CN101124245A - Neonatal Fc receptor (FcRn)-binding polypeptide variants, dimeric Fc binding proteins and methods related thereto - Google Patents

Abstract

The compositions and methods of the present invention are based, in part, on our discovery that an effector function mediated by an Fc-containing polypeptide can be altered by modifying one or more amino acid residues within the polypeptide (by, for example, electrostatic optimization). The polypeptides that can be generated according to the methods of the invention are highly variable, and they can include antibodies and fusion proteins that contain an Fc region or a biologically active portion thereof.

Description

Neonatal Fc receptor (FcRn)-in conjunction with polypeptide variants, dimeric Fc binding proteins and methods involving thereof

Related application

The application requires interests that submit to, that be entitled as the U.S. Provisional Application series number 60/519,744 of " antibody and the variant thereof that contain the constant region that changes " on November 12nd, 2003.The interests of that the application also requires to submit on November 12nd, 2003, as to be entitled as " Fc receptors bind polypeptide, optimize deutero-pH specificity variant and uses thereof " U. S. application series number 60/519,743 by static.The interests of the U. S. application series number 60/519,733 that the application also requires to submit on November 12nd, 2003 is entitled as " by building the mutant that defines can the mediator carrying out the static mould in the people Fc structural domain of antibody half life ".The application also relates to the PCT application XXXXXXXX that submits to same date with the application, be entitled as " Fc γ receptors bind polypeptide variants and methods involving thereof ".The complete content of all these applications is incorporated into herein as a reference.

Background technology

Many biological procedureses are mediated by a kind of protein and another kind of proteinic special interaction.For example, enzyme is specifically in conjunction with the protein of its substrate, and part (for example neurotransmitter and hormone) substantive information takes place from the cytotropic transmission of cell during with their connection receptors bind.The interaction that takes place in the immunne response situation is the most attracting interaction, and wherein antibody (being also referred to as immunoglobulin (Ig)) produces the phylactic power defensive power with the allogenic material of giving health antagonism can causing infection or disease.Antibody contain specifically with antigen and with " effect " cell (for example phagocytic cell) on the distinct structural domain of acceptor interaction.Although conjugated antigen is useful (endogenous target interacts because this can prevent antigen and its), efficient immune is destroyed antigen.Therefore, the most effective antibody is those antibody with structural domain of mediation high-affinity antigen bonded structural domain and mediation net effect subfunction.

Natural antibody is the allos tetramer normally; They contain two identical light (L) chains and two identical weights (H) chain that link together by disulfide linkage.Every heavy chain has the back with several constant regions (CH ₁, CH ₂And CH ₃) a variable region (V _H), and every light chain has the back with a variable region (V of a constant region only _L).The constant region of light chain is alignd V with first constant region of heavy chain _LWith V _HAlignment.The reason that the variable region is so named is that some amino acid in the variable region is extensively different between antibody.These variable regions are also referred to as complementary determining region (CDR), are responsible for the binding specificity of each specific antibodies to its specific antigen.3 CDR are contained in each variable region, and these CDR (are called framework region, FR) separately by the zone of high conservative.CDR in every chain abuts against together by the FR district, and participates in the formation of the antigen site of antibody with the CDR from other chain.

Constant region is not participated in antibody directly and is combined with antigenic, but is based on the combination of itself and cell receptor or complement molecule and mediates various effector functions.According to the aminoacid sequence of CH, antibody or immunoglobulin (Ig) can be dispensed to dissimilar (A, D, E, G and M).The most frequently used therapeutic antibodies is " G " type (that is, they is IgG).These types can further be divided.For example, IgG antibody further is divided into isotype IgG1, IgG2, IgG3 and IgG4.The crystalline structure in human IgG Fc district has been determined (Deisenhofer, Biochemistry 20:2361-2370,1981; For the explanation in Fc district, referring to U.S. Patent number 6,242, Fig. 1 of 195).

Fc district mediation effector function, these functions are divided into two classes.The first kind is to be independent of the antigen combination and the function of generation; These function endowings in circulation persistence and stride the ability (seeing Ward and Ghetie, Therapeutic Immunology 2:77-94,1995) that barrier cell shifts by transcytosis.Second class is antibody combines the back running with antigen a function; These functions relate to complement cascade or carry the participation of the cell of Fc acceptor (FcR).FcR is the cell surface receptor of specialization on the hematopoietic cell, the removal (by the phagocytic immunity mixture) of the pathogenic agent of its mediation lining antibody, and the red corpuscle of mediation lining corresponding antibodies and the cracking of various other cellular targets (for example tumour cell).Cracking is by the cytotoxicity (ADCC of antibody dependent cellular mediation; See Van de Winkel and Anderson, J.Leuk.Biol.49:511-24,1991) take place.FcR based on it to the specificity of immunoglobulin (Ig) isotype and define.For example, the Fc acceptor of IgG antibody is called Fc γ R.

Another Fc acceptor FcRn regulates the serum half-life of IgG.The increase of Fc (polypeptide that perhaps contains Fc) transformation period or reduce and can be respectively the increase or the minimizing of the avidity of FcRn (neonatal Fc receptor (neonatal Fcreceptor)) to be reflected (Ghetic etc. by Fc district, Nature Biotechnol.15:637-640,1997; Kim etc., Eur.J.Immunol.24:542-548,1994; (J.Immunol.169:5171-5180,2002) such as Della ' Acqua.FcRn is consistent in conjunction with the biological action of rescue antibody in the FcRn autolysosome Degradation of the dependency of affinity and serum half-life and proposition.In addition, FcRn is transferred to fetus with IgG from parent.

These surfaces go up different effects by FcRn in cell and the mediation of the ability of transcellular transport bonded IgG.It is believed that by the endotheliocyte internalization, and the acid endosome of targeted cells and lysosome are with degraded in the antibody self-circulation under normal circumstances.FcRn can the acid pH (＜6.5) of endosome down binding antibody Fc district, merge with endothelial cell membrane, and, rescue antibody thus and make it to exempt from destruction at the neutral pH (＞7.4) of blood flow release antibody down.When the serum antibody level reduces, can obtain more FcRn molecule and be used for the IgG combination, rescue the IgG of increasing amount thus.On the contrary, if the serum IgG level rises, then FcRn becomes saturated, will be increased by the ratio of the antibody of internalization and degraded thus (Ghetie and Ward, Annu.Rev.Immunol. (2000), 18:739-66).

Consistent with above model, if the polypeptide that expection changes its to neonatal Fc receptor in conjunction with the affinity increase have the longer transformation period.On the contrary, if the polypeptide that changes expects then that to the reducing in conjunction with affinity of neonatal Fc receptor it has the short transformation period.

Monoclonal antibody (mAb) has been used for the treatment of disease (King and Adair, Curr.Opin.Drug Discovery Dev.2:110-117,1999 of human patients at present; Vaswani and Hamilton, Ann.Allergy Asthma Immunol.81:105-119,1998; With Hollinger and Hoogenboom, Nature Biotechnol.16:1015-1016,1998).But this is not to say that the treatment based on antibody is successful fully at present; In some cases, the limited cycling time of therapeutical agent and/or low bioavailability cause the patient of relatively low percentage ratio that the treatment based on antibody is shown fully replying, perhaps in other cases, because circulating half-life that prolongs or the toxicity that is exposed to due to the non-target tissue may make antibody can not be used as therapeutical agent.

Therefore, the antibody (containing the polypeptide of Fc, for example fusion rotein) that needs antigen dependent/non-dependent effector function to be customized with other according to the desired use of antibody.Similarly, need to allow prediction can cause the method (avoiding depending on the heavy journey of correcting one's mistakes while trying thus) of the antibody sequence change that antigen dependent/non-dependent effector function changes.In some cases, may expect to increase the transformation period of antibody.For example, have the protein therapeutic agent of the transformation period of increase in the blood and have cyclical administration that side by side reduces medicine or the advantage that alternatively reduces drug dose.This antibody-like also has the advantage of increase to the contact of disease site (for example tumour).On the contrary, the protein therapeutic agent expection with transformation period of minimizing will have lower toxicity, and observed effectiveness when having kept using the not change medicine of higher and the worse dosage of tolerance.These therapeutical agents and their method of preparation will have great benefit.

Summary of the invention

(for example the present invention relates in for example Fc district or its FcRn binding fragment, to have polypeptide that the specific amino acids alternate changes, the polypeptide that in the IgG constant region, has amino acid replacement), the described alternative change that causes antigen dependent/non-dependent effector function (for example, circulating half-life).The present invention also provides the method and the method for utilizing its conduct based on proteinic therapeutical agent of the polypeptide of this change of preparation.The present invention also provides new FcRn form and application method thereof.

The present invention at least in part based on the human Fc district (especially, Fc district from IgG antibody) evaluation of particular amino acid residue in the constant domain (Fc), described amino-acid residue will change the circulating half-life of antigen the dependent/non-dependent effector function, particularly antibody of antibody after changing by one or more amino acid mutations.Therefore, the present invention relates to contain the polypeptide in all or part of Fc district, for example antibody and fusion rotein, wherein said Fc district undergos mutation at one or more amino-acid residues place, thereby increases or reduce the antigen dependent/non-dependent effector function of described polypeptide.

The present invention also provides the technology of identifying the expectation amino acid mutation and the method for preparing the polypeptide that comprises this sudden change.Described method comprises that the branch submodule builds, and its amino acid that can be used for the predicted amino acid sequence changes, and changes combine (for example, the strengthening or reduction) with Fc acceptor (for example human newborn Fc acceptor) thus.Usually, described method start from " initial " or " target " polypeptide or contain with the mixture of FcRn bonded first polypeptide (for example, crystalline structure or homology model), the modification of first polypeptide afterwards causes " second " or " change " polypeptide different with first polypeptide so that have better performance in specific treatment or diagnostic use.For example, second polypeptide can more effectively be carried out one or more antigen dependency effector functions (for example transformation period of Gai Bianing).Mould is built on computers, and (in silico) carries out.

On the one hand, the present invention relates to comprise at least the polypeptide of change of the FcRn bound fraction in Fc district, wherein said polypeptide is compared with initial polypeptide and is comprised at least one sudden change, and wherein said at least one sudden change is selected from:

Be replaced by charged amino acid 248 of EU amino acid positions;

Be replaced by positively charged amino acid 249 of EU amino acid positions;

Be replaced by polare Aminosaeren or Methionin 251 of EU amino acid positions;

Be replaced by polare Aminosaeren 252 of EU amino acid positions;

Be replaced by polare Aminosaeren 255 of EU amino acid positions;

Be replaced by Methionin 256 of EU amino acid positions;

Be replaced by charged amino acid 257 of EU amino acid positions;

Be replaced by polare Aminosaeren or charged amino acid 258 of EU amino acid positions;

Substitute 277 of EU amino acid positions;

Be replaced by charged amino acid 279 of EU amino acid positions;

Be replaced by charged amino acid 280 of EU amino acid positions;

Be replaced by charged amino acid or glutamine 281 of EU amino acid positions;

Be replaced by charged amino acid 282 of EU amino acid positions;

Be replaced by polare Aminosaeren or charged amino acid 284 of EU amino acid positions;

Be replaced by positively charged amino acid, polare Aminosaeren or aspartic acid 285 of EU amino acid positions;

Be replaced by L-glutamic acid, Threonine or methionine(Met) 286 of EU amino acid positions;

Be replaced by polare Aminosaeren or charged amino acid 287 of EU amino acid positions;

Be replaced by charged amino acid 288 of EU amino acid positions;

Substitute 289 of EU amino acid positions;

Be replaced by polare Aminosaeren or charged amino acid 304 of EU amino acid positions;

Be replaced by polare Aminosaeren or charged amino acid 305 of EU amino acid positions;

Substitute 306 of EU amino acid positions;

Be replaced by polar or charged amino acid 307 of EU amino acid positions;

Be replaced by charged amino acid 308 of EU amino acid positions;

Be replaced by charged amino acid 309 of EU amino acid positions;

Be replaced by charged amino acid or polare Aminosaeren 310 of EU amino acid positions;

Be replaced by positively charged amino acid 311 of EU amino acid positions;

Be replaced by positively charged amino acid or polare Aminosaeren 312 of EU amino acid positions;

Be replaced by charged amino acid 313 of EU amino acid positions;

Be replaced by charged amino acid 315 of EU amino acid positions;

Be replaced by positively charged amino acid 316 of EU amino acid positions;

Be replaced by charged amino acid or polare Aminosaeren 317 of EU amino acid positions;

Be replaced by charged amino acid 340 of EU amino acid positions;

Be replaced by polare Aminosaeren or charged amino acid 343 of EU amino acid positions;

Be replaced by leucine 344 of EU amino acid positions;

Be replaced by polare Aminosaeren or charged amino acid 345 of EU amino acid positions;

Be replaced by polare Aminosaeren or charged amino acid 376 of EU amino acid positions;

Be replaced by Serine 378 of EU amino acid positions;

Be replaced by charged amino acid 383 of EU amino acid positions;

Be replaced by charged amino acid 385 of EU amino acid positions;

Be replaced by electronegative amino acid 389 of EU amino acid positions;

Be replaced by charged amino acid 424 of EU amino acid positions;

Be replaced by charged amino acid 426 of EU amino acid positions;

Be replaced by polare Aminosaeren or charged amino acid 430 of EU amino acid positions;

Be replaced by charged amino acid 431 of EU amino acid positions;

Be replaced by polare Aminosaeren 432 of EU amino acid positions;

Be replaced by Methionin, arginine or leucine 434 of EU amino acid positions;

Be replaced by electronegative amino acid 436 of EU amino acid positions; With

Be replaced by charged amino acid 438 of EU amino acid positions.

On the other hand, the present invention relates to comprise at least the polypeptide of change of the FcRn bound fraction in Fc district, wherein said polypeptide is compared with initial polypeptide and is comprised at least one sudden change, and wherein said at least one sudden change is selected from:

The Methionin of 248 of EU amino acid positions is replaced by charged amino acid;

The aspartic acid of 249 of EU amino acid positions is replaced by positively charged amino acid;

The leucine of 251 of EU amino acid positions is replaced by polare Aminosaeren or Methionin;

Substitute the methionine(Met) of 252 of EU amino acid positions;

The arginine of 255 of EU amino acid positions is replaced by polare Aminosaeren;

The Threonine of 256 of EU amino acid positions is replaced by Methionin;

The proline(Pro) of 257 of EU amino acid positions is replaced by charged amino acid;

The L-glutamic acid of 258 of EU amino acid positions is replaced by polare Aminosaeren or charged amino acid;

Substitute the tryptophane of 277 of EU amino acid positions;

The Xie Ansuan of 279 of EU amino acid positions is replaced by charged amino acid;

The aspartic acid of 280 of EU amino acid positions is replaced by charged amino acid;

The glycine of 281 of EU amino acid positions is replaced by charged amino acid or glutamine;

The Xie Ansuan of 282 of EU amino acid positions is replaced by charged amino acid;

The Xie Ansuan of 284 of EU amino acid positions is replaced by polare Aminosaeren or charged amino acid;

The Histidine or the L-Ala of 285 of EU amino acid positions are replaced by charged amino acid or polare Aminosaeren;

The Histidine or the L-Ala of 285 of EU amino acid positions are replaced by positively charged amino acid, polare Aminosaeren or aspartic acid;

The l-asparagine or the Methionin of 286 of EU amino acid positions are replaced by L-glutamic acid, Threonine or methionine(Met);

The L-Ala of 287 of EU amino acid positions is replaced by polare Aminosaeren or charged amino acid;

The Methionin of 288 of EU amino acid positions is replaced by charged amino acid;

Substitute the Threonine of 289 of EU amino acid positions;

The Serine of 304 of EU amino acid positions is replaced by polare Aminosaeren or charged amino acid;

The Xie Ansuan of 305 of EU amino acid positions is replaced by polare Aminosaeren or charged amino acid;

Substitute the leucine or the Xie Ansuan of 306 of EU amino acid positions;

The Threonine or the Xie Ansuan of 307 of EU amino acid positions are replaced by polar or charged amino acid;

The Xie Ansuan of 308 of EU amino acid positions is replaced by charged amino acid;

The leucine of 309 of EU amino acid positions is replaced by charged amino acid;

The Histidine of 310 of EU amino acid positions is replaced by charged amino acid or polare Aminosaeren;

The glutamine of 311 of EU amino acid positions is replaced by positively charged amino acid;

The aspartic acid or the leucine of 312 of EU amino acid positions are replaced by positively charged amino acid or polare Aminosaeren;

The l-asparagine of 313 of EU amino acid positions is replaced by charged amino acid;

The l-asparagine of 315 of EU amino acid positions is replaced by charged amino acid;

The l-asparagine of 316 of EU amino acid positions is replaced by positively charged amino acid;

The Methionin of 317 of EU amino acid positions is replaced by charged amino acid or polare Aminosaeren;

The Methionin of 340 of EU amino acid positions is replaced by charged amino acid;

The proline(Pro) of 343 of EU amino acid positions is replaced by polare Aminosaeren or charged amino acid;

The arginine of 344 of EU amino acid positions is replaced by leucine;

The L-glutamic acid of 345 of EU amino acid positions is replaced by polare Aminosaeren or charged amino acid;

The aspartic acid of 376 of EU amino acid positions is replaced by polare Aminosaeren or charged amino acid;

The L-Ala of 378 of EU amino acid positions is replaced by Serine;

The Serine of 383 of EU amino acid positions is replaced by charged amino acid;

The glycine of 385 of EU amino acid positions is replaced by charged amino acid;

The l-asparagine of 389 of EU amino acid positions is replaced by electronegative amino acid;

The Serine of 424 of EU amino acid positions is replaced by charged amino acid;

The Serine of 426 of EU amino acid positions is replaced by charged amino acid;

The L-glutamic acid of 430 of EU amino acid positions is replaced by polare Aminosaeren or charged amino acid;

The leucine of 431 of EU amino acid positions is replaced by charged amino acid;

The Histidine of 432 of EU amino acid positions is replaced by polare Aminosaeren;

The l-asparagine of 434 of EU amino acid positions is replaced by Methionin, arginine or leucine;

The tyrosine of 436 of EU amino acid positions is replaced by electronegative amino acid; With

The glutamine of 438 of EU amino acid positions is replaced by charged amino acid.

On the other hand, the present invention relates to the polypeptide of the change of claim 1 or 2, wherein the amino acid replacement of at least one position in the EU amino acid position 277,289,306,344 or 378 is charged amino acid, polare Aminosaeren or nonpolar amino acid.

In the embodiment, charged amino acid is electronegative amino acid.

In the embodiment, electronegative amino acid is selected from aspartic acid and L-glutamic acid.

In the embodiment, charged amino acid is positively charged amino acid.

In the embodiment, positively charged amino acid is selected from arginine, Histidine and Methionin.

In the embodiment, positively charged amino acid is Methionin.

In the embodiment, polare Aminosaeren is selected from: methionine(Met), phenylalanine, tryptophane, Serine, Threonine, tyrosine, l-asparagine, glutamine and halfcystine.

In the embodiment, nonpolar amino acid is selected from: L-Ala, leucine, Isoleucine, Xie Ansuan, glycine and proline(Pro).

In the embodiment, the polypeptide of change is antibody or its fragment.

In the embodiment, the polypeptide of change is a fused protein.

In the embodiment, Fc district or its FcRn bound fraction derive from human antibodies.

In the embodiment, polypeptide comprises complete Fc district.

In the embodiment, initial polypeptide comprises the aminoacid sequence of SEQ ID NO:2.

In the embodiment, Fc district or its Fc bound fraction are the IgG isotypes.

In the embodiment, the IgG isotype is the IgG1 hypotype.

In the embodiment, polypeptide is at V _LOr V _HComplementary determining region (CDR) in comprise one or more inhuman amino-acid residues.

In the embodiment, polypeptide is in conjunction with (a) antigen and (b) FcR.

In the embodiment, antigen is tumor associated antigen.

In the embodiment, polypeptide is in conjunction with (a) part and (b) FcR.

In the embodiment, FcR is FcRn.

In the embodiment, polypeptide is to be different from combining with FcR in conjunction with affinity of the initial polypeptide that do not contain sudden change.

In the embodiment, the polypeptide of described change high about 1.5 times to about 100 times compared with the beginning polypeptide in conjunction with affinity.

In the embodiment, the polypeptide of described change low about 1.5 times to about 100 times compared with the beginning polypeptide in conjunction with affinity.

