and intraperitoneal, intramuscular, subcutaneous, intracavity, percutaneous is used by injection of normal position and infusion, but also can be used by the peristaltic pump mode.For example, comprise that the therapeutic combination of the present invention as the integrin antagonist passes through the vein mode usually, for example use with the unit dose injection.

Therapeutic combination of the present invention comprises physiology carrier that can tolerate and the related agents described herein as active component that is dissolved or dispersed in wherein.

As used herein, term " pharmaceutically acceptable " refers to compositions, carrier, diluent and the reagent of the following material of table, and described material can be applied to mammal and can not produce the physiological effect do not expected as feeling sick, and is dizzy, regurgitation etc.It is well-known to those skilled in the art wherein dissolving or having disperseed the preparation of drug combination of active component, so need not limit on the basis of preparation.Typically, this compositions can be made into injection such as liquid solution or suspension, still, also can be made into the solid form that is suitable for forming before use solution or suspension in liquid.Preparation also can carry out emulsifying.Active component and its amount can be applicable to the pharmaceutically acceptable of Therapeutic Method described herein and with the mixed with excipients of active component compatibility.

Appropriate excipients is, for example, and water, saline, glucose, glycerol, ethanol etc. and these combination.In addition, if necessary, compositions can also comprise auxiliary substance such as the wetting agent or the emulsifying agent of increased active component effectiveness in a small amount, pH buffer agent etc.The pharmaceutically acceptable salt that can comprise described composition in the therapeutic combination of the present invention.Pharmaceutically acceptable salt comprises acid-addition salts (with the free amine group group salify of polypeptide), and described acid is mineral acid, for example, and hydrochloric acid or phosphoric acid, or as acetic acid, tartaric acid, organic acid such as mandelic acid.Also can be from inorganic base, for example, sodium, potassium, ammonium, the hydroxide of calcium or ferrum, and organic base such as 2-aminopropane., Trimethylamine, the 2-ethylaminoethanol, histidine, procaines etc. obtain the salt that forms with the free carboxy group.In cyclic peptide α v antagonist formulation, especially preferably use HCl salt.The last carrier that tolerates of physiology is that those skilled in the art is known.The example of liquid phase carrier is an aseptic aqueous solution, and it can only contain active component and water maybe can also contain buffer agent for example the sodium phosphate of physiology pH value, normal saline or both, as phosphate-buffered saline.

In addition, aqueous carrier can contain more than one buffer salt and such as salt such as sodium chloride and potassium chloride, glucose, Polyethylene Glycol and other solutes.Fluid composition also can contain water or anhydrous liquid phase.The example of these other liquid phases of class has glycerol, vegetable oil such as Oleum Gossypii semen and water fat liquor.

Typically, for example, for form is the immunotherapeutic agent of ErbB (ErbB1) receptor blocking bi-specific antibody, integrin receptor blocking antibody or antibody fragment or antibody conjugates or anti-vegf receptor blocking antibody, segment or conjugate, the treatment effective dose is when using in the compositions that physiology can tolerate, be enough to make plasma concentration to reach about 0.01 microgram (μ g) every milliliter (ml) to about 100 μ g/ml, preferred about 1 μ g/ml is about 5 μ g/ml extremely, usually the amount of about 5 μ g/ml.In other words, in the using once a day or repeatedly of a day or many days, dosage can be at about 0.1mg/kg to about 300mg/kg, and preferably about 0.2mg/kg is about 200mg/kg extremely, and most preferably from about 0.5mg/kg extremely changes between about 20mg/kg.When immunotherapeutic agent was the fragment of monoclonal antibody or conjugate form, its consumption can be adjusted with respect to the ratio of the quality of whole antibody according to the quality of fragment/conjugate at an easy rate.Preferred blood plasma molar concentration is extremely about 5 mMs (mM) of about 2 micromoles (μ M), preferred about 100 μ M to 1mM antibody antagonisies.

