CN101511386A - Methods of treating brain tumors with antibodies - Google Patents

Abstract

The application is directed toward a method of treating a brain tumor in a patient comprising systemically administering a monoclonal antibody.

Description

Method with treating brain tumors with antibodies

The cross reference of related application

The application is a non-provisional application, and 60/751092 the priority submitted in 15th of the 60/687118 and 2005 year December that requires to submit on June 2nd, 2005, and both all are incorporated herein reference as general purpose in full with it these.

The statement of government rights

Work described in the application provides the part fund by fund number (Grants) 1R43CA101283-01A1 and the RO1 NS32148 of National Institutes of Health (National Institutes ofHealth).U.S. government has certain right in the present invention.

Invention field

The present invention relates to use treating brain tumors with antibodies, relate more specifically to, for example, use in the combination also and the mab treatment cerebral tumor of hepatocyte growth factor.

Background of invention

Human hepatocyte growth factor (HGF) is the multi-functional heterologous polypeptides that is produced by mesenchymal cell.Shown that HGF stimulates blood vessel to take place, form takes place and moves (motogenesis) takes place, and the growth of dissimilar cells and dispersion (Bussolino etc., J.Cell.Biol.119:629,1992; Zarnegar and Michalopoulos, J.Cell.Biol.129:1177,1995; Matsumoto etc., Ciba.Found.Symp.212:198,1997; Birchmeier andGherardi, Trends Cell.Biol.8:404,1998; Xin etc., Am.J.Pathol.158:1111,2001).The multi-effective active of HGF is by its receptor, that is, by proto-oncogene cMet coding to stride film tyrosine kinase mediated.Except regulating multiple normal cell function, HGF and receptor c-Met thereof also show the initiation with tumor, invade and transfer relevant (Jeffers etc., J.Mol.Med.74:505,1996; Comoglio and Trusolino, J.Clin.Invest.109:857,2002).HGF/cMet comprises and originates from lung at various people's entity tumors, colon, and rectum, stomach, kidney, ovary, skin, coexpression in the tumor of multiple myeloma and parathyroid tissue is generally overexpression (Prat etc., Int.J.Cancer 49:323,1991; Chan etc., Oncogene2:593,1988; Weidner etc., Am.J.Respir.Cell.Mol.Biol.8:229,1993; Derksen etc., Blood 99:1405,2002).For these tumors, HGF has played autocrine (Rong etc., Proc.Natl.Acad.Sci.USA91:4731,1994; Koochekpour etc., CancerRes.57:5391,1997) and paracrine growth factor (Weidner etc., Am.J.Respir.Cell.Mol.Biol.8:229,1993) and the effect of anti-apoptotic regulator (Gao etc., J.Biol.Chem.276:47257,2001).Therefore, the antagonism molecule of blocking-up HGF-cMet approach, for example antibody may have the potentiality of extensive anticancer therapy.

HGF is the albumen of 102kDa, its sequence and structure and plasminogen and other thrombin similar (Nakamura etc., Nature342:440,1989; Weidner etc., Am.J.Respir.Cell.Mol.Biol.8:229,1993, it all is incorporated herein by reference).Synthetic people HGF (preproHGF) as 728 amino acid precursors, it is cracked into single stranded form (proHGF) (Nakamura etc., Nature 342:440,1989 of non-activity in cell; Rosen etc., J.Cell.Biol.127:1783,1994).After exocytosis, the proHGF cracking produces the heterodimer molecule (Nakamura etc., Nature 342:440,1989 that are connected with disulfide bond be made up of α-subunit and β-subunit of biologically active; Naldini etc., EMBO J.11:4825,1992).α-subunit contains 440 residues (69kDa, glycosylation), is made up of N-terminal hairpin domain and 4 winding (kringle) domain.The B-subunit contains 234 residues (34kDa), and has the serine protease spline structure territory that lacks proteolytic activity.The HGF cracking is that receptor activation is necessary, but is not necessary (Hartmann etc., Proc.Natl.Acad.Sci.USA 89:11574,1992 of receptors bind; Lokker etc., J.Biol.Chem.268:17145,1992).HGF contains 4 N-glycosylation sites of inferring, wherein 1 in α-subunit, other 3 in β-subunit.HGF has the cell-specific binding site of 2 uniquenesses: the high-affinity of cMet receptor (low-affinity (Kd=10-9M) binding site of binding site of Kd=2 * 10-10M) and heparin sulfate Dan Baijutang (HSPG), all be present in (Naldini etc. on cell surface and the extracellular matrix, Oncogene6:501,1991; Bardelli etc., J.Biotechnol.37:109,1994; Sakata etc., J.Biol.Chem., 272:9457,1997).NK2 (surrounding the N-end of α-subunit and the albumen of preceding two winding (kringle) domain) can satisfy the needs that combine and activate mobility's signal cascade with cMet, yet, mitogenesis is replied but needs full-length proteins (Weidner etc., Am.J.Respir.Cell.Mol.Biol.8:229,1993).HSPG by with the N-of HGF terminal interact be bonded to HGF (Aoyama, etc., Biochem.36:10286,1997; Sakata, etc., J.Biol.Chem.272:9457,1997).The interactional supposition effect of HSPG-HGF comprises the bioavailability that strengthens HGF, biological activity and oligomerization (Bardelli, etc., J.Biotechnol.37:109,1994; Zioncheck etc., J.Biol.Chem.270:16871,1995).

CMet is a member of IV albuminoid tyrosine kinase receptor family.Total length cMet gene is cloned, and is accredited as cMet proto-oncogene (Cooper etc., Nature 311:29,1984; Park etc., Proc.Natl.Acad.Sci.USA 84:6379,1987).Having synthesized the cMet receptor at first is strand, and the glycosylated precursor p170 of part (MET) is (Park etc., Proc.Natl.Acad.Sci.USA 84:6379,1987 (Fig. 1); Giordano etc., Nature 339:155,1989; Giordano etc., Oncogene 4:1383,1989; Bardelli etc., J.Biotechnol.37:109,1994).Through after the further glycosylation, this albumen becomes the heterodimer maturation protein (1385 aminoacid) of the 190kDa that is made up of β-subunit of the α-subunit (residue 1-307) of 50kDa and 145kDa through Proteolytic enzyme.The Cytoplasm tyrosine kinase domain of β-subunit participates in signal transduction.

Studied effective antagonistic molecule that several distinct methods remove to attempt to obtain HGF/cMET: the HGF albumen of truncate, for example (N-end structure territory adds winding (kringle) domain 1 to NK1; Lokker etc., J.Biol.Chem.268:17145,1993), (N-end structure territory adds winding (kringle) domain 1 and 2 to NK2; Chan etc., Science 254:1382,1991) and NK4 (N-end structure territory adds 4 winding (kringle) domain; Kuba etc., Cancer Res.60:6737,2000), and anti-cMet mAb (Dodge, Master's thesis, San Francisco State University (San Francisco State University), 1998).