In the embodiment, the polypeptide of described change demonstrates a kind of in conjunction with affinity to FcR at a pH, and demonstrates a kind of different from affinity to FcR at the 2nd pH.

In the embodiment, the polypeptide of described change in conjunction with affinity when the pH than at the 2nd pH the time high about 1.5 times to about 100 times.

In the embodiment, the antibody of described change in conjunction with affinity when the pH than at the 2nd pH the time low about 1.5 times to about 100 times.

In the embodiment, the polypeptide of change demonstrates when being administered to the patient and the different circulating half-life of initial polypeptide that does not conform to sudden change.

In the embodiment, the transformation period of the polypeptide of change than about 1 hour of the long half time of the initial polypeptide that does not contain sudden change to about 1 week.

In the embodiment, the transformation period of the polypeptide of change is lacked about 1 hour to about 1 week than the transformation period of the initial polypeptide that does not conform to sudden change.

In the embodiment, the polypeptide conjugated protein A or the G of change.

In the embodiment, relate to the polypeptide of the change of claim 1 or 2.

In the embodiment, the nucleotide sequence of the polypeptide of the present invention relates to encode claim 1 or 2.

In the embodiment, described nucleic acid is expression vector.

In the embodiment, expression vector is in host cell.

In the embodiment, the method that the present invention relates to treat the patient who suffers from disease, described method comprise the polypeptide of using the change of the FcRn bound fraction that comprises the Fc district at least to the patient, and wherein said polypeptide comprises at least one sudden change that is selected from down group:

Be replaced by L-glutamic acid 284 of EU amino acid positions;

Be replaced by L-glutamic acid 285 of EU amino acid positions;

Be replaced by aspartic acid 286 of EU amino acid positions;

Be replaced by L-glutamic acid or aspartic acid 288 of EU amino acid positions;

Be replaced by L-glutamic acid 290 of EU amino acid positions; With

Be replaced by aspartic acid 304 of EU amino acid positions,

Wherein, the polypeptide of change is compared with the initial polypeptide that does not conform to sudden change and is demonstrated longer circulating half-life.

In the embodiment, the method that the present invention relates to treat the patient who suffers from disease, described method comprise the polypeptide that uses the change of the FcRn bound fraction that comprises the Fc district at least to the patient, and described polypeptide comprises at least one sudden change that is selected from down group:

The Xie Ansuan of 284 of EU amino acid positions is replaced by L-glutamic acid;

The Histidine of 285 of EU amino acid positions is replaced by L-glutamic acid;

The l-asparagine of 286 of EU amino acid positions is replaced by aspartic acid;

The Methionin of 288 of EU amino acid positions is replaced by L-glutamic acid or aspartic acid;

The Methionin of 290 of EU amino acid positions is replaced by L-glutamic acid; With

The Serine of 304 of EU amino acid positions is replaced by aspartic acid,

Wherein, the polypeptide of change is compared with the initial polypeptide that does not contain sudden change and is had longer circulating half-life.

In the embodiment, the method that the present invention relates to treat the patient who suffers from illness, described method comprise the polypeptide of using the change of the FcRn bound fraction that comprises the Fc district at least to the patient, and described polypeptide comprises at least one sudden change that is selected from down group:

248 of EU amino acid positions are replaced by aspartic acid;

249 of EU amino acid positions are replaced by arginine or Methionin;

250 of EU amino acid positions are replaced by arginine or Methionin;

251 of EU amino acid positions are replaced by arginine, Methionin or l-asparagine;

252 of EU amino acid positions are replaced by Serine or Threonine;

254 of EU amino acid positions are replaced by Serine or Threonine;

256 of EU amino acid positions are replaced by arginine, L-glutamic acid, perhaps Methionin;

255 of EU amino acid positions are replaced by leucine, aspartic acid or methionine(Met);

260 of EU amino acid positions are replaced by Methionin;

257 of EU amino acid positions are replaced by arginine, aspartic acid, L-glutamic acid or Methionin;

277 of EU amino acid positions are replaced by arginine, aspartic acid, glutamine or Methionin;

279 of EU amino acid positions are replaced by L-glutamic acid;

281 of EU amino acid positions are replaced by glutamine;

282 of EU amino acid positions are replaced by arginine, aspartic acid, L-glutamic acid or Methionin;

287 of EU amino acid positions are replaced by aspartic acid, L-glutamic acid, Methionin, proline(Pro) or Threonine;

284 of EU amino acid positions are replaced by aspartic acid;

285 of EU amino acid positions are replaced by aspartic acid or phenylalanine;

286 of EU amino acid positions are replaced by L-glutamic acid or methionine(Met);

288 of EU amino acid positions are replaced by aspartic acid;

290 of EU amino acid positions are replaced by aspartic acid;

304 of EU amino acid positions are replaced by aspartic acid or L-glutamic acid;

305 of EU amino acid positions are replaced by arginine;

306 of EU amino acid positions are replaced by arginine, aspartic acid, L-glutamic acid or Methionin;

307 of EU amino acid positions are replaced by arginine, aspartic acid or L-glutamic acid;

309 of EU amino acid positions are replaced by arginine, aspartic acid, Methionin or L-glutamic acid;

310 of EU amino acid positions are replaced by arginine, leucine, Methionin or l-asparagine;

312 of EU amino acid positions are replaced by arginine, l-asparagine or Methionin;

313 of EU amino acid positions are replaced by aspartic acid, arginine or Methionin;

315 of EU amino acid positions are replaced by aspartic acid or L-glutamic acid;

343 of EU amino acid positions are replaced by glutamine or Methionin;

345 of EU amino acid positions are replaced by arginine or glutamine;

374 of EU amino acid positions are replaced by arginine, Methionin or leucine;

376 of EU amino acid positions are replaced by l-asparagine;

426 of EU amino acid positions are replaced by arginine, aspartic acid or L-glutamic acid;

428 of EU amino acid positions are replaced by arginine, glutamine or Methionin;

430 of EU amino acid positions are replaced by Methionin;

431 of EU amino acid positions are replaced by proline(Pro);

432 of EU amino acid positions are replaced by arginine;

434 of EU amino acid positions are replaced by leucine or Methionin; With

438 of EU amino acid positions are replaced by L-glutamic acid,

Wherein, the polypeptide of change is compared with the initial polypeptide that does not conform to sudden change and is had shorter circulating half-life.

On the other hand, the present invention relates to treat the patient's who suffers from illness method, described method comprises the polypeptide of using the change of the FcRn bound fraction that comprises the Fc district at least to the patient, and described polypeptide comprises at least one sudden change that is selected from down group:

The Methionin of 248 of EU amino acid positions is replaced by aspartic acid;

The aspartic acid of 249 of EU amino acid positions is replaced by arginine or Methionin;

The Threonine of 250 of EU amino acid positions is replaced by arginine or Methionin;

The leucine of 251 of EU amino acid positions is replaced by arginine, Methionin or l-asparagine;

The methionine(Met) of 252 of EU amino acid positions is replaced by Serine or Threonine;

The methionine(Met) of 254 of EU amino acid positions is replaced by Serine or Threonine;

The Threonine of 256 of EU amino acid positions is replaced by arginine, L-glutamic acid or Methionin;

The arginine of 255 of EU amino acid positions is replaced by leucine, aspartic acid or methionine(Met);

The Threonine of 260 of EU amino acid positions is replaced by Methionin;

The proline(Pro) of 257 of EU amino acid positions is replaced by arginine, aspartic acid, L-glutamic acid or Methionin;

The tryptophane of 277 of EU amino acid positions is replaced by arginine, aspartic acid, glutamine, perhaps Methionin;

The Xie Ansuan of 279 of EU amino acid positions is replaced by L-glutamic acid;

The glycine of 281 of EU amino acid positions is replaced by glutamine;

The Xie Ansuan of 282 of EU amino acid positions is replaced by arginine, aspartic acid, L-glutamic acid or Methionin;

The L-Ala of 287 of EU amino acid positions is replaced by aspartic acid, L-glutamic acid, Methionin, proline(Pro) or Threonine;

The Xie Ansuan of 284 of EU amino acid positions is replaced by aspartic acid;

The Histidine of 285 of EU amino acid positions is replaced by aspartic acid or phenylalanine;

The l-asparagine of 286 of EU amino acid positions is replaced by L-glutamic acid or methionine(Met);

The Methionin of 288 of EU amino acid positions is replaced by aspartic acid;

The Methionin of 290 of EU amino acid positions is replaced by aspartic acid;

The Serine of 304 of EU amino acid positions is replaced by aspartic acid or L-glutamic acid;

The Xie Ansuan of 305 of EU amino acid positions is replaced by arginine;

The leucine of 306 of EU amino acid positions is replaced by arginine, aspartic acid, L-glutamic acid or Methionin;

The Threonine of 307 of EU amino acid positions is replaced by arginine, aspartic acid or L-glutamic acid;

The leucine of 309 of EU amino acid positions is replaced by arginine, aspartic acid, Methionin or L-glutamic acid;

The Histidine of 310 of EU amino acid positions is replaced by arginine, leucine, Methionin or l-asparagine;

The aspartic acid of 312 of EU amino acid positions is replaced by arginine, l-asparagine, perhaps Methionin;

The tryptophane of 313 of EU amino acid positions is replaced by aspartic acid, arginine or Methionin;

The l-asparagine of 315 of EU amino acid positions is replaced by aspartic acid or L-glutamic acid;

The proline(Pro) of 343 of EU amino acid positions is replaced by glutamine or Methionin;

The L-glutamic acid of 345 of EU amino acid positions is replaced by arginine or glutamine;

The proline(Pro) of 374 of EU amino acid positions is replaced by arginine, Methionin or leucine;

The aspartic acid 376 of EU amino acid position position is replaced by l-asparagine;

The Serine of 426 of EU amino acid positions is replaced by arginine, aspartic acid or L-glutamic acid;

The methionine(Met) of 428 of EU amino acid positions is replaced by arginine, glutamine, perhaps Methionin;

The L-glutamic acid of 430 of EU amino acid positions is replaced by Methionin;

The L-Ala of 431 of EU amino acid positions is replaced by proline(Pro);

The leucine of 432 of EU amino acid positions is replaced by arginine;

The l-asparagine of 434 of EU amino acid positions is replaced by leucine or Methionin; With

The glutamine of 438 of EU amino acid positions is replaced by L-glutamic acid,

Wherein, the polypeptide of change is compared with the initial polypeptide that does not contain sudden change and is demonstrated shorter circulating half-life.

In the embodiment, the present invention relates to prepare the method for polypeptide of the change of claim 1 or 2, described method comprises:

(a) with the nucleic acid molecule transfectional cell of nucleotide sequence of the polypeptide that comprises the described change of encoding; With

(b) be somebody's turn to do the polypeptide that changes from described cell or cell conditioned medium liquid purifying.

In the embodiment, the present invention relates to prepare the method for the antibody of claim 11 or 13, described method comprises:

(a) provide the variable region (V of the light chain that comprises encoding said antibody _L) and constant region (C _L) first nucleic acid molecule of nucleotide sequence;

(b) provide the variable region (V of the heavy chain that comprises encoding said antibody _H) and constant region (CH ₁, CH ₂And CH ₃) second nucleic acid molecule of nucleotide sequence;

(c) under the condition of the antibody expression of the described change of light chain that allows to comprise described coding and heavy chain, with the first and second nucleic acid molecule transfectional cells; With

(d) from described cell or the described antibody of cell conditioned medium liquid purifying.

In the embodiment, the present invention relates to comprise the dimeric Fc binding proteins of first and second polypeptide chains, wherein said first and second polypeptide chains respectively comprise the Fc plot structure territory that at least one and Fc binding domains can be operatively connected.

In the embodiment, described Fc structural domain the combining of being suddenlyd change with reduction or elimination and FcRn.

In the embodiment, described first and second polypeptide chains are covalently bound.

In the embodiment, the Fc binding domains comprises the extracellular domain of FcRn.

In the embodiment, the Fc binding domains combines with β2Wei Qiudanbai.

In the embodiment, the Fc binding domains derives from people FcRn.

In the embodiment, the described conjugated protein aminoacid sequence shown in the SEQ ID NO:10 that comprises.

In the embodiment, the present invention relates to measure the method for the polypeptide of the FcRn bound fraction that comprises the Fc district at least at the binding affinity (affinity) of FcR, described method comprises that the polypeptide that makes the FcRn bound fraction that comprises the Fc district at least contacts with the dimeric Fc binding proteins of claim 44, and determines this interactional avidity.

In the embodiment, the present invention relates to from the library of the polypeptide of the FcRn bound fraction that comprises the Fc district at least screening has the binding affinity of change to FcRn the method for polypeptide, this method comprises:

(a) the library member is contacted with the dimeric Fc binding proteins of claim 44; With

(b) measure the binding affinity of polypeptide to dimeric Fc binding proteins; With

(c) selection has the polypeptide of the binding affinity of change to FcRn.

In the embodiment, the present invention relates to the method for polypeptide that from polypeptide mixture purifying comprises the FcRn bound fraction in Fc district at least, this method comprises mixture is loaded on the affinity column of the dimeric Fc binding proteins matter that contains claim 44, wash-out comprises the polypeptide of the FcRn bound fraction in Fc district at least, thus this polypeptide of purifying.

In the embodiment, when a pH, described mixture is loaded on the affinity column, and when the 2nd pH from the affinity column the described polypeptide of wash-out.

In the embodiment, polypeptide is unchangeability in purge process.

In the embodiment, the present invention relates to identify with initial polypeptide compare the method that FcRn has the polypeptide that combines affinity of change that this method comprises:

The associated change in conjunction with free energy of performance of the space of the amino acid whose best charge distribution of initial polypeptide and initial polypeptide when (a) determining to be combined in FcRn in the solvent;

(b) identify at least one candidate amino acid residue position when changing initial polypeptide and combine to be finished in the initial polypeptide in conjunction with free energy with FcRn; With

(c) identify the amino acid of selecting at this amino acid position, so that cause FcRn is had the polypeptide in conjunction with the change of affinity (affinity) of change after in this initial polypeptide, mixing this sudden change.

In the embodiment, described method comprises that also the amino acid that will select mixes in the initial polypeptide.

In the embodiment, described method comprises that also the polypeptide of the change of calculating the described Fc of containing compares the variation in conjunction with free energy in conjunction with FcRn the time with initial polypeptide.

In the embodiment, described calculation procedure at first is included in the computer patrix and builds up the sudden change of beginning in the polypeptide, the variation of calculations incorporated free energy then.

In the embodiment, described calculation procedure is used at least a definite method that is selected from down group: use the static bound energy determined based on the method for Poisson-Boltzmann formula and Fan Dewaershi bound energy, and use determine bound energy based on the solvent-accessible surface Method for Area.

In the embodiment, sudden change is an amino acid replacement.

In the embodiment, amino acid replacement causes mixing the elected amino acid that has different electric charges with candidate amino acid.

In the embodiment, sudden change increased the polypeptide of the change that contains Fc and FcRn in solvent in conjunction with the time in conjunction with free energy, the polypeptide that has reduced the change that contains Fc thus to FcRn in conjunction with affinity.

In the embodiment, sudden change reduced the polypeptide of the change that contains Fc and FcRn in solvent in conjunction with the time in conjunction with free energy, the polypeptide that has increased the change that contains Fc thus to FcRn in conjunction with affinity.

On the one hand, the present invention relates to identify that two different pH levels FcRn is had the method in conjunction with the polypeptide of change affinity, that contain Fc of change, described method comprises:

(a) determine the space performance of the amino acid whose best charge distribution of initial polypeptide when a pH level combines with FcRn and the associated change in conjunction with free energy of initial polypeptide in solvent;

(b) determine the space performance of the amino acid whose best charge distribution of initial polypeptide when the 2nd pH level combines with FcRn and the associated change in conjunction with free energy of initial polypeptide in solvent;

(c) by the residue of charge distribution, the different charge distribution of the demonstration under the first and second pH levels relatively, identifying has at least one candidate amino acid residue position in conjunction with free energy when changing initial polypeptide and combine with FcRn to be finished in the initial polypeptide; With

(d) select to be positioned at the elected amino acid of described amino acid position, so as should elected amino acid to mix cause behind the initial polypeptide to FcRn have change in conjunction with polypeptide affinity, that contain Fc, that change.

In one embodiment, a pH is about 7.4.

In the embodiment, the affinity of polypeptide when a pH than at the 2nd pH the time high about 1.5 times to about 100 times.

The details of one or more embodiments of the present invention will be set forth in the accompanying drawings and the description below.Based on this description and accompanying drawing and claim, other features, objects and advantages of the present invention will be conspicuous.The content of all patents, patent application and other reference of quoting in this specification sheets intactly is incorporated herein by reference hereby.

The accompanying drawing summary

Figure 1A shows the dna sequence dna as the ripe huCBE11 heavy chain of the initial polypeptide in the inventive method.This dna sequence dna is encoded in the pEAG1787 expression vector.Figure 1B shows the aminoacid sequence of the ripe huCBE11 heavy chain of prediction.

Fig. 2 shows the aminoacid sequence as the Fc district of the huCBE11 heavy chain of the initial polypeptide in the inventive method.By the EU numbering in Kabat, indicate amino position.

Fig. 3 A shows the dna sequence dna of huCBE11 light chain.This dna sequence dna is encoded in the pEAG1754 expression vector.Fig. 3 B shows the aminoacid sequence of the huCBE11 light chain of prediction.

Fig. 4 A shows the dna sequence dna of people's beta-2 microglobulin.This dna sequence dna is encoded in the pEAG1761 expression vector.Fig. 3 B shows the aminoacid sequence of people's beta-2 microglobulin of prediction.

The nucleotide sequence of the cDNA of Fig. 5 A code displaying people FcRn/Fc/ fusion rotein.This dna sequence dna is encoded in the pEAG1761 expression vector.Fig. 3 B shows the aminoacid sequence of this FcRn/Fc/ fusion rotein of prediction.

Fig. 6 shows the result that use obtains in conjunction with affinity based on the FcRn of the polypeptide of the test evaluation change of the present invention of FRET.Indicate with the arrow that refers to downwards in the sudden change that aspect affinity, has measurable increase (H285E, N286D, K290E, and S304D).In conjunction with the arrow indication of the significantly reduced sudden change of affinity (N434L) to refer to left.

Fig. 7 shows the structure that the FcRn of the polypeptide that uses the change of the present invention of AlphaScreen test evaluation obtains in conjunction with affinity.Indicate with the arrow that refers to downwards in conjunction with the sudden change (V284E, H285E, N286D, and K290E) that affinity can increase with measuring.In conjunction with the arrow indication of the significantly reduced sudden change of affinity (Q438E) to refer to left.

Fig. 8 shows the structural models in the Fc district of the huCBE11 heavy chain that uses among the present invention.Specific " going up mutant (up mutant) " (V284E with enhanced FcRn affinity, H285E, N286E, K290E) and specific " following mutant (down mutant) " (V282E with FcRn affinity of reduction, M252T, N434L, position Q438L) has been done to indicate with respect to other structural domain of FcRn.

A figure among Fig. 9 shows the position of FcRn coupling collar, and this ring aspartic acid 280 that relevant FcRn structural domain shows from Fig. 9 extends to Threonine 299.B figure shows position (for example, the 243F of relevant FcRn structural domain residue in 15 dust FcRn zone of action; 244P; 245P; 246K; 247P; 248K; 249D; 250T; 251L; 252M; 253I; 254S; 255R; 256T; 257P; 258E; 259V; 260T; 261C; 275F; 276N; 277W; 278Y; 279V; 280D; 282V; 283E; 284V; 285H; 286N; 287A; 288K; 289T; 290K; 291P; 292R; 293E; 302V; 303V; 304S; 305V; 306L; 307T; 308V; 309L; 310H; 311Q; 312D; 313W; 314L; 315N; 316G; 317K; 318E; 319Y; 336I; 337S; 338K; 339A; 340K; 341G; 342Q; 343P; 344R; 345E; 346P; 347Q; 348V; 367C; 369V; 372F; 373Y; 374P; 375S; 376D; 377I; 378A; 379V; 380E; 381W; 382E; 383S; 384N; 385G; 386Q; 387P; 388E; 389N; 391Y; 393T; 408S; 424S; 425C; 426S; 427V; 428M; 429H; 430E; 431A; 432L; 433H; 434N; 435H; 436Y; 437T; 438Q; 439K; And 440S).