Medicament of the present invention for the biomolecule that belongs to non-immunotherapeutical peptide or protein and peptide or other similar sizes, its treatment effectively amount typically is such polypeptide amount, be enough to make plasma concentration to reach about 0.1 microgram (μ g) every milliliter (ml) to about 200 μ g/ml when promptly using in the compositions that physiology can tolerate, preferred about 1 μ g/ml is to the amount of about 150 μ g/ml.Polypeptide according to every mole of 500 gram masses of having an appointment calculates, and preferred blood plasma molar concentration is that about 2 micromoles (μ M) arrive about 5 mMs (mM), preferred about 100 μ M to 1mM polypeptide antagonists.

For being preferably chemical cytotoxic agent of the present invention or chemotherapeutics (neither immunotherapeutic agent, neither non-immunotherapeutical peptide/albumen) activating agent, its typical doses is per kilogram of body weight 10mg to 1000mg every day, and preferred about 20 to 200mg, and more preferably 50 to 100mg.

" pharmaceutical composition " of the present invention can comprise the medicament (" complementary therapy ") that can reduce or avoid following the side reaction of conjoint therapy appearance of the present invention; it includes but not limited to; as reduce the medicament of toxicity of anticancer agents effect, inhibitors of bone resorption for example, Cardioprotective medicine.Described assistant medicament can prevent or reduce chemotherapy, the incidence rate of the nausea and vomiting that radiotherapy or operation bring, or reduce and use the infection probability that the bone marrow depression cancer therapy drug brings.Assistant medicament is that those skilled in the art are known.In addition, immunotherapeutic agent of the present invention can also and adjuvant such as BCG and immune system stimulant use together.And compositions can comprise and contains the radioactive label isotope with cytotoxic effect or the immunotherapeutic agent or the chemotherapeutics of other cytotoxic agents such as cytotoxic peptide (for example cytokine) or cytotoxic drug etc.

Term is used for the treatment of tumor or neoplasm metastasis " pharmaceutical kit ", is meant a kind of packing and normally, the operation instructions of medicament in tumor and neoplasm metastasis Therapeutic Method.Medicament in the test kit of the present invention generally is formulated into the therapeutic combination that this place is described, and therefore can adopt any form that is suitable for placement in the test kit.These forms can comprise preparations such as liquid, powder, tablet, suspension, so that treatment molecule of the present invention is provided, and preferred resisting-ErbB1 antibody.These medicaments can provide in each the independent container that is suitable for using separately according to the inventive method, or can be combined in the compositions and provide in the single container in this packing.Can contain the pharmaceutical quantities that is enough to use one or many in the packing according to Therapeutic Method described herein.Test kit of the present invention also comprises " operation instruction " of contained material in the packing.

Embodiment

Embodiment 1:

F (AB ') the 2-fragment of preparation MAB 425 and MAB 225

Adopt limited proteolysis that anti-egfr antibodies humanization MAb 425 and chimeric MAb225 are converted into F (ab ') 2 fragments.Each antibody all must be optimized the generation of F (ab ') 2 antibody.The general approach that is used for this preparation is as follows.

The pepsin cracking is best to two antibody, yet the papain cracking also is suitable for.Remove residual complete antibody and Fc fragment from the protein A agarose column.F (ab ') 2 segmental productive rates are near 100%.F (ab ') 2 fragments can be stored in-20 ℃, in long-time, not lose any activity.

General approach:

Growth fermentation → centrifugal → ultrafiltration → protein A chromatography → dialysis/ultrafiltration → cracking → protein A chromatography → dialysis/ultrafiltration → F (ab ') 2 products

Detailed content:

PBS，pH：7.4

Protein content: 5.06mg/ml (Pierce).

Reagent:Two hydration citric acid trisodiums, citric acid, three (methylol) aminomethane, pepsin, glycine, sodium chloride

Buffer:

0.1M sodium citrate buffer pH 3.5,

The 10mg/mL pepsin, in sodium citrate buffer pH 3.5,

1M?Tris?pH?11

1.5M glycine+3M NaCI pH 8.9

0.1M citric acid pH 2.5

The pepsin digestion process:

By at the 0.1M sodium citrate, among the pH:3.5 MAb dialysed overnight is adjusted pH and buffer conditions.With pepsin with 1: the ratio of 33w/w adds in the immunoglobulin of dialysing.

