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CN101432015A - Functional antibodies - Google Patents

️Wed May 13 2009

CN101432015A - Functional antibodies - Google Patents

Functional antibodies Download PDF

Info

Publication number

CN101432015A

CN101432015A CNA2007800111267A CN200780011126A CN101432015A CN 101432015 A CN101432015 A CN 101432015A CN A2007800111267 A CNA2007800111267 A CN A2007800111267A CN 200780011126 A CN200780011126 A CN 200780011126A CN 101432015 A CN101432015 A CN 101432015A Authority

China

Prior art keywords

antigen

antibody

domain

binding proteins

binding site

Prior art date

2006-02-15

Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)

Pending

Application number

CNA2007800111267A

Other languages

Chinese (zh)

Inventor

朱祯平

Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)

ImClone LLC

Original Assignee

ImClone Systems Inc

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2006-02-15

Filing date

2007-02-15

Publication date

2009-05-13

2007-02-15 Application filed by ImClone Systems Inc filed Critical ImClone Systems Inc

2009-05-13 Publication of CN101432015A publication Critical patent/CN101432015A/en

Status Pending legal-status Critical Current

Links

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Abstract

The invention is directed to novel antibodies, which comprise single domain binding sites. The antibodies can be bivalent or multivalent, and can be bispecific. The invention is further directed to monospecific and bispecific antibodies that bind to mPDGFRa. The antibodies can be administered alone or in combination with anti- angiogenic or anti-neoplastic drugs.

Description

Functional antibodies

The cross reference of related application

The application requires the U. S. application No.60/773 of application on February 15th, 2006, and 994 priority, this application are introduced for referencial use in full.

Invention field

The present invention relates to new antibody, it comprises single structure territory binding site.Above-mentioned antibody can be bivalence or multivalent antibody, and can be bispecific or polyspecific.The present invention has also disclosed in conjunction with the bi-specific antibody of cell surface receptor protein and multi-specificity antibody.When giving object separately or with angiogenesis inhibitor or antitumorigenic drug regimen, above-mentioned antibody can be used for suppressing tumor growth and suppresses the hyperplasia disease.

Background of invention

Bi-specific antibody (BsAb) is based on the molecule of immunoglobulin (Ig), and it is in conjunction with two different epi-positions on the identical or different antigen.Laboratory and early stage clinical research have shown that BsAb can have remarkable meaning in treatment of cancer, for example by making cytotoxic agent such as effector lymphocyte, radionuclide, medicine and toxin target tumor cell (Weiner et al., 1997, Cancer Immunol.Immunother.45:190-2.; Van Spriel et al., 2000, Immuol.Today 21:391-7; Segal et al., 2000, J.Immunol.Methods 248:1-6), perhaps by biologic activity (the Lu et al. of while two different tumor target position of targeting (perhaps epi-position) to strengthen single Antybody therapy method, 1999, J.Immunol.Methods 230:159-71; Lu et al., 2001, Cancer Res.61:7002-8.; Lu et al., 2002, J.Immunol.Methods 267:213-26).Development is to carry out clinical research (Carter et al. by the material that the traditional method that comprises hybridoma and chemical bond method is difficult to produce sufficient amount and quality based on a major obstacle in the Therapeutic Method of BsAb, 1995, J.Hematotherapy 4:463-70).The IgG light chain of two different series and the coexpression of heavy chain can produce many light chains and heavy chain is right, wherein only a pair of be the functional bispecific heterodimer of wishing (Suresh et al., 1986, MethodsEnzymol.121:210-28).On the other hand, two IgG or its segmental chemical crosslinking are normally invalid, and can cause losing antibody activity (Zhu et al., 1994, Cancer Lett.86:127-34).In these two kinds of methods, the purification of BsAb from the non-functional material is lower usually than difficulty and output usually, described non-functional material for example is non-homogeneous Ig light chain and the homodimer of heavy chain and the heterodimer of mispairing that produces by hybridoma technology, and derive from chemically combined poly aggregation (Cao et al., 1998, Bioconj.Chem.9:635-44).

In order to improve effect, developed many recombination methods and produced BsAb, as antibody fragment form (Carter et al., 1995; Pluckthun et al., 1997, Immunotechology 3:83-105; Todorovska et al., 2001, J.Immunol.Methods 248:47-66) and total length IgG form (Carter, 2001, J.Immunol.Methods 248:7-15).For example, the generation of homology total length IgG sample BsAb realizes effective I g C by so-called " knobs-into-holes " engineering method _H3 domain heterodimerizations (Ridgway et al., 1996, Protein Eng.9:617-21; Merchant etal., 1998, Nat.Biotech.16:677-81), and by not homospecific two strand Fv (scFv) fusion (Zhuang et al., 2000, Protein Eng.13:361-7 on the N or C-terminal of total length IgG molecule; Coloma and Morrison, 1997, Nat.Biotechnol.15:159-63) realize.The following structure of BsAbs promptly uses or does not use flexible joint heredity to merge two strand Fv (scFv) or Fab fragment (Mallender et al., 1994, J.Biol.Chem.269:199-206; Mack et al., 1995, Proc.Natl.Acad.Sci.USA.92:7021-5; Zapata et al., 1995, Protein Eng.8:1057-62), through dimerization device such as leucine zipper (Kostelny et al., 1992, J.Immunol.148:1547-53; De Kruif et al., 1996, J.Biol.Chem.271:7630-4) and Ig C _L/ C _H1 domain (Muller et al., 1998, FEBS Lett.422:259-64); Through bivalent antibody (Holliger etal., 1993, Proc.Nat.Acad.Sci.USA.90:6444-8; Zhu et al., 1996, Bio/Technology (NY) 14:192-6); Fab-scFv merges (Lu et al., 2002; Schoonjans etal., 2000, J.Immunol.165:7050-7); And little antibody formation (miniantibodyformats) (Pack et al., 1992, Biochemistry 31:1579-84; Pack et al., 1993, Bio/Technology 11:1271-7).In most applications, these recombination methods cause bivalent, bispecific antibodies to produce, and it is monovalent for each target antigen.Further, the IgG sample bi-specific antibody example that comprises functional Fc domain is limited.

Summary of the invention

The invention provides new bi-specific antibody, it comprises single variable domains (sVD) antigen binding site.Antibody of the present invention also can comprise such antigen binding site, and it also comprises antibody Fvs except the sVD antigen binding site.

Antibody of the present invention can be specific to any antigen.In one embodiment, antibodies cell surface antigen of the present invention, described antigen can be receptor tyrosine kinase (comprise but the non-PDGFR of being limited to, VEGFR1, VEGFR2, EGFR).In another embodiment, the part of antibodies cell surface receptor of the present invention.In a preferred embodiment, antibody of the present invention has in the receptor and activity.The present invention further provides the materia medica compositions of described antibody.

The invention provides and suppress the activated method of one or more receptor tyrosine kinase.Tumor growth in the mammal can be treated or suppresses by the antibody of the present invention that gives this mammal effective dose.Antibody of the present invention can with combine other cell surface antigen (comprising for example RTK) or the cytokine antibody co-administered of (comprising for example RTK part).In certain embodiments, method of the present invention also comprises and gives described mammal antitumor agent or comprise chemotherapeutics for example and/or the Therapeutic Method of radiotherapy.In another embodiment, the invention provides for example psoriatic method of non-cancer hyperplasia disease in the treatment mammal.

The accompanying drawing summary

Fig. 1 is the example schematic of the bispecific antigen-binding proteins based on single domain antibody of the present invention.Described single structure territory (being called VH2 in the drawings) can be mixed by N-terminal or the C-terminal that is blended in other antibody structure territory.

Fig. 2 illustrates expression and the purification of mPDGFR α-specificity Fab.Described Fab expresses in escherichia coli host HB2151 cell, by affinitive layer purification, and analyzes by SDS-PAGE.A) have the heavy chain (VH-CH1) of similar molecular weight and " standard " mPDGFR α-specificity Fab of light chain (VL-CL).B) there be or do not exist 1F2 and 1F9Fab fragment and the standard Fab contrast that lacks the VL domain under the DTT condition.The unit of molecular weight marker is a kilodalton.

Fig. 3 illustrates combination and the blocking-up of the Fab of purification and measures.A) Fab of purification and mice PDGFR α's quantitatively combines ELISA.At first the Fab with various amounts is being incubated in the 96 hole flat boards with mPDGFR α-Fc fusion rotein bag quilt in advance, resists with rabbit subsequently-Anti-Human-insulation of κ antibody HRP conjugate.Quantize by adding peroxidase substrate with the bonded antibody-HRP of flat board, read absorbance at A450nm.B) Fab of purification is to mPDGFR α and the bonded inhibition of fixed PDGF-AA.The Fab of various amounts and the mPDGFR α/Fc of fixed amount are incubated in solution.Mixture is incubated in 96 hole flat boards with PDGF-AA bag quilt, subsequently with anti-people-Fc antibody-HRP conjugate insulation.Quantize bonded mPDGFR α by adding peroxidase substrate then, read absorbance at A450nm.The data that illustrate are meansigma methods ± SD of duplicate sample.

Fig. 4 illustrates the structure of unit price of the present invention and bivalence Fab.

Fig. 5 illustrates expression and the purification of bivalence mPDGFR alpha specific Fab.Described Fab expresses in escherichia coli host HB2151 cell, by affinitive layer purification, and analyzes by SDS-PAGE.Contrast bivalence 1F2-2H Fab and standard Fab (5C5) under non-reduced condition.The unit of molecular weight marker is a kilodalton.

Fig. 6 illustrates measuring in conjunction with blocking-up of antibody purified.A) anti--mPDGFR α unit price of purification and bivalence 1F2Fab antibody is quantitative in conjunction with measuring.The antibody of various amounts is incubated in the 96 hole flat boards of using mPDGFR α/Fc bag quilt in advance, subsequently with Anti-Human-K antibody HRP conjugate insulation.Quantize to read absorbance at A450nm by adding peroxidase substrate then in conjunction with dull and stereotyped antibody-HRP.B) the Fab antibody of purification is to mPDGFR α and the bonded inhibition of fixed PDGF-AA.The antibody of various amounts and the mPDGFR α of fixed amount-Fc are incubated, this mixture are transferred in advance wrap in the flat board of quilt with PDGF-AA.After the washing, should flat board and Anti-Human-Fc antibody-HRP conjugate insulation.Quantize bonded mPDGFR α by adding peroxidase substrate, read absorbance at A450nm.The data that illustrate are meansigma methods ± SD of duplicate sample.2B4 is the antibody at mice VEGFR2 (F1k1).

Fig. 7 provides the sketch map based on the total length IgG of 1F2.1F2VH is expressed as the fusion rotein that merges to CL and/or CH, provide tetravalence (A group: MW～150,000) or bivalence (B group and C organize: MW～125,000) antibody.

Fig. 8 illustrates the expression and the purification of anti-mPDGFR Alpha antibodies.In right group, carrying out handling antibody with DTT before the electrophoresis.

Fig. 9 illustrates the combination and the part blocking-up of antibody purified and measures.A) quantitative anti--mPDGFR Alpha antibodies in conjunction with purification.The antibody of various amounts is incubated in the 96 hole flat boards of using mPDGFR α/Fc bag quilt in advance, subsequently with anti-people-κ antibody HRP conjugate insulation.Quantize to read absorbance at A450nm by adding peroxidase substrate in conjunction with dull and stereotyped antibody-HRP.B) antibody purified is to mPDGFR α and the bonded inhibition of fixed PDGF-AA.With the antibody of various amounts and the mPDGFR α of fixed amount/Fc insulation, be transferred in advance and wrap in the flat board of quilt with PDGF-AA.Behind the washing flat board, itself and Anti-Human-Fc antibody-HRP conjugate are incubated.Quantize bonded mPDGFR α by adding peroxidase substrate then, read absorbance at A450nm.Meansigma methods ± the SD of the duplicate sample of data representation.2B4 is the antibody at mice VEGFR2 (F1k1).