Figure 10 shows that biotinylated hIgG and different concns by 3,6 and 9 μ g/ml are coated on combining of hFcRnFc on the elisa plate.The concentration of biotinylated hIgG by shown in change, and with 1: 20,000 concentration is used the HPR-Streptavidin, and develops with standard scheme.After the termination reaction, read the absorbancy of 450nm.The hFcRnFc of 9 μ g/ml () and 6 μ g/ml (o) shows no change on binding curve, and 3 μ g/ml (Δ) hFcRnFc demonstrate reduction and the combining of the hIgG bag quilt.Negative control (◇) is presented at the background combination of Streptavidin HRP under the non-existent situation of hFcRnFc.

Detailed Description Of The Invention

The present invention is at least in part based on the evaluation to following polypeptide, described polypeptide (antibody and melting for example Hop protein matter) comprise Fc district at least one who neonatal Fc receptor (FcRn) is demonstrated the combination of change Individual part (for example constant region of immunoglobulin (Ig) such as IgG1). The polypeptide of this change and wild type peptide phase Than that demonstrate increase or that reduce and combination FcRn, therefore in serum, have respectively increase or The half-life of reducing. The Fc district expection that FcRn is had the affinity of raising will have longer serum Half-life, the mammal animal the long half-lift that this quasi-molecule can having for the polypeptide that expectation is used In the methods for the treatment of, for example be used for the treatment of chronic disease or illness. On the contrary, the FcRn knot that has reduction The Fc region variants expection of closing affinity has the short half-life, and this quasi-molecule also can be used, for example, in The circulation timei that shortens may be in the favourable situation to administration, for example, be used for examining in the body Be broken into picture or be used for initial polypeptide has toxic and side effect when the circulation midium or long term exists situation. Tool There is the FcRn of reduction also to have the possibility of littler leap placenta in conjunction with the Fc region variants of affinity, Disease or the illness that also can be used for the treatment of thus the pregnant woman. In addition, may expect the FcRn combination that reduces Other application of affinity comprises those application of expected location brain, kidney and/or liver. Exemplary at one In the embodiment, the polypeptide of change of the present invention demonstrates the transhipment of striding glomerular epithelium from vascular system Reduce. In another embodiment, the polypeptide of the present invention's change demonstrates from brain and strides blood-brain barrier (BBB) The transhipment in intravasation gap reduces.

The invention still further relates to the method and the method for using this polypeptide of the polypeptide of this change of preparation. The present invention The new model and the using method thereof that also relate to FcRn.

Various aspects of the present invention describe in further detail in following trifle:

I. definition

Term " protein ", " polypeptide " and " peptide " in this article can Alternates. Protein can comprise One or more natural amino acids or alpha-non-natural amino acid.

" initial polypeptide " or " first polypeptide " is the polypeptide that comprises following amino acid sequence, this amino acid order Modify and have with the polypeptide that changes or modify in the few one or more Fc disclosed herein district of Lieque Different effector functions. Initial polypeptide is the natural or people who comprises Fc district or its FcRn bound fraction Worker's polypeptide. Initial polypeptide can comprise natural Fc region sequence or have earlier already present amino acid sequence Modify the Fc district of (for example add, lack and/or substitute). Initial polypeptide of the present invention advances by shown in this paper Row modify with regulate (increase or reduce) and FcRn in conjunction with affinity.

Herein, term " polypeptide of change " or " second polypeptide " refer to be included in the Fc district and contain at least one The polypeptide of the non-natural Fc bound fraction of individual sudden change. When the polypeptide of mentioning change demonstrates the " effect of change Subfunction " time, the polypeptide that means described change is compared with initial polypeptide and is conducive to more or less that it is a kind of Or multiple (and possibly, but optional, all) effector function.

Herein, term " Fc district " comprises the amino acid sequence that derives from the heavy chain of antibody constant region. The Fc district The part of heavy chain of antibody constant region, (according to the EU index, about from the papain cleavage site 216) the hinge area N that locates end is initial, comprises hinge, CH2 and CH3 domain.

Initial polypeptide can comprise a part that mediates the Fc district of being combined with FcRn at least. For example, exist In the embodiment, initial polypeptide is antibody or Fc fusion. Herein, " fusion " Refer to comprise the chimeric polyeptides of first amino acid sequence that is connected with second amino acid sequence, wherein said the One be connected amino acid sequence and in natural situation, do not connect. For example, fused protein can comprise volume The amino of at least one part (for example giving the Fc district part in conjunction with character with FcRn) in code Fc district (for example the ligand binding domains of acceptor or part is subjected to for acid sequence and coding NIg polypeptide The body binding structural domain) amino acid sequence. These amino acid sequences are present in protein separately usually In, but in fused polypeptide, be pulled together; Perhaps they can under normal circumstances be present in same In the protein, but in fused polypeptide, be placed on the new arrangement position. Fused protein can By for example chemical synthesis or by making up and translate the multinuclear by these peptide districts of expectation relation coding Thuja acid produces.

Herein, term " connection ", " fusion " and " fusion " are used interchangeably. These terms refer to two Perhaps plural element or composition are by any mode (comprising chemically conjugated or recombination method) Link together. " frame in merge " or " being operatively connected " refer to two or more reading frames (ORF) with Keep the mode of the proper reading frame of these original ORF to connect to form a continuous longer ORF. Therefore, the recombinant fusion protein of gained is the single protein that contains two or more sections, wherein Described section corresponding to the polypeptide of original ORF coding (these sections under normal circumstances not natural as This connection). Although the reading frame of preparation is continuous at whole fusion section thus, these districts Duan Keneng is separated by for example frame nipple sequence physically or on the space.

In the embodiment, polypeptide of the present invention comprises immunoglobulin (Ig) antigen binding site or acceptor branch Be responsible for the part of receptors bind in the son in the part of responsible ligand binding or the ligand molecular.

Herein, term " effector function " refer to Fc district or its part binding immunoassay system protein and/ Or cell and mediate the functional capabilities of various biological effects. Effector function can be that antigen relies on Property or the antigen dependent/non-dependent.

Herein, the make a comment or criticism antibody of being everlasting of term " antigen dependence effector function " lures after in conjunction with corresponding antigens The effector function of leading. Typical antigen dependence effector function (for example, comprises conjugated complement protein C1q) ability. For example, the combination in complement C1 component and Fc district can activate classical complement system, Cause the cracking of opsonic action and cytopathy substance, this process is called CDC (CDCC). The activation of complement also stimulates inflammatory reaction, and also may relate to the autoimmunity hypersensitivity.

Other antigen dependence effector function is subjected to by some Fc on its Fc district and the cell by antibody The combination of body (" FcR ") mediates. Have multiple to the different antibodies type, comprise IgG (the γ acceptor, perhaps Ig γ R), IgE (epsilon receptor, or Ig ε R), IgA (α acceptor, or Ig α R) and IgM (μ acceptor, or Ig μ R), Has specific Fc acceptor. The Fc acceptor is many important in conjunction with triggering on antibody and the cell surface The different biological reaction comprises endocytosis, the particle of antibody sandwich or the gulping down of microorganism of immune complex Food and destroy (be also referred to as AD Φ, or ADCP), immune complex removing, kill Hinder the coated target cell of cell antagonist cracking (cytotoxicity that is called the antibody dependent cellular mediation, Adjusting, the immunoglobulin (Ig) product of or ADCC), the release of inflammatory mediator, immune system cell activation Placenta shift and control.

Some Fc acceptor, Fc γ acceptor (Fc γ R) is in cancellation or strengthen immunity and playing the part of key aspect raising The role. Fc γ R is expressed on the leucocyte, by three dissimilar compositions: Fc γ RI, Fc γ RII and Fc γ RIII. The Fc district of IgG Immunoglobulin Isotype (Gessner etc., Ann.Hematol., (1998), 76: 231-48). On the structure, these Fc γ R all are members of immunoglobulin superfamily, have in conjunction with IgG The α chain, this α chain has the outer part of the born of the same parents that are made up of two or three Ig spline structure territories. People Fc γ RI (CD64) be expressed on the person monocytic cell, show high affine to monomer I gG1, IgG3 and IgG4 Power is in conjunction with (Ka=10⁸-10 ⁹M ^-1). People Fc γ RII (CD32) and Fc γ RIII (CD16) are to IgG1 and IgG3 Have low-affinity (Ka＜10⁷M ^-1), and only can in conjunction with complex form or the polymer form this A little IgG isotypes. In addition, Fc γ RII and Fc γ RIII type comprise " A " and " B " form. Fc γ RIIa (CD32a) To be combined in macrophage, NK cell and some T by membrane spaning domain thin with Fc γ RIIIa (CD16a) Cellular surface, and Fc γ RIIb (CD32b) and Fc γ RIIIb (CD16b) are by glypican (GPI) Anchor optionally is combined in the cell surface of granulocyte (for example neutrophil cell). People Fc γ RI, Fc γ RII With the corresponding mouse homologue of Fc γ RIII be Fc γ RIIa, Fc γ RIIb/1 and Fc γ R1o.

Herein, term " antigen dependent/non-dependent effector function " refers to no matter whether antibody is corresponding in conjunction with it Antigen all can be by the effector function of antibody induction. Typical antigen dependent/non-dependent effector function bag Draw together cell traffic, circulating half-life and the clearance rate of immunoglobulin (Ig). The Fc acceptor of structure uniqueness, " neonatal Fc receptor " or " FcRn " is also referred to as and remedies acceptor (salvage receptor), regulating this A little function aspects have key effect. Preferably, the FcRn of polypeptide of the present invention institute combination is people FcRn.

Different from the Fc γ R of contactin, people FcRn structurally is similar to the I type The polypeptide of ajor histocompatibility compound (MHC) (Ghetie and Ward, Immunology Today, (1997), 18 (12): 592-8). FcRn is typically with the heterodimer formal representation, and this heterodimer is by striding The compound that film α chain or heavy chain and solubility β chain or light chain (β2-microglobulin) form forms. FcRn With I type MHC molecule 22-29% sequence homogeneity is arranged, have the MHC peptide combination of non-functional form Ditch (Simister and Mostov, Nature, (1989), 337:184-7). The same with MHC, the α of FcRn Chain is made up of three extracellular domains (α 1, and α 2, and α 3), and the kytoplasm tail of a weak point fixes on the cell table with protein anchor Face. FcR binding site interaction in α 1 and α 2 domains and the antibody Fc district (Raghavan etc., Immunity, (1994), 1:303-15).

FcRn expresses in mammiferous placenta materna or yolk bag, and participates in IgG from parent Transfer to fetus. FcRn also expresses in the rodentine small intestine of new life, and FcRn participates in herein Maternal immunoglobulin G is striden the epithelial transfer of brush border from colostrum or the breast taken in. FcRn also is expressed in many In many other tissues of species, and in the various endothelial cell line. Its also HAS's endothelium, Express in muscle vascular system and the hepatic sinusoid. It is believed that FcRn passes through to reclaim in conjunction with IgG and with IgG To serum, also participate in keeping circulating half-life or the serum levels of IgG. The knot of FcRn and IgG molecule Closing is that strictly pH is dependent, has best combination less than 7.0 pH the time.

Herein, term " half-life " refers to the polypeptide biological half life in vivo of the specific Fc of containing. Half-life can be by disposing the half that is administered to the experimenter from the circulation of animal and/or other tissue Measuring the needed time represents. Made up the given polypeptide that contains Fc in the form with the function of time Clearance curve the time, curve is two-phase normally, has α phase fast and long β mutually. α phase allusion quotation The balance of Fc polypeptide between blood vessel internal clearance and blood vessel external series gap that the expression of type ground is used, it partly The size that depends on polypeptide. β typically represents the metabolism of Fc polypeptide in the blood vessel internal clearance mutually. Therefore, In preferred embodiments, term used herein " half-life " refers to the half-life of the Fc polypeptide of β phase. The typical β phase half-life of human antibodies is 21 days.

Herein, term " sudden change " comprise amino acid whose in the initial polypeptide substitute, add or disappearance to obtain The polypeptide that changes.

" amino acid replacement " refer to in the predetermined amino acid sequence (amino acid sequence of initial polypeptide) at least An existing amino acid residue is replaced by another different " displacement " amino acid residue. Described one or more The displacement residue can be " natural amino acid residue " (that is, the genetic codon coding), and is selected from: third ammonia Acid (A); Arginine (R); Asparagine (N); Aspartic acid (D); Cysteine (C); Glutamine (Q); Glutamic acid (E); Glycine (G); Histidine (H); Isoleucine (I); Leucine (L); Lysine (K); Methionine (M); Phenylalanine (F); Proline (P); Serine (S); Threonine (T); Tryptophan (W); Tyrosine (Y); And valine (V). Be also included within this with one or more alpha-non-natural amino acid residues are alternative In the definition of the amino acid replacement of literary composition. " alpha-non-natural amino acid residue " refers to non-natural amino acid listed above Residue, can be in polypeptide chain the residue of covalent bond adjacent amino acid residue. Alpha-non-natural amino acid is residual The example of base comprises nor-leucine, ornithine, norvaline, homoserine and other amino acid residue Analog is such as middle those that describe of the Meth.Enzym.202:301-336 such as Ellman (1991). For The preparation these alpha-non-natural amino acid residues, can use such as Science 244:182 such as Noren (1989) and Ellman etc., the method for the same quoted passage. In brief, these methods comprise and use non-natural amino In-vitro transcription and this RNA of translation after the acid residue chemical activation inhibition type tRNA.

Herein, term " nonpolar " comprise amino acid with uncharged side chain (for example, A, L, I, V, G, P). These amino acid participate in hydrophobic interaction usually.

Herein, term " polarity " comprises having zero net charge but have non-zero in the different piece of its side chain The amino acid (for example, M, F, W, S, Y, N, Q, C) of Partial charge. These amino acid can participate in dredging Water-based interacts and electrostatic interaction.

Herein, term " charged " is included in the amino acid that can have non-zero net charge on the side chain (for example, R, K, H, E, D). These amino acid can participate in hydrophobic interaction and static is done mutually With.

" amino acid insertion " refers in the predetermined amino acid sequence of at least one amino acid incorporation. Although insert logical Often be the insertion of one or more amino acid residues, but can carry out the present invention's bigger " peptide insertion ", example As insert about 3 to about 5 or even reach about 10 amino acid residues. As above disclosed, The residue that inserts can be natural or non-natural.

" amino acid deletions " refers to remove at least one amino acid residue from predetermined amino acid sequence.

Herein, term " enough big spatial volumes " comprises having the side chain that occupies 3 bigger dimension spaces Amino acid. Exemplary amino acid with side chain chemistry of enough large space volumes comprise tyrosine, Tryptophan, arginine, lysine, histidine, glutamic acid, glutamine and methionine or its Analog or analogies.

Herein, the surface area of the atom of term " solvent-accessible surface area " abutment solvent molecule. The solvent-accessible surface area can use method well known in the art to calculate. In brief, atom or If when one group of atom can contact with solvent (water) the molecule generation Fan Dewaershi of prescribed level, decided Justice is to reach. The Fan Dewaershi contact is to cause maximum the permission when solvent molecule rolls along protein The position at the center of solvent molecule during contact.

Term " in conjunction with affinity " comprises in this article the intensity of binding interactions and comprises thus reality In conjunction with affinity and apparent in conjunction with affinity. Actual is association rate and dissociation rate in conjunction with affinity The ratio. Therefore, give or the affinity of optimizing integration comprises and changes one of these compositions or both are to obtain Aspiration level in conjunction with affinity. Apparent affinity can comprise for example interactional affinity (avidity).

Term " in conjunction with free energy " or " free energy of combination " comprise the meaning of its this area approval in this article Justice, and especially be applied to Fc-Fc acceptor interaction in the solvent. Reduction in conjunction with free energy will strengthen the parent Closing property, and will reduce affinity in conjunction with the increase of free energy.

Term " binding structural domain " or " binding site " refer in this article in the polypeptide that mediation and target molecule are (for example Antigen, part, acceptor, substrate or inhibitor) one or more zones of specific binding. Example The property binding structural domain comprises the receptors bind domain of antibody variable region, part, the ligand binding of acceptor Domain or enzymatic structure territory. Term " ligand binding domains " (for example refers to any natural receptor in this article Cell surface receptor) or kept at least qualitatively ligand binding capacity and preferably corresponding natural receptor Any zone or the derivative of natural receptor of BA. Term " receptors bind domain " is at this The literary composition any native ligand of middle finger or kept at least qualitatively receptor binding capacity and preferably corresponding Any zone or the derivative of the native ligand of the BA of native ligand. An embodiment In, polypeptide has at least one molecule that orientation is reduced or eliminate (cell surface antigen or can for example Dissolubility antigen) has specific binding structural domain. In preferred embodiments, binding structural domain is Antigen binding site.

In a preferred embodiment, to comprise at least one binding site (for example, anti-for polypeptide of the present invention Former binding site, receptor binding site or ligand binding site). In the embodiment, the present invention is many Peptide comprises at least two binding sites. In the embodiment, polypeptide of the present invention comprises three in conjunction with the position The point. In another embodiment, polypeptide of the present invention comprises the four combinations site.

Polypeptide of the present invention can be monomer or polymer. For example, in the embodiment, the present invention is many Peptide is dimer. In the embodiment, dimer of the present invention is homodimer, comprises two phases Monomer subunit together. In the embodiment, dimer of the present invention is heterodimer, comprises two Different monomer subunits. Dimeric subunit can comprise one or more polypeptide chain. For example, one In the embodiment, dimer comprises at least two polypeptide chains. In the embodiment, dimer comprises Article two, polypeptide chain. In another embodiment, dimer comprises four polypeptide chains (for example, at antibody molecule Situation under).

When term " exposure " amino acid residue comprises in the polypeptide in being present in solvent in this article, its At least a portion on surface is exposed to the amino acid residue of solvent to a certain extent. Preferably, expose Amino acid residue to be that at least about 1/3rd side chain surface area is exposed to the amino acid of solvent residual Base. Can obtain several different methods and be used for determining whether residue exposes, and comprises molecular model or polypeptide Construction analysis.

Term " variant ", " polypeptide of change ", " polypeptide of modification ", " contain the amino acid whose many of modification Peptide " and similar term comprise in this article having the amino acid sequence different from the amino acid of initial polypeptide Polypeptide. Typically, this type of polypeptide has one or more sudden changes, for example one or more amino acid Residue is substituted by other amino acid residue or one or more amino acid residues takes place and inserts or lack Lose. Preferably, polypeptide comprises the amino acid sequence of at least a portion that contains non-natural Fc district. Should Variant is inevitable to have with initial antibody and is less than 100% sequence homogeneity or similitude. Preferred real at one Execute in the scheme, the amino acid sequence that variant has with initial polypeptide has about 75% to the ammonia that is less than 100% The amino acid sequence of base acid sequence homogeneity or similitude, more preferably about 80% to be less than 100%, More preferably about 85% to be less than 100%, more preferably about 90% to be less than 100%, optimum Selection of land about 95% is to the amino acid homogeneity or the similitude that are less than 100%. In the embodiment, There is an amino acid difference between initial antibody and the modified antibodies of the present invention. Same about this sequence One property or similitude are defined as in this article at sequence alignment and introduce where necessary breach to reach maximum Behind the sequence homogeneity percentage, the amino acid residue identical with the initial amino acid residue (that is, in the candidate sequence Identical residue) percentage. Modified polypeptide of the present invention can be expressed, perhaps alternatively can Carrying out mould on the computer builds.

Phrase " candidate amino acid residue position " is included in the amino that identifies in the polypeptide of the present invention herein The acid position, wherein the candidate amino acid position substitute by mould build, prediction or empirical find can Change, disappearance, insert or with the FcRn binding affinity of adjusting polypeptide behind another amino acid replacement.

Term " selected amino acid " is used in reference in this article by what the inventive method was selected and is used for conduct Replacement amino acid mixes the amino acid residue of candidate amino acid position in the polypeptide. In the embodiment, Candidate amino acid residue position be elected to amino acid replacement so that static in the combination of Fc-FcRn compound Role reduces or increases in the free energy.

Term " antibody " comprises natural the resisting that obtains from the animal that produces antibody or produce in this article Body. For example, antibody can be from rodent for example mouse, rat, gerbil jird, hamster or cavy; From than large animal for example rabbit, cat or dog; From usually as the animal of penkeeping (for example pig, ox, horse, Sheep or goat); Or obtain from primate (comprising people and non-human primate) or produce Antibody. Term " antibody " also comprises the immunoglobulin molecules of immunoglobulin molecules and modification, for example Contain the antigen binding site of conjugated antigen (with antigen immune reaction) and mediate in the Fc district at least and The molecule of the part of FcRn combination. Herein, term " antibody " also comprises at least one that comprises the Fc district The modification of part or synthetic antibody molecule.