Recently, people such as Cao (Proc.Natl Acad.Sci.USA.98:7443,2001, it is incorporated herein by reference) report, the co-administered growth that can suppress the subcutaneous glioma xenograft of mice of 3 kinds of mAb and HGF.WO 2005/017107 A2 report, list is handled the growth that can suppress the subcutaneous glioma xenograft of mice with anti-HGFmAb, and this patent is incorporated herein the reference as general purpose in full.Yet the general that these publications all do not solve anti-HGF or other mAb is used the problem (Rich etc., Nat.Rev.Drug Discov.3:430,2004) that whether can suppress growth of tumor in the brain that blood brain barrier causes obstacle.In fact, previous observed antibody systemic treatment has been limited vascular permeability by attribution to the ineffectivity of central nervous system (CNS) tumor, even CNS shifts (Bendell etc., Cancer 97:2972,2003).

Therefore, exist demand to the method for the general administering therapeutic cerebral tumor by mAb.The present invention satisfies this demand and other demand.

Summary of the invention

In one embodiment, the invention provides the method for the cerebral tumor of the general administering therapeutic patient by mAb.The cerebral tumor can be a glioma, as astrocytoma, for example, glioblastoma.Use and can for example pass through, vein, intramuscular or subcutaneous route are carried out.In preferred embodiments, in the mAb hepatocyte growth factor (HGF) and mAb, for example, humanization L2G7mAb.In a further preferred embodiment, mAb, for example, the general of the anti-HGF mAb that neutralizes is used and is used to induce disappearing of the cerebral tumor.

Description of drawings

Fig. 1. combination and the blocking-up of the anti-HGF mAb that measures by ELISA are active.A. for combination, with mAb capture to bag by on the elisa plate of goat anti-mouse IgG, with the BSA sealing, and hatch with HGF-Flag (1 μ g/ml), then with HRP-M2 anti--FlagmAb (Invitrogen) hatches.B. combine with Met-Fc for blocking-up HGF-Flag, plate wrapped quilt with the anti-human IgG of goat-Fc, seal, hatch with Met-Fc (2 μ g/ml) with BSA, then with HGF-Flag (1 μ g/ml)+/-anti-HGF mAb hatches.Detect bonded HGF-Flag with the anti-Flag mAb of HRP-M2.

Fig. 2 .mAb L2G7 is to the dispersion of HGF, mitogenesis, the active blocking effect of angiogenesis and anti-apoptotic.A. as described in document (Cao etc., Proc.Natl Acad.Sci.USA.98:7443,2001), stimulated mdck cell (ATCC) 2 days with 50ng/ml HGF+/-10 μ g/ml L2G7.With cell with violet staining after, under 100 times amplification, take pictures.B.Mv1Lu mink lung epithelial cell (ATCC; 5 * 10 ⁴Individual cell/ml) hatch 24h in the contrast mAb (mIgG) of the serum-free DMEM that contains or do not contain HGF (50ng/ml) and L2G7 or isotype coupling adds the 3H-thymidine, and 6h measures the cell proliferation level.C. as described in document (Xin etc., Am.J.Pathol.158,1111,2001), HUVEC (CAMBREX; 10 ⁴Individual cell/100 μ l/ holes) in the EBM-2/0.1%FCS that contains or do not contain HGF (50ng/ml) and L2G7 or contrast mAb, hatches 72h,, measure the propagation level by adding WST-1.D. as described in document (Xin etc., Am.J.Pathol.158,1111,2001), HUVEC (6 * 10 ⁴Individual cell/100 μ l/ holes) in DMEM/ glue, covers with 100 μ l/ holes, the EMB-2/0.1%FCS/0.1%BSA that contains or do not contain 200ng/ml HGF+/-20 μ g/ml L2G7.After hatching 48h, fixed cell is used Toluidine blue staining, and takes pictures under 40 times amplification.E. as document (Fan etc., Oncogene 24:1749,2005) as described in, U87 tumor cell in serum-free DMEM with or without HGF (20ng/ml)+/-mAb L2G7 (20 μ g/ml) or isotype control antibodies (mIgG) handle 48h, use anti-Fas mAb CH-11 (Upstate Biotechnology then, 40ng/ml) handle 24h,, measure cell survival rate by adding WST-1.B, among c and the e, numerical value is meansigma methods ± s.d.

Fig. 3 .L2G7 is to the inhibition of glioma xenograft or disappear.As described in document (Kimet al, Nature 362:841,1993), to NIH III Beige/Nude mouse subcutaneous transplanting U118 (A) or U87 (B) neuroglial cytoma, monitoring tumor size.Reach about 50mm in the tumor size ³After, each organizes mice (n=6 or 7) as indicated, and 2 intraperitoneal give contrast mAb (mIgG) or the PBS that (i.p.) 50 or 100 μ g L2G7 or 100 μ g isotypes mate weekly; First day of representing to handle of arrow.Numerical value is mean tumour volume ± s.e.m.C. as described in document (Abounader etc., FASEB J.16,108,2002) to caudatum/shell intracranial injection U87 tumor cell (10 of Scid/beige mice ⁵Individual/mice).As shown by arrows, begin and end at the 5th day and the 52nd day respectively, each organize mice (n=10) weekly 2 intraperitoneal (i.p.) use 100 μ g L2G7 or PBS, the monitoring survival rate.Survival research adopts the Kaplan-Meier curve to analyze.D.3 the usefulness of dosage is after 2 intraperitoneal are used 100 μ g L2G7 or PBS weekly, at the 21st day sacrificed representative mice such as document (Abounader etc., FASEB J.16,108,2002) brain section of preparation as described in has shown the size of U87 intracranial transplantation thing.E. handle the 29th day after 3 times pretreated the 18th day of beginning with L2G7, the intracranial U87 gross tumor volume of individual mice.F. pretreated the 18th day and handle with L2G7 or contrast mAb after the 29th day, the brain section of representative mice.

Fig. 4. have the histologic analysis of the mouse brain section of U87 intracranial transplantation thing.With 3 weekly behind the L2G7 or tumor that control treatment has been set up of 2 dosage, put to death mice.To pour into fixed frozen section H﹠amp; E dyes, antibody and index shown in quantitatively determining with computer assistant images analytic process.A. detect the anti-Ki67 (DAKO) of proliferative cell.B. detect the anti-laminin (LifeTechnologies) of blood vessel.C. detect the antibody (Cell Signaling Technology) of the cracked caspase-3 of apoptosis of tumor cells reaction.

Detailed Description Of The Invention

The invention provides by among the HGF and mAb or for other cell factor, For example growth factor, or cell surface protein, for example general of the antibody of cytokine receptor The method of administering therapeutic brain tumor. Although be not that enforcement of the present invention institute is essential to the understanding of mechanism , but we think that success of the present invention is at least in part owing to defective blood in the tumour Brain barrier and make antibody enter brain tumor from blood.