Under 37 ℃, in water-bath, continue to stir the mixture.After 75 minutes, add 7ml 1M Tris liquid and stop digestion.In this step, the pH of reaction should be set at about 8.5.This mixture is gone to the protein A post so that remove residual IgG and/or the Fc fragment then.

Albumen-A-agarose:

The pepsin digestion thing is added Balanced albumen-A-agarose column, return baseline until chromatogram with the level pad washing.Collect the direct current fraction, in Amicon chamber (Membrane YM 30),, dialyse with PBS pH:7.4 then volume-diminished.Antibody with the 0.1M citric acid pH:2.5 potential pollutant of eluting such as Fc fragment or unmodified from albumen-A-agarose column.

Chromatography condition:

Post bed size 5cm * 2.5cm.Estimate that 1ml protein A agarose can be in conjunction with 10mgIgG, with 1.5M glycine+3M NaCl, pH:8.9 balance columns, flow velocity: 60ml/h detects: OD 280nm, 0.2/2.0Abs-Range, Uvicord SI1, record chart speed: 0.1mm/min, each fraction is collected 5ml.

The productive rate of F (ab ') 2 goods (Pierce)

Consider that Fc-part probably accounts for the productive rate of F (ab ') 2 goods of 1/3rd, two kinds of antibody of molecule near 100%.The concentration of sample should be 6-7mg/ml.Monitor the purity of F (ab ') 2 goods with SDS-PAGE.

Embodiment 2: preparation bi-specific antibody BAB＜425,225 〉

Employing Brennan etc. (Science, 1985,229,81-83) described method generates bi-specific antibody with the segmental chemistry reorganization of IgG.Each step of amending method of the present invention is as follows:

F (ab ') 2 products → be reduced to Fab ' → gel chromatography → derive → gel chromatography → put together → gel chromatography → ultrafiltration → aseptic filtration → BAbs

Two species specificity F (ab '), 2 fragments all are converted into Fab '.The success of puting together step depends on selects suitable Fab ' fragment to be used for the Ai Ermanshi modification.For the BAb that comes from MAb 225/MAb 425,＜225〉component modified.After importing these modifications, the productive rate of individual BAb is 20-30%.Anti-225Fab ' modifies with ellman's reagent, and is puted together on 425 specificity Fab '.Reclaim bi-specific antibody by gel filtration.

Detailed content:

In this step, two kinds of antibody all reduce so that produce Fab ' fragment with DTT.

Before puting together, modify the Fab ' that comes from MAb 225 with ellman's reagent.But also can modify the Fab ' that comes from MAb 425 with ellman's reagent.

Fragment:

(i)F(ab′<425>)2 7.4mg/ml

(ii)F(ab′<225>)2 6.9mg/ml

Solution/buffer:

PBS pH 7.4, PBS+0.65M NaCl+2.5mM EDTA, pH 7.4,51mM dithiothreitol, DTT among the PBS, the 0.1M sodium phosphate buffer, pH 8.0,35mM ellman's reagent in the 0.1M sodium phosphate buffer, pH 8.0,250mM EDTA, pH7.4

Reagent:

1, the 4-dithiothreitol, DTT, ellman's reagent (Elman ' s reagent), sodium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, EDTA, Titriplex III, Superdex 200 (26/60) Pharmacia,

The synthetic first step:

Preparation MAb＜225〉Fab '-TNB.

6550μl?F(ab′)2MAb?22540mg+65.4μl?DTT?51mM+65.4μl?EDTA250mM.

The final concentration of DTT is 0.5mM, and EDTA is 2.5mM.

Cover reactant with argon, under 30 ℃, in water-bath, continue to stir to hatch 40 minutes.After hatching, add 1120 μ l ellman's reagents in reactant mixture, this step has reversibly been blocked the free SH group among the gained Fab '.