Herein, term " hinge area " comprises connection CH1 domain and CH2 domain in the heavy chain molecule Part, for example, according to the EU numbering system from about 216 to 230. This hinge area comprises greatly About 25 residues, and have flexibility, allow thus two N end antigen binding domains independently mobile. Hinge Sequence can be subdivided into three different structure territories: upper, the lower hinge arrangement territory of neutralization (Roux etc., J. Immunol.1998,161:4083).

Herein, term " CH2 domain " comprises in the heavy chain molecule from for example about 231 of EU positions Extend to 340 part. The CH2 domain is unique, because it is not tight with another domain Pairing. On the contrary, branch's sugar chain of two N connections is got involved two CH2 of complete natural IgG molecule Between the domain.

Herein, term " CH3 domain " comprises in the heavy chain molecule from the extension of the N of CH2 domain end The part of about 110 residues (for example about residue is 341 to 446, the EU numbering system). The CH3 domain typically forms the C end parts of antibody. Yet, in some immunoglobulin (Ig)s, can To extend extra domain from the CH3 domain to form C end parts (for example, the IgM of this molecule The μ chain and the CH4 domain in the δ chain of IgE).

In the embodiment, in " the FcRn coupling collar " in Fc district, carry out one or more amino acid and dash forward Become. The FcRn coupling collar is by 280,281,282,283,284,285,286,287,288,289,290,291, 292,293,294,295,296,297,298 and 299 amino acids residues (according to the EU numbering) form. This ring is described among Fig. 9.

In another embodiment, in 15  FcRn " contact zone ", carry out one or more amino acid mutations. Herein, term 15  FcRn " contact zone " comprise that the residue that is positioned at such as upper/lower positions (also listed and be used for this The exemplary amino acid of a little positions): 243F; 244P; 245P; 246K; 247P; 248K; 249D; 250T; 251L; 252M; 253I; 254S; 255R; 256T; 257P; 258E; 259V; 260T; 261C; 275F; 276N; 277W; 278Y; 279V; 280D; 282V; 283E; 284V; 285H; 286N; 287A; 288K; 289T; 290K; 291P; 292R; 293E; 302V; 303V; 304S; 305V; 306L; 307T; 308V; 309L; 310H; 311Q; 312D; 313W; 314L; 315N; 316G; 317K; 318E; 319Y; 336I; 337S; 338K; 339A; 340K; 341G; 342Q; 343P; 344R; 345E; 346P; 347Q; 348V; 367C; 369V; 372F; 373Y; 374P; 375S; 376D; 377I; 378A; 379V; 380E; 381W; 382E; 383S; 384N; 385G; 386Q; 387P; 388E; 389N; 391Y; 393T; 408S; 424S; 425C; 426S; 427V; 428M; 429H; 430E; 431A; 432L; 433H; 434N; 435H; 436Y; 437T; 438Q; 439K; And 440S (EU numbering).

Refer to implement the computer of all or certain operations described herein when mentioning " computational analysis " herein Implementation. This process comprise to the output device of user's exhibition information (for example, CRT monitor, LCD, Printer, communication device such as modem, audio frequency output etc.). This computer implementation is not limit In specific computer platform, specific processor or specific high-level programming language.

Term " structure " or " structured data " comprise in this article by atom, molecule, compound, amino Acid residue or its part and big molecule or its part and especially in solvent, be combined with antigen That occupy, known, prediction and/or the three-dimensional space position that mould is built of polypeptide. Can use multiple Be used for the method in molecule/atomic level evaluation and/or predict, for example X-ray crystallography, NMR The structure mould is built etc.

Phrase " the space performance of best CHARGE DISTRIBUTION " comprises for Fc district or Fc-FcRn multiple in this article The compound mould is built electron distributions, and the initial polypeptide of wherein being combined with respect to initial polypeptide and/or with FcRn Know and/or the performance of CHARGE DISTRIBUTION that mould is built, optimize (minimizing) static at polypeptide when FcRn is combined Contribution in the free energy. The mould of best CHARGE DISTRIBUTION is built and can be realized by program on computers, Wherein incorporate into the structure of Fc district known and/or that mould is built or Fc-FcRn compound described by input Program. Can use the continuous phantom of reaction to build (response continuum modeling) (for example, linearity The Poisson-Boltzmann formula) the electrostatical binding free energy of compound in solvent is expressed as Fc Solvation item, Fc-FcRn interact and FcRn desolvation item and. Program on this computer Be characterised in that can be in the performance mould of the present invention CHARGE DISTRIBUTION of building CHARGE DISTRIBUTION the time incorporate into one pole, Bipolar and four utmost point items, on this computer program allow to estimate widely the polypeptide amino acid residue in the Fc district or The solvation of its part during taking place not in conjunction with attitude and between in conjunction with attitude to change/desolvation energy. It is right that the method for space performance that mould is built the best CHARGE DISTRIBUTION of polypeptide-FcRn compound can also be incorporated into The mould of Fan Dewaershi power, solvent-accessible surface area power etc. is built.

Term " solvent " comprises this area approval implication of its broad sense in this article, refer to wherein dissolve and/or Any liquid that has polypeptide of the present invention. Preferably, solvent is the biocompatibility solvent. Preferably Solvent comprises PBS, serum etc.

Preferred initial polypeptide comprises the amino acid sequence that derives from human antibodies. " derive from " and specify polypeptide Or the polypeptide of source species or the origin that amino acid sequence refers to this polypeptide. Preferably, derive from specific The polypeptide of beginning polypeptide or amino acid sequence or amino acid sequence have with homing sequence or its part basically (wherein said part is made up of at least 10 to 20 amino acid identical amino acid sequence, preferably At least 20-30 amino acid, more preferably 30-50 amino acid at least), perhaps having can be by this The field those of ordinary skill is accredited as the amino acid sequence that originates from homing sequence according to alternate manner. Example Can comprise one or more from another mammalian species such as, the polypeptide that derives from human polypeptide Amino acid. For example, can comprise in the polypeptide of the present invention primate heavy chain part, hinge fraction, Or binding site. Perhaps, initial polypeptide for example can exist in the antigen binding site of antibody (CDR) One or more mouse source amino acid. Preferred polypeptide of the present invention does not have immunogenicity.

Term " PEGization part ", " polyalkylene glycol moiety " or " peg moiety " comprise poly-(alkylidene) two The alcoholic compound or derivatives thereof, it can have or not have coupling agent or be spread out by coupling or activated partial Biochemical (for example, have sulfydryl, trifluoromethane sulfonic acid ester, trifluoro esilate (tresylate), azacyclo-Propane, oxirane or preferably have the maleimide amine moiety, for example PEG-maleimide). Other suitable poly-(alkylidene) diol compound includes but not limited to dimaleoyl imino one methoxyl group The PEG polypropylene glycol of PEG, activation and the charged or neutral polymer such as Types Below: Glucan, colominic acid or other polymer based on sugar, amino acid whose polymer, And biotin derivative.

Term " funtion part " comprises the part of preferably adding desired function to variant polypeptide. Preferably, Add function and significantly not change the inherence expectation of polypeptide active, for example in the situation of antibody, aobvious Change the antigen-binding activity of this molecule outstandingly. Variant polypeptide of the present invention can comprise one or more merits The energy part, these parts can be identical or different. The example of useful funtion part includes but not limited to But PEGization part, enclosure portion test section, diagnosis of partial and treatment part. The exemplary inspection Survey part and comprise fluorescence part, radio isotope part, radiopaque part etc. Exemplary diagnosis Part comprises the part that is applicable to whether the indication that discloses disease or illness exists. The exemplary treatment part Comprise for example antiinflammatory, anticancer, anti-neurodegeneration agent and anti-infective. Funtion part also can tool In the above-mentioned functions one or more are arranged. Other useful funtion part is known in the art and describes As follows.

Herein, term " anticancer " or " chemotherapeutics " comprise the life of infringement neoplastic cell or tumour cell Long and/or propagation and can for reducing, suppress or destroy the medicament of malignant tumour. The example of this type of medicament Son includes but not limited to cytostatics, alkylating reagent, antibiotic, cytotoxicity nucleosides, microtubule Protein binding agent, hormone and hormone antagonist etc. Can play retardance or slow down immunoreactive cell Or any medicament of the effect of malignant cell growth all within the scope of the present invention.

Term " carrier " or " expression vector " refer to root in this article for the purpose of specification and claim According to the present invention as expect polynucleotides introducings cell and making these expectation polynucleotides at cells The carrier of carrier. It is known to those skilled in the art that this carrier can easily be selected from: plasmid, Bacteriophage, virus and retrovirus. Usually, the carrier compatible with the present invention comprise selected marker, Suitable restriction site be beneficial to expect gene cloning and enter eucaryon or prokaryotic and/or The ability that copies therein.

Term " host cell " refers to the cell of suppressed by vector conversion, and wherein said carrier utilizes recombinant DNA Technique construction and at least one heterologous gene of encoding. Describing from the recombinant host method of separating protein The time, unless clear regulation separately, otherwise term " cell " and " cell culture " can Alternate with finger The source of protein. In other words, reclaim protein from " cell " and can refer to intact cell from centrifugation, Perhaps reclaim from the cell culture medium that contains culture medium and suspension cell.

Herein, " tumor associated antigen " refers to usually relevant with tumour cell (that is, with the normal cell phase Ratio is with identical or exist more) any antigen. This antigen can be tomour specific relatively The property, its expression is limited to the malignant cell surface, but this antigen also may reside in non-malignant cell On. In the embodiment, the polypeptide of change of the present invention is combined with tumor associated antigen. Therefore, The initial polypeptide of the present invention can derive from, produce from or create any from the Tumor-assaciated molecular reaction A kind of antibody.

Herein, term " pernicious " refers to tumour or the cancer of non-benign. Herein, " cancer " comprises regulating Or Growth of Cells out of control be the malignant tumour of feature. Exemplary cancer comprises: cancer knurl, sarcoma, white Blood disease and lymthoma. Term " cancer " comprises primary malignant tumor, and (for example, its cell is not yet to the experimenter The malignant tumour of non-original tumour on-site site migration in the health) and second malignant neoplasm (for example,---tumour cell is to being different from on-site second site migration of original tumour---draws by metastases The malignant tumour that rises).

Herein, phrase " will have benefited from the experimenter who uses of polypeptide " and comprise executing from polypeptide of the present invention With middle acquisition positive treatment or prevention result's experimenter, for example mammalian subject. This paper is open The example of useful purposes of polypeptide comprise, for example, detect the antigen that polypeptide identifies and (for example, be used for Diagnostic method) or with polypeptide treat to reduce or eliminate the target that polypeptide is identified. For example, at one In the embodiment, the experimenter can be by soluble molecule or granulin molecule (for example toxin or pathogen) self-loopa Or the minimizing in the serum or removing and be benefited, perhaps by cell (for example tumour cell) colony of expressing target Reduce or eliminate and be benefited. As herein in greater detail, polypeptide can make with the non-form of puting together With, perhaps can for example put together with medicine, prodrug, label or isotope.

II. the polypeptide that contains Fc that is used for modification

In the embodiment, the initial polypeptide of the present invention comprises at least is enough to give the FcRn knot in the Fc district Close the part of character. The part of being combined with FcRn in the Fc district comprise IgG1 about 282 to 438 Amino acids (EU numbering). Amino acid position in the Fc district is in this article according to EU index number system Numbering (is seen Kabat etc., " Sequences of Proteins of Immunological Interest ", U.S.Dept. Health and Human Services, 5^thEdition, 1991). " the EU index among the Kabat " refers to the human IgG1 The residue numbering of EU antibody.

Fc of the present invention district is human origin preferably. The encode nucleosides in exemplary Fc district (human IgG1 district) Acid sequence is presented among the SEQ ID NO:1, by the nucleotide sequence coded amino of SEQ ID NO:1 Acid sequence is presented among the SEQ ID NO:2. The amino acid sequence that has also provided the Fc district in the following table 1 is used for Illustrate amino acid whose EU numbering.

Table 1: human IgG1 CH2 and CH3 domain

CH2 domain (EU position 231-340)

231  APELLGG

238  PSVFLFPPKP

248  KDTLMISRTP

258  EVTCVVVDVS

268  HEDPEVKFNW

278  YVDGVEVHNA

288  KTKPREEQYN

298  STYRVVSVLT

308  VLHQDWLNGK

318  EYKCKVSNKA

328  LPAPIEKTIS

338  KAK

CH3 domain (EU position 341-446)

341  GQPREPQ

348  VYTLPPSRDE

358  LTKNQVSLTC

368  LVKGFYPSDI

378  AVEWESNGQP

388  ENNYKTTPPV

398  LDSDGSFFLY

408  SKLTVDKSRW

418  QQGNVFSCSV

428  MHEALHNHYT

438  QKSLSLSPG

In the embodiment, the initial polypeptide of the present invention comprises the 282-340 position of CH2 domain at least Amino acid. In another embodiment, the initial polypeptide of the present invention comprises whole CH2 domain (according to EU Numbering, about 231-340 amino acids in antibody Fc district). In another embodiment, the present invention is initial Polypeptide comprises at least the CH2 domain and at least one hinge area (is numbered the antibody Fc district according to EU About 216-230 amino acids) and the CH3 domain (according to EU numbering, the antibody Fc district approximately The 341-446 amino acids). In the embodiment, the initial polypeptide of the present invention comprises CH2 and CH3 knot The structure territory. In the embodiment, the initial polypeptide of the present invention comprises hinge, CH2 and CH3 domain. In the embodiment, the initial polypeptide of the present invention comprises the sequence shown in the SEQ ID NO:2. The Fc district Or its FcRn bound fraction can derive from any isotype (comprising IgG1, IgG2, IgG3 and IgG4) Heavy chain. In the embodiment, use human homogeneous type IgG1.

The domain that consists of the Fc district of initial polypeptide can derive from different immunoglobulin molecules. For example, Polypeptide can comprise the CH2 domain that derives from the IgG1 molecule and derive from the hinge area of IgG3 molecule. In another example, initial polypeptide can comprise part and derive from the IgG1 molecule and partly derive from IgG3 Hinge area. In another example, initial polypeptide can comprise and partly derives from IgG1 molecule and part Derive from the chimeric hinge of IgG4 molecule. As above-mentioned, it will be appreciated by the skilled addressee that can To modify initial Fc domain (for example in the non-FcRn bound fraction of this molecule) thus so that it is at ammonia Be different from the natural antibody molecule on the base acid sequence.

The initial polypeptide of the present invention can comprise at least one Fc district or its FcRn bound fraction. Preferably The initial polypeptide of the present invention also comprises at least one binding structural domain, for example antigen binding structural domain, acceptor Binding structural domain or ligand binding domains. In the embodiment, initial polypeptide comprises at least one Binding structural domain and at least one Fc part. In the embodiment, initial polypeptide is by two combination knots Structure territory and two Fc partly form.

In the embodiment, the present invention initial polypeptide has at least one to the mediation biological effect Target molecule (for example, can be in conjunction with the part of cell surface receptor or cell surface that can binding partner Acceptor) the specific binding structural domain of tool, this binding structural domain with at least one Fc part with feminine gender Or positive signal passes to cell. In the embodiment, initial polypeptide have at least one to want Antigen (for example, cell surface antigen or soluble antigen) the tool specific knot of target to reduce or eliminate Close domain and at least one Fc district or its FcRn bound fraction.

A. antibody

In the embodiment, the initial polypeptide of the present invention is antibody. Use scheme known in the art, example As, preferably (for example, the Tumor-assaciated of purifying is anti-by subcutaneous or intraperitoneal injection related antigen repeatedly Former or contain cell or the cell extract of this antigen) and adjuvant in mammal, produce antibody. This is exempted from Epidemic disease typically causes the immunity that comprises that self-activating splenocyte or lymphocyte produce antigen reactivity antibody Reply.

Be in the embodiment of antibody at the polypeptide that contains Fc, antibody can be monoclonal antibody or many grams Grand antibody. The method for preparing monoclonal antibody known a period of time (see, for example, Kohler and Milstein, Nature, 256:495-497,1975), the equally also known immunoglobulin (Ig) of will encoding The technology that DNA stably introduces among the myeloma cell (is seen, such as Oi etc., Proc.Natl.Acad.Sci. USA80:6351-6355,1983). These technology comprise mutagenesis in vitro and DNA transfection, so that can Also can be for the preparation of the polypeptide that is used for the inventive method or thus many to make up recombination immunoglobulin The polypeptide (for example, therapeutic and diagnostic antibody) that peptide produces. Below further describe the preparation method, carry Body and host.

Initial polypeptide used among the present invention can be at non-human mammal, for example mouse, cavy, spirit Grow in class animal, rabbit or the rat by producing with antigen or this animal of its fragment immunity. See Harlow﹠ Lane, the same quoted passage is for all purposes are incorporated herein by reference. Although gained antibody can be from animal blood Gather in the crops to provide the polyclonal antibody goods in clear, but usually expect from spleen, lymph node or peripheral blood, to divide From monoclonal antibody (MAb) goods of single lymphocyte so that homogeneity to be provided. Typically use rabbit or cavy The preparation polyclonal antibody. Mouse is typically for the preparation of monoclonal antibody. Monoclonal antibody can for Fragment, by antigen fragment is injected in the mouse, preparation " hybridoma " and the acquisition of screening hybridoma be special The antibody of ground conjugated antigen is with preparation. This method of knowing (Kohler etc. (1975), Nature, 256: 495) in, will be from the relatively short-life of the mouse of having injected antigen or the lymphocyte that will certainly die and immortality Tumor cell line (for example, myeloma cell line) merge, produce thus immortality and can produce heredity Hybrid cell or " hybridoma " of the B cell antibody of coding. By selecting, dilute and again cultivating Contain each individual cells strain of the specific gene that forms monospecific antibody, become each single the gained hybrid separation The heredity strain. These hereditary strains produce the homogeneity antibody for expectation antigen, and this antibody is because its pure heredity The blood lineage is known as " monoclonal ".

The hybridoma that inoculation and cultivation prepare thus in appropriate media, described medium optimization Contain one or more and suppress parent myeloma cell's growth of not merging or the material of surviving. This area The technical staff understands, can obtain to be used to form, to select and cultivate from multiple channel by commercial sources The reagent of hybridoma, clone and culture medium, and ripe foundation of standard scheme. Usually, Analyze to cultivate the culture medium of hybridoma, with the generation of the monoclonal antibody that checks anti-expectation antigen. Preferably, the binding specificity of the monoclonal antibody of hybridoma generation is by immunoprecipitation or external Test, for example radiommunoassay (RIA) or enzyme linked immunosorbent assay (ELISA) (ELISA) are determined. Identify Generation can be passed through after having the hybridoma of the antibody of expecting specificity, affinity and/or activity The limiting dilution method is carried out subclone also by standard method (Goding, Monoclonal to these clones Antibodies:Principles and Practice, pp59-103 (Academic Press, 1986)) cultivate. Also Being appreciated that can be from culture medium, ascites or serum, by conventional purification process, for example, egg White matter A, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography are separated these subclones The monoclonal antibody of secretion.

Randomly, can select to be combined with the specific region of antigen or expectation fragment but not with its of antigen The antibody of its non-overlapping fragment combination. This rear selection can be by measuring one of antibody and antigen Combination and definite which kind of deletion mutant of Lieque mistake mutant are combined to realize with antibody. In conjunction with can Estimate by for example Western trace or ELISA. Demonstrate the minimal segment with the antibody specific bond Be defined as the epi-position of this antibody. Perhaps, can utilize competition experiments to determine epitope specificity, in competition Measured antibody and be combined with antigen with reference to antibody competition in the test. When measured antibody with reference to antibody competition The time, then the epi-position of their institute's combinations is identical, perhaps fully approaches the consequently combination of an antibody and disturbs in addition The combination of one antibody.