1. antibody

Antibody is very large compound molecule with complex internal structure (molecular weight is about 150,000, or contains 1320 aminoacid of having an appointment).The natural antibody molecule contains two pairs of identical polypeptide chains, and every pair contains a light chain and a heavy chain.Every light chain and heavy chain are made up of two zones successively: participate in distinguishing in conjunction with variable (" V ") district of target antigen with constant (" C ") of immune other component interaction.The variable region of light chain and heavy chain folds in three dimensions, forms the variable region of conjugated antigen (for example, the receptor of cell surface).In the light or variable region of heavy chain, 3 short-movie sections (average length is 10 aminoacid) that are called as complementary determining region (" CDRs ") are arranged at each.6 CDRs in the antibody variable territory (3 from light chain, 3 from heavy chain) fold in three dimensions, form the antibody combining site of real locking target antigen.The position of CDRs and length are by explication.Kabat, E. etc., Sequences of Proteins of Immunological Interest, U.S.Department ofHealth and Human Services, 1983, the 1987. variable region parts that are not included among the CDRs are called as framework, and it forms the environment of CDRs.

Therefore monoclonal antibody (mAb) is the antibody of single molecular species, does not comprise by extracting serum and the polyclonal antibody that produces to animal (for example rodent, rabbit or goat) injections of antigens with from animal.Humanized antibody is that genetic engineering (monoclonal) is changed antibody, and wherein the CDRs from mouse antibodies (" donor antibody ", it also can be a rat, hamster or other similar species) is transplanted on people's antibody (" receptor antibody ").Humanized antibody also can prepare (for example, Pascalis etc., J.Immunol.169:3076,2002) with the imperfect CDRs of mouse antibodies.Therefore, humanized antibody is CDRs and the variable region framework of people's antibody and the antibody of constant region that has from donor antibody.In addition, in order to keep high binding affinity, can adopt in two other structural details at least one.Referring to, U.S. Patent No. 5,530,101 and 5,585,089, it all is incorporated herein by reference, and both provide detailed guidance for the structure of humanized antibody.

In first structural detail,, select and the variable region of heavy chain framework of donor antibody has the framework of the humanized antibody variable region of heavy chain of maximal sequence homogeneity (between 65% and 95%) by in many known person antibody, suitably selecting receptor antibody.In second structural detail, when making up humanized antibody,, be used for aminoacid in the selected people's receptor antibody of the corresponding amino acid replacement framework (except the CDRs) of donor antibody according to ad hoc rule.Particularly, replaced amino acid whose selection is a foundation with itself and the interactional ability of CDRs in the framework.For example, when measuring in three dimensions, replaced aminoacid can be close to the CDR in the donor antibody sequence, perhaps in humanized antibody in the 4-6 dust of CDR.

Chimeric antibody is the variable region of wherein mice (or other rodent) antibody and the bonded antibody of constant region of people's antibody; Its structure that relies on genetic engineering to carry out is known.This antibody-like derives from the binding specificity that has kept mouse antibodies under people's the condition about 2/3.The immunogenicity of the non-human sequence's who exists in the chimeric and humanized antibody of mice ratio prompting chimeric antibody is in the middle of mice and humanized antibody.The genetically engineered antibody of comparing the immunogenicity reduction with mouse antibodies of other type comprises with phage display method (Dower etc., WO91/17271; McCafferty etc., WO92/001047; Winter, WO92/20791; And Winter, FEBS Lett.23:92,1998, it all is incorporated herein by reference) or with transgenic animal (Lonberg etc., WO93/12227; Kucherlapati WO91/10741, it all is incorporated herein by reference) people's antibody of preparation.

Term used herein " proper manners " antibody is meant that the major part of the aminoacid sequence of one or two chain (for example, about 50% or more) wherein derives from human immunoglobulin gene's mAb.Therefore, proper manners antibody includes but not limited to chimeric antibody, humanized antibody and people's antibody.The antibody of " immunogenicity reduction " that this paper uses is to be desirably in to have the immunogenic antibody that significantly is lower than mouse antibodies when human patients is used.Such antibody comprises chimeric antibody, humanized antibody and people's antibody, and by determining B or t cell epitope in the replacement mouse antibodies, for example, the specific amino acid of the residue that exposes and the antibody (Padlan, Mol.Immunol.28:489,1991) that makes." genetically engineered " antibody that this paper uses is under the help of recombinant DNA technology, its gene is fabricated or places non-natural environment, and (for example, the people's gene in mice or on the phage therefore) antibody do not comprise, for example, the mice mAb that adopts conventional hybridization tumor technology to make.

The epi-position of mAb is and bonded its antigenic zone of mAb.If two kinds of antibody all suppress (blocking-up) another kind of antibody competitively and combine with antigenic, then these two kinds of antibody and identical or eclipsed epi-position combination.That is, CBA (referring to, for example, Junghans etc., Cancer Res.50:1495,1990, it is incorporated herein by reference) in when measuring, 1 times, 5 times, 10 times, 20 times or 100 times of excessive a kind of antibody suppress at least 50% of another kind of antibody, preferred 75%, 90% even 99% combination.Perhaps, if nearly all bonded amino acid mutation that reduces or eliminates a kind of antibody can both reduce or eliminate the combination of another kind of antibody in the antigen, then these two kinds of antibody have identical epi-position.If the bonded amino acid mutation that has some to reduce or eliminate a kind of antibody reduces or eliminates the combination of another kind of antibody, then these two kinds of antibody have eclipsed epi-position.

2. in and anti-HGF antibody

With the bonded monoclonal antibody of HGF (mAb) (that is, anti-HGF mAb), if its part in conjunction with or suppress one or more biological activitys of HGF fully, then think in it and HGF, or neutral (that is, when mAb is used as single agents).The biological characteristics of the HGF that neutralizing antibody can suppress has HGF and its cMet receptors bind, and to cause some cell line, for example Madin-Darby dog kidney (MDCK) cell disperses; Stimulate some cell, comprise hepatocyte, 4MBr-5 monkey epithelial cell and various human tumor cells propagation (that is mitogenesis); Perhaps, for example, form by stimulating human vascular endothelial (HUVEC) propagation or pipe, or the ability that stimulates blood vessel to take place when inducing of blood vessel being measured when being applied to chick chorioallantoic membrane (CAM).The antibody that uses among the present invention preferably with people HGF, that is, be the protein binding of gene bank (GenBank) sequential coding of D90334 by accession number.Similarly, at the neutralization of any cytokine or cytokine receptor, that is, antagonistic antibodies can suppress combining of cytokine and receptor and/or suppress signal through the cytotropic transmission of cytokine.If cytokine is a somatomedin, then such antibody can suppress the cell proliferation by this cytokine induction.