Figure 10 provides the sketch map in conjunction with the selected tetravalence bi-specific antibody based on 1F2 of mPDGFR α and flk-1.A) IgG-sample antibody is called 1F2/scFv2B4.1F2 V _HBe expressed as the fusant that merges to CL, 2B4 is expressed as and merges to C _HThe scFv fusant.B) IgG-sample antibody is called 1F2-2B4.1F2 VH is expressed as and 2B4V _LThe fusant that amino terminal merges.

Figure 11 illustrates based on the expression of the bi-specific antibody of 1F2 and purification.Described antibody is expressed in mammalian cell, by affinitive layer purification, and analyzes by SDS-PAGE.Before electrophoresis with/need not handle described antibody by (+/-) DTT.The unit of molecular weight marker is a kilodalton.

Figure 12 illustrates the quantitative combination of anti--mPDGFR α * anti--Flk-1 bi-specific antibody of purification and measures.A) at first the antibody of various amounts is incubated in the 96 hole flat boards of using mPDGFR α/Fc bag quilt in advance, subsequently with Anti-Human-κ antibody HRP conjugate insulation.B) at first the antibody of various amounts is being incubated in the 96 hole flat boards with Flk-1/Fc bag quilt in advance, subsequently with anti-people κ antibody HRP conjugate insulation.The data that illustrate are meansigma methods ± SD of duplicate sample.

The blocking-up of Figure 13 illustrates the bispecific of purification anti--mPDGFR α * anti--Flk-1 bi-specific antibody is measured.A) bi-specific antibody of purification is to the bonded inhibition of mPDGFR α and fixed PDGF-AA.B) bi-specific antibody of purification is to the bonded inhibition of Flk-1 and fixed VEGF165.In these two mensuration, the antibody of various amounts and the receptor of fixed amount (mPDGFR α/Fc or Flk-1) are incubated in solution, be transferred in the flat board of using part (PDGF-AA or VEGF165) bag quilt in advance.Then with flat board and Anti-Human-Fc antibody-HRP conjugate insulation.Quantize mPDGFR α by adding peroxidase substrate, read absorbance at A450nm.The data that illustrate are meansigma methods ± SD of duplicate sample.

Figure 14 illustrates the inhibitory action of bispecific 1F2-2B4IgG to the receptor phosphorylation of PDGF and VEGF stimulation.At first eEnd.1 cell and various antibody are incubated 30 minutes at 37 ℃, stimulated 15 minutes at 37 ℃ with VEGF or PDGF subsequently.After lysis, by reaching insulation immunoprecipitation receptor from the cell lysate supernatant of ProA/G-sepharose 4B subsequently with anti--mPDGFR α or anti--mVEGFR2 antibody.On 4-12% Nupage Bis-Tris gel, differentiate sedimentary receptor protein, be transferred on the polyvinylidene fluoride film.Use anti--phosphotyrosine antibody-HRP conjugate on trace, to detect phosphoric acid-mVEGFR2 and phosphoric acid-mPDGFR α.The total receptor protein of application of sample on gel uses the TPPA of mPDGFR α or mVEGFR2.

Figure 15 illustrates the proteic V of some Fab that identified _HAnd V _LThe aminoacid sequence of domain.Fab1E10,1A11,3B2,1C10,3G7,1F9 and 1F2 differentiate at the screening in conjunction with the Fab-phage of mice PDGFR α.Fab2B4 differentiates at the screening in conjunction with the Fab-phage of mice VEGFR2.^: frameshift mutation; *: the termination codon that frameshift mutation produces; @...@: in-frame disappearance.

Detailed Description Of The Invention

The invention provides the new antibodies that comprises single variable domains (sVD) antigen binding site. The present invention also provides antigen-binding proteins, and it comprises the Fv sample antigen binding site of single domain antigen binding site and two domains.

Single domain antigen binding site and immunoglobulin variable domain (V for example_HOr V_L) similar, but there is not second antigen binding structural domain (corresponding V for example_LOr V_H) condition under can specific binding antigen. Perhaps, this single domain in fact can not associate with corresponding binding structural domain is stable. Yet, single domain antibody can with both comprised V_LDomain also comprises V_HThe compatibility that the antibody of domain is similar and affinity conjugated antigen. In addition, the same with natural antibody, single domain antibody can be blocked receptor-ligand binding. Therefore, the antibody of the phage display library of hybridoma, the transgenic mice of expressing people's antibody and Fab or scFv binding structural domain is used for identical purpose to the antibody of the present invention that comprises the single domain binding site with for example deriving from.

Antibody of the present invention is immunoglobulin-like antibody in some respects. 1) the allos tetramer that formed by two different polypeptide chains (each polypeptide chain is comprised of two different polypeptide chains) of the antibody of the present invention of IgG sample. 2) described different polypeptide chain associates and can for example pass through disulfide bond in the same manner heavy chain and the constant region of light chain of the naturally occurring immunoglobulin (Ig) of covalently bound standard. 3) one of described polypeptide chain can be stablized self-association in the mode of naturally occurring heavy chain immunoglobulin. For example, one of described polypeptide chain can comprise the C corresponding to naturally occurring immunoglobulin (Ig)_H2、C _H3 or C_HOne or more domain of 4. In a preferred embodiment, antibody of the present invention comprises the constant domain structure of naturally occurring IgG.

A huge advantage of immunoglobulin spline structure is antibody chain natural pairing when expressing.Different with more existing tetramer antibody, do not need further processing can obtain the homogeneity antibody preparations.

Antigen-binding proteins of the present invention comprises antigen binding site, and these antigen binding sites are single variable domains (sVDs).In certain embodiments of the invention, the sVD binding site replaces a variable domains of IgG sample antibody, and scFv replaces another variable domains.The example of this embodiment is described in Figure 1A.In other embodiments of the present invention, the sVD binding site engages (graft) at the N-terminal or the C-terminal of IgG heavy chain or light chain with IgG antibody.The example of this embodiment is described in Figure 1B.SVD also can mix in other position of immunoglobulin-like antibody.For example, sVD can be attached to C _HAnd/or C _LThe C-terminal of constant domain.In addition, the replacement by making up sVD binding site shown in Figure 1 and at C _HAnd/or C _LThe C-terminal of constant domain adds the sVD binding site, can produce for specific antigen polyspecific and/or polyvalent IgG-sample antibody.

As known in the art, Fv forms (V for example by two domains _HAnd V _LDomain).In the antibody of standard, the V of Fv _HAnd V _LAlthough associating, domain forms antigen binding site, whether direct-connected, but each connects with the antibody constant region that is connected.The VH of Fc and VL domain also can be connected to form strand Fv (scFv) with synthetic linker.

Antibody specificity is meant the selectivity identification of antibody for antigenic defined epitope.For example, natural antibody is a monospecific.Bi-specific antibody (BsAb) is the antibody with two kinds of different antigen-binding specificities or site.When antigen-binding proteins had more than one specificitys, the epi-position that is identified can be relevant with single antigen or an above antigen.

The natural antibody molecule is made up of with two identical light chains two identical heavy chains.Each light chain is all covalently bound by intrachain disulfide bond and heavy chain.Two heavy chains further are connected to each other by a plurality of disulfide bond at hinge region.Each chain folding is domain (approximately 110-125 aminoacid) and the structure with similar size, but their function differences.Light chain comprises a variable domains (V _L) and a constant domain (C _L).Heavy chain comprises a variable domains (V _H), and comprise three or four constant domain (C according to the classification or the isotype of antibody _H1, C _H2, C _H3 and C _H4).In mice and people, isotype is IgA, IgD, IgE, IgG and IgM, and IgA and IgG further are further divided into subclass or hypotype.By V _LAnd V _HThe antibody moiety that domain is formed is known as Fv, and it forms antigen binding site.Strand Fv (scFv) is the protein through through engineering approaches, contains V on a polypeptide chain _LDomain and V _HDomain, the N-terminal of one of them domain and the C-terminal of another domain are by the flexible joint combination.Fab is meant by V _L, V _H, C _LAnd C _HThe antibody moiety that 1 domain is formed.＂

Variable domains demonstrate an antibody with next, particularly be positioned at the aminoacid sequence transmutability of the antibody of antigen binding site.At V _LAnd V _HIn respectively found three zones, be called " hypervariable region ", " complementary determining region (CDR) ".

Fc is the title that comprises the antibody moiety of paired heavy chain constant domain.At IgG ₁In the antibody, for example Fc comprises C _H2 and C _H3 domains.The Fc of IgA or IgM antibody further comprises the CH4 domain.Fc is relevant with the cytotoxicity and the antibody dependent cellular cytotoxicity of Fc receptors bind, complement-mediated.For natural antibody such as IgA and IgM, they are the proteinic complex of a plurality of IgG samples, and complex forms needs the Fc constant domain.

At last, " hinge " distinguished the Fab and the Fc part of antibody, provides Fab relative to each other and with respect to the activeness of Fc, and comprised that a plurality of disulfide bond are with covalently bound two heavy chains.

Antibody of the present invention has the characteristics combination of hope.At first, they are homogeneity basically.By design, the mispairing of heavy chain of antibody and light chain is significantly reduced or is eliminated.For example, some bi-specific antibodys utilize two different heavy chains that two species specificity are provided.When this heavy chain is arranged in the IgG type molecule, have four kinds of combinations.Wherein two kinds of heavy chains by mispairing are formed, and product is a monospecific thus.In antibody of the present invention, mispairing is eliminated basically.

Second feature of antibody of the present invention is that it is a bivalence for every kind of binding specificity.Many bi-specific antibodys are monovalent for each antibody combining site that comprises.This is significant for antibody function, because bivalence allows bonded concertedness, and binding affinity significantly increases with respect to the molecule that comprises the former specific binding site of monoclonal antibody.

The 3rd advantage of antibody of the present invention is exist to form the Fc zone of natural antibody (IgG for example ₁The C of antibody _H2 and/or C _H3) and one or more heavy chain constant domain of other antibody function is provided.In addition, a plurality of binding structural domains and constant domain are separated, and the function that is provided by constant domain is not weakened thus.The constant domain function comprises in conjunction with some additional molecule (for example in conjunction with cell surface and soluble Fc receptor, J chain at IgA and IgM associates, S albumen at IgA), activating complement approach (CDC, CDC), by some different leukocyte groups to the identification of the bonded antibody of target cell (cytotoxicity of antibody dependent cellular mediation, ADCC) and opsonic action (enhancing phagocytosis).Fc heavy chain constant domain also can be given the serum half-life of increase.

Proteinic the 4th advantage of the present invention is to obtain no requirement (NR) in the complete product in external processing.Although reset with manual type, each domain all has the permission natural feature that system's invading the exterior reaches in biology.For example, bi-specific antibody can be expressed in protokaryon and eukaryotic expression system.The protein that produces is bispecific on substantially.

The sVD antigen binding site that is used for antibody of the present invention can obtain by several different methods with the binding site that contains the Fv district.The V of selected binding structural domain _HAnd/or V _LThe aminoacid sequence of part can derive from naturally occurring antibody, perhaps selecting or modifying, screening in conjunction with feature, perhaps selection according to hope.For example, V _HAnd/or V _LDomain can directly derive from the monoclonal antibody in conjunction with feature with hope.Perhaps, V _HAnd/or V _LDomain can derive from the mammiferous V gene order library of selection.The element in this library is expressed V _HAnd/or V _LThe combination at random of domain, and had those elements of hope in conjunction with feature with discriminating by the antigen selection of any hope.Particularly preferably be people V gene library.This method for screening is known in the art.Perhaps, from the V in selected inhuman source _HAnd/or V _LDomain can be impregnated in the chimeric antibody that comprises people's constant domain.For example, for administration of human, that may wish is to use the antibody with one or more functional human constant domain, wherein V _HAnd V _LDomain is selected from inhuman source.In order to maximize the immunogenicity of constant domain correlation function or reduction antibody, preferred people's constant domain.

Perhaps, the V domain can be by " humanization ".The structure of humanized variable domains is that the aminoacid sequence that wherein comprises one or more complementary determining region (CDRs) in inhuman source combines with people's framework region (FR).For example, see Jones, P.T.et al., 1996, Nature 321,522-25; Riechman, L.et al., 1988, Nature 332, and the U.S. Patent No. 5,530,101 of 323-27 and Queen etc. is described.Humanized construct is used in particular for eliminating disadvantageous immunogenicity feature, for example is used for the treatment of in people's the situation in the antigen binding structural domain expection from inhuman source.Variable domains has the attach structure homology, allows to differentiate easily the amino acid residue corresponding to CDR and FR in the variable domains.See for example Kabat, E.A., et al., 1991, Sequences of Proteins of Immunological Interest.5thed.National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD is described.Therefore, participating in the bonded aminoacid of antigen is easy to be differentiated.In addition, developed the method for antigen affinity that keeps or strengthen the humanization binding structural domain of the CDR that comprises joint.A kind of mode is that the external framework residue that will influence described CDR district conformation is included in the receptor variable domains.Another kind of mode be with external CDR be bonded on the most homologous people's variable domains in described external variable region on.Queen，C.et?al.，1989，Proc.Natl.Acad.ScL?USA?86，10029-33。By at first using each FR sequence of overlapping primer amplification of the CDR sequence that comprises hope, reach connection gained gene segment in amplified reaction subsequently, be engaged with on the different FR CDR most convenient.CDR is bonded on the different variable domains can further replace such amino acid residue, and the CDR in these residues and the aminoacid sequence is adjacent or pile up (packed) in the folding varistructure domain structure of the conformation that influences CDR at CDR.Humanized variable domains of the present invention therefore comprise the people's domain that comprises one or more inhuman CDR and wherein additionally replaced or replace with keep or enhancing in conjunction with the domain of feature.

Antibody of the present invention also can use the residue that is exposed to the surface by displacement to reduce immunogenic variable domains, with generation seem at the autoimmune system antibody (Padlan, E.A., 1991, MoI.Immunol.28,489-98).Antibody modify by this method do not lose affinity (Roguska et al., 1994, Proc.Natl.Acad.ScL USA 91,969-973).Because near amino acid whose inner accumulation the antigen binding site remains unchanged, and therefore kept affinity.The replacement of the residue that is exposed to the surface that reduces immunogenicity according to the present invention and carry out does not mean that CDR residue or the influence replacement in conjunction with the adjacent residues of feature.

Usually can preferably use is people's variable domains basically.People's binding structural domain can derive from phage display library, wherein the combination of people's heavy chain and light chain variable domain be showed on the surface of filobactivirus (see for example McCafferty et al., 1990, Nature 348,552-54; Aujame et al., 1997, Human Antibodies 8,155-68).The combination typical case of variable domains is showed on the filobactivirus with Fab or scFv form.At the variable domains combined sorting library of the antigen with hope in conjunction with feature.Preferred single structure territory and variable domains combination demonstrate for the antigenic high affinity of selecting and for the cross reactivity seldom of other related antigen.(see for example Griffiths et al. by screening numerous antibody fragments, 1994, EMBOJ.13,3245-60), separate diversified high affinity binding structural domain, expection some of them domain has inferior nanomole (sub-nanomolar) affinity for the antigen of hope.

Perhaps, people's binding structural domain can derive from wherein import the people Ig gene segment do not reset and wherein endogenous mouse Ig gene in the transgenic animal of inactivation (referring to Bruggemann and Taussig, 1997, Curr.Opin.Biotechnol.8,455-58).Preferred transgenic animal contain greatly continuous Ig gene segment (the Mendez et al. that size surpasses 1Mb, 1997, Nature Genet.15,146-56), but the people Mab of medium affinity can produce from the transgenic animal that contain less locus and (see for example Wagner et al., 1994, Eur.J.Immunol.42,2672-81; Green et al., 1994, Nature Genet.7,13-21).

The sVD binding site can derive from antigenic specificity Fv zone, and (it had both comprised V _HDomain also comprises V _LDomain).Normally, can show that the binding affinity in Fv zone and specificity are mainly caused by one of variable domains.Perhaps, scFv can directly obtain.The direct sources of sVD comprises mammal (for example camel), and its natural expression only contains V _HThe antibody of domain also is configured to the phage display library of only expressing single variable domains.For example, people's domain antibodies phage display library commercially available from Domantis (Cambridge, UK).As this paper institute illustration, the sVD binding site that is specific to PDGFR α derives from and contains V _LThe Fab library of the variant that domain has lacked naturally.

In physiology's immunne response, the sudden change of the antibody gene of expression and selection cause target antigen is had the production of antibodies of high affinity.Mix the V in the antibody of the present invention _HAnd V _LDomain can be carried out external sudden change similarly, and the row filter program of going forward side by side obtains the variant of high affinity.Therefore, binding structural domain of the present invention comprises in conjunction with feature by direct sudden change or those domains of being improved by the affinity maturation method.Affinity and specificity can by the sudden change CDR and the screening have the expection feature antigen binding site and modified or improved (see for example Yang et al., 1995, J.MoI.Bio.254,392-403).The main aminoacid in conjunction with determiner that those skilled in the art understand as single domain antibody can be in the CDR that Kabat describes, but can comprise other residue and for example as be embedded in V _H-V _LThe V of heterodimer _H-V _LResidue in the interface.CDR or determine that bonded other residue is suddenlyd change in many ways.A kind of mode is each residue of randomization or residue combination, so that in the identical antigen binding site group of others, finds all 20 aminoacid or its hypotypes at ad-hoc location.Perhaps, by the fallibility PCR method in CDR residue scope induced mutation (see for example Hawkins et al., 1992, J.MoI.Bio.226,889-96).The Vector for Phage Display that contains the gene (for example heavy chain and/or chain variable region gene) of the binding structural domain of encoding can in colibacillary mutant strain, breed (see for example Low et al., 1996, J.MoI.Bio.250,359-68).These method of mutagenesis are examples of numerous methods well known by persons skilled in the art.

Each variable domains of antibody of the present invention can be complete heavy chain immunoglobulin or light chain variable domain, perhaps can be functional equivalents or its mutant or the derivant of naturally occurring domain, or for example at the synthetic domain of external use as technique construction as described in the WO 93/11236 (Medical Research Council/Griffiths et al.).For example, can mix corresponding to the domain that lacks one or more amino acid whose antibody variable territory.Important identification mark is that each variable domains combines the ability that forms antigen binding site with the complementary structure territory.

Antigen binding site of the present invention has any epi-position, antigenic site or combination of proteins site.Interested especially is the antibody that is used for the treatment of disease.In the preferred antibody and receptor protein, as participating in the receptor that angiogenesis and/or tumor form.In and receptor be the intrinsic kinase activity inactivation of instigating the receptor transduction signal.Reliably in the receptor and to measure be the inhibition of receptor phosphorylation.The invention is not restricted in any specific receptor and mechanism.Some possible mechanism comprise that the outer binding structural domain of the born of the same parents that prevent part and receptor combines, and prevent the Dimerized or oligomerize of receptor.Yet, do not get rid of other mechanism.

The neutralization of receptor activation can be carried out in external or body in the sample of endotheliocyte or non-endotheliocyte such as tumor cell.The neutralization of the receptor activation in the sample of expression of receptor cell comprises cell is contacted with antibody of the present invention.External, before in cell sample, adding VEGF, simultaneously or afterwards cell is contacted with antibody.In vivo, contact with receptor by giving mammal antibody of the present invention.Give mammiferous method comprise for example oral, intravenous gives, intraperitoneal gives, subcutaneous give or intramuscular gives.

The example of this receptor comprises but non-platelet derived growth factor receptor (PDGF-R), vegf receptor (for example VEGFR-2/KDR/Flk-1, VEGFR1/Flt-1, VEGFR3/Flt-4), EGF-R ELISA (EGFR), the IGF-1 (IGFR) etc. of being limited to.The example of other non-limiting receptor tyrosine kinase comprises Flt-4, HER2/neu, Tek and Tie2.

Other factor that can be used as the regulator of interior angiogenesis of body and/or tumor growth comprises fibroblast growth factor (FGF) and nerve growth factor (NGF).Corresponding receptor is fibroblast growth factor acceptor (FGF-R) and trk C (NGFR).Participating in cell migration, form change and invasive another receptor is macrophage stimulating protein receptor (MSP-R or RON).Interested receptor comprises that people's albumen reaches from other mammiferous homologue.

Antibody of the present invention can mix the Ig antigen binding structural domain in any source.Above the antibody of listed receptor for example be known, and be to be used for antibody V of the present invention _HAnd V _LThe source of domain.The example that is specific to the scFv variable region binding structural domain of KDR comprises for example IMC-1C11 (V _HNucleotide and aminoacid sequence: SEQ ID NO:1 and 2; V _LNucleotide and aminoacid sequence: SEQ IDNO:3 and 4) (see WO 00/44777), IMC-2C6 (V _HNucleotide and aminoacid sequence: SEQ IDNO:5 and 6; V _LNucleotide and aminoacid sequence: SEQ ID NO:7 and 8) (see WO 03/075840) and IMC-1121 (V _HNucleotide and aminoacid sequence: SEQ ID NO:5 and 6; V _LNucleotide and aminoacid sequence: SEQ ID NO:9 and 10) VH that (sees WO 03/075840) and VL domain.The example that is specific to the binding structural domain of Flt-1 comprises 6.12 (V _HNucleotide and aminoacid sequence: SEQID NO:11 and 12; V _LNucleotide and aminoacid sequence: SEQ ID NO:13 and 14) and IMC-18F1 (V _HNucleotide and aminoacid sequence: SEQ ID NO:27 and 28; V _LNucleotide and aminoacid sequence: SEQ ID NO:29 and 30).

The binding structural domain that is specific to EGFR for example comprises

(Cetuximab; IMC-C225) (V _HNucleotide and aminoacid sequence: SEQ ID NO:15 and 16; V _LNucleotide and aminoacid sequence: SEQ ID NO:17 and 18), disclose as WO 96/40210; And IMC11F8 (V _HNucleotide and aminoacid sequence: SEQ ID NO:19 and 20; V _LNucleotide and aminoacid sequence: SEQ IDNO:21 and 22).The example that is specific to the binding structural domain of IGFR is IMC-A12 (V _HNucleotide and aminoacid sequence: SEQ ID NO:23 and 24; V _LNucleotide and aminoacid sequence: SEQ ID NO:25 and 26).Antibody (seeing WO 2005/037235) in conjunction with the FGF receptor comprises for example FR1-H7 (V _HNucleotide and aminoacid sequence: SEQ ID NO:31 and 32; V _LNucleotide and aminoacid sequence: SEQ ID NO:33 and 34), FR1-A1 (V _HNucleotide and aminoacid sequence: SEQ ID NO:35 and 36; V _LNucleotide and aminoacid sequence: SEQ ID NO:37 and 38), and FR1-4H (V _HNucleotide and aminoacid sequence: SEQ ID NO:39 and 40; V _LNucleotide and aminoacid sequence: SEQ ID NO:41 and 42).Antibody (seeing WO 2005/120557) in conjunction with RON or MSP-R comprises IMC-41A10 (V _HNucleotide and aminoacid sequence: SEQ ID NO:43 and 44; V _LNucleotide and aminoacid sequence: SEQ ID NO:45 and 46) and IMC-41B12 (V _HNucleotide and aminoacid sequence: SEQ ID NO:47 and 48; V _LNucleotide and aminoacid sequence: SEQ ID NO:49 and 50).Antibody in conjunction with PDGFR α comprises for example 3G3 and 7G11 (Loizos et al., 2005, MoI.Cancer Ther.4:369).

Further, the part as the CDR zone of listed binding structural domain can be mixed and is used for producing protein-bonded binding structural domain as herein described above.

In some receptor listed above the preferred antibodies two kinds.In embodiments of the invention, the bispecific antigen-binding proteins is in conjunction with the also activation of two kinds of different receptor tyrosine kinases of blocking-up participation angiogenesis.In such embodiment, described antibodies PDGFR and vegf receptor, for example VEGFR2/Flk-1/KDR.In another such embodiment, described antibodies KDR and FLT-I.

In another embodiment, antibodies HER2 of the present invention and EGFR.In another preferred embodiment, antibodies EGFR of the present invention and IGFR.

In another embodiment, antigen-binding proteins of the present invention is in conjunction with EGFR and VEGFR.In a preferred embodiment, described VEGFR is VEGFR2.This antibody can be used for blocking the epithelial stimulation of blood vessel, and this realizes by EGFR and the conduction of VEGFR disabling signal.This occurs in the situation of angiogenesis particularly useful replying the EGFR part of tumor cell secretion, particularly TGF α.

Antibody of the present invention can be used for making antigen on the target cell and the antigen crosslinking on the immune system effector lymphocyte.This can be used for for example promoting the immunne response at the cell that has interested specific antigen on cell surface.According to the present invention, immune system effector lymphocyte comprises the T cell of antigen-specific sexual cell such as active cell immunne response and NK cell (NK) cell of non-specific cell such as macrophage, neutrophilic granulocyte and mediated cell immunne response.

Antibody of the present invention can have the binding site of any cell surface antigen of immune system effector lymphocyte.This cell surface antigen comprises for example cytokine and lymphokine receptor, Fc receptor, CD3, CD16, CD28, CD32 and CD64.Except the antigen binding site that sVD provides, bi-specific antibody also can comprise the binding site that Fv provides.This Fv can derive from aforementioned antigenic antibody, derive from combinatorial library, and obtains by other method well known in the art.The bi-specific antibody that is specific to cytokine and lymphokine receptor also can comprise the binding site that comprises corresponding to the aminoacid sequence of all or part native ligand of receptor.For example, be in the situation of IL-2 receptor at cell surface antigen, bi-specific antibody of the present invention can have the antigen binding site that comprises corresponding to the aminoacid sequence of IL-2.Other cytokine and lymphokine comprise for example interleukin such as interleukin 4 (IL-4) and interleukin 5 (IL-5), and colony stimulating factor (CSF) is as granulocyte-macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF).

Antibody of the present invention can produce by expressing two polypeptide chains, and they comprise at least one single structure territory antigen binding site together.These two polypeptide chains all comprise can be Dimerized at least one heavy chain constant domain (C for example _H2 and/or C _H3).Use comprises the DNA construct of antibacterial secretory signal sequence at the section start of each polypeptide chain, can produce antibody in escherichia coli expediently.Many antibacterial signal sequences known in the art.Preferred signal sequence is from the pelB gene of Erwinia carotovora.The dna fragmentation of encoding antibody can be advanced in the carrier of for example end user's cytomegalovirus promoter (HCMV) and enhancer with high level expression in mammalian cell by the clone, described mammalian cell for example is CHO, NS0, COS-7 and PER.C6 cell, and the cell line such as lymphoma, myeloma or the hybridoma that come from lymph (are seen for example Bendig, et al, U.S.Patent 5,840,299; Maeda, et al. (1991) Hum.Antibod.Hybridomas 2,124-34).

Selected marker is to be coded in surviving or the essential proteinic gene of growing through transformed host cells of growing in the selective medium.Typical selected marker coding as following protein: (a) give to antibiotic or other toxin resistance of ampicillin, neomycin, methotrexate or tetracycline for example, (b) complementary auxotroph defective, perhaps (c) provides the critical nutrients of non-availability from compound culture medium, the gene of the bacillus cereus D-alanine racemase of for example encoding.Useful especially selected marker is given the methotrexate resistance.For example, by in the culture medium of the competitive antagonist methotrexate (Mtx) that contains DHFR, cultivating all transformants, select the cell of gene transformation at first to be differentiated with DHFR.Proper host cell is Chinese hamster ovary (CHO) cell of the active defective of DHFR when using wild type DHFR, its preparation and enrichment procedure such as Urlaub and Chasin (1980) Proc.Natl.Acad.Sd.USA77, and 4216 is described.Cell transformed is exposed under the methotrexate of increase level then.Cause a plurality of copies of DHFR gene synthetic like this, and follow a plurality of copies of DNA of other DNA of comprising described expression vector such as encoding said antibody or antibody fragment synthetic.

When hope was expressed gene construct in yeast, the example that is used for the suitable selection gene of yeast was the trp1 gene that exists among the yeast plasmid YRp7.Stinchcomb?et?al.(1979)Nature，282，39；Kingsman?et?al.(1979)Gene1，141。The trp1 gene provides at the selected marker that lacks the yeast mutant of energy for growth in tryptophan, for example ATCC No.44076 or PEP4-1.Jones(1977)Genetics?85，12。Then, the existence of trp1 infringement provides by there not being growth under the condition of tryptophan to detect the effective environment of conversion in the yeast host cell genome.Similarly, the yeast strains of Leu2-defective (ATCC 20,622 or 38,626) is by the known plasmid complementation of carrying the Leu2 gene.

In liquid culture medium, cultivate transformed host cells by means known in the art, contain assimilable carbon source for example carbohydrate such as glucose or lactose in the described culture medium, nitrogenous source is aminoacid, peptide, protein or its catabolite such as peptone for example, ammonium salts etc. reach for example carbonate of sulfate, phosphate and/or sodium, potassium, magnesium and calcium of inorganic salt.Described culture medium further contains material such as the trace element that for example promotes growth, for example ferrum, zinc, manganese etc.

The antibody of binding growth factor receptor preferably can be blocked the active activation of receptor tyrosine kinase (RTK).Tyrosine kinase suppresses to use the method for knowing to be determined, for example determines by the spontaneous phosphorylation level of measurement reorganization kinases receptors and/or the phosphorylation of natural or synthetic.Therefore, phosphorylation assay can be used for determining RTK antagonist of the present invention.Phosphorylation can for example be used the antibody test that is specific to phosphotyrosine in ELISA or on the western trace.Some assay methods of tyrosine kinase activity are at Panek et al., and J.Pharmacol.Exp.Thera. (1997) 283:1433-44 and Batley et al. describe among Life Sci. (1998) 62:143-50.

In addition, the method that detects protein expression can be used for determining the RTK antagonist that wherein measured protein expression is mediated by RTK.These methods comprise the immunohistochemical method (IHC) that detects protein expression, detect the fluorescence in situ hybridization method (FISH) of gene amplification, competitive radioligand binding assay, solid-phase matrix engram technology such as Northern and Southern trace, reverse transcriptase-polymerase chain reaction (RT-PCR) and ELISA.See for example Grandis et al., Cancer, (1996) 78:1284-92; Shimizu et al., Japan J.Cancer Res., (1994) 85:567-71; Sauter et al., Am.J.Path., (1996) 148:1047-53; Collins, Glia, (1995) 15:289-96; Radinsky etal., Clin.Cancer Res., (1995) 1:19-31; Petrides et al., Cancer Res., (1990) 50:3934-39; Hoffmann et al., Anticancer Res., (1997) 17:4419-26; Wikstrand etal., Cancer Res., (1995) 55:3140-48 is described.

The bonded ability of antibody blocking part can be for example by measuring in external competitive assays.In this mensuration, the part of immobilization RTK (for example EGF of EGFR) carries out combination and measures to determine antibody competition inhibition RTK and the bonded effectiveness of immobilized part.

Algoscopy also can be used for determining the RTK antagonist in the body.For example, suppress can be by using the mitogenesis algoscopy observation that the receptors ligand stimulated cells is carried out under the condition that has with the unrestraint agent for receptor tyrosine kinase.For example, (American type culture collection (ATCC), Rockville MD) can be used for measuring EGFR and suppress the A431 cell that EGF stimulates.Another kind method comprises that use for example is injected into the inhibition situation of the growth of tumour cell of the human tumor cells detection expression EGFR in the mice.See U.S. Patent No. 6,365,157 (Rockwell et al.) are described.

Preferred antibody of the present invention has bispecific, and can be simultaneously in conjunction with two synantigens not.Described different antigen can be positioned on the different cells or be positioned on the same cell.Antigenic crosslinked can illustrating external for example combines the first antigenic surface of solids by providing, and adds to be specific to the first antigenic bi-specific antibody and also to be specific to described protein-bonded second antigen, detects the bonded second antigenic existence.

Preferred antibody of the present invention can be blocked the interaction between two receptors and its part separately.For example, the antibody that is specific to KDR and Flt-1 suppresses inductive cell migration of VEGF and the inductive cell migration of P1GF.In the bi-specific antibody the more independent parental generation antibody of two kinds of specific combinations of receptors bind suppress aspect the cell migration can be more effective (see for example Zhu, Z., WO 2004/003211).

Compare with monospecific antibody, bi-specific antibody can be the stronger inhibitor of cell function.For example, the propagation of VEGF stimulated cells function such as endotheliocyte and VEGF-and the inductive human leukemia cell's of P1GF-migration can more effectively be suppressed by bi-specific antibody, even reduces for the affinity of or these two target antigens.Being specific to the two antibody of (unit price) KDR and Flt-1 can be than more effectively suppress VEGF or the inductive cell migration of P1GF (WO 2004/003211) at the arbitrary monospecific scFv of target antigen.

In another embodiment, antibody with EGFR (or Her2/neu) and IGFR bispecific is used for and EFG-and the receptor activation of IGF-stimulation and the signal transduction in downstream, and described bi-specific antibody can be in conjunction with the interaction of these two receptors and blocking-up and its ligands specific.Observe the arbitrary stimulation of EGFR or IGFR and all cause common downstream signal transduction molecule to comprise the activation of Akt and p44/42 (for example phosphorylation), although activation degree difference.In some tumor cells, the inhibition of EGFR function can compensate by the just adjusting of other growth factor receptors signal pathway, particularly stimulates by IGFR to compensate.With use in conjunction with a kind of receptor and not exclusively to block the treatment that the antibody of the phosphorylation of Akt or p44/42 carries out opposite, with tumor cell be incubated the two phosphorylation in conjunction with the two antibody of EGFR and IGFR with blocking-up Akt and p44/42.Therefore, the inhibition of IGFR signal causes tumor growth to suppress and increases the sensitivity of tumor cell for some therapeutic agents.

Also observe use in conjunction with other RTK for example the antibody of RON to the inhibition of this common signal transductory cascade composition phosphorylation.Therefore, antigen-binding proteins can be used for usually treating and is characterised in that by a plurality ofs' transduction pathway and activates the cell growth cause or the tumor disease that transforms.

Antibody of the present invention can be used for treating multiple proliferative disease.For example, the tumor that the invention provides the treatment expression and stimulate by more than one receptor tyrosine kinases.Receptor for stimulating by more than one can cause uncontrolled growth, and its blocking-up for independent every kind of receptor is insensitive.Perhaps, the stimulation of second receptor can increase the stimulation of replying first receptor and the activation that observes.Perhaps, the effect of single receptor can be doubled.In the situation on each, in the situation of the antigen-binding proteins that has two kinds of receptors of blocking-up, observe the inhibition to tumor growth of remarkable improvement.

Antibody of the present invention can be used for treating the disease of receptor for stimulating by EGFR paracrine and/or autocrine loop.For example, the tumor of expressing EGFR is for the EGF characteristic sensitivity that exists in its environment, and can further be subjected to EGF that tumor produces or the stimulation of TGF-α.Though do not wish to be subjected to the constraint of any specific mechanism, can comprise disease or the pathological changes that tumor growth is for example stimulated with the disease of method of the present invention treatment or prevention or pathological changes.Therefore method of the present invention can effectively be treated the entity tumor of not vascularization, perhaps the abundant tumor of vascularization not as yet.

Some antibody of the present invention can be used for suppressing the angiogenesis with the hyperplasia disease association.For example, by the relevant angiogenesis of blocking-up tumor, can suppress tumor growth.In one embodiment, the tumor vascular RTK of antibodies also suppresses tumor and produces angiogenic part (being VEGF), its also in conjunction with the vegf receptor of vascular cell blood vessel to suppress the propagation of this cell.In different embodiments, a plurality of vegf receptors of antibodies, blocking VEGF or other VEGFR part (for example P1GF) are in conjunction with the vegf receptor more than a type thus.

Treatable tumor comprises primary tumo(u)r and metastatic tumour, and the tumor of refractory.The tumor of refractory comprises all reactionless or have a tumor of resistance for the combination of independent chemotherapeutics, independent antibody, independent radiotherapy or these Therapeutic Method.The tumor of refractory comprises that also the material that seems by such suppresses, but after stopping to treat 5 years at the most, sometimes until the tumor of 10 years or longer time back recurrence.But described tumor normal level is expressed EGFR or other RTK, and perhaps it can cross expression RTK, for example is at least 10,100 or 1000 times of normal level.

Express EGFR and comprise cancer, glioma, sarcoma, adenocarcinoma, sarcoadenoma and adenoma by the example of the tumor of the ligand stimulation of EGFR.This tumor in fact can take place at any position of body, for example takes place at mammary gland, the heart, lung, small intestinal, colon, spleen, kidney, bladder, head and neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, cervix uteri or liver.Treatable some tumors of expressing EGFR that observed comprise but non-colorectum and the head and neck tumor of being limited to according to the present invention, head and neck squamous cell carcinoma particularly, the cerebral tumor such as glioma, and lung, mammary gland, pancreas, esophagus, bladder, kidney, ovary, cervix uteri and prostatic tumor.The tumor non-limitative example that observes (promptly unadjusted) receptor tyrosine kinase activity with constitutive activity comprises glioma, nonsmall-cell lung cancer, ovarian cancer and carcinoma of prostate.Other tumor example comprises the cancer of Kaposi ' s sarcoma, cns tumor, neuroblastoma, blood capillary blastoma, meningioma and metastatic encephaloma, melanoma, gastrointestinal and kidney and sarcoma, rhabdomyosarcoma, spongioblastoma, preferred pleomorphism spongioblastoma, and leiomyosarcoma.Expressing excessively of other RTK can produce similar growth defect.For example great majority shift osteocarcinoma from prostate, mammary gland or lung transfer.Tumor of prostate can be hormonal dependent at first, but this dependent forfeiture is consistent with the stimulation that the IGFR of the cell that moves to bone of walking around mediates.

Antibody of the present invention also can be used for treating the hyperplasia disease except tumor, comprises the antibody of the present invention that gives the mammal effective dose.As disclosed herein, the hyperplasia disease is meant the pathological changes that is caused by the undue growth of the non-cancerous cell of expressing EGFR family member or other tyrosine kinase receptor.The excessive cell that is produced by the disease of hyper-proliferative is expressed RTK or perhaps it cross expression RTK at normal level.

The example of hyperplasia disease comprises psoriasis, actinic keratosis and seborrheic keratosis, wart, cicatrix, reaches eczema.Also comprise because the hyperplasia disease that viral infection causes, for example papilloma virus infection.For example, there are many different variations and the order of severity in psoriasis.Feature that dissimilar psoriasises shows such as pus sample blister (psoriasis pustulosa), skin seriously come off (erythrodermic psoriasis), water droplet sample speckle (drop psoriasis pustulosa (guttae psoriasis)) and smooth muscle (smooth) inflammation infringement (seborrhiasis).The present invention expect the treatment all types of psoriasises (for example psoriasis vulgaris, psoriasis pustulosa, erythrodermic psoriasis, arthropathic psoriasis, parapsoriasis, palmoplantar pustulosis (palmoplantar pustulosis)).

According to the present invention, antibody can be synthetic and combine through chemistry or biology with other material such as antitumor or angiogenesis inhibitor material, with the treatment disease.The antitumor agent that is connected with antibody comprises any preparation that destroys or damage the tumor or the tumor in the cellular environment of this antibodies of this antibodies.For example, antitumor agent is toxic agents such as chemotherapeutics or radiosiotope.Use conventional method that chemotherapeutics and antibodies (are seen for example Hermentin and Seiler (1988) Behring Inst.Mitt.82,197-215), comprised by peptide and non-peptide linker and carry out combination.

Antibody of the present invention also can be used in vivo producing agent and be connected with the external detectable signal of diagnosing.Described signal produces agent and produces and can measure by Electro-Magnetic Release Unit usually by the signal of external equipment measurement.It is enzyme or chromophore that most of signals produce material, perhaps emitting fluorescence, phosphorescence or chemiluminescence.Chromophore comprises the dyestuff of the light that is absorbed in ultraviolet light or visible-range, perhaps can be the substrate or the catabolite of enzymic catalytic reaction.

The present invention further comprises the application that antibody of the present invention and treatment or diagnostic agent are formed second class grade chemical.For example, will be connected with antibody of the present invention in conjunction with a right member.Antitumor agent is combined another right member's combination with this, thus and the site of guiding antibodies.In a preferred embodiment, with biotin and antibodies of the present invention, thereby provide target position for providing with avidin or the bonded antitumor agent of Streptavidin or other composition.Perhaps, biotin or other this composition are connected with antibody of the present invention, and in diagnostic system for example with the albumen of making reports, wherein detectable signal produces and combines with avidin or Streptavidin.

Antibody can give with the combination of one or more suitable adjuvant, and described adjuvant for example is cytokine (for example IL-10 and IL-13), or other immunostimulant, for example but non-ly be limited to chemotactic factor, antigen that tumor is relevant, and peptide.Yet, should recognize to give the progress that antibody promptly is enough to prevent, suppress or reduce with the treatment effective means tumor separately.

In certain embodiments, hope is that another kind of antigen-binding proteins in conjunction with the bonded antibody combination binding partner of the present invention of RTK and block ligand gives object.The part binding antibody is known in the art, for example comprise anti-VEGF (

Bevacizumab).

Antibody of the present invention also is used for the Therapeutic Method that makes up, by with antitumor agent such as chemotherapeutics or radiosiotope combination medicine-feeding.Suitable chemotherapeutics is that those skilled in the art are known, comprise irinotecan (irinotecan) (CPT-11), anthracene nucleus class preparation (for example daunomycin and amycin), methotrexate, vindesine (vindesine), neocarzinostain NCS, cisplatin, chlorambucil, cytosine arabinoside, 5-fluorouracil, (left-handed) melphalan, ricin and calicheamicin.Antibody and angiogenesis inhibitor or antitumor agent give the patient with the amount of effective inhibition angiogenesis and/or reduction tumor growth.Antibody also can give with other therapeutic scheme combination, for example makes up with radiotherapy.The example of combined therapy is seen for example U.S. Patent No. 6,217,866 (Schlessinger et al.) (anti-EGFR-antibodies combination antitumor agent); WO 99/60023 (Waksal et al.) (anti-EGFR-antibodies combination radiotherapy) is described.

Can use any suitable antitumor agent, for example the combination of chemotherapeutics, radiotherapy or these therapies.Known in the art or the antitumor agent assessed is based on its target position or binding mode and classify.For example, alkylating agent comprises but non-cisplatin, cyclophosphamide, (left-handed) melphalan and the dacarbazine of being limited to.The example of antimetabolite comprises but non-amycin, daunomycin and the paclitaxel (paclitaxel) of being limited to, gemcitabine (gemcitabine), and topoisomerase enzyme inhibitor irinotecan (irinotecan) (CPT-11), amino camptothecin (aminocamptothecin), camptothecine (camptothecin), DX-8951f, and topotecan (topoisomerase I) and etoposide (etoposide) are (VP-16) and teniposide (teniposide) (VM-26) (topoisomerase II).For radiotherapy, radioactive source can be the outside ((brachytherapy-BT) of outside outer radiotherapy-EBRT) or inside for the patient of treatment.This classification can be used for selecting the antitumor agent that uses.For example, observed in conjunction with the antibody of IGFR perhaps effective especially when giving with topoisomerase enzyme inhibitor.

The dosage that gives antitumor agent depends on multiple factor, comprises the tumor type and the seriousness of for example drug type, treatment and gives the approach of medicine.Yet, should emphasize that the present invention is non-and be limited to any given dose.

In combined therapy, antibody before beginning with the treatment of another kind of material, during or give afterwards, and combination in any gives, promptly before the treatment of beginning antitumor agent and during, before and afterwards, during and afterwards, perhaps before, during and give afterwards.For example for treatment tumor or tumor disease, can be before beginning radiotherapy 1-30 days, preferred 3-20 days, more preferably gave in 5-12 days.In a preferred embodiment of the invention, chemotherapy be Antybody therapy simultaneously, before or give subsequently.

In the present invention, can use any suitable method or approach to give antibody of the present invention, optional antitumor agent, receptor antagonist or the other medicines composition of giving jointly.For example, the antitumor agent scheme of using according to the present invention comprises any scheme of be sure oing to be suitable for most treating the patient tumors pathological changes.Different malignant tumor can need to use specificity antineoplastic antibody and specificity antineoplastic agent, and this will determine according to the patient.That the approach of giving comprises is for example oral, intravenous gives, intraperitoneal gives, subcutaneous give or intramuscular gives.The dosage of the antitumor agent that gives depends on multiple factor, comprise antitumor agent for example type, treatment tumor type and seriousness and give the approach of antitumor agent.Yet, should emphasize that the present invention is non-and be limited to any specific method that gives or approach.

Those skilled in the art understand antibody of the present invention with the purpose of prevention or treatment when being used for mammal, will give with the form that extra packet contains the compositions of medicine acceptable carrier.Suitable medicine acceptable carrier comprises for example following one or more carrier: water, saline, phosphate buffered saline (PBS), glucose, glycerol, ethanol etc., with and the combination.The medicine acceptable carrier can further comprise a spot of auxiliary substance such as humidizer or emulsifying agent, antiseptic or buffer, and it strengthens protein-bonded shelf-life or effect.As known in the art, can be formulated as composition for injection, with provide active component after giving mammal fast, continue or delay d to discharge.

The present invention also comprises the test kit that suppresses tumor growth and/or angiogenesis or treat other disease, and described test kit comprises the antibody of the present invention for the treatment of effective dose.Preferred people or humanized antibody.This test kit can further contain any appropriate antagonist, for example participate in that tumor takes place or the another kind of growth factor receptors of angiogenesis (for example EGFR, VEGFR-1/Flt-1, VEGFR-2/Flk-1/KDR, IGFR, PDGFR, NGFR, FGFR etc., as above-mentioned).Perhaps or in addition, test kit of the present invention can further comprise antitumor agent.The example of suitable antitumor agent is described in this article among the present invention.Test kit of the present invention can further comprise adjuvant; The example is also described hereinbefore.

Antibody of the present invention is also included within the scope of the present invention with external research or the application in the diagnostic method in vivo, and this is known in the art.Described diagnostic method comprises the test kit that contains antibody of the present invention.

Therefore, receptor binding antibodies of the present invention can be used in vivo with the external research of carrying out, diagnosis, prevention or Therapeutic Method in, this is well known to those skilled in the art.Certainly, those skilled in the art understand and expection can change the principle of the present invention that this paper discloses, and this modification comprises within the scope of the invention.All lists of references of mentioning in the literary composition are all incorporated into for referencial use with its full content.

Embodiment

The further illustration of following embodiment the present invention, but the scope that do not limit the present invention in any way.Gene about carrier and plasmid construction, coded polypeptide inserts in this carrier and the plasmid, plasmid imports in the host cell and gene and the expression of gene outcome and definite detailed description can derive from numerous publications, comprise Sambrook, J et al., (1989) Molecular Cloning:A LaboratoryManual, 2 ^NdEd., Cold Spring Harbor Laboratory Press; And Coligan, J.et al. (1994) Current Protocols in Immunology, Wiley ﹠amp; Sons is incorporated herein by reference.

From phage display Fab library, select the anti-mPDGFR Alpha antibodies of people to use big natural human Fab's phage display library (to contain 3.7 * 10 from Dyax ¹⁰Individual clone) selects anti-mPDGFR α-Fc albumen (R﹠amp; D Systems (Minneapolis, antibody MN).(100 μ l) grows to logarithmic (log) phase in the 2YTAG of 20ml culture medium with library stock solution, with the rescue of M13K07 helper phage, spends the night 30 ℃ of amplifications in 2YTAK culture medium (2YT that contains 100 μ g/ml ampicillin and 50 μ g/ml kanamycin).The phage prepared product precipitates in 4%PEG, 0.5M NaCl.The phage prepared product is resuspended among the 1ml 3% skimmed milk/PBS that contains the incoherent human IgG of 240 μ g, 37 ℃ of insulations 1 hour, with the blocking-up non-specific binding and with the combining of the proteic Fc-labelling of mPDGFR α.

In the protein of selecting the reduction (being respectively 50,10 and 2 μ g) on the test tube immobilized mPDGFR α-Fc is carried out three times with bag and selected circulation.For each selection circulation, at first with the Maxisorp Star test tube of mPDGFR α-Fc-bag quilt (Nunc, Rosklide, DenMark) with 3% skimmed milk/PBS 37 ℃ of blocking-up 1 hour, be incubated 1 hour with described phage prepared product in room temperature then.Test tube is with PBST (PBS that contains 0.1% Tween 20) washing 15 times, subsequently with PBS washing 15 times.100mM triethylamine (Sigma) solution that uses the 1ml prepared fresh was the phage of room temperature elution of bound 10 minutes.The TG1 cell of the phage of eluting and 10ml mid-log phase is incubated 30 minutes in 37 ℃ of static insulations 30 minutes and vibration.The TG1 cell that precipitation infects is plated on three 2YTAG flat boards, 30 ℃ of incubated overnight.All colonies of growing on the flat board are scraped the 2YTA culture medium that places 3-5ml, mix (final concentration: 10%), be divided into equal portions, and preserve with glycerol in-80 ℃.For selection circulation subsequently, will increase from previous selection circulation phage stock solution (100 μ l), and be used for aforesaid selection, use the mPDGFR α-Fc of reduction to wrap by Maxisorp Star test tube.

In order to estimate to import phage, before selecting, 10 μ l phagies are used to infect the TG1 cell, titration on the 2YTAG flat board then.For the phage of estimating from select, to reclaim, the cell that the TG1 of 10 μ l is infected and phage titration on the 2YTAG flat board through above-mentioned option program eluting.Calculate each output of selecting circulation to reclaim, although the mPDGFR α-Fc of bag quilt reduces, output increases (circulation for the first time: 1 * 10 in successive circulation ^-6%; Circulation for the second time: 4 * 10 ^-6%; Circulation for the third time: 2 * 10 ^-4%).

Have combination and block active antibody in the second time with after selecting circulation for the third time by the ELISA screening, picked at random is from 190 clones of each circulation, and is active with blocking-up by phage E LISA and the two detection combination of FabELISA.In brief, each TG1 clone that picked at random reclaims after selecting circulation for the second time and for the third time grows in 96 hole flat boards at 37 ℃.In order to produce phage, save cell with the M13K07 helper phage as above-mentioned.In order to produce soluble Fab, cell is incubated in the 2YTA of the IPTG that contains 1mM culture medium.In order to carry out, the phage prepared product was blocked 1 hour in room temperature with the 18% skimmed milk/PBS of the cells and supernatant that contains soluble Fab with 1/6 volume in conjunction with ELISA.Phage prepared product or cells and supernatant with blocking-up added in the 96 hole microtitre flat boards (Nunc) of using mPDGFR α-Fc (1 μ g/ml, 50 μ l, spend the night by 4 ℃) bag quilt then, room temperature insulation 1 hour.After room temperature insulation 1 hour, should wash 3 times with PBST by flat board.

In order to carry out phage E LISA, with flat board with-M13 phage antibody-HRP conjugate (Amersham Biosciences) insulation.In order to carry out Fab ELISA, flat board is incubated with Anti-Human-Fab antibody-HRP conjugate (Jackson ImmunoResearch Laboratory).After three washings, (KPL, Gaithersburg MD) add lustre to, and (Molecular Device, Sunnyvale CA) measure absorbance at 450nm to use the microplate reader by adding the TMB peroxidase substrate.Also carried out being used for ELISA, in library screening, differentiated to be Fc specific antibody in conjunction with person (binder) to eliminate in conjunction with 1C11 IgG.In conjunction with in measuring, is male the clone of the picking of the selection back second time more than 77% with selecting the clone of the recovery of back 99% for the third time at mPDGFR α, points out the high efficiency of option program.

In order to block ELISA, phage prepared product or the Fab culture supernatant of 50 μ l are mixed with the mPDGFR α-Fc (0.5 μ g/ml) of fixed amount, room temperature insulation 30 minutes.Then mixture is transferred to and uses rhPDGF-AA (0.5 μ g/ml in advance; R﹠amp; D Systems, Minneapolis is MN) in the 96 hole flat boards of bag quilt, room temperature insulation 1 hour.For quantitative bonded mPDGFR α-Fc albumen, with flat board and the anti-people of rabbit-Fc antibody-HRP conjugate room temperature insulation 1 hour, with after scouring three times and add the TMS peroxidase substrate.Bonded mPDGFR α-Fc albumen quantizes by reading absorbance at 450nm.Blocking-up is active to be represented by being reduced by the detected ELISA signal of anti-people-Fc antibody-HRP conjugate.It is active that about 4.2% the clone in conjunction with mPDGFR α illustrates the PDGF-AA blocking-up.

Based on the blocking-up measurement result, select 14 clones (comprise non-blocking-up clone (3F3) in contrast) further to study at first in conjunction with the person.Separate phagemid dna, determine DNA sequence by the dideoxy nucleotide sequencing.The bio information website of use Andrew CR.Martin ' s Bioinformatics Group (

Www.bioinf.org.uk

) and MagAlign of DNAstar to V _HAnd V _LGene is classified and is arranged.

The analysis of anti--mPDGFR Alpha antibodies of selecting demonstrates the antibody (table 1) of 9 kinds of uniquenesses in 14 clones.Except 1C10 and 1F2, find no identical V _HOr V _LInteresting ground, 4 have the active antibody of strong blocking-up and have incomplete light chain.Clone 1F2 and 1F9 have in-frame V _LDisappearance (leader peptide sequences, C subsequently _LSequence).Clone 1C10 and the total identical V of 1F2 _HGene.Yet, because the V of 1C10 _LCoded sequence comprises termination codon, thus C _LDo not expressed.Clone 3G7 comprises and is positioned at V _LThe termination codon of 5 ' end of gene.It is active that 1C10 and 3G7 all show strong blocking-up.Fab expresses and seems very low in conjunction with activity, but the bad combination of anti-people-Fab second class grade chemical can be described.

Nucleotide and the aminoacid sequence of Fab are as follows: lE10 V _HDomain: SEQ ID NO:51 and 52; 1E10 V _LDomain: SEQ ID NO:53 and 54; 1A12 V _HDomain: SEQ ID NO:55 and 56; 1A12 V _LDomain: SEQ ID NO:57 and 58; 3B2 V _HDomain: SEQ ID NO:59 and 60; 3B2 V _LDomain: SEQ ID NO:61 and 62; 1C10 V _HDomain: SEQ ID NO:63 and 64; 1C10 V _LDomain: SEQ ID NO:65 and 66; 3G7VH domain: SEQ ID NO:67 and 68; 3G7 V _LDomain nucleotide sequence: SEQ ID NO:69; 3G7 V _LDomain: QAW; 1F9 V _HDomain: SEQ ID NO:70 and 71; 1F9 V _LDomain: SEQ ID NO:72 and 73; 1F2 V _HDomain: SEQ ID NO:74 and 75; 1F2 V _LDomain: SEQ ID NO:76 and 77.Figure 15 shows the position in each domain (comprising the truncate of VL or the disappearance of 3G7,1F9 and 1F2) and CDR zone.

6 clones' Fab fragment is expressed in escherichia coli HB2151 host cell, by affinity chromatography Protein G column purification.The clone's of each selection phasmid is used to transform the non-escherichia coli host HB2151 cell of preventing.The segmental expression of Fab is by inducing cell in the 2YTA of the IPTG that contains 1mM culture medium among the HB2151 30 ℃ of cultivations.The pericentral siphon extract of cell is as preparation as described in the Lu (x).Soluble Fab albumen uses the protein G post to carry out purification according to manufacturer (Amersham Biosciences) guidance.For purity and the molecular weight of checking prepared product, with antibody purified at NuPAGE ^TMElectrophoresis in the 4-12% Bis-Tris gel (Invitrogen), and pass through SimplyBlue ^TMSafeStain (Invitrogen) solution-dyed is observed.

The conjugated protein of purification analyzed (Fig. 2) by SDS-PAGE.The molecular weight that has the Fab of complete light chain shown in Fig. 2 A is about 50,000.On the contrary, compare with standard control Fab (Fig. 2 B), the unreduced fragment of 1F2 and 1F9 produces little band (DTT:MW～37,500).Under reducing condition (+DTT), show two bands, the band of top is consistent with the VH-CH1 fragment of normal size, the band of below with have CL fragment (MW～12,500) but lack the light chain unanimity of variable domains.

The segmental antigen of solvable Fab that combines and the blocking-up of mPDGFR α/PDGF-AA is contrasted in quantity 6 anti-mPDGFR α clones of anti--mPDGFR Alpha antibodies and mPDGFR α in conjunction with render a service and blocking-up mPDGFR α/PDGF-AA interaction in ability.In conjunction with in measuring, at first with Fab in the 96 hole flat boards of using mPDGFR α/Fc fusion rotein (1 μ g/ml) bag quilt in advance room temperature insulation 1 hour, subsequently with rabbit anti--Anti-Human-Fab antibody HRP conjugate insulation 1 hour.Quantize dull and stereotyped bonded antibody-HRP by adding peroxidase substrate then.Consistent with initial The selection result (table 1), 1F2Fab than all other Fab albumen more effectively in conjunction with mPDGFR α (Fig. 3 A).

The binding kinetics of various resisting-mPDGFR Alpha antibodies is determined by surface plasma resonance technology, uses BIAcore 3000 biosensors, and (Uppsala Sweden) assesses for Biacore, Inc. to use BIA appraisal procedure 2.0.Affinity constant K d calculates with the ratio of dissociation yield (koff)/combination rate (kon).The numerical value of report is meansigma methods ± S.E. (table 2) that at least twice (for Fab) and three times (for IgG) measure.With consistent in conjunction with measuring, sVD antibody 1F2 has the more high-affinity (K of about 0.5nM _d), higher more than 23 times than the normal Fab (1E10) that selects.

In table 2,1F2-2H Fab is the bivalence Fab through through engineering approaches that contains second identical VH domain, is expressed as the fusant with CL.The Fab of described bivalence is also passed through the Protein G chromatography purification at expression in escherichia coli.1F2-2H Fab to purification carries out the single protein band that SDS-PAGE analyzes an about 50kD of demonstration, to standard Fab fragment similar (Fig. 5).The affinity of this bivalence Fab is 79.5pM, and the affinity of unit price 1F2Fab is 418pM by contrast, affinity is shown strengthens 5.2 times (table 2).

In blocking-up is measured, the mPDGFR α/Fc fusant of not commensurability antibody and fixed amount be incubated 30 minutes in solution, this mixture is transferred to wrap in the 96 hole flat boards of quilt then and is incubated 1 hour with rhPDGF-AA.The quantity of bonded mPDGFR α is determined by quantizing bonded Anti-Human-Fc-Ab.The powerful blocking-up of described 1F2 Fab mPDGFR α/PDGF-AA interacts (Fig. 3 B), and the IC50 value is 12nM, and under the contrast, the IC50 value of 1F9,1E10 and 1A11 is respectively 57,140 and 220nM.Waiting on the mole foundation, bivalence Fab all illustrates medium enhanced activity (Fig. 6) in conjunction with mPDGFR α and the interaction of blocking-up receptor/ligand.

The clone's that the clone of total length IgG, expression and purification will be selected Fab changes total length IgG into, and this realizes among the heavy chain expression carrier pDFc and light chain expression vector pLC κ by VH and VL domain being cloned respectively into.PDFc has cloning site Hind III and Nhe I between leader peptide sequences and CH1.PLC κ has cloning site Hind III and BsiW I between leader peptide and CL.In brief, the gene of coding VH and VL is increased from phagemid dna by reverse transcription-pcr, and the clone advances in its expression vector separately.Then, heavy chain and light chain expression vector (VH-pDFc and VL-pLC κ) cotransfection are advanced in the COS-7 cell, as previously mentioned transient expression.After transfection, collected in 48 hours and 96 hours and the set cell culture medium.By the affinity chromatography, use albumen-A post to instruct antibody purification from cells and supernatant according to manufacturer (Amersham Biosciences).After the function that confirms IgG, with V _HAnd V _LExpression of gene is advanced in the single expression vector the clone, and transfection is advanced in the COS-7 cell.Carry out antibody purification as mentioned above.

From the 1F2 Ig that only contains a VH, produce three IgG sample construct (see figure 7)s, comprise having 4 V _HTetravalence 1F2-2H IgG (A), and have and C _HOr C _LBe expressed as the 1F2 V of fusant _HBivalence 1F2 (B and C).The plasmid of expressing light chain and heavy chain is used for cotransfection COS-7 cell, to carry out transient expression.Use the expression of cells and supernatant by ELISA monitoring antibody.The expression of 1F2-2H is lower than two bivalence 1F2 antibody all the time, is about 1/4th of bivalence 1F2 antibody.Use the antibody of protein A column purification by affinity chromatography from cells and supernatant.

Clone 1E10 is the blocking-up person (blocker) with normal antibody composition, also changes it into total length IgG.At first with the V of 1E10 _HAnd V _LGene is cloned respectively among the pDFc and pLC κ.As above-mentioned, after carrying out dna sequencing and confirming that 1E10 IgG is in conjunction with activity, with the V that expresses _H/ V _LThe clone is advanced in the single expression vector, be used for transfection COS-7 cell then, carry out antibody purification subsequently.

Analyze antibody purified to determine the purity and the molecular weight of prepared product by SDS-PAGE.Bivalent antibody 1F2-CH/CL and 1F2-CL/CH molecular weight MW are～125,000, and its migration velocity is faster than normal IgG (1E10 and 2B4; Fig. 8 left side).When handling antibody with DTT before electrophoresis (Fig. 8 right side), heavy chain and light chain dissociate.Antibody 1F2-CH/CL has the band of the close top relevant with heavy chain, and is similar with 2B4 to 1E10, and the lower band of fast transferring has the animal migration (MW～12,500, only CL) near expection.On the other hand, antibody 1F2-CL/CH illustrates lower band, the expression light chain animal migration similar to 1E10 with 2B4, the band of top only with C _H1-Fc polypeptide (MW～37,500) is relevant.

Total length is anti--and the combination of mPDGFR Alpha antibodies contrasts 1F2 variant antibody 1F2-2H IgG, the 1F2-CH/CL of all three purification and 1F2-CL/CH combines mPDGFR α with antibody 1E10 affinity with blocking-up is active by ELISA.The antibody of various amounts is incubated in the ELISA flat board with mPDGFR α/Fc bag quilt, detects the bonded antibody of mPDGFR α by Anti-Human-κ antibody HRP conjugate then.Although ELISA result shows 1F2-2H IgG and has four binding sites that 1F2-2H still is lower than about 8 times of two bivalence 1F2 IgGs (1F2-CH/CL and 1F2-CL/CH) (Fig. 9 A) in conjunction with activity.On the other hand, by ELISA (Fig. 9 A) and BlAcore analysis (table 2) these two bivalence 1F2 (1F2-CH/CL and 1F2-CL/CH) are shown and have extremely similar binding affinity.The affinity of bivalence 1F2IgG increases about order of magnitude than its parental generation 1F2 Fab, and 1E10 IgG increases by 20 times (table 2) than its Fab.In a word, these two bivalence 1F2 IgG exceed about order of magnitude (Fig. 9 A and table 2) with the binding affinity of mPDGFR α than 1E10IgG.

Also carried out quantitative blocking-up and measured-mPDGFR Alpha antibodies anti-to assess.As mentioned above, the antibody of various amounts and the mPDGFR α/Fc fusant of fixed amount are incubated 30 minutes in solution, then this mixture are transferred to insulation in the 96 hole flat boards of rhPDGF-AA bag quilt 1 hour.Then by quantizing the quantity that bonded Anti-Human-Fc-Ab determines bonded mPDGFRa.With consistent in conjunction with determination data, it is better blocking-up person that bivalence 1F2-CH/CL and 1F2-CL/CH and 1F2-2H IgG compare with 1E10 IgG, and the IC50 value of 1F2-CH/CL, 1F2-CL/CH, 1F2-2H IgG and 1E10 IgG is respectively 3.2,2.7,17 and 9.6nM (Fig. 9 B).

Bi-specific antibody is anti--and Flk-1 antibody 2B4 also separates the phage library from Dyax Fab, uses basic as above-mentioned identical method is carried out, and difference is to use mVEGFR2-Fc fusion rotein bag by Maxisorp Star test tube.The Fab of 2B4 is specific to Flk-1 and blocks Flk-1/VEGF ₁₆₅Interact.The V of Fab 2B4 _HAnd V _LDomain is respectively shown in SEQ ID NO:79 and 81.Figure 15 illustrates the position in each domain and CDR zone.Change 2B4Fab into total length IgG1 as above-mentioned.The binding kinetics of 2B4 Fab and IgG is analyzed by BlAcore and is determined.The binding affinity of 2B4 Fab and IgG and mVEGFR2 is respectively 6.7 ± 3.0nM and 0.39 ± 0.1nM.2B4 IgG blocking VEGF R2/VEGF ₁₆₅Interact, the IC50 value is about 3.5nM (Figure 13 B).

Based on the bi-specific antibody of domain (anti--the anti-Flt-1 of mPDGFR α x) structure--the bi-specific antibody based on domain makes up in two ways: scFv-form and Fab-form (Figure 10, A and B).In the scFv-form, at first use the VH of overlapping PCR assembling coding 2B4 and the PCR fragment of VL gene.The terminal NH2 with 2B4VH of the COOH of 2B4VL is terminal to connect (Figure 10 A) by 5-aminoacid joint (Ala-Ser-Thr-Lys-Gly derives from the N-terminal of CL domain).Gained gene code 2B4 VL-VH connects the clone with it by Hind III/Nhe I and advances in the pDFc carrier, to express scFv2B4-CH fusant (being made up of 2B4 VL-2B4VH-CH1-CH2-CH3).Then with the scFv2B4-CH expression vector with as the pairing of above-mentioned 1F2VH-CL expression vector, by cotransfection COS-7 cell with transient expression.

In the Fab-form, at first use the V of overlapping PCR assembling 1F2 _HThe V of gene and 2B4 _LGene.In this fusion, 1F2 V _HCOOH terminal with 2B4 V _LAmino terminal by 5-aminoacid joint from C _H5 ' the terminal connection.Then 1F2 VH-2B4VL encoding gene is cloned among the carrier pLC κ to express 1F2 VH-2B4VL-CL-fusion rotein by HindIII/BsiW I site.2B4 VH gene clone is advanced in the pDFc carrier to express VH.The plasmid of expressing light chain and heavy chain matches shown in Figure 10 B, is used for cotransfection COS-7 cell with transient expression.

After the dual combination activity that detects described clone by ELISA, the Ig expression construct is combined in the single expression vector, use the IgG of the COS-7 cell transient expression design of transfection then.As above-mentioned from cells and supernatant antibody purification.

Produce (100-200ml cell culture) based on the expression of the bi-specific antibody of domain and purification 1F2/scFv2B4 and 1F2-2B4 antibody with transient expression by dying at the COS-7 transit cell.Use the protein A post by affinity chromatography antibody purification from cells and supernatant.

Bi-specific antibody of purification (1F2/scFv2B4 and 1F2-2B4) and parental generation antibody 1F2 thereof and 2B4 analyze (Fig. 8) by SDS-PAGE.Compare with 2B4IgG, (being correlated with than low migration and its high molecular (～175,000) DTT), bivalence 1F2-CH/CL moves～125 untreated bi-specific antibody, 000 molecular weight position.After handling with DTT, two compositions of this of each antibody of these four kinds of antibody are all independently differentiated.With standard I gG (2B4) contrast, the low band of bi-specific antibody 1F2/scFv2B4 (VH-CL fusant) is corresponding to standard I gG light chain.The band of top and scFv-heavy chain fusion rotein (MW～62,500) are relevant.On the other hand, the band of 1F2-2B4 top is corresponding to the heavy chain of standard I gG (2B4), and lower band arrives (MW～37,500) at the position detection except VH-light chain fusant.

Based on the dual specificity of the bi-specific antibody of domain in crosslinked mensuration, at first bispecific 1F2-2B4 antibody and monospecific 1F2-CH/CL and 2B4 antibody and mPDGFR α or mVEGFR2 are incubated in solution, move to then in the microtitre flat board with second receptor (being respectively mVEGFR2 or mPDGFR α) bag quilt, the biotin labeled polyclonal antibody with first receptor is incubated subsequently.Only BsAb rather than parental generation monospecific 2B4 IgG can crosslinked this two target receptors that are connected with 1F2-CH/CL.

Antigen based on the bi-specific antibody of domain is determined in conjunction with rendeing a service at immobilized mPDGFR α and Flk-1.ELISA illustrates bi-specific antibody 1F2/scFv2B4 and 1F2-2B4 in conjunction with mPDGFR α and Flk-1, but is not and parental generation antibody 1F2 and 2B4 same effectively (Figure 12).Yet in both of these case, the binding ratio 1F2/scFv2B4 of 1F2-2B4 antibody is slightly more effective.As desired, Flk-1-specific antibody 2B4 is not in conjunction with mPDGFR α (Figure 12 A), and mPDGFR alpha specific antibody 1F2 is not also in conjunction with Flk-1 (Figure 12 B).The binding kinetics of bi-specific antibody and mPDGFR α and Flk-1 uses the BIAcore device to determine (table 3) by the surface ion resonance technique.Consistent with the result who observes by ELISA, to compare with 1F2/scFv2B4 antibody, the affinity of 1F2-2B4 antibody and mPDGFR α and Flk-1 is all higher.

1F2/scFv2B4		5.1±2.6E ⁵	9.3±1.75E ^-5	2.2±0.75E ^-10
1F2-2B4		2.4±1.5E ⁵	7.0±2.0E ^-5	4.1±1.8E ^-10

Figure 13 illustrates these two kinds of bi-specific antibodys and all suppresses mPDGFR α in conjunction with immobilized PDGF-AA (Figure 13 A), estimates to be respectively 9.9nM and 25.3nM for the IC50 of 1F2/scFv2B4 and 1F2-2B4.Described antibody is also blocked Flk-1 in conjunction with immobilized VEGF ₁₆₅(Figure 13 B), the IC50 value is respectively 9.5nM and 19.5nM.As institute expectedly, 2B4 interacts for mPDGFR α/PDGF-AA does not have influence, and 1F2 is for Flk-1/VEGF ₁₆₅Interact and do not have influence.

Although compare with monospecific bivalent molecule separately, the mPDGF α combination and the bonded affinity of mVEGFR2 of bi-specific antibody are lower, but when checking 1F2-CH/CL and 2B4 IgG (table 3) in conjunction with the receptor at cell surface expression on the eEnd.1 cell (express mPDGFR α and mVEGFR2 the two cell), BsAb illustrates than the higher effectiveness of arbitrary parental generation monospecific bivalent antibody.Average fluorescent strength on the cell (MFI) is respectively 15.7,31 and 36.9 for 1F2-CH/CL (mPDGFR α combination), 2B4 IgG (mVEGFR2 combination) and 1F2-2B4IgG (mPDGFR α and mVEGFR2 combination).

The expression of inductive activated inhibition mVEGFR2 of the part of mVEGFR2 and mPDGFR and mPDGFR α confirms in the eEnd.1 cell by facs analysis.With cell and 2B4IgG, 1F2-CH/CL or 1F2-2B4IgG (10 μ g/ml) 4 ℃ of insulations 1 hour, subsequently with the anti-people Fc of PE labelled goat antibody (Jackson ImmunoResearch Lab.) insulation 1 hour, at GuavaEasycyte System (Guava Technologies, Inc.Hayward CA) goes up analysis.

In order to check receptor phosphorylation, the eEnd.1 cell is plated on the 6cm culture dish, grow to 70-80% and be paved with rate, afterwards with cell washed twice in PBS, overnight incubation in serum-free medium.At first cell and various antibody are incubated 30 minutes at 37 ℃, stimulated 15 minutes at 37 ℃ with VEGF or PDGF-AA subsequently.(50mM Tris-HCl, pH 7.4,150mMNaCl, 1% TritonX-100,1mM EDTA, 1mM Phenylmethanesulfonyl fluoride, 0.5mM Na at cell pyrolysis liquid for cell ₃VO ₄, 1 μ g/ml leupeptin, 1 μ g/ml pepsin inhibitor and 1 μ g/ml aprotinin) in cracking 1 hour, subsequently with lysate 12,000rpm is centrifugal 10 minutes at 4 ℃.Use anti--mPDGFR α (eBioscience, San Diego, CA) or anti--mVEGFR2 antibody (Santa CruzBiotech, Santa Cruz, CA) from the cell lysate supernatant, precipitate receptor, add the ProA/G-sepharose 4B (Santa Cruz Biotech) of 20 μ l subsequently.Sedimentary receptor protein is gone up at 4-12% NupageBis-Tris gel (Invitrogen) and is differentiated, and is transferred on the polyvinylidene fluoride film.Use anti--phosphoric acid-tyrosine antibody-HRP conjugate (Santa Cruz Biotech) on trace, to detect phosphoric acid-mVEGFR2 and phosphoric acid-mPDGFR α.Use the antibody (all deriving from Santa CruzBiotech) of mPDGFR α or mVEGFR2 to measure the total receptor protein of application of sample on gel.

As shown in figure 14, mPDGFR α and mVEGFR2 receptor phosphorylation that BsAb had both suppressed PDGF-and stimulated also suppress mPDGFR α and the mVEGFR2 receptor phosphorylation that VEGF-stimulates, and the monospecific parental generation antibody is only blocked the activation by the single receptor of its related ligand stimulation.In contrast, anti-EGFR-antibodies C225 does not have any effect for the activation of arbitrary receptor of ligand stimulation.

Sequence table

＜110〉Imclone Systems Inc. (ImClone Systems, Inc.)

Zhenping，Zhu

＜120〉functional antibodies

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Claims (37)

1. antigen-binding proteins that comprises the complex of two first polypeptide and two second polypeptide,

Each first polypeptide all has the immunoglobulin light chain constant of being positioned at domain (C _LDomain) first antigen binding site of N-terminal, described C _LDomain can with first constant domain of heavy chain immunoglobulin (C _H1 domain) the stable association,

Each second polypeptide all has the C of being positioned at _HSecond antigen binding site of the N-terminal of 1 domain, described C _HConnect behind 1 domain and can stablize self associating one or more heavy chain constant domain;

Wherein at least one described first antigen binding site and described second antigen binding site are single variable domains (sVD).

2. the antigen-binding proteins of claim 1, wherein said first and second antigen binding site all are single variable domains (sVDs).

3. the antigen-binding proteins of claim 1, a site in wherein said first and second antigen binding site is single variable domains (sVD), another site in described first and second antigen binding site is strand Fv (scFv).

4. antigen-binding proteins that comprises the complex of two first polypeptide and two second polypeptide,

Each first polypeptide all comprises the heavy chain immunoglobulin of being made up of variable domains and constant domain,

Each second polypeptide all comprises the light chain immunoglobulin of being made up of variable domains and constant domain,

Wherein these two first polypeptide and this two second polypeptide are stable associates, and forms the immunoglobulin-like molecule,

The variable domains of wherein said heavy chain immunoglobulin and the variable domains of described light chain immunoglobulin are stable associates, and forms first antigen binding site,

Wherein said first or second polypeptide or this two peptide species further are included in second strand variable domains (sVD) antigen binding site of N-terminal or C-terminal.

5. each antigen binding site of claim 1-4, wherein said first antigen binding site has different specificitys with described second antigen binding site.

6. the antigen binding site of claim 5, wherein said different specificity are at the epi-position that is positioned on the synantigen not.

7. the antigen binding site of claim 5, wherein said different specificity is at the epi-position that is positioned on the same antigen.

8. claim 1 and 2 each antigen-binding proteins, wherein said first antigen binding site has homospecificity mutually with second antigen binding site.

9. each antigen-binding proteins of claim 1-4, wherein said constant domain is people's constant domain.

10. each antigen-binding proteins of claim 1-4, wherein said single variable domains is the people.

11. each antigen-binding proteins of claim 1-4, it is in conjunction with the Fc receptor.

12. each antigen-binding proteins of claim 1-4, it produces CDC (CDC).

13. each antigen-binding proteins of claim 1-4, it produces the cytotoxicity (ADCC) of antibody dependent cellular mediation.

14. each antigen-binding proteins of claim 1-4, it is connected with antitumor agent.

15. each antigen-binding proteins of claim 1-4, it is connected with the generation agent of detectable signal.

16. each antigen-binding proteins of claim 1-4, at least one site-specific in wherein said first and second antigen binding site is in receptor tyrosine kinase (RTK).

17. the antigen-binding proteins of claim 16, its block ligand combines with receptor tyrosine kinase.

18. the antigen-binding proteins of claim 16 is in it and the activation of receptor tyrosine kinase.

19. the antigen-binding proteins of claim 16, it suppresses the signal transduction by receptor tyrosine kinase.

20. the antigen-binding proteins of claim 16, wherein said receptor tyrosine kinase is the people.

21. the antigen-binding proteins of claim 16, wherein said receptor tyrosine kinase are selected from the group that PDGFR α, VEGFR1, VEGFR2, VEGFR3, EGFR, HER2, IGFR, FGFR, NGFR, RON, Tek and Tie2 form.

22. the antigen-binding proteins of claim 16, a site-specific in wherein said first and second antigen binding site be in PDGFR α, another site in described first and second antigen binding site is specific to VEGFR2.

23. the antigen-binding proteins of claim 16, a site-specific in wherein said first and second antigen binding site be in VEGFR1, another site in described first and second antigen binding site is specific to VEGFR2.

24. the antigen-binding proteins of claim 16, a site-specific in wherein said first and second antigen binding site be in IGFR, another site in described first and second antigen binding site is specific to EGFR or HER2.

25. a neutralization receptor tyrosine kinase activated method, comprise with in being enough to and each antigen-binding proteins of the claim 1-4 that is specific to described receptor tyrosine kinase of the amount of receptor activation handle cell.

26. one kind is suppressed the method that blood vessel takes place, comprise with in being enough to and each antigen-binding proteins of the claim 1-4 of the amount of receptor activation handle mammal.

27. a method that reduces tumor growth comprises with each antigen-binding proteins of the claim 1-4 of the amount that is enough to reduce tumor growth and handles mammal.

28. claim 26 and 27 each methods, wherein said antigen-binding proteins are in conjunction with PDGFR α and in conjunction with VEGFR2, and suppress that the inductive PDGFR α of part activates and the inductive VEGFR2 activation of part.

29. claim 26 and 27 each methods, wherein said antigen-binding proteins are in conjunction with VEGFR1 and in conjunction with VEGFR2, and suppress that the inductive VEGFR1 of part activates and the inductive VEGFR2 activation of part.

30. the method for claim 27, wherein said antigen-binding proteins are in conjunction with EGFR and in conjunction with IGFR, and the inductive EGFR of inhibition part activates and the inductive IGFR of part activates.

31. the method for claim 27, wherein said antigen-binding proteins are in conjunction with HER2 and in conjunction with IGFR, and the inductive HER2 of inhibition part activates and the inductive IGFR of part activates.

32. the method for claim 27, it further comprises the antitumor agent that gives effective dose.

33. a method that produces antigen-binding proteins comprises:

A) time and the following construct of mode coexpression in host cell to be enough to make that described expression of polypeptides and described antibody form:

A kind of DNA construct of reorganization, its coding has first polypeptide of first antigen binding site of the N-terminal that is positioned at immunoglobulin light chain constant domain (CL domain), and described CL domain can be stablized first constant domain of association heavy chain immunoglobulin (C _H1 domain), reach

A kind of DNA construct of reorganization, its coding has the C of being positioned at _HSecond polypeptide of second antigen binding site of the N-terminal of 1 domain, described C _HConnect behind 1 domain and can stablize self associating one or more heavy chain constant domain,

At least one site in wherein said first antigen binding site and described second antigen binding site is single variable domains (sVD); And

B) reclaim described antigen-binding proteins.

34. the method for claim 33, wherein said construct are positioned on the identical DNA expression vector.

35. the method for claim 33, wherein said construct are positioned on the different DNA expression vectors.

36. the method for claim 33, wherein said host cell are bacterial cell, yeast cells or mammalian cell.

37. the method for claim 33, wherein said antibody is secreted from described host cell.

CNA2007800111267A 2006-02-15 2007-02-15 Functional antibodies Pending CN101432015A (en)

Applications Claiming Priority (2)

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US60/773,994		2006-02-15

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JP (1)	JP2009526857A (en)
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CN (1)	CN101432015A (en)
AU (1)	AU2007215013A1 (en)
BR (1)	BRPI0707824A2 (en)
CA (1)	CA2638794A1 (en)
EA (1)	EA200870265A1 (en)
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- 2007-02-15 WO PCT/US2007/004051 patent/WO2007095338A2/en active Application Filing
- 2007-02-15 MX MX2008010561A patent/MX2008010561A/en not_active Application Discontinuation
- 2007-02-15 KR KR1020087022202A patent/KR20080106245A/en not_active Application Discontinuation
- 2007-02-15 CA CA002638794A patent/CA2638794A1/en not_active Abandoned
- 2007-02-15 EP EP07750859A patent/EP1986684A2/en not_active Withdrawn
- 2007-02-15 EA EA200870265A patent/EA200870265A1/en unknown
- 2007-02-15 CN CNA2007800111267A patent/CN101432015A/en active Pending
- 2007-02-15 AU AU2007215013A patent/AU2007215013A1/en not_active Abandoned
- 2007-02-15 BR BRPI0707824-2A patent/BRPI0707824A2/en not_active Application Discontinuation
- 2007-02-15 JP JP2008555359A patent/JP2009526857A/en not_active Ceased
2008
- 2008-09-02 NO NO20083785A patent/NO20083785L/en not_active Application Discontinuation

Cited By (6)

* Cited by examiner, † Cited by third party

Publication number	Priority date	Publication date	Assignee	Title
CN102574919A (en) *	2009-09-29	2012-07-11	株式会社绿十字	Antibodies specifically binding to the epidermal growth factor receptor
CN102574919B (en) *	2009-09-29	2014-12-10	株式会社绿十字	Antibodies specifically binding to the epidermal growth factor receptor
CN105524176A (en) *	2010-05-21	2016-04-27	梅里麦克制药股份有限公司	Bi-specific fusion proteins
CN105524176B (en) *	2010-05-21	2021-03-19	银溪制药股份有限公司	Bispecific fusion proteins
CN108610417A (en) *	2018-04-28	2018-10-02	暨南大学	Anti-tetanus toxin neutralizing antibody, preparation method and the usage
CN108610417B (en) *	2018-04-28	2021-01-26	暨南大学	Anti-tetanus toxin neutralizing antibody, its preparation method and use

Also Published As

Publication number	Publication date
JP2009526857A (en)	2009-07-23
NO20083785L (en)	2008-11-17
AU2007215013A1 (en)	2007-08-23
WO2007095338A2 (en)	2007-08-23
CA2638794A1 (en)	2007-08-23
EA200870265A1 (en)	2009-02-27
KR20080106245A (en)	2008-12-04
MX2008010561A (en)	2009-03-02
BRPI0707824A2 (en)	2011-05-10
WO2007095338A3 (en)	2008-04-17
EP1986684A2 (en)	2008-11-05

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Legal Events

Date	Code	Title	Description
2009-05-13	C06	Publication
2009-05-13	PB01	Publication
2009-07-08	C10	Entry into substantive examination
2009-07-08	SE01	Entry into force of request for substantive examination
2012-05-23	C02	Deemed withdrawal of patent application after publication (patent law 2001)
2012-05-23	WD01	Invention patent application deemed withdrawn after publication	Open date: 20090513

CN101432015A - Functional antibodies - Google Patents