In another embodiment, the DNA of coding expectation monoclonal antibody can easily use routine side Method (for example, by use can be specifically in conjunction with the gene of the heavy chain of coding mouse source antibody and light chain Oligonucleotide probe) separation and order-checking. That separate and hybridoma subclone are this DNA Preferred source. In case after separating, this DNA can put into expression vector, turns to expression vector then Dye protokaryon or eukaryotic host cell, for example Escherichia coli (E.coli) cell, ape COS cell, Chinese storehouse Mouse ovary (CHO) cell or before this carrier transfection, do not produce the myeloma cell of immunoglobulin (Ig). More special , can not use DNA (can synthesize as described herein) clone's constant region of this separation Equal the U.S. Patent number submitted to January 25 nineteen ninety-five with variable region sequences so that according to Newman 5,658,570 described Dispersal risks, the document is incorporated into herein as a reference. Basically, this need to be from choosing Extract RNA in the cell of selecting, change into cDNA and use the Ig primer amplified. Be applicable to The primer of this purpose also is described in U.S. Patent number 5,658, in 570. Just as discussed in more detail below, The transformant of expression expectation antibody can be cultured to relatively a large amount of, thereby clinical and commercial immunity is provided The globulin supply.

Those skilled in the art also will understand, encoding antibody or antibody fragment (for example antigen binding site) DNA also can derive from the antibody phage library, for example use pd bacteriophage or Fd phasmid skill Art. Illustrative methods is seen for example EP 368 684 B1; United States Patent (USP) 5,969,108, Hoogenboom, H.R. with Chames.2000.Immunol.Today 21:371; The 2002.Nat.Med. such as Nagy 8:801; The 2001.Proc.Natl.Acad.Sci.USA 98:2682 such as Huie; The 2002.J. such as Lui Mol.Biol.315:1063, all these documents are all incorporated into herein as a reference. Several parts of publication (examples As, Marks etc., Bio/Technology 10:779-783 (1992)) described by chain reorganization, Combination is infected and the interior restructuring of body (making up the strategy of big phage library) produces high affinity human antibody. In another embodiment, can use ribosomal display replace bacteriophage as display platform (see, for example, The 2000.Nat.Biotechnol.18:1287 such as Hanes; The 2001.Proc.Natl. such as Wilson Acad.Sci.USA 98:3750; Or the 2001 J.Immunol.Methods 248:31 such as Irving). In an embodiment again, can screen the cell surface library to obtain antibody (Boder etc., 2000, Proc. Natl.Acad.Sci.USA 97:10701; Daugherty etc., 2000 J.Immunol.Methods 243: 211). These methods provide and can replace conventional hybridization knurl technology for separating of with anti-with the rear clone monoclonal The alternative technique of body.

Other embodiment of the present invention is included in the transgenic animals that can not produce endogenous immunoglobulin Produce in (for example mouse) people's antibody or basically people's antibody (for example see U.S. Patent number 6,075,181, 5,939,598,5,591,669 and 5,589,369, all are all incorporated into herein as a reference). For example, Described, homozygous deletion heavy chain of antibody bonding pad will cause pressing down fully in chimeric germ line mutation mouse The generation of endogenous antibody processed. Human immunoglobulin gene's series is transferred in this germ line mutation mouse To cause after antigen is attacked, producing people's antibody. Another of use SCID mouse generation people antibody be the side preferably Method is disclosed in United States Patent (USP) 5,811, and in 524, this document is incorporated into herein as a reference. Significantly, with this The relevant inhereditary material of a little people's antibody also can separate and operates by described herein.

Another height effective method of preparation recombinant antibodies is disclosed in Newman, Biotechnology, 10:1455-1460 (1992). Particularly, this technology causes producing primateization antibody, this antibody Contain monkey variable region and human constant region. This list of references is intactly incorporated into herein as a reference. In addition, This technology also is described in the U.S. Patent number 5,658,570,5,693,780 and 5,756,096 of common transfer In, all these patents are incorporated into herein as a reference.

In another embodiment, can select lymphocyte and separate variable gene by micromanipulation. For example, can be from the mammal of immunity separating peripheral blood mononuclear cells, and in vitro culture about 7 My god. The special IgG that can the screening and culturing thing meets screening criteria with detection. Separation is thin from positive hole Born of the same parents. Can utilize FACS or divide the B cell by in the hemolytic plaque assay of complement-mediated, identifying B cell from each single generation Ig. Can will produce by micromanipulation the B cell adding examination of IgG In the pipe, and can use for example RT-PCR amplification V_HAnd V_LGene. Can be with V_HAnd V_LGene Be cloned in the antibody expression vector and transfectional cell (for example eucaryon or prokaryotic) in order to express.

In addition, can also obtain to can be used for producing from multiple separate sources the genetic sequence of polypeptide of the present invention. For example, such as above a large amount of discussion, can obtain the various mankind with the obtainable preservation thing of public form Antibody gene. Many antibody sequences and antibody coding gene announce, and can be from these sequences Utilize the suitable antibody gene of technology chemical synthesis known in the art. With the present invention's fit in this respect The oligonucleotides synthetic technology be well known to those skilled in the art, and can use several commerce from Moving synthesizer is implemented. In addition, can by the synthetic merchant's of commercial DNA service, obtain coding herein The dna sequence dna of described a few class heavy chain and light chain. The inhereditary material that uses aforementioned any method to obtain is right After can change or synthetic to provide or to obtain polypeptide of the present invention.

Variable domains and constant domain can separately be cloned, for example use the PCR and The primer of selecting to be used for amplification purpose domain is cloned. In addition, many antibody variables territories and The sequence of constant domain is known, and these domains can use method well known in the art synthetic. For example, can select to have the specific effect subfunction (or lack specific effect subfunction) or have specific repairing Be decorated with and reduce immunogenic constant region domain. Perhaps, can be from the variable base from selected animal Because of the sequence library, obtain variable domains. Can express the structure of random combine with the expectation antigen selection Territory (for example, V_HAnd V_LDomain) library has the element in conjunction with feature of expectation with evaluation. This type of screening technique is well known in the art. For example, can be with all antibody gene constituent clones To bacteriophage lambda expression vector (Huse, WD etc. (1989), Science, 2476:1275). In addition, can Screen the from the teeth outwards cell of expressing antibodies (Francisco etc. (1994), PNAS, 90:10444; Georgiou etc. (1997), Nat.Biotech., 15:29; Boder and Wittrup (1997) Nat. Biotechnol.15:553; Boder etc. (2000), PNAS, 97:10701; Daugtherty, P. etc. (2000) J.Immunol.Methods.243:211) or virus (for example, Hoogenboom, HR. (1998), Immunotechnology 4:1; Winter etc. (1994) .Annu.Rev.Immunol.12:433; Griffiths, AD. (1998) .Curr.Opin.Biotechnol.9:102). Those skilled in the art are also clear, The DNA of encoding antibody domain also can derive from the antibody phage library, for example uses the pd phagocytosis Body or Fd phasmid technology. Illustrative methods is referring to for example, EP 368 684 B1; U.S.Pat.No. 5,969,108; Hoogenboom etc. (2000) Immunol.Today 21:371; Nagy etc. (2002) Nat.Med.8:801; Huie etc. (2001), PNAS, 98:2682; Lui etc. (2002), J.Mol.Biol. 315:1063, all these documents are all incorporated into herein as a reference. Several parts of publication (for example, Marks Deng, Bio/Technology 10:779-783 (1992)) described by chain reorganization, combination infect and The restructuring conduct makes up a kind of strategy of big phage library to produce high affinity human antibody in the body. Another In the embodiment, can use ribosomal display replace bacteriophage as display platform (see, for example, Hanes, Deng (1998), PNAS 95:14130; Hanes and Pluckthun. (1999), Curr.Top.Microbiol. Immunol.243:107; He and Taussig. (1997), Nuc.Acids Res., 25:5132; Hanes etc. (2000), Nat.Biotechnol.18:1287; Wilson etc. (2001), PNAS, 98:3750; Or Irving etc. (2001) J.Immunol.Methods 248:31).

The preferred library that is used for screening is people's variable gene library. Also can use from inhuman source V_LAnd V_HDomain. The library can be inmature, come from immunity the experimenter's or semi-synthetic (Hoogenboom and Winter. (1992) .J.Mol.Biol.227:381; Griffiths etc. (1995) EMBO J.13:3245; (1995) .J.Mol.Biol.248:97 such as de Kruif; Barbas etc. (1992), PNAS, 89:4457). In one embodiment, can suddenly change immunoglobulin domains to produce Nucleic acid molecules library (Thompson etc. (1996), J.Mol.Biol.256:77 with bigger heterogeneity; Lamminmaki etc. (1999), J.Mol.Biol.291:589; Caldwell and Joyce. (1992), PCR Methods Appl.2:28; Caldwell and Joyce. (1994), PCR Methods Appl.3:S136). Can select the high-affinity variant by the Application standard screening technique. In another embodiment, can, for example Use utilizes technology known in the art from the information of crystal structure, changes V_HAnd V_LSequence is to increase Add antibody affinity.

Perhaps, can use technology well known to those skilled in the art, selection and cultivation generation antibody Clone. These technical descriptions are in various laboratory manuals and main publication. In this regard, as Followingly be applicable to technical description of the present invention at Current Protocols in Immunology, Coligan etc., Eds., Green Publishing Associates and Wiley-Interscience, John Wiley and Sons, New York (1991), this document intactly (comprises appendix) and incorporates into herein as a reference.

In addition, significantly, the scope of the invention comprises that also antigen is in conjunction with all allele of dna sequence dna Variant, variant and mutant.

As the well-known, can be from initial hybridoma or from the cell of other conversion, by mark Accurate technology, for example guanidinium isothiocyanate extracts and precipitation reaches subsequently centrifugal or chromatography, isolation of RNA. Current During prestige, can from total RNA, pass through for example chromatography on oligo dT cellulose of standard technique, separate MRNA. Suitable technology is well known in the art.

In the embodiment, can use reverse transcriptase and archaeal dna polymerase, according to well-known process, Simultaneously or prepare dividually the cDNA of encoding antibody light chain and heavy chain. Can utilize total constant region primer Perhaps utilize based on the heavy chain of publishing and the more special primer of light chain DNA and amino acid sequence, start PCR. As discussed above, can also use PCR to separate the dna clone of encoding antibody light chain and heavy chain. In the case, can utilize total primer or bigger homologous probe, for example the mouse constant region is visited Pin, the screening library.

Can use technology known in the art DNA isolation from cell, plasmid DNA typically, and The technology of knowing according to standard (elaborates at for example relevant with recombinant DNA technology above-mentioned reference literary composition In offering) carry out restricted mapping and order-checking. Certainly, can be according to the present invention at separation method or subsequently In the process of analyzing on any some synthetic DNA. In many situations, reported in the document for The immunoreactivity antibody of each of these antigens.

In another embodiment, the combination of initial polypeptide and antibody causes antigen from for example tissue or certainly follows Reduce or eliminate in the ring. In another embodiment, initial polypeptide has at least one to antigen-specific Binding structural domain, it can be for detection of (for example, detection of contamination or the diagnosis disease of existing of target molecule Disease or illness). In an embodiment again, the initial polypeptide of the present invention comprises at least one binding site, This binding site makes specific site among this molecular targeted experimenter (for example target tumor cell or blood clotting Piece).

In the embodiment, the initial polypeptide of the present invention can be exempted from one or more antigen related antigens The epidemic disease reaction. For example, in order to treat cancer or neoplasia, the antigen binding structural domain of polypeptide preferably with choosing Fixed tumor associated antigen combination. Consider number and the phase of the antigen reported relevant with neoplasia Close the number of antibody, it is multiple complete anti-that those skilled in the art will understand that polypeptide of the present invention can derive from Any in the body. More generally, in the present invention useful initial antibody can available from or come Come from and anyly can (comprise in the document first with the antibody relevant with selected situation or the antibody of labeled reactant Those antibody of having reported). In addition, for generation of the initial antibody of polypeptide disclosed in this invention or its Fragment can be mouse, the people, chimeric, humanized, non-human primate or primate Animalization. The exemplary oncologic related antigen of used initial antibody combination among the present invention for example comprises, Pan B antigen is (for example, at pernicious and non-malignant B cell (for example B cell in the non-Hodgkin's lymphoma) The CD20 that finds on the surface) and pan T cellular antigens (for example, CD2, CD3, CD5, CD6, CD7). Other exemplary oncologic related antigen includes but not limited to MAGE-1, MAGE-3, and MUC-1, HPV 16, HPV E6﹠E7, TAG-72, CEA, α-Lewis^y, L6-antigen, CD19, CD22, CD25, CD30, CD33, CD37, CD44, CD52, CD56, mesothelin, PSMA, HLA-DR, the EGF acceptor, Vegf receptor and HER2 acceptor.

The antibody with the tumor associated antigen reaction of report can be by described change herein to provide in the past The polypeptide of change of the present invention. Can comprise with the exemplary target antibody of tumor associated antigen reaction: 2B8, Lym1, Lym2, LL2, Her2, B1, BR96, MB1, BH3, B4, B72.3,5E8, B3F6,5E10, α-CD33, α-CanAg, α-CD56, α-CD44v6, α-Lewis, and α-CD30.

More particularly, exemplary target antibody includes but not limited to 2B8 and C2B8 (Zevalin^And Rituxan^, IDEC Pharmaceuticals Corp., San Diego), Lym1 and Lym2 (Techniclone), LL2 (Immunomedics Corp., New Jersey), Trastuzumab (Herceptin^，Genentech Inc.，South San Francisco)，Tositumomab(Bexxar ^， Coulter Pharm.，San Francisco)，Alemtzumab(Campath ^，Millennium Pharmaceuticals，Cambridge)，Gemtuzumab ozogamicin(Mylotarg ^， Wyeth-Ayerst，Philadelphia)，Cetuximab(Erbitux ^，Imclone Systems，New York)， Bevacizumab(Avastin ^, Genentech Inc., South San Francisco), BR96, BL22, LMB9, LMB2, MB1, BH3, B4, B72.3 (Cytogen Corp.), SS1 (NeoPharm), CC49 (National Cancer Institute), Cantuzumab mertansine (ImmunoGen, Cambridge), MNL 2704 (Milleneum Pharmaceuticals, Cambridge), Bivatuzumab mertansine (Boehringer Ingelheim, Germany), Trastuzumab-DM1 (Genentech, South San Francisco), My9-6-DM1 (ImmunoGen, Cabridge), SGN-10 ,-15 ,-25 and-35 (Seattle Genetics, Seattle) and 5E10 (University of Iowa). In preferred embodiments, The initial antibody of the present invention and the antibody of tightly enumerating above are in conjunction with identical tumor associated antigen. Especially excellent The choosing embodiment in, polypeptide derives from Y2B8, C2B8, CC49 and C5E10 or and Y2B8, C2B8, CC49 and C5E10 be in conjunction with same antigen, even more preferably, comprise the anti-of domain disappearance Body (that is, Δ CH2 antibody).

In first preferred embodiment, initial antibody and Rituxan^In conjunction with identical tumor associated antigen. Rituxan^(being also referred to as rituximab, IDEC-C2B8 and C2B8) is that first FDA approval is used for controlling Treat the lymphadenomatous monoclonal antibody of human B cell and (see U.S. Patent number 5,843,439; 5,776,456 Hes 5,736,137, all incorporate into herein as a reference). Y2B8 (the 2B8 of 90Y mark; Zevalin^ Ibritumomab tiuxetan) is the mouse source starting material of C2B8. Rituxan^It is chimeric anti-CD20 list Clonal antibody, it has growth inhibition and it is reported can some lymphoma cell line of external sensitization The cells apoptosis susceptible that it is caused chemotherapeutics. This antibody in conjunction with people's complement, has effectively Strong FcR combination can be passed through complement-dependent (CDC) and antibody dependent (ADCC) mechanism at body Effectively kill and wound human lymphocyte (Reff etc., Blood 83:435-445 (1994)) outward. Art technology people The member understands, C2B8 or the 2B8 dimer variant (homodimer or heterodimer) synthetic according to the disclosure Can partly put together according to the inventive method and effector, pernicious swollen to be provided at treatment performance CD20+ Have during the patient of knurl even the modified antibodies of bigger effect.

In other preferred embodiment of the present invention, the initial polypeptide of the present invention derive from CC49 or with CC49 is in conjunction with identical tumor associated antigen. CC49 is in conjunction with people's tumor associated antigen TAG-72, this antigen Some tumour cell (especially LS174T tumor cell line) surface conjunction with human origin. LS174T[American type culture collection (ATCC herein) numbering CL188] be The variant of LS180 (ATCC No.CL187) adenocarcinoma of colon system.

In addition, also understand, developed many mouse source Dan Ke that TAG-72 had binding specificity Grand antibody. Wherein a kind of monoclonal antibody is called B72.3, and it is by hybridoma B72.3 (ATCC No. HB-8108) mouse IgG 1 that produces. B72.3 is that end user's breast cancer extract is developed as immunogene First generation monoclonal antibody (see Colcher etc., Proc.Natl.Acad.Sci. (USA), 78: 3199-3203 (1981); With U.S. Patent number 4,522,918 and 4,612,282, all documents are all incorporated into Herein as a reference). The monoclonal antibody of other anti-TAG-72 is called " CC " (for colon cancer). As Schlom etc. (U.S. Patent number 5,512,443 is incorporated into herein as a reference) are described, and the CC monoclonal is to make Use the second generation mouse resource monoclonal family by means of the TAG-72 preparation of B72.3 purifying. Because they are right The binding affinity that TAG-72 is relatively good, in the ATCC preservation following CC antibody, these antibody Lower with limitation acquisition: CC49 (ATCC No.HB 9459) can required; CC 83 (ATCC No. HB 9453); CC46 (ATCC No.HB 9458); CC92 (ATTCC No.HB 9454); CC30 (ATCC No.HB 9457); CC11 (ATCC No.9455); And CC15 (ATCC No.HB 9460). U.S.P.N.5,512,443 also instructions can be used by recombinant DNA technology known in the art For example human constant region (Fc) domain is replaced the mouse constant region, and these disclosed antibody are changed over chimeric shape Formula. Except disclosing mouse and chimeric anti-TAG-72 antibody, Schlom etc. have also prepared the people source (it is special to be disclosed in the U.S. to change CC49 antibody variants (being disclosed among the PCT/US00/25552) and strand construct In sharp number 5,892,019), all these documents are also incorporated into herein as a reference. Those skilled in the art To understand, all aforementioned antibody, construct or recombinant and variant thereof all can be synthesize and be used for Provide according to polypeptide of the present invention.

Except anti-TAG-72 antibody discussed above, different groups have also reported the disappearance domain The structure of CC49 and B72.3 antibody and part sign (for example, the Cancer Biotherapy such as Calvo, 8 (1): 95-109 (1993), the Int.J.Cancer 53:97-103 (1993) such as Slavin-Chiorini and The Cancer.Res.55:5957-5967 such as Slavin-Chiorini (1995).

In the embodiment, the initial polypeptide of the present invention and CD23 (United States Patent (USP) 6,011,138) combination. In a preferred embodiment, the initial polypeptide of the present invention and 5E8 antibody are in conjunction with identical epi-position. Another In the embodiment, the initial polypeptide of the present invention comprises at least one, and (for example 5E8 is anti-from anti-CD23 antibody Body) CDR.

In a preferred embodiment, the initial antibody of the present invention and CRIPTO-I antigen (WO 02/088170A2 or WO03/083041A2) combination. In a preferred embodiment, this Bright polypeptide and B3F6 antibody are in conjunction with identical epi-position. In an embodiment again, polypeptide of the present invention comprise to Lack one from anti-CRIPTO-I antibody, for example CDR of B3F6 antibody.

Other embodiment of the present invention comprise derive from C5E10 or with C5E10 in conjunction with identical tumour phase Close the modified antibodies of antigen. Such as what set forth in common application 09/104,717 co-pending, C5E10 knows The antibody of not about 115kDa glycoprotein determinant, seemingly tumor of prostate is thin for this glycoprotein determinant Born of the same parents systems (for example, DU145, PC3 or ND1) are special. Therefore, with the present invention's associating, specifically Can use individually in conjunction with the polypeptide of identical tumor associated antigen with C5E10 antibody or by this Bright method and effector are partly puted together, and repairing of the treatment that can be used for improving the neoplasia disease is provided thus The decorations polypeptide. In especially preferred embodiment, initial polypeptide derives from has the ATCC preserving number The C5E10 antibody that the hybridoma cell line of PTA-865 is secreted or comprise the institute of this C5E10 antibody Have or the incomplete antigen land. The gained polypeptide then can be according to method and therapeutic effects herein Subdivision (seeing following) is puted together and is administered to the patient who suffers from prostate cancer.

B. antibody variants

Except natural antibody, the initial antibody of the present invention can also comprise non-natural immunoreactivity sheet Section or part.