When analyzing by described method of embodiment or means known in the art, in using among the present invention and mAb usually for example 0.01,0.1,0.5,1,2,5,10,20 or the concentration of 50 μ g/ml under suppress cytokine, for example, the pact at least 50% of HGF, preferred 75%, more preferably 90% or 95% even 99%, the biological function of 100% (almost completely) (for example, stimulate proliferation or blood vessel take place) most preferably from about.The measurement of inhibition degree in the just enough complete stimulating organism activity of the amount of used cytokine, is carried out when perhaps being 0.05,0.1,0.5,1,3 or 10 μ g/ml usually.Preferably at least 50%, 75%, 90% 95% or the mol ratio that is suppressed at antibody and cytokine almost completely be 0.5 *, 1 *, 2 *, 3 *, 5 * or 10 * time reach.Preferred mAb is neutral,, can suppress biological activity during as single agents that is, still, in certain methods, has used two kinds of mAb together, suppresses to produce.MAb a kind of in the above listed biological activity of neutralization just most preferably, but several; For this purpose, when the single agents in and all bioactive anti-HGF mAb of HGF be called as " neutral fully ", and such mAb is most preferred.The mAb that uses among the present invention preferably has specificity to HGF, that is, its not with the HGF associated protein, for example, fibroblast growth factor (FGF) and VEGF (VEGF) combination are perhaps only with low-down degree combination.MAb has at least 10 usually ⁷M ^-1, preferred 10 ⁸M ^-1Or higher, most preferably 10 ⁹M ^-1Or higher, even 10 ¹⁰M ^-1Or higher binding affinity (Ka).

Employed mAb comprises the antibody (2 light chain and 2 heavy chains) of natural tetramer form among the present invention, and can be any known isotype IgG, IgA, IgM, IgD and IgE and hypotype thereof, that is, and human IgG1, IgG2, IgG3, IgG4 and mice IgG1, IgG2a, IgG2b and IgG3.MAb also means and comprises antibody fragment, Fv for example, Fab and F (ab ') 2; Difunctional hybrid antibody (for example, Lanzavecchia etc., Eur.J.Immunol.17:105,1987), single-chain antibody (Huston etc., Proc.Natl.Acad.Sci.USA85:5879,1988; Bird etc., Science 242:423,1988); And the antibody (for example, U.S. Patent No. 5,624,821) that changed of constant region.MAb derives from animal (for example, mice, rat, hamster or chicken), perhaps can be genetically engineered product.Rodent mAb prepares by standard method well known in the art, described standard method comprises through intraperitoneal, the HGF in the proper adjuvant is used or be applied in to intravenous in the foot pad, carry out multiple immunity, extracting spleen or lymph-node cell then, is to merge with the immortalized cell that suits, and then selects the hybridoma of generation and the bonded antibody of HGF, for example, referring to following embodiment.Used chimeric and humanization mAb in the preferred embodiments of the invention by above-mentioned means known in the art preparation.The people's antibody that makes by for example phage display or transgenic mice method also be preferred (referring to, for example, above-mentioned Dower etc., McCafferty etc., Winter, Lonberg etc., Kucherlapati).More broadly, proper manners antibody defined herein, antibody and genetically engineered antibody that immunogenicity reduces all are preferred.

The anti-HGF mAb L2G7 (be preserved in American type culture collection according to budapest treaty, ATCC number is PTA-5162) that neutralizes is the preferred example that is used for mAb of the present invention.Deposita will be in the preservation of authorized depositary institution, and, after the request of nearest release sample is received by depositary institution at least 5 years, deposit at least 30 years in the future, or in the implementation period of relevant patent, be as the criterion with the longest time, if undergo mutation, can not survive or destroy, deposita all will be replaced.All all will can not cancel after patent application comes forth the restriction of the availability of these cell lines the public with recalling.Have or eclipsed epi-position identical with L2G7 in and mAb other example is provided.The variant of L2G7, L2G7 for example chimeric or the humanization form is particularly preferred.In external or body inner analysis described herein, combine with HGF with the L2G7 competition, and in the mAb of HGF also be preferred.Other variant of L2G7, for example with L2G7 in the variable region amino acid sequence (when for example, comparing by the Kabat numbering system; Kabat etc., above institute's quoted passage is offered), at least on CDRs, have 90%, 95% or 99% homogeneity, and the mAb that keeps its functional character, perhaps because a small amount of function irrelevance amino acid replacement (for example, conservative substitution), disappearance or insert and the mAb that is different from L2G7 also can use in the present invention.Other preferred mAb comprises proper manners mAb defined herein, mAb and genetically engineered mAb that immunogenicity reduces.

Any amino acid replacement of illustrated immunoglobulin all is preferably the conservative amino acid displacement.For amino acid replacement being divided into the purpose of conservative or non-conservative, aminoacid can be classified as follows: I class (hydrophobic side chain): met, ala, val, leu, ile; II class (neutral hydrophilic side chain): cys, ser, thr; III class (acid side-chain): asp, glu; IV class (basic side chain): asn, gln, his, lys, arg; V class (influencing the residue of chain orientation): gly, pro; And VI class (aromatic series side chain): trp, tyr, phe.Conservative substitution comprises with the displacement between amino acid.Non-conservation displacement member and the another kind of amino acid whose member by certain class of amino acid exchange and carry out.

Other is preferred for mAb of the present invention and comprises all anti--HGF mAb that describe among US2005/0019327A1 or the WO2005/017107A2, no matter it is clearly described by title or sequence, still (, two pieces of applications of being quoted all being incorporated herein by reference) of the relation hint of the mAb that clearly describes by description or with institute for its disclosure and all other purposes about antibody.Particularly preferred mAb has by called after 1.24.1,1.29.1,1.60.1,1.61.3,1.74.3,1.75.1,2.4.4,2.12.1, the mAb that weight that the hybridoma of 2.40.1 and 3.10.1 produces and that provided by the SEQ ID NO 24-43 of WO2005/017107 A2 respectively and light chain variable region sequence limit; MAb with each CDRs identical with listed any mAb; Each variable region light and variable region of heavy chain and listed mAb has at least 90%, 95% or 99% homogeneity, perhaps only because of insignificant amino acid replacement, lacks or inserts and mAb discrepant with it; With the HGF epi-position bonded mAb identical with any listed mAb, and all mAb of containing by claim 1-94 wherein.It is definite that sequence homogeneity adopts the Kabat numbering convention to compare between immunoglobulin variable domain sequence.