The final concentration of ellman's reagent is 5.0mM in the reaction.Under room temperature, reactant mixture was stirred 30 minutes, so that block all SH groups.The color of reactant mixture becomes yellow from clarification.By Superdex 200 (26/60) posts reactant mixture is carried out purification with PBS+0.65M NaCl+2.5mM edta buffer liquid, so that Fab ' molecule that reductive Ai Ermanshi is modified separates with the reagent of surplus with potential pollutant such as unreduced F (ab ') 2, Fab '.Merge the Fab-TNB fraction, cover, before coupling reaction, be kept on ice with argon.

Synthetic second step:

Preparation MAb＜425〉Fab '.

6135μl?F(ab′)2MAb?42540mg+80μl?EDTA?250mM+80μl?DTT?51mM。

Before the startup of reaction should almost not finished early than the Fab-TNB preparation.The reaction final concentration is 0.5mM DTT and 2.5mM EDTA.Cover reactant mixture with argon, hatched 40 minutes in 30 ℃.Immediately reactant mixture is moved to after hatching on Balanced Superdex 200 (26/60) posts, adopt PBS+0.65M NaCl+2.5mM EDTA pH 7.4 buffer that Fab ' is separated with DTT with the F that is not reduced (ab ') 2.Buffer and collecting tubule are saturated to prevent free SH radical oxidation with argon respectively.The fraction that will comprise Fab ' directly is collected in to be used in the saturated test tube of argon.

Synthetic the 3rd step:

Fab '＜425〉and Fab '＜225-the puting together of TNB

Coupling reaction: 32.5ml MAb 225Fab '-TNB, 0.9mg/ml, 31.6mg+23.5mlMAb 425Fab ' 1.5mg/ml, 34.8mg.This Fab ' and this Fab '-TNB antibody is merged, and volume is reduced to about 5ml (use argon) in that the Amicon that comprises YM 10 films is indoor.Cover this reactant mixture with argon, continue to stir in 4 ℃ and spend the night.Conjugate is crossed Superdex 200 (26/60) posts carry out purification, buffer and post all use helium saturated.Reclaim bispecific F (ab ') 2, (peak 1).Peak 1: the bi-specific antibody of purification (166-187ml), peak 2: residual Fab '.

For verification sample identity and purity, sample is added to irreducibility 10%SDS-Page gel.The BAb of purification＜425,225 in this exemplary embodiments〉productive rate of F (ab ') 2 is 11mg (16.7%).

Claims (10)

1. One kind can with bi-specific antibody or its fragment of EGF receptors bind, described antibody or its fragment comprise with bonded first antigen binding site of first epi-position of described EGF receptor and with the second epi-position bonded second different antigen binding sites of described EGF receptor, wherein said first antigen binding site comes from MAb 425 humanized, chimeric or Mus, described second antigen binding site comes from MAb 225 humanized, chimeric or Mus, and described first and second antigen binding sites all with identical EGF acceptor molecule on different epi-position combinations.
2. 2. according to the bi-specific antibody of claim 1, wherein said different epi-positions be positioned at described EGF receptor native ligand in conjunction with the territory.
3. 3. according to the bi-specific antibody of claim 1, at least one in the wherein said epi-position be positioned at described EGF receptor native ligand in conjunction with the territory.
4. 4. according to the bi-specific antibody of claim 1, wherein the native ligand of first or second antigen binding site and described EGF acceptor molecule combines epi-position combination in the territory.
5. 5. according to the bi-specific antibody of claim 1, wherein the native ligand of first and second antigen binding sites and described EGF acceptor molecule combines epi-position combination in the territory.
6. 6. come from the bispecific antibody fragment of the bi-specific antibody of claim 1, wherein fragment is F (ab ') 2.
7. 7. immunoconjugates, it comprises according to the bi-specific antibody of claim 1 or its fragment, this antibody or fragment directly or by linkers in C-terminal and cytokine fusion.
8. 8. pharmaceutical composition that is used for the treatment of cancer, it comprises the bi-specific antibody of claim 1, and randomly pharmaceutically acceptable carrier, diluent or excipient.
9. 9. the pharmaceutical composition of claim 8, it further comprises chemotherapeutics or cytokine.
10. 10. the pharmaceutical composition of the bi-specific antibody of claim 1 or claim 8 is used for the treatment of purposes in the medicine of relevant tumor of EGF receptor and neoplasm metastasis in preparation.