In another embodiment, the weight chain variable of the antigen binding structural domain of target antibody of the present invention part and Light chain variable partly is present in the same polypeptide, for example is present in single-chain antibody (ScFv) or miniantibody (minibody) (see for example the US patent No. 5,837,821 or WO 94/09817A1). Miniantibody is By the dimer molecule that two polypeptide chains form, every polypeptide chain contains a ScFv molecule and (comprises one The single polypeptide of individual or a plurality of antigen binding sites, for example, by flexible joint with utilize is connected peptide with The V that the CH3 domain merges_HThe V that domain connects_LDomain). The ScFv molecule can be with V_H-joint-V_LDirection or V_L-joint-V_HDirection makes up. Connect and compose the V of antigen binding site_LAnd V_HStructure The flexible hinge in territory preferably comprises about 10 to about 50 amino acid residues. Be used for showing of this purpose Example connection peptide is (Gly4Ser) 3 (Huston etc. (1988), PNAS, 85:5879). Other connects peptide Well known in the art.

The method for preparing single-chain antibody is well known in the art, such as (1989) such as Ho, and Gene, 77:51; Bird etc. (1988), Science 242:423; Pantoliano etc. (1991), Biochemistry 30:10117; Milenic etc. (1991), Cancer Research, 51:6363; Takkinen etc. (1991), Protein Engineering 4:837. Miniantibody can (be seen, for example U.S. by the method for using this area to describe State's patent 5,837,821 or WO94/09817A1) make up the ScFv composition and connect peptide-CH₃Composition and making Standby. These compositions can separate from different plasmids with the form of restriction fragment, connect also again then Be cloned in the appropriate carrier. Can utilize the correct assembling of restrictive diges-tion and dna sequence analysis checking. In the embodiment, miniantibody of the present invention comprises the connection peptide. In the embodiment, this connects Connect peptide and comprise the Gly/Ser joint, for example, GGGSSGGGSGG.

In another embodiment, can make up the tetravalence miniantibody. Except (for example, using flexible joint Has amino acid sequence (G4S)₄G3AS) connect outside two ScFv molecules, the tetravalence miniantibody can by The mode identical with miniantibody makes up.

In another embodiment, the initial antibody of the present invention comprises double-chain antibody (diabody). Double-chain antibody with The scFv molecular mimicry connects but usually have short (being less than 10, preferably 1-5) amino acid residue Head connects two variable domains, so that the V on same polypeptide chain_LAnd V_HDomain can not take place Interact. On the contrary, the V of a polypeptide chain_LAnd V_HV on domain and second polypeptide chain_HAnd V_LDomain (respectively) interaction (WO 02/02781).

In another embodiment, the initial antibody of the present invention comprises operationally and is connected with the FcR bound fraction The immunoreactivity fragment or its part (for example, scFv molecule, miniantibody, tetravalence miniantibody or Double-chain antibody). In an exemplary, the FcR bound fraction is whole Fc district.

In another embodiment, at least one antigen binding structural domain tool catalytic of initial antibody (Shokat and Schultz. (1990) .Annu.Rev.Immunol.8:335). Has catalytic in conjunction with special The property the antigen binding structural domain can use technology known in the art preparation (for example to see U.S. Patent number 6,590,080, U.S. Patent number 5,658,753). The catalytic binding specificity can by many with for Enzyme and identify be used for stablize those machine-processed similar basic machines that transition state reduces free energy of activation thus System works. For example, can place best general acidity and alkaline residue lives in order to participate in catalysis Catalytic reaction in the property site; Can form the enzyme-substrate intermediate of covalency; Catalytic antibody also can tool Have reacting suitable orientation and increasing at least 7 order of magnitude (Fersht etc. of reactant valid density (1968), J.Am.Chem.Soc.90:5833) also greatly reduce thus the entropy of chemical reaction. At last, Catalytic antibody can conversion of substrate in conjunction with the time energy that obtains so that reactant to the knot that is similar to transition state The structure distortion.

Can be by use with the molecule of complementary electric charge as immunogene, so that acidity or alkaline residue are drawn Enter in the antigen binding site. This technology has been proved successfully to utilize and has contained positively charged ammonium The haptens initiation antibody of ion (Shokat etc., (1988), Chem.Int.Ed.Engl.27:269-271). In other method, can cause antibody is similar to the transition state of expected response with Stabilized size, shape The compound of shape and electric charge (that is, transition state analog). See U.S. Patent number 4,792,446 and the U.S. special Sharp number 4,963,355, these two parts of patents have been described application transition state analog immune animal and have been produced catalysis Antibody. These two parts of patents are incorporated herein by reference hereby. These molecules can be used as one of immunoconjugates Part, for example with the immunogenic carrier molecule, for example KLH uses together.

In the embodiment, the initial antibody of the present invention is two special. Two special moleculars can be with two Individual different target site, for example on the identical target molecule or different target molecules on the target site combination. For example, In the situation of antibody, two special moleculars can with for example same antigen or two two on the synantigen not Individual different epi-position combination. Two special moleculars can be used for for example diagnosing and treating application. For example, they Can be with immobilized enzyme in immunoassays, to use. They are all right, for example by with the Tumor-assaciated branch Son and detectable label (for example closely the chelating agent of binding radioactivity nucleic) combination are used for diagnosing and controlling Treat cancer. Two special moleculars are all right, for example (for example pass through by making cytotoxicity point to special target With pathogen or tumour cell and cytotoxicity triggering molecule, for example φt cell receptor combination), be used for the people The class treatment. Two special moleculars can also be as for example fibrinolytic agent or vaccine adjuvant.

The example of two specific bond molecules comprises having at least two two for the arm of tumor-cell antigen The specific bond molecule; Has at least one for the arm of tumor-cell antigen and at least one is for cell Toxicity trigger the arm of molecule two specific bond molecules (for example the anti-malignant B cell of anti-CD3/ (1D10), resist The anti-p185.sup.HER2 of CD3/, the anti-p97 of anti-CD3/, the anti-clear-cell carcinoma of anti-CD3/, anti-CD3/ resist OVCAR-3, the anti-melanotropin analog of anti-CD3/L-D1 (inhibitor against colon carcinoma cells), anti-CD3/, anti-EGF Acceptor/anti-CD3, the anti-CAMA1 of anti-CD3/, the anti-CD19 of anti-CD3/, anti-CD3/MoV18, anti-N-CAM (NCAM)/anti-CD3, anti-FABP (FBP)/anti-CD3, anti-general Cancer associated antigen (anti-pan carcinoma associated antigen) (AMOC-31)/anti-CD3); Have At least one arm of being combined with tumour antigen specifically and at least one arm of being combined with toxin two special Binding molecule (for example, anti-saporin/anti-Id-1, the anti-saporin of anti-CD22/, the anti-saporin of anti-CD7/, anti-The anti-saporin of CD38/, the anti-ricin A of anti-CEA/ chain, anti-interferon alpha (IFN-α)/anti-hybridoma is only Special type, the anti-vinca alkaloids of anti-CEA/); The two specific bond molecules that are used for the prodrug of invertase activation (for example, (this enzymatic mitomycin phosphate prodrug changes into silk and splits mould anti-CD30/ alkali resistance phosphatase Plain alcohol)); Can be as two specific bond molecules of fibrinolytic agent (for example, antifibrin/resist Tissue plasminogen activator (tPA), the former activator of antifibrin/antiurokinase type fibrinolysin (uPA)); Make immune complex be oriented to two specific bond molecules (anti-low-density lipoprotein for example of cell surface receptor (LDL)); Two specific bond molecules (for example, the anti-CD3/ anti-herpes simplex that is used for the treatment of infectious diseases Virus (HSV), anti-φt cell receptor: CD3 compound/resisiting influenza virus, the anti-HIV of anti-Fc γ R/); With In external or body, detect tumour two specific bond molecules (for example the anti-EOTUBE of anti-CEA/, resist The anti-DPTA of CEA/, anti-p185HER2/ antihapten); Two specific bond molecules as vaccine adjuvant (are seen Fanger etc., the same quoted passage); With as two specific bond molecules of diagnostic tool (anti-rabbit igg/anti-for example Ferritin, anti-horseradish peroxidase (HRP)/antihormones, anti-somatostatin/anti-Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2, The anti-FITC of anti-HRP/, the anti-beta galactosidase of anti-CEA/ (seeing Nolan etc., the same quoted passage)). Three spies The example of xenoantibody comprises the anti-CD37 of the anti-CD4/ of anti-CD3/, the anti-CD37 of the anti-CD5/ of anti-CD3/ and anti-The anti-CD37 of the anti-CD8/ of CD3/.

In the preferred embodiment, the two special moleculars of the present invention are combined with CRIPTO-1.

Two special moleculars can be unit prices for every species specificity, perhaps can be to every species specificity It is multivalence. For example, antibody molecule or fusion can comprise one with first target molecule anti-The binding site of answering and a binding site that reacts with second target molecule, perhaps it can comprise two Binding site and two binding sites that react with second target molecule with the reaction of first target molecule. Preparation The method of two special moleculars is well known in the art. For example, it is two special to use recombinant technique to produce Molecule. Exemplary techniques for generation of two special moleculars is (for example, Kontermann known in the art Deng, Methods in Molecular Biology Vol.248: antibody engineering: method and scheme (Antibody Engineering:Methods and Protocols) .Pp 227-242 US 2003/0207346 A1 and The list of references of wherein quoting). In the embodiment, use such as at US 2003/0207346A1 With the method for describing among US patent 5,821,333 and the US2004/0058400, the preparation polymer is two special Molecule.

Phrase " how special fusion " refers to have at least two binding specificities and (that is, has made up herein The binding structural domain of two or more parts or acceptor) fusion (seeing above definition). Many Special fusion can be assembled into heterodimer, heterotrimer or the different tetramer, basically can join See WO89/02922 (on April 6th, 1989 open), EP 314,317 (announcement on May 3rd, 1989), U.S. Patent number 5,116, disclosing in 964 (on May 2nd, 1992 authorized). Preferred how special fusion egg Two special in vain. The example of bispecific fusion protein comprise CD4-IgG/TNF acceptor-IgG and CD4-IgG/L-selects albumen-IgG. The molecule of mentioning at last united lymphocyte homing receptor (LHR, L-selects albumen) the lymph node binding function and the HIV binding function of CD4, can be potentially be used for pre-Anti-or treatment HIV infection, associated conditions or as diagnosticum.

The target binding site of many specific bond of the present invention molecule can be easily by the ordinary skill people The member selects. Limit never in any form, the exemplary combination site comprise a kind of of tumour antigen or Multiple epi-position. Other exemplary target molecule comprises that for example heparin sulfate, growth factor or its acceptor are (for example EGF-R ELISA, IGF-1, HGF (HGF/SF) are subjected to One or more epi-positions body) (see, for example, the Proc.Natl.Acad.Sci 2001.98:7443 such as Cao; The 2004.J.Biol.Chem.279:2856 such as Lu).

In another embodiment, the antigen binding structural domain of initial antibody is by no V_LThere is lower stable V in chain_HDomain for example derives from the V of camelids_HDomain forms (Hamers-Casterman etc. (1993) .Nature, 363:446; Desmyter etc. (1996) .Nat.Struct.Biol.3:803; Decanniere etc. (1999) .Structure, 7:361; Davies etc. (1996) .Protein Eng., 9:531; Kortt etc. (1995) .J.Protein Chem., 14:167).

Technology known in the art be can use, inhuman initial antibody or its fragment or domain changed, To reduce its immunogenicity. Humanized initial polypeptide is the initial polypeptide that derives from non-human protein's matter, This polypeptide has kept or has basically kept the character of initial antibody, but has lower in human body Immunogenicity. For the initial antibody of humanization, can realize by the whole bag of tricks, comprise that (a) is with complete Inhuman variable domains grafting on human constant region to produce chimeric target antibody; (b) with one or more non-At least a portion grafting of people's complementary determining region (CDR) is on people's framework region and constant region and keep and not Keep crucial framework residue; (c) transplant complete inhuman variable domains, but by the displacement surface Residue carries out " covering up " with the proper manners part to it. These methods are disclosed in (1984) such as Morrison, PNAS.81:6851-5; Morrison etc., (1988), Adv.Immunol.44:65-92; Verhoeyen etc., (1988), Science 239:1534-1536; Padlan, (1991), Molec.Immun.28:489-498; Padlan, (1994), Molec.Immun.31:169-217; And U.S.Pat.Nos.5,585,089, 5,693,761 and 5,693,762, all these documents all intactly are incorporated herein by reference hereby.

Also can make and spend the immunogenicity that immunization reduces initial antibody. Herein, term " goes to exempt from Epidemic disease " comprise change antibody with modify t cell epitope (see, WO9852976A1 for example, WO0034317A2). For example, analysis is from the V of initial antibody_HAnd V_LSequence " is done from each V district Figure " human T-cell's epi-position, thereby show epi-position in this sequence with respect to complementary determining region (CDR) and The position of its Key residues. Each single t cell epitope of analyzing among the t cell epitope figure is minimum to identify The optional amino acid that risk causes antibody activity to change substitutes. Design a series of optional V_HAnd V_LOrder Row mix these sequences in a series of polypeptide of the present invention subsequently to comprise the amino acid replacement combination, with Carry out functional check. Usually, preparation and determination methods 12-24 variant antibody. Afterwards, will comprise modification V and complete heavy chain and the light chain gene in people C district be cloned in the expression vector, the gained plasmid is introduced Clone is to produce complete antibody. In suitable biochemistry and biological test, compare these then Antibody is identified best variant.

In the embodiment, initial polypeptide comprises chimeric antibody. In the application's context, term " embedding Close antibody " refer to immunoreactivity zone or site available from or from first species and constant region (can be Whole, part or modify according to the present invention) available from any antibody of second species. In preferred enforcement In the scheme, target calmodulin binding domain CaM or site are from inhuman source (for example mouse), and constant region is the people. Preferably Ground is by replacing at least in part one or more CDR also where necessary by partly replacing framework District and change sequence change the heavy chain of target antibody and the variable domains in the light chain. Although CDR comes The antibody in source can be with the target antibody that framework region is originated same type or or even identical subclass, but Can expect CDR from dissimilar antibody, and preferably from the antibody of different plant species. For The antigen binding capacity of a variable domains is transferred to another variable domains, with all CDR Being replaced as may from whole CDR of donor variable region. On the contrary, may only need to turn to Move for active necessary those residues of keeping binding structural domain. Consider U.S. Patent number The explanation of setting forth in 5,585,089,5,693,761 and 5,693,762 is by implementing routine test or logical Cross the immunogenic functional antibodies that hit and miss experiment obtains to have reduction, fully the art technology people In member's the limit of power.

In preferred embodiments, the initial polypeptide of the present invention does not cause harmful immune response in human body. Those skilled in the art will understand, also can prepare chimeric initial polypeptide. In this application, term " embedding Close beginning antibody " refer to immunoreactivity zone or site available from or from first species and constant region is (passable Be complete, part or modify according to the present invention) available from any initial antibody of second species. In the preferred embodiment, target calmodulin binding domain CaM or site are from inhuman source (for example mouse), and constant region is the people . Although the immunogene specificity of variable region is not subjected to the impact in its source usually, human constant region recently Constant region from inhuman source has the littler possibility that causes immune response in people experimenter.

C. fusion

The initial polypeptide of the present invention also can be the fusion that comprises at least the FcRn bound fraction in Fc district. Preferably, fusion of the present invention comprises binding structural domain (comprising at least one binding site). The present invention Fusion can be two specially (to have for binding site of first target with for second target Target second binding site), perhaps can be (the having two binding sites for same target) of multivalence.

The example of the fusion of reporting in the document comprises φt cell receptor (Gascoigne etc., Proc.Natl. Acad.Sci.USA 84:2936-2940 (1987)); CD4 (Capon etc., Nature 337:525-531 (1989); Traunecker etc., Nature 339:68-70 (1989); Zettmeissl etc., DNA Cell Biol. USA 9:347-353 (1990); With Byrn etc., Nature 344:667-670 (1990)); L-selects albumen (homing receptor) (Watson etc., J.Cell.Biol.110:2221-2229 (1990); With Watson etc., Nature 349:164-167 (1991)); CD44 (Aruffo etc., Cell 61:1303-1313 (1990)); CD28 and B7 (Linsley etc., J.Exp.Med.173:721-730 (1991)); CTLA-4 (Lisley etc., J.Exp.Med.174:561-569 (1991)); CD22 (Stamenkovic etc., Cell 66:1133-1144 (1991)); TNF acceptor (Ashkenazi etc., Proc.Natl.Acad.Sci.USA 88:10535-10539 (1991); Lesslauer etc., Eur.J.Immunol.27:2883-2886 (1991); With Peppel etc., J. Exp.Med.174:1483-1489 (1991)); With IgE acceptor (Ridgway and Gorman, J.Cell.Biol. Vol.115, Abstract No.1448 (1991)) fusions.

Usually, the C of binding structural domain end merges with the N end in Fc district, and alternative cell anchorage zone. Example As, preferably before fusion the part of inactivation or disappearance and receptors bind any stride film district or lipid or Phosphatide anchor recognition sequence. The DNA of coding part or ligand binding moiety utilizes Restriction Enzyme in the coding phase Hope 5 ' and 3 ' terminal or close this 5 ' and 3 ' terminal position of the DNA of ORF section cut. Gained Dna fragmentation can easily insert among the DNA of encoding heavy chain constant region then. The essence that merges Really the position can be selected by rule of thumb in order to optimize the secretion of soluble fusion protein matter or in conjunction with feature. So After, the DNA transfection of encoding fusion protein is used for expressing to host cell.

In the embodiment, fusion comprises binding structural domain (for example, the acceptor of part or acceptor Extracellular domain (ECD)) be connected Fc part and synthetic connection peptide randomly. An enforcement side In the case, when preparation fused protein of the present invention, with the nucleic acid of coding part or receptors bind domain Nucleic acid with C end and coding Fc district N end merges. The fusion of N end also is fine. Can also implement with The fusion of the C end of constant region fc part or heavy chain CH1 or light chain respective regions near the N end Merge.

In the embodiment, the Fc district of fusion comprises basically complete antibody Fc district, rises Start from the hinge area (about 216 of the lucky upstream of papain cleavage site that chemically defines IgG Fc Position EU numbering residue, first residue of taking CH is 114) and end at its C end. The exact position of merging is not crucial; Concrete site is known, and can be through selecting with excellent The BA of chemoattractant molecule, secretion or in conjunction with feature. The method for preparing fusion be this area Know.

For bispecific fusion protein, fusion can be assembled into polymer, especially different dimerization Body or the different tetramer. Usually, the immunoglobulin (Ig) of these assemblings has known cellular construction. Base This four chain structure unit are existence forms of IgG, IgD and IgE. At the immune ball of HMW more Four chain elements repeat in the albumen; IgM is four bases to link together by disulfide bond usually The pentamer form of this unit exists. IgA globulin and the IgG globulin also can be at serum occasionally In exist with the polymer form. For polymeric situation, each of four unit all can be phase With with different.

Other exemplary part and its acceptor that can be included in the fusion of the present invention comprise following:

I) cell factor and cytokine receptor

Cell factor has pleiotropic effects to Proliferation of lymphocytes, differentiation and functional activation. Various Cell factor or its receptor binding moiety all can be used for fusion of the present invention. Exemplary cells because of Attached bag draw together interleukin (for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-11, IL-12, IL-13, and IL-18), colony stimulating factor (CSFs) (for example, granulocyte CSF (G-CSF), GM CSF (GM-CSF) and monocyte macrophage CSF (M-CSF)), swollen Tumor necrosis factor (TNF) α and β, and interferon such as interferon-' alpha ', β, or γ (U.S. Patent number 4,925,793 With 4,929,554).

Cytokine receptor typically is made up of the special α chain of part and common β chain. Cell factor The example of acceptor comprises GM-CSF, IL-3 (U.S. Patent number 5,639,605), IL-4 (U.S. Patent number 5,599,905), IL-5 (U.S. Patent number 5,453,491), the acceptor of IFN γ (EP0240975), and TNF Receptor family (for example, TNFR-1 (EP 417,563), TNFR-2 (EP 417,014) lymphotoxin-beta is subjected to Body).