In other embodiments, be used for the present invention, that is, one or more that are used for using the mAb that treats the cerebral tumor and following somatomedin by the general of mAb combine: vascular endothelial cell growth factor (VEGF); Neurotrophic factor, nerve growth factor (NGF) for example, Brain Derived Neurotrophic Factor (BDNF) or NT-3; Transforming growth factor, for example TGF-α or TGF-β (TGF-β 1 and/or TGF-β 2); Platelet derived growth factor (PDGF); Epidermal growth factor (EGF); Heregulin; Epiregulin; Emphiregulin; Neuroregulation element (NRG-1 α and/or NRG-1 β, NRG-2 α and/or NRG-2 β, NRG-3 or NRG-4), insulin like growth factor (IGF-1 and IGF-2); Or in preferred embodiments, fibroblast growth factor (FGF), particularly acid FGF (FGF-1), most preferably basic FGF (FGF-2) perhaps is FGF-n, wherein n is any numeral of 3-23.Usually, such mAb is that neutralization is right.In other embodiments, being used for mAb of the present invention combines with the cell receptor of any or multiple above-mentioned somatomedin.

Being used for natural mAb of the present invention can produce from its hybridoma.Genetically engineered mAb, for example, chimeric or humanization mAb can express by multiple methods known in the art.For example, encoding its gene light and heavy chain V district can be synthetic by eclipsed oligonucleotide, inserts with available C district then essential regulatory region is provided, for example, promoter, enhancer, the expression vector in polyadenylation site etc. (for example, can available from Invitrogen).The preferred CMV promoter-enhancer that uses.Expression vector can be used various known methods then, for example lipofection or electroporation transfection are to multiple mammal cell line, for example CHO or unproductive (non-producing) myeloma, comprise Sp2/0 and NS0, and the cell of expressing the antibody of selecting by suitable antibiotic.Referring to, for example, U.S. Patent No. 5,530,101.In the commercialization bioreactor, can produce more substantial antibody by cultivating cell.

Be used for mAb of the present invention or other antibody in case express, just can be according to this area standardization program, microfiltration for example, ultrafiltration, protein A or G affinity chromatograph, molecular-exclusion chromatography, anion-exchange chromatography, cation-exchange chromatography and/or carry out purification based on affinity chromatograph of other form of organic dyestuff etc.For pharmaceutical use, preferred homogeneity is at least about 90% or 95% remarkable pure antibody, and most preferably homogeneity is 98% or 99% or higher.

3. Therapeutic Method

In preferred embodiments, the invention provides the method for the treatment of with the pharmaceutical preparation that comprises mAb described herein.The pharmaceutical preparation of antibody comprises the acceptable carrier on the pharmacology of lyophilizing or aqueous solution form, and the optional mAb that contains in excipient or the stabilizing agent.Acceptable carrier, excipient or stabilizing agent to the receptor avirulence, and comprise buffer under used dosage and concentration, for example pH is generally 5.0-8.0, the phosphate of the most common 6.0-7.0, citrate or acetate buffer; Salt, sodium chloride for example, potassium chloride etc. ooze so that wait; Antioxidant, antiseptic, low molecular weight polypeptide, protein, hydrophilic polymer, polysorbate80 for example, aminoacid, carbohydrate, chelating agen, sugar and other well known to a person skilled in the art standard analysis (Remington ' s Pharmaceutical Science 16th edition, Osol, A.Ed.1980).MAb is usually with 1-100mg/ml, and for example, the concentration of 10mg/ml exists.MAb can also be wrapped in carrier, for example in the liposome.

In a further preferred embodiment, the invention provides by mAb, for example, neutralization is anti--HGF mAb or at the method for the general administering therapeutic patient with brain tumors of the antibody of cytokine or its receptor.The patient is the people preferably, but also can be any mammal.Here, general is used the approach of using that is meant, by this approach, mAb can enter blood circulation comprehensively, and therefore enters biological organs, comprises cerebral vessels.In other words, mAb uses in the blood brain barrier periphery.The example that general is used comprises venoclysis or bolus injection, or intramuscular or subcutaneous or intraperitoneal are used.Yet general is used and is not comprised directly to tumor or organ, for example injects in brain or film around it or the cerebrospinal fluid.Venoclysis can be carried out in being as short as 15min, but more commonly carries out 30min or 1,2,3 hours even more than 4 hours or 4 hours.Institute is enough to cure to dosage, alleviates or suppresses by sanatory further developing (" dose therapeutically effective ") to small part.Dose therapeutically effective preferably causes disappearing of tumor, more preferably causes the elimination of tumor.Dose therapeutically effective is generally the 0.1-5mg/kg body weight, for example, and 1,2,3 or 4mg/kg, but also can be as high as 10mg/kg, even 15 or 20mg/kg.Unit dose that can also administration of fixed, for example, 50,100,200,500 or 1000mg, perhaps, dosage can be based on patient's surface area, for example, 100mg/m ²Cure being enough to, alleviate or the dose therapeutically effective that suppresses to be used by the sanatory frequency that further develops is called as effective therapeutic scheme to small part.Such scheme optimization causes disappearing of tumor, more preferably causes the elimination of tumor.Usually using 1-8 (for example, 1,2,3,4,5,6,7 or 8 times) dosage treats cancer, but also can use the dosage more than 10,20 or 20.MAb can be once a day, and weekly twice, weekly, week about once, every month is once, and perhaps according to for example half life of mAb, with 1 week, in 2 weeks, in 4 weeks, in 8 weeks, some of 3-6 month or longer time are used at interval.Multiple therapeutic process also is possible, and this is chronic using.

Method of the present invention, for example, mAb, as anti-HGF mAb, particularly L2G7 and variant thereof, the general that comprises humanization L2G7 is used and can be used for treating all cerebral tumors, comprising: meningioma; Glioma comprises ependymoma, oligodendroglioma and all types of astrocytoma (low potential malignancy, anaplastic and glioblastoma multiforme or simple glioblastoma (simply glioblastoma)); Medullary substance blastoma (medullablastomas), ganglioglioma, schwannoma, chordoma; And child's primary brain tumors, comprise the former ectoderm tumor of going crazy.Primary brain tumors (that is, originating from brain) and Secondary cases or metastatic brain tumor can be by method treatments of the present invention.Express Met and/or HGF, particularly the Met of level rising and/or the cerebral tumor of HGF are particularly suitable for by the anti-HGF antibody that neutralizes, and for example the general of L2G7 or its variant is used and treated.

