CN1993146A - Antibody-drug conjugates and methods - Google Patents

Abstract

The present invention relates to antibody-drug conjugate compounds of Formula I: Ab-(L-D)<p> where one or more maytansinoid drug moieties (D) are covalently linked by L to an antibody (Ab) which binds to an ErbB receptor, or which binds to one or more tumor-associated antigens or cell-surface receptors. These compounds may be used in methods of diagnosis or treatment of cancer, and other diseases and disorders.

Description

Antibody-drug conjugates and method

The non-provisional application that this submits to according to 37 CFR § 1.53 (b), according to 35 USC § 119 (e), require the US provisional application 60/616 of US provisional application submission October 5 in 60/576,517,2004 of submission on June 1st, 2004,098 rights and interests, these all whole addings as a reference.

Field that the present invention belongs to

The present invention relates generally to have the chemical compound of active anticancer, relate more particularly to and chemotherapy maytansinoid medicine or toxin conjugated antibody.The invention still further relates to and utilize that the antibody-drug coupling compound is external, the method for original position and in-vivo diagnostic or treatment mammalian cell or related diseases disease of science.

Background of invention

Set up the monoclonal antibody therapy that is used for the disorderly patient's targeted therapy of cancer, immunology and angiogenesis.Using antibody-drug conjugates (ADC) is immune conjugate local delivery cytotoxicity or cytostatics, promptly kills and wounds or suppress medicine (Payne, G. (2003) the Cancer Cell 3:207-212 of tumor cell in the treatment of cancer; Trail etc. (2003) Cancer Immunol.Immunother.52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv.Drug Del.Rev.26:151-172; US4975278) allow in theory the drug moiety targeted delivery to tumor, and at the cell inner accumulation of tumor, using these non-coupling medicaments in this general may cause normal cell and the unacceptable toxic level (Baldwin etc. of tumor cell that will remove, (1986) Lancet pp. (Mar.15,1986): 603-05; Thorpe, (1985) " Antibody Carriers Of Cytotoxic Agents In Cancer Therapy:AReview; " in Monoclonal Antibodies ' 84:Biological And Clinical Applications, A.Pinchera etc. (volume), pp.475-506).What therefore will seek is maximum effect while toxicity minimum.Design and the effort that improves ADC concentrate on the selectivity of monoclonal antibody (mAbs) and medicine-be connected and medicine-release characteristics.Be used for these tactful polyclonal antibodies and monoclonal antibody and all have report (Rowland etc., (1986) Cancer Immunol.Immunother.21:183-87).The medicine that is used for these methods comprises daunorubicin, amycin, methotrexate, mitomycin, neocarzinostain NCS (Takahashi etc. (1988) Cancer 61:881-888) and vindesine (Rowland etc., (1986) are the same).The toxin that is used for antibody-toxin conjugated thing comprises bacteriotoxin such as diphtheria toxin, diphtherotoxin, phytotoxin such as Ricin (US4753894; US 5629197; US 4958009; US 4956453), micromolecule toxin such as geldanamycin (geldanamycin) (Mandler etc. (2000) J.of the Nat.Cancer Inst.92 (19): 1573-1581; Mandler etc. (2000) Bioorganic ﹠amp; Med.Chem.Letters 10:1025-1028; Mandler etc. (2002) Bioconjugate Chem.13:786-791), maytansinoid (maytansinoids) (EP1391213; Liu etc., (1996) Proc.Natl.Acad.Sci.USA 93:8618-8623) and calicheamicin (calicheamicin) (Lode etc. (1998) Cancer Res.58:2928; Hinman etc. (1993) Cancer Res.53:3336-3342).Described toxin may be by comprising that tubulin combination, DNA combination or topoisomerase suppress mechanism and influence its cytotoxicity and cell depression effect.When being coupled to large-scale antibody or protein acceptor part, some cytotoxic drugs tend to inactivation or activity reduces.

(ibritumomab tiuxetan, Biogen/Idec), it is by the antigenic Mus IgG1 of anti-CD20 kappa monoclonal antibody with by thiourea linker (linker)-chelating agen is bonded to have ratified antibody-radiosiotope conjugate ZEVALIN_ ¹¹¹In or ⁹⁰The y radiosiotope is formed (Wiseman etc. (2000) Eur.J.Nucl.Med.27 (7): 766-77; Wiseman etc. (2002) Blood 99 (12): 4336-42; Witzig etc. (2002) J.Clin.Oncol.20 (10): 2453-63; Witzig etc. (2002) J.Clin.Oncol.20 (15): 3262-69).2000, ratified the antibody-drug conjugates MYLOTARG that forms by the hu CD33 antibody that is connected to calicheamicin ^TM(gemtuzumab ozogamicin WyethPharmaceuticals), is used for that (Drugs of theFuture (2000) 25 (7): 686 by the injection for curing acute myeloid leukemia; US 4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001).The antibody-drug conjugates Cantuzumab mertansine (Immunogen, Inc.) (Xie etc. (2004) the J.of Pharm.and Exp.Ther.308 (3): 1073-1082 that form by the huC242 antibody that is connected to maytansinoid drug moiety DM1 by disulphide joint SPP; Tolcher etc. (2003) J.Clin.Oncology21 (2): 211-222; US 5208020) carried out that the cancer of CanAg is expressed in treatment such as I phase of colon cancer, cancer of pancreas, gastric cancer and other cancer tests.MLN-2704 (Millennium Pharm., BZLBiologics, Immunogen Inc.) antibody-drug conjugates of being made up of anti-prostate specific membrane antigen (PSMA) monoclonal antibody that is connected to maytansinoid drug moiety DM1 is being developed to be used for effective treatment of tumor of prostate.Identical maytansinoid drug moiety DM1 is connected to the mouse monoclonal antibody TA.1 (Chari etc. (1992) Cancer Research 52:127-131) of mice by non-disulphide joint (non-disulfide linker) SMCC.This conjugate is renderd a service little 200 times than corresponding disulphide joint conjugate according to reports.Think that the SMCC joint is in described conjugate " can not be cracked " (also can referring to: US 4981979).Reported the HERCEPTIN_ (trastuzumab (trastuzumab) (WO 2005/037992) that is connected to DM1 by SMCC.

Be used for the effort of the effective cell target thing of cancer diagnosis and treatment in searching, researcher has been managed to have identified and has been striden film or tumor relative polypeptide, compare with one or more normal non-cancerous cell, these polypeptide are expressed on the surface of one or more particular type cancerous cell specifically.Usually, and compare on the surface of non-cancerous cell, these tumor relative polypeptides are expressed on the cancerous cell surface more.Identify that these tumor relevant cell surface antigen polypeptide are that tumor associated antigen (TAA) has produced by the target cancer cell specifically based on Antybody therapy and carries out destructive ability.

Set up the monoclonal antibody therapy of the targeted therapy that is used for the disorderly patient of cancer, immunologic derangement and angiogenesis.The example of successful Antybody therapy is the deutero-Humanized monoclonal antibodies HERCEPTIN_ of recombinant DNA (trastuzumab (trastuzumab)), in the test based on cell, it optionally is attached to the extracellular region (ErbB2) of human epidermal growth factor receptor 2's albumen HER2 with high affinity (Kd=5nM), and (US 5821337; US 6054297; US 6407213; US 6639055; (1985) Science 230:1132-9 such as Coussens L; (1989) Science 244:707-12 such as Slamon DJ).Trastuzumab is the IgG1 kappa antibody that comprises people's framework region, has the murine antibody complementary determining region (4D5) in conjunction with HER2.Trastuzumab is in conjunction with HER2 antigen, so the growth of anticancer.Because trastuzumab is a humanized antibody, so it minimizes any HAMA reaction among the patient.This anti-HER 2 humanized antibody is by mammalian cell (Chinese hamster ovary (Chinese Hamster Ovary, CHO)) suspension culture production.The transmembrane receptor protein of HER2 (or c-erbB2) proto-oncogene coding 185kDa, this transmembrane receptor protein is structurally relevant with EGF-R ELISA.In the primary breast cancer of 25%-30%, observe HER2 albumen and cross expression, can utilize based on the fixedly tumor blocking-up assess and determine HER2 albumen of SABC and cross expression (Press MF waits (1993) Cancer Res 53:4960-70).Show that in testing in vitro and animal, trastuzumab suppressed to express the propagation of human tumor cells of HER2, and (Hudziak RM waits (1989) Mol Cell Biol 9:1165-72; Lewis GD, et al (1993) CancerImmunol Immunother; 37:255-63; Baselga J, et al (1998) Cancer Res.58:2825-2831).Trastuzumab is the cell toxicant that antibody relies on, ADCC, mediators (HotalingTE waits (1996) [summary] .Proc.Annual Meeting Am Assoc Cancer Res; 37:471; Pegram MD waits (1997) [summary] .Proc Am Assoc Cancer Res; 38:602; Sliwkowski etc. (1999) Seminars in Oncology 26 (4), Suppl 12:60-70; Yarden Y. and Sliwkowski, M. (2001) Nature Reviews:Molecular Cell Biology, MacmillanMagazines, Ltd., Vol.2:127-137).Clinically, it is activated that HERCEPTIN_ crosses among the expression metastatic breast cancer patient at ErbB2-, and described patient had before accepted a large amount of anticancer therapy (Baselga etc., (1996) J.Clin.Oncol.14:737-744).Cross the breakthrough of expressing among the patient with breast cancer though HERCEPTIN is treatment ErbB2-, described patient had before accepted anticancer therapy widely, in this colony Most patients to HERCEPTIN treatment do not react or react faint.Therefore, significant need is developed the cancer therapy of other HER2 guiding clinically, is used to suffer from HER2-and crosses expressing tumor or express other relevant disease with HER2, and HERCEPTIN is treated the patient's who does not react or react faint treatment.Except HER2, targeted therapies also might utilize other tumor associated antigen.

General introduction

The invention provides new chemical compound with anticancer activity of cell biology.Described chemical compound can suppress the mammal tumor growth, and can be used for treating the human cancer patient.

The present invention relates to therapeutic antibodies-drug conjugates (ADC) chemical compound is sent, transports, accumulated or remains in the cell.The present invention relates more specifically to obtain the active metabolite molecule of high concentration in cancerous cell.Can realize targeting in the cell at the method and the chemical compound of cell inner accumulation or maintenance by allowing bioactive agents.This effective targeting is applicable to multiple therapeutics preparation and method.

Obtained surprising discovery, promptly antibody-drug conjugates causes enhanced external tiring and the interior effect of body, and described antibody-drug conjugates has the stable non-disulphide joint group that the maytansinoid drug moiety is connected in antibody.In addition, with respect to some disulphide joint conjugate, described antibody-drug conjugates shows unexpected safer result in vivo.

Antibody-drug conjugates (ADC) chemical compound comprises the antibody that is covalently attached to one or more maytansinoid drug moieties by joint.ADC can represent with formula I:

Ab-(L-D) _p I

Wherein one or more maytansinoid drug moieties (D) are covalently bound to antibody (Ab) by L.Ab is in conjunction with the ErbB receptor, perhaps in conjunction with the antibody of one or more tumor associated antigens or cell surface receptor.Joint L can be stable outside the extracellular is born of the same parents.Joint L, maytansinoid drug moiety D or joint and maytansinoid drug moiety (L-D) together do not comprise disulphide group (disulfide group).

In one embodiment, the drug moiety of real mass (substantial amount) when antibody-drug conjugates enters the cell with cell surface receptor just by described antibody cracking, wherein said cell surface receptor is specific to the antibody of antibody-drug conjugates, and described drug moiety when antibody-drug conjugates enters described cell really by described antibody cracking.

In another embodiment, ADC is specifically in conjunction with the receptor by the ErbB gene code, as EGFR, HER2, HER3 and HER4.ADC can be specifically in conjunction with the ectodomain of HER2 receptor.ADC can suppress the growth of the tumor cell of expression HER2 receptor.

In another embodiment, the antibody of formula I (Ab) is humanized antibody such as huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (trastuzumab).

Another aspect of the invention is pharmaceutical composition, it comprises formula I chemical compound or its pharmaceutically acceptable salt or solvate and pharmaceutically acceptable diluent, carrier or excipient.

Provide drug regimen (pharmaceutical combination) on the other hand, second kind of chemical compound that it comprises formula I chemical compound and has anticancer property or other therapeutics effect.

The diagnosis and the therapeutics purposes that comprise chemical compound disclosed herein and compositions on the other hand.

On the other hand for killing and wounding or suppress the method for tumor cell or cancer cell multiplication, it comprises with killing and wounding or suppress tumor cell or the effective a certain amount of antibody-drug conjugates of cancer cell multiplication, or its pharmaceutically acceptable salt or solvate are handled described cell.

Be the treatment method for cancer on the other hand, it is included as the preparation that the patient uses formula I chemical compound.A kind of method is used for the treatment of mammalian cancer, and wherein said cancer is expressed as feature with the mistake of ErbB receptor.Described mammal is optional not to react the anti-ErbB Antybody therapy of non-coupling, or reacts faint.Described method is included as the antibody-drug coupling compound of described administration treatment effective dose.

Be the method for the growth of tumour cell that suppressed to express growth factor receptors on the other hand, described growth factor receptors is selected from HER2 receptor and EGF receptor, described method is included as the patient and uses specifically antibody-drug coupling compound and chemotherapeutics in conjunction with described growth factor receptors, and wherein said antibody-drug conjugates and described chemotherapeutics are used with the amount of effective inhibition patient tumors cell growth respectively.

On the other hand for treating the method for human patients, described human patients is responsive or be diagnosed as to suffer from the ErbB2 receptor and cross the disease that is expressed as feature to cross the disease that is expressed as feature with the ErbB2 receptor, and described method comprises the antibody-drug coupling compound of using formula I and the combination of chemotherapeutics.

For detecting the method for testing of cancerous cell, comprising on the other hand: cell is contacted with the antibody-drug coupling compound, and measure the combination degree of antibody-drug coupling compound and cell.

Relate to the method that screening is used for the treatment of the ADC drug candidate of disease or disorder on the other hand, wherein disease or disorder are expressed as feature with the mistake of HER2.

Comprise that on the other hand goods are test kit, comprise antibody-drug conjugates, container and instruct the package insert or the label for the treatment of.

Comprise on the other hand with the treatment of antibody-drug coupling compound and cross disease or the disorderly method that is expressed as feature with HER2 among the patient.

Comprise manufacture method, preparation method, synthetic method, coupling method and the purification process of antibody-drug coupling compound on the other hand and be used for the preparation of described antibody-drug coupling compound, synthetic and link coupled intermediate.

The accompanying drawing summary

Fig. 1 shows the body outer cell proliferation test that the SK-BR-3 cell handled with antibody-drug conjugates carries out :--trastuzumab-SPP-DM1 ,-△-trastuzumab-SPDP-DM1 and-zero-trastuzumab-SMCC-DM1.

Fig. 2 shows the body outer cell proliferation test that the BT-474 cell handled with antibody-drug conjugates carries out :--trastuzumab-SPP-DM1 ,-△-trastuzumab-SPDP-DM1 and-zero-trastuzumab-SMCC-DM1.

Fig. 3 shows the body outer cell proliferation test that the MCF7 cell handled with antibody-drug conjugates carries out :--trastuzumab-SPP-DM1 ,-△-trastuzumab-SPDP-DM1 and-zero-trastuzumab-SMCC-DM1.

Fig. 4 shows the body outer cell proliferation test that the MDA-MB-468 cell handled with antibody-drug conjugates carries out :--trastuzumab-SPP-DM1 ,-△-trastuzumab-SPDP-DM1 and-zero-trastuzumab-SMCC-DM1.

Fig. 5 shows the serum clearance rate of the relative trastuzumab-SPP-DM1 of trastuzumab-SMCC-DM1 in the cream-coloured nude mice of no tumor, and six time points in 7 days (taking medicine back 5 minutes, 1 hour, 6 hours, 24 hours, 72,168 hours) are measured conjugate and total antibody serum concentration.

Fig. 6 shows the stability that following conjugate is passed in time in the no tumor nude mice: trastuzumab-SPDP-DM1, trastuzumab-SPP-DM1, trastuzumab-SPP-DM3, trastuzumab-SPP-DM4 and trastuzumab-SMCC-DM1, six time points (taking medicine back 5 minutes, 1 hour, 6 hours, 24 hours, 72,168 hours) are measured serum-concentration in 7 days.

After Fig. 7 display process 7 days, there is and do not have the serum-concentration measured value of total trastuzumab/trastuzumab-SMCC-DM1 and total trastuzumab/trastuzumab-SPP-DM1 in the mice of tumor.

Fig. 8 is shown as 4 and is tried the plasma concentration clearance rate that rat uses behind 10mg/kg trastuzumab-SPP-DM1 and study.Measured total antibody and trastuzumab-SPP-DM1 concentration.(tr=trastuzumab)

Fig. 9 is shown as 4 and is tried the plasma concentration clearance rate that rat uses behind 10mg/kg trastuzumab-SMCC-DM1 and study.Measured total antibody and trastuzumab-SMCC-DM1 concentration.

Figure 10 shows that as time passes the mice mean tumour volume that according to dosage gives following medicine changes: carrier (PBS pH 6.5), trastuzumab-SPP-DM1 (370 μ g DM1/m ²) and trastuzumab-SMCC-DM1 (330 μ g DM1/m ²), wherein dosage refers to the dosage of the DM1 that used.

Figure 11 shows that as time passes the nude mouse mean tumour volume that according to dosage gave the Fo5 tumor allograft of following medicine in the 0th day changes: carrier (PBS pH 6.5), 10mg/kg trastuzumab-SIAB-DM1 (3.4DM1/Ab; 168 μ g DM1/kg) and 10mg/kg trastuzumab-SMCC-DM1 (3.2DM1/Ab; 158 μ g DM1/kg), wherein dosage refers to the dosage of the antibody-drug conjugates used.

Figure 12 shows as time passes, (7 every group of the cream-coloured nude mices of MMTV-Her2 Fo5 of single injection carrier (PBS pH 6.5), 10mg/kg trastuzumab-SPP-DM1,10mg/kg trastuzumab-SPP-DM4,10mg/kg trastuzumab-SPP-DM3 and 10mg/kg trastuzumab-SMCC-DM1, tumor is all arranged, and mean tumour volume Ti=7) changes.

Figure 13 is presented in the HER2-Fo5 tumor, time that the gross tumor volume of carrier (PBS pH 6.5), trastuzumab-SPP-DM1, trastuzumab-SPP-DM4, trastuzumab-SPP-DM3 and trastuzumab-SMCC-DM1 doubles and the analysis of logarithm cell killing.

Fig. 14 shows as time passes, according to dosage gives the body weight change of the rat of following medicine: carrier (10mM sodium succinate, 100mg/mL sucrose, 0.1%Tween 20, pH 5.0), trastuzumab-SPP-DM1 (1860 μ g DM1/m ²), trastuzumab-SMCC-DM1 (1860 μ g DM1/m ²), trastuzumab-SMCC-DM1 (3260 μ g DM1/m ²) and do not have DM1 (650 μ g/m ²).

Figure 15 shows as time passes, according to dosage give the liver functional test value of every liter of AST unit recording in the rat model of following medicine: carrier (10mM sodium succinate, 100mg/mL sucrose, 0.1%Tween 20, pH 5.0), trastuzumab-SPP-DM1 (22.3mg/kg), trastuzumab-SMCC-DM1 (10mg/kg), trastuzumab-SMCC-DM1 (25mg/kg), trastuzumab-SMCC-DM1 (50mg/kg) and do not have DM1.

Figure 16 shows as time passes, the safety spectrum of PLT unit in the every liter of cell that according to dosage gives to record in the rat model of following medicine: carrier (10mM sodium succinate, 100mg/mL sucrose, 0.1%Tween 20, pH 5.0), trastuzumab-SPP-DM1 (22.3mg/kg), trastuzumab-SMCC-DM1 (10mg/kg), trastuzumab-SMCC-DM1 (25mg/kg), trastuzumab-SMCC-DM1 (50mg/kg) and do not have DM1.

Figure 17 shows the body outer cell proliferation test that HT1080EphB2 (C8) cell handled with antibody-drug conjugates carries out :-▲-anti-EphB2R 2H9-SPP-DM1 and-_-anti-EphB2R2H9-SMCC-DM1.

The detailed description of exemplary

To at length work out reference for particular of the present invention now, set forth embodiment with subsidiary structure and general formula.Though will describe the present invention in conjunction with cited embodiment, and will appreciate that them and do not mean that to limit the invention to those embodiments.On the contrary, the present invention will be contained all replacement schemes, modification scheme and equivalent, and these replacement schemes, modification scheme and equivalent are included in the defined scope of the present invention of claim.

Those skilled in the art will appreciate that with many methods and material similar or that be equal to described herein and can be used in enforcement the present invention.The present invention never is limited to described method and material.

Unless otherwise defined, technology used herein and scientific terminology have the identical meaning with those skilled in the art's common sense, and with Singleton etc., (1994) Dictionary ofMicrobiology and Molecular Biology, 2nd Ed., J.Wiley ﹠amp; Sons, New York, NY; And Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, New York unanimity.

Definition

Except as otherwise noted, following term used herein and phrase will have following implication:

When this paper used trade (brand) name, the applicant was intended to comprise independently the similar medicine and the active pharmaceutical ingredient of described trade (brand) name product formulation, described trade (brand) name product.

This paper term " antibody " uses with broad sense, (for example contain monoclonal antibody, polyclonal antibody, dimer, polymer, multi-specificity antibody especially, bi-specific antibody) and antibody fragment, as long as they demonstrate required biologic activity (Miller etc. (2003) Jour.of Immunology170:4854-4861).Antibody can be antibody Mus, the people, humanized, chimeric or that come from other species.Antibody is by the albumen that can discern and produce in conjunction with the immune system of specific antigen.(Janeway, C., Travers, P., Walport, M., Shlomchik (2001) Immuno Biology, 5thEd., Garland Publishing, New York). target antigen generally has the many binding sites by the CDR identification of multiple antibody, is also referred to as epi-position.Every kind all has different structures in conjunction with the antibody of different epi-positions specifically.Therefore, a kind of antigen can have more than one corresponding antibodies.Antibody comprises the immunocompetence part of total length immunoglobulin molecules or total length immunoglobulin molecules, promptly comprise immunity specifically in conjunction with antigenic antigen binding site or its a part of molecule of interesting target, this target includes but not limited to the cell of the autoimmune antibody that cancerous cell or production are relevant with autoimmune disease.Immunoglobulin disclosed herein can be the immunoglobulin molecules of any kind (for example, IgG, IgE, IgM, IgD and IgA), class (for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.Described immunoglobulin can come from any species.Yet an aspect, described immunoglobulin are people, Mus or rabbit source.

" antibody fragment " comprises the part of full length antibody, is generally its antigen combination or variable region.The example of antibody fragment comprises Fab, Fab ', F (ab ') ₂, and Fv fragment; Double antibody; Linear antibody; Fragment, antiidiotype (anti-Id) antibody, CDR (complementary determining region) and above-mentioned any immunity of being produced by the Fab expression library is specifically in conjunction with epi-position-binding fragment, the single-chain antibody molecule of cancer cell antigen, virus antigen or microbial antigen; And the multi-specificity antibody that forms by antibody fragment.

Term monoclonal antibody used herein refers to the antibody that the antibody colony by homogeneity basically obtains, and promptly except the sudden change of the natural generation that may exist on a small quantity, the single antibody of forming described colony is identical.Monoclonal antibody is a high special, at single antigen site.In addition, and comprise that the polyclonal antibody goods at the different antibodies of different determinants (epi-position) are opposite, every kind of monoclonal antibody is at the single determinant on the antigen.Except its specificity, the monoclonal antibody advantage is that they can be synthesized but are not polluted by other antibody.Described modifier " monoclonal " refers to the feature of the antibody that obtains from the antibody colony of homogeneity basically, can not be interpreted as and need produce described antibody with any ad hoc approach.For example, monoclonal antibody used according to the invention can prepare by hybridoma method, and described hybridoma method is described first by (1975) Nature 256:495 such as Kohler, perhaps can pass through recombinant DNA method (referring to, US 4816567) preparation.For example, monoclonal antibody also can be utilized (1991) Nature such as Clackson, 352:624-628; Marks etc. (1991) J.Mol.Biol., the technology that 222:581-597 describes is separated by phage antibody library.

Monoclonal antibody is particularly including " chimeric " antibody herein, and the fragment of these antibody, wherein the part of heavy chain and/or light chain is equal to or homology with the corresponding sequence that comes from specific species or belong in the antibody of specific antibodies class or subclass, and the remainder of chain is equal to or homology with the corresponding sequence that comes from other species or belong in the antibody of other antibody class or subclass, as long as they show required biologic activity (US4816567; And Morrison et al (1984) Proc.Natl.Acad.Sci.USA, 81:6851-6855).Interested herein chimeric antibody comprises " primatesization " antibody, and it comprises variable region antigen binding sequence and the human constant region sequence that comes from non-human primates (for example, old century monkey or ape (old world Monkey or Ape)).

" complete antibody " for comprising VL and VH domain herein, and the antibody of constant region of light chain (CL) and CH CH1, CH2 and CH3.Constant region can be native sequences constant region (for example, the natural sequence constant region of people) or its aminoacid sequence variant.Complete antibody can have one or more " effector functions ", promptly is attributable to those biologic activity in antibody Fc district (native sequences Fc district or aminoacid sequence variant Fc district).The example of antibody mediated effect device function comprises the C1q combination; The cell toxicant that complement relies on; The Fc receptors bind; The cell-mediated cell toxicant (ADCC) that antibody relies on; Phagocytosis; Negative adjusting with cell surface receptor such as B-cell receptor and BCR.

According to the aminoacid sequence of its CH, complete antibody can be appointed as different classifications.Five kinds of main complete antibody classifications are arranged: IgA, IgD, IgE, IgG and IgM wherein severally can be further divided into subclass (isotype), for example IgG1, IgG2, IgG3, IgG4, IgA and IgA2.CH corresponding to the different antibodies classification is called α, δ, ε, γ and μ.The subunit structure and the three-dimensional conformation of different classes of immunoglobulin are known.

" ErbB receptor " is receptor protein tyrosine kinase, and it belongs to the ErbB receptor family, and this family is the important mediators of cell growth, differentiation and survival.The ErbB receptor family comprises four kinds of different members, comprise EGF-R ELISA (EGFR, ErbB1, HER1), HER2 (ErbB2 or p185 ^Neu), HER3 (ErbB3) and HER4 (ErbB4 or tyro2).Personnel selection breast tumor cell line SKBR3 has identified one group of anti--ErbB2 antibody (Hudziak et al (1989) Mol.Cell.Biol.9 (3): 1165-1172.Obtained maximum inhibition with the antibody that is called 4D5, the percentage ratio that 4D5 suppresses cell proliferation is 56%.In this test, other antibody in this group is reduced to more low degree with cell proliferation.Find that further antibody 4D5 crosses ErbB2 and expresses the cytotoxic effect sensitivity (US5677171) of breast tumor cell line to TNF-α.Fendly et al (1990) Cancer Research 50:1550-1558; Kotts et al. (1990) In Vitro 26 (3): 59A; Sarup et al. (1991) Growth Regulation 1:72-82; Shepard etal.J. (1991) Clin.Immunol.11 (3): 117-127; Kumar et al. (1991) Mol.Cell.Biol.11 (2): 979-986; Lewis et al. (1993) Cancer Immunol.Immunother.37:255-263; Pietras et al. (1994) Oncogene 9:1829-1838; Vitetta et al. (1994) Cancer Research54:5301-5309; Sliwkowski et al. (1994) J.Biol.Chem.269 (20): 14661-14665; Scott et al. (1991) J.Biol.Chem.266:14300-5; D ' souza et al.Proc.Natl.Acad.Sci. (1994) 91:7202-7206; Lewis et al. (1996) Cancer Research 56:1457-1465; The middle anti-ErbB antibody of discussing such as Hudziak have further been identified among and Schaefer et al. (1997) the Oncogene 15:1385-1394.

Franklin et al (2004) Cancer Cell 5:317-328; Tagliabue et al (1991) Int.J.Cancer 47:933-937; McKenzie et al (1989) Oncogene 4:543-548; Maier etal (1991) Cancer Res.51:5361-5369; Bacus et al (1990) MolecularCarcinogenesis 3:350-362; Stancovski et al (1991) PNAS (USA) 88:8691-8695; Bacus et al (1992) Cancer Research 52:2580-2589; Xu et al (1993) Int.J.Cancer53:401-408; WO94/00136; Kasprzyk et al (1992) Cancer Research 52:2771-2776; Hancock et al (1991) Cancer Res.51:4575-4580; Shawver et al (1994) Cancer Res.54:1367-1373; Arteaga et al (1994) Cancer Res.54:3758-3765; Harwerth etal (1992) J.Biol.Chem.267:15160-15167; US 5783186; Other anti-ErbB antibody with different qualities has been described among and Klapper et al (1997) the Oncogene 14:2099-2109.

Two kinds of other ErbB receptor family members have been identified in the screening of sequence homogeneity; ErbB3 (US5,183,884; US 5,480, and 968; Kraus et al (1989) PNAS (USA) 86:9193-9197) and ErbB4 (EP 599274; Plowman et al (1993) Proc.Natl.Acad.Sci.USA, 90:1746-1750; And Plowman et al (1993) Nature 366:473-475).These two kinds of receptors are fastened at least some breast cancer cells and are all shown enhanced expression.

The ErbB receptor can comprise ectodomain usually, and it can be in conjunction with the ErbB part; The lipotropy membrane spaning domain; Tyrosine kinase domain in the conservative born of the same parents; With the carboxyl terminal signal structure territory of containing several tyrosine residues that can phosphorylation.The ErbB receptor can be " native sequences " ErbB receptor or its " aminoacid sequence variant ".The ErbB receptor can be native sequences people ErbB receptor.Therefore, " ErbB receptor family member " is EGFR (ErbB1), ErbB2, ErbB3, ErbB4 or current known or any other ErbB receptor that will identify in the future.

Term " ErbB1 ", " EGF-R ELISA ", " EGFR " and " HER1 " are used interchangeably herein, for example refer to disclosed EGFR among Carpenter et al (1987) Ann.Rev.Biochem.56:881-914, the mutant form that comprises its natural generation (for example, Humphrey et al., (1990) PNAS (USA), the deletion mutant EGFR among the 87:4207-4211).Term erbB1 refer to the to encode gene of EGFR protein.For example, Murthy et al (1987) Arch.Biochem.Biophys. has described the antibody at HER1 among 252:549-560 and the WO 95/25167.

Term " ERRP ", " EGF-receptor associated protein(RAP) ", " EGFR associated protein " and " EGF-R ELISA associated protein " are used interchangeably herein, for example refer to disclosed ERRP among the US 6399743 and US2003/0096373.

Wording " ErbB2 " and " HER2 " are used interchangeably herein, refer to for example Semba et al (1985) PNAS (USA), the people HER2 albumen of describing among 82:6497-6501 and Yamamoto et al (1986) Nature, 319:230-234 (Genbank accession number X03363).Term " erbB2 " refer to the to encode gene of people ErbB2, " neu " refer to the to encode gene of rat p185neu.

" ErbB3 " and " HER3 " refers to for example US 5183884; US 5480968; Disclosed receptor polypeptides among Kraus et al (1989) PNAS (USA) 86:9193-9197.At the antibody of ErbB3 is that known in the art (US 5183884; US 5480968; WO 97/35885).

Term " ErbB4 " and " HER4 " refer to for example EP patent application 599,274 herein; Plowman etal., Proc.Natl.Acad.Sci.USA, 90:1746-1750 (1993); With Plowman et al., Nature, disclosed receptor polypeptides among the 366:473-475 (1993) comprises its isotype, and is for example disclosed among the WO 99/19488.Antibody at HER4 has for example been described among the WO 02/18444.

The antibody of ErbB receptor can obtain from many sources commercial, comprises for example Santa CruzBiotechnology, Inc., California, USA.

" ErbB part " meaning is the polypeptide of combination and/or activation ErbB receptor.The ErbB part can be native sequences people ErbB part such as epidermal growth factor (EGF) (Savage et al (1972) J.Biol.Chem., 247:7612-7621); Transforming growth factor alpha (TGF-α) (Marquardt et al (1984) Science 223:1079-1082); Amphiregulin (amphiregulin) is also referred to as schwannoma (schwanoma) or horn cell autocrine growth factor (keratinocyte autocrine growthfactor) (Shoyab et al (1989) Science 243:1074-1076; Kimura et al (1990) Nature348:257-260; Cook et al (1991) Mol.Cell.Biol., 11:2547-2557); β cytokines (betacellulin) (Shing et al (1993) Science 259:1604-1607; Sasada et al (1993) Biochem.Biophys.Res.Commun.190:1173); Heparin associative list skin growth factor (HB-EGF) (Higashiyama et al (1991) Science 251:936-939); Epiregulin (Toyoda etal (1995) J.Biol.Chem.270:7495-7500; Komurasaki et al (1997) Oncogene15:2841-2848); Transfer albumen (heregulin) (referring to following); Neuregulin-2 (NRG-2) (Carraway et al., Nature, 387:512-516 (1997)); Neuregulin-3 (NRG-3) (Zhang et al (1997) Proc.Natl.Acad.Sci., 94:9562-9567); Neuregulin-4 (NRG-4) (Harari et al (1999) Oncogene, 18:2681-89) or cripto (CR-1) (Kannan et al (1997) J.Biol.Chem., 272 (6): 3330-3335).ErbB part in conjunction with EGFR comprises EGF, TGF-α, amphiregulin, β cytokines, HB-EGF and epiregulin.ErbB part in conjunction with ErbB3 comprises accent albumen.Can comprise β cytokines, epiregulin, HB-EGF, NRG-2, NRG-3, NRG-4 and accent albumen in conjunction with the ErbB part of ErbB4.The ErbB part also can be synthetic ErbB part.Synthetic part can be specific to specific ErbB receptor, perhaps can discern specific ErbB receptor complex.The example of synthetic ligands be synthetic accent albumen/EGF chimera biregulin (referring to, for example, Jones et al (1999) FEBS Letters, 447:227-231 adds it as a reference).

" transferring albumen (Heregulin) " (HRG) refers to by US 5641869 or the disclosed accent protein gene product of Marchionni et al (1993) Nature 362:312-318 encoded polypeptides.Transfer proteic example to comprise and transfer protein-alpha, transfer albumen-β 1, transfer albumen-β 2 and transfer albumen-β 3 (Holmes et al (1992) Science256:1205-1210; With US 5641869); Neu differentiation factor (NDF) (Peles et al (1992) Cell 69:205-216); Acetyl choline receptor inducing activity (ARIA) (Falls et al (1993) Cell 72:801-815); Glial growth factor (GGFs) (Marchionni et al (1993) Nature, 362:312-318); Sensation and motor neuron derivative factor (SMDF) (Ho et al (1995) J.Biol.Chem.270:14523-14532); γ-accent albumen (Schaefer et al (1997) Oncogene, 15:1385-1394).This term comprises the biological active fragment and/or the aminoacid sequence variant of native sequences HRG polypeptide, as its EGF spline structure territory fragment (for example, HRG β 1177-244).

" the different oligomer of ErbB " is for comprising the non-covalent bonded oligomer of at least two kinds of different ErbB receptors." ErbB dimer " is for comprising the non-covalent in conjunction with oligomer of two kinds of different ErbB receptors.The cellular exposure of expressing two or more ErbB receptors can form this species complex when the ErbB part.ErbB oligomer for example, as the ErbB dimer, can be as Sliwkowski et al (1994) J.Biol.Chem., 269 (20): described in the 14661-14665, separate with SDS-PAGE by immunoprecipitation.The example of this ErbB oligomeric body comprises EGFR-ErbB2 (being also referred to as HER1/HER2), ErbB2-ErbB3 (HER2/HER3) and ErbB3-ErbB4 (HER3/HER4) complex.In addition, ErbB oligomeric body can comprise two or more ErbB2 receptors with different ErbB receptors such as ErbB3, ErbB4 or EGFR (ErbB1) combination.Other protein such as cytokine receptor subunit (for example, gp130) can be included in this oligomeric body.

" ligand activation of ErbB receptor " means and combines institute's Mediated Signal Transduction (for example, caused by the intracellular kinase domain of ErbB receptor phosphorylation tyrosine residue in ErbB receptor or the substrate polypeptide) by ErbB part and the ErbB oligomeric body that comprises interested ErbB receptor.This can relate to combining of ErbB part and ErbB oligomeric body usually; this is in conjunction with the kinase domain of one or more ErbB receptors in the activation oligomeric body, and causes the phosphorylation of tyrosine residue in the phosphorylation of tyrosine residue in one or more ErbB receptors and/or other substrate polypeptide thus.Can test quantitative ErbB receptor activation with multiple tyrosine phosphorylation.

" native sequences " polypeptide is for having and the polypeptide that comes from the identical aminoacid sequence of natural polypeptide (for example, ErbB receptor or ErbB part).Such native sequences polypeptide can separate from nature, perhaps can be by reorganization or synthesizing mean production.Therefore, the native sequences polypeptide can have the human polypeptides, Mus polypeptide of natural generation or from the amino acid sequence of polypeptide of any other mammalian species.

Term " aminoacid sequence variant " refers to have the polypeptide that is different from the native sequences amino acid sequence of polypeptide to a certain extent.Usually, at least one receptors bind domain of aminoacid sequence variant and natural ErbB part or sequence homogeneity at least about 70% will be arranged with at least one ligand binding domains of natural ErbB receptor, perhaps with such receptor or ligand binding domains at least about 80%, or at least about 90% homology.Described aminoacid sequence variant has replacement, deletion and/or inserts on some positions in the aminoacid sequence of natural acid sequence.

" sequence homogeneity " is defined as aligned sequences and introduces the room in case of necessity with after obtaining maximum sequence homogeneity percentage ratio, the percent of identical residue in the aminoacid sequence variant.The method and computer program that is used for described comparison is well known in the art.A kind of such computer program is Genentech, " Align 2, " of Inc. design, and it is committed to U.S. Copyright Bureau in December in 1991 10 days in company with customer documentation, Washington, DC 20559.

The reaction of " the cell-mediated cell toxicant that antibody relies on " and the mediation of " ADCC " phalangeal cell, wherein express the non-specific cell toxic cell (for example NK cell (NK) cell, neutrophil cell and macrophage) of Fc receptor (FcRs) and discern bonded antibody on the target cell, cause the target cell cracking subsequently.The main cell NK cell of mediation ADCC is only expressed Fc γ RIII, and monocytes Fc γ RI, Fc γ RII and Fc γ RIII.The expression of FcR is summarized in Ravetch and Kinet on the hematopoietic cell, Annu.Rev.Immunol, 9:457-92 (1991) the 464th page table 3.Be the ADCC activity of assessment molecules of interest, (US 55003621 can to carry out external ADCC test; US 5821337).The effector lymphocyte who can be used for this test comprises PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) and NK cell (NK) cell.Perhaps, or in addition, can body in the ADCC activity of assessment molecules of interest, for example as Clynes etal (1998) PNAS (USA), among the 95:652-656 in the disclosed animal model.

" maytansinoid drug moiety " meaning is the substructure with antibody-drug conjugates of maytansine compound structure.Maytansine separates (US 3896111) from East Africa shrub tingia Folium Mayteni hookeri (Maytenus serrata) at first.Found afterwards that certain micro-organisms also produced maytansinoid such as maytansinol (maytansinol) and C-3 maytansinol ester (US 4151042).Synthetic maytansinol and maytansinol analog have been reported.Referring to United States Patent (USP) 4137230; 4248870; 4256746; 4260608; 4265814; 4294757; 4307016; 4308268; 4308269; 4309428; 4313946; 4315929; 4317821; 4322348; 433 1598; 4361650; 4364866; 4424219; 4450254; 4362663; With 4371533, and Kawai et al (1984) Chem.Pharm.Bull.3441-3451), all special the adding as a reference.

Term " Fc receptor " or " FcR " are used to describe the receptor in binding antibody Fc district, as native sequences people FcR.FcR can comprise receptor Fc γ RI, Fc γ RII and Fc γ RIII subclass in conjunction with IgG antibody (gamma receptor), comprises the allelic variant of these receptors and the different splicing forms of these receptors.Fc γ RII receptor comprises Fc γ RIIA (" activated receptor ") and Fc γ RIIB (" inhibition receptor "), and they have similar aminoacid sequence, and are mainly variant in its Cytoplasm domain.Activated receptor Fc γ RIIA comprises the activation motif (ITAM) based on immunity receptor tyrosine in its Cytoplasm domain.Inhibition receptor Fc γ RIIB comprises the inhibition motif (ITIM) based on immunity receptor tyrosine in its Cytoplasm domain.(referring to summary M.in Da_ron, Annu.Rev.Immunol., 15:203-234 (1997)).Ravetch and Kinet, Annu.Rev.Immunol., 9:457-92 (1991); Capel et al (1994) Immunomethods, 4:25-34; And de Haas et al (1995) J.Lab.Clin.Med. has summarized FcRs among the 126:330-41.Term " FcR " comprises other FcR herein, also comprises those that will identify in the future.Described term also comprises neonate receptor FcRn, and it is responsible for the transfer (Guyer et al (1976) J.Immunol.117:587 and Kim et al (1994) J.Immunol.24:249) of parent IgGs to fetus.

" cell toxicant that complement relies on " or " CDC " refer to the ability of molecule cracking target thing in the presence of complement.The complement activation approach is attached to the compound molecule of related antigen (for example antibody) by first assembly (C1q) of complement system and is started.In order to assess complement activation, can carry out CDC test, described in Gazzano-Santoro et al (1996) J.Immunol.Methods for example, 202:163.

" natural antibody " is generally about 150,000 daltonian different tetramer glycoproteins, is made up of with two identical weights (H) chain two identical light (L) chains.Every light chain is connected in heavy chain by a covalent disulfide bonds, yet the number of disulfide bond changes in different immunoglobulin isotype heavy chains.Every heavy chain and light chain also have rule intrachain disulfide bond at interval.Every heavy chain one end has variable region (VH), next is many constant regions.Every kind of light chain at one end has variable region (VL), has constant region at its 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 variable region of light chain and heavy chain is arranged side by side.It is believed that particular amino acid residue forms the contact surface between light chain and the variable region of heavy chain.

Term " variable " refers to that some part difference in sequence of variable region in the antibody is very big, is used for combination and the specificity of every kind of specific antibodies to its specific antigen.Yet transmutability is not equally distributed in the variable region of all antibody.In light chain and variable region of heavy chain, attention all concentrates on three fragments that are called the hypervariable region.The conservative more part in variable region is called framework region (FRs).The variable region of natural heavy chain and light chain respectively comprises four FRs, adopts β-sheet conformation in a large number, links to each other by three hypervariable regions, and it forms the ring that connects β-lamellar structure, constitutes the part of β-lamellar structure sometimes.Hypervariable region in every chain very closely combines by FRs, and make contributions (referring to Kabat et al (1991) Sequences ofProteins of Immunological Interest for the formation of the antigen-binding site of antibody with hypervariable region from another chain, 5th Ed.Public Health Service, NationalInstitutes of Health, Bethesda, MD).Constant region does not directly relate to antibody and combines with antigenic, but shows multiple effector functions, for example participation of antibody in the cell toxicant (ADCC) of antibody dependence.

Refer to be responsible for the amino acid residue of the bonded antibody of antigen when using term " hypervariable region " herein.Described hypervariable region comprises usually from the amino acid residue of " complementary determining region " or " CDR " (for example, 31-35 (H1), 50-65 (H2) and the 95-102 (H3) in the residue 24-34 (L1) in the variable region of light chain, 50-56 (L2) and 89-97 (L3) and the variable region of heavy chain; Kabat et al is the same) and/or from those residues (for example, 26-32 (H1), 53-55 (H2) and 96-101 (H3) in the residue 26-32 (L1) in the variable region of light chain, 50-52 (L2) and 91-96 (L3) and the variable region of heavy chain of " hypermutation ring "; Chothia and Lesk (1987) J.Mol.Biol., 196:901-917)." framework region " or " FR " residue is those variable region residues except the hypervariable region residue of definition herein.

The papain digestion of antibody produces two identical Fabs, is called " Fab " fragment, and every all has single antigen-binding site and residual " Fc " fragment, and its title has reflected that it is easy to crystalline ability.Pepsin generation F (ab ') 2 fragments, it has two antigen-binding sites, still can coupled antigen.

" Fv " is the minimum antibody fragment that comprises complete antigen identification and antigen-binding site.This zone by a heavy chain and variable region of light chain closely, non-covalent bonded dimer forms.Determined the lip-deep antigen-binding site of VH-VL dimer with three interactional conformations in hypervariable region of this each variable region just.Six hypervariable regions are given the antibody antigen binding specificity jointly.Yet, even if single variable region half of Fv of three hypervariable regions of antigenic specificity (or only comprise) also has the ability of identification and conjugated antigen, though its affinity is lower than complete binding site.

The Fab fragment also comprises the constant region of light chain and first constant region (CH1) of heavy chain.Fab ' fragment is different with the Fab fragment, and its carboxyl terminal in heavy chain CH1 district has added several residues, comprises one or more cysteine from the antibody hinge region.The Fab ' that the cysteine residues of constant region is had at least one free mercapto herein is called Fab '-SH.Originally F (ab ') 2 antibody fragments produce with the right form of Fab ' fragment, and described Fab ' fragment is to having hinge cysteine between Fab ' fragment.Other chemical coupling thing of antibody fragment also is known.

Based on the aminoacid sequence of its constant region, can be appointed as a kind of in two kinds of clear types of determining that are called kappa (κ) and lambda (λ) from " light chain " of the antibody of any invertebrate species.

" strand Fv " or " scFv " mean strand variable region antibody fragment, and it comprises the VH and the VL domain of antibody, and wherein these domains are present in the single polypeptide chain.Described Fv polypeptide can further comprise peptide linker between VH and VL domain, it can make scFv form antigen in conjunction with required structure (Pl ü ckthun in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., Springer-Verlag, New York, pp.269-315 (1994).WO 93/16185; US 5571894; Anti-ErbB antibody scFv fragment has been described among the US 5587458.

Term " double antibody (diabodies) " refers to have the little antibody fragment of two antigen-binding sites, and described fragment comprises the variable region of heavy chain (VH) that is connected to variable region of light chain (VL) in the same polypeptide chain (VH-VL).Do not allow two paired catenation sequences of domain on same the chain by using too short, force the complementary structure territory of described domain and another chain to be matched, produce two antigen-binding sites.EP404 for example, 097; WO 93/11161; With Hollinger et al (1993) Proc.Natl.Acad.Sci.USA, double antibody has been described in more detail among the 90:6444-6448.

Inhuman (for example, Rodents) antibody of " humanization " form is the chimeric antibody that comprises the minmal sequence that comes from non-human immunoglobulin.To a great extent, by the metathetical human normal immunoglobulin of residue (receptor antibody) from inhuman species (donor antibody) hypervariable region, described inhuman species are for for example having mice, rat, rabbit or the non-human primates of required specificity, affinity and ability by the residue that wherein comes the autoreceptor hypervariable region for humanized antibody.In some cases, human normal immunoglobulin's framework region (FR) residue is replaced by corresponding inhuman residue.In addition, humanized antibody can comprise undiscovered residue in receptor antibody or the donor antibody.Having made up these modifies with the further antibody performance of improving.Generally speaking, humanized antibody can comprise all or at least one and two variable regions typically basically, wherein all or all basically hypermutation rings are corresponding to those hypermutation rings of non-human immunoglobulin, and all or all basically FRs are the FRs of human normal immunoglobulin's sequence.The optional at least a portion that also will comprise constant region for immunoglobulin (Fc) (normally human normal immunoglobulin's constant region) of humanized antibody.More details are referring to Jones et al (1986) Nature, 321:522-525; Riechmann etal (1988) Nature 332:323-329; And Presta, (1992) Curr.Op.Struct.Biol., 2:593-596.

Humanization anti-ErbB antibody comprises huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and the huMAb4D5-8 (HERCEPTIN_, trastuzumab) that describes in US 5821337 table 3 of (the special adding as a reference) herein; Humanization 520C9 (WO 93/21319) and humanization 2C4 antibody.

" isolating " antibody refers to differentiate and the antibody that separates and/or reclaim from its natural surroundings component.The contaminant component of its natural surroundings refers to disturb the diagnosis of antibody or the material of therapeutic use, may comprise enzyme, hormone and other protein or nonprotein solute.Described antibody can be purified to (1) when measuring with the Lowry method in the weight of antibody greater than 95%, reach the degree of at least 15 residues that are enough to obtain N-terminal or internal amino acid sequence when (2) using revolving cup (spinning cup) protein sequencer to measure, perhaps (3) carry out SDS-PAGE under reproducibility or irreducibility condition, dye with Coomassie blue or silver and reach homogenizing.Because at least a component of antibody natural surroundings will not exist, thereby isolated antibody comprises the original position antibody in the reconstitution cell.Yet isolated antibody will be by at least one purification step preparation usually.

" its combination " interested antigen, for example, the antigenic antibody of ErbB2 refers to can be with enough affinity in conjunction with this antigen, so that described antibody can be used for the antibody of the described antigenic cell of targeted expression.Wherein said antibody refers to the antibody in conjunction with ErbB2, and is opposite with other ErbB receptor, and it usually will be preferentially in conjunction with ErbB2, also refers to not take place with other protein such as EGFR, ErbB3 or ErbB4 the antibody of obvious cross reaction.In such embodiments, analyze or radioimmunoprecipitation (RIA) when measuring with fluorescent activation cell sorting (FACS), the degree of these non-ErbB2 protein of the described antibodies cell surface protein of endogenous receptor (for example, in conjunction with) will be less than 10%.Sometimes, significant cross reaction can not take place with rat neu albumen in anti-ErbB antibody, and for example, Schecter et al (1984) Nature 312:513 and Drebin et al (1984) Nature are described in the 312:545-548.

The antibody of the ligand activation of " blocking-up " ErbB receptor reduces or stops this activation, wherein compare with monoclonal antibody 4D5, described antibody can more effectively be blocked the ligand activation of ErbB receptor basically, and is for example, approximately the same effective with monoclonal antibody 7F3 or 2C4 or its Fab fragment.For example, the antibody of the ligand activation of blocking-up ErbB receptor can be than the antibody of the high about 50-100% of effectiveness of 4D5 aspect the formation of blocking-up ErbB oligomeric body.The blocking-up of the ligand activation of ErbB receptor can take place by any way, for example, by disturbing: part in conjunction with ErbB receptor, ErbB complex form, the phosphorylation of tyrosine kinase residue in the ErbB complex in the tyrosine kinase activity of ErbB receptor and/or the ErbB receptor or that cause by the ErbB receptor.

Has appointment antibody, as monoclonal antibody 2C4 (Omnitarg, Genentech, the antibody of Inc.) " biological property " refers to have the antibody of one or more biological characteristicses of described antibody, (for example, other antibody ErbB2) differentiates described biological characteristics with combining same antigen with it.For example, the antibody with biological property of 2C4 can be blocked the HRG activation of the ErbB oligomeric body that comprises ErbB2 and ErbB3, ErbB1 or ErbB4; Blocking-up comprises EGF, TGF-α, HB-EGF, epiregulin and/or the amphiregulin activation of the ErbB receptor of EGFR and ErbB2; The activation of EGF, the TGF-α of blocking-up MAPK and/or HRG mediation; And/or as the combination of 2C4, in conjunction with the identical epi-position in the ErbB2 ectodomain (for example, blocking-up monoclonal antibody 2C4 is in conjunction with the antibody of ErbB2).

Unless otherwise, wording " monoclonal antibody 2C4 " refer to have following example Mus 2C4 antibody or come from the antibody of the antigen of Mus 2C4 antibody in conjunction with residue.For example, monoclonal antibody 2C4 can be for mouse monoclonal antibody 2C4 or its variant, and as humanized antibody 2C4, its antigen with mouse monoclonal antibody 2C4 is in conjunction with amino acid residue (WO 01/00245).Unless otherwise, when using wording " rhuMAb 2C4 " herein, refer to comprise the variable region of light chain (VL) that is respectively SEQ ID Nos.3 and 4 and the antibody of variable region of heavy chain (VH) sequence, described variable region of light chain (VL) and variable region of heavy chain (VH) sequence are fused to people's light chain and heavy chain IgG1 (non-A allotype) the constant region sequence of choosing wantonly by Chinese hamster ovary (CHO) cell (WO 01/00245) expression.

Unless otherwise, term " monoclonal antibody 4D5 " refer to have Mus 4D5 antibody (ATCC CRL10463) or come from the antibody of the antigen of Mus 4D5 antibody in conjunction with residue.For example, monoclonal antibody 4D5 can be for mouse monoclonal antibody 4D5 or its variant, and as humanization 4D5, its antigen with mouse monoclonal antibody 4D5 is in conjunction with residue.Exemplary humanization 4D5 antibody comprises huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and the huMAb4D5-8 (HERCEPTIN_) among the US 5821337.

" growth inhibitor " refers to for example express the chemical compound or the compositions of the growth of cancer cells of ErbB external or suppress cell in vivo.Therefore, described growth inhibitor can be the remarkable growth inhibitor that reduces the percent of S phase ErbB express cell.The example of growth inhibitor comprises the reagent (making it in non-S phase position) that the blocking-up cell cycle carries out, as induce G1 to stagnate and reagent (TheMolecular Basis of Cancer that the M-phase stagnates, Mendelsohn and Israel, eds., Chapter 1, entitled " Cell cycle regulation; oncogenes; and antineoplastic drugs " by Murakami etal. (WB Saunders:Philadelphia, 1995), especially is p.13).The example of " growth inhibited " antibody refers in conjunction with ErbB2 and suppressed to express those antibody of the growth of cancer cells of ErbB2.Antibody concentration at about 0.5 to 30 μ g/ml, growth inhibited anti-ErbB antibody can suppress the growth of Sk-Br-3 breast tumor cell in the cell culture 20% or more or more than 50% (for example, from about 50% to about 100%), wherein measures growth inhibited (US5677171) after six days at the described antibody of Sk-Br-3 cells contacting.

The antibody of " inducing cell death " instructs the activation cell to become the antibody of non-living cells.Described cell is generally the cell of expressing the ErbB2 receptor, especially crosses the cell of expressing the ErbB2 receptor.Described cell can be a cancerous cell, for example, and mammary gland, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas or bladder cell.External, described cell can be Sk-Br-3, BT474, Calu 3, MDA-MB-453, MDA-MB-361 or SKOV3 cell.Can be when not having complement and immune effector cell the external test cell death, to distinguish the inductive cell death of cell toxicant (CDC) that the cell-mediated cell toxicant (ADCC) that relied on by antibody or complement rely on.Therefore, can and under the situation of no immune effector cell, carry out the cell death test with heat-killed serum (promptly not having complement).Whether can inducing cell death for measuring described antibody, can assess forfeiture with respect to the film integrality of untreated cell, the forfeiture of described film integrality by iodate third ingot (PI), trypan blue (referring to Moore et al (1995) Cytotechnology, 17:1-11) or 7AAD absorb assessment.The antibody of inducing cell death refers to absorb those antibody (vide infra) of inducing PI to absorb in the test at the PI of BT474 cell.

The antibody of " cell death inducing " refers to induce the antibody of programmed cell death, and it forms (being called the apoptosis body) mensuration by annexin V combination, dna fragmentationization, cell shrinkage, endoplasmic reticulum expansion, cell breakage and/or membrane vesicle.Described cell was generally the cell of expressing the ErbB2 receptor, comprised tumor cell, for example, and mammary gland, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas or bladder cell.External, described cell can be Sk-Br-3, BT474, Calu 3 cells, MDA-MB-453, MDA-MB-361 or SKOV3 cell.

Therapeutics treatment and prevention or prevention measure are all represented in term " treatment (treat) " or " treatment (treatment) ", and wherein purpose is to stop or slow down (alleviating) undesirable physiology changes or disorderly, as the development of cancer or spread.At purpose of the present invention, useful or required clinical effectiveness include but not limited to the weakening of the alleviating of symptom, disease degree, disease condition stable (promptly not worsening), progression of disease delay or slow down, the improvement of disease condition or mitigation and remission (no matter be part or whole), no matter be detectable or undetectable." treatment (Treatment) " can also refer to the survival of the prolongation that desired survival is compared when not receiving treatment.Need the patient of treatment to comprise that those have suffered from described disease or disorderly and suffer from described disease or disorderly patient easily, perhaps will prevent its disease or those disorderly patients.

" disorderly (disorder) " refers to and will benefit from any disease of treatment of the present invention.This comprises chronic and acute disorder or disease, comprises the pathology disease that those make the described disorder of mammal easy infection.The non-limitative example of the disorder that this place will be treated comprises optimum and malignant tumor; Leukemia and lymph malignant disease, particularly mammary gland, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas, prostate or bladder cancer; Neuron, neuroglia, spider cell, hypothalamus and other body of gland, macrophage (macrophagal), epithelium, substrate and blastaea (blastocoelic) disorder; And inflammatory, blood vessel generation disease and immunology disease.The exemplary disorder according to the present invention for the treatment of is a solid malignant.

Term " treatment effective dose " refers to effectively to treat the amount of mammalian diseases or disorderly medicine.With regard to cancer, the treatment effective dose of medicine can: (i) reduce the quantity of cancerous cell; (ii) reduce the tumor size; (iii) the cancerous cell infiltration in the organ to the periphery suppressed, block, slow down to a certain degree and preferred the termination; (iv) suppress (promptly slowing down) neoplasm metastasis to a certain degree and preferably stopping; (v) suppress tumor growth; And/or (vi) with the sx of one or more and related to cancer to a certain degree.When reaching medicine and can stop growth and/or kill the degree of cancerous cell of existence, it may be that cell suppresses and/or Cytotoxic.In animal model, can be by using physical measurement tumor during the course of disease behind the ADC, and the part by measuring tumor and disappear fully and assess effect.For treatment of cancer, for example can make progress the time by assess disease (time to disease progression) (TTP) and/or assaying reaction rate (responserate) (RR) measure effect.

Term " bioavailability " refers to be administered to system's utilizability (that is blood/plasma level) of patient's specified rate medicine.Bioavailability is the absoluteness term, refers to that medicine arrives the measured value of body circulation time (speed) and total amount (degree) from the dosage forms of using.

Term " cancer " and " cancer " refer to or describe the mammalian physiology disease that typically is grown to feature with unconfined cell." tumor " comprises one or more cancerous cell.The example of cancer includes but not limited to cancer (carcinoma), lymphoma, blastoma (blastoma), sarcoma and leukemia or lymph malignant disease.The example more specifically of this cancer comprises that squamous cell carcinoma (for example, the epithelium squamous cell carcinoma), pulmonary carcinoma, comprise small cell lung cancer, nonsmall-cell lung cancer (" NSCLC "), adenocarcinoma of lung and squamous cell lung carcinoma (squamous carcinoma), peritoneal cancer, hepatocarcinoma, stomach (gastric) or stomach (stomach) cancer comprise human primary gastrointestinal cancers, gastrointestinal stromal tumors (gastrointestinal stromal tumor) (GIST), cancer of pancreas, glioblastoma (glioblastoma), cervical cancer (cervical cancer), ovarian cancer, hepatocarcinoma (liver cancer), bladder cancer, hepatoma (hepatoma), breast carcinoma, colon cancer, rectal cancer, colorectal carcinoma, endometrium or uterus carcinoma, salivary-gland carcinoma, kidney or kidney (kidney or renal) cancer, carcinoma of prostate, cancer of vagina, thyroid carcinoma, hepatocarcinoma (hepatic carcinoma), anus cancer, carcinoma of penis, and head and neck cancer.

" express the cancer of ErbB " and refer to be included in the cancer that there is the proteic cell of ErbB in its cell surface." cancer of expression ErbB2 " refers to produce at its cell surface the ErbB2 of enough levels, so anti-ErbB antibody can combine and have the cancer at described treatment for cancer effect with it.

" cross express " receptor for example cancer of ErbB receptor refers to compare with the non-cancerous cell of homologue type, has the cancer of the described receptor of obvious higher level at its cell surface.This cross to express can by gene amplification or enhanced transcribe or translate due to.The ErbB receptor is crossed expression and can be in diagnosis or prognosis test be measured by the ErbB protein level that improves on the assessment cell surface and (for example, test by SABC; IHC).Perhaps, or in addition, can be by for example fluorescence in situ hybridization (FISH; Referring to WO 98/45479), southern trace or polymerase chain reaction (PCR) technology such as real-time quantitative PCR (RT-PCR) measure the level of the nucleic acid of coding ErbB in the cell.Cross expressing of ErbB part can be on the diagnostics be measured by the level of part described in the assess patient (or encode its nucleic acid), for example, in tumor biopsy or by above-mentioned multiple diagnostic test such as IHC, FISH, southern trace, PCR or body build-in test.Also can by measure released antigen (for example, the ErbB ectodomain) in biofluid such as the serum study the ErbB receptor cross expression (referring to, for example, US4933294; WO 91/05264; US 5401638; With Sias et al (1990) J.Immunol.Methods132:73-80).Except above-mentioned test, those of skill in the art can also adopt multiple other body build-in test.For example, the intravital cell of patient can be contacted with antibody, described antibody is chosen wantonly and is used detectable labelling, for example, the labelled with radioisotope mistake can be assessed combining of antibody and patient's cell, for example, by the radioactivity external scan, perhaps by analyzing slicer, the patient of contacted described antibody before this slicer is taken from.

By the HER2 molecule copy number corresponding to each cellular expression being carried out the SABC scoring was the tumor rating of expressing HER2, and can carry out biochemical measurement: 0=0-10,000 copy/cell, 1+=is at least about 200,000 copy/cell, 2+=is at least about 500,000 copy/cells, the about 1-2 of 3+=* 10 ⁶Copy/cell.HER2 crosses to express with the 3+ level and causes tyrosine kinase not rely on activation (the Hudziak et al. of part, (1987) Proc.Natl.Acad.Sci.USA 84:7159-7163), occur in about 30% the breast carcinoma, and in these patients, there is not recurrence survival period (relapse-freesurvival) and overall survival phase (overal survival) minimizing (Slamon et al (1989) Science244:707-712; Slamon et al (1987) Science, 235:177-182).On the contrary, " not being expressed as feature with the mistake of ErbB2 receptor " cancer is in diagnostic test, compares with the non-cancerous cell of homologue type, does not express the cancer of the ErbB2 receptor that is higher than normal level.Mus monoclonal Anti-HER 2 suppresses the growth of breast cancer cell line, and described breast cancer cell line is with 2+ and 3+ (each cell 1-2 * 10 ⁶The HER2 receptor) level is crossed expression HER2, but the cell of expressing more low-level HER2 is not had activity (Lewis etal (1993) Cancer Immunol.Immunother.37:255-263).Based on this observed result, with antibody 4D5 humanization (huMAb4D5-8, rhuMAb HER2, US 5821337; Carter et al (1992) Proc.Natl.Acad.Sci.USA 89:4285-4289), and at patient with breast cancer's body build-in test, described patient with breast cancer's tumor is crossed expression HER2, but it gets along with behind conventional chemotherapy (Cobleigh etal (1999) J.Clin.Oncol.17:2639-2648).In this test, the Most patients tumor is with 3+ horizontal expression HER2, and a part is 2+.

The cancer that " do not rely on hormone " does not depend on the cancer of the existence of hormone for its propagation, and wherein said hormone is in conjunction with the receptor of being expressed by cancerous cell.Implementing to weaken in the tumor or near the tumor when pharmacology of hormone concentration or surgical operation strategy, such cancer does not experience clinical degeneration (regression).The example that does not rely on the cancer of hormone comprises and does not rely on androgenic carcinoma of prostate, do not rely on estrogenic breast carcinoma, carcinoma of endometrium and ovarian cancer.Such cancer can begin with the tumor that hormone relies on, and in hormone antagonist treatment back from the hormone-sensitive stage development to hormone toleration tumor.

The material that term used herein " cytotoxic reagent " refers to suppress or stop the cell function and/or causes cytoclasis.Described term (for example will comprise radiosiotope ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁵³Sm, ²¹²Bi, ³²P, ⁶⁰The radiosiotope of C and Lu), the enzyme activity toxin of chemotherapeutics and toxin such as micromolecule toxin or antibacterial, fungus, plant or animal origin, comprise its synthetic analogues and derivant.

" chemotherapeutics " is the chemical compound that is used for treatment of cancer.

This definition " chemotherapeutics " also comprise (i) rise to regulate or inhibitory hormone to the antihormone of function of tumor, such as estrogen antagonist and selective estrogen receptor modulators (SERM), comprise for example tamoxifen (tamoxifen) (comprising the NOLVADEX_ tamoxifen), raloxifene (raloxifene), droloxifene (droloxifene), 4-trans-Hydroxytamoxifen, bent former times sweet smell (trioxifene) difficult to understand, that Lip river former times sweet smell (keoxifene), LY117018, Ao Nasi ketone (onapristone) and FARESTON_ toremifene (toremifene); (ii) be suppressed at virtue enzyme (aromatase) inhibitor of the virtue enzyme of regulating the estrogen generation among the adrenal gland, such as for example 4 (5)-imidazoles, aminoglutethimide (aminoglutethimide), MEGASE_ megestrol acetate (megestrol acetate), AROMASIN_ exemestane (exemestane), Formestane (formestanie), fadrozole (fadrozole), RIVISOR_ R 83842 (vorozole), FEMARA_ letrozole (letrozole) and ARIMIDEX_ arimidex (anastrozole); (iii) androgen antagonist is such as Drogenil (flutamide), nilutamide (nilutamide), than Ka Mite (bicalutamide), leuprorelin (leuprolide) and goserelin (goserelin); And troxacitabine (troxacitabine) (1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitor; (v) kinases inhibitor; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotide, the signal that particularly suppresses to relate to abnormal cell proliferation by way of in the antisense oligonucleotide of gene expression, such as for example PKC-α, Raf and H-Ras; (viii) ribozyme is such as vegf expression inhibitor (as the ANGIOZYME_ ribozyme) and HER2 expression inhibitor; (ix) vaccine, such as the gene therapy vaccine, for example ALLOVECTIN_ vaccine, LEUVECTIN_ vaccine and VAXID_ vaccine; PROLEUKIN_rIL-2; LURTOTECAN_ topoisomerase 1 inhibitor; ABARELIX_rmRH; (x) anti-angiogenic rebirth agent such as bevacizumab (bevacizumab) (AVASTIN_, Genentech); And (xi) above-mentioned any pharmaceutically acceptable salt, acid or derivant.

Kinases inhibitor comprises tyrosine kinase inhibitor, and it suppresses the tyrosine kinase activity of tyrosine kinase such as ErbB receptor to a certain extent.The example of tyrosine kinase inhibitor comprise the EGFR-targeted drug as: (i) in conjunction with the antibody of EGFR, comprise MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCCCRL 8509) (referring to, US 4943533, Mendelsohn et al.) and variant, as 225 (C225 or Cetuximab of chimericization; ERBITUX_ is Imclone) with reformation type (reshaped) people 225 (H225) (WO 96/40210, Imclone Systems Inc.); Antibody (US 5212290) in conjunction with II type sudden change EGFR; Humanization and chimeric antibody (US 5891996) in conjunction with EGFR; With people's antibody, as ABX-EGF (WO 98/50433) in conjunction with EGFR; (ii) with the link coupled anti-egfr antibodies of cytotoxic reagent (EP 659439A2); Comprise ZD1839 or Gefitinib (IRESSA with micromolecule in conjunction with EGFR ^TMAstra Zeneca), Erlotinib HCl (CP-358774, TARCEVA ^TMGenentech/OSI) and AG1478, (SU 5271 for AG1571; Sugen), quinazoline such as PD153035,4-(3-chloro aminobenzen) quinazoline (4-(3-chloroanilino) quinazoline), Pyridopyrimidine (pyridopyrimidines), pyrimidopyrimidines, pyrrolopyrimidine (pyrrolopyrimidines), as CGP 59326, CGP 60261 and CGP 62706, and pyrazolyl pyrimidine (pyrazolopyrimidine), 4-(phenylamino)-7H-pyrrolo-[2,3-d] pyrimidine (4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines), curcumin curcumin (two Resina Ferulae acyl methane (diferuloyl methane), 4,5-two (4-fluoro aniline) phthalimide) (4,5-bis (4-fluoroanilino) phthalimide), the tyrphostines that contains nitrothiophene (nitrothiophene) part; PD-0183805 (Wamer-Lambert); Antisense molecule (for example, with ErbB-code nucleic acid bonded those); 1,4-Benzodiazine (quinoxalines) (US 5804396); Tryphostins (US 5804396); ZD6474 (Astra Zeneca); PTK-787 (Novartis/Schering AG); Pan-ErbB inhibitor such as CI-1033 (Pfizer); (ISIS 3521 for Affinitac; Isis/Lilly); Imatinib mesylate (Imatinibmesylate) (Gleevac; Novartis); PKI 166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxanib (Sugen); ZD6474 (AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone); Perhaps as US 5804396; WO 99/09016 (American Cyanamid); WO98/43960 (American Cyanamid); WO 97/38983 (Warner Lambert); WO99/06378 (Warner Lambert); WO 99/06396 (Warner Lambert); WO96/30347 (Pfizer, Inc); WO 96/33978 (Zeneca); WO 96/3397 (Zeneca); And described in the WO96/33980 (Zeneca).

" anti-angiogenic rebirth agent " refers to block to a certain extent or disturb the chemical compound of vascular development.The anti-angiogenic rebirth factor can be the micromolecule or the antibody of for example binding growth factor or growth factor receptors, and described somatomedin or growth factor receptors relate to the promotion angiogenesis.Exemplary anti-angiogenic rebirth agent be in conjunction with the antibody of VEGF (VEGF) such as bevacizumab (bevacizumab) (AVASTIN_, Genentech).

Term " cytokine " " be the proteinic common name that discharges by a kind of cell colony, it acts on another kind of cell as the iuntercellular mediators.The example of this cytokine is lymphokine, monokine and traditional polypeptide hormone.What be included in described cytokine is growth hormone such as human growth hormone, N-methionyl-human growth hormone (N-methionyl human growth hormone) and bovine growth hormone; Parathyroid hormone; Thyroxine (thyroxine); Insulin; Proinsulin; Relaxin (relaxin); Relaxation precipitinogen (prorelaxin); Glycoprotein hormones such as follicle stimulating hormone (follicle stimulating hormone) (FSH), thyrotropin (thyroid stimulating hormone) (TSH) and interstitialcellstimulating hormone (ICSH) (luteinizing hormone) (LH); Liver growth factor; Fibroblast growth factor; Prolactin antagonist; Galactagogin; Tumor necrosis factor-alpha and-β; Miao Le-inhibiting substances (mullerian-inhibitingsubstance); Mice promoting sexual gland hormone related peptides (mouse gonadotropin-associated peptide); Inhibin (inhibin); Activin (activin); VEGF; Integrin; Thrombopoietin (thrombopoietin) (TPO); Nerve growth factor such as NGF-β; PDGF; Transforming growth factor (TGFs) is as TGF-α and TGF-β; Insulin like growth factor-1 and-II; Erythropoietin (EPO); The osteogenic induction factor (osteoinductive factors); Interferon such as interferon-' alpha ' ,-β and-γ; Colony stimulating factor (CSFs) is as macrophage-CSF (M-CSF); Granulocyte-macrophage-CSF (GM-CSF); And granulocyte-CSF (G-CSF); Interleukin (ILs) is as IL-1, IL-1 α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; Tumor necrosis factor such as TNF-α or TNF-β; And other polypeptide factor comprises LIF and kit part (kitligand) (KL).Term cytokine used herein comprise natural origin or from the protein of reconstitution cell culture and the biologic activity equivalent of native sequences cytokine.

The used term of the application " prodrug (prodrug) " refers to the precursor or the derivative form of pharmaceutically active substances, and the cytotoxicity that itself and parent's medicine compare tumor cell is lower, can or change the active parent's form that has more into by enzyme effect activation.Referring to, for example, Wilman, " Prodrugs in CancerChemotherapy " Biochemical Society Transactions, 14, pp.375-382,615thMeeting Belfast (1986) and Stella et al., " Prodrugs:A Chemical Approach toTargeted Drug Delivery; " Directed Drug Delivery, Borchardt et al., (ed.), pp.247-267, Humana Press (1985).Prodrug of the present invention includes but not limited to phosphorous hydrochlorate (ester) prodrug, contain sulfo-phosphate (ester) prodrug, sulfur-bearing hydrochlorate (ester) prodrug, contain the peptide prodrug, the prodrug that D-is amino acid modified, the glycosylation prodrug, the prodrug that contains beta-lactam, contain the prodrug of the optional benzene acetamide oxide (phenoxyacetamide) that replaces or contain the prodrug of choosing the phenyl acetamide (phenylacetamide) that replaces wantonly, 5-flurocytosine and can be converted into other 5-floxuridine prodrug that has more active cytotoxicity free drug.The example that is used for cytotoxic drug of the present invention for prodrug forms of can deriving includes but not limited to those above-mentioned chemotherapeutics.

" liposome " is by can be used for that medicine (as disclosed anti-ErbB antibody and the optional chemotherapeutics of this paper) is delivered to the capsule foaming material that mammiferous polytype lipid, phospholipid and/or surfactant are formed.The composition of liposome is arranged in the double-decker usually, is similar to biomembranous lipid and arranges.

Term " package insert " is used in reference to the description that is included in traditionally in the commercial packing of treatment product, and it comprises relevant indication, usage, consumption, method of administration, incompatible information and/or relates to the warning that this treatment product uses.

" heart protective agent " refers to stop or weaken the chemical compound or the compositions of myocardial dysfunction (cardiomyopathy and/or congestive heart failure), described myocardial dysfunction be that patient's drug administration such as anthracene nucleus class (anthracycline) antibiotic and/or anti-ErbB antibody are relevant.Described heart protective agent can for example be blocked or weaken the cardiotoxicity effect and/or the prevention of free radical mediated or weaken the oxidative stress damage.The example of the heart protective agent that this definition comprised comprises iron chelating agent dexrazoxane (dexrazoxane) (ICRF-187) (Seifert et al., The Annals of Pharmacotherapy, 28:1063-1072 (1994)); Lipid lowering agent and/or antioxidant such as probucol (probucol) (Singalet al., J.Mol.Cell Cardiol., 27:1055-1063 (1995)); Amifostine (amineothiot 2-[(3-aminopropyl) amino] ethyl mercaptan-dihydrogen phosphoric acid ester (amifostine (aminothiol2-[(3-aminopropyl) amino] ethanethiol-dihydrogen phosphate ester), be also referred to as WR-2721 and be called the dephosphorization acid cell absorpting form of WR-1065) and S-3-(3-methyl aminopropyl amino) propyl dithiocarbamate phosphoric acid (S-3-(3-methylaminopropylamino) propylphosphorothioicacid), (WR-151327), referring to Green et al., (1994) Cancer Research, 54:738-741; Digoxin (digoxin) (Bristow, M.R.ed. (1980) Drug-Induced Heart Disease.NewYork:Elsevier 191-215); Beta-blocker such as metoprolol (metoprolol) (Hjalmarsonet al (1994) Drugs 47:Suppl 4:31-9; With Shaddy et al (1995) Am.Heart J., 129:197-9); Vitamin E; Ascorbic acid (vitamin C); Free radical scavenger such as oleanolic acid (oleanolic acid), ursolic acid (ursolic acid) and N-acetylcystein (NAC); Rotation hold back (spin trapping) chemical compound such as α-phenyl-tert-butylnitrone (alpha-phenyl-tert-butyl nitrone) (PBN) (Paracchini et al (1993) Anticancer Res., 13:1607-1612); Selenizing organic compound (selenoorganic compounds) is as P251 (Elbesen); Or the like.

" isolating " nucleic acid molecules refers to be identified and isolated nucleic acid molecule by the nucleic acid molecules of at least a pollution, and is relevant with it usually in the antibody nucleic acid of natural origin.Isolated nucleic acid molecule refers to the form of non-occurring in nature discovery or the nucleic acid molecules of composition.Therefore isolated nucleic acid molecule refers to be different from the nucleic acid molecules that exists in the n cell.Yet isolated nucleic acid molecule comprises the nucleic acid molecules that comprises in the cell of common expressing antibodies, and for example, in described cell, described nucleic acid molecules is in the chromosome position different with n cell.

" alkyl " refers to C ₁-C ₁₈Hydrocarbon is just comprising, secondary, uncle or ring carbon atom.The example of alkyl comprises C1-C8 Hydrocarbon part, such as but not limited to: methyl (Me ,-CH ₃), ethyl (Et ,-CH ₂CH ₃), 1-propyl group (n-Pr, n-propyl group ,-CH ₂CH ₂CH ₃), 2-propyl group (i-Pr, i-propyl group ,-CH (CH ₃) ₂), 1-butyl (n-Bu, n-butyl ,-CH ₂CH ₂CH ₂CH ₃), 2-methyl isophthalic acid-propyl group (i-Bu, i-butyl ,-CH ₂CH (CH ₃) ₂), 2-butyl (s-Bu, s-butyl ,-CH (CH ₃) CH ₂CH ₃), 2-methyl-2-propyl group (t-Bu, t-butyl ,-C (CH ₃) ₃), 1-amyl group (n-amyl group ,-CH ₂CH ₂CH ₂CH ₂CH ₃), 2-amyl group (CH (CH ₃) CH ₂CH ₂CH ₃), 3-amyl group (CH (CH ₂CH ₃) ₂), 2-methyl-2-butyl (C (CH ₃) ₂CH ₂CH ₃), 3-methyl-2-butyl (CH (CH ₃) CH (CH ₃) ₂), 3-methyl isophthalic acid-butyl (CH ₂CH ₂CH (CH ₃) ₂), 2-methyl-1-butene base (CH ₂CH (CH ₃) CH ₂CH ₃), 1-hexyl (CH ₂CH ₂CH ₂CH ₂CH ₂CH ₃), 2-hexyl (CH (CH ₃) CH ₂CH ₂CH ₂CH ₃), 3-hexyl (CH (CH ₂CH ₃) (CH ₂CH ₂CH ₃)), 2-methyl-2-amyl group (C (CH ₃) ₂CH ₂CH ₂CH ₃), 3-methyl-2-amyl group (CH (CH ₃) CH (CH ₃) CH ₂CH ₃), 4-methyl-2-amyl group (CH (CH ₃) CH ₂CH (CH ₃) ₂), 3-methyl-3-amyl group (C (CH ₃) (CH ₂CH ₃) ₂), 2-methyl-3-amyl group (CH (CH ₂CH ₃) CH (CH ₃) ₂), 2,3-dimethyl-2-butyl (C (CH ₃) ₂CH (CH ₃) ₂), 3,3-dimethyl-2-butyl (CH (CH ₃) C (CH ₃) ₃

" alkenyl (alkenyl) " refers to C ₂-C ₁₈Hydrocarbon is just comprising, secondary, uncle or ring carbon atom, has at least one unsaturated site, i.e. carbon-to-carbon sp ²Two keys.The example of alkenyl comprises C ₂-C ₈Hydrocarbon part is such as but not limited to ethylene or vinyl (CH=CH ₂), pi-allyl (CH ₂CH=CH ₂), cyclopentenyl (C ₅H ₇) and 5-hexenyl (CH ₂CH ₂CH ₂CH ₂CH=CH ₂).

" alkynyl (alkynyl) " refers to C ₂-C ₁₈Hydrocarbon is just comprising, secondary, uncle or ring carbon atom, has at least one unsaturated site, i.e. carbon-to-carbon sp triple bond.The example of alkynyl comprises that C2-C8 Hydrocarbon part is such as but not limited to acetylene (C ≡ CH) and propargyl (CH ₂C ≡ CH).

" alkylidene (Alkylene) " refers to saturated, side chain or the straight chain or the ring-type hydrocarbon group of 1-18 carbon atom, and having two monoradical centers, derives and obtains by remove two hydrogen atoms from the different carbon atoms of the same of parent's alkane or two in described monoradical center.Typical alkylidene comprises C ₁-C ₈Hydrocarbon part is such as but not limited to methylene (CH ₂-) 1,2-ethyl (CH ₂CH ₂-), 1,3-propyl group (CH ₂CH ₂CH ₂-), 1,4-butyl (CH ₂CH ₂CH ₂CH ₂-) or the like.

" alkylene group (alkenylene) " refers to unsaturated, side chain or the straight chain or the ring-type hydrocarbon group of 2-18 carbon atom, and having two monoradical centers, derives and obtains by remove two hydrogen atoms from the different carbon atoms of the same of parent's alkene or two in described monoradical center.Typical alkylene group comprises C ₂-C ₈Hydrocarbon part such as but not limited to: 1,2-ethylene (CH (CH-).

" alkynylene (Alkynylene) " refers to unsaturated, side chain or the straight chain or the ring-type hydrocarbon group of 2-18 carbon atom, and having two monoradical centers, derives and obtains by remove two hydrogen atoms from the different carbon atoms of the same of parent's alkynes or two in described monoradical center.Typical alkynylene comprises C ₂-C ₈Hydrocarbon part is such as but not limited to acetylene (C ≡ C-) propargyl (CH ₂C ≡ C-) and 4-pentynyl (CH ₂CH ₂CH ₂C ≡ C-).

" aryl " alone or in combination refers to the unit price aromatic hydrocarbon group of 6-20 carbon atom, and it obtains by deriving from hydrogen atom of single carbon atom removal of parent's aromatic rings system.Aryl can comprise one, two or three rings, and wherein this ring can hang (pendent) mode and be attached together, and for example biphenyl maybe can be condensed, for example naphthalene or anthracene.Some aromatic hydrocarbon groups are represented with the exemplary configurations of " Ar ".Typical aromatic hydrocarbon group comprises C ₆-C ₁₂The Hydrocarbon part is such as but not limited to the group that comes from benzene, substituted benzene, naphthalene, anthracene, biphenyl or the like.

" aralkyl (arylalkyl) " refers to the non-annularity alkyl, wherein with carbon atom, typically with end or sp ³A hydrogen atom of carbon atom bonding is replaced by aryl.Typical aralkyl includes but not limited to benzyl, 2-diphenylphosphino ethane-1-base, 2-styrene-1-base, menaphthyl, 2-naphthalene ethane-1-base, 2-naphthalene acetylene-1-base, naphthalene phenyl (naphthobenzyl), 2-naphthalene vinylbenzene-1-base (2-naphthophenylethan-1-yl) or the like.Aralkyl comprises 6 to 20 carbon atoms, and for example the moieties of aralkyl comprises alkanoyl (alkanyl), alkenyl or the alkynyl of 1 to 6 carbon atom, and aryl moiety is 5 to 14 carbon atoms.

" fragrant Heterocyclylalkyl (heteroarylalkyl) " refers to the non-annularity alkyl, wherein with carbon atom, typically with end or sp ³A hydrogen atom of carbon atom bonding is replaced by fragrant heterocycle.Typical fragrant Heterocyclylalkyl includes but not limited to 2-benzimidazole methylene (2-benzimidazolylmethyl), 2-furan ethyl or the like.The virtue Heterocyclylalkyl comprises 6 to 20 carbon atoms, and for example the moieties of fragrant Heterocyclylalkyl comprises alkanyl, alkenyl or the alkynyl of 1 to 6 carbon atom, and fragrant heterocyclic moiety is 5 to 14 carbon atoms, and 1 to 3 hetero atom is selected from N, O, P and S.The fragrant heterocyclic moiety of virtue Heterocyclylalkyl can be monocycle (2 to 6 carbon atoms) with 3 to 7 ring memberses or bicyclo-(4 to 9 carbon atoms with 7 to 10 ring memberses, with 1 to 3 hetero atom that is selected from N, O, P and S), for example: bicyclo-[4,5], [5,5], [5,6] or [6,6] system.

Alkyl, alkylene, aryl, aralkyl and fragrant Heterocyclylalkyl can be substituted, and wherein one or more hydrogen atoms are substituted base respectively independently and replace.Typical substituent group includes but not limited to-X ,-R ,-O ^-,-OR ,-SR ,-S ^-,-NR ₂,-NR ₃,=NR ,-CX ₃,-CN ,-OCN ,-SCN ,-N=C=O ,-NCS ,-NO ,-NO ₂,=N ₂,-N ₃,-NC (=O) R ,-C (=O) R ,-C (=O) NR ₂,-SO ₃ ^-,-SO ₃H ,-S (=O) ₂R ,-OS (=O) ₂OR ,-S (=O) ₂NR ,-S (=O) R ,-OP (=O) (OR) ₂,-P (=O) (OR) ₂,-PO ^- ₃,-PO ₃H ₂,-C (=O) R ,-C (=O) X ,-C (=S) R ,-CO ₂R ,-CO ₂ ^-,-C (=S) OR ,-C (=O) SR ,-C (=S) SR ,-C (=O) NR ₂,-C (=S) NR ₂,-C (=NR) NR ₂, wherein each X is halogen: F, Cl, Br or I independently; Each R is H, C independently ₁-C ₁₈Alkyl, C ₆-C ₂₀Aryl, C ₃-C ₁₄Heterocycle protecting group or prodrug moiety.Above-mentioned alkylene, alkylidene and alkynylene can be substituted too.

" fragrant heterocycle " is also referred to as heterocycle or heterocyclic radical (heterocyclyl) finger ring shape system group, and wherein one or more annular atomses are hetero atoms, for example nitrogen, oxygen and sulfur.Heteroaromatic group comprises 5 to 14 carbon atoms and 1 to 3 hetero atom that is selected from N, O, P and S.The fragrant heterocyclic moiety of virtue Heterocyclylalkyl can be monocycle with 3 to 7 ring memberses (2 to 6 carbon atoms and be selected from 1 to 3 hetero atom of N, O, P and S) or bicyclo-(4 to 9 carbon atoms with 7 to 10 ring memberses, with 1 to 3 hetero atom that is selected from N, O, P and S), for example: bicyclo-[4,5], [5,5], [5,6] or [6,6] system.Heteroaromatic compounds is described in Paquette, Leo A.; " Principles of Modern HeterocyclicChemistry " (W.A.Benjamin, New York, 1968), particularly the 1st, 3,4,6,7 and 9 chapters; " The Chemistry of Heterocyclic Compounds, A series of Monographs " (John Wiley ﹠amp; Sons, New York, 1950 so far), particularly the 13rd, 14,16,19 and 28 roll up; And J.Am.Chem.Soc. (1960) 82:5566.

" joint " or " connection " refers to comprise antibody is covalently attached to the covalent bond of drug moiety or the chemical part of atomic link.In many embodiments, joint is represented with L.Joint comprises divalent group such as alkylene, arlydene, heterocycle arlydene (heteroarylene), such as :-(CR ₂) _nO (CR ₂) _n-, alkoxyl (for example polyethylene oxygen, PEG, polymethylene oxygen) and alkylamino (for example polyethylene ammonia, Jeffamine ^TM) repetitive; And two acid esters and amide, comprise succinate, succinamide, benzilate, malonate and caproamide.

Term " chirality " molecule refers to have the molecule that the mirror image companion can not overlapping (superimposability) characteristic, and term " achiral " molecule refers to the molecule that the mirror image companion can overlapping (superimposable).

Term " stereoisomer " refers to have identical chemical composition, but on atom or group spatial arrangements different chemical compound.

" non-corresponding isomer " refers to have two or more chiral centres, and its molecule is not the stereoisomer of mirror image each other.Non-corresponding isomer has different physical characteristics, for example fusing point, boiling point, spectral characteristic and reactivity.Non-corresponding mixture of isomers can be separated under Analytical high resolution method such as electrophoresis and chromatography.

" enantiomer " refers to be two stereoisomers of the chemical compound of non-superimposable mirror image each other.

Spatial chemistry used herein definition and convention be generally according to S.P.Parker, Ed., McGraw-HillDictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; And Eliel, E.and Wilen, S., Stereochemistry of Organic Compounds (1994) JohnWiley ﹠amp; Sons, Inc., New York.Many organic compound exist with the optically active form, and promptly they have the ability of Plane of rotation polarized light flat.In describing activity of optically active compounds, prefix D and L or R and S are used to represent the absolute configuration of the molecule of relevant its chiral centre.Prefix d and l or (+) and (-) are used to indicate the direction of rotation of the linearly polarized light that is caused by described chemical compound, and (-) or l refer to that chemical compound is left-handed.The chemical compound of prefixing (+) or d is dextral.For given chemical constitution, except they are that these stereoisomers are identical the mirror image each other.Particular stereoisomer also can be described as enantiomer, and this mixture of isomers is commonly referred to mixture of enantiomers.50: 50 mixture of enantiomers is called racemic mixture or racemate, and it can never take place in stereo selectivity or stereospecific chemical reaction or the process.Term " racemic mixture " and " racemate " refer to the molar mixture that waits of two class enantiomer, and it lacks optically active.

Phrase used herein " pharmaceutically acceptable salt, " refers to the pharmaceutically acceptable organic or inorganic salt of ADC.Exemplary salt includes but not limited to sulphuric acid, citric acid, acetic acid, oxalic acid, chloride, bromide, iodide, nitric acid, bisulfate, phosphoric acid, acid phosphoric acid, the .gamma.-pyridinecarboxylic acid, lactic acid, salicylic acid, acid citric acid, tartaric acid, oleic acid, tannic acid, pantothenic acid, the Tartaric acid hydrogen salt, ascorbic acid, succinic acid, maleic acid, gentisinate, Fumaric acid, gluconic acid, glucuronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethyl sulfonic acid, benzenesulfonic acid, p-methyl benzenesulfonic acid, and pamoic acid (promptly 1,1 '-methylene-two-(2-hydroxyl-3-naphthoic acid)) salt.Pharmaceutically acceptable salt can relate to and comprise another kind of molecule such as acetic acid ion, succinic acid ion or other gegenion.Described gegenion can be any organic or inorganic part of the electric charge on the stable matrix chemical compound.In addition, pharmaceutically acceptable salt can have the charge atom of surpassing in its structure.Wherein the multi-charge atom is that the example of the part of pharmaceutically-acceptable salts may have a plurality of gegenions.Therefore, pharmaceutically acceptable salt may have one or more charge atoms and/or one or more gegenion.

" pharmaceutically acceptable solvate " refers to combining of one or more solvent molecules and ADC.The example that forms the solvent of pharmaceutically acceptable solvate includes but not limited to water, isopropyl alcohol, ethanol, methanol, DMSO, ethyl acetate, acetic acid and ethanolamine.

Antibody-drug conjugates

Chemical compound of the present invention comprises those chemical compounds with active anticancer effectiveness.Particularly, described chemical compound comprises the link coupled antibody that promptly is covalently attached to drug moiety by joint, and wherein said medicine has cytotoxicity or cell depression effect when not being coupled to antibody.Like this, adjusted by the biologic activity that is coupled to the described drug moiety of antibody.Antibody-drug conjugates of the present invention (ADC) can be optionally be delivered to tumor tissues with the cytotoxic reagent of effective dose, can obtain stronger selectivity thus, promptly can reach the effect of expectation than low dosage.

In one embodiment, compare with corresponding maytansinoid chemical compound is only arranged, the bioavailability of born of the same parents' intracellular metabolite thing of ADC or ADC improves in the mammal.Simultaneously, compare with corresponding antibody (antibody of ADC, no drug moiety or joint) is only arranged, the bioavailability of born of the same parents' intracellular metabolite thing of ADC or ADC improves in the mammal.

In one embodiment, just from the antibody cracking, wherein said cell has the special cell surface receptor of antibody of antibody-drug conjugates to the maytansinoid drug moiety of ADC when antibody-drug conjugates is attached to cell surface receptor or enters cell.After antibody-drug conjugates entered described cell, drug moiety can be from described antibody cracking.In mammalian body, the maytansinoid drug moiety can be by zymetology effect, hydrolysis, oxidation or other mechanism, in cell from the antibody cracking of born of the same parents' intracellular metabolite thing of described chemical compound or described chemical compound.For example, never mean the present invention is limited to the specific mechanism of action that the sulphur atom of the maytansinoid drug moiety of ADC can be oxidized to sulfone or sulfoxide radicals simultaneously.In cell, the proton that is attached on the carbon of sulfone and sulfoxide can removed under general catalytic action or zymetology catalytic action, and causes that beta-eliminates cataclasis, and this cataclasis is from antibody cracking and the separate drug part of ADC.Perhaps, other electron withdraw group in the joint such as amide, antibody or drug moiety can cause similarly cracked/cracking mechanism in cell.

Antibody-drug conjugates (ADC) can be represented with formula I:

Ab-(L-D) _p I

Or its pharmaceutically acceptable salt or solvate, wherein:

Ab is in conjunction with the ErbB receptor, and perhaps in conjunction with the antibody of one or more tumor associated antigens or cell surface receptor, described cell surface receptor is selected from (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor (bone morphogenetic proteinreceptor)-IB type, Genbank accession number NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

(3) STEAP1 (prostate is striden film epithelium antigen (six transmembrane epithelialantigen), Genbank accession number NM_012449 for six times);

(4) 0772P (CA125, MUC16, Genbank accession number AF361486);

(5) MPF (MPF, MSLN, SMR, megalokaryocyte intensifier (megakaryocytepotentiating factor), mesothelin, Genbank accession number NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family (solute carrierfamily) 34 (sodium phosphates), the member 2, phosphate cotransporter albumen (sodium-dependentphosphate transporter) 3b that II type sodium relies on, Genbank accession number NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, brain signal albumen (Semaphorin) 5b Hlog, the sema domain, seven thrombospondins repeat (seven thrombospondin repeats) (1 type and class 1 type), membrane spaning domain (TM) and short Cytoplasm domain, (brain signal albumen) 5B, Genbank accession number AB040878);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);

(9) ETBR (Endothelin (Endothelin) Type B receptor, Genbank accession number AY275463);

(10) MSG783 (RNF124, imaginary albumen (hypothetical protein) FLJ20315, Genbank accession number NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, relating to prostate cancers because of 1, the carcinoma of prostate associated protein 1, prostaticly stride film epithelium antigen 2 for six times, stride film prostatein (six transmembrane prostate protein), Genbank accession number AF455138 six times);

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, the potential cationic channel of transient receptor (transient receptor potential cation channel), subfamily M (subfamily M), member 4 (member 4), Genbank accession number NM_017636);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma derivative growth factor (teratocarcinoma-derived growth factor), Genbank accession number NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/ dust crust virus (Epstein Barr virus) receptor) or Hs.73792 Genbank accession number M26004);

(15) CD79b (CD79B, CD79 β, IGb (immunoglobulin be correlated with beta (immuneoglobulin-associated beta), B29, Genbank accession number NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (phosphatase anchorin (the phosphatase anchor protein) 1a that contains the SH2 domain), SPAP1B, SPAP1C, Genbank accession number NM_030764);

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20R α (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22) EphB2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763;

(26) BAFF-R (B cell activation factor receptor, BlyS receptor 3, BR3, NP_443177.1);

(27) CD22 (B-cell receptor CD22-B isotype (isoform), NP-001762.1);

(28) CD79a (CD79A, CD79 α, the relevant alpha (immuneoglobulin-associated alpha) of immunoglobulin, also form complex with Ig M molecule from the teeth outwards with Ig beta (CD79B) covalent interaction, transduction relates to the B cell-specific proteins of the signal of B cell differentiation, Genbank accession number NP_001774.1);

(29) CXCR5 (Bai Jiteshi (the lymphoma receptor 1 of Burkitt ' s), by the activatory g protein coupled receptor of CXCL13 chemotactic factor, in lymphocyte migration and humoral defense, play a role, infect and may in AIDS, lymphoma, myeloma and leukemia, play a role Genbank accession number NP_001707.1 at HIV-2);

(30) HLA-DOB (the Beta subunit of MHC II quasi-molecule (Ia antigen), its binding peptide also is presented to the CD4+T lymphocyte, Genbank accession number NP_002111.1 with it);

(31) P2X5 (purinoceptor (purinergic receptor) P2X ligand-gated ion channel (ligand-gated ion channel) 5, ion channel by the outer ATP gate of born of the same parents, may relate to synapse transmission and neural newborn, its defective may cause IDI's Pathophysiology situation (pathophysilolgy of idiopathic detrusor instability), Genbank accession number NP_002552.2);

(32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

(33) LY64 (lymphocyte antigen 64 (RP105), be rich in the multiple I type of leucine memebrane protein (LRR) family, regulate B cell activation and apoptosis, the forfeiture of function is relevant with the enhancing of Patients with SLE disease activity, Genbank accession number NP_005573.1);

(34) FcRH1 (Fc receptor-like protein 1, the immunoglobulin Fc domain receptor of inferring comprise C2 type Ig sample and ITAM domain, may work Genbank accession number NP_443170.1 in the bone-marrow-derived lymphocyte differentiation);

(35) (transposition (translocation) related immune globulin superfamily receptors 2, the immunity receptor of inferring may work in B cell development and lymphoma generation IRTA2; The gene imbalance that is caused by transposition betides in some B cell malignant diseases Genbank accession number NP_112571.1); And

(36) (the transmembrane protein polysaccharide of inferring transfers albumen (heregulin) family relevant with follistatin (follistatin) with the EGF/ of somatomedin, Genbank accession number AF179274 to TENB2;

Condition is that described antibody is not TA.1.

L is non-disulphide joint.L includes but not limited to structure:

Wherein wave is represented covalently bound with Ab and D;

X is:

R is H or C independently ₁-C ₆Alkyl; And n is 1 to 12;

D is the maytansinoid drug moiety.Maytansinoid includes but not limited to structure:

Wherein wave is represented covalently bound with L;

R is H or C independently ₁-C ₆Alkyl; And

M is 1,2 or 3.

The ratio of medicine and antibody or drug loading are represented with the p of formula I chemical compound.Drug loading value p is 1 to 8.Formula I chemical compound comprises all mixture of the antibody-drug conjugates of different loads and connection, and wherein 1,2,3,4,5,6,7 and 8 drug moiety is covalently attached to described antibody.

In another embodiment, Ab is the antibody in conjunction with one or more tumor associated antigens or cell surface receptor, and described tumor associated antigen or cell surface receptor are selected from (1)-(16) and (18)-(36), promptly not in conjunction with the ErbB receptor, comprise HER2.

Antibody

In its scope, the antibody unit of formula I (Ab-) comprises and receptor, antigen or the combination of other acceptance part or reactive relevant or compound any antibody unit that described receptor, antigen or other acceptance part are relevant with given cell population of interest.Antibody can be any albumen or albumen sample molecule, and it combines with the cell colony part that want therapeutics or biology modify, compound or reaction.An aspect, antibody unit play a role and the maytansinoid drug moiety are delivered to the specific target cells group of antibody unit and its reaction.Such antibody includes but not limited to large molecular weight protein such as full length antibody and antibody fragment.

The antibody that comprises antibody-drug conjugates of the present invention keeps preferably that it is natural, the antigen binding capacity of wild type homologue.So, antibody of the present invention can conjugated antigen, preferably combination specifically.Such antigen comprises for example tumor associated antigen (TAA), cell surface receptor protein and other cell surface molecule, cell survival regulatory factor, cell proliferation regulatory factor, relevant (for example with tissue development or differentiation, known or suspect on function, help tissue development or differentiation) molecule, lymphokine, cytokine, relate to Cycle Regulation molecule, relate to molecule that blood vessel takes place and relevant with angiogenesis (for example, known or suspect on function, help the blood vessel generation) molecule.Tumor associated antigen can be the differentiation bunch factor (that is CD albumen).Antibody of the present invention can antigen bonded with it can be the member of the subgroup of one of above-mentioned classification, and other subgroup of wherein said classification comprises have distinguishing characteristics other molecule/antigen of (for interested antigen).

In one embodiment, the antibody of antibody-drug conjugates (ADC) is specifically in conjunction with the receptor by the ErbB gene code.Described antibody can be specifically in conjunction with the ErbB receptor that is selected from EGFR, HER2, HER3 and HER4.ADC can be specifically in conjunction with the ectodomain (ECD) of HER2 and suppressed to express the growth of the tumor cell of HER2 receptor.The antibody of ADC can be monoclonal antibody, for example mouse monoclonal antibody, chimeric antibody or humanized antibody.Humanized antibody can be huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 or huMAb4D5-8 (trastuzumab).Described antibody can be antibody fragment, for example the Fab fragment.

In the antibody-drug conjugates of formula I (ADC) and antibody that can be used in treatment of cancer includes but not limited to the antibody of anti-cell surface receptor and tumor associated antigen (TAA).Such tumor associated antigen is known in the art, can prepare to be used to produce antibody with method well known in the art and information.Be used for the effort of the effective cell target thing of cancer diagnosis and treatment in searching, researcher has been managed to have identified and has been striden film or tumor relative polypeptide, compare with one or more normal non-cancerous cell, these polypeptide are expressed on the surface of one or more particular type cancerous cell specifically.Usually, and compare on the surface of non-cancerous cell, these tumor relative polypeptides are expressed on the cancerous cell surface more.Identify that these tumor relevant cell surface antigen polypeptide have produced by the target cancer cell specifically based on Antybody therapy and carry out destructive ability.

The example of TAA includes but not limited to following listed tumor associated antigen (1)-(36).For simplicity, below list relevant these antigenic all information known in the art, comprise title, substitute title, Genbank accession number and original reference, follow the nucleic acid and the protein sequence of NCBI (NCBI) and differentiate convention.Nucleic acid and protein sequence corresponding to TAA (1)-(36) can obtain in public database such as GenBank.The tumor associated antigen of antibody target comprises with respect to the sequence of identifying in the cited references, have all aminoacid sequence variant and isotypes at least about 70%, 80%, 85%, 90% or 95% sequence homogeneity, perhaps show the biological characteristics identical with TAA or all aminoacid sequence variant and isotypes of feature basically, wherein said TAA has the sequence of finding in the cited references.For example, have usually the TAA of the variant sequence of binding antibody specifically, described antibody is specifically in conjunction with the TAA with listed corresponding sequence.Sequence in the list of references that this paper quotes especially and content are special to add as a reference.

Tumor associated antigen (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-IB type, Genbank accession number NM_001203)

Ten Dijke, P., et al Science 264 (5155): 101-104 (1994), Oncogene14 (11): 1377-1382 (1997)); WO2004063362 (claim 2); WO2003042661 (claim 12); US2003134790-A1 (38-39 page or leaf); WO2002102235 (claim 13; The 296th page); WO2003055443 (91-92 page or leaf); (embodiment 2 for WO200299122; The 528-530 page or leaf); WO2003029421 (claim 6); WO2003024392 (claim 2; Figure 112); WO200298358 (claim 1; The 183rd page); WO200254940 (100-101 page or leaf); WO200259377 (349-350 page or leaf); WO200230268 (claim 27; The 376th page); WO200148204 (embodiment; Fig. 4)

NP_001194 bone morphogenetic protein receptor, IB/pid type=NP_001194.1-

Cross reference: MIM:603248; NP_001194.1; AY065994

(2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486)

Biochem.Biophys.Res.Commun.255 (2), 283-288 (1999), Nature395 (6699): 288-291 (1998), Gaugitsch, H.W., et al (1992) J.Biol.Chem.267 (16): 11267-11273); WO2004048938 (embodiment 2); WO2004032842 (EXAMPLE IV); WO2003042661 (claim 12); WO2003016475 (claim 1); WO200278524 (embodiment 2); WO200299074 (claim 19; The 127-129 page or leaf); WO200286443 (claim 27; 222nd, 393 pages); WO2003003906 (claim 10; The 293rd page); WO200264798 (claim 33; The 93-95 page or leaf); WO200014228 (claim 5; The 133-136 page or leaf); US2003224454 (Fig. 3); WO2003025138 (claim 12; The 150th page); US 20050107595; US 20050106644;

NP_003477 solute carrier family 7 (day ion amino acid transport protein, y+ system), member 5/pid=NP_003477.3-people (Homo Sapiens) cross reference: MIM:600182; NP_003477.3; NM_015923; NM_003486_1

(3) STEAP1 (prostatic six times stride the film epithelium antigen, Genbank accession number NM_012449)

Cancer Res.61 (15), 5857-5860 (2001), Hubert, R.S., et al (1999) Proc.Natl.Acad.Sci.U.S.A.96 (25): 14523-14528); WO2004065577 (claim 6); WO2004027049 (Fig. 1 L); EP1394274 (embodiment 11); WO2004016225 (claim 2); WO2003042661 (claim 12); US2003157089 (embodiment 5); US2003185830 (embodiment 5); US2003064397 (Fig. 2); (embodiment 5 for WO200289747; The 618-619 page or leaf); (embodiment 9 for WO2003022995; Figure 13 A, embodiment 53; The 173rd page, embodiment 2; Fig. 2 A);

NP_036581 strides the film epithelium antigen prostatic six times

Cross reference: MIM:604415; NP_036581.1; NM_012449_1

(4) 0772P (CA125, MUC16, Genbank accession number AF361486)

J.Biol.Chem.276 (29): 27371-27375 (2001)); WO2004045553 (claim 14); WO200292836 (claim 6; Figure 12); WO200283866 (claim 15; The 116-121); US2003124140 (embodiment 16); US2003091580 (claim 6); WO200206317 (claim 6; The 400-408 page or leaf);

Cross reference: GI:34501467; AAK74120.3; AF361486_1

(5) MPF (MPF, MSLN, SMR, megalokaryocyte intensifier, mesothelin, Genbank accession number NM_005823)

Yamaguchi, N., et al Biol.Chem.269 (2), 805-808 (1994), Proc.Natl.Acad.Sci.U.S.A.96 (20): 11531-11536 (1999), Proc.Natl.Acad.Sci.U.S.A.93 (1): 136-140 (1996), J.Biol.Chem.270 (37): 21984-21990 (1995)); WO2003101283 (claim 14); (WO2002102235 (claim 13; The 287-288 page or leaf); WO2002101075 (claim 4; The 308-309 page or leaf); WO200271928 (320-321 page or leaf); WO9410312 (52-57 page or leaf);

Cross reference: MIM:601051; NP_005814.2; NM_005823_1

(6) Napi3b (member 2 for NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), the phosphoric acid transport protein 3b that II type sodium relies on, genbank accession number NM_006424)

J.Biol.Chem.277 (22): 19665-19672 (2002), Genomics62 (2): 281-284 (1999), Feild, J.A., et al (1999) Biochem.Biophys.Res.Commun.258 (3): 578-582); WO2004022778 (claim 2); EP1394274 (embodiment 11); WO2002102235 (claim 13; The 326th page); EP875569 (claim 1; The 17-19 page or leaf); WO200157188 (claim 20; The 329th page); WO2004032842 (EXAMPLE IV); WO200175177 (claim 24; The 139-140 page or leaf);

Cross reference: MIM:604217; NP_006415.1; NM_006424_1

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, brain signal albumen (semaphorin) 5b Hlog, the sema domain, seven thrombospondins repeat (1 type and class 1 type), membrane spaning domain (TM) and short Cytoplasm domain, (brain signal albumen) 5B, Genbank accession number AB040878)

Nagase T., et al (2000) DNA Res.7 (2): 143-150); WO2004000997 (claim 1); WO2003003984 (claim 1); WO200206339 (claim 1; The 50th page); WO200188133 (claim 1; 41-43, the 48-58 page or leaf); WO2003054152 (claim 20); WO2003101400 (claim 11);

Accession number: Q9P283; EMBL; AB040878; BAA95969.1.Genew; HGNC:10737;

(8) PSCAhlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628); Ross et al (2002) Cancer Res.62:2546-2553; US2003129192 (claim 2); US2004044180 (claim 12); US2004044179 (claim 11); US2003096961 (claim 11); US2003232056 (embodiment 5); WO2003105758 (claim 12); US2003206918 (embodiment 5); EP1347046 (claim 1); WO2003025148 (claim 20);

Cross reference: GI:37182378; AAQ88991.1; AY358628_1

(9) ETBR (endothelin receptor B, Genbank accession number AY275463);

Nakamuta M., et al Biochem.Biophys.Res.Commun.177,34-39,1991; Ogawa Y., et al Biochem.Biophys.Res.Commun.178,248-255,1991; Arai H., et al Jpn.Circ.J.56,1303-1307,1992; Arai H., et al J.Biol.Chem.268,3463-3470,1993; Sakamoto A., Yanagisawa M., et al Biochem.Biophys.Res.Commun.178,656-663,1991; Elshourbagy N.A., et al J.Biol.Chem.268,3873-3879,1993; Haendler B., et al J.Cardiovasc.Pharmacol.20, s1-S4,1992; Tsutsumi M., et al Gene 228,43-49,1999; Strausberg R.L., et al Proc.Natl.Acad.Sci.U.S.A.99,16899-16903,2002; Bourgeois C., et al J.Clin.Endocrinol.Metab.82,3116-3123,1997; Okamoto Y, et al Biol.Chem.272,21589-21596,1997; Verheij J.B., et al Am.J.Med.Genet.108,223-225,2002; Hofstra R.M.W., et al Eur.J.Hum.Genet.5,180-185,1997; Puffenberger E.G, et al Cell 79,1257-1266,1994; Attie T., et al, Hum.Mol.Genet.4,2407-2409,1995; Auricchio A., et al Hum.Mol.Genet.5:351-354,1996; Amiel J., et al Hum.Mol.Genet.5,355-357,1996; Hofstra R.M.W., et al Nat.Genet.12,445-447,1996; Svensson P.J., et al Hum.Genet.103,145-148,1998; Fuchs S., et al Mol.Med.7,115-124,2001; Pingault V., et al (2002) Hum.Genet.111,198-206; WO2004045516 (claim 1); WO2004048938 (embodiment 2); WO2004040000 (claim 151); WO2003087768 (claim 1); WO2003016475 (claim 1); WO2003016475 (claim 1); WO200261087 (Fig. 1); WO2003016494 (Fig. 6); WO2003025138 (claim 12; The 144th page); WO200198351 (claim 1; The 124-125 page or leaf); EP522868 (claim 8; Fig. 2); WO200177172 (claim 1; The 297-299 page or leaf); US2003109676; US6518404 (Fig. 3); US5773223 (claim 1a; The 31-34 hurdle); WO2004001004;

(10) MSG783 (RNF124, imaginary albumen (hypothetical protein) FLJ20315, Genbank accession number NM_017763);

WO2003104275 (claim 1); WO2004046342 (embodiment 2); WO2003042661 (claim 12); WO2003083074 (claim 14; Page 61); WO2003018621 (claim 1); WO2003024392 (claim 2; Figure 93); WO200166689 (embodiment 6);

Cross reference: LocusID:54894; NP_060233.2; NM_017763_1

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, relating to prostate cancers is because of 1, and the carcinoma of prostate associated protein 1 is striden film epithelium antigen 2 prostatic six times, stride the film prostatein six times, Genbank accession number AF455138)

Lab.Invest.82 (11): 1573-1582 (2002)); WO2003087306; US2003064397 (claim 1; Fig. 1); WO200272596 (claim 13; The 54-55 page or leaf); WO200172962 (claim 1; Fig. 4 B); WO2003104270 (claim 11); WO2003104270 (claim 16); US2004005598 (claim 22); WO2003042661 (claim 12); US2003060612 (claim 12; Figure 10); WO200226822 (claim 23; Fig. 2); WO200216429 (claim 12; Figure 10);

Cross reference: GI:22655488; AAN04080.1; AF455138_1

(12) TrpM4 (subfamily M, the member 4, Genbank accession number NM_017636 for BR22450, FLJ20041, TRPM4, TRPM4B, the potential cationic channel of transient receptor)

Xu, X.Z., et al Proc.Natl.Acad.Sci.U.S.A.98 (19): 10692-10697 (2001), Cell 109 (3): 397-407 (2002), J.Biol.Chem.278 (33): 30813-30820 (2003)); US2003143557 (claim 4); WO200040614 (claim 14; The 100-103 page or leaf); W0200210382 (claim 1; Fig. 9 A); WO2003042661 (claim 12); WO200230268 (claim 27; The 391st page); US2003219806 (claim 4); WO200162794 (claim 14; Figure 1A-D);

Cross reference: MIM:606936; NP_060106.2; NM_017636_1

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma derivative growth factor, Genbank accession number NP_003203 or NM_003212)

Ciccodicola, A., et al EMBO is (7): 1987-1991 (1989) J.8, Am.J.Hum.Genet.49 (3): 555-565 (1991)); US2003224411 (claim 1); WO2003083041 (embodiment 1); WO2003034984 (claim 12); WO200288170 (claim 2; The 52-53 page or leaf); WO2003024392 (claim 2; Figure 58); WO200216413 (claim 1; 94-95,105 pages); WO200222808 (claim 2; Fig. 1); (embodiment 2 for US5854399; The 17-18 hurdle); US5792616 (Fig. 2);

Cross reference: MIM:187395; NP_003203.1; NM_003212_1.

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/ dust crust virus receptor) or Hs.73792Genbank accession number M26004)

Fujisaku et al (1989) J.Biol.Chem.264 (4): 2118-2125); Weis J.J., et al J.Exp.Med.167,1047-1066,1988; Moore M., et al Proc.Natl.Acad.Sci.U.S.A.84,9194-9198,1987; Barel M., et al Mol.Immunol.35,1025-1031,1998; Weis J.J., et al Proc.Natl.Acad.Sci.U.S.A.83,5639-5643,1986; Sinha S.K., et al (1993) J.Immunol.150,5311-5320; WO2004045520 (embodiment 4); US2004005538 (embodiment 1); WO2003062401 (claim 9); WO2004045520 (embodiment 4); WO9102536 (Fig. 9 .1-9.9); WO2004020595 (claim 1);

Accession number: P20023; Q13866; Q14212; EMBL; M26004; AAA35786.1.

(15) CD79b (CD79B, CD79 β, IGb (immunoglobulin be correlated with beta), B29, Genbank accession number NM_000626 or 11038674)

Proc.Natl.Acad.Sci.U.S.A. (2003) 100 (7): 4126-4131, Blood (2002) 100 (9): 3068-3076, Muller et al (1992) Eur.J.Immunol.22 (6): 1621-1625); WO2004016225 (claim 2, Figure 140); WO2003087768, US2004101874 (claim 1, the 102nd page); WO2003062401 (claim 9); WO200278524 (embodiment 2); US2002150573 (claim 5, the 15th page); US5644033; WO2003048202 (claim 1, the 306th and 309 page); WO 99/558658, US6534482 (claim 13, Figure 17 A/B); WO200055351 (claim 11,1145-1146 page or leaf);

Cross reference: MIM:147245; NP_000617.1; NM_000626_1

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (the phosphatase anchorin 1a that contains the SH2 domain), SPAP1B, SPAP1C, Genbank accession number NM_030764, AY358130)

Genome Res.13 (10): 2265-2270 (2003), Immunogenetics 54 (2): 87-95 (2002), Blood 99 (8): 2662-2669 (2002), Proc.Natl.Acad.Sci.U.S.A.98 (17): 9772-9777 (2001), Xu, M.J., et al (2001) Biochem.Biophys.Res.Commun.280 (3): 768-775; WO2004016225 (claim 2); WO2003077836; WO200138490 (claim 5; Figure 18 D-1-18D-2); WO2003097803 (claim 12); WO2003089624 (claim 25);

Cross reference: MIM:606509; NP_110391.2; NM_030764_1

(17) HER2 (ErbB2, Genbank accession number M11730)

Coussens L., et al Science (1985) 230 (4730): 1132-1139); Yamamoto T., etal Nature 319,230-234,1986; Semba K., et al Proc.Natl.Acad.Sci.U.S.A.82,6497-6501,1985; Swiercz J.M., et al J.Cell Biol.165,869-880,2004; KuhnsJJ., et al J.Biol.Chem.274,36422-36427,1999; Cho H.-S., et al Nature 421,756-760,2003; Ehsani A., et al (1993) Genomics 15,426-429; WO2004048938 (embodiment 2); WO2004027049 (Fig. 1 I); WO2004009622; WO2003081210; WO2003089904 (claim 9); WO2003016475 (claim 1); US2003118592; WO2003008537 (claim 1); WO2003055439 (claim 29; Figure 1A-B); WO2003025228 (claim 37; Fig. 5 C); (embodiment 13 for WO200222636; The 95-107 page or leaf); WO200212341 (claim 68; Fig. 7); WO200213847 (the 71-74); WO200214503 (114-117 page or leaf); WO200153463 (claim 2; The 41-46 page or leaf); WO200141787 (the 15th page); WO200044899 (claim 52; Fig. 7); WO200020579 (claim 3; Fig. 2); US5869445 (claim 3; The 1-38 hurdle); WO9630514 (claim 2; The 56-61 page or leaf); EP1439393 (claim 7); WO2004043361 (claim 7); WO2004022709; (embodiment 3 for WO200100244; Fig. 4);

Accession number: P04626; EMBL; M11767; AAA35808.1.EMBL; M11761; AAA35808.1

(18) NCA (CEACAM6, Genbank accession number M18728);

Barnett T., et al Genomics 3,59-66,1988; Tawaragi Y, et al Biochem.Biophys.Res.Commun.150,89-96,1988; Strausberg R.L., et al Proc.Natl.Acad.Sci.U.S.A.99:16899-16903,2002; WO2004063709; EP1439393 (claim 7); WO2004044178 (embodiment 4); WO2004031238; WO2003042661 (claim 12); WO200278524 (embodiment 2); WO200286443 (claim 27; The 427th page); WO200260317 (claim 2);

Accession number: P40199; Q14920; EMBL; M29541; AAA59915.1.EMBL; M18728;

(19) MDP (DPEP1, Genbank accession number BC017023)

Proc.Natl.Acad.Sci.U.S.A.99 (26): 16899-16903 (2002)); WO2003016475 (claim 1); WO200264798 (claim 33; The 85-87); JP05003790 (Fig. 6-8); WO9946284 (Fig. 9);

Cross reference: MIM:179780; AAH17023.1; BC017023_1

(20) IL20R α (IL20Ra, ZCYTOR7, Genbank accession number AF184971);

Clark H.F., et al Genome Res.13,2265-2270,2003; Mungall A.J., et alNature 425,805-811,2003; Blumberg H., et al Cell 104,9-19,2001; Dumoutier L., et al J.Immunol.167,3545-3549,2001; Parrish-Novak J., et al J.Biol.Chem.277,47517-47523,2002; Pletnev S., et al (2003) Biochemistry42:12617-12624; Sheikh F., et al (2004) J.Immunol.172,2006-2010; EP1394274 (embodiment 11); US2004005320 (embodiment 5); WO2003029262 (74-75 page or leaf); WO2003002717 (claim 2; The 63rd page); WO200222153 (45-47 page or leaf); US2002042366 (20-21 page or leaf); WO200146261 (57-59 page or leaf); WO200146232 (63-65 page or leaf); WO9837193 (claim 1; The 55-59 page or leaf);

Accession number: Q9UHF4; Q6UWA9; Q96SH8; EMBL; AF184971; AAF01320.1.

(21) Brevican (BCAN, BEHAB, Genbank accession number AF229053)

Gary S.C., et al Gene 256,139-147,2000; Clark H.F., et al Genome Res.13,2265-2270,2003; Strausberg R.L., et al Proc.Natl.Acad.Sci.U.S.A.99,16899-16903,2002; US2003186372 (claim 11); US2003186373 (claim 11); US2003119131 (claim 1; Figure 52); US2003119122 (claim 1; Figure 52); US2003119126 (claim 1); US2003119121 (claim 1; Figure 52); US2003119129 (claim 1); US2003119130 (claim 1); US2003119128 (claim 1; Figure 52); US2003119125 (claim 1); WO2003016475 (claim 1); WO200202634 (claim 1);

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5, Genbank accession number NM_004442)

Chan, J.and Watt, V.M., Oncogene 6 (6), 1057-1061 (1991) Oncogene10 (5): 897-905 (1995), Annu.Rev.Neurosci.21:309-345 (1998), Int.Rev.Cytol.196:177-244 (2000)); WO2003042661 (claim 12); WO200053216 (claim 1; The 41st page); WO2004065576 (claim 1); WO2004020583 (claim 9); WO2003004529 (128-132 page or leaf); WO200053216 (claim 1; The 42nd page);

Cross reference: MIM:600997; NP_004433.2; NM_004442_1

(23) ASLG659 (B7h, Genbank accession number AX092328)

US20040101899 (claim 2); WO2003104399 (claim 11); WO2004000221 (Fig. 3); US2003165504 (claim 1); US2003124140 (embodiment 2); US2003065143 (Figure 60); WO2002102235 (claim 13; The 299th page); US2003091580 (embodiment 2); WO200210187 (claim 6; Figure 10); WO200194641 (claim 12; Fig. 7 b); WO200202624 (claim 13; Figure 1A-1B); US2002034749 (claim 54; The 45-46 page or leaf); (embodiment 2 for WO200206317; The 320-321 page or leaf, claim 34; The 321-322 page or leaf); WO200271928 (468-469 page or leaf); (embodiment 1 for WO200202587; Fig. 1); (embodiment 3 for WO200140269; Pages 190-192); (embodiment 2 for WO200036107; The 205-207); WO2004053079 (claim 12); WO2003004989 (claim 1); WO200271928 (233-234,452-453 page or leaf); WO0116318;

(24) PSCA (prostate stem cell antigen precursor, Genbank accession number AJ297436)

Reiter R.E., et al Proc.Natl.Acad.Sci.U.S.A.95,1735-1740,1998; Gu Z., et al Oncogene 19,1288-1296,2000; Biochem.Biophys.Res.Commun. (2000) 275 (3): 783-788; WO2004022709; EP1394274 (embodiment 11); US2004018553 (claim 17); WO2003008537 (claim 1); WO200281646 (claim 1; The 164th page); WO2003003906 (claim 10; The 288th page); (embodiment 1 for WO200140309; Figure 17); (embodiment 1 for US2001055751; Fig. 1 b); WO200032752 (claim 18; Fig. 1); WO9851805 (claim 17; The 97th page); WO9851824 (claim 10; The 94th page); WO9840403 (claim 2; Figure 1B);

Accession number: O43653; EMBL; AF043498; AAC39607.1.

(25) GEDA (Genbank accession number AY260763);

AAP14954 lipoma (lipoma) HMGIC fusion partner sample albumen/pid=AAP14954.1-people

Species: homo sapiens (people)

WO2003054152 (claim 20); WO2003000842 (claim 1);

WO2003023013 (embodiment 3, claim 20); US2003194704 (claim 45);

Cross reference: GI:30102449; AAP14954.1; AY260763_1

(26) BAFF-R (B cell activation factor receptor, BlyS receptor 3, BR3, Genbank accession number: AF116456); BAFF receptor/pid=NP_443177.1-homo sapiens Thompson, J.S., et al Science 293 (5537), 2108-2111 (2001); WO2004058309; WO2004011611; WO2003045422 (embodiment; The 32-33 page or leaf); WO2003014294 (claim 35; Fig. 6 B); WO2003035846 (claim 70; The 615-616 page or leaf); WO200294852 (136-137 hurdle); WO200238766 (claim 3; The 133rd page); (embodiment 3 for WO200224909; Fig. 3);

Cross reference: MIM:606269; NP_443177.1; NM_052945_1; AF132600

(27) CD22 (B-cell receptor CD22-B isotype, BL-CAM, Lyb-8, Lyb8, SIGLEC-2, FLJ22814, Genbank accession number: AK026467);

Wilson et al (1991) J.Exp.Med.173:137-146; WO2003072036 (claim 1; Fig. 1);

Cross reference: MIM:107266; NP_001762.1; NM_001771_1

(28) CD79a (CD79A, CD79 α, the alpha that immunoglobulin is relevant, with Igbeta (CD79B) covalent interaction, form complex from the teeth outwards with Ig M molecule, transduction relates to the B cell-specific proteins of the signal of B cell differentiation) PROTEIN SEQUENCE Fullmpggpgv...dvqlekp (1..226; 226aa), pI:4.84, MW:25028TM:2[P] GeneChromosome:19q13.2, Genbank accession number: NP_001774.10)

WO2003088808, US20030228319; WO2003062401 (claim 9); US2002150573 (claim 4,13-14 page or leaf); WO9958658 (claim 13, Figure 16); WO9207574 (Fig. 1); US5644033; Ha et al (1992) J.Immunol.148 (5): 1526-1531; Mueller et al (1992) Eur.J.Biochem.22:1621-1625; Hashimoto et al (1994) Immunogenetics 40 (4): 287-295; Preud ' homme etal (1992) Clin.Exp.Immunol.90 (1): 141-146; Yu et al (1992) J.Immunol.148 (2) 633-637; Sakaguchi et al (1988) EMBO is (11): 3457-3464 J.7;

(29) CXCR5 (Burkitt lymphoma receptor 1, by the activatory g protein coupled receptor of CXCL13 chemotactic factor, in lymphocyte migration and humoral defense, play a role, infect and perhaps in AIDS, lymphoma, myeloma and leukemia generation, work at HIV-2) PROTEINSEQUENCE Full mnypltl...atslttf (1..372; 372aa), pI:8.54MW:41959TM:7[P] Gene Chromosome:11q23.3, Genbank accession number: NP_001707.1)

WO2004040000; WO2004015426; US2003105292 (embodiment 2); US6555339 (embodiment 2); WO200261087 (Fig. 1); WO200157188 (claim 20, the 269th page); WO200172830 (12-13 page or leaf); WO200022129 (embodiment 1, the 152-153 page or leaf, embodiment 2, the 254-256 pages or leaves); WO9928468 (claim 1, the 38th page); US5440021 (embodiment 2, the 49-52 hurdles); WO9428931 (56-58 page or leaf); WO9217497 (claim 7, Fig. 5); Dobner et al (1992) Eur.J.Immunol.22:2795-2799; Barella et al (1995) Biochem.J.309:773-779;

(30) HLA-DOB (the Beta subunit of MHC II quasi-molecule (Ia antigen), its binding peptide also is presented to the CD4+T lymphocyte with it) PROTEIN SEQUENCE Fullmgsgwvp...vllpqsc (1..273; 273aa, pI:6.56MW:30820TM:1[P] GeneChromosome:6p21.3, Genbank accession number: NP_002111.1)

Tonnelle et al (1985) EMBO is (11): 2839-2847 J.4; Jonsson et al (1989) Immunogenetics 29 (6): 411-413; Beck et al (1992) J.Mol.Biol.228:433-441; Strausberg et al (2002) Proc.Natl.Acad.Sci USA 99:16899-16903; Servenius etal (1987) J.Biol.Chem.262:8759-8766; Beck et al (1996) J.Mol.Biol.255:1-13; Naruse et al (2002) Tissue Antigens 59:512-519; WO9958658 (claim 13, Figure 15); US6153408 (35-38 hurdle); US5976551 (168-170 hurdle); US6011146 (145-146 hurdle); Kasahara et al (1989) Immunogenetics 30 (1): 66-68; Larhammar etal (1985) J.Biol.Chem.260 (26): 14111-14119;

(31) P2X5 (purinoceptor P2X ligand-gated ion channel 5, ion channel by the outer ATP gate of born of the same parents, may relate to synapse transmission and neural the generation, its defective may cause IDI's Pathophysiology situation) PROTEIN SEQUENCE Full mgqagck...lephrst (1..422; 422aa), pI:7.63, MW:47206TM:1[P] Gene Chromosome:17p13.3, Genbank accession number: NP_002552.2)

Le et al (1997) FEBS Lett.418 (1-2): 195-199; WO2004047749; WO2003072035 (claim 10); Touchman et al (2000) Genome Res.10:165-173; WO200222660 (claim 20); WO2003093444 (claim 1); WO2003087768 (claim 1); WO2003029277 (the 82nd page);

(32) CD72 (B cell differentiation antigen CD72, Lyb-2) PROTEIN SEQUENCE Fullmaeaity...tafrfpd (1..359; 359 aa), pI:8.66, MW:40225TM:1[P] GeneChromosome:9p13.3, Genbank accession number: NP_001773.1)

WO2004042346 (claim 65); WO2003026493 (51-52,57-58 page or leaf); WO200075655 (105-106 page or leaf); Von Hoegen et al (1990) J.Immunol.144 (12): 4870-4877; Strausberg et al (2002) Proc.Natl.Acad.Sci USA99:16899-16903;

(33) LY64 (lymphocyte antigen 64 (RP105), be rich in the I type memebrane protein family that leucine repeats (LRR), regulate B cell activation and apoptosis, the forfeiture of function strengthens relevant with the Patients with SLE disease activity) PROTEIN SEQUENCE Full mafdvsc...rwkyqhi (1..661; 661aa), pI:6.20, MW:74147TM:1[P] Gene Chromosome:5q12, Genbank accession number: NP_005573.1)

US2002193567; WO9707198 (claim 11, the 39-42); Miura et al (1996) Genomics 38 (3): 299-304; Miura et al (1998) Blood 92:2815-2822; WO2003083047; WO9744452 (claim 8,57-61 page or leaf); WO200012130 (24-26 page or leaf);

(34) FcRH1 (it comprises C2 type Ig sample and ITAM domain for Fc receptor-like protein 1, the immunoglobulin receptor that the Fc domain is inferred, and may in the bone-marrow-derived lymphocyte differentiation effect be arranged) PROTEINSEQUENCE Full mlprlll...vdyedam (1..429; 429aa), pI:5.28, MW:46925TM:1[P] Gene Chromosome:1q21-1q22, Genbank accession number: NP_443170.1)

WO2003077836; WO200138490 (claim 6, Figure 18 E-1-18-E-2); Davis etal (2001) Proc.Natl.Acad.Sci USA 98 (17): 9772-9777; WO2003089624 (claim 8); EP1347046 (claim 1); WO2003089624 (claim 7);

(35) IRTA2 (the immunoglobulin superfamily receptor 2 that transposition is relevant, the effective immunity receptor of inferring of possibility in B cell development and lymphoma generation; Relevant with the gene that causes by the transposition imbalance in occurring in some B cell malignant diseases) PROTEIN SEQUENCE Fullmllwvil...assaphr (1..977; 977aa), pI:6.88MW:106468TM:1[P] GeneChromosome:1q21, Genbank accession number: Human:AF343662, AF343663, AF343664, AF343665, AF369794, AF397453, AK090423, AK090475, AL834187, AY358085; Mouse:AK089756, AY158090, AY506558; NP_112571.1

WO2003024392 (claim 2, Figure 97); Nakayama et al (2000) Biochem.Biophys.Res.Commun.277 (1): 124-127; WO2003077836; WO200138490 (claim 3, Figure 18 B-1-18B-2);

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, the transmembrane protein polysaccharide of inferring, the EGF/ that relates to somatomedin and follistatin (follistatin) transfers protein family) PROTEINSEQUENCE Full mvlwesp...rastrli (1..374; 374 aa, NCBI accession number: AAD55776, AAF91397, AAG49451, NCBI RefSeq:NP_057276; NCBI Gene:23671; OMIM:605734; SwissProt Q9UIK5; Genbank accession number: AF179274, AY358907, CAF85723, CQ782436

WO2004074320 (SEQ ID NO 810); JP2004113151 ( SEQ ID NOS 2,4,8); WO2003042661 (SEQ ID NO 580); WO2003009814 (SEQ ID NO 411); EP1295944 (69-70 page or leaf); WO200230268 (the 329th page); WO200190304 (SEQ IDNO 2706); US2004249130; US2004022727; WO2004063355; US2004197325; US2003232350; US2004005563; US2003124579; US 6410506; US 66420061; Horie et al (2000) Genomics 67:146-152; Uchida et al (1999) Biochem.Biophys.Res.Commun.266:593-602; Liang et al (2000) Cancer Res.60:4907-12; Glynne-Jones et al (2001) Int J Cancer.Oct 15; 94 (2): 178-84.

The production of antibody

Many methods have been used for manufacture order clonal antibody (MAbs).Hybridoma technology refers to produce the cloned cell line of single antibody class type, uses many species to comprise mice (Mus), hamster, rat and people's cell.Preparation MAbs comprises that it is recombinant DNA technology that other method of chimeric and humanized antibody is used genetic engineering.

Can be by repeatedly subcutaneous (sc) or intraperitoneal (ip) injection related antigen and adjuvant prepare polyclonal antibody in animal body.Monoclonal antibody is obtained by the antibody colony of homogeneity in fact, and promptly except the sudden change of the natural generation that may exist on a small quantity, the single antibody of forming described colony is identical.

Production (Kozbor, J.Immunol., 133:3001 (1984) that human myeloma and mice-people's hybridoma cell line is used for human monoclonal antibodies have also been described; With Brodeur et al.Monoclonal AntibodyProduction Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., NewYork, 1987)).Tested and wherein cultivated the culture medium that hybridoma is arranged, be used for production at described antigenic monoclonal antibody.Can be by immuno-precipitation or by the external binding specificity of measuring the monoclonal antibody of producing in conjunction with test as radioimmunoassay (RIA) or enzyme linked immunological absorption test (ELISA) by hybridoma.For example can pass through Munson et al (1980) Anal.Biochem., the binding affinity of the Scatchard assay determination monoclonal antibody of 107:220.

Utilize conventional program (for example, by use can be specifically in conjunction with the oligonucleotide probe of coding murine antibody heavy chain and light chain) can separate the DNA of the monoclonal antibody of encode at an easy rate and check order.Described hybridoma is as the source of this DNA.In case separated DNA, can be placed in the expression vector, be transfected into then and originally do not produce in the host cell such as Bacillus coli cells, ape COS cell, Chinese hamster ovary (CHO) cell or myeloma cell of antibody protein, (US 2005/0048572 to obtain monoclonal antibody synthetic in recombinant host cell; US 2004/0229310).The DNA of relevant encoding antibody recombinant expressed summary in antibacterial comprises Skerra et al (1993) Curr.Opinion in Immunol., 5:256-262 and Pl ü ckthun (1992) Immunol.Revs., 130:151-188.

In another embodiment, can separate monoclonal antibody or antibody fragment from the antibody phage library, described antibody phage library McCafferty et al (1990) Nature 348:552-554; Clackson et al (1991) Nature 352:624-628; The technology that and Marks et al (1991) J.Mol.Biol., 222:581-597 describe obtains, these document descriptions separate Mus and people's antibody respectively with phage library.Publication has afterwards been described the production of high affinity (nM scope) people's antibody, it replaces (Marks et al (1992) Bio/Technology by chain, 10:779-783) and as reorganization in combination infection that makes up very big phage library strategy and the body (Waterhouse et al (1993) Nuc.Acids.Res. 21:2265-2266) carries out.Therefore, these technology are the feasibility substitute technologies of separating traditional monoclonal antibody hybridoma technology of monoclonal antibody.

Also can modifying DNA, for example replace homology Mus sequence (US4816567 by personnel selection heavy chain and constant region of light chain; With Morrison et al (1984) Proc.Natl Acad.Sci.USA, 81:6851), perhaps by covalently boundly modifying the coded sequence of NIg polypeptide is all or part of to immunoglobulin coding sequence.

Typically, the constant region that replaces antibody with such NIg polypeptide, the variable region that perhaps replaces an antigen-binding site of antibody, form chimeric bivalent antibody, it comprises antigen-binding site with antigenic specificity and another antigen-binding site with different antigenic specificities.

Description is according to the exemplary production technology of the antibody (Ab) that uses in the antibody-drug conjugates of the present invention (ADC).The production of antibody be will set forth with reference to anti-ErbB antibody, but to those skilled in the art, other member of ErbB receptor family and the antibody of any other receptor or tumor associated antigen or target thing clearly can be produced and modify in a similar manner.

The ErbB2 antigen that is used to produce antibody can be ErbB2 ectodomain or its part that comprises required epi-position of for example soluble form.Perhaps, at the cell of its cell surface expression ErbB2, for example through transforming the NIH-3T3 cell of expressing ErbB2; Or cancerous cell line such as Sk-Br-3 cell (Stancovskiet al (1991) PNAS (USA) 88:8691-8695) can be used for producing antibody.The ErbB2 of other form that is used to produce antibody is apparent to those skilled in the art.

Embodiment 1 describes the production of exemplary humanization anti-ErbB antibody.Humanized antibody can for example comprise the inhuman hypervariable region residue that is incorporated into people variable region heavy chain domain, and can further be included in the framework region (FR) that is selected from down the group site replaces: utilization Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed.Public Health Service, National Institutes ofHealth, Bethesda, 69H, 71H and the 73H of the variable region numbering system that provides among the MD (1991).In one embodiment, humanized antibody comprises FR at site 69H, 71H and 73H two or all sites and replaces.

As humanized replacement scheme, can produce people's antibody.For example, it is possible producing transgenic animal (for example, mice) now, and under the situation that does not have endogenous immunoglobulin to produce, described transgenic animal can produce a complete set (full repertoire) people's antibody when immunity.(Jakobovits et al(1993)Proc.Natl.Acad.Sci.USA，90：2551；Jakobovits et al(1993)Nature 362：255-258；Bruggermann et al(1993)Year in Immuno.7：33；and US 5591669；US 5589369；US 5545807)。

Perhaps, display technique of bacteriophage (McCafferty et al (1990) Nature 348:552-553) can be used for from immunoglobulin variable (V) a complete set of gene produced in vitro people's antibody in district and antibody fragment from non-immune donor.(Johnson，Kevin S.and Chiswell，David J.(1993)Current Opinion inStructural Biology 3：564-571)。A complete set of V gene can be made up, and the various antigens antibody of (comprising autoantigen) (Marks et al (1991) J.Mol.Biol.222:581-597 can be separated basically from non-immune people's donor; Griffith et al (1993) EMBO J.12:725-734; US 5565332; US 5573905).Also can (US 5567610 by external activatory B cells produce people antibody; US5229275).People's anti-ErbB antibody (US 5772997 and WO 97/00271) has been described.

Developed the technology of multiple production antibody fragment.Traditionally, these fragments by the proteolytic digestion of complete antibody derive (referring to, for example, Morimoto et al (1992) Joumal ofBiochemical and Biophysical Methods 24:107-117; With Brennan et al (1985) Science, 229:81).Also can directly produce antibody fragment by recombinant host cell and antibody phage discussed above library.Perhaps, can directly reclaim Fab '-SH fragment and carry out chemical coupling to form F (ab ') from escherichia coli ₂Fragment (Carter et al (1992) Bio/Technology 10:163-167).According to another kind of method, can directly from the recombinant host cell culture, separate F (ab ') ₂Fragment.Other technology of producing antibody fragment will be conspicuous for those of skill in the art.In other embodiments, selected antibody is strand Fv fragment (scFv).Referring to WO 93/16185; US 5571894; And US 5587458.For example, antibody fragment also can be " a linear antibody ", for example described in the US 5641870.This linear antibody fragment can be monospecific or bispecific.

The bi-specific antibody (Millstein etal (1983), Nature 305:537-539) that has at the binding specificity of at least two different epi-positions can be in conjunction with proteic two the different epi-positions of ErbB2.Other this antibody can be with the binding site combination of ErbB2 binding site and EGFR, ErbB3 and/or ErbB4.Perhaps, the arm of anti-ErbB can with excited molecule on combining leukocyte such as the Fc receptor (Fc γ R) of TXi Baoshouti molecule (for example CD2 or CD3) or IgG, arm combination as Fc γ RI (CD64), Fc γ RII (CD32) and Fc γ RIII (CD16) concentrates on the cell of expressing ErbB2 with the defense mechanism with cell.Bi-specific antibody can be used for that also cytotoxic reagent is positioned to express the cell of ErbB2, and (WO 96/16673; US 5837234; WO98/02463; US 5821337).(WO 93/08829 to have disclosed the purification process of bi-specific antibody; Traunecker et al (1991) EMBO J.10:3655-3659; WO 94/04690; Suresh et al (1986) Methods in Enzymology121:210; US 5731168).Can utilize leucine zipper (Kostelny et al (1992) J.Immunol.148 (5): 1547-1553) and strand Fv (sFv) dimer (Gruber et al (1994) J.Immunol.152:5368) produce bi-specific antibody.

Also described the technology of producing bi-specific antibody from antibody fragment, for example utilized complete antibody to produce F (ab ') through proteolytic cleavage in its position ₂Segmental chemical bond (Brennan et al (1985) Science 229:81).Can reclaim Fab '-SH fragment and chemical coupling to form bi-specific antibody (Shalaby et al (1 992) J.Exp.Med.175:217-225 from escherichia coli." double antibody " technology provides the alternative method (Hollinger et al (1993) Proc.Natl.Acad.Sci.USA 90:6444-6448) of preparation bispecific antibody fragment.

Can prepare antibody above bivalence.Nucleic acid that can be by the encoding antibody polypeptide chain recombinant expressed produces the antibody of multivalence " Octopus " sample with three or more antigen-binding sites and two or more variable regions at an easy rate, and (US 2002/0004586; WO 01/77342).For example, can prepare three-specific antibody (Tutt et al (1991) J.Immunol.147:60).

Can expect the amino acid sequence modifications of antibody.For example, expection is in conjunction with the antibody mutation body of tumor associated antigen and binding affinity and/or other biological characteristics of many isotypes raising antibody.Introduce the aminoacid sequence variant that suitable nucleotide changes or synthesize by peptide preparation antibody by nucleic acid to encoding antibody.This modification comprises, for example, deletes residue in the aminoacid sequence of antibody and/or inserts residue in the aminoacid sequence of antibody and/or replace residue in the antibody aminoacid sequence.The combination in any of implementing deletion, insert and replacing is to obtain final construct, so that described final construct has required feature.Aminoacid changes the translation post-treatment that also may change antibody, for example changes the number or the position of glycosylation site.

Be used to identify that the specific residue of antibody or the process useful in zone are called " alanine scanning mutagenesis ", wherein said specific residue or zone are preferred mutation position (Cunningham and Wells (1989) Science, 244:1081-1085, (for example amino acid residue or one group of target residue have wherein been identified, charged residue such as arg, asp, his, lys and glu), and with neutral or electronegative aminoacid, for example alanine or poly-alanine replace, to optimize aminoacid and antigenic interaction.Aminoacid sequence inserts the amino comprise from a residue to the polypeptide that comprises 100 or more residues and/or the fusion of carboxyl terminal, and inserts in the sequence of single or multiple amino acid residues.The terminal example that inserts comprises anti-ErbB antibody with N end methinyl residue or the antibody that is blended in the cytotoxicity polypeptide.Other of anti-ErbB antibody molecule inserts the fusions that variant comprises enzyme (for example for ADEPT:Tietze etal (2003) Current Pharm.Design 9:2155-2175) or polypeptide and anti-ErbB antibody N-or C-end, described polypeptide increases the serum half-life of antibody, for example albumin-binding peptide.

Blood plasma-protein binding may be to improve the effective means of the pharmacokinetic properties of short-lived molecule.Albumin is rich in protein in the blood plasma.Serum albumin binding peptide (ABP) can change the proteic pharmacodynamics of active structure domain of fusion, comprises the change that tissue absorbs, penetrates and spread.Can select suitable serum albumin peptide binding sequence to regulate these drug effect parameters (US20040001827) by specificity.By the phage display Screening and Identification a series of albumin binding peptides (Dennis etal (2002) " Albumin Binding As A General Strategy For Improving ThePharmacokinetics Of Proteins " J Biol Chem.277:35035-35043; WO 01/45746).Chemical compound of the present invention comprises: (i) Table III and IV among Dennis et al (2002) the J Biol Chem.277:35035-35043, the 35038th page; (ii) [0076] SEQ ID NOS:9-22 among the US 20040001827; The (iii) ABP sequence of 12-13 page or leaf among the WO 01/45746, SEQ ID NOS:z1-z14 instruction, these all add as a reference herein.

Usually change aminoacid sequence by changing basic nucleotide sequence.The nucleic acid molecules of encoding antibody aminoacid sequence variant prepares by several different methods known in the art.These methods include but not limited to separate (under the situation of the aminoacid sequence variant of natural generation) or the variant of the previous preparation by oligonucleotide mediated (or fixed point) mutation, PCR mutation and antagonist or the cassette mutagenesis that unmanifest pattern is carried out from natural origin.Most interested replacement mutation position comprises the hypervariable region, but can expect that also FR changes.

The substance of antibody biological characteristics is modified by selecting to replace and is finished, described selection replaces keeping the structure that (a) replaces polypeptide backbone in the zone, for example lamella or helical conformation, (b) electric charge at molecular targets position or hydrophobicity, or (c) side chain size influence the aspect significant difference.Based on common side chain characteristic the residue of natural generation is divided into following group:

(1) hydrophobic: nor-leucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) tart: asp, glu;

(4) alkalescence: asn, gln, his, lys, arg;

(5) influence the residue of chain direction: gly, pro; With

(6) aromatic: trp, tyr, phe.

Non-conservation replace will inevitably follow with in these classifications wherein the member of a class exchange another kind of.

Usually also can replace any cysteine residues that does not relate to the correct conformation of keeping antibody, with the oxidation stability of improving molecule and stop unusual coupling with serine.Otherwise, the cysteine key can be added to antibody to improve its stability (when particularly antibody is antibody fragment such as Fv fragment).

In order to increase the serum half-life of antibody, for example can be incorporated in the antibody (especially antibody fragment) as remedying the receptors bind epi-position as described in the US 5739277.The epi-position in IgG molecule (for example, IgG1, IgG2, IgG3 or IgG4) Fc district " remedied the receptors bind epi-position " and refer in term used herein, and it is responsible for strengthening serum half-life (US 2003/0190311, US6821505 in the body of IgG molecule; US 6165745; US 5624821; US 5648260; US 6165745; US 5834597).

The glycosylation variant of antibody refers to the wherein reformed variant of glycosylation pattern of antibody.Change and to refer to delete the carbohydrate part found in one or more antibody, add one or more carbohydrates parts, change glycosylated composition (glycosylation pattern) or glycosylated degree to antibody.

Antibody can be the conservative site in its constant region glycosylated (N-connects or O-connects) (Hse et al (1997) J.Biol.Chem.272:9062-9070; Jefferis and Lund, (1997) Chem.Immunol.65:111-128; Wright and Morrison, (1997) TibTECH 15:26-32).The oligosaccharide side chain of immunoglobulin influences proteinic function (Boyd et al (1996) Mol.Immunol.32:1311-1318; Wittwe and Howard, (1990) Biochem.29:4175-4180), and the intramolecular interaction between the glycoprotein part, this intramolecular interaction can influence described conformation and the three-dimensional surface of glycoprotein (Hefferis and Lund, supra are provided; Wyss and Wagner (1996) Current Opin.Biotech.7:409-416).Based on the specific recognition structure, oligosaccharide also can be used for given glycoprotein is guided to specific molecular (Malhotra et al (1995) Nature Med.1:237-243; Umana et al (1999) Nature Biotech.17:176-180).Remove oligosaccharide and can optimize antigen combination and other characteristic (Boyd et al (1996) Mol.Immunol.32:1311-1318) of antibody.

The glycosylated factor of influence comprises growth pattern, culture medium prescription, culture density, oxidation, pH, purification scheme or the like (US 5047335 during the antibody recombinant production; US 5510261; US 5278299).Can for example use endoglycosidase H (Endo H) from the glycosylation of glycoprotein by Enzymology method removal glycosylation or particular type.In addition, can genetically engineered recombinant host cell, for example making it in processing particular type polysaccharide is deficiency.These technology and similar techniques are well known in the art.

Can analyze the glycosylation structure of antibody at an easy rate by the conventional art of carbohydrate analysis, described technology comprises agglutinin chromatography, NMR, mass spectral analysis, HPLC, GPC, monosaccharide composition analysis, zymetology digests and HPAEC-PAD continuously, wherein HPAEC-PAD uses high pH anion-exchange chromatography to separate oligosaccharide based on electric charge.The method that discharges oligosaccharide for the purpose of analyzing also is known, includes but not limited to zymetology processing (generally using peptide-N-glycosidase F/ inscribe-beta galactosidase to carry out), utilizes the caustic alkali environment to eliminate with main release O-syndeton and utilizes anhydrous hydrazine chemical method release N-to be connected oligosaccharide with O-.

The maytansinoid drug moiety

The maytansine chemical compound suppresses cell proliferation (Remillard et al (1975) Science 189:1002-1005 by the formation that suppresses the polymerization microtubule of tubulin during the mitosis; US 5208020).Maytansine and maytansinoid are highly Cytotoxic, but its serious systemic side effects has limited its clinical practice in treatment of cancer greatly, and described serious systemic side effects is mainly owing to the tumor-selective a little less than it.Because to the serious side effects of central nervous system's stomach function regulating intestinal system, (Issel et al (1978) Can.Treatment.Rev.5:199-207) interrupted in the clinical trial of maytansine.

The maytansinoid drug moiety is attractive drug moiety in antibody-drug conjugates, because they: (i) by fermentation or chemical modification, by fermented product derive prepare relatively easy, (ii) be easy to use functional group's derivatization, described functional group is adapted to pass through non-disulphide joint and is coupled to antibody, (iii) stable and (iv) effective in blood plasma to kinds of tumor cells system.

The maytansine chemical compound that is suitable for as the maytansinoid drug moiety is well known in the art, it can separate from natural origin according to known method, with gene engineering production (referring to Yuet al (2002) PNAS 99:7968-7973), perhaps according to known method synthetic preparation maytansinol and maytansinol analog.

Exemplary maytansinoid drug moiety comprises aromatic rings that those have modification, as: C-19-dechlorination (dechloro) (US 4256746) (by the Li-Al hydrogen thing reduction preparation of ansamitocin P2); C-20-hydroxyl (or C-20-demethyl)+/-C-19-dechlorination (United States Patent (USP) 4361650 and 4307016) (by preparing) with streptomycete (Streptomyces) or actinomycetes (Actinomyces) demethylation or with the LAH dechlorination; With C-20-de-methoxy, C-20-acyloxy (OCOR) ,+/-dechlorination (US4294757) (by with the preparation of acyl chlorides acidylate), and those of modification are arranged in other site.

Exemplary maytansinoid drug moiety also comprises those with following modification: C-9-SH, by maytansinol and H ₂S or P ₂S ₅(US 4424219) prepared in reaction; C-14-alkoxyl-methyl (de-methoxy/CH2 OR) (US 4331598); C-14-methylol or acyloxy methyl (CH2OH or CH2OAc) prepare from Nocard's bacillus (Nocardia) (US 4450254); C-15-hydroxyl/acyloxy is by transforming maytansinol preparation (US 4364866) with streptomycete; The C-15-methoxyl group separates (US 4313946 and US 4315929) from trewianudiflora (Trewianudiflora); The C-18-N-demethylation is by preparing (US 4362663 and US 4322348) with streptomycete to the maytansinol demethylation; And 4, the 5-deoxidation by the titanous chloride ./LAH reduction preparation (US 4371533) of maytansinol.

Known to the type that connects, the many sites on the maytansine chemical compound can be used as connection site.For example, for forming ester bond, the C-14 site that has the C-3 site of hydroxyl, modifies by methylol, all be fit to by the C-15 site of hydroxyl modified and C-20 site with hydroxyl.

Maytansinoid drug moiety (D) comprises those with following structure:

Wherein wave is represented the covalently bound of the sulphur atom of D and antibody-drug conjugates (ADC) joint (L).R can be H or C independently ₁-C ₆Alkyl.Alkylidene (alkylene) chain that amide groups is connected in sulphur atom can be methyl (methanyl), ethyl group (ethanyl) or propyl group, be that m is 1,2 or 3 (US633410,5208020, Chari et al (1992) Cancer Res.52:127-131; Liu et al (1996) Proc.Natl.Acad.Sci 93:8618-8623).

All stereoisomers of maytansinoid drug moiety all are the chemical compounds of the present invention of expection, i.e. any combination of the chiral carbon position R of D and S conformation.In one embodiment, maytansinoid drug moiety (D) will have following stereochemical structure:

The concrete scheme of D comprises:

DM1 (N ^{2 '}-Tuo acetyl-N ^{2 '}-(3-sulfydryl-1-oxopropyl)-maytansine) (CR wherein ₂) _m=CH ₂CH ₂

DM3 (N ^{2 '}-deacetylation-N- ^{2 '}(4-sulfydryl-1-oxo amyl group)-maytansine) (CR wherein ₂) _m=CH ₂CH ₂CH (CH ₃);

DM4 (N ^{2 '}-deacetylation-N ^{2 '}-(4-methyl-4-sulfydryl-1-oxo amyl group)-maytansine) (CR wherein ₂) _m=CH ₂CH ₂C (CH ₃) ₂:

Heating rate (US 2004/0235840 A1) in the sterically hindered born of the same parents that may influence ADC that alkyl such as methyl provide near the carbon the sulphur atom of DM3 and DM4.So variable alkyl unit (CR ₂) _mMay influence external and interior (petency), effect (efficacy) and the safety/toxicity renderd a service of body.

Joint

Joint L does not comprise disulfide group (disulfide group) by covalent bond, and antibody is connected to drug moiety.Joint is difunctional or multi-functional part, and it can be used for connecting one or more drug moieties (D) and antibody unit (Ab), with the antibody-drug conjugates (ADC) that forms formula I.Can prepare antibody-drug conjugates (ADC) with the joint of reactive functionality easily with bound drug and binding antibody.The cysteine mercaptan or the amine of antibody (Ab), for example N-end or amino acid side chain such as lysine can form key with the functional group of joint reagent, drug moiety or medicine-joint reagent.

Joint is stable in the extracellular preferably.Before transhipment or being delivered in the cell, antibody-drug conjugates (ADC) is preferably stable and complete, and promptly antibody still is connected to drug moiety.Joint is stable outside target cell, and can be with certain effect speed (efficacious rate) cracking in cell.Effectively joint is incited somebody to action: the specificity binding characteristic that (i) keeps antibody; (ii) allow to send in the cell of conjugate or drug moiety; (iii) keep stable and complete, promptly sent or be transported to the just cracking of its target location up to conjugate; The cytotoxicity, cell killing effect or the cell depression effect that (iv) keep the maytansinoid drug moiety.The stability of ADC can and separate by standard analytical techniques such as mass spectrometry, HPLC/and analytical technology LC/MS measures.

The covalently bound joint that requires of antibody and drug moiety has two reactive functionality, is bivalence on reactive meaning promptly.The bivalence joint reagent such as peptide, nucleic acid, medicine, toxin, antibody, hapten and the reporter molecule group that can be used for connecting two or more functions or biologic activity part are known, described method has been described and by conjugate (Hermanson, GT. (1996) the Bioconjugate Techniques of its acquisition; Academic Press:New York, p234-242).

Joint can have the following structures of being selected from:

Wherein wave is represented covalently bound with Ab and D with any direction; X can have the following structure of any direction:

Wherein R is H or C independently ₁-C ₆Alkyl; And n is 1 to 12; Y can have the following structure of any direction:

Wherein R is H or C independently ₁-C ₆Alkyl; And n is 1 to 12.

For example, joint can have the structure of the following SMCC of being called:

In another embodiment, joint (L) has following structure:

Wherein wave is represented covalently bound with Ab and D with any direction.

For example, joint can have the structure of the following SIAB of being called:

In another embodiment, joint (L) has following structure:

In another embodiment, joint can be conditioned dissolubility or reactive group replaces.For example, sulphonic acid ester (sulfonate) substituent group can strengthen the water solublity of reagent and promote joint reagent and the coupling reaction of antibody or drug moiety, perhaps promote the coupling reaction of Ab-L and D or D-L and Ab, this depends on the employed synthetic route of preparation ADC.

In another embodiment, joint has the reactive functional groups that contains nucleophilic group, and promptly the electrophilic group that exists on the antagonist is reactive.Useful electrophilic group includes but not limited to the carbonyl of aldehyde and ketone on the antibody.The hetero atom of joint nucleophilic group can react with the electrophilic group on the antibody, forms covalent bond with the antibody unit.Available nucleophilic group includes but not limited to hydrazides, oxime, amino, hydrazine, thiosemicarbazones, hydrazinecarboxylate and aryl hydrazide (arylhydrazide) on the joint.Electrophilic group on the antibody provides the site easily that is connected with joint.

Joint can be the joint that comprises the peptide of one or more aminoacid units.Peptide linker reagent can be by solid phase or liquid phase synthesizing method at automatic synthesizer such as Rainin Symphony PeptideSynthesizer (Protein Technologies, Inc., Tucson, AZ), or Model 433 (AppliedBiosystems, Foster City, CA) go up preparation (E.Schr_der and K.L ü bke, The Peptides, volume 1, pp 76-136 (1965) Academic Press), this is that chemistry of peptides comprises t-BOC chemistry (Geiser et al " Automation of solid-phase peptide synthesis " in MacromolecularSequencing and Synthesis, Alan R.Liss, Inc., 1988, pp.199-218) with Fmoc/HBTU chemistry (Fields, G.and Noble, R. (1990) " Solid phase peptide synthesis utilizing9-fluoroenylmethoxycarbonyl amino acids ", Int.J.Peptide Protein Res.35:161-214) field is known.

Described chemical compound refers on wording but is not limited to ADC:BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo group (sulfo)-EMCS, sulfo group (sulfo)-GMBS, sulfo group-KMUS, sulfo group-MBS, sulfo group-SIAB, sulfo group-SMCC and sulfo group-SMPB and SVSB (succinimido-(4-vinyl sulfone(Remzaol) benzoate) with the preparation of following cross-linking agent, and comprises two-maleimide reagent: DTME, BMB, BMDB, BMH, BMOE, BM (PEO) ₃, and BM (PEO) ₄, its commercial can be from Pierce Biotechnology, Inc., Customer ServiceDepartment, P.O.Box 117, Rockford, IL.61105 U.S.A, U.S.A 1-800-874-3723, International+815-968-0747 obtains.Referring to the 467-498 page or leaf, 2003-2004 ApplicationsHandbook and Catalog.Two-maleimide reagent allows the free sulphur alcohol radical of antibody cysteine residues to be connected in drug moiety, labelling or the joint intermediate that contains mercaptan with sequential or form simultaneously.Except that maleimide, comprise iodoacetamide, acetbromamide, vinylpyridine, disulphide, pyridyl disulfide, isocyanates and isothiocyanate with other functional group of the mercapto of antibody, maytansinoid drug moiety or joint intermediate reaction.

BM(PEO) ₃ BM(PEO) ₄

Wherein DM1 has following structure by exemplary antibodies-drug conjugates that the BMPEO joint is connected to the mercapto of trastuzumab:

Wherein Tr is a trastuzumab; N is 0,1 or 2; And p is 1,2,3 or 4.

Available joint reagent also can obtain via other commercial source, as Molecular BiosciencesInc. (Boulder, CO), perhaps according to Toki et al (2002) J.Org.Chem.67:1866-1872; US6214345 to Firestone et al; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583; The method of describing among the and WO 04/032828 is synthetic.

Joint can be for surpassing a drug moiety is covalently attached to antibody by branch, multifunctional access head part dendroid type fittings (Sun et al (2002) Bioorganic; Medicinal ChemistryLetters 12:2213-2215; Sun et al (2003) Bioorganic ﹠amp; Medicinal Chemistry11:1761-1768; King et al (2002) Tetrahedron Letters 43:1987-1990).The dendroid joint can improve the mol ratio of medicine and antibody, i.e. the ratio of load, and its effectiveness with ADC is relevant.Like this, on the antibody that only has a reactive cysteine mercapto, can connect many drug moieties by the dendroid joint.

The following exemplary concrete form of dendroid joint reagent allow up to nine nucleophilic drug moiety reagent by with ethyl chloride chlormethine functional group reactions by coupling:

Drug loading

In the molecule of formula I, represent drug loading with p, represent the average number of the maytansinoid medicine of each antibody load.Drug loading can be 1 to 8 medicine of each antibody (Ab) (D), and promptly wherein 1,2,3,4,5,6,7 and 8 drug moiety is covalently attached to antibody.The composition of the ADC of formula I comprises the set with the antibody of 1 to 8 drug coupling.In the ADC goods from coupling reaction, the medicine average number of each antibody can be identified by traditional method such as mass spectrometry method, ELISA test, electrophoresis and HPLC.Also can measure the quantitative distribution of ADC according to p.Can measure p meansigma methods (Hamblett et al (2004) Clinical CancerRes.10:7063-7070 in the specific ADC goods by ELISA; Sanderson et al (2005) Clinical Cancer Res.11:843-852).Yet p (medicine) value distributes and can not distinguish by the detection limit of antibody-antigen combination and ELISA.Simultaneously, the ELISA test of detection antibody-drug conjugates is not measured drug moiety and is connected in antibody in where, as heavy chain or light chain segments or particular amino acid residue.In some cases, can be by realize separation, purification and the evaluation of homogeneity ADC as means such as reversed-phase HPLC or electrophoresis, wherein p is the particular value from ADC and other medicines load.

For some antibody-drug conjugates, p may be subject to the connection site number on the antibody.For example, the site of connection is a cysteine mercaptan, and in above-mentioned exemplary, antibody may only have one or several cysteine mercapto, and one or several abundant reactive mercapto perhaps may only be arranged, and joint can connect by this mercapto.Higher drug loading, for example p＞5 may cause the forfeiture of gathering, insolubility, toxicity or the cell permeability of some antibody-drug conjugates.

Typically, during the coupling reaction, the drug moiety that is coupled to antibody is less than theoretical maximum.Antibody can comprise for example many not with the lysine residue of medicine-joint intermediate (D-L) or joint reagent reacting.Have only the strongest reactive lysine group can with the reactive joint reagent reacting of amine.Equally, have only the strongest reactive cysteine mercapto can with thiol-reactive joint reagent reacting.Usually, even if having, antibody can not comprise many free reactive cysteine mercaptos that can be connected to drug moiety yet.Most of cysteine mercaptan residues in the antibody chemical compound all exist with the disulfide bond form, must reduce under incomplete or complete reducing condition with Reducing agent such as dithiothreitol, DTT (DTT) or TCEP.In addition, antibody must place under the degeneration condition to expose reactive nucleophilic group such as lysine or cysteine.The load of ADC (medicine/antibody ratio) can be controlled in several different modes, comprise: (i) restriction is with respect to the medicine-joint intermediate (D-L) of antibody or the molar excess number of joint reagent, the reducing condition of the incomplete or restriction that (ii) limits coupling reaction time or temperature and (iii) modify at cysteine mercaptan.

Surpass a nucleophilic or electrophilic group and medicine-joint intermediate or joint reagent reacting in the antibody, afterwards with the drug moiety reagent reacting, products therefrom is the mixture of ADC chemical compound then, and it for example has the distributions that are connected in the drug moiety of antibody such as 1,2,3.Liquid-phase chromatography method such as polymer anti-phase (PLRP) and hydrophobic interaction (HIC) can be by the chemical compounds in the drug loading value separating mixture.Can separate ADC goods (" Effect of drugloading on the pharmacology; pharmacokinetics; and toxicity of an anti-CD30antibody-drug conjugate " with single medicine load value (p), Hamblett, K.J., et al, Abstract No.624, AmericanAssociation for Cancer Research; 2004 Annual Meeting, March 27-31,2004, Proceedings of the AACR, Volume 45, and March 2004; " Controlling the Locationof Drug Attachment in Antibody-Drug Conjugates ", Alley, S.C., et al, AbstractNo.627, American Association for Cancer Research; 2004 Annual Meeting, March 27-31,2004, Proceedings of the AACR, Volume 45, March 2004).Yet these single load value ADCs may remain heterogeneous mixture, because drug moiety can be connected in different loci on the antibody via joint.

The preparation of antibody-drug conjugates

The ADC of formula I can pass through several routes, use organic chemical reactions well known by persons skilled in the art, condition and reagent preparation, comprise: (1) antibody nucleophilic group or electrophilic group and bivalence joint reagent reacting, antibody-joint intermediate A b-L that formation is connected by covalent bond is afterwards with activatory drug moiety D reaction; (2) nucleophilic group of drug moiety or electrophilic group and joint reagent reacting form the medicine-joint intermediate D-L that is connected by covalent bond, and nucleophilic group or the electrophilic group with antibody reacts afterwards.Can utilize multiple antibody, drug moiety and joint to use coupling method (1) and (2), with the antibody-drug conjugates of preparation formula I.

Nucleophilic group on the antibody includes but not limited to: (i) N end is amino, (ii) side chain amino, and lysine for example, (iii) side chain mercapto, for example cysteine and (iv) sugared hydroxyl or amino, wherein antibody is glycosylated.Ammonia, mercaptan and hydroxyl are nucleophilic, can form covalent bond with the electrophilic group reaction on blank area (linker moieties) and the joint reagent, described electrophilic group comprises: (i) active ester such as NHS ester, HOBt ester, haloformate (haloformates) and acid halide; (ii) alkyl and benzyl halogenide such as Haloacetamide; (iii) aldehyde, ketone, carboxyl and maleimide base group.Some antibody has reducible interchain disulfide bond, i.e. the cysteine bridge.Antibody can be by using Reducing agent such as DTT (Cleland ' s reagent, dithiothreitol, DTT) or TCEP (three (2-carboxyethyl) phosphonium salt hydrochlorate (tris (2-carboxyethyl) phosphine hydrochloride); Getz et al (1999) Anal.Biochem.Vol 273:73-80; Soltec Ventures, Beverly, it is reactive MA) handling and make it for the coupling with joint reagent.Like this, each cysteine disulphide bridges will form two reactive mercaptan nucleophilic things in theory.Can other nucleophilic group be introduced in the antibody by lysine and 2-imino group thiophene (Traut ' s reagent) reaction, described reaction is converted into mercaptan with amine.

Also can partly produce antibody-drug conjugates to introduce electrophilic by modified antibodies, the electrophilic part can with the nucleophilic displacement of fluorine radical reaction on joint reagent or the medicine.For example can form the aldehydes or ketones base with the sugar of periodate oxidation agent oxidation glycosylated antibodies, the aldehydes or ketones base can react with the amido of joint reagent or drug moiety (drug moieties).Gained imines Schiff's base group can form stable key, or can for example form stable amine key by the borohydride reagents reduction.In one embodiment, the carbohydrate part of glycosylated antibodies can produce carbonyl (aldehyde and ketone) with beta-Galactose oxidase or sodium metaperiodate (sodiummeta-periodate) reaction in protein, carbonyl can with suitable radical reaction (Hermanson, GT. (1996) the Bioconjugate Techniques on the medicine; AcademicPress:New York, p234-242).In another embodiment, the protein that comprises N terminal filament propylhomoserin or threonine residues can react with sodium metaperiodate, causes aldehyde to replace first aminoacid (Geoghegan ﹠amp; Stroh, (1992) Bioconjugate Chem.3:138-146; US 5362852).Such aldehyde can react with drug moiety or joint nucleopilic reagent.

Equally, nucleophilic group on the drug moiety includes but not limited to: ammonia, mercaptan, hydroxyl, hydrazides, oxime, hydrazine, thiosemicarbazones, hydrazinecarboxylate and aryl hydrazide (arylhydrazide) group, these groups can form covalent bond with the electrophilic group reaction on blank area and the joint reagent, and described electrophilic group comprises: (i) active ester such as NHS ester, HOBt ester, haloformate and acid halide; (ii) alkyl and benzyl halogenide such as Haloacetamide; (iii) aldehyde, ketone, carboxyl and maleimide base group.

Maytansine can for example change May-SSCH3 into, it can be reduced to free mercaptan May-SH, and form maytansinoid-antibody mediated immunity conjugate together with antibody response (Chari et al (1992) Cancer Research 52:127-131) and the disulphide joint modified.(WO 04/016801 to have reported the antibody-maytansinoid conjugate that forms with the disulphide joint; US6884874; US 2004/039176 A1; WO 03/068144; US 2004/001838 A1; US6441163; US 5208020; US 5416064; WO 01/024763).Disulphide joint SPP makes up with joint reagent N-butanimide 4-(2-pyridine sulfo-) valeric acid.Antibody-SPP-DM1 conjugate is represented with following structure:

In order to contrast, tested disulphide (S-S) joint antibody-drug conjugates with non-disulphide joint ADC of the present invention.Press embodiment 3 preparation trastuzumab-SPP-DM1 (Ranson, M.andSliwkowski M. (2002) Oncology 63 (suppl 1): 17-24).Also tested the disulphide antibody-drug conjugates: trastuzumab (trastuzumab)-SPDP-DM1, trastuzumab (trastuzumab)-SPP-DM3 and trastuzumab-SPP-DM4, it has following structure:

Trastuzumab-SPDP-DM1

Trastuzumab-SPP-DM3

Trastuzumab-SPP-DM4

ADC of the present invention comprises SMCC joint and DM1 maytansinoid drug moiety, represents with Ab-SMCC-DM1:

The concrete form of Ab-SMCC-DM1 is trastuzumab-SMCC-DM1, and wherein p is 1,2,3 or 4 (WO 2005/037992 for Ab=trastuzumab, Tr).The another kind of concrete form of ADC be trastuzumab-SIAB-DM1 (trastuzumab=Tr), it has following structure:

Screening at the antibody-drug conjugates (ADC) of tumor associated antigen and cell surface receptor

Transgenic animal and cell line especially can be used for screening the antibody-drug conjugates (ADC) with preventative or therapeutic treatment disease or disorderly potentiality, and described disease or disorder relate to tumor associated antigen and for example expression (US 6632979) excessively of HER2 of cell surface receptor.Screen available ADC and may be related to the candidate ADC that transgenic animal are used range of doses, and in disease or the disorderly influence of different time points test ADC to being assessed.Perhaps, or in addition, can be before being exposed to the disease inducement or use described medicine with it simultaneously, if applicable.Candidate ADC can be under culture medium or high flux screening array continuously also respectively, perhaps screening simultaneously.The speed of screening ADC only is subjected to synthesis rate or comprises the restriction of the screening technique speed of detection/mensuration/data analysis, and the screening of ADC is used for the application of the preventative or therapeutic treatment of disease or disorder.

The concrete scheme of screening technique comprises that (a) is transplanted to cell the non-human animal from stable breast cancer cell line, is the ability that forms from transplanted cells for described non-human animal uses the ADC drug candidate and (c) measures drug candidate inhibition tumor (b).The invention still further relates to the method for screening the candidate ADC that is used for the treatment of disease or disorder, described disease or disorder are expressed as feature with the mistake of receptor protein, described method comprises (a) ability that the cell of the breast cancer cell line of self-stabilization contacts with drug candidate and (b) assessment candidate ADC suppresses the stable cell lines growth in the future.

A concrete scheme of screening technique comprises (a) ability that the cell of the breast cancer cell line of self-stabilization contacts with the ADC drug candidate and (b) assessment candidate ADC blocks the ligand activation of HER2 in the future.Another embodiment has been assessed candidate ADC blocking-up and has been transferred the bonded ability of albumen (heregulin).In another embodiment, assessed the ability of the tyrosine phosphorylation of candidate ADC block ligand stimulation.

The concrete scheme of another of screening technique comprises that the cell of the breast cancer cell line of self-stabilization in (a) future contacts and (b) assess the ability of candidate ADC inducing cell death with the ADC drug candidate.In one embodiment, assessed the apoptosis-induced ability of candidate ADC.

Another embodiment of screening technique comprises that (a) is for for example crossing expression natural human protein in its mammary glandular cell, for example HER2 or its segmental transgenic nonhuman mammal are used the ADC drug candidate, to encode natural human protein or its segmental nucleotide sequence with the proteinic biologic activity of natural human of wherein this transgene mammal stably is incorporated in its genome, described nucleotide sequence is operably connected to the transcription regulating nucleotide sequence of its expression of guiding, and grow antagonist and treat the tumor that for example Anti-HER 2 is reactionless or reaction is very weak, for example mammary neoplasms perhaps is applied to the non-human mammal that has from the tumor of described transgenic nonhuman mammal transplanting; (b) assessment candidate ADC is to target disease or disorderly influence.Without limitation, described disease or disorder can be the cancers that HER2-crosses expression, as mammary gland, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas and bladder cancer.Described cancer can be with each cell at least about 500,000 copies, perhaps each cell is at least about 2,000,000 copy is expressed the breast carcinoma of HER2.For example can assess the ability of ADC drug candidate inducing cell death and/or apoptosis with method of testing well known in the art and described below.

In one embodiment, by using the candidate ADC of range of doses for transgenic animal, and assess described animal as time passes the physiological reaction of described chemical compound is screened candidate ADC.It can be oral using, and perhaps by suitable injection, this depends on the chemical property of the chemical compound that will assess.Sometimes, to use described chemical compound together may be suitable to the participant cofactor (co-factor) that strengthens compound efficacy.Be used to screen the chemical compound that can be used for treating different syndromes if come from the cell line that is subjected to the preliminary operation genetic animal, crossing of wherein said disease and some tumor-associated antigen protein or cell surface receptor expressed, for example HER2-crosses to express and is correlated with, then add test-compound to cell culture medium, and use the cell effect of suitable biochemistry and/or histology's testing evaluation as time passes described chemical compound in the suitable time.Sometimes, together compound of interest to be applied to described culture medium may be suitable to the participant cofactor that strengthens compound efficacy.

Therefore, the invention provides the method for testing that is used to identify ADC, the HER2 albumen of described ADC also combination mistake of targeting expression specifically, proteic existence of HER2 and unusual cell function associated, and the pathogeny of mammary glandular cell hypertrophy relevant with the breast tumor generation cause of disease and/or differentiation is relevant.

In order to identify the ADC of the ligand activation of blocking ErbB (for example ErbB2) receptor, can measure compounds block ErbB part with express ErbB (ErbB2) receptor (for example be in the coupling of another kind of ErbB receptor in, wherein interested described ErbB receptor and described another kind of ErbB receptor formation ErbB oligomeric body) the bonded ability of cell.For example, the transgenic animal of expressing HER2 separate and transfection can be incubated i.e. cultivation with ADC with the cell of expressing another kind of ErbB receptor (HER2 therewith forms the oligomeric body) from crossing, and contact with the ErbB part of labelling then.Can assess the ability of the ErbB receptors bind in compounds block part and the ErbB oligomeric body then.

For example, can carry out monolayer culture on ice with 24 hole flat boards, come evaluate candidate ADC suppress to transfer combining of albumen (HRG) and breast tumor cell line, described cell line is crossed expression HER2, and by transgenic nonhuman mammal (for example mice) foundation of this paper.The anti-ErbB monoclonal antibody can be added each aperture to and is incubated 30 minutes.Can add then ¹²⁵The rHRG β 1 of I-labelling _177-224(25,000cpm), can continue to be incubated 4 to 16 hours.Dose-effect curve can be formulated, and the IC of compound of interest can be calculated ₅₀Value.

Perhaps, or in addition, can assess the tyrosine phosphorylation of the ErbB ligand stimulation of ADC blocking-up ErbB receptor, described ErbB receptor is present in the ErbB oligomeric body.For example, the cell line of setting up from transgenic animal can be incubated with being tried ADC herein, uses the tyrosine phosphorylation activity of anti-phosphorylated tyrosine monoclonal antibody (its optional and detectable label coupling) test ErbB ligand-dependent then.The kinases receptors activation test of describing among the US 5766863 also can be used for measuring ErbB receptor activation and chemical compound to active blocking-up.

In one embodiment, can as described belowly screen ADC in the MCF7 cell basically, the HRG that described ADC suppresses the p180 tyrosine phosphorylation stimulates.For example, the cell line of setting up from the HER2 transgenic animal can join the 24 hole flat boards, chemical compound can be added to each aperture and be incubated 30 minutes in room temperature; Can be with rHRG β _1177-244Add each aperture to the 0.2nM final concentration, can continue to be incubated about 8 minutes.Culture medium can be come cessation reaction by adding 100 μ l SDS sample buffers (5%SDS, 25mM DTT and 25mM Tris-HCl, pH 6.8) from each aperture sucking-off.Each sample (25 μ l) can be gone up electrophoresis at 4-12% gradient gel (Novex), and electrophoretic transfer is to polyvinylidene fluoride film then.Anti-phosphorylated tyrosine (with 1 μ g/ml) immunoblotting is developed, can pass through the quantitative Mr-180 of reflection density algoscopy, the intensity of 000 dominant response band.Assessment is KIRA (kinases receptors activation) test (Sadick et al (1998) Jour.of Pharm.and Biomed.Anal.1-9) to the alternative method of the inhibition of receptor phosphorylation.In this test, some anti-HER 2 monoclonal antibodies of generally acknowledging can be used as positive control, and known described monoclonal antibody suppresses the p180 tyrosine phosphorylation that HRG stimulates.Can formulate the dose-effect curve of the inhibition of the p180 tyrosine phosphorylation that HRG is stimulated, described inhibition records by the reflection density algoscopy, can also calculate the IC of compound of interest ₅₀

Also can assess and be tried the growth inhibitory effect (Schaefer et al (1997) Oncogene 15:1385-1394) of ADC the cell line that comes from the HER2 transgenic animal.According to this test, can handle cell 4 days with the test-compound of variable concentrations, and dye with Gentian Violet or redox dye Alamar Blue.Be incubated the growth inhibitory effect that can show this cell line with described chemical compound, it is similar to the monoclonal antibody 2C4 effect shown to the MDA-MB-175 cell (Schaefer et al., the same).In another embodiment, external source HRG can obviously not reverse this inhibition.

For identifying the growth inhibited ADC chemical compound of targeting HER2 specifically, can screen the HER2-that suppresses to come from transgenic animal and cross the ADC (US 5677171) that expresses growth of cancer cells.According to this test, the HER2 overexpressing cell is incubated in 1: 1 mixture of F12 and DMEM culture medium, wherein is added with 10% hyclone, glutamine and penicillin streptomycin.Cell adds test-compound with 20,000 cells (2mls/35mm ware) bed board in the 35mm Tissue Culture Dish with variable concentrations.After six days, use electronics COULTER ^TMThe cell number that the cell counter statistics is compared with untreated cell.Those ADC of the about 20-100% of cell growth inhibiting or about 50-100% can be chosen as the growth inhibited chemical compound.

For selecting the ADC of inducing cell death, the film integrality that is absorbed indication by for example PI, trypan blue or 7AAD is lost, can be with respect to the contrast assessment.PI absorbs test to be used from galactophore of transgenic animal tumor tissues isolated cells.According to this test, (MEM): among the Ham ' s F-12 (50: 50), described culture medium is added with 10% hot deactivation FBS (Hyclone) and 2mM L-glutaminate to cell culture in Dulbecco ' s Modified EagleMedium.Like this, described test is implemented under the situation that does not have complement and immune effector cell.Cell is with 3 * 10 ⁶The density of every culture dish is seeded in 100 * 20mm culture dish, makes it to spend the night to adhere to.Removing culture medium then also only changes with fresh culture or with the culture medium that contains the variable concentrations chemical compound.The described cell 3 day time of insulation.After each the processing, wash cell monolayer with PBS, and by the trypsinization attachment removal.Cell was centrifugal 5 minutes of 4 ℃ of 1200rpm then, and precipitation is resuspended in the ice-cold Ca of 3ml ²⁺Binding buffer liquid (10mM Hepes, pH 7.4,140mM NaCl, 2.5mM CaCl ₂) in, be distributed in 35mm band medicated cap coarse filter 12 * 75mm test tube (every pipe 1ml, each processed group 3 pipe), to remove cell clot.Add PI (10 μ g/ml) toward test tube then.Sample can be used FACSCAN ^TMFlow cytometer and FACSCONVERT ^TMCellQuest software (BectonDickinson) is analyzed.Absorb those chemical compounds of inducing the remarkable cell death level of statistics that record by PI and can be chosen as the cell death inducing compounds.

In order to select apoptosis-induced chemical compound, use from the cell of galactophore of transgenic animal tumor tissues foundation and implement annexin in conjunction with test.As earlier paragraphs is discussed, with cell culture and be seeded in the culture dish.Removing culture medium then also only changes with fresh culture or with the culture medium that contains 10 μ g/ml antibody-drug conjugates (ADC).After three days the soak, wash cell monolayer, and separate by trypsinization and to adhere to PBS.Centrifuge cell is resuspended in Ca then ²⁺In the binding buffer liquid, and be distributed to and be used for cell death test in the above-mentioned test tube.Annexin (annexin V-FITC) (the 1 μ g/ml) for example that adds labelling then in the test tube.Sample can be used FACSCAN ^TMFlow cytometer and FACSCONVERT ^TMCellQuest software (Becton Dickinson) is analyzed.For contrast, induce on the statistics remarkable annexin to be chosen as the apoptosis induction chemical compound in conjunction with those chemical compounds of level.

The body outer cell proliferation test

Usually, by the cytotoxicity or the cell inhibitory activity of mensuration antibody-drug conjugates as described below (ADC): in cell culture medium, the mammalian cell that will have tumor associated antigen or receptor protein contacts with the antibody of ADC; About 6 hours of cultured cell is to about 5 days time; Measure cell viability (viability).Test is used to measure viability, the i.e. propagation (IC of ADC based on cells in vitro ₅₀), cytotoxicity (EC ₅₀) and apoptosis induction (Caspase activation).

The vitro efficacy of antibody-drug conjugates is by cell proliferation measurements determination (Fig. 1-4).CellTiter-Glo ^_Luminescent Cell Viability Assay is can be at (the PromegaCorp. of commercial utilization, Madison, WI) homogeneous phase test (homogeneous assay) method, (US 5583024 based on coleoptera (Coleoptera) luciferase recombinant expressed for they; US 5674713; US5700670).The test of this cell proliferation quantitatively is a living cells number in the fundamental measurement culture with existing ATP's, and ATP is indicator (Crouch et al (1993) J.Immunol.Meth.160:81-88 of metabolic activity cell; US 6602677).CellTiter-Glo ^_Test is implemented in 96 orifice plates, makes it to be easy to carry out automatization's high flux screening (HTS) (Cree (1995) AntiCancer Drugs 6:398-404).The homophase method of testing relates to directly with single agents (CellTiter-Glo ^_Reagent) add the cell of cultivation in being supplemented with the culture medium of serum to.Do not need the cell flushing, remove culture medium and a plurality of liquid step of moving.Adding reagent and mixing back 10 minutes, described system surveys 15 cells/well in the array of 384-hole.

The form of homogeneous phase " interpolation-mixing-mensuration " causes lysis, and the luminous signal that is directly proportional with the amount of existing ATP of generation.The amount of ATP directly is directly proportional with the cell number that exists in the culture.CellTiter-Glo ^_Test produces " wide variety of glow-type (glow-type) " luminous signal, and it is produced by luciferase reaction, has the half-life greater than five hours usually, and this depends on employed cell type and culture medium.Living cells reflects with relative light unit (RLU).Substrate B eetle fluorescein is by reorganization LUC Photinus pyralis LUC Photinus pyralis FL oxidative deamination, follows ATP to be converted into AMP simultaneously and produces photon.The half-life that prolongs has been eliminated the needs of use reagent syringe (reagent injector), and the motility that many flat boards are continuous or batch mode is handled is provided.Can with multiple porous array for example 96 or 384 hole arrays use the test of this cell proliferation.Can be with photometer or CCD camera imaging device recording data.Provide fluorescence output with relative light unit (RLU), As time goes on it measure.

At four kinds of different breast tumor cell lines, killed and wounded measurements determination the anti-proliferative effect of three kinds of antibody-drug conjugates (Fig. 1-4) by above-mentioned cell proliferation, cell in vitro.Fig. 1 shows, treatment S K-BR-3 (HER2 3+) breast tumor cell is after 3 days, the effectiveness measured value when improving trastuzumab-SPP-DM1, trastuzumab-SPDP-DM1 and trastuzumab-SMCC-DM1 concentration gradually.Fig. 2 shows, treatments B T-474 (HER2 3+) breast tumor cell is after 3 days, the effectiveness measured value when improving trastuzumab-SPP-DM1, trastuzumab-SPDP-DM1 and trastuzumab-SMCC-DM1 concentration gradually.Fig. 3 shows, handles MCF7 (HER2 is low) breast tumor cell after 3 days, the effectiveness measured value when improving trastuzumab-SPP-DM1, trastuzumab-SPDP-DM and trastuzumab-SMCC-DM1 concentration gradually.Fig. 4 shows, handles MDA-MB-468 (HER2 feminine gender) breast tumor cell after 3 days, the effectiveness measured value when improving trastuzumab-SPP-DM1, trastuzumab-SPDP-DM1 and trastuzumab-SMCC-DM1 concentration gradually.

Determined that known mistake expresses the SK-BR-3 of HER2 receptor protein and the IC of BT-474 ₅₀Value.For 6 tests at the SK-BR-3 cell, many conjugates, trastuzumab-SPP-DM1 (2.8 DM1 of each trastuzumab (medicine/Ab)) obtains the average IC of 14.4 μ g/ml ₅₀, its scope is 9.1 to 22.3 μ g/ml, at 4 tests of BT-474 cell, obtains the average IC of 51.7 μ g/ml ₅₀, its scope is 28.7 to 63.1 μ g/ml.At 4 tests of SK-BR-3 cell, many conjugate trastuzumab-SMCC-DM1 (2.7 DM1 of each trastuzumab (medicine/Ab)) obtain the average IC of 15.2 μ g/ml ₅₀, its scope is 12.6 to 18.8 μ g/ml, at 2 tests of BT-474 cell, obtains the average IC of 94.9 μ g/ml ₅₀, its scope is 75.2 to 114.6 μ g/ml.Cell MCF7 and the MDA-MB-468 non-activity of described conjugate to only expressing HER2.

Shown that with the Raji cell the intensive cell in vitro of anti-CD19-SMCC-DM1 kills and wounds (IC ₅₀=＜0.25 μ g/ml), wherein in the Raji cell naked antibody and the contrast ADC, trastuzumab-SMCC-DM1 do not show influence.Shown that with the Ramos cell anti-CD79a-SMCC-DM1 and the intensive cell in vitro of anti-CD79b-SMCC-DM1 kill and wound (IC ₅₀=＜0.25 μ g/ml), wherein in the Raji cell naked antibody and the contrast ADC, trastuzumab-SMCC-DM1 do not show influence.

Figure 17 shows the body outer cell proliferation test (embodiment 5) utilize HT1080EphB2 (C8) cell to implement, and described HT1080EphB2 (C8) cell is with anti-EphB2R 2H9 antibody-drug conjugates: 2H9-SPP-DM1 (IC ₅₀80ng/ml) and 2H9-SMCC-DM1 (IC ₅₀50ng/ml) handled.

Serum clearance rate and stability in the mice body

Naked be used to first test (nude naive) (transplants and accepts tumor) serum clearance rate and the stability of having studied ADC in the mice by external source.Fig. 5 shows that no trastuzumab-SMCC-DM1 tumor do not have the serum clearance rate in the cream-coloured nude mice of trastuzumab-SPP-DM1 tumor relatively, 7 day time in six time point determining conjugates and total antibody serum concentration.The cracking that total antibody and the ADC difference on amount shows joint and antibody separating from its DM1 part.As shown in higher coupling antibody serum concentration was in the 7th day the time, the time that the ADC that SMCC-connects is kept perfectly in vivo will be longer than the conjugate that SPP-connects.

Fig. 6 shows the stability that following conjugate is passed in time in the no tumor nude mice: trastuzumab-SPDP-DM1, trastuzumab-SPP-DM1, trastuzumab-SPP-DM3, trastuzumab-SPP-DM4 and trastuzumab-SMCC-DM1, six time point determining serum-concentrations in 7 days.The ADC that has the SMCC joint in vivo is more stable than the conjugate that is connected by SPP or SPDP, although trastuzumab-SMCC-DM1 has approximately and disulphide conjugate (the most hindered sulfideconjugate) stability that trastuzumab-SPP-DM4 is identical of obstruction.

Test shown in Fig. 5 and 6 is carried out in no tumor nude mice.Yet in having the nude mice of tumor, the conjugate that is connected with SPP is compared, and the conjugate that SMCC connects shows identical enhanced stability.As shown in Figure 7, handle after 7 days, initial trastuzumab-SMCC-DM1 of about 72% still is a conjugate, and after handling 7 days, have only initial trastuzumab-SPP-DM1 of about 10% still to be conjugate, shown trastuzumab-SMCC-DM1 conjugate enhanced stability in having the nude mice of tumor of non-zymetology cleavable.

Serum clearance rate and stability in the rat body

Fig. 8 and 9 shows disulphide joint ADC (trastuzumab-SPP-DM1) and non-disulphide joint ADC (trastuzumab-SMCC-DM1) relative stability in rat and clearance rate collection of illustrative plates.The parameter of research comprises:

Parameter	Trastuzumab-SPP-DM1	Trastuzumab-SMCC-DM1
			Vd(ml/kg	41	41
Clearance rate (ml/ days/kg)	52	15
			T 1/ 2alpha	0.09	0.15
T 1/ 2beta	0.7	0.85
			T 1/ 2Gamma (my god)	2.6	5.5

Compare with disulphide joint (disulfide linker) ADC trastuzumab-SPP-DM1 (Fig. 8), non-disulphide joint ADC trastuzumab-SMCC-DM1 (Fig. 9) shows better stability in rat blood serum.

Effect in the body

Can be by allograft or the xenograft of in rodent, implanting cancerous cell and the effect of measuring antibody-drug conjugates of the present invention with ADC processing tumor.Depend on antibody binding specificity, dosage regimen and the other factors of the receptor that exists on cell line, ADC and the cancerous cell, expected results is different.Body internal strength effectiveness high expressed HER2 transgenic explant (explant) mouse model of anti-HER2 ADC is measured.From Fo5 mmtv transgenic mice breeding allograft, described Fo5 mmtv transgenic mice is treated reactionless to HERCEPTIN or is reacted very weak.The experimenter handles once with ADC, and monitoring 3-6 week, to measure the time that tumor doubling time, cell killing logarithm and tumor are dwindled.Follow the tracks of dose-response and carry out a plurality of dosetests.

Tumor is easy to take place in the transgenic mice of expressing sudden change activated form neu, sudden change activated form neu is the rat homologue of HER2, but crossing the HER2 that expresses in breast carcinoma does not suddenly change, and expressing in the transgenic mice of not mutated HER2 the intensity much smaller (Webster et al (1994) Semin.Cancer Biol.5:69-76) that tumor forms excessively.

Form in order to strengthen tumor with not mutated HER2, produce transgenic mice with HER2 cDNA plasmid, wherein in HER2 cDNA plasmid, deleted upstream ATG, to stop translation initial at this upstream ATG codon place, otherwise it will reduce the frequency (for example, referring to Child et al (1999) J.Biol.Chem.274:24335-24341) that the HER2 start codon real from the downstream starts translation.In addition, add chimeric intron to 5 ' end, also will strengthen expression (Neuberger andWilliams (1988) Nucleic Acids Res.16:6713 as previous report; Buchman and Berg (1988) Mol.Cell.Biol.8:4395; Brinster et al (1988) Proc.Natl.Acad.Sci.USA 85:836).Described chimeric intron comes from the Promega carrier, pCI-neo mammalian expression vector (bp 890-1022).CDNA 3 ' end one side is human growth hormone's exon 4 and 5 and poly adenosine sequence.In addition, used the FVB mice, because this strain system produces more responsive to tumor.Promoter from MMTV-LTR is used for guaranteeing that tissue specificity HER2 expresses at mammary gland.For animal is fed AIN 76A feedstuff to strengthen the sensitivity (Rao et al (1997) Breast Cancer Res.and Treatment45:149-158) that tumor is formed.

Figure 10-13 shows that ADC has intensive anti-tumor activity in the allograft of HER2 positive tumor (Fo5), and described HER2 positive tumor (Fo5) originally occurs in the MMTV-HER2 transgenic mice.In this model, only antibody (for example trastuzumab) does not have notable antitumor activity (Erickson et al US 6632979).Shown in Figure 10 and 11, compare with contrast (carrier (vehicle)) level of growth, handle with ADC and blocked growth of tumor.Handle with trastuzumab-SMCC-DM1 and trastuzumab-SIAB-DM1 conjugate, tumor growth slows down at most.Shown in Figure 10,12 and 13, trastuzumab-SMCC-DM1 conjugate slows down greater than the conjugate with SPP joint tumor growth, promptly more effective, no matter be measure the time that tumor doubles in nude mice or measure corresponding to shown in the doubling time measured value logarithm cell killing (Log Cell Kill) (Figure 13).

Measured effect in the body of anti-CD22 ADC with the mouse tumor xenograft models.The 1st day (have indicated except), quantitatively give test group mouse anti CD22 antibody-drug conjugates or naked (naked) antibody once, eight SCID mices of each test group, every mice has 2,000 ten thousand Bjab-luc (the Bjab cell of expressing luciferase) xenograft tumor cells (embodiment 8).

ADC or Ab	μg DM1/m2	The Abmg/kg mice	Average drug loading (p)	MTD (my god)
					Tr-SMCC-DM1	200	4.2	3.2	3
7A2-SMCC-DM1	200	3.8	3.6	6
					5E8-SMCC-DM1	200	3.8	3.6	10
RFB4-SMCC-DM1	200	3.2	4.25	18
					RFB4-SMCC-DM1 (in the 1st, 7,14 day administration 3X)	405	10	2.75	55
7A2	-	4	-	3
					5E8	-	4	-	3
RFB4	-	4	-	3

Measured the time (MTD, average tumor doubling time) of tumor size doubles.(trastuzumab-SMCC-DM1), three kinds of naked anti-CD22 antibody do not show effect basically with respect to the ADC of non-specific binding.Corresponding conjugate all shows the effect of tangible retardance tumor growth.With the effect that RFB4-SMCC-DM1 has established multiple dosing, wherein the mice MTD for single-dose is 18 days, and is 55 days for the mice MTD that was administered three times in the 1st, 7 and 14 day.Tumor disappears in all 8 mices that occur in three administration groups fully.Have in the SCID mice of 2,000 ten thousand Bjab-luc xenograft tumor cells every mice, use single dose (400 μ g DM1/m2) after 7 days, wherein antibody is that (trastuzumab-SMCC-DM1) shows the delay of dwindling of initial tumor volume or tumor growth with respect to contrast for other anti-CD22-SMCC-DM1 conjugate of 12F7,9A8,8C9,8G10,3F11,10D2,6C9,14D1 and 11H10.Have in the SCID mice of 500 ten thousand Ramos RA1 xenograft tumor cells every mice, use single dose (200 μ g DM1/m2) after 11 days, (trastuzumab-SMCC-DM1) also is being effective aspect the retardance tumor growth with respect to contrast for anti-CD22 conjugate RFB4-SMCC-DM1,5E8-SMCC-DM1 and 7A2-SMCC-DM1.

On the test group of ten the every group SCID mices with Bjab-luc xenograft, studied conjugate RFB4-SMCC-DM1 (embodiment 8) with three kinds of different drug loadings.Low (1.95) and medium (3.7) drug loading conjugate shows the effect of tangible retardance tumor growth respectively, and MTD is about 15 days.High capacity (6.75) conjugate does not demonstrate the effect that obviously is different from contrast conjugate GP120-SMCC-DM1 or naked antibody RFB4.

Anti-CD22 ADC or Ab	μg DM1/m2	Ab mg/kg mice	Average drug loading (p)	MTD (my god)
					RFB4	-	10	-	3
RFB4-SMCC-DM1 (low load)	144	5	1.95	15
					RFB4-SMCC-DM1 (medium load)	273	5	3.7	15
RFB4-SMCC-DM1 (high capacity)	497	5	6.75	3
					GP120-SMCC-DM1 (high capacity)	449	5	6.1	3

Anti-CD19-SMCC-DM1 and anti-CD22-SMCC-DM1 conjugate do not show activity in vivo in Raji cell mouse tumor xenogeneic graft model.Other anti-CD19 and anti-CD22 conjugate can have activity in vivo at other cancerous cell tumor model.

Measured the interior effect of body of anti-CD79a (alpha) and anti-CD79b (beta) ADC with the mouse tumor xenograft models.The mouse test group administration that had 2,000 ten thousand Biab-luc xenograft tumor cells with the sample among the embodiment 8 after the following harmony in the exterior in the 1st day for every group of 8 SCID mices, every mice.

ADC, Ab or contrast	μg DM1/m2	Ab mg/kg mice	Average drug loading (p)	MTD (my god)
					PBS (buffer contrast)	-	-	-	3.5
Anti-GP120	-	3.2	-	3.5
					The anti-CD79b of SN8	-	3.1	-	4
The anti-CD79b of 17A7	-	3.1	-	4
					The anti-CD79a of 8H9	-	4.0	-	3
Anti-GP120-SMCC-DM1	200	3.2	4.2	3.5
					The anti-CD79b-SMCC-DM1 of SN8	200	3.1	4.4	＞7
The anti-CD79b-SMCC-DM1 of 17A7	200	3.1	4.4	＞7
					The anti-CD79a-SMCC-DM1 of 8H9	200	4.0	3.4	＞7

After 7 days, the anti-CD79b-SMCC-DM1 of conjugate SN8, the anti-CD79b-SMCC-DM1 of 17A7 and the anti-CD79a-SMCC-DM1 of 8H9 all demonstrate dwindle (the average 160mm3) of initial tumor volume.In the test group of 8 mices, the anti-CD79b-SMCC-DM1 of conjugate SN8 obtains part and disappears (PR) in 4 animals, obtain disappear fully (CR) in 2 animals.The anti-CD79b-SMCC-DM1 of conjugate 17A7 obtains CR in 1 animal.The anti-CD79a-SMCC-DM1 of conjugate 8H9 obtains PR in 2 animals, obtain CR in 1 animal.Have in the CB17 ICR SCID mice of 2,000 ten thousand Bjab-luc xenograft tumor cells every mice, use single dose (192 μ g DM1/m2) after 8 days, wherein antibody is that (trastuzumab-SMCC-DM1) shows the retardance of dwindling of initial tumor volume or tumor growth with respect to contrast for other anti-CD79b-SMCC-DM1 conjugate of 2F2,5C3,7H7,8D11,15E4 and 16C11.

Measured the dose response effect of the mice of using anti-CD79b-SMCC-DM1.The mouse test group administration that had 2,000 ten thousand Bjab-luc xenograft tumor cells with the sample in the following table (embodiment 8) in the 1st day for every group of 8 SCID mices, every mice.Anti-CD79b-SMCC-DM1 is with 0.5,2.0 and the horizontal administration of 3.64mg Ab/kg mice.

ADC, Ab or contrast	μg DM1/m2	The Abmg/kg mice	MTD (my god)
				Vehicle Control (PBS)	-		4
Anti-CD79b-SMCC-DM1	32	0.5	10
				Anti-CD79b-SMCC-DM1	130	2.0	35
Anti-CD79b-SMCC-DM1	236	3.64	＞70

Measured effect in the body of anti-tenb 2 ADC with the mouse tumor xenograft models.TENB2 is a tumor antigen, has shown that TENB2 almost only expresses and cross expression (Glynne-Jones et al (2001) Int J Cancer.Oct 15 in human prostate in the human prostate tumor; 94 (2): 178-84).PC3-TVA-919cv1:5 is for expressing the PC-3 of high-level TENB2.

Volume with every mice 0.2ml is nude mouse subcutaneous injection 500 ten thousand PC3-TVA-919 high expresseds or medium express cell.With cell suspension in HBSS.When the average tumor size reaches 100-200mm ³The time, mice is divided into 8 groups at random, every group has 8-10 mice respectively, and the single IV that gives following sample handles (embodiment 8).

ADC or contrast	μg DM1/m2	Ab mg/kg mice	Medium expression MTD (my god)	High expressed MTD (my god)
					PBS (vehicle Control)	-	-	30	22
Mouse-anti-artemisiifolia (ragweed)-SPP-DM1 (negative control)	373	6.68	18	22
					Mouse-anti-artemisiifolia-SMCC-DM1 (negative control)	373	9.34	14	18
Mouse-anti TENB2:3146-SPP-DM1	373	7.34	58	35
					Mouse-anti TENB2:3146-SMCC-DM1	373	10.6	44	19
Inosculating antibody TENB2-SPP-DM1	373	8.7	57	43
					Inosculating antibody TENB2-SMCC-DM1	373	7.63	43	22

With respect to negative control and vehicle Control, mouse-anti TENB2-DM1 conjugate shows the antitumor efficacy at the PC3-TENB2 tumor.With respect to negative control and vehicle Control, the anti-NaPi3b-SMCC-DM1 conjugate of Mus 10H1 does not show the antitumor efficacy at the PC3-NaPi3b tumor.Other antibody variants of anti-NaPi3b conjugate can have the activity in vivo at PC3-NaPi3b tumor or other cancerous cell line.

Rodent toxicity

In the acute toxicity rat model, assessed the contrast " carrier (vehicle) " of antibody-drug conjugates and no ADC.By handle female Sprague-Dawley rat with ADC, observe and analyze influence subsequently to multiple organ, so studied the toxicity of ADC.Based on total observed result (body weight), clinical pathology mathematic(al) parameter (serum chemistry and hematology) and histopathology, can observe, identify and measure the toxicity of ADC.Discovery is being equal to dosage level, compares with trastuzumab-SPP-DM1, and trastuzumab-SMCC-DM1 is relevant with littler acute toxicity.

Single injection (two dosage: 1860 and 3260 μ gDM1/m by trastuzumab-SMCC-DM1 ²), with disulphide ADC, trastuzumab-SPP-DM1 (two dosage: 1860 and 3260 μ gDM1/m ²), the single injection (the 0th day) of free DM1 maytansine (mercaptan) and control vector compares, and adolescence female rats (100-125gms) has been implemented 5-days studies on acute toxicity.Measure body weight every day.Carried out clinical chemistry, sero-enzyme and analysis of Hematology Changes in the 3rd day and the 5th day; Complete obduction by histopathological evaluation is drawn a conclusion together.The toxicity signal comprises the clinical observation of body weight loss.

After quantitatively giving ADC, for the animal that only gives carrier, it is total the indicating with generality of whole body or local toxicity that the weight of animals loss or body weight change.Figure 14 show 5 day the time body weight (gram) variation.The rat of accepting disulphide ADC, trastuzumab-SPP-DM1 shows tangible, dose-dependent toxicity, and this lethality and losing weight during than low dosage during by higher dosage displays.On the contrary, the rat of accepting trastuzumab-SMCC-DM1 has increased body weight, with respect to the rat that gives the placebo carrier, gives not show than the rat of low dosage the reduction of the speed that increases weight.Give the rat of higher level trastuzumab-SMCC-DM1, comparing with free DM1 cytotoxin has also increased body weight.

Increase and hepatic necrosis by the increasing of liver enzyme, mitosis and apoptosis sign number have been measured hepatotoxicity.Get involved by leukocyte, main granulocyte (neutrophil cell) and/or platelet loss and lymphatic organ, i.e. atrophy or apoptosis activity observation are to the toxicity of blood lymph (Hematolymphoid) system.Toxicity also shows as increase and the degenerative enteritis (degenerative entercolitis) of gastrointestinal disease if any silk division and apoptosis sign number.

The enzyme index of the hepatic injury of being studied comprises:

AST (aspartate transaminase)

-location: Cytoplasm; Liver, heart, skeletal muscle, kidney

-liver: 7000: 1 blood plasma ratio

-T1/2:17 hour

ALT (alanine aminotransferase)

-location: Cytoplasm; Liver, kidney, heart, skeletal muscle

-liver: 3000: 1 blood plasma ratio (plasma ratio)

-T1/2:42 hour; Change round the clock

GGT (g-glutamyl transferase)

-location: plasma membrane with cell of high secretion or absorbability; Liver, kidney, intestinal

The weak prediction thing of-hepatic injury; Usually in bile duct disease, raise

Three kinds of enzymes of said determination are not liver specificities.Find that ADC of the present invention causes instantaneous, the slight rising of liver enzyme ALT and AST and instantaneous skein cell to reduce (Figure 15).Do not observe peripheral blood granulocyte or hematoblastic influence (Figure 16).

Figure 15 and 16 shows, in this five days studies on acute toxicity, the rat that is exposed to 22.3mg/kg trastuzumab-SPP-DM1 (the 2nd group) demonstrates the most serious clinical toxicity.These animals demonstrate and the most serious lose weight, the rising of liver functional test value, leukocyte and thrombocytopenia and at the adenoid morphology toxicity of hemopoietic sign.Resulting result compares with previous use 25mg/kg Research on dose, and toxic degree is similar.On the contrary, respectively with 10 and the dosage of 25mg/kg to give the 3rd group of trastuzumab-SMCC-DM1 and the 4th treated animal can not be that the basis differentiates with clinical pathology and weight data with the animal of vehicle treated.In morphology, show in these animal liver that the MF number slightly increases, yet peripheral lymph and hemopoietic tissue are in normal range.

The animal of the 5th group of 50mg/kg trastuzumab-SMCC-DM1 demonstrates the toxicity sign.Yet except that a kind of liver functional test (ALT), the toxicity order of severity is less than accepting 50% and animal trastuzumab-SPP-DM1 same medicine dosage (the 2nd group).At about identical dosage, trastuzumab-SMCC-DM1 (the 4th group, 22.3mg/kg) show trastuzumab-SPP-DM1 of about 25% (the 2nd group, AST level 25mg/kg).To the 5th day of this research, the 5th treated animal showed weight increase (after the 3rd day and of short duration body weight loss in the 4th day), and serum bilirubin reduces and platelet count rising (Figure 15).

The animal (the 6th group) that is exposed to free maytansinoid DM1 demonstrates and the identical toxicity pattern of handling with the trastuzumab conjugate of animal.The medication amount of 10mg/kg trastuzumab-SPP-DM1 dosage that the dosage of this free DM1 gives in being equivalent to before study.The toxicity order of severity in the 6th treated animal sees less than the 2nd treated animal, but greater than in the animal of handling with 10mg/kg trastuzumab-SPP-DM1, being seen before.Seem that rehabilitation is quite fast: in the animal of handling with free maytansine, the spleen section shows that immature hemopoietic becomes dosis refracta to increase; Simultaneously, during by the 5th day, in the 6th treated animal, show that LFTs and clinical blood mathematic(al) parameter obviously are tending towards the trend of normalization.

Macaque toxicity/safety

Can assess toxicity and the safety of the ADC that is applied to macaque (Cynomolgus monkey).Carried out toxicity/safety research of antibody-drug conjugates trastuzumab-SMCC-DM1 with macaque.Studied trastuzumab-SMCC-DM1 that three groups of monkeys use with assessment intravenous injection toxicity with respect to contrast (carrier), the dosage of the trastuzumab-SMCC-DM1 that is injected progressively raises.The 1st group (4 experimenters) only accepted carrier (PBS, pH 6.5, promptly remove the preparation of ADC) at the 1st day and the 22nd day, after this carried out obduction in the 36th day.The 2nd group (4 experimenters) accepted trastuzumab-SMCC-DM1 4900 μ g/m2 in the 1st day and the 22nd day.The 3rd group (4 experimenters) accepted trastuzumab-SMCC-DM1 7200 μ g/m2 in the 22nd day.

Infer hepatotoxicity by the liver enzyme measured value that raises in the rodent toxicity research.Quantitatively give (the 2nd group: 4900 μ g/m of macaque carrier (the 1st group) and trastuzumab-SMCC-DM1 ²The 3rd group: 7200 μ g/m ²).Liver enzyme AST, platelet count, leukocyte, absolute neutrophil cell, erythrocyte and reticulocyte and 2 IV dosage regimens of having measured in macaque at trastuzumab-SMCC-DM1 are done contrast (embodiment 10).

Using of antibody-drug conjugates pharmaceutical preparation

Therapeutic antibodies-drug conjugates (ADC) can be used by any approach that is suitable for the disease that will treat.Typically, parenteral is in infusion, subcutaneous, intramuscular, intravenous, Intradermal, the sheath, injects in (bolus), the tumor injection fast or ADC (Shire et al (2004) J.Pharm.Sciences 93 (6): 1390-1402) is used in epidural.The pharmaceutical preparation of therapeutic antibodies-drug conjugates (ADC) typically is used for the parenteral dispenser in company with pharmaceutically acceptable parenteral preparing carriers, and is injectable unit dosage form.Have that the antibody-drug conjugates (ADC) of required purity is optional to be mixed with pharmaceutically acceptable diluent, carrier, excipient or stabilizing agent, can be lyophilized formulations or aqueous solution form (Remington ' s Pharmaceutical Sciences (1980) 16th edition, Osol, A.Ed.).

Acceptable parenteral vehicle, diluent, carrier, excipient and stabilizing agent are nontoxic at used dosage and concentration to the experimenter, and it comprises buffer such as phosphate, citrate and other organic acid; Antioxidant comprises ascorbic acid and methionine; Antiseptic (as eight the last of the ten Heavenly stems dimethyl benzyl ammonium chloride; Hexamethonium chloride; Benzalkonium chloride, benzethonium chloride; Phenol, butyl or benzyl alcohol; Alkyl parabens class such as methyl or propyl group metagin; Catechol; Resorcinol; Hexalin; The 3-amylalcohol; And metacresol); Low-molecular-weight (less than about 10 residues) polypeptide; Protein is as serum albumin, gelatin or immunoglobulin; Hydrophilic polymer such as polyvinylpyrrolidone; Aminoacid such as glycine, glutamine, agedoite, histidine, arginine or lysine; Monosaccharide, disaccharide and other carbohydrate comprise glucose, mannose or dextrin; Chelating agen such as EDTA; Sugar is as sucrose, mannitol, trehalose or sorbitol; Salifiable counter ion of shape such as sodium; Metal composite (for example zinc-protein complex); And/or non-ionic surface active agent such as TWEEN ^TM, PLURONICS ^TMOr Polyethylene Glycol (PEG).For example, freeze dried anti-ErbB antibody preparation has been described among the WO 97/04801, the special adding as a reference herein.The exemplary formulation of ADC such as trastuzumab-SMCC-DM1 comprise about 100mg/ml trehalose (2-(methylol)-6-[3,4,5-trihydroxy-6-(methylol) Pentamethylene oxide .-2-yl] oxygen-Pentamethylene oxide .-3,4, the 5-triol; C ₁₂H ₂₂O ₁₁CAS 99-20-7 number) and about 0.1%TWEEN ^TM20 (multi-sorbitol esters 20; Lauric acid 2-[2-[3,4-two (2-hydroxy ethoxy) oxolane-2-yl]-2-(2-hydroxy ethoxy) ethyoxyl] ethyl ester; C ₂₆H ₅₀O ₁₀CAS 9005-64-5 number), pH is about 6.

The pharmaceutical preparation of therapeutic antibodies-drug conjugates (ADC) can comprise a certain amount of drug moiety (D), antibody-joint intermediate (Ab-L) and/or medicine-joint intermediate (D-L) of not reacting, its be in the ADC preparation process the incomplete purification of excess reagent, impurity and side-product with separate; Perhaps in the ADC storing process in batches or be subjected in the preparation ADC compositions storing process time/result of temperature effect hydrolysis or degraded.For example, but ADC preparation trastuzumab-SMCC-DM1 can comprise the free drug DM1 of detection limit.Perhaps, or in addition, but it can comprise the medicine-joint intermediate DM1-SMCC of detection limit.Perhaps, or in addition, but it can comprise the antibody trastuzumab of detection limit.The exemplary formulation of trastuzumab-SMCC-DM1 can comprise the DM1-SMCC until 10% molar equivalent.Unexpectedly, through body outer cell proliferation measurements determination (embodiment 5), for SK-BR-3 and BT-474 breast cancer cell, with free drug DM1 (IC ₅₀0.0045 μ M) compare DM1-SMCC (IC ₅₀0.05 μ M) effectiveness is approximately little 20 times aspect cell killing.

For example, also can active pharmaceutical ingredient be packaged in the prepared microcapsule by condensation technique or interfacial polymerization, for example be packaged in respectively in hydroxy methocel or gelatin-microcapsule and poly-(methyl methacrylate) microcapsule, be packaged in gluey drug delivery system (for example, liposome, albumin microsphere, microemulsion, nano-particle and Nano capsule) or be packaged in the bulky grain emulsion (macroemulsion).These technology are disclosed in Remington ' s Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980).

Can prepare the slow release goods.The example of suitable slow release goods comprises the solid-state hydrophobic polymer semi permeability substrate that comprises ADC, and its mesostroma is the shaped particles product form, for example thin film or microcapsule.The example of sustained-release matrix comprises polyester, hydrogel (for example, poly-(2-ethoxy-acrylic acid methyl ester .) or polyvinyl alcohol, polyactide (US 3773919), L-glutamic acid and the copolymer of gamma-ethyl-L-glutamic acid, nondegradable ethylene-vinyl acetate, degradable poly lactic coglycolic acid such as LUPRON DEPO ^TM(Injectable microspheres of forming by poly lactic coglycolic acid and leuprorelin acetate) and poly--D-(-)-3-hydroxybutyric acid.

The preparation that is used for using in the body must be aseptic, and this is easy to filter realization by sterile filtration membrane.

Described preparation comprises that those are suitable for the preparation of aforementioned drug delivery route.Described preparation can provide with unit dosage form easily, can prepare by the method that any pharmaceutical field is known.Technology and preparation be found in usually Remington Pharmaceutical Sciences (Mack Publishing Co., Easton, PA).Such method comprises active component and the carrier-bound step of forming one or more helper components.Substantially, active component and liquid carrier or solid-state carrier or both homogeneous in small, broken bits are closely combined, then, if necessary,, make described preparation thus product shaping.

Water slurry comprises the active material (ADC) with mixed with excipients, and described excipient is suitable for producing water slurry.Such excipient comprises suspending agent, as sodium carboxymethyl cellulose, croscarmellose, polyvidone, methylcellulose, hydroxypropyl emthylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth and Radix Acaciae senegalis, with the phospholipid of dispersion or wetting agent such as natural generation (for example, lecithin), the condensation product of epoxyalkane (ethylene oxide) and fatty acid (for example, Myrj 45), the condensation product of oxirane and long-chain fatty alcohol (for example, heptadecaethyleneoxycetanol), oxirane and the condensation product (for example, polyoxyethylene sorbitol monoleate) that comes from the partial ester of fatty acid and hexitan.Water slurry can also comprise one or more antiseptic such as ethyl or n-pro-pyl p-hydroxy-benzoic acid ester, one or more coloring agent, one or more flavoring agents and one or more sweeting agents, as sucrose or glucide.

The pharmaceutical composition of ADC can be for sterile injectable goods form, as the water or the oleagenous suspension of sterile injectable.This suspension can utilize the preparation of those suitable dispersants already mentioned above or wetting agent and suspending agent according to known technique.Described sterile injectable goods also can be for being dissolved in the nontoxic parenteral acceptable diluent or the sterile injectable solution or the suspension of solvent, as be dissolved in 1, the solution of 3-butane-glycol or make freeze-dried powder.Availablely accept carrier and solvent is water, RingerShi solution and isotonic sodium chlorrde solution.In addition, aseptic fixed oil can be used as solvent or suspension media traditionally.For this purpose, can use the fixed oil of any gentleness, comprise synthetic list or diacylglycerol fat.In addition, fatty acid such as oleic acid can be used for the preparation of injectable product equally.

Can will change according to main body of being treated and specific application mode with the amount of carrier material combination with the active component of production single dose form.For example, the aqueous solution that is used for intravenous infusion can comprise the active component of about 3 to the 500 μ g of every ml soln, so that can produce the infusion of about 30mL/hr speed of suitable volumes.Can finish subcutaneous (fast notes (bolus)) with the concentration of about 1.5ml or littler cumulative volume and the every ml of about 100mg ADC uses.For the frequent also ADC of chronic administration of needs, can make subcutaneouly, as syringe or automated injection device technology by pre-filling.

As general recommendation, the pharmaceutically initial effective dose of each dosage of ADC of being used will be in about 0.01-100mg/kg scope, and promptly about 0.1 to 20mg/kg weight in patients every day, wherein the typical initial range of compound used therefor is about 0.3 to 15mg/kg/ day.For example, can be the human patients dosed administration originally with the every kg weight in patients of about 1.5mg ADC.Described dosage can progressively be elevated to maximum tolerated dose (MTD).Dosage regimen can be per approximately 3 weeks, but according to disease of being diagnosed or reaction, and described scheme can be more frequently or be more not frequent.In therapeutic process, described dosage can further be adjusted to and be equal to or less than MTD, its can be safely cyclical administration repeatedly, 4 times or more times according to appointment.

The preparation that is suitable for the parenteral dispenser comprises water and non-water aseptic parenteral solution, and it can comprise antioxidant, buffer, antibacterial and make described preparation and the isoosmotic solute of blood of desiring the experimenter; With water and non-water sterile suspensions, it can comprise suspending agent and thickening agent.

Although common disapprove oral protein therapeutic agent, because limited intestinal (gut) absorption, hydrolysis or degeneration cause low bioavailability, can be prepared as capsule, cachet or the tablet of dispersive unit as the ADC that all comprises the amount of pre-determining but be suitable for oral ADC preparation.

Described preparation can be packaged in unit dose or the multi-dose container, for example in Mi Feng ampoule bottle and the phial, can be kept under cryodesiccated (freeze dried) condition, this condition only need be added aseptic liquid carrier before will using, and for example water is used for injection at once.Instant injection and suspension are from sterilized powder, granule and the pharmaceutical tablet of described type before.Exemplary unit dose formulations comprises the dosage or unit sub-doses every day (unit daily sub-dose) or its suitable part every day of active component.

The present invention further provides veterinary compositions, it comprises at least a active component as defined above together with veterinary's carrier.Veterinary's carrier is the material that is used for the applying said compositions purpose, and it can be solid-state, liquid or gas material, and it also should be inert or acceptable in veterinary field in addition, and can be compatible with described active component.These veterinary's compositionss can parenteral, oral or use by any other required approach.

The antibody-drug conjugates treatment

Antibody-drug conjugates of the present invention will can be used for treating multiple disease or disorder, as cancer and autoimmune disorder.Exemplary disease or disorder comprise optimum or malignant tumor; Leukemia and lymph malignant disease; Other disease such as neuron, neuroglia, spider cell, hypothalamus, body of gland, macrophage (macrophagal), epithelium, substrate, blastaea (blastocoelic), inflammatory, angiogenesis and amynologic disease.To the responsive cancer of ADC treatment comprise with specific tumors related antigen or cell surface receptor for example HER2 cross those cancers that are expressed as feature.

The ADC chemical compound of identifying in animal model and the test based on cell can further have among the high primates of tumor and the human clinical trial to be tested.The human clinical trial can be designed as be similar to the test HER2 cross among the metastatic breast cancer patient of expression, the clinical trial of the effect of anti-HER 2 monoclonal antibody HERCEPTIN, wherein said patient had before accepted the significant antitumor treatment, reported as Baselga et al. (1996) J.Clin.Oncol.14:737-744.Can design clinical trial with assessment ADC and known treatment scheme, as radiation and/or relate to the combined effect (Pegram et al (1999) Oncogene 18:2241-2251) of chemotherapy of known chemotherapeutics and/or cytotoxic reagent.

Usually, the disease that treat or disorder are cancers.The example of the cancer that this place will be treated includes but not limited to cancer (carcinoma), lymphoma, blastoma (blastoma), sarcoma (sarcoma) and leukemia or lymph malignant disease.This cancer example more specifically comprises that squamous cell carcinoma (for example, squamous cell cancer), pulmonary carcinoma, comprise small cell lung cancer, nonsmall-cell lung cancer (" NSCLC "), adenocarcinoma of lung and squamous cell lung carcinoma, peritoneal cancer, hepatocarcinoma, stomach or stomach cancer comprise human primary gastrointestinal cancers, gastrointestinal stromal tumors (GIST), cancer of pancreas, glioblastoma, cervical cancer, ovarian cancer, the liver cancer, bladder cancer, hepatoma (hepatoma), breast carcinoma, colon cancer, rectal cancer, colorectal carcinoma, endometrium or uterus carcinoma, the glandula cancer, kidney or renal carcinoma, carcinoma of prostate, cancer of vagina, thyroid carcinoma, hepatocarcinoma (hepatic carcinoma), anus cancer, carcinoma of penis, and head and neck cancer.

It can be by ErbB receptor a kind of cancer of identifying of the overactivity of HER2 for example that cancer will be treated in this place.This overactivity may express or generate owing to crossing of ErbB receptor or ErbB part to be increased.In one embodiment, will diagnose or whether prognosis test is feature with the overactivity of ErbB receptor with the cancer of measuring the patient.For example, in cancer, can measure the ErbB gene amplification of ErbB receptor and/or expression excessively.This area has multiple being used to measure the method for testing that this amplification/mistake is expressed, and comprises above-mentioned IHC, FISH and released antigen test.Perhaps, or in addition, can measure in the tumor or the ErbB part relevant according to known method, as the level of TGF-alpha with tumor.Such test can be surveyed the protein in the sample that will test and/or encode its nucleic acid.In one embodiment, the ErbB ligand level in the tumor can utilize SABC (IHC) to measure; Referring to, for example, Scher et al. (1995) Clin.Cancer Research 1:545-550.Perhaps, or in addition, can assess the level of ErbB part code nucleic acid in the sample that will test; For example by FISH, southern trace or round pcr.In one embodiment, can analyze ErbB2 with IHC and cross expression, for example use HERCEPTEST (Dako).Can be to paraffin-embedded tissue section carrying out IHC test from the tumor slicer, the ErbB2 protein staining strength criterion of institute's foundation is as follows: mark 0, do not observe dyeing or in less than 10% tumor cell, observe film dyeing; Mark 1+ detects the fuzzy/film dyeing that can distinguish dimly in surpassing 10% tumor cell, described cell is some film dyeing only; Mark 2+ observes the weak complete film dyeing to moderate in surpassing 10% tumor cell; Mark 3+ observes moderate to intensive complete film dyeing in surpassing 10% tumor cell.ErbB2 cross express the assessment mark be 0 or those tumors of 1+ can be accredited as and do not cross expression ErbB2, and mark to be those tumors of 2+ or 3+ can be accredited as expression ErbB2.

Perhaps, or in addition, can carry out the FISH test as INFORM to tumor tissues formalin fixed, paraffin-embedded ^TM(Ventana Co., Ariz.) or PATHVISION ^TM(Vysis, 111.), ErbB2 crosses the degree (if any) of expression in the tumor to measure.

In addition, ErbB receptor or ErbB part cross expression or amplification can utilize the in-vivo diagnostic testing evaluation, for example by using, and from the external scan patient in conjunction with the molecule that will detect and the molecule (as antibody) that has detectable marker (for example radiosiotope) to locate described labelling.

Be prevention or treatment disease, the suitable dose of ADC will depend on type, the disease of the disease that will treat as defined above the order of severity and the course of disease, use whether described molecule is used to prevent or therapeutic purposes, former treatment, patient's medical history and to the reaction of described antibody and attending doctor's judgement.Described molecule is suitable for once or is applied to the patient in a series of treatment.According to the type and the order of severity of disease, no matter be that for example one or many is used separately, or continuous infusion, the initial candidate dosage that is applied to the patient all is the molecule of about 1 μ g/kg to 15mg/kg (for example 0.1-20mg/kg).According to above-mentioned factor, typically every day, dosage can be about 1 μ g/kg to 100mg/kg or more.The exemplary dose of ADC in the patient to be applied about 0.1 to about 10mg/kg weight in patients scope.

For several days or repetitive administration for more time, according to described disease, described treatment will last till always that required disease symptoms obtains desired inhibition.The exemplary dose dosage regimen comprises the original upload dosage of using about 4mg/kg, the maintenance dose of about weekly afterwards 2mg/kg anti-ErbB antibody.Also can use other dosage regimen.The progress of this treatment can be easily by conventional art and method of testing monitoring.

Therapeutic alliance

Antibody-drug conjugates (ADC) can be combined into medicine composition with second kind of chemical compound with anticancer property or be combined as the dosage regimen of conjoint therapy.Second kind of chemical compound of described medicine composition or dosage regimen preferably have with the combination in the complementary activity of ADC, in order to avoid they have a negative impact mutually.

Second kind of chemical compound can be chemotherapeutics, cytotoxic reagent, cytokine, growth inhibitor, antihormone agent, aromatase inhibitor, kinases inhibitor, lipid kinase inhibitors, androgen antagonist, antisense oligonucleotide, ribozyme, gene therapeutic vaccine, anti-angiogenic rebirth agent and/or heart protective agent.Such molecule is fit to provide so that predeterminated target is effectively measured in composition of medicine.The pharmaceutical composition that comprises ADC also can have the chemotherapeutics such as the tubulin for the treatment of effective dose and form inhibitor, topoisomerase enzyme inhibitor or DNA conjugate.

Perhaps, or in addition, second kind of chemical compound can be the antibody in conjunction with the ligand activation of ErbB2 and blocking-up ErbB receptor.Second antibody can be monoclonal antibody 2C4 or humanization 2C4 " Omnitarg " (WO 01/00245).Second antibody can with cytotoxic agent or chemotherapeutics, for example maytansinoid, auristatin, calicheamicin or 1,8 two-naphthalimide part coupling.For example, may be desirably in the antibody that further provides in a kind of preparation or the dosage regimen in conjunction with EGFR, ErbB2, ErbB3, ErbB4 or blood vessel endothelial factor (VEGF).

Other therapeutic scheme can make up with the anticarcinogen of using according to the present invention's evaluation.Combined therapy can be used as scheme simultaneously or order and uses.When order when using, described combination can twice or repeatedly use in use.Use co-administered comprising simultaneously, and it utilizes preparation or single pharmaceutical preparation separately, also comprises with any order continuous administration, wherein preferably has two kinds of (or owning) active agents and bring into play the period of its biologic activity simultaneously.

In one embodiment, relate to anticarcinogen and one or more chemotherapeutics or the growth inhibitor that combined administration is identified herein with ADC treatment of the present invention, comprise the cocktail sample mixture of using different chemotherapeutics jointly, choose wantonly and treat with anti-ErbB antibody such as trastuzumab.Chemotherapeutics comprises hydrochloric acid Erlotinib (CP-358774, TARCEVA ^TMGenentech/OSI), taxane (as paclitaxel and Docetaxel) and/or anthracycline antibiotics.The preparation of this chemotherapeutics and dosage regimen can use according to the explanation of manufacturer, perhaps according to the rule of thumb determined use of those of skill in the art.Chemotherapy Service Ed., M.C.Perry, Williams ﹠amp; Wilkins, Baltimore has also described the preparation and the dosage regimen of this chemotherapeutics among the Md. (1992).

Anticarcinogen can make up with the hormone antagonist chemical compound; For example estrogen antagonist chemical compound such as tamoxifen; Anti-Progesterone such as onapristone (EP 616812); Perhaps androgen antagonist such as flutamide (flutamide) wherein use with the known dosage of these molecules.The cancer that will treat for the irrelevant cancer of hormone in, described patient can accept the hormone antagonist treatment in advance, and, cancer become with hormone irrelevant after, can use anti-ErbB antibody (and optional other reagent described herein) for the patient.For the patient uses heart protective agent (with prevention or reduce and treat relevant myocardial dysfunction) or one or more cytokines may be useful jointly.Except above-mentioned Therapeutic Method, can implement the surgical removal cancerous cell to the patient, and/or implement X-ray therapy.

The suitable dose of any above-mentioned reagent of using jointly is those dosage of present use, and because the synergy (synergism) of the new reagent of identifying and other chemotherapeutics or treatment, described dosage can decrease.

Described combined therapy can provide " synergism " and confirm " synergistic ", and promptly the effect that is obtained when together using described active component is greater than respectively with the summation of the effect of described chemical compound acquisition.When described active component: (1) is prepared jointly and is used simultaneously or send with the unit dose formulations form of combination; (2) alternately or parallel sending as different preparations; Or (3) can obtain cooperative effect when using by some other scheme.When sending with rotational therapy, chemical compound is used or sent to order, for example when separately injecting, can obtain cooperative effect in different syringes.Substantially, during alternating treatment, order is the various active component of continuous administration effective dose, and in combined therapy, two or more active component of effective dose are used together.

The metabolite of antibody-drug conjugates

The interior metabolism product of ADC chemical compound described herein also falls within the scope of the present invention, and described scope comprises until such product and is new and is non-obvious degree with respect to prior art.Such product can for example come from oxidation, reduction, hydrolysis, amidatioon, esterification, zymetology cracking of institute's administered compound or the like.Correspondingly, the present invention includes the new and non-obvious chemical compound of producing by described method, this method comprises chemical compound of the present invention is contacted with mammal that the time that is contacted is enough to produce its metabolite.

Can by the preparation labelled with radioisotope (for example ¹⁴C or ³H) ADC, with can detect dosage (for example greater than about 0.5mg/kg) with its parenteral administration in animal such as rat, mice, Cavia porcellus, monkey or be applied to the people, the time (typically about 30 seconds to 30 hours) that metabolism is enough to take place is provided and separates its converted product from urine, blood or other biological sample and identify metabolite.These products are easy to separate, because they are through labelling (other the antibody that passes through to utilize the epi-position that retains in can be in conjunction with metabolite separates).Measure the metabolite structure example in a usual manner as analyzing by MS, LC/MS or NMR.Substantially, the analysis of metabolite is finished in the mode identical with conventional medicament metabolism research well known to those skilled in the art.Described converted product is as long as their discoveries in vivo just can be used in the diagnostic test of ADC compounds for treating administration.

Metabolite comprises cracked product in the ADC body, and wherein cracking occurs in any key that drug moiety is connected to antibody.Therefore the metabolic cracking can produce naked antibody (naked antibody) or antibody fragment.The antibody metabolite can be connected to a part or the whole joint of joint.The metabolic cracking also may cause producing the part of drug moiety (drug moiety) or this drug moiety.The metabolite of drug moiety can be connected to a part or the whole joint of joint.

Goods

In another embodiment, provide goods or " test kit " that comprises ADC and be used for the treatment of the material of above-mentioned disorder.Described goods comprise label or package insert on container and the container or that link to each other with container.Suitable containers comprises for example bottle, phial, syringe or blister-pack.Described container can be made of multiple material such as glass or plastics.Described container is equipped with the treatment effective antibody-drug conjugates of selected disease (ADC) compositions, can have aseptic access port (for example, described container can be intravenous solution bag or phial, and described phial has the stopper of available subcutaneous injection needle penetration).At least a active agent is ADC in the compositions.Label or package insert indicate described compositions and are used for the treatment of selected disease such as cancer.In one embodiment, label or package insert indicate the compositions that comprises in conjunction with the antibody of ErbB2 and can be used for treating cancer, and described cancer is expressed the ErbB receptor that is selected from EGF-R ELISA (EGFR), ErbB2, ErbB3 and ErbB4.In addition, label or package insert can indicate the patient that will treat and suffer from cancer, and described cancer is a feature with the ErbB receptor overactivity that is selected from EGFR, ErbB2, ErbB3 or ErbB4.For example, described cancer can be for crossing a kind of cancer of expressing these receptors and/or crossing one of expression ErbB part (as TGF-α).Label or package insert also can indicate described compositions and can be used for treating cancer, and wherein said cancer is not expressed as feature with the mistake of ErbB2 receptor.In other embodiments, package insert can indicate cancer, carcinoma of prostate, colon cancer or the colorectal carcinoma that the ADC compositions also can be used for the treatment of and hormone has nothing to do.

Described goods can comprise first container that (a) wherein contains chemical compound, and wherein said chemical compound comprises ADC of the present invention, and wherein the antibody of ADC is in conjunction with ErbB2 and suppressed to express the first antibody of the growth of cancer cells of ErbB2; (b) wherein contain second container of chemical compound, wherein said chemical compound comprises the second antibody in conjunction with the ligand activation of ErbB2 and blocking-up ErbB receptor, or the conjugate of this second antibody and maytansinoid.Goods in this embodiment can further comprise package insert, and described package insert indicates first kind and second kind of chemical compound and can be used for treating cancer.Perhaps, or in addition, described goods can further comprise second (or 3rd) container, and this container comprises pharmaceutically acceptable buffer, as injection bacteriostatic water (BWFI), phosphate buffered saline (PBS), RingerShi solution and dextrose solution.It may further include from other required material of viewpoint of commercial and user, comprises other buffer, diluent, filter, syringe needle and syringe.

Embodiment

Production, evaluation and the humanization of embodiment 1-anti-ErbB monoclonal antibody 4D5

Specificity in conjunction with the mouse monoclonal antibody 4D5 of ErbB2 ectodomain as production as described in Fendly et al (1990) the Cancer Research 50:1550-1558.Briefly, with the NIH 3T3/HER2-3 of phosphate buffered saline (PBS) (PBS) results that contain 25mM EDTA as production as described in Hudziak et al (1987) Proc.Natl.Acad.Sci. (USA) 84:7158-7163 ₄₀₀Cell (expresses about 1 * 10 ⁵And be used for immune BALB/c mouse individual ErbB2 molecule/cell).Be 10 among the mice i.p. injection 0.5ml PBS in the 0th, 2,5 and 7 weeks ⁷Individual cell.Inject (WGA) the ErbB2 film extract of purification of wheat germ agglutinin-agarose gel (wheat germ agglutinin-sepharose) in the 9th and 13 weeks for mice i.p., described mice has immunoprecipitation ³²The antiserum of the ErbB2 of p labelling.After this ErbB2 goods of i.v. injection 0.1ml are that X63-Ag8.653 merges with splenocyte and mouse myeloma.Screen the hybridoma supernatant according to ErbB2-in conjunction with usefulness ELISA and radioimmunoprecipitation.

Mouse monoclonal antibody 4D5 adopts " gene conversion mutation (gene conversion mutagenesis) " tactful humanization, as described in US 5821337, herein with the special adding of its full content as a reference.Below the Humanized monoclonal antibodies 4D5 that uses in the test is called huMAb4D5-8.This antibody is the IgG1 isotype.

The purification of embodiment 2-trastuzumab

Contain 440mg HERCEPTIN_ (huMAb4D5-8, rhuMAb HER2 with one, US5821337) antibody) phial is dissolved in 50mL MES buffer (25mM MES, 50mM NaCl, pH 5.6) in, last sample is at balance is crossed in same buffer cation exchange column (on the Sepharose S, 15cm * 1.7cm).Use same buffer (5 pillar volumes) flushing pillar then.By the NaCl concentration of buffer is brought up to 200mM eluting trastuzumab.Collection comprises the fraction of described antibody, is diluted to 10mg/ml, carries out dialysis in the buffer that contains 50mm potassium phosphate, 50mM NaCl, 2mM EDTA, pH6.5.

Embodiment 3-antibody-drug conjugates: the preparation of trastuzumab-SPP-DM1:

With the derive trastuzumab of purification of N-butanimide-4-(2-pyridine sulfo-) valerate, to introduce dithio pyridine (dithiopyridyl) base.Handle trastuzumab with SPP (5.3 molar equivalents in the 2.3mL ethanol), (376.0mg 8mg/ml) is dissolved in the kaliumphosphate buffer (pH 6.5) of the 44.7mL 50mM that contains NaCl (50mM) and EDTA (1mM) trastuzumab.After environment temperature is incubated 90 minutes in argon, the Sephadex G25 pillar gel filtration of reactant mixture by crossing with 35mM sodium citrate, 154mM NaCl, 2mM EDTA balance.Collect and test the fraction that contains antibody.Measure the degree of modification of antibody as mentioned above.(response rate of trastuzumab-SPP-Py) is 337mg (89.7%) to modified antibodies, and wherein each antibody (P ') connects 4.5 releasable 2-sulfo-pyridine radicals.

With above-mentioned 35mM sodium citrate buffer solution, pH 6.5 trastuzumab-SPP-Py (337.0mg, releasable 2-sulfo-pyridine (thiopyridine) base of 9.5 μ mols) is diluted to final concentration 2.5mg/mL.Add the DM1 (N that is dissolved in the 3.0mM dimethyl acetylamide (DMA, 3%v/v in the final reacting mixture) to antibody-solutions then ^{2 '}-deacetylation-N ^{2 '}-(3-sulfydryl-1-oxo propionyl)-maytansine) (1.7 equivalents, 16.1 μ mols), its structure as shown in Figure 1.Being reflected at environment temperature carried out 20 hours in argon.

With sample on the reactant liquor the Sephacryl S300 solvent resistant column of crossing with 35mM sodium citrate, 154mM NaCl, pH 6.5 balances (5.0cm * 90.0cm, 1.77L) on.Flow velocity is 5.0mL/min, has collected 65 fraction (each 20.0mL).Main peak is divided into center (Fig. 3) with No. 47 levels.Main peak comprises monomer trastuzumab-SPP-DM1.Collect and test fraction 44-51.Measure the number of the DM1 drug molecule that each antibody molecule is connected (p ') by measuring 252nm and the light absorption value at 280nm place, be found to be 3.7 drug molecules of each antibody molecule.

Embodiment 4-antibody-drug conjugates: the preparation of trastuzumab-SMCC-DM1:

With (butanimide 4-(N-maleimide methyl) cyclohexane extraction-1-carboxylate, SMCC, PierceBiotechnology, Inc) the derive trastuzumab of purification is to introduce the SMCC joint.As described in the embodiment 2 from HERCEPTIN_ purification trastuzumab, and with the SMCC of 7.5 to 10 molar equivalents (20mM among DMSO or the DMA (dimethyl acetylamide) 6.7mg/mL) handles with the exchange of 20mg/mL buffering in 50mM potassium phosphate/50mM sodium chloride/2mM EDTA, pH 6.5.Stir under the environment temperature in argon after 2 to 4 hours, reactant mixture filters by the Sephadex G25 pillar of crossing with 50mM potassium phosphate/50mM sodium chloride/2mM EDTA, pH 6.5 balances.Perhaps, use 30mM citrate and 150mM sodium chloride in pH 6 gel filtration reactant mixtures.Collect and test the fraction that contains antibody.The response rate of trastuzumab-SMCC is 88%.

More than the medicine of Huo Deing-joint intermediate trastuzumab-SMCC is diluted to final concentration 10mg/mL with 50mM potassium phosphate/50mM sodium chloride/2mM EDTA, pH 6.5, (1.7 equivalents have 5 SMCC/ trastuzumabs, 7.37mg/ml) reaction with the solution of 10mM DM1 in dimethyl acetylamide.Under the environment temperature in argon stirring reaction liquid 4 to about 16 hours.(1.5 * 4.9cm) filter the coupling reaction mixture by Sephadex G25 solvent resistant column in pH 6.5 with 1 * PBS.Perhaps, use 10mM succinate and 150mM sodium chloride in pH 5 gel filtration reactant mixtures.The DM1/ trastuzumab is 3.1 than (p), and its light absorption value by 252nm and 280nm place records.Medicine also can be measured by mass spectral analysis than (p) with antibody.Also can monitor coupling by sds polyacrylamide gel electrophoresis.Can assemble by the laser light scattering analysis and evaluation.

Antibody-SMCC-DM1 conjugate:

Follow this scheme, prepared other antibody-drug conjugates, comprising with SMCC joint and DM1 drug moiety:

Antibody-drug conjugates (ADC)	Antibody (Ab)	Antigen	Average drug loading (DM1/Ab)
				10H-SMCC-DM1	10H1 (Mus) resists-NaPi3b	NaPi3b	5.0
2H9-SMCC-DM1	2H9 Mab	Anti--EphB2R	4.0
				Artemisiifolia (ragweed)-SMCC-DM1	Artemisiifolia		2.7
Anti-tenb 2 .SMCC-DM1	Anti--TENB2-3416#2 mice MAb	TENB2	2.5
				Anti--TENB2 20D1 Fc chimera-SMCC-DM1	Chimeric people mouse anti-TENB2	TENB2	3.32
Anti-CD19-SMCC-DM1	Anti-CD19 (mIgG1 isotype)	CD19	5.05
				2H7-SMCC-DM1	Anti-CD20 (mIgG2a isotype)	CD20	2.44,3.84 (two batches)
CD20 LC-SMCC-DM1	Anti-CD20 (Rituxan_)	CD20
				HB5-SMCC-DM1	Anti-CD21 (mIgG2a isotype)	CD21	4.05
RFB4-SMCC-DM1	Anti-CD22	CD22	(1.95-6.75 many batches)
				Cy34.1.2-SMCC-DM1	Anti-CD22	CD22	2.70
12F7.1.5-SMCC-DM1	Anti-CD22	CD22	5.90
				9A8.1.1-SMCC-DM1	Anti-CD22	CD22	5.00
8C9.1.2-SMCC-DM1	Anti-CD22	CD22	2.90
				14B3.3.1-SMCC-DM1	Anti-CD22	CD22	5.20
8G10.4.2-SMCC-DM1	Anti-CD22	CD22	3.60
				2B4.1.4-SMCC-DM1	Anti-CD22	CD22	5.20
7A2.4.1-SMCC-DM1	Anti-CD22	CD22	3.60
				4H3.2.2-SMCC-DM1	Anti-CD22	CD22	3.60
5E8.1.8-SMCC-DM1	Anti-CD22	CD22	3.60
				3F11.2.1-SMCC-DM1	Anti-CD22	CD22	4.50
6C9.1.3-SMCC-DM1	Anti-CD22	CD22	3.70
				10D2.4.3-SMCC-DM1	Anti-CD22	CD22	3.65
10D6.8.1-SMCC-DM1	Anti-CD72	CD72	2.90
				ZL7-SMCC-DM1	Anti-CD79a (alpha)	CD79a	3.60
5C3-SMCC-DM1	Anti-CD79a (alpha)	CD79a	1.85
				6G1-SMCC-DM1	Anti-CD79a (alpha)	CD79a	3.50

7H7-SMCC-DM1	Anti-CD79a (alpha)	CD79a	3.25
				8D11-SMCC-DM1	Anti-CD79a (alpha)	CD79a	3.60
8H9-SMCC-DM1	Anti-CD79a (alpha)	CD79a	3.40
				11E5-SMCC-DM1	Anti-CD79a (alpha)	CD79a	3.60
15E4-SMCC-DM1	Anti-CD79a (alpha)	CD79a	3.25
				16C11-SMCC-DM1	Anti-CD79a (alpha)	CD79a	2.50
2F2-SMCC-DM1	Anti-CD79b (beta)	CD79b	3.20
				17A7-SMCC-DM1	Anti-CD79b (beta)	CD79b	4.40
SN8-SMCC-DM1	Anti-CD79b (beta)	CD79b	4.40,0.80 (two batches)
				1F9-SMCC-DM1	Anti-FcRH1	FcRH1	4.20
7A2-SMCC-DM1	Anti-FcRH2	FcRH2	4.20,3.95 (two batches)
				7G7-SMCC-DM1	Anti-FcRH2	FcRH2	3.80
c11D6-SMCC-DM1	Anti-FcRH2	FcRH2	2.20
				51505.111-SMCC-DM1	Anti-CXCR5	CXCR5	6.10
12C7 Fc chimera-SMCC-DM1	Anti-Brevican	brevican	4.60
				H2：PSCA hlog-SMCC-DM1	Anti-PSCA hlog	PSCA	3
H6：xPSCAhlog-SMCC-DM1	Anti-PSCA hlog	PSCA	2.6
				xFcRH5：3909 7D11.1.1-SMCC-DM1	Anti-IRTA2	IRTA2	2.6
xFCRH5.7D11-SMCC-DM1	Anti-IRTA2	IRTA2	4.4
				11D10 LC-SMCC-DM1	Mouse anti-CA 0772P MAb	CA 0772P MUC16
xNCA LC-SMCC-DM1	Mouse anti-CEACAM6 MAb	CEACAM6 NCA

Antibody-BMPEO-DM1 conjugate:

For carrying out the cysteine coupling, can in the presence of EDTA, go back original antibody with Reducing agent such as dithiothreitol, DTT (DTT) or TCEP.37 ℃ are descended insulation after about 1 hour, with the 100mM potassium phosphate pH are transferred to be about 7.Modify reductive antibody with two-maleimide reagent BM (PEO) 4 (Pierce Chemical), on the antibody surface, stay unreacted dimaleoyl imino.This can by BM (PEO) 4 is dissolved in 50% ethanol/water mixture to concentration be 10mM, and add the antibody-solutions that contains in the phosphate buffered saline (PBS) that concentration is approximately 1.6mg/ml (10 μ M) to ten times of excessive molal quantitys, make it to react 1 hour and form Ab-BMPEO and realize.At 30mM citrate, pH 6, (the HiTrap post Pharmacia) is removed excessive BM (PEO) 4 to also have in the buffer of 150mMNaCl gel filtration.The DM1 of about 10 times of molar excess is dissolved in the dimethyl acetylamide (DMA), and adds the Ab-BMPEO intermediate to.Dimethyl formamide (DMF) also can be used for dissolved substance part reagent.At gel filtration or carry out dialysis among the PBS remove nonreactive DM1 before, the reactant mixture reaction is spent the night.In PBS on the S200 post gel filtration to remove high molecular weight aggregates and the Ab-BMPEO-DM1 of purification be provided.

Follow this scheme, prepared other antibody-drug conjugates, comprising with BMPEO joint and DM1 drug moiety:

Antibody-drug conjugates (ADC)	Antibody (Ab)	Antigen	Average drug loading (DM1/Ab)
				Trastuzumab-BM (PEO)-DM1	Trastuzumab (Herceptin_)	HER2	2.94
CD120-BMPEO4-DM1	Mouse anti-GP120 MAb	GP120						2
				RFB4-BMPEO4-DM1	Anti-CD22	CD22	3.7 and 4.25 (two batches)

The test of embodiment 5-body outer cell proliferation

Use effect (the Promega Corp.Technical Bulletin TB288 of following scheme by cell proliferation measurements determination ADC; Mendoza et al (2002) Cancer Res.62:5485-5488):

1. 100 μ l in the culture medium are contained about 10 ⁴The cell culture sample aliquot of individual cell (SKBR-3, BT474, MCF7 or MDA-MB-468) is placed in each hole of 96-hole zone of opacity wallboard.

2. prepared and contained culture medium and acellular control wells.

3.ADC add test hole to and be incubated 3-5 days.

4. with dull and stereotyped balance about 30 minutes to room temperature.

5. add with each hole in the identical CellTiter-Glo reagent of cell culture medium volume that exists.

6. content mixes 2 minutes with the inducing cell cracking on the orbital oscillation device.

7. flat board at room temperature is incubated 10 minutes to stablize fluorescence signal.

8. write down luminous and be reflected in the chart according to the relative flat light emission of RLU=.

Embodiment 6-mice serum clearance rate and stability

Six groups of cream-coloured sudden changes, nude mice (no tumor) have been studied, every group of four animals.The 0th day, except the vehicle group of only accepting carrier, every mice all accepted to be dissolved in the 2mg/kg single dose ADC in the 200 μ l aqueous carriers.Under narcotism, collect blood in each time point after the administration (5 minutes, 1 hour, 6 hours, 24 hours, 72 hours and 168 hours) by cardiac puncture.Separation of serum is measured antibody and ADC.

The 1st group: carrier (PBS, pH 6.5, no ADC)

The 2nd group: trastuzumab-SMCC-DM1

The 3rd group: trastuzumab-SPP-DM1

The 4th group: trastuzumab-SPDP-DM1

The 5th group: trastuzumab-SPP-DM3

The 5th group: trastuzumab-SPP-DM4

Serum stability in the embodiment 7-rat

Six dosage groups of 6 Sprague-Dawley rats of each dosage group (every 100-125gms) have been studied.In the 0th day, be carrier, 10mg/kg trastuzumab-SPP-DM1 or the 10mg/kg trastuzumab-SMCC-DM1 that animal is used single IV dosage by a lateral tail vein with the volume dose of 10ml/kg.0 (before the administration) after administration, 10 and 30 minutes; 1,2,4,8,24 and 36 hour; Collect about 300 μ l whole bloods with each time point of 2,3,4,7,14,21,28 days.

Effect in the embodiment 8-gross tumor volume body

High expressed HER2 transgenic explant mice:

The animal that is suitable for the transgenic test can be from the normal business source as Taconic (Germantown, N.Y.) acquisition.Many strains are all is suitable, but owing to the more hypersensitivity that tumor is formed, the FVB female mice is preferred.FVB is male to be used for copulation, and vasectomized CD.1 kind Mus is used to stimulate pseudopregnancy.Vasectomized mice can obtain from any commercial supplier.Creating mice (Founders) cultivates with the FVB mice or with 129/BL6xFVB p53 heterozygous mice.P53 allele heterozygosity mice is used for increasing potentially tumor and forms.Yet it is unnecessary that this has been proved to be.Therefore, some F1 are to mix strain system for tumor.Create mouse tumor and have only FVB.Obtained six and created mice, it has some growing tumors, does not have the young baby.

By single trastuzumab-DM1 maytansinoid conjugate (10mg/kg dosage) injection for curing the animal of tumor (from the allograft of Fo5 mmtv transgenic mice amplification) is arranged, assess gross tumor volume in 20 days after the injection.

Bjab-luc xenograft SCID mice:

The test group that every group of eight to ten SCID mice, every mice have 2,000 ten thousand Bjab-luc xenograft tumor cells gave antibody-drug conjugates or naked antibody in the 1st day.The group of eight mices is all tested with anti-CD22 ADC, naked anti-CD22 antibody and contrast for every group.Contrast ADC (trastuzumab-SMCC-DM1; Load: DM1/ trastuzumab=3.2) be not specificity junction mixture, use that the result is that to double average time be about 3 days to tumor with 200 μ gDM1/m2,4.2 trastuzumabs/kg mice.Anti-CD22 conjugate 7A2-SMCC-DM1 (load: DM1/Ab=3.6), 5E8-SMCC-DM1 (load: DM1/Ab=3.6) and RFB4-SMCC-DM1 (load: DM1/Ab=4.3).Naked antibody 7A2,5E8 and RFB4 give with the 4mg/kg mice.

Embodiment 9-is to the toxicity of rat

Assessed the acute toxicity spectrum of the trastuzumab-SPP-DM1 (disulphide joint) that compares with free DM1 and trastuzumab-SMCC-DM1 (non-disulphide joint).The 1st day injection animal,, and carried out complete obduction in the 5th day in complete chemistry and hematology's collection of illustrative plates of the 3rd day and the 5th day acquisition baseline position.For each group, use three animals to undertissue at random: breastbone, liver, kidney, thymus, spleen, intestine and small intestine carry out the conventional organization credit and analyse.Test group is as follows:

The 1st group: carrier (10mM sodium succinate, 100mg/mL sucrose, 0.1%Tween 20, pH5.0)

The 2nd group: trastuzumab-SPP-DM1,22.3mg/kg

The 3rd group: trastuzumab-SMCC-DM1,10mg/kg

The 4th group: trastuzumab-SMCC-DM1,25mg/kg

The 5th group: trastuzumab-SMCC-DM1,50mg/kg

The 6th group: free DM1,160 μ g/kg

Embodiment 10-is to toxicity/safety of macaque

Three groups of each four (2 male, and 2 female) young machin (Macacafascicularis) of group (macaque (Cynomolgus monkey)) have been studied.

The 1st group: (4 animals) only accepted carrier (PBS, pH 6.5, promptly remove the preparation of ADC) at the 1st day and the 22nd day, after this carried out obduction in the 36th day.

The 2nd group: (4 animals) accepted trastuzumab-SMCC-DM1 4900 μ g/m2 in the 1st day and the 22nd day.

The 3rd group: (4 animals) accepted trastuzumab-SMCC-DM1 7200 μ g/m2 in the 22nd day.

Dosed administration represents with the surface area of animal so that corresponding with other species, promptly no matter what species represent with dosage μ g/m2, and therefore can between species, compare.Trastuzumab-SMCC-DM1 the preparation that is used for the 2nd group and the 3rd group research comprises PBS, 5.4mM sodium phosphate, 4.2mM potassium phosphate, 140mM sodium chloride, and pH 6.5.

Before first time dosed administration after (domestication) and the administration for the first time after the 3rd, 7,11 and 14 day (the 1st and 2 group) and the administration for the second time the 3rd, 7,11,14 and 21 day (the 1st, 2 and 3 group) collect blood and be used for analysis of Hematology Changes.Erythrocyte (RBC) and platelet (PLT) counting pass through light scattering determining.Leukocyte (WBC) counting is measured by peroxidase/basophilic leukocyte method.Reticulocyte count is by utilizing the light scattering determining of the dye of positive ion.Cell counting is measured on Advia 120 instruments.ALT (alanine aminotransferase) and AST (aspartate transaminase) pass through UV/NADH with U/L; The IFCC methodology adopts Total Ab ELISA-ECD/GxhuFc-HRP.Conj.Ab ELISA-xDM1/ECD-Bio/SA-HRP measurements determination on Olympus AU400 instrument.

All patents, patent application and the list of references of quoting as proof in the whole description all known adding as a reference.

Claims (67)

1. the antibody-drug coupling compound comprises the antibody that is covalently attached to one or more maytansinoid drug moieties by joint, and described chemical compound has formula I:

Ab-(L-D) _p I

Or its pharmaceutically acceptable salt or solvate, wherein:

Ab is in conjunction with the ErbB receptor, and perhaps in conjunction with the antibody of one or more tumor associated antigens or cell surface receptor, described tumor associated antigen or cell surface receptor are selected from (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-IB type, Genbank accession number NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

(3) STEAP1 (prostate is striden the film epithelium antigen six times, Genbank accession number NM_012449);

(4) 0772P (CA125, MUC16, Genbank accession number AF361486);

(5) MPF (MPF, MSLN, SMR, megalokaryocyte intensifier, mesothelin, Genbank accession number NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), the member 2, the phosphate cotransporter albumen 3b that II type sodium relies on, Genbank accession number NM 006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, brain signal albumen 5b Hlog, the sema domain, seven thrombospondins repeat (1 type and class 1 type), membrane spaning domain (TM) and short Cytoplasm domain, (brain signal albumen) 5B, Genbank accession number AB040878);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);

(9) ETBR (endothelin receptor B, Genbank accession number AY275463);

(10) MSG783 (RNF124, imaginary albumen FLJ20315, Genbank accession number NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, relating to prostate cancers because of 1, the carcinoma of prostate associated protein 1, prostaticly stride film epithelium antigen 2 for six times, stride film prostatein, Genbank accession number AF455138 six times);

(12) TrpM4 (member 4, Genbank accession number NM_017636 for BR22450, FLJ20041, TRPM4, TRPM4B, the potential cationic channel of transient receptor, subfamily M);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma derivative growth factor, Genbank accession number NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/ dust crust virus receptor) or Hs.73792Genbank accession number M26004);

(15) CD79b (CD79B, CD79 β, IGb (immunoglobulin be correlated with beta), B29, Genbank accession number NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (the phosphatase anchorin 1a that contains the SH2 domain), SPAP1B, SPAP1C, Genbank accession number NM_030764);

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20R α (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22) EphB2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763;

(26) BAFF-R (B cell activation factor receptor, BlyS receptor 3, BR3, NP_443177.1);

(27) CD22 (B-cell receptor CD22-B isotype, NP-001762.1);

(28) CD79a (CD79A, CD79 α, the immunoglobulin alpha that is correlated with, with Ig beta (CD79B) covalent interaction and form the B cell-specific proteins that complex, transduction relate to the signal of B cell differentiation, Genbank accession number NP_001774.1 with Ig M molecule from the teeth outwards);

(29) CXCR5 (Burkitt lymphoma receptor 1, by the activatory g protein coupled receptor of CXCL13 chemotactic factor, in lymphocyte migration and humoral defense, play a role, infect and may in AIDS, lymphoma, myeloma and leukemic generation, play a role Genbank accession number NP_001707.1 at HIV-2);

(30) HLA-DOB (the Beta subunit of MHC II quasi-molecule (Ia antigen), its binding peptide also is presented to the CD4+T lymphocyte, Genbank accession number NP_002111.1 with it);

(31) P2X5 (purinoceptor P2X ligand-gated ion channel 5, ion channel by the outer ATP gate of born of the same parents, may relate to synapse transmission and neural the generation, its defective may cause IDI's Pathophysiology situation, Genbank accession number NP_002552.2);

(32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

(33) LY64 (lymphocyte antigen 64 (RP105), be rich in the I type memebrane protein family that leucine repeats (LRR), regulate B cell activation and apoptosis, the forfeiture of function is relevant with the enhancing of Patients with SLE disease activity, Genbank accession number NP_005573.1);

(34) FcRH1 (Fc receptor-like protein 1, immunoglobulin Fc domain infer receptor, comprise C2 type Ig sample and ITAM domain, may in the bone-marrow-derived lymphocyte differentiation, work Genbank accession number NP_443170.1);

(35) (transposition related immune globulin superfamily receptors 2, the immunity receptor of inferring may work in B cell development and lymphoma generation IRTA2; The gene imbalance that is caused by transposition betides in some B cell malignant diseases Genbank accession number NP_112571.1); And

(36) TENB2 (the transmembrane protein polysaccharide of inferring, relevant with the accent protein family and the follistatin of the EGF/ somatomedin of somatomedin, Genbank accession number AF179274;

Condition is that described antibody is not TA.1;

L is non-disulphide joint;

D is the maytansinoid drug moiety; And

P is 1 to 8.

2. the antibody-drug coupling compound of claim 1, wherein Ab is in conjunction with being selected from (1)-(16) and one or more tumor associated antigens of (18)-(36) or the antibody of cell surface receptor.

3. the antibody-drug coupling compound of claim 1 or claim 2, wherein L is the joint that is selected from following structure:

Wherein wave is represented covalently bound with Ab and D;

X is:

-(CH ₂) _n-，-(CH ₂CH ₂O) _n-

Y is:

R is H or C independently ₁-C ₆Alkyl; And n is 1 to 12.

4. the antibody-drug coupling compound of arbitrary aforementioned claim, wherein D is selected from structure:

Wherein wave is represented covalently bound with L,

R is H or C independently ₁-C ₆Alkyl, and

M is 1,2 or 3.

5. the antibody-drug coupling compound of claim 4, wherein m is 2, and R is H.

6. the antibody-drug coupling compound of arbitrary aforementioned claim, wherein the maytansinoid drug moiety is DM1, has structure:

7. the antibody-drug coupling compound of claim 3, it has structure:

8. the antibody-drug coupling compound of claim 7, it has structure:

9. the antibody-drug coupling compound of claim 3, it has structure:

10. the antibody-drug coupling compound of claim 1, it has structure:

11. the antibody-drug conjugates of claim 10, wherein Ab is a trastuzumab, and p is 1,2,3 or 4.

12. the antibody-drug coupling compound of claim 1, it has structure:

Wherein Tr is a trastuzumab, and p is 1,2,3 or 4.

13. the antibody-drug coupling compound of claim 1, it has structure:

Wherein n is 0,1 or 2; And p is 1,2,3 or 4.

14. the antibody-drug coupling compound of claim 13, wherein Ab is a trastuzumab.

15. the antibody-drug coupling compound of claim 1, wherein p is 1,2,3 or 4.

16. the antibody-drug coupling compound of claim 1, wherein said antibodies is by the receptor of ErbB gene code.

17. the antibody-drug coupling compound of claim 16, wherein said receptor is selected from EGFR, HER2, HER3 and HER4.

18. the antibody-drug coupling compound of claim 17, wherein said antibody are specifically in conjunction with the HER2 receptor.

19. the antibody-drug coupling compound of claim 1, it is specifically in conjunction with the ectodomain of HER2 receptor and suppressed to express the growth of the tumor cell of HER2 receptor.

20. the antibody-drug coupling compound of arbitrary aforementioned claim, wherein said antibody is selected from monoclonal antibody, antibody fragment, chimeric antibody and humanized antibody.

21. the antibody-drug coupling compound of claim 1, wherein said antibody is for being selected from the humanized antibody of huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).

22. the antibody-drug coupling compound of claim 21, wherein said antibody are huMAb4D5-8 (trastuzumab).

23. the antibody-drug conjugates of claim 20, wherein said antibody are the Fab fragment.

24. the antibody-drug coupling compound of claim 1, wherein said antibody is connected in joint by the cysteine mercaptan of antibody.

25. the antibody-drug coupling compound of claim 24, wherein p is 1,2,3 or 4.

26. comprise the antibody-drug coupling compound of claim 1 or claim 2 or the pharmaceutical composition of its pharmaceutically acceptable salt and pharmaceutically acceptable diluent, carrier or excipient.

27. the pharmaceutical composition of claim 26, it further comprises the chemotherapeutics for the treatment of effective dose, and described chemotherapeutics is selected from: Erlotinib, Bortezomib, fulvestrant, Sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, Oxaliplatin, 5-FU, formyl tetrahydrofolic acid, rapamycin, Lapatinib, Lonafarnib, Sorafenib and Gefitinib.

28. the pharmaceutical composition of claim 26, it further comprises the anti-angiogenic rebirth agent for the treatment of effective dose.

29. the pharmaceutical composition of claim 26, it further comprises the bevacizumab for the treatment of effective dose.

30. suppress the method for cell proliferation, comprise with the mammalian cell in the antibody-drug coupling compound processing cell culture medium of claim 1 or claim 2, suppress the propagation of cell thus.

31. the method for claim 30, wherein said mammalian cell have the HER2 receptor protein at the antibody-drug coupling compound.

32. the method for claim 31, wherein said mammalian cell is a breast tumor cell.

33. the method for claim 30, wherein said antibody-drug coupling compound cytotoxicity is stronger than the maytansinoid chemical compound of the maytansinoid part that comprises the antibody-drug coupling compound.

34. the method for claim 30, wherein said antibody-drug conjugates is apoptosis-induced.

35. the treatment method for cancer is included as the patient and uses the antibody-drug coupling compound of claim 1 or claim 2 and the preparation of pharmaceutically acceptable diluent, carrier or excipient.

36. the method for claim 35, wherein said cancer is selected from mammary gland, ovary, stomach, endometrium, salivary gland, lung, kidney, colon, colorectum, thyroid, pancreas, prostate and bladder cancer.

37. the method for claim 36, wherein said cancer is for crossing the breast carcinoma of expressing ErbB2 with 2+ or higher level.

38. the method for claim 35, wherein the amount of the antibody-drug coupling compound of using for the patient at every dose about 0.1 to about 10mg/kg weight in patients scope.

39. the method for claim 35 is wherein with the interval administration of antibodies-drug conjugates in about three weeks.

40. the method for claim 35 is wherein by infusion administration of antibodies-drug conjugates.

41. the method for claim 35, wherein said antibody-drug conjugates is prepared with pharmaceutically acceptable parenteral carrier.

42. the method for claim 41, wherein said antibody-drug conjugates is formulated as injectable unit dosage form.

43. the method for claim 42, wherein intravenous administration of antibodies-drug conjugates.

44. the method for claim 35 is wherein with the combination of antibody-drug coupling compound, for the patient uses growth inhibited antibody.

45. the method for claim 35 is wherein with antibody-drug coupling compound combination, for the patient uses in conjunction with ErbB2 and blocks second kind of antibody of the ligand activation of ErbB receptor.

46. the method for claim 45, wherein second kind of antibody comprises monoclonal antibody 2C4 or humanization 2C4.

47. the method for claim 46, wherein said second kind of antibody and cytotoxic reagent coupling.

48. the method for claim 35, wherein make up with the antibody-drug coupling compound, for the patient uses chemotherapeutics, wherein said chemotherapeutics is selected from: Erlotinib, Bortezomib, fulvestrant, Sutent, letrozole, imatinib mesylate, PTK787/ZK 222584, Oxaliplatin, 5-FU, formyl tetrahydrofolic acid, rapamycin, Lapatinib, Lonafamib, Sorafenib and Gefitinib.

49. the method for claim 35, wherein with the combination of antibody-drug coupling compound, for the patient uses the anti-angiogenic rebirth agent, wherein said anti-angiogenic rebirth agent is selected from bevacizumab.

50. suppressed to express the method for the growth of tumour cell of growth factor receptors, be included as antibody-drug conjugates and chemotherapeutics that the patient uses claim 1, described antibody-drug conjugates is specifically in conjunction with the growth factor receptors that is selected from HER2 or EGF, and wherein said antibody-drug conjugates and described chemotherapeutics are used with the amount of effective inhibition patient tumors cell growth respectively.

51. the method for treatment human patients, described human patients is to crossing the disease sensitivity that is expressed as feature or suffering from described disease after diagnosing with the ErbB2 receptor, described method comprises antibody-drug conjugates and the chemotherapeutics or the growth inhibitor of co-administered claim 1.

52. the method for claim 51, wherein the antibody of antibody-drug conjugates is anti-ErbB antibody.

53. the method for claim 52, wherein said anti-ErbB antibody has the biological property of 4D5 monoclonal antibody.

54. the method for claim 52, the epi-position that wherein said anti-ErbB antibodies is substantially the same with the 4D5 monoclonal antibody.

55. the method for claim 52, wherein said anti-ErbB antibody is selected from humanized antibody: huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7 and huMAb4D5-8 (trastuzumab).

56. be used to detect the method for testing of cancerous cell, comprise:

(a) cell is contacted with the antibody-drug coupling compound of claim 1 or claim 2; With

(b) measure the degree of antibody-drug coupling compound in conjunction with cell.

57. the method for testing of claim 56, wherein said cell is a breast tumor cell.

58. the method for testing of claim 56, wherein said combination degree detects by the level of measuring the ErbB-code nucleic acid with fluorescence in situ hybridization (FISH).

59. the method for testing of claim 56, wherein said combination degree is measured by SABC (IHC).

60. goods comprise:

The antibody-drug coupling compound of claim 1 or claim 2;

Container; With

Package insert or label, it indicates described chemical compound and can be used for treating with the ErbB receptor and cross the cancer that is expressed as feature.

Can be used for treating and cross the cancer that is expressed as feature 61. the goods of claim 60, wherein said package insert or label indicate described chemical compound with the ErbB2 receptor.

62. the goods of claim 61, wherein said cancer are breast carcinoma.

63. the goods of claim 62, wherein said cancer is expressed as feature with the mistake of ErbB2 receptor 2+ or above level.

64. prepare the method for antibody-drug coupling compound, comprise by joint antibody is covalently attached to one or more maytansinoid drug moieties that described chemical compound has formula I:

Ab-(L-D) _p I

Or its pharmaceutically acceptable salt or solvate, wherein:

Ab is in conjunction with the ErbB receptor, and perhaps in conjunction with the antibody of one or more tumor associated antigens or cell surface receptor, described tumor associated antigen or cell surface receptor are selected from (1)-(36):

(1) BMPR1B (bone morphogenetic protein receptor-IB type, Genbank accession number NM_001203);

(2) E16 (LAT1, SLC7A5, Genbank accession number NM_003486);

(3) STEAP1 (prostate is striden the film epithelium antigen six times, Genbank accession number NM_012449);

(4) 0772P (CA125, MUC16, Genbank accession number AF361486);

(5) MPF (MPF, MSLN, SMR, megalokaryocyte intensifier, mesothelin, Genbank accession number NM_005823);

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodium phosphate), the member 2, the phosphate cotransporter albumen 3b that II type sodium relies on, Genbank accession number NM_006424);

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, brain signal albumen 5b Hlog, the sema domain, seven thrombospondins repeat (1 type and class 1 type), membrane spaning domain (TM) and short Cytoplasm domain, (brain signal albumen) 5B, Genbank accession number AB040878);

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, PIKEN cDNA2700050C12, RIKEN cDNA 2700050C12 gene, Genbank accession number AY358628);

(9) ETBR (endothelin receptor B, Genbank accession number AY275463);

(10) MSG783 (RNF124, imaginary albumen (hypothetical protein) FLJ20315, Genbank accession number NM_017763);

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, relating to prostate cancers because of 1, the carcinoma of prostate associated protein 1, prostaticly stride film epithelium antigen 2 for six times, stride film prostatein, Genbank accession number AF455138 six times);

(12) TrpM4 (member 4, Genbank accession number NM_017636 for BR22450, FLJ20041, TRPM4, TRPM4B, the potential cationic channel of transient receptor, subfamily M);

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma derivative growth factor, Genbank accession number NP_003203 or NM_003212);

(14) CD21 (CR2 (complement receptor 2) or C3DR (C3d/ dust crust virus receptor) or Hs.73792Genbank accession number M26004);

(15) CD79b (CD79B, CD79 β, IGb (immunoglobulin be correlated with beta), B29, Genbank accession number NM_000626);

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (the phosphatase anchorin 1a that contains the SH2 domain), SPAP1B, SPAP1C, Genbank accession number NM_030764);

(17) HER2 (Genbank accession number M11730);

(18) NCA (Genbank accession number M18728);

(19) MDP (Genbank accession number BC017023);

(20) IL20R α (Genbank accession number AF184971);

(21) Brevican (Genbank accession number AF229053);

(22) EphB2R (Genbank accession number NM_004442);

(23) ASLG659 (Genbank accession number AX092328);

(24) PSCA (Genbank accession number AJ297436);

(25) GEDA (Genbank accession number AY260763;

(26) BAFF-R (B cell activation factor receptor, BlyS receptor 3, BR3, NP_443177.1);

(27) CD22 (B-cell receptor CD22-B isotype, NP-001762.1);

(28) CD79a (CD79A, CD79 α, the immunoglobulin alpha that is correlated with is with Ig beta (CD79B) covalent interaction and form complex with Ig M molecule from the teeth outwards, transduction relates to the B cell-specific proteins of the signal of B cell differentiation, Genbank accession number NP_001774.1);

(29) CXCR5 (Burkitt lymphoma receptor 1, by the activatory g protein coupled receptor of CXCL13 chemotactic factor, in lymphocyte migration and humoral defense, play a role, infect and may in AIDS, lymphoma, myeloma and leukemia, play a role Genbank accession number NP_001707.1 at HIV-2);

(30) HLA-DOB (the Beta subunit of MHC II quasi-molecule (Ia antigen), its binding peptide also is presented to the CD4+T lymphocyte, Genbank accession number NP_002111.1 with it);

(31) P2X5 (purinoceptor P2X ligand-gated ion channel 5, ion channel by the outer ATP gate of born of the same parents, may relate to synapse transmission and neuranagenesis, its defective may cause IDI's Pathophysiology situation, Genbank accession number NP_002552.2);

(32) CD72 (B cell differentiation antigen CD72, Lyb-2, Genbank accession number NP_001773.1);

(33) LY64 (lymphocyte antigen 64 (RP105), be rich in the I type memebrane protein family that leucine repeats (LRR), regulate B cell activation and apoptosis, the forfeiture of function is relevant with the enhancing of Patients with SLE disease activity, Genbank accession number NP_005573.1);

(34) FcRH1 (Fc receptor-like protein 1, the immunoglobulin Fc domain of inferring comprise C2 type Ig sample and ITAM domain, may work Genbank accession number NP_443170.1 in the bone-marrow-derived lymphocyte differentiation);

(35) (transposition related immune globulin superfamily receptors 2, the immunity receptor of inferring may work in B cell development and lymphoma generation IRTA2; The gene imbalance that is caused by transposition betides in some B cell malignant diseases Genbank accession number NP_112571.1); And

(36) (the transmembrane protein polysaccharide of inferring transfers protein family relevant with follistatin with the EGF/ of somatomedin, Genbank accession number AF179274 to TENB2;

Condition is that described antibody is not TA.1;

L is the joint that is selected from following structure:

Wherein wave is represented covalently bound with Ab and D;

X is:

-(CH ₂) _n-，-(CH ₂CH ₂O) _n-

Y is:

Wherein R is H or C independently ₁-C ₆Alkyl; And n is 1 to 12;

D is the maytansinoid drug moiety that is selected from following structure:

Wherein wave is represented covalently bound with L; R is H or C independently ₁-C ₆Alkyl; M is 1,2 or 3; And p is 1 to 8;

Wherein said method comprises:

Make Ab and joint reagent reacting form antibody-joint intermediate A b-L, make Ab-L and drug moiety D reaction form antibody-drug conjugates then; Perhaps

Make drug moiety D and joint reagent reacting form medicine-joint intermediate D-L, make D-L and Ab reaction form antibody-drug conjugates then.

65. the method for claim 64, wherein Ab is selected from (1)-(16) and one or more tumor associated antigens of (18)-(36) or the antibody of cell surface receptor for combination.

66. the method for claim 64 or claim 65, wherein said joint reagent is SMCC.

67. the method for claim 64 or claim 65, wherein said joint reagent is for being selected from DTME, BMB, BMDB, BMH, BMOE, BM (PEO) ₃And BM (PEO) ₄Two-maleimide reagent.

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