Ii) attachment proteins

Adhesion molecule is to allow cell embrane-associated protein interact with each other. Various attachment proteinses, bag Draw together leucocyte homing receptor and cell adhesion molecule, or its receptor binding moiety, all can mix this In the bright fusion. The leucocyte homing receptor comprises Jie being expressed between inflammatory phase on the leukocyte surface Lead β-1 integrin (for example, VLA-1,2,3,4,5 and 6) of being combined with extracellular matrix components and With β 2 integrins of cell adhesion molecule (CAM) combination on the blood vessel endothelium (for example, LFA-1, LPAM-1, CR3 and CR4). The example of CAM comprises ICAM-1, ICAM-2, VCAM-1 and MAdCAM-1. Other CAM comprises those that select protein family, comprises CD62L, L-Select albumen and CD62P.

Iii) chemotactic factor (CF)

Chemotactic factor (CF)---chemotactic protein that stimulates leucocyte to move to sites of infection---also can mix In the fusion of the present invention. The example of chemotactic factor (CF) comprise macrophage inflammatory protein (MIP-1-α and MIP-1-β), neutrophil cell chemotactic factor (CF) and RANTES (activated adjusting, normally thin by T The cellular expression secretion).

Iv) growth factor and growth factor receptors

Growth factor or its acceptor (or its receptor binding moiety or ligand binding moiety) can mix this In the bright fusion. The example of growth factor comprises VEGF (VEGF) and its isotype (U.S. Patent number 5,194,596); Fibroblast growth factor (FGF) comprises aFGF and bFGF; Atrial natriuretic peptide (ANF); LGF (HGFs; U.S. Patent number 5,227,158 and 6,099,841), Neurotrophic factor is the bone neurotrophic factor (BDNF) of deriving for example, NT-3, and-4 ,-5, Or-6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factor NGF-β for example, platelet-derived Growth factor (PDGF) (U.S.Pat.Nos.4,889,919,4,845,075,5,910,574 and 5,877,016); TGF (TGF) is TGF-α and TGF-β (WO 90/14359) for example, bone Inducible factor comprises BMP (BMP); Insulin like growth factor-1 and-II (IGF-I and IGF-II; U.S. Patent number 6,403,764 and 6,506,874); Hematopoietin (EPO); Do thin Intracellular cytokine (SCF), TPO (c-Mpl part) and Wnt polypeptide (U.S. Patent number 6,159,462).

The example that can be used as the growth factor receptors of receptor targeted domain of the present invention comprises that EGF is subjected to Body; Vegf receptor (for example, Flt1 or Flk1/KDR), pdgf receptor (WO 90/14425); HGF Acceptor (U.S. Patent number 5,648,273 and 5,686,292) and neurotrophin receptor comprise in conjunction with NGF, The low affinity receptor (LNGFR) of BNDF and NT-3 is also referred to as p75^NTROr p75, and as acceptor EGFR-TK trk family member's high affinity receptor (e.g.trkA, trkB (EP 455,460), trkC (EP 522,530)).

V) hormone

It is sharp to comprise that as the example of the growth hormone of target agent in the fusion of the present invention feritin, people grow Plain (HGH; U.S. Patent number 5,834,598), N-methionyl human growth hormone (HGH); BGH; Somatotropin releasing factor; Parathyroid hormone (PTH); Thyroid-stimulating hormone (TSH) (TSH); Thyroid gland Plain; Proinsulin and insulin (U.S. Patent number 5,157,021 and 6,576,608); FSH (FSH), calcitonin, lutropin (LH), leptin, hyperglycemic factor; Bombesin (bombesin); Somatotropin; The Mullerian mortifier; Relaxain and relaxation precipitinogen; The promoting sexual gland hormone related peptide; Urge Breast is plain; Human placental lactogen; OB albumen; With the mullerian mortifier.

Vi) clotting factor

The exemplary blood clotting factor that is used as the target agent in fusion of the present invention comprises clotting factor (for example factor V, VII, VIII, X, IX, XI, XII and XIII, the von Wilebrand factor); Tissue factor (U.S. Patent number 5,346,991,5,349,991,5,726,147); Fibrin ferment and factor; The fiber egg White and fibrinogen; Fibrinolysin and plasminogen; Activator of plasminogen, for example urokinase or people urine Or tissue plasminogen activator (t-PA).

Other exemplary fused albumen is instructed in for example WO0069913A1 and WO0040615A2. Another example molecule that can be included in the fusion of the present invention is IGSF9. Can use this area The technology of knowing (seeing for example U.S. Patent number 5,116,964 and 5,225,538) preparation fusion.

III. identify the method for the candidate amino acid that is used for modification

The invention provides in the Fc district (or its FcRn bound fraction) that identifies the initial polypeptide that contains Fc special Decide the method for amino acid residue, wherein said amino acid residue changes by sudden change (for example amino acid replacement) Expection will cause to the adjusting of FcRn binding affinity with to the half-life of described polypeptide in serum after becoming Adjusting.

Described method comprises molecule or computer modeling, and this mould is built and can be used for prediction Fc district's adjusting (example As strengthening or reducing) amino acid change of FcRn combination. Usually, these methods originate in " first " or " initial " polypeptide or contain its compound (for example crystal structure or homology model), and cause because FcRn Binding affinity is conditioned and " second " or " change " or " modification " polypeptide different from first polypeptide, The polypeptide of this modification has better performance in particular treatment or diagnostic application. Described mould build can Carry out on the computer.

These methods can comprise one or more steps. For example, described method can comprise provides target The structure of the compound of Fc polypeptide and FcRn or corresponding data. In another or later step, the method Can be included in identify in the Fc district of initial polypeptide can be modified (for example sudden change) and predicted can impact Polypeptide is to definite residue or one group of residue (that is, candidate amino acid) of the binding affinity of FcRn.

The preferred sudden change of introducing in initial polypeptide Fc district comprises the antigen dependent/non-dependent that changes initial polypeptide Those sudden changes of effector function (for example half-life). In the embodiment, sudden change does not damage initial many Any other existing effector function of peptide (for example, antigen, part or receptors bind or Fc mediation Effector function (effector function of non-binding FcRn)) or in its desired use, it is caused and cuts A little less than. Therefore, the many advantages in other advantage that the Fc district provides are preferably kept in the sudden change of introducing. For example, The polypeptide that contains Fc usually has the ADCC function. This important cell killing activity has brachymemma The antibody construction thing in Fc district in will partially or even wholly lose. Keep Fc dependence ADCC merit Can, may be important in some applications, because it can cause the cell killing effect, thereby increase Exhaust the machine-processed anticarcinogen that works or the effectiveness of other medicines by the ADCC dependence by force.

In preferred embodiments, the polypeptide of change of the present invention contains and does not abolish or more preferably uncomfortable Save other expectation immunological effect subfunction of initial polypeptide or the sudden change of receptors bind function. Especially excellent In the embodiment of choosing, the polypeptide of change contains the polypeptide that does not change this change is combined albumen with following Fc The sudden change of the combination of matter, wherein said Fc is in conjunction with the purifying of albumen for the polypeptide that can be beneficial to this change Fc is in conjunction with albumen, especially staphylococcal protein A or G. Fc is upper to be responsible for being combined with a-protein The site is (Deisenhofer J.1981, Biochemistry, April 28 known in the art; 20 (9): 2361-70).

A. based on the analysis of sequence

In the embodiment, based on the Fc district of initial polypeptide from have different FcRn binding affinities Mammal Fc district sequence relatively, predict potential change site. The sequence in comparison Fc district will Enter initial polypeptide from one or more corresponding amino acid replacement with different sequences in conjunction with character In the Fc district.

In the embodiment, when expecting than short-half-life, from the immunity of irrelevant mammalian species Select corresponding amino acid in the globulin, wherein said immunoglobulin (Ig) shows lower FcRn acceptor parent And power. In another embodiment, when expectation than when long half-lift, from irrelevant mammalian species Select homologous amino acid in the immunoglobulin (Ig), wherein said immunoglobulin (Ig) shows higher FcRn and is subjected to Body affinity.

For example, rabbit Fc district shows and people Fc district (especially in contacting the zone of hFcRn) The high level homology. In addition, rabbit igg than human IgG more tightly in conjunction with hFcRn (Ober etc., Int. Immunol.13:1551-1559,2001). Therefore, in an exemplary, potential changes The displacement point is accredited as in polypeptide Fc district to be finished (for example in people Fc district) and has expectation and give birth to Thing is learned those different residues of polypeptide (for example rabbit Fc district) of character, and with in the human IgG1 Fc district these One or more one or more corresponding ammonia that are replaced by from rabbit igg 1 Fc district in the amino acid residue The sour residue of base.

For example, chimeric protein will show in people Fc specific by rabbit Fc amino acid replacement Amino acid or amino acid combination. (be presented at from the contact-making surface of people Fc and neonatal Fc receptor (hFcRn) In the homology model of ratIgG2a ratFcRn crystal structure exploitation), determine these rabbit Fc amino acid. Can will be defined as being present in people Fc then: the specific amino acids in the people FcRn contact structures territory is from people's order Row change over the rabbit sequence. Perhaps, one in these residues of people Fc can be replaced by with a plurality of Corresponding amino acid residue from the immunoglobulin (Ig) of cavy same type.

Exemplary change site comprises EU position 280,281,282,283,284,285,288,289,290, 305,307,308,309,315,340,344 and 378.

More particularly, polypeptide of the present invention can contain the amino acid mutation that at least one is selected from lower group: root According to the EU numbering system, (wherein, the D indication treats that in described EU position (278) sudden change is (logical to Asp280Asn Cross alternative) amino acid position, N indicates and waits to be substituted into this position to form the amino acid of the polypeptide that changes), Gly281Glu, Val282Glu, Glu283Gln, His285Arg, Asn286Thr, Lys288Arg, Thr289Pro, Lys290Pro, Val305Thr, Thr307Pro, Val308Ile, Leu309Thr, Asn315Arg, Lys340Arg, Arg344Leu, Ala378Ser, Ser383Lys, Glu386Lys, Pro387Ala, and Asn389Asp.

B. conformational analysis

In another embodiment, identify that the method for target amino acid comprises analysis (for example, visual observation or meter Calculation and Analysis) initial polypeptide (polypeptide that for example, contains Fc) and/or with Fc acceptor (for example FcRn) combination Initial polypeptide.

The three-dimensional structure of protein affects its biologically active and stability, and this structure can be with multiple Method is determined or is predicted. Usually, empirical approach is used the physical biochemistry analysis. Perhaps, Can be by one or more homologous proteins (or protein complex) with known three-dimensional structure 3 d structure model is set up, the prediction tertiary structure. X-ray crystallography may be the most familiar the determining of people The method of protein structure (therefore, term " crystal structure " can be used for alternative terms " structure ") (for example, The crystal structure in human IgG1 Fc district determined (Disenhofer etc., Biochemistry (1981), 20: 2361-70)), but also can use garden dichroism, light scattering, perhaps pass through the suction of measuring radiation energy Receipts and release are estimated. Other useful technology comprises neutron diffraction, nuclear magnetic resonance (NMR) and homology Mould is built. All these methods all are well known by persons skilled in the art, and they are described in fully Standard textbook (is seen Physical Chemistry for example, 4^thEd., W.J.Moore, Prentiss-Hall, N, J., 1972, or Physical Biochemistry, K.E.Van Holde, Prentiss-Hall, N.J.1971)) and In many publications. Can use in these technology any to determine the Fc district, contain the Fc district (or its The structure of the compound of the polypeptide FcRn bound fraction) or this polypeptide and FcRn (can be analyzed then The amino acid that this structure substitutes with prediction) and/or can use described technology so that method to be provided The information of one or more steps (for example, described below those).

Form the existing report of method of the crystal of antibody, antibody fragment or scFv-antigenic compound, example As the report of van den Elsen etc. (Proc.Natl.Acad.Sci.USA 96:13679-13684,1999, Hereby incorporate into herein as a reference). Can utilize these technology known in the art to determine to contain FcRn With the structure of the compound that contains the Fc polypeptide, in order to analyze according to the inventive method. Perhaps, can Easily from commerce or public's database, for example Protein Data Bank obtains the composite structure of publishing Or corresponding data. In addition, recently also obtained rat FcRn and contain single FcRn binding site The eutectic structure of heterodimer rat Fc (2.8 ) (for example, Martin etc., Molecular Cell, (2001), 7:867-77). When structure (for example x-ray structure) or its corresponding data the unknown of compound maybe can't obtain When getting, can utilize and adopt related complex (for example compound from another species or homology ligand/receptor Thing) homology model. For example, can use the crystal structure mould of rat Fc-FcRn compound to build people Fc Interaction with FcRn.

Can estimate the data corresponding to the Fc/FcRn compound, to determine potential change site. Another In the embodiment, the inventive method comprise analysis (for example, structural analysis or computational analysis) free (that is, Unconjugated) contain the Fc polypeptide and be combined with FcRn contain conformational difference between the Fc polypeptide.

C. static optimization

The basic calculating formula that is used for enforcement the inventive method is provided at for example U.S. Patent number 6,230,102 In (its content hereby intactly incorporate among the application as a reference). In the embodiment, according to calculating The result who analyzes the electrostatic force between polypeptide and FcRn and obtain, preferably according to describe herein this Bright discrete standard (discrete criteria) or rule change (or " modification ") polypeptide. This computational analysis is fair Permitted to predict the best CHARGE DISTRIBUTION in the polypeptide receptor compound, in computer system, represented CHARGE DISTRIBUTION A kind of mode be to represent with one group multipole (multipole). Perhaps, can be by being positioned at the polypeptide atom One group of point charge of position, the expression CHARGE DISTRIBUTION. In case determined CHARGE DISTRIBUTION (preferably, best electricity Lotus distributes) after, can modified polypeptide with coupling, or coupling better, this CHARGE DISTRIBUTION.

Can by implementing U.S. Patent number 6,230, calculate (or such as Tidor and Lee, J. described in 102 Chem.Phys.106:8681,1997; Kangas and Tidor, J.Chem.Phys.109:7522,1998 Described in calculate) computer implemented method, mediate described computational analysis. Can be to this computer Program carry out adaptability revision with the truth of considering polypeptide-FcRn combination (and with other method not Consider that with, the inventive method for example polypeptide and FcRn are at solvent (for example, aqueous solvent such as water, phosphoric acid The salt solution of salt buffer (PBS), blood plasma or blood) in conjunction with the time solvent, long-range electrostatic interaction and dielectric Effect). This computer implemented method can be for the identification of the following modification to this polypeptide structure, described repairing Decorations will cause making electrostatic interaction to the tribute in conjunction with free energy of modified polypeptide and FcRn at the polypeptide of modifying Offer the CHARGE DISTRIBUTION that minimizes (for unmodified (" initial ") polypeptide). Typically, implement herein The computer system of described operation (seeing in more detail U.S. Patent number 6,230,102) (or device) will comprise The output device from information to the user (for example, CRT monitor, LCD, printer, the communication device that show Such as modem, audio frequency output etc.). In addition, can the operation part ground of the inventive method will be carried out Or fully give the medium that suits in electronic installation, to use, in order to carry out this instruction. Therefore, originally Inventive method can be revised as and comprise software (for example, computer-readable instruction) and hardware (for example, calculating Machine, robot and chip) high throughput method. This computer implemented method is not limited to certain computer and puts down Platform, par-ticular processor or specific high-level programming language. Useful method is seen U.S. Patent number 6,230,102, More detailed description is seen Lee and Tidor (J.Chem.Phys.106:8681-8690,1997), all literary compositions Offer and hereby incorporate into herein as a reference.

Can be by following application rule of the present invention. In order to regulate the FcRn binding affinity of polypeptide, example As, to reduce, to improve or to rebuild this combination, at first obtain basic sequence and/or structured data.

In the embodiment, the candidate amino acid residue can be selected from through determining to have suboptimum or the best Those residues of binding affinity. Alternatively or additionally, can in the Fc district, be close to and have Those residues of the residue of good or suboptimum binding affinity are selected target amino acid residues. Typically, head Use earlier electrostatic charge to optimize to determine in the Fc district position (Lee and Tidor, the J. of suboptimum for combination Chem.Phys, 106:8681-8690,1997; Kangas and Tidor, J.Chem.Phys.109: 7522-7545,1998).

Then, further computational is analyzed one or more sudden changes (that is, modifying). Based on these calculating, To having the modified polypeptide subset of one or more modifications of rule according to the present invention, determine combination then Affinity.

Use non-individual body electrostatics model (continuum electrostatics model), can be at polypeptide Fc In amino acid whose each side chain on implement electrostatic charge optimization. Electric charge optimization gives the electric charge of former subcenter, But always do not produce actual sudden change. Therefore, can carry out taking turns electric charge optimization, apply various therebetween Constraints is to represent the natural side chain feature of destination locations. For example, can for-1,0 and+1 clean Side-chain charges implement to be optimized, and additional therebetween do not have atomic charge to surpass particular value (0.85 electronics for example The unit of charge) constraints. Increase based on the potential of electrostatical binding free energy of observing in optimizing then Add, determine candidate amino acid side chain position and modify at the residue of these positions.

Can calculate from natural residue turns to complete uncharged side chain isostere (that is, to have phase With shape but on the atom no electric charge or have the residue of Partial charge) in conjunction with free energy difference (unit: Kcal/mol). The binding affinity of negative indication predicting increases.

When in conjunction with free energy difference be favourable (Δ G＜-0.3kcal/mol) and with from natural residue to fully not Charged side chain isostere (that is, has identical shaped but no electric charge or have the part electricity on the atom When the transformation residue of lotus) is correlated with, from amino acid whose set with non-polar sidechain (A for example, C, I, L, M, F, P, V) middle selection modification.

The combination that best CHARGE DISTRIBUTION in using side chain and-1 clean side-chain charges can obtain is free Can difference be favourable (Δ G＜-0.3kcal/mol) time, from having the amino acid whose set of electronegative side chain Select in (for example D, E) to modify.

Similarly, the best CHARGE DISTRIBUTION in using side chain and+1 clean side-chain charges can obtain In conjunction with free energy difference be favourable (Δ G＜-0.3kcal/mol) time, from having the amino of positively charged side chain Select to modify in the set (for example R, H, K) of acid.

At last, the best CHARGE DISTRIBUTION in using side chain and 0 the clean side-chain charges knot that can obtain Close free energy difference and be favourable (Δ G＜-0.3kcal/mol) time, from adding C, G, having of M and F is not with Select in the amino acid whose set of charge polarity side chain (for example N, C, Q, G, H, M, F, S, T, W, Y) Modify.

As described herein, can build on computers designed modified polypeptide, and recomputate In conjunction with energy. Can use the dihedral angle increment of 60 degree, different by in CHARMM, implementing rotation The scanning of structure body dihedral angle makes up the side chain of modifying, with the position of determining that each side chain is wished most. Then For wild type (initial) and sudden change (modification) compound, use the Poisson-Boltzmann electrostatic energy With the extraneous term that is used for Fan Dewaershi energy and hidden surface area, calculations incorporated energy.

Then, for modifying resulting result by calculating these computationals, can be on demand, example As after repeating the method subsequently, reappraise on computers, perhaps by extra experiment structure/merit Can data supply information. The rule that can be divided into following several classes allows to carry out several predictions:

1) in conjunction with FcRn the time, repaiied by the hidden residue place, interaction interface of part on the polypeptide Decorations (improving interaction by forming hydrogen bond);

2) the following polar residues on the modified polypeptide, described polar residues in conjunction with the time by hidden and thus Pay a price for desolvation but do not form any direct electrostatic interaction (usually by repairing with acceptor Adorn into the wild type residue and have the hydrophobic residue of similar shape or can cause favourable by interpolation The residue of electrostatic interaction, to be improved); With

3) the following surface residue on the modified polypeptide, described surface residue is present in the incomplementarity electromotive force The zone of (uncomplementary potential). These modifications are considered to improve polypeptide and FcRn Between long-range electrostatic interaction and do not upset the accumulative facies mutual effect of combination interface.

So implement, rule of the present invention allows successfully to predict the change of affinity (for example to reduce and increase Add), side chain modifies. These discoveries can be divided into three major types modifies. The first kind modify relate to be positioned at anti-The interface residue that can form hydrogen bond that former upper charged group is crossed over; Equations of The Second Kind relate in conjunction with the time Payment desolvation punishment but do not cause anti-electrostatic interaction (back elctrostatic interaction) Hidden polar residues; Relate to long-range electrostatic interaction with the 3rd class.

First kind modification is determined by checking basic physical/chemical factor, because these residue essence Unsaturated hydrogen gametophyte upper and antigen forms hydrogen bond. Different from other method, the present invention's rule allows Carry out mysterious residue and modify, wherein desolvated cost is allowed to overweight useful mutual work Use energy.