In preferred embodiments, mAb and other anticancer therapy co-administered (that is, and before other anticancer therapy, during or use afterwards).For example, mAb, for example anti-HGF mAb, as L2G7 and variant thereof can with any one or more oncology chemotherapeutics known to the skilled, for example, alkylating agent, as carmustine, chlorambucil, cisplatin, carboplatin, oxiplatin, procarbazine and cyclophosphamide; Antimetabolite, as fluorouracil, fluorodeoxyuridine, fludarabine, gemcitabine, methotrexate and hydroxyurea; Natural product comprises plant alkaloid and antibiotic, as bleomycin, and doxorubicin, daunorubicin, idarubicin, etoposide, mitomycin, mitoxantrone, vinblastine, vincristine and Taxol (paclitaxel) or related compound, as Clearly approval is used for the medicament of the cerebral tumor, comprises the temozolomide and contains carmustine Thin slice; And other medicines, comprise irinotecan and

And all approveds of listing among WO 2005/017107 A2 (it is incorporated herein by reference) and experimental anticarcinogen are used together.MAb is can be in standard chemical therapeutic scheme for example co-administered with 1,2,3 kinds or these medicaments more than 3 kinds.Other can comprise biological product with the medicament that anti-HGF mAb uses, and as monoclonal antibody, comprises the antigenic Herceptin at HER2 ^TM, at the Avastin of VEGF ^TM, the antibody of EGF receptor, as

Or anti-FGF mAb, and micromolecule anti-angiogenic agent or EGF receptor antagonist medicine, as

With

In addition, mAb can with the radiotherapy of arbitrary form, comprise the outer light beam irradiation, conformal intensity modulated radiotherapy (Intensity Modulated.RadiationTherapy) (IMRT) and any type of radiosurgery comprises gamma knife, matches vigorous cutter (Cyberknife), linear accelerator (Linac), and interstitial radiotherapy's (for example, implanting radioactive particle, the GliaSite ball) uses together.

Although in a preferred embodiment of the invention, mAb does not combine with any other medicament or yoke closes, in other embodiments, mAb can with radiosiotope, chemotherapeutic agent or prodrug or toxin conjugate close.For example, its can with emission α, β and/or gamma-ray radiosiotope, for example, 90Y, iodine isotope is as 131I, or bismuth isotope, as 212Bi or 214Bi; Plant or bacterioprotein toxin are as ricin or Pseudomonas exotoxin or its segment, as PE40; The micromolecule toxin, as with calicheamicin, auristatin or maytansine are relevant or by its derived compounds; Chemotherapeutic agent is as amycin (doxorubin) or any above listed other chemotherapeutics combinations.With these medicaments and the bonded method of mAb is well known to a person skilled in the art.

MAb, for example, the anti-HGF mAb that neutralizes uses as the general of L2G7 or its variant, (for example randomly add other treatment, chemotherapy or radiotherapy), compare with the contrast scheme that does not give mAb, can increase and (for example have some cerebral tumor, glioblastoma) patient's intermediate value progresson free survival or total time-to-live reach at least 30% or 40%, preferred 50%, 60%-70%, even 100% or more.If anti--using of HGF mAb is accompanied by other treatment, for example chemotherapy or radiotherapy also comprises described other treatment in the then contrast scheme.Not not placebo or do not have particular treatment if the using of anti--HGF mAb followed other treatment, then contrast scheme.In addition, perhaps the two selects a ground, mAb, for example, the anti-HGF mAb that neutralizes uses as the general of L2G7 or its variant, (for example add other treatment, chemotherapy or radiotherapy), compare with the contrast scheme of not using mAb mentioned above, can improve complete response rate (the disappearing fully of tumor of the patient with some cerebral tumor, promptly, alleviate), the part response rate (reply the part that is meant the tumor size and dwindle, for example by patient's part, dwindle at least 30% or 50%) or the target response rate (fully+and part) reach at least 30% or 40%, preferred 50%, 60%-70%, even 90% or higher.The variation of the tumor size that treatment is replied can be passed through MRI, mensuration such as CT scan.

Similarly, for example, according to described in the embodiment 2 hereinafter like that (for example to intracranial xenotransplantation human glioma's animal, immunodeficient mouse is as nude mice or SCID mice) when general is used, neutralize anti-HGF mAb or anti-FGF mAb or other mAb will prolong the intermediate value survival period of animal at least about 25 or 30 or 40 days, preferred 50,60 or 70 days or longer, and such prolongation is significant statistically.This point even still correct when at least 5 or 18 days or longer time after beginning of will treating is deferred to tumor cell transplantation.In addition, such treatment will make tumor on average dwindle at least 25%, and will be preferred 50%, even 75%; And, will be less than with 50% of the mean tumour volume of the animal of contrast treatment with the mean tumour volume of the animal of mAb treatment, even 25% or 10%.The measurement in 21 or 29 days behind tumor cell transplantation usually of tumor size.

Normally, in clinical trial (for example, the II phase, II/III phase or III phase test), pass through mAb, for example, using of anti-HGF mAb treated, and adds that randomly the patient of other treatment compares with the patient that acceptance does not contain the contrast scheme of this antibody, the increase of its aforementioned intermediate value progresson free survival phase and/or response rate is significant statistically, for example, significant level is that p equals 0.05 or 0.01, even 0.001.Fully and the part response rate according to the objective criterion of using always in the cancer clinical trial, for example, list or the objective criterion of admitting is measured by American National ICR and/or U.S. food Drug Administration.

Embodiment

1. the generation of anti-HGF mAb and vitro characteristics

The exploitation to complete neutral anti-HGF mAb L2G7 is described among U.S. Patent application publication No.US 2005/0019327 A1, and it is incorporated herein by reference.In a word, inject extensive immune Balb/c mice with recombined human HGF through the foot pad, and produce hybridoma by it by conventional method.Produce by traditional recombinant technique by merging to the HGF (HGF-Flag) of Flag peptide and merging the chimeric fusion protein of forming to the Met extracellular domain (Met-Fc) in human IgG1 Fc district, and be used for determining that anti-HGF mAb suppresses the ability of HGF and its Met receptors bind.Fig. 1 a has shown the independent anti-HGF mAb of 3 kinds of each self-identifying different table positions captures HGF in solution ability.Although judge by binding ability, between the centre, it is unique one and blocks HGF-Flag and the bonded mAb of Met-Fc (Fig. 1 b) fully in ELISA IgG2a mAb L2G7 to the affinity of HGF.MAb L2G7 has specificity to HGF, because it shows not and other somatomedin, and as VEGF, FGF or EGF combination.

MAb L2G7 blocking-up HGF points out it may suppress all by the inductive cell response of HGF with the bonded ability of Met, but this supposition needs checking, because the α of HGF and β-subunit mediate different activity (Lokker etc., EMBO J.11:2503,1992; Hartmann etc., Proc.Natl Acad.Sci.USA 89:11574,1992).One of HGF by its α-subunit mediation, and the important biomolecule activity that obtains its alternative title " dispersion factor " thus is the dispersive ability of inducing cell.Fig. 2 a shows that L2G7 can suppress the epithelial dispersion by the inductive MDCK of HGF fully, a kind ofly is widely used in the quantitative bioanalysis of HGF dispersed activity.The important biomolecule activity of passing through its β-subunit mediation of HGF is the mitogenesis effect to some cell type.Fig. 2 b shows that L2G7 suppresses inductive 3H-thymidine the mixing in Mv 1 Lu mink lung epithelial cell by HGF fully when the mol ratio of mAb and HGF is 1:1.Therefore, mAbL2G7 blocking-up by HGF is inductive can be by the biological activity of α and β-HGF subunit decision.

Blood vessel is that the growth of entity tumor is necessary.HGF is strong effectively angiogenesis factor (Grant etc., Proc.Natl Acad.Sci.USA 90:1937,1993), tumor level and the human malignant lesion of HGF, comprise gliomatous vessel density be correlated with (Schmidt etc., Int.J.Cancer84:10,1999).HGF can also stimulate other angiogenesis factor, as the generation of VEGF, and can strengthen by the inductive blood vessel generation of VEGF (Xin etc., Am.J.Pathol.158,1111,2001).With blood vessel relevant preceding two steps taking place is that endothelial cell proliferation and microtubule form.Therefore in three-dimensional collagen gel, determined the influence of L2G7 to the inductive Human umbilical vein endothelial cells of HGF (HUVEC) propagation and blood vessel sample microtubule (vessel-like tubule) formation.L2G7 finishes during for 1.5:1 in the mol ratio of mAb and HGF and has suppressed HGF (50ng/ml is 72h) to the stimulation (Fig. 2 c) of HUVEC propagation.Be suspended in HUVEC in the three-dimensional collagen gel in that (200ng/ml 48h) has formed interconnective branch microtubule reticular system after stimulating, and adds cell that L2G7 handles with HGF and then demonstrates seldom or do not have this microtubule to form (Fig. 2 d) with HGF.Therefore, the L2G7 blocking-up is by the propagation and the morphology aspect of the inductive blood vessel generation of HGF.

HGF protection tumor cell avoids taking place comprising the inductive apoptosis death of the DNA damage agent that is usually used in treatment of cancer (Bowers etc., Cancer Res.60:4277,2000 by various ways; Fan etc., Oncogene 24:1749,2005).Most of people's glioblastoma cellular expression death receptor FAS, thus make its external to apoptosis sensitivity (Weller etc., J.Clin.Invest.94:954,1994) by anti-FAS antibody induction.Therefore, determined the influence to the cytoprotection of the U87 neuroglial cytoma through apoptosis anti-FAS mAb CH-11 handled of L2G7 to the HGF mediation.The U87 cell is after CH-11 handles (24h), survival rate drops to about 45% of undressed contrast, this effect by in advance with HGF incubated cell and being reversed fully in the presence of irrelevant isotype control antibodies, but in the presence of L2G7 by HGF reverse (Fig. 2 e).

2. the effect of anti-HGF mAb in glioma xenotransplantation tumor model

The ability of the multiple short tumor promotion of L2G7 blocking-up HGF points out this mAb at least HGF+/Met+ people's tumor to be had anti-tumor activity.Most of glioma show expresses Met and HGF (Rosen etc., Int.J.Cancer 67:248,1996).For glioma cell line U87 and U118, confirmed the expression of Met by flow cytometry, and adopted the specific ELISA of HGF in 7 days the supernatant that is paved with old culture, to detect the HGF of about 20-35ng/ml.Determined the antitumor action of L2G7 in the nude mouse model of U118 that has set up and the subcutaneous xenograft of U87.As (1993, it is incorporated herein by reference for Kim etc., Nature362:841) as described in the document, reach about 50mm in the tumor size ³After, use 2 times with the L2G7 intraperitoneal weekly.Under the dosage of per injection 100 μ g (about 5mg/kg), L2G7 has suppressed the U118 growth of tumor fully, and (Fig. 3 a).In U87 graft model, per injection 50 μ g or 100 μ g L2G7 have not only suppressed tumor growth, and the actual tumor regression (Fig. 3 b) that caused.Contrast mAb (per injection 100 μ g) compares with the PBS contrast and has only slightly suppressed tumor growth.The growth no effect of L2G7 to expressing Met but not secreting the U251 glioma xenograft of HGF.The result proves in these bodies, as the L2G7 of single agents by blocking the active tumor growth that stops of HGF specifically.

Subsequently, in the mice of setting up intracranial U87 glioma xenograft, checked the effectiveness of L2G7.By caudatum/shell stereotaxis (stereotactic) injection to the right, implant U87 people's glioblastoma cell (100,000 cell/animals) to mice.Implant the back and used L2G7 (100 μ g/ injection, intraperitoneal is used, 2 times weekly) in the 5th day to the 52nd day, significant prolongation animal dis current (Fig. 3 c).In control mice, the intermediate value survival period is 39 days, and during by the 41st day, all mices all die from carrying out property (progressive) tumor.By contrast, all have all lived through the 70th day through mices that L2G7 handles, and 80% mice lived through the 90th day, stop mAb and handle back 7 weeks (Fig. 3 c).In the mice of putting to death in the 21st day behind 3 dosage L2G7, control tumor is than the big (6.6+2.7mm more than 10 times of the tumor of handling through L2G7 ³To 0.54+0.17mm ³) (Fig. 3 d).

For the effectiveness of test mAb under exacting terms more, the time started of in similar experiment L2G7 being handled has been deferred to the 18th day.The early stage execution subgroup mice (every group of n=5) of processing procedure, by adopting the computer-aided image analysis method by measuring through H﹠amp; The tumor cross-sectional area of the painted brain section of E is quantitatively determined gross tumor volume.L2G7 induced significantly the disappearing of tumor (Fig. 3 e, f).Particularly, handling the volume of tumor in the time of the 18th day before is 26.7+2.5mm ³(scope: 19.5-54mm ³, intermediate value: 27.9mm ³).At the 29th day, that is, after the L2G7 of 3 dosage, gross tumor volume only was 11.7+5.0mm ³(scope: 0-26.2mm ³, intermediate value 7.5mm ³), therefore, tumor has in fact disappeared or has on average dwindled 50% or more.The volume of tumor in the time of the 29th day of handling through the paired contrast of isotype mAb is 134.3+22.0mm ³(scope: 71.2-196.8mm ³, intermediate value 128mm ³).Therefore, the tumor growth of handling through contrast mAb nearly 5 times, average external volume is bigger 12 times than the tumor of handling through L2G7.In the mice (every group of n=10) of not putting to death, the intermediate value survival period of control mice is 32 days, and all dead by the 42nd day, and mice none death before the 46th day of handling through L2G7, and L2G7 makes the intermediate value survival period be deferred to the 61st day.Therefore, L2G7 has induced the tumor regression of the very high mice of tumor load.

For the antitumor action of research L2G7 may mechanism, the tissue slice of intracranial tumor has been carried out more detailed analysis (Fig. 4).After the L2G7 of 3 dosage, (vessel density takes place in tumor cell proliferation (Ki-67 index) and blood vessel, promptly, account for the percentage ratio of tumor area with the painted tumor vascular area of anti-laminin) descended 51% and 62% respectively, and the apoptotic index of the quantitative tumor cell of quantity by activated form caspase-3 positive cell has increased by 6 times.Soon the remarkable tumor regression that takes place after beginning L2G7 treatment has shown and has been similar to observed cell death reaction (Chuntharapai etc. in human colon tumor Colo 205 xenografts that anti-death receptor 4 (TRAIL1) mAb handles with agonist, J.Immunol.166:4891,2001).

This paper results reported is the cerebral tumor to not in conjunction with the remarkable example of the mAb reaction of toxin or radionuclide.As a comparison, in subcutaneous heteroplastic transplantation model, almost completely suppress U87 and the gliomatous growth phase ratio of U118 with mAb L2G7, anti-VEGF Mus mAb A4.6.1 only suppresses the G55 human glioma's of about 50-60% growth (Kim etc., Nature362:841,1993), described A4.6.1 had produced medicine Avastin through humanization afterwards In intracranial coordination tumor model, the general of transplanting the anti-VEGF mAb carry out simultaneously with the G55 neuroglial cytoma is used and is only made the animal dis current prolong 2-3 week (Rubenstein etc., Neoplasia2:306,2000).Similarly, the general of the mAb of EGF receptor variant is used and has moderately been prolonged intracranial transplantation substantially and have the intermediate value survival period of mice of the neuroglial cytoma of transfection EGF receptor variant (to extend to 21 days from 13 days, or extended to 19 days, but there is an example to extend to 58 days from 19 days from 13 days; Mishima etc., Cancer Res.61:4349,2001).Yet, the using with xenotransplantation of mAb that act on of these appropriateness begins simultaneously, or situation about beginning soon after xenotransplantation is issued to, therefore caused by the beginning that postpones the xenograft vascularization at least in part probably, the beginning of described delay xenograft vascularization is the incident of impossible targeting in the patient who has the cerebral tumor.By contrast, the general of anti-HGF mAbL2G7 is used, even after transplanting the 5th day, even the 18th day, still postpone survival period under the situation about using when tumor is set up well, and caused tumor regression, therefore meet the situation of human patients.

The remarkable antitumor action of mAb L2G7 is likely the multi-functional characteristic owing to the uniqueness of its molecular target HGF, that is, and and mitogenesis, angiogenesis and cytoprotective characteristic (Birchmeier etc., Nat.Rev.Mol.Cell Biol.4:915,2003; Trusolino etc., Nat.Rev.Cancer4:289,2002).The ability that L2G7 induces glioma to disappear has hinted the cell death reaction that may cause because of the apoptosis or the inductive cytoprotective approach of deactivation HGF of Fas mediation; described apoptosis is bonded to the Met blocking-up by HGF; described cytoprotective approach relates to phosphatidylinositol 3-kinase; Akt and NF-kappaB intermediate (Fan etc.; Oncogene 24:1749,2005).The L2G7 of the ability indication systemic delivery of the cytoprotective of L2G7 blocking-up HGF and angiogenesis function has strengthened being used for the treatment of at present the cell toxicant form of malignant brain tumor, as gamma-rays and chemotherapy.

Although invention has been described for the REFERENCE TO RELATED embodiment preferred, will be appreciated that, can carry out various modifications under the condition of the present invention not deviating from.

For general purpose, all publications of being quoted, patent and patent application are all with its full text, and to seem single publication, patent also is incorporated herein by reference for general purpose is incorporated herein by reference the same degree separately with patent application is concrete.

Claims (23)

1. the method for the treatment patient cerebral tumor comprises to the patient with brain tumors general and uses monoclonal antibody (mAb), and treats the cerebral tumor thus.

2. the mAb that the process of claim 1 wherein is a chimeric antibody, humanized antibody or people's antibody.

3. the mAb that the process of claim 1 wherein is the anti-HGF mAb of neutralization.

4. the method for claim 2, mAb wherein is humanization L2G7 mAb.

5. the mAb that the process of claim 1 wherein uses through intravenous.

6. the cerebral tumor that the process of claim 1 wherein is a glioma.

7. the method for claim 6, the cerebral tumor wherein is a glioblastoma.

8. the patient who the process of claim 1 wherein is the people.

9. the patient who the process of claim 1 wherein is also through radiation therapy treatment.

10. the mAb that the process of claim 1 wherein uses with one or more other active anticancer medicines.

11. the method for claim 1, mAb wherein be selected from following somatomedin and combine: vascular endothelial cell growth factor (VEGF), nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), NT-3, transforming growth factor (TGF)-α (TGF-α), TGF-β 1, TGF-β 2, platelet derived growth factor (PDGF), epidermal growth factor (EGF), heregulin, epiregulin, emphiregulin, neuroregulation element (NRG)-1 α (NRG-1 α), NRG-1 β, NRG-2 α, NRG-2 β, NRG-3, NRG-4, insulin like growth factor (IGF)-1 (IGF-1), IGF-2, acid fibroblast growth factor (FGF) (FGF-1), basic FGF (FGF-2) and FGF-n, wherein n is arbitrary numeral of 3-23.

12. cause the method that patient's cerebral tumor disappears, comprise to the patient with brain tumors general and use monoclonal antibody (mAb), and cause that thus the cerebral tumor disappears.

13. the method for claim 12, mAb wherein is a chimeric antibody, humanized antibody or people's antibody.

14. the method for claim 12, mAb wherein is the anti-HGF mAb of neutralization.

15. the method for claim 13, mAb wherein is humanization L2G7 mAb.

16. the method for claim 12, mAb wherein uses through intravenous.

17. the method for claim 12, the cerebral tumor wherein is an astrocytoma.

18. the method for claim 17, the cerebral tumor wherein is a glioblastoma.

19. the method for claim 12, disappearing wherein is to disappear fully.

20. the method for claim 12 also comprises and uses the radiation therapy treatment patient.

21. the method for claim 12, mAb wherein uses with one or more other active anticancer medicines.

22. the method for claim 12, mAb wherein be selected from following somatomedin and combine: vascular endothelial cell growth factor (VEGF), nerve growth factor (NGF), Brain Derived Neurotrophic Factor (BDNF), NT-3, transforming growth factor (TGF)-α (TGF-α), TGF-β 1, TGF-β 2, platelet derived growth factor (PDGF), epidermal growth factor (EGF), heregulin, epiregulin, emphiregulin, neuroregulation element (NRG)-1 α (NRG-1 α), NRG-1 β, NRG-2 α, NRG-2 β, NRG-3, NRG-4, insulin like growth factor (IGF)-1 (IGF-1), IGF-2, acid fibroblast growth factor (FGF) (FGF-1), basic FGF (FGF-2) and FGF-n, wherein n is arbitrary numeral of 3-23.

23. neutral anti-HGF antibody is used for application by the medicine of the general administering therapeutic cerebral tumor in preparation.

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