CN101484186A - Treatment of tumors in pediatric patients with epidermal growth factor receptor antagonists - Google Patents

Abstract

Combination therapy methods of treating pediatric tumors by administering an EGFR antagonist and a chemotherapeutic agent are disclosed. The methods also include treating refractory pediatric tumors.

Description

With the intravital tumor of epidermal growth factor receptor antagonists treatment infant patient

The cross reference of relevant application

The application requires to submit on July 27th, 2006, application number is the interests of 60/833,487 U.S. Provisional Patent Application, and its full content is herein incorporated in the introducing mode.

Technical field

The present invention relates to the intravital tumor of administering drug combinations treatment infant patient by EGFR antagonist and chemotherapeutant.

Background technology

Calendar year 2001, have the child of 11,900 examples and the teenager below 20 years old to be diagnosed as cancer in the U.S., and about 2,200 examples are died from this disease.Entity tumor occupies about 30% of child's cases of cancer.Invasive cerebral nervous system cancer is occupied 17% of all infant cancers, is only second to acute lymphoblastic leukemia.The cerebroma confirmed cases of half are virulent approximately.Other common child's cancers comprise ewing's sarcoma, leukemia, neuroblastoma, osteosarcoma, rhabdomyosarcoma, soft tissue sarcoma and nephroblastoma.

A major obstacle of treatment child cerebroma is that brain is still being grown rapidly, is vulnerable to from such as radiotherapy or the toxic injury of chemotherapy.Other classes child treatment for cancer also faces same obstacle.Therefore, need the new therapy of treatment infant patient in-vivo tumour.

EGF-R ELISA (EGFR) family expresses or crosses and is expressed in the various various cancers, and is usually directed to the formation of tumor.EGFR family comprises EGF receptor (EGFR is also referred to as erbB-1/HER1), HER2 (being also referred to as c-neu/erB-2), erbB-3/HER3 and erbB-4/HER4.For example, EGFR and HER2 are considered to play crucial effects in the process of regulate tumor cell growth and existence.Particularly, EGFR is survived, is prevented that several paths of apoptosis, dedifferentiation, transfer (comprising cell migration and invasion and attack) are associated with influence.In comprising head and neck cancer, colorectal carcinoma, cancer of pancreas, ovarian cancer, renal cell carcinoma, nonsmall-cell lung cancer and gliomatous those cancers, expressing EGFR is a topmost part.If the multiple cancer in these cancers is not diagnosed out in early days, its prognosis is bad, and is limited for the treatment of terminal illness.

There is various EGFR inhibitor to be used for the treatment of the certain cancers of these cancers in the clinical research at present.One of them example is (cetuximab) (immune clone System Co., Ltd makes), it is chimeric (people/Mus) monoclonal antibody, block ligand combines, prevents the activation of receptor and the growth that suppresses cultured cell with EGFR.Another example is ABX-EGF, and it is a kind of complete human monoclonal antibodies at EGFR, it is reported, and the combination of its transforming growth factor capable of blocking (TFG-a) and epidermal growth factor (EGF), this is two kinds of known and bonded parts of EGFR.

(trastuzumab) be a kind of humanized antibody of granting the positive metastatic breast cancer of treatment HER2, it is designed to target and hinders HER2 albumen and cross the function of expression.

In addition, carrying out clinical research aspect the different micromolecule EGFR inhibitor at present.An example of tyrosine kinase inhibitor is IRESSA ^TMIt is a kind of micromolecule EGFR tyrosine kinase inhibitor, it is reported the activity that can suppress the EGFR tyrosine kinase, it has cytostatic effect to the human cancer cell who expresses in the functional EGFR scope, can suppress the hypertrophy of tumor cell by raising p27.

Summary of the invention

In some embodiments, the invention provides a kind of by the infant patient being treated to suppress the method for infant patient's tumor growth in vivo with the EGFR antagonist of effective dose and chemotherapeutant.One preferred embodiment in, this EGFR antagonist is that specificity is in conjunction with the EGFR ectodomain and its active EGFR antibody that neutralizes.One preferred embodiment in, this chemotherapeutant is an Irinotecan.

The specific embodiment

The invention provides a kind of by giving effective dose the EGFR antagonist and chemotherapeutant with the method for treatment infant patient tumor growth in vivo.Infant patient of the present invention is the patient to 18 years old from birth.Treatment comprises: (1) prevention is easily suffered from this disease but is not suffered from or this disease takes place for the patient that shows this disease symptoms, for example prevents the outbreak of clinical symptoms; (2) suppress growth of tumor, for example block its development; Or (3) tumor remission, for example impel disappearing of tumor symptom.Suppress tumor growth and comprise and slow down or stop growing, also comprise and impel tumor regression.The effective dose of treatment disease is meant that this amount is enough to effectively treat the above-mentioned disease that limits when the patient's administration to this treatment of needs.

According to the tumor that the present invention treated is to express any tumor of EGFR.This tumor comprises the blastocyte tumor, and it comprises hepatoblastoma and neuroblastoma; Carcinoma comprises adenocarcinoma; The neuroglia cancer comprises ependymoma, astrocytoma, mesoglioma and mixed nerve glioma; Sarcoma comprises rhabdomyosarcoma and sarcoadenoma; And adenoma.This tumor can appear at whole body everywhere substantially, comprises for example breast, heart, lung, esophagus, small intestinal, colon, rectum, stomach, spleen, kidney, bladder, head and neck, larynx, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, cervix uteri or liver.One preferred embodiment in, this tumor is nervous system neoplasms such as glioma and neuroblastoma.The tumor of being treated comprises constitutional and metastatic tumo(u)r, also comprises malignant tumor." malignant tumor " comprises the tumor of not replying or resisting treatment with chemotherapeutant, antibody, radiation or its combined therapy to single.Malignant tumor also comprise by these pharmaceutical treatments seem be inhibited but after stopping to treat to 5 years, have up to 10 years or the tumor of long time recurrence more.

According to EGFR antagonist of the present invention can be antagonist in extracellular antagonist or the cell, and can use more than one antagonist.The extracellular antagonist includes but not limited to the bonded biomolecule of protein or other and EGFR.In some embodiments of the present invention, the extracellular antagonist combines with the ectodomain of EGFR, thus suppress that EGFR combines with its one or more parts and/or in the part induced activity of EGFR.The EGFR part comprises EGF, TFG-α, amphiregulin (Amphirgulin), heparin conjunction type EGF-R ELISA (HB-EGF) and β cytokines.Extracellular EGFR antagonist also can comprise the material that suppresses to have the EGFR dimer of other EGFR receptor subunits (being the EGFR homodimer) or have the heterodimer of other growth factor receptorses (for example HER2).

In preferred embodiment, the EGFR antagonist is to combine with EGFR and stop the bonded antibody of part.An example of EGFR antibody is that (the GenBank registration number: 1NQLA), it is a kind of chimeric (people/Mus) IgG monoclonal antibody to Cetuximab (IMC-C225).Referring to for example United States Patent (USP) 4,943,533 (people such as Mendelsohn), United States Patent (USP) 6,217,866 (people such as Schlessinger), U.S. Patent application 08/973,065 (people such as Goldstein) and 09/635,974 (Teufel), WO 99/60023 (people such as Waksal) and WO 00/69459, above-mentioned all contents are incorporated herein by reference.Cetuximab combines with EGFR specifically, and hinders binding partner such as EGF.Cetuximab Fab contains Cetuximab Fab fragment, i.e. variable region sequences of the heavy chain of murine antibody M225 and light chain (U. S. application 2004/00612 is incorporated herein by reference) and IgG 1 C _H1 heavy chain and κ constant region of light chain.(Cetuximab comprises all three kinds of IgG1 CH.) Cetuximab heavy chain CDR zone has following sequence: have the CDR1 zone (SEQ ID NO:1) of N Y G V H sequence, the CDR2 zone (SEQ ID NO:2) that has V I W S G GN T D Y N T P F T S sequence and the CDR3 zone (SEQ ID NO:3) that has A L TY Y D Y E F A Y sequence.Cetuximab light chain CDR zone has following sequence: have the CDR1 zone (SEQ ID NO:4) of R A S Q S I G T N I H sequence, the CDR3 zone (SEQ ID NO:6) that has the CDR2 zone (SEQ ID NO:5) of Y A S E S I S sequence and have Q Q N N N W P T T sequence.

Another example of EGFR antibody is ABX-EGF, and it is the complete IgG specific to EGFR ₂Monoclonal antibody.ABX-EGF has the height specificity in conjunction with EGFR, can hinder combining of EGFR and two ligands, EGF and TGF-α.Summary 1102 referring to people such as for example Figlin were done in the 37 the ASCO annual meeting that San Francisco is held in 12-15 day May calendar year 2001 is incorporated herein by reference.Be called the ABX-EGF sequence and the sign thereof of cloning E7.6.3 in the past and be disclosed in United States Patent (USP) 6,235,883 (U.S. Abgenix company limiteies) the 28th hurdle the 62nd walks to the 29th hurdle the 36th row and diagram 29-34, be incorporated herein by reference (also referring to people such as Yang, " Critical Rev.Oncol./Hematol. ", 38 (1): 17-23,2001, be incorporated herein by reference).

Another example of EGFR antibody is

(trastuzumab), it is a dna homology recombinant humanized monoclonal antibody, the high affinity of the ectodomain ground selective binding of its (Kd is 5nM) and human EGFR 2 protein HER2 aspect the cellular level analysis.This antibody is IgG ₁-κ type, it contains the human skeleton district that has with the complementary zone of the bonded murine antibody of HER2 (4D5) decision.Referring to international monopoly publication WO 01/89566 (Mass), be incorporated herein by reference.

Other EGFR antibody comprise: EMD72000 (Merck KGaA company), and it is the humanization form of mouse-anti EGFR monoclonal antibody EMD55900; H-R3 (TheraCIM), human monocloned antibody against EGFR; Y10, mouse monoclonal antibody once was considered as resisting the Mus autoploid of people EGFRvIII variant; And MDX-447 (Medarex).Referring to United States Patent (USP) 5,558,864 (people such as Bendig), 5,884,093 (people such as Kettleborough) and 5,891,996 (people such as Mateo deAcosta del Rio), all the elements are incorporated herein by reference.

Extracellular EGFR antagonist can be biomolecule, but is generally micromolecule, such as directly acting on EGFR cytoplasmic structure territory to suppress the synthetase inhibitors of EGFR medium signal transduction.An example of micromolecule EGFR antagonist is IRESSA ^TM(ZD1939), it is to suppress quinoline miaow quinoline (quinozaline) derivant of EGFR as simulation ATP.Referring to United States Patent (USP) 5,616, the page 4 (Zeneca company limited) of 582 (Zeneca company limiteies), WO 96/33980 (Zeneca company limited), the also summary 5 that can be done in the 37 the ASCO annual meeting that San Francisco is held in 12-15 day May calendar year 2001 referring to people such as Rowinsky, the also summary 1712 that can be done in the 37 the ASCO annual meeting that San Francisco is held in 12-15 day May calendar year 2001 referring to people such as Anido.Another example of micromolecule EGFR antagonist is

(OSI-774), it is 4-(anilino-of replacement) quinoline miaow quinoline derivant [6, two (2-methoxyl group-ethyoxyl)-quinazolines of 7--4-yl]-(3-acetenyl-phenyl) amine hydrochlorate] the EGFR inhibitor.For example page 2 the 12nd walks to page 4 the 34th row and the 19th page of 14-17 is capable referring to WO 96/30347 (Pfizer company limited).Also can be referring to people such as Moyer, " Cancer Res. ", 57:4838-48 (1997), people such as Pollack, " J.Pharmacol ", 291:739-48 (1999).

Play a role to cause p27 to regulate cell cycle arrest by the phosphorylation of inhibition EGFR and the PI3/Akt and MAP (the former activated protein of mitogen) the signal transduction of kinases path in downstream thereof.The summary of being done in the 37 the ASCO annual meeting that San Francisco is held in 12-15 day May calendar year 2001 referring to people such as Hidalgo 281.

Also report has other micromolecule that suppress EGFR, and wherein many being considered to has specificity to the EGFR tyrosine kinase domain.Some examples of these micromolecule EGFR antagonist are addressed in WO 91/116051, WO 96/30347, WO 96/33980, WO 97/27199 (Zeneca company limited), WO 97/30034 (Zeneca company limited), WO97/42187 (Zeneca company limited), WO 97/49688 (Pfizer company limited), WO 98/33798 (WarnerLambert company), WO 00/18761 (American Cyanamid company) and WO00/31048 (Warner Lambert company).The example of specificity micromolecule EGFR anticaking agents comprises C1-1033 (Pfizer), it is quinoline miaow quinoline (N-[4-(3-chloro-4-fluoro-aniline)-7-(3-morphine quinoline-4-base-propoxyl group)-quinazoline-6-yl]-acrylamide) inhibitor of tyrosine kinase, particularly EGFR addresses in the 8th page of 22-6 of WO 00/31048 capable; PKI-166 (Novartis), it is the pyrrolopyrimidine inhibitor of EGFR and addresses in the 10-12 page or leaf of WO 97/27199; GW2016 (Glaxo SmithKline), it is the inhibitor of EGFR and HER2; EKB569 (Wyeth) reports that it can suppress the growth that the tumor cell of expressing EGFR or HER2 is crossed in the inside and outside; AG-1478 (Tryphostin), it is the quinoline miaow quinoline micromolecule that suppresses from EGFR and erB-2 signal; AG-1478 (Sugen), but it is the Double bottom thing inhibitor of also Profilin kinase c K2; PD153035 (Parke-Davis), report can suppress EGFR kinase activity and tumor growth, bring out the cultured cell apoptosis, strengthen the cytotoxicity of cytotoxin chemotherapeutant; SPM-924 (Schwarz Pharma), the tyrosine kinase inhibitor of targeted therapy carcinoma of prostate; CP-546,989 (OSIPharmaceuticals) are reported as the inhibitor that comes from the treatment entity tumor; ADL-681, the EGFR inhibitors of kinases of targeted therapy tumor; PD 158780, and it is that report is to suppress the pyrrolopyrimidine of the tumor growth rate of A4431 tumour transplatation in the Mus body; CP-358,774, it is to suppress the quinoline miaow quinoline of the autophosphorylation of HN5 tumour transplatation in the Mus body; ZD1839, it is that report has the quinoline miaow quinoline that the Mus in-vivo tumour is transplanted the anti-tumor activity of pattern, comprising carcinoma vulvae, NSCLC cancer, carcinoma of prostate, ovarian cancer, colorectal cancer; CGP 59326A, it is the pyrrolopyrimidine that report suppresses EGFR positive tumor establiss in the Mus body; PD 165557 (Pfizer); CGP54211 and CGP53353 (Novartis), they are the inferior nitrogen phthalimides (dianilnophthalimides) of dianil.The EGFR tyrosine kinase inhibitor of natural generation comprises genistein, Antibiotic TAN 420F, quercetin and tabulation mycin.

Report with suppress EGFR and therefore the further micromolecule within the scope of the invention be following molecule: such as addressing in United States Patent (USP) 5,679 tricyclic compound of 683 chemical compound; Such as addressing in United States Patent (USP) 5,616 the quinoline miaow quinoline derivant of 582 chemical compound; Such as addressing in United States Patent (USP) 5,196 benzazolyl compounds of 446 chemical compound.

Should be understood to, the used useful micromolecule of the present invention is the EGFR inhibitor, but needn't be specific to EGFR fully.

In a preferred embodiment, the EGFR antagonist is meant the anti-egfr antibodies with one or more following performances:

1) this antibody and the external structure territory of EGFR combine and suppress the combination of part.For example, by utilizing direct combination calibrating purification or that the film binding partner carries out can measure its inhibition.In this embodiment, antibody among the present invention or fragment wherein are preferably so that (EGF combines with EGFR TFG-a) equally by force as the native ligand of EGFR at least.

2) in this antibody and EGFR.The combining activity and the receptor phosphorylation that has excited tyrosine kinase and/or relate to other proteic phosphorylations of unlike signal path of the ectodomain of part and EGFR outside.The neutralization of EGFR comprises with normal one or more the relevant active inhibition of signal transduction, weakens, inactivation and/or interruption.Cell component can be measured in the body, exsomatize or external neutralization after for example utilizing tissue, cultured cell or purification.

The neutral a kind of mensuration of EGFR is to suppress the activity of receptor tyrosine kinase.Utilize known method can examine and determine the inhibition of tyrosine kinase; For example, by measuring recombinate the autophosphorylation level of kinases receptors and/or the phosphorylation of natural or synthetic substrate.Therefore, the phosphorylation calibrating is used for measuring the neutralizing antibody of content of the present invention.For example in ELISA mensuration or immunoblotting, utilize antibody to be specific to phosphorylated tyrosine and can detect phosphorylation.Some detections of relevant tyrosine kinase activity are addressed in people such as Panek, people such as " J.Pharmacol.Exp.Thera. " 283:1433-44 (1997) and Batley, " Life Sci. " 62:143-50 (1998).Antibody of the present invention cause the tyrosine phosphorylation of the EGFR in the cell of replying part reduce at least about 75%, preferably at least about 85% and more preferably at least about 90%.

Neutral another kind of mensuration of EGFR is the phosphorylation that suppresses EGFR downstream substrate.Thereby, can determine the phosphorylation level of MEK and ERK.The degree that weakens of phosphorylation is at least about 40%, and can be at least about 60% or at least about 80%.

In addition, can utilize the detection method that detects protein expression to measure the EGFR neutralization, wherein regulate the protein of being measured by the EGFR tyrosine kinase activity.These methods comprise the immunohistochemistry (IHC) that detects protein expression, fluorescence in situ hybridization (FISH), the competitive radioligand binding assay that detects gene amplification, the solid matrix engram technology is such as RNA and southern blotting technique method, reverse transcriptase-polymerase chain reaction (RT-PCR) and ELISA.Referring to for example, people such as Grandis, " Cancer ", 78:1284-92 (1996); People such as Shimizu, " Japan J.Cancer Res. ", 85:567-71 (1994); People such as Sauter, " Am.J.Path. ", 148:1047-53 (1996); " Collins, Glia " 15:289-96 (1995); People such as Radinsky, " Clin.Cancer Res " .1:19-31 (1995); People such as Petrides, " Cancer Res. " 50:3934-39 (1990); People such as Hoffmann, " Anticancer Res. " 17:4419-26 (1997); People such as Wikstrand, " Cancer Res. " 55:3140-48 (1995).

Stripped calibrating also can be used for measuring the EGFR neutralization.For example can be observed the cell line receptor tyrosine kinase by the short cell division calibrating that utilizes the cell line that receptors ligand excited under the situation that has and do not exist inhibitor suppresses.An example of this short cell division calibrating is the short cell division calibrating of 3T3 cell (being derived from 3T3 (clone A31-714) cell of U.S.'s cell bank of Manassas, Virginia, The United States state)).Other method relates to test and suppresses EGFR expressing tumor cell or the transfection growth with the cell of expressing EGFR.The tumor cell that utilizes tumor model for example to be injected into the intravital people of Mus also can be observed inhibition.

Antibody of the present invention is not limited to the neutral any specific mechanism of EGFR.Anti-egfr antibodies of the present invention can carry out combined outside with the EGFR cell surface receptor, hinder binding partner and follow-up through the associated tyrosine kinase mediated signal transduction of receptor and avoid EGFR and the signal transduction cascade in the proteinic phosphorylation in other downstreams.

3) this antibody is to modulated EGFR.The quantity that is present in the EGFR of cell surface depends on the generation of receptor protein, internalization, degraded.The quantity of the EGFR that the internalization by detecting receptor or the molecule of receptors bind can determine on the cell surface to be had indirectly.For example, can determine the internalization of receptor by the cell of tape label antibody contact expression EGFR.Then membrane-bound antibody is separated, collects, counts.By dissolved cell and detect the antibody of the marker determination internalization in the lysate.

Another approach is directly to measure the acceptor quantity that is stored on the cell, then with anti-egfr antibodies or other mass treatment, and the fluorescence-activated cell sorting detection method of the staining cell by being used for the EGFR surface expression for example.Cultivate staining cell down and measure fluorescence intensity in time at 37 ℃.In contrast, part dyeing colony cultivates (receptor internalization stops under this condition) down at 4 ℃.

Another measure of regulating downwards is to reduce total receptor protein number of being deposited in the cell, and the degraded of reflection inner recipient.Thereby, will cause the minimizing of total cell EGFR with the used antibody treatment cell of the present invention (particularly tumor cell).In a preferred implementation, this minimizing degree is at least about 70%, more preferably at least about 80%, even more preferably at least about 90%.

In order to treat the human experimenter, the antibody among the present invention is preferably human.Alternatively, this antibody can stem from non-human primates or other mammals or humanization or chimeric antibody.

Antibody fragment of the present invention can generate by the DNA of whole antibody of cracking or expression encode fragment.By by people such as Lamoyi, " J.Immunol.Methods ", 56:235-243 (1983) and the described method of " Parham, J.Immunol. " 131:2895-2902 (1983) can prepare antibody fragment.This fragment can comprise Fab fragment or F (ab ＇) ₂Segmental one or both.This fragment also can comprise single-chain fragment variable region antibody, i.e. ScFv antibody, dimer or other antibody fragments.The method that produces this functionally equivalent is disclosed in PCT application WO 93/21319, European patent application EP 239400, PCT application WO 89/09622, European patent application EP 338745 and the European patent application EP 332424.

The vehicle that is used for the present invention transforms and the preferred host cell of antibody expression is the mammalian cell of COS-7 cell, Chinese hamster ovary (CHO) cell for example, and is derived from the lymphocyte series such as lymphoma, myeloma (for example NSO) or hybrid tumor.Optionally use other eucaryons host such as yeast.

The host cell that is transformed can be cultivated by method known in the art in the liquid medium in the assimilated source of containing carbon as described below: carbon (carbohydrate such as glucose or lactose), nitrogen (aminoacid, polypeptide, protein or catabolite such as peptone, ammonium salt or its analog) and inorganic salt (carbonate of sulfate, phosphate and/or sodium, potassium, magnesium and calcium).This medium further comprises for example growth helping matter, such as trace element for example ferrum, zinc, magnesium etc.

Can be from isolate the high-affinity anti-egfr antibodies the present invention by the phage antibody library of human heavy chain and chain variable region gene structure.For example, can obtain variable domains of the present invention from the peripheral blood lymphocyte that contains the variable region gene of recombinating.Alternatively, never homology can obtain the variable domains part such as CDR and FW zone, and can recombinate.In addition, variable domains part (for example FW zone) can be synthetic consensus sequence.

For example can obtain antibody of the present invention and antibody fragment from abiogenous antibody or Fab or scFv phage library.Can be understood as, for from containing V _HAnd V _LThe antibody in territory makes single domain antibody, can expect that some amino acid surrogates of CDRs outside strengthen combination, express or dissolving.For example, can expect that modification is not hidden in V _H-V _LAmino acid residue in the interface.

And then the transgenic mouse (for example from San Jose Medarex Kunming mouse) that utilize to produce human immunoglobulin gamma heavy chain and κ light chain hands over the tumor technology can obtain antibody and antibody fragment (Harlow ﹠amp among the present invention by standard; Lane, ed., antibody: laboratory manual, cold spring harbor laboratory, 211-213 (1998) is herein incorporated by introducing).In preferred embodiment, the substantial portion that produces genomic human antibodies is inserted in the musculus cdna group, and compensation produces the deficiency of endogenous murine antibody.Utilize the PDGFRa (usually in Freund's complete adjuvant) that has exciting agent as required that this kind mice is carried out subcutaneous (s.c) immunity.Immunization method is known in the art.

Spendable antibody comprises the antigen-binding fragment of complete immunoglobulin, immunoglobulin and comprises the antigen-binding proteins of the antigen-binding fragment that comprises immunoglobulin among the present invention.The antigen-binding fragment of immunoglobulin comprises for example Fab, Fab ＇ and F (ab ＇) ₂Develop other antibody formations that keep binding specificity but have other characteristics that to expect and comprised for example bispecific, multivalence (more than two binding sites), compact size (for example separately in conjunction with the territory).

Single-chain antibody lack some or all constant region of deutero-whole antibody.Therefore, they can overcome and use the more related problems of whole antibody.For example, single-chain antibody is considered to not have certain interaction do not expected between CH and the other biological molecule.In addition, single-chain antibody is more much smaller than whole antibody, and has better permeability than whole antibody, thereby allows single-chain antibody more effectively to locate and in conjunction with the targeting antigen binding site.And, to compare with whole antibody, undersized relatively single-chain antibody makes itself littler probability ground evoke undesired immunoreation in the receptor.

Each strand has a V by the first peptide linker covalent bonding _HWith a V _LA plurality of single-chain antibodies of domain can be by at least one or a plurality of peptide linker covalent bonding to form the polyvalent single-chain antibody that can be monospecific or polyspecific.Each chain of multivalence single-chain antibody comprises variable light chain segments and variable heavy chain fragment, and is connected by other chains of peptide linker and at least one.This peptide linker is made up of at least ten five amino acid residues.The maximum number of the sour residue of base is about 100.

Two single-chain antibody formation dimers capable of being combined also can be called the bivalence dimer.Dimer has two chains and two binding sites, and can be monospecific or bispecific.This dimeric each chain comprises and V _LThe V that domain connects _HDomain.Paired joint is connected between this domain and short the domain that is enough to prevent on the same chain, thereby impels between the complementary structure territory on the different chains and match to create this two antigen binding sites again.

Three single-chain antibody formation trimers capable of being combined are also referred to as the trivalent trimer.Trimer can by with V _LDomain or V _HThe V that the carboxyl terminal of domain directly combines _LDomain or V _HThe amino-terminal end of domain constitutes, promptly without any joint sequence.This trimer has three Fv head ends of the polypeptide that ring-type shape mode from beginning to end arranges.This is trimerical may configuration to be planar structure, and three binding sites are positioned at a plane, form 120 ° to each other.Trimer can be monospecific, bispecific or tri-specific.

Therefore, antibody of the present invention and fragment thereof include but not limited to bonded antibody, bivalence fragment such as the F (ab ＇) that forms naturally of specific and antigen ₂, unit price fragment such as Fab, single-chain antibody, strand Fv (scFv), single domain antibody, multivalence single-chain antibody, dimer, trimer etc.

According to the present invention, EGFR antagonist and one or more antitumor drug administering drug combinations.Just as used herein, this term " antitumor " medicine does not comprise the EGFR antagonist, unless other situations are refered in particular to.Can use any suitable antitumor drug, such as chemotherapeutant, radiation or its combination.This antitumor drug can be alkylating agent or antimetabolite.The example of alkylating agent includes but not limited to cisplatin, cyclophosphamide, melphalan and dacarbazine.The example of antimetabolite includes but not limited to amycin, daunorubicin (dunorubicin), paclitaxel and gemcitabine.

In a preferred implementation, this antitumor drug is a chemotherapeutant.Preferred chemotherapeutant comprises amifostine, cisplatin, dacarbazine (DTIC), dactinomycin, dichloromethyldiethylamine (chlormethine), streptozocin, cyclophosphamide, card chlorine mustard (BCNU), chlormethine (CCNU), adriamycin (amycin), Evacet (doxil), gemcitabine (gemzar), daunorubicin, daunorubicin liposome (daunoxome), the methylbenzyl hydrazine, mitomycin, cytosine arabinoside, etoposide, 9-methylpteroylglutamic acid, fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), Docetaxel (taxotere), aldesleukin, Radix Asparagi amidase, busulfan, carbo, cladribine, camptothecine, CPT-11,10-hydroxyl-7-ethyl-camptothecine (SN38), dacarbazine, uridnine, fludarabine, hydroxyurea, ifosfamide, comply with as than crisp (idarubicui), mesna, interferon-ALPHA, interferon beta, Irinotecan, mitoxantrone, topotecan, leuprorelin, megestrol, melphalan, mercapto (base) purine, plicamycin, mitotane, pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin, zitazonium, teniposide, testolactone, thioguanine, thiophene replaces group,, uracil mustard, vinorelbine, chlorambucil and above-mentioned combination.In a preferred embodiment, this chemotherapeutant is an Irinotecan.

According to dosage comprise the administration of the antitumor drug of chemotherapeutant and/or antimetabolite according to package insert and/or patient's clinical response on time.This dosage is conspicuous for the person of ordinary skill of the art, need not improper experiment.For example, preferably Irinotecan being carried out the recommended dose scope is about 50mg/m ²To about 350mg/m ²Administration, but this dosage can or high or low, this will depend on patient's clinical response.The dosage range of each administration of other drug and/or therapeutic scheme is about 1mg/m ²To about 1,000mg/m ²Clinical experience based on suitable clinical trial protocol and/or those skilled in the art is implemented radiotherapy.

In therapeutic alliance, begin with before another Drug therapy, during or carry out the administration of EGFR antagonist afterwards, also can be its any compound mode, promptly before the beginning antitumor drug treatment and during, before and afterwards, during and afterwards or before, during and afterwards.For example, before the beginning radiotherapy, can 1 day to 30 days, preferred 3 days to 20 days, more preferably carry out the administration of EGFR antibody between 5 days to 12 days.In preferred implementation of the present invention, chemotherapy can be implemented simultaneously with Antybody therapy, perhaps more preferably implements chemotherapy after Antybody therapy.

In the present invention, can utilize any appropriate method or approach to carry out the administration of antagonist of the present invention, and selectively, with antitumor drug and/or other acceptor inhibitor administering drug combinations.Scheme according to antitumor drug that the present invention utilizes comprises any scheme that is considered to be most suited to treat patient tumors.Different tumors need be used specific anti-tumour antibody and specific antitumor drug, implement according to patient's tolerance basis.That route of administration comprises is for example oral, intravenous administration, intraperitoneal administration, subcutaneous injection administration or administered intramuscular.The dosage of antagonist depends on all multifactor, comprise antagonist type for example, the route of administration of the tumor type for the treatment of and seriousness and antagonist.But should be emphasized that the present invention is not limited to any specific administration method or approach.

It should be appreciated by those skilled in the art that the therapeutic dose and the frequency depend on the dosis tolerata of individual patient and the pharmacology and the pharmacokinetics character of used blocker or inhibitor.Ideal situation is to realize the saturability pharmacokinetics of used medicine.The loading dose scope of anti-egfr antibodies can be for example about 10mg/m ²To about 1000mg/m ², preferably about 200mg/m ²To about 400mg/m ²After this be several additional day dosage or weekly dose scope, for example about 200mg/m ²To about 400mg/m ²Monitoring is implemented in the side effect that the patient showed, when this side effect is serious, stopped treatment.

Those skilled in the art will be appreciated that also how the monitor treatment process is to determine effective dose.For example, a kind of approach is monitoring MRI, CT or other brain inspections.

Embodiment

Embodiment 1: following embodiment discloses the method by the administering drug combinations treatment malignant entity tumor of Irinotecan and EGFR antibody.

From year March in August, 2005 to 2006, carry out the administration of the Cetuximab of Irinotecan and various dose level for 20 infant patients at least 8 weeks to suffering from malignant entity tumor and life expectancy.With following tumor type the patient be divided into two groups (age be 1-12 year be the A of colony, the age be 13-18 year be the B of colony): brain stem glioma/astrocytoma (10), hepatoblastoma, osteosarcoma (1), ependymoma (1), neuroblastoma (1), rhabdomyosarcoma (1) and other tumors (5).The dosage of Irinotecan is 60 minutes infusion 20mg/m ²/ day, 5 days weekly, totally two weeks, per 21 days courses of treatment.Following table 1 is dosage and the experimenter's quantity that has dose-limiting toxicity (DLTs):

Table 1

Colony	Dosage level	Cetuximab (mg/m 2/ day, 1 day, 8 days, 15 days)	Irinotecan (mg/m 2/ day, 1-5 days and 8-12 days)	Experimenter's quantity	The experimenter's quantity that has DLT (dose-limiting toxicity)
						A	1	75	20	6	1 (3rd level diarrhoea)
A	2	150	20	6	2 (the 4th grade of neutrophils reduces, 3rd level diarrhoea)
						B	1	75	20	8	1 (3rd level diarrhoea)

Because two patients among six patients of the A of colony, dosage level 2 have the experience of DLTs, are considered to be relevant to Irinotecan, and Irinotecan progressively is downgraded to 16mg/m ²A patient experience among the B of colony the 3rd level infusion reaction, thereby stop the treatment.Other viewed toxicity comprise the 1st grade of erythra.

An experimenter who suffers from the higher nerve glioma (A group, dosage level 1) of EGFR feminine gender realizes that part alleviates and be at present in the cycle 13.An experimenter who suffers from neuroblastoma (A group, dosage level 1) experiences slight alleviation and experiences the cycle 9 at present.The best that seven experimenters have average 3.4 months stable disease is replied (between less than two months to the time range more than seven months).

In a word, the combination of Irinotecan and Cetuximab demonstrates desirable anti-tumor activity in malignant entity tumor.

Above-mentioned explanation of having set forth and embodiment only are used to describe the present invention, do not think limitation of the present invention.Can think each disclosed viewpoint of the present invention and embodiment for independently or with the combination of other viewpoints of the present invention, embodiment and modification.And when some features in the embodiments of the present invention only showed in some diagrams, these features can be incorporated in the represented embodiment of other diagrams, still are within protection scope of the present invention simultaneously.In addition, unless specific in other situations, any method step of the present invention does not limit the certain order of any enforcement.Those skilled in the art will find apparent that the modification of the disclosed embodiment that merges spirit of the present invention and purport, this modification all within the scope of the present invention.And whole reference contents of all references are herein incorporated, with for referencial use.

Sequence table

＜110〉Imclone Systems Inc. (IMCLONE SYSTEMS, INC.)

＜120〉with the intravital tumor of EGF-R ELISA anticaking agents treatment infant

<130>1017.52210-PCT

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<150>60/833,487

<151>2006-07-27

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＜170〉PatentIn is 3.3 editions

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Claims (14)

1. method that suppresses infant patient's tumor growth in vivo, described method comprise epidermal growth factor (EGFR) antagonist and the chemotherapeutant treatment infant patient with effective dose.

2. method according to claim 1 is characterized in that, described tumor is a malignant tumor.

3. method according to claim 1 is characterized in that, described tumor is a glioma.

4. method according to claim 1 is characterized in that, described tumor is a neuroblastoma.

5. method according to claim 1 is characterized in that, described antagonist is antibody or fragment wherein.

6. method according to claim 5 is characterized in that, described antibody or fragment wherein are specific to be combined with the EGFR ectodomain.

7. method according to claim 5 is characterized in that, the specific inhibition of described antibody or fragment wherein EGFR part combine with EGFR and neutralize wherein activity.

8. method according to claim 5 is characterized in that, described antibody is Cetuximab.

9. method according to claim 5 is characterized in that, described antibody or fragment wherein are monoclonal.

10. method according to claim 5 is characterized in that, described antibody or fragment wherein are chimeric.

11. method according to claim 5 is characterized in that, described antibody or fragment wherein are humanized.

12. method according to claim 5 is characterized in that, described antibody or fragment wherein are human.

13. method according to claim 1 is characterized in that, described chemotherapeutant is an Irinotecan.

14. method according to claim 1, this method further comprise the infant patient is implemented radiotherapy.