Equations of The Second Kind is modified and represent another group and modify because the energy that increases mainly by elimination disadvantageous go molten Agent turns into and keeps apolar interaction and cause.

The 3rd class is modified and is related to long-range interaction, and it is affine that these interactions show remarkable increase The potentiality of power. The modification of these types is especially significant, because they directly do not contact with antigen Therefore and less upset the meticulous interaction at polypeptide-FcRn interface.

Therefore, after according to the side chain chemistry of these rules for the expectation of candidate amino acid location positioning, Can be for example by substituting, insert or disappearance (see herein further describe), modify or change this (these) residue position.

For the peptide modified rule, what attention can also be in best CHARGE DISTRIBUTION is initial except above During calculating, (and follow-up) add some decisive factor, for example solvent effect.

In the embodiment, based on the electric charge optimization data, select to change the effector function of target antibody Preferred sudden change (for example amino acid replacement), and can be by can be at different pH in the prediction Fc district Level---for example, the neutral pH of about 7.2-7.4; The acid pH of about 3.0-5.0 (such as 4.0); Or The about alkaline pH of 8.0-10.9 (for example 9.0)---change the knot of (for example strengthen or reduce) and Fc acceptor The amino acid that closes changes (for example substituting) and identifies described sudden change.

Electric charge optimization causes one group of best electric charge at the atom center, but does not produce actual sudden change prompting. One Dawn can change based on the result that electric charge is optimized (for example sudden change) after using above method to determine that electric charge is optimized One or more target amino acid residues or any adjacent amino acid residue in the polypeptide (for example are positioned at CH2 In the FcRn coupling collar in domain or Fc district or residue on every side). In this process, analyze best electricity Lotus distributes, and selects than the sudden change of present residue closer to optimal cases. For example, can select with At present residue Comparatively speaking mate, coupling or closer to the amino acid replacement of optimal cases better. Can select one or more sudden change, in order to obtain best CHARGE DISTRIBUTION. Can pass through logarithm According to observing or by the computational analysis data, selecting preferred sudden change.

At present, the software that is used for inspection electrostatic force can be built best CHARGE DISTRIBUTION by mould, and the user can afterwards Which kind of determine amino acid replacement or change to improve this distribution. Therefore, these steps (for example, check mould The best CHARGE DISTRIBUTION of building and the sequence modification that is identified for improving the antigen combination) be, perhaps can be, The part of present method required for protection. Yet, soft owing to can easily modify this in future Part makes program comprise selection to amino acid replacement (or change), thus may only need to check Output rusults and Carry out suggested change (or, if the expectation, its certain variation).

In the embodiment, can be by can be in different pH levels (for example, greatly in the prediction Fc district The neutral pH of about 7.2-7.4; The acid pH of about 3.0-5.0 (such as 4.0); Or about 8.0-10.9 (for example 9.0) alkaline pH) amino acid that changes (for example strengthen or reduce) and the combination of Fc acceptor (for example, changes Substitute), identify to be used for alternative amino acid.

In the embodiment, the present invention relates to regulate the polypeptide of the FcRn bound fraction that comprises the Fc district In the method for the binding affinity of two different pH levels and FcRn, described method comprises: determine many Peptide and FcRn in solvent when a pH horizontal integration sky of the best CHARGE DISTRIBUTION of polypeptide amino acid Between performance and relevant in conjunction with Gibbs free; Determine polypeptide and FcRn in solvent in the 2nd pH The space of the best CHARGE DISTRIBUTION of polypeptide amino acid performance and relevant in conjunction with free energy during horizontal integration Change; These CHARGE DISTRIBUTION are identified to show different electric charge branches in the first and second pH levels based on the comparison In the residue of cloth, the polypeptide to be finished when changing polypeptide in conjunction with FcRn in conjunction with at least one of free energy Candidate amino acid residue position; Be used for advancing at described amino acid position with the amino acid residue of selecting to select Row substitutes, so that polypeptide is regulated the affinity of FcRn after substituting this elected amino acid residue.

In the embodiment, a pH is about 7.4. In the embodiment, the 2nd pH is big About 6.0.

In the embodiment, with the neutral amino acid of the initial polypeptide of charged amino acid replacement. In another embodiment, the neutral amino acid of initial polypeptide is with another neutral amino acid replacement. In another embodiment, the amino acid of initial polypeptide (for example uncharged or electronegative amino acid) With positively charged amino acid replacement. Positively charged amino acid comprises histidine, lysine, asparagus fern Acid amides. In another embodiment, the amino acid of initial polypeptide is (for example uncharged or positively charged Amino acid) with electronegative amino acid replacement. Electronegative amino acid comprises aspartate (asparagus fern Propylhomoserin) and glutamate (glutamic acid). In another embodiment, the amino acid of initial polypeptide (for example, band Negative electrical charge or positively charged amino acid) use uncharged amino acid replacement. In another embodiment, The amino acid of initial polypeptide (for example, uncharged amino acid) is with having the not charged of different CHARGE DISTRIBUTION The lotus amino acid replacement.

In certain embodiments, after introducing amino acid in the polypeptide that is changing, the amino that this substitutes Acid changes the electric charge of polypeptide, so that the polypeptide of this change has the net charge different with initial polypeptide. In some other embodiment, after amino acid is introduced in the polypeptide of change, the amino acid that this substitutes Do not change the electric charge of polypeptide, so that the polypeptide of this change has identical net charge but tool with initial polypeptide Different CHARGE DISTRIBUTION is arranged.

In another embodiment, amino acid is divided into following three groups: (1) has the non-of uncharged side chain Polar amino acid (for example, A, L, I, V, G, P). These amino acid participate in hydrophobic interaction usually; (2) has zero net charge but the different piece of side chain has the polar amino acid (example of non-zero Partial charge As, M, F, W, S, Y, N, Q, C). These amino acid can participate in hydrophobic interaction and static mutually Mutual effect. (3) can side chain have non-zero net charge charge residue (R for example, K, H, E, D). These amino acid can participate in hydrophobic interaction and electrostatic interaction.

In the embodiment, at least one sudden change that changes the interactional affinity of polypeptide-Fc is the sudden change from one of following three types:

(1) changes interaction interface or FcRn and away from the incomplementarity static between the polypeptide at this interface The sudden change of the CHARGE DISTRIBUTION in the gesture zone. These changes can comprise: in polarity, nonpolar and charged Substitute between organizing on the lotus amino acid (this is with the position of chop and change Partial charge); And at polarity amino Substitute in acid is organized and in the charge residue group, condition is that (for example, C is at SG for their change CHARGE DISTRIBUTION Have the part negative electrical charge on the atom and have part positive charge at the HG atom. And N is at SG and HD Have part positive charge on the atom, and have the part negative electrical charge at ND and OD atom; Then, use C Alternative N will change CHARGE DISTRIBUTION). For example, in the embodiment, (have zero only with polar amino acid Electric charge but atom in side chain has Partial charge) (all are former in the side chain to substitute nonpolar amino acid Have zero charge on the son) or vice versa;

(2) on the sudden change antibody in conjunction with the time by hidden and pay thus desolvation punishment (pay Desolvation penalty) (in conjunction with the time desolventizing energy cost) but do not form any favourable with FcRn Polarity or the charged residue of electrostatic interaction. In the case, by being mutated into not and solvent Interact and thus in conjunction with the time do not pay the nonpolar amino acid of desolvation punishment, and obtain to change Kind.

(3) sudden change changes the dielectric that molecular shape also affects the medium between polypeptide and FcRn thus The surface residue of character. Because ratio of solvent protein has higher screening ability (dielectric constant), so electricity Lotus can produce stronger interaction by protein than passing through solvent. Therefore, fill out with protein side chain Fill (or removing) space between the electric charge on polypeptide and the FcRn and will regulate the mutual work of these electric charges With. These sudden changes comprise the ammonia that carries out with the replacer who has with the difform side chain of original amino acid Base acid substitutes the (all changes the variation between isostere: V to T, D to N, N To D, L to D, L to N, D to L, N to L, Q to E and E to Q). For non-This group on the polar amino acid substitutes, and this phenomenon will be to electrostatic interaction between polypeptide and the FcRn Unique impact.

In a particular, the polypeptide of change is included in corresponding to the EU position that is selected from lower group The substituting of amino acid position: 248,249,250,251,252,254,256,255,260,257,277, 281,282,287,284,285,286,288,290; 304,305,306,307,309,310,312,313, 315,343,374,426,428,430,431,432,434, or 438.

At one more particularly in the embodiment, the polypeptide of change can comprise the arbitrary of following sudden change or Any combination (and until owning): 248 are replaced by aspartic acid in the EU position; In EU position 249 The position is replaced by arginine or lysine; 250 are replaced by arginine or lysine in the EU position; At EU 251 of positions are replaced by arginine, lysine or asparagine; 252 are replaced by silk in the EU position Propylhomoserin or threonine; 254 are replaced by serine or threonine in the EU position; In the EU position 256 Be replaced by arginine, glutamic acid or lysine; 255 are replaced by leucine, asparagus fern ammonia in the EU position Acid or methionine; 260 are replaced by lysine in the EU position; Be replaced by smart ammonia in EU position 257 Acid, aspartic acid, glutamic acid or lysine; 277 are replaced by arginine, asparagus fern ammonia in the EU position Acid, glutamine or lysine; 279 are replaced by glutamic acid in the EU position; In EU position 281 The position is replaced by glutamine; In the EU position 282 be replaced by arginine, aspartic acid, glutamic acid, Or lysine; In the EU position 287 be replaced by aspartic acid, glutamic acid, lysine, proline or Threonine; 284 are replaced by aspartic acid or glutamic acid in the EU position; 285 substitute in the EU position Be aspartic acid, glutamic acid or phenylalanine; 286 are replaced by aspartic acid, glutamic acid in the EU position Or methionine; 288 are replaced by aspartic acid or glutamic acid in the EU position; In the EU position 290 Be replaced by aspartic acid or glutamic acid; 304 are replaced by aspartic acid or glutamic acid in the EU position; At EU 305 of positions are replaced by arginine; 306 are replaced by arginine, aspartic acid, paddy in the EU position Propylhomoserin or lysine; 307 are replaced by arginine, aspartic acid or glutamic acid in the EU position; 309 are replaced by arginine, aspartic acid, lysine or glutamic acid; 310 are replaced by in the EU position Arginine, leucine, lysine or asparagine; 312 are replaced by arginine, sky in the EU position Winter acid amides or lysine; 313 are replaced by aspartic acid, arginine or lysine in the EU position; 315 of EU positions are replaced by aspartic acid or glutamic acid; 343 are replaced by glutamine in the EU position Or lysine; 345 are replaced by arginine or glutamine in the EU position; 374 are replaced in the EU position On behalf of arginine, lysine or leucine; 376 are replaced by asparagine in the EU position; In the EU position Put 426 and be replaced by arginine, aspartic acid or glutamic acid; 428 are replaced by essence in the EU position Propylhomoserin, glutamine or lysine; 430 are replaced by lysine in the EU position; In EU position 431 The position is replaced by proline; 432 are replaced by arginine in the EU position; 434 are replaced by in the EU position Leucine or lysine; Or in the EU position 438 be replaced by glutamic acid.

Introduce substituting at one more particularly in the embodiment in the Fc district of IgG1, this substitutes choosing From one of following sudden change: K248D, D249R, D249K, T250R, T250K, L251R, L251K, L25 1N, M252S, M252T, M254S, M254T, T256R, T256E, T256K, R255D, R255L, R255M, T260K, T260R, T260K, T260Q, P257R, P257D, P257E, P257K, W277R, W277D, W277Q, W277K, V279E, G281Q, V282R, V282D, V282E, V282K, V282E, H287D, A287E, A287K, A287P, A287T, V284D, V284E, A287E, H287D, H285E, H285F, N286D, N286E, N286M, K288D, K288E, K290D, K290E, S304D, S304E, V305R, V306E, L306R, L306D, L306E, L306K, V307E, T307R, T307D, L309R, L309D, L309E, L309K, H310R, H310N, H310L, H310K, L312K, D312R, D312N, D312K, N313R, W313D, N313K, W313K, N315D, N315E, P343Q, P343K, E345R, P374R, P374L, P374K, D376N, S426R, S426D, S426E, E430K, A431P, L432R, N434K, N434L, or Q438E.

D. side chain is heavily piled up

In another embodiment, select the method for preferred amino acid replacement to be included in to contain FcRn and The upper side chain of using of structure (for example, crystal structure or model) that contains the compound of Fc polypeptide is heavily piled up technology (sidechain repacking). Heavily pile up in the calculating at side chain, can calculate modification target residue, and Calculate to estimate gained Fc polypeptide mutant in the stability in conformation that FcRn is combined. Side chain is heavily piled The long-pending row that produce by hierarchical arrangement that calculate have the stability (that is, the intramolecular energy of change) of change Variant.

In another embodiment, the method for selecting preferred amino acid to substitute is included in and contains two polypeptide (examples As, contain polypeptide and the FcRn of Fc) the upper application side of structure (for example, crystal structure or model) of compound Chain is heavily piled up technology. Then, can select to cause the receptors bind affinity expected to change (for example, increases Add or reduce) sudden change be used for experiment and express.

The quantity of calculating the protein mutant of estimating can be very huge, because each variable amino The acid position can be mutated into all 20 kinds of standard amino acids. Be used for Calculation results is carried out classification The example calculation algorithm comprises that terminal is eliminated (dead-end elimination) and tree-search algorithm (is seen example As, Lasters etc. (Protein Eng.8:815-822,1995), Looger and Hellinga (J.Mol.Biol. 307:429-445,2001) and Dahiyat and Mayo (Protein Sci.5:895-903,1996)).

E.3-D show

In the embodiment, three-dimensional structure and/or mould that can visual analysis polypeptide-FcRn compound Type (for example using 3-D molecular imaging device) is conducive to or is unfavorable for the prominent of specific molecular conformation with prediction Become.

In the embodiment, sudden change is by the amino acid residue that contains Fc polypeptide and FcRn in sudden change Between introduce extra contact, cause containing the affinity increase of polypeptide with the Fc acceptor of Fc. This can With the polypeptide by for example will containing Fc than p1 amino acid side chain (for example, alanine, glycine, silk Propylhomoserin, aspartic acid, asparagine, valine, cysteine) be replaced by than big amino acid side chain (example As, methionine, tryptophan, phenylalanine, tyrosine, leucine, isoleucine, lysine, Arginine, glutamine, glutamic acid, proline, threonine, histidine) realize. Another enforcement In the scheme, sudden change contains the amino acid that contacts with FcRn in the polypeptide of Fc by change, causes containing The polypeptide of Fc reduces the affinity of Fc acceptor. This can be for example by will containing Fc polypeptide Big amino acid side chain (for example, methionine, tryptophan, phenylalanine, tyrosine, leucine, different Leucine, lysine, arginine, glutamine, glutamic acid, proline, threonine, histidine) Be replaced by than p1 amino acid side chain (for example, alanine, glycine, serine, aspartic acid, asparagus fern Acid amides, valine, cysteine) obtain.

In the embodiment, in FcRn coupling collar district, carry out one or more sudden changes. The FcRn knot Cyclization comprises amino acid residue 280,281,282,283,284,285,286,287,288,289,290, 291,292,293,294,295,296,297,298 and 299 (according to the EU numberings). Shown this among Fig. 1 Ring. In the embodiment, in being selected from lower group amino acid, implement one or more sudden changes: 280,281, 282,283,284,285,286,287,288,289,291,292,293,294,295,296,297,298 With 299 (according to the EU numberings). In the embodiment, one or more in the FcRn coupling collar Amino acid whose sudden change causes reducing with the binding affinity of FcRn. Preferably, in the FcRn coupling collar One or more amino acid whose sudden change cause the FcRn binding affinity to increase. In the embodiment, The present invention relates to increase the method for the half-life of polypeptide of the present invention, comprise in the sudden change FcRn coupling collar extremely Few amino acid residue. In another embodiment, the present invention relates to be used for the treatment of patient's composition, Wherein said patient will have benefited from that polypeptide with the change of the half-life with increase of the present invention carries out Treatment.

In another embodiment, in 15  FcRn contact zones, for example (also listing usefulness with upper/lower positions Exemplary amino acid in these positions) one or more carries out one or more sudden changes: 243F; 244 P; 245P; 246K; 247P; 248K; 249D; 250T; 251L; 252M; 253I; 254S; 255R; 256T; 257P; 258E; 259V; 260T; 261C; 275F; 276N; 277W; 278Y; 279V; 280D; 282V; 283E; 284V; 285H; 286N; 287A; 288K; 289T; 290K; 291P; 292R; 293E; 302V; 303V; 304S; 305V; 306L; 307T; 308V; 309L; 310H; 311Q; 312D; 313W; 314L; 315N; 316G; 317K; 318E; 319Y; 336I; 337S; 338K; 339A; 340K; 341G; 342Q; 343P; 344R; 345E; 346P; 347Q; 348V; 367C; 369V; 372F; 373Y; 374P; 375S; 376D; 377I; 378A; 379V; 380E; 381W; 382E; 383S; 384N; 385G; 386Q; 387P; 388E; 389N; 391Y; 393T; 408S; 424S; 425C; 426S; 427V; 428M; 429H; 430E; 431A; 432L; 433H; 434N; 435H; 436Y; 437T; 438Q; 439K; And 440S (according to the EU numbering).

F. elutriation FcR mutant library

In another embodiment, can use polymer FcRn acceptor of the present invention (that is, to advance one herein The FcRn-Fc fusion that step is described in detail), analyze the member's of any obtainable Fc polypeptide libraries Effector function changes. For example, can use polymer Fc acceptor screening of the present invention or " elutriation " sudden change The phage library of the polypeptide that contains Fc. Phage display library typically comprises phage particle, This phage particle is expressed the polypeptide that contains Fc of this sudden change or Fc district or containing of they and is inserted in The zone of the sudden change of the polynucleotide sequence in this phage genome or part. Therefore, bacteriophage literary composition The storehouse can comprise following phage particle therein, and this phage particle is expressed the institute of the Fc-polypeptide of sudden change Each member that constituent or combinatorial libraries are arranged. The bacteriophage of using in these methods typically Filobactivirus comprises fd and M13. The typically conduct and virus capsid protein (example of Fc part of sudden change As, phage gene III or VIII protein) fusions be expressed in phage surface. The preparation phagocytosis The method in body library be known in the art (see, Brinkman etc., J.Immunol.Methods, (1995), 182:41-50; Ames etc., J.Immunol.Methods, (1995), 184:177-86; Kettleborough Deng, (1994), 24:952-8; Persic etc., Gene, (1997), 187:9-18; Burton etc., Advances In Immunol., (1994), 57:191-280. Perhaps, can screen existing phage display library. Can The library that obtains comprises the library of the Fc polypeptide of the change of describing among the WO 02/060919.

Of course, for example the polymer FcRn of usage flag (is incorporated into or is fixed in for example pearl of solid phase surface On), select or identify the bacteriophage that is expressed as follows the Fc district, described Fc district and polymer Fc of the present invention Acceptor is to be higher or lower than the affinity generation combination of initial polypeptide. Also can in the FACS sorting, use This bacteriophage is with the cell in the Fc district of select expressing higher affine form.

Use these methods, can detect the Fc molecule that changes form on the FcRn binding affinity Change, and select to have the binding affinity increase of expectation or those molecules that reduce.

G. further optimize the FcRn binding affinity

Can rebuild the polypeptide of the change of the inventive method generation, and it is done further to change to advance one Step joint FcR is in conjunction with (for example, with further increase or further reduction combination). Therefore, can be upper State and implement extra step after the step, comprise, for example: (a) obtain corresponding to change or " second " The data of the structure of the compound of polypeptide and acceptor; (b) (we can be called " volume with it to use these data Outer data " come with the data separation with using that in first " wheel ", obtains making it), determine more than second The performance that the following additional charge of peptide constant region distributes, wherein said additional charge distribute so that static is done Be used in the minimum in conjunction with contribution in the free energy of second polypeptide and acceptor; (c) express and the of receptors bind Three polypeptide, the 3rd polypeptide has the sequence that is different from second polypeptide at least one amino acid residue. In addition, can use experience be the information that provides of further optimizing in conjunction with data. Can carry out again volume Outer many wheels optimization.

IV. change the method for polypeptide

After obtaining expectation sudden change pending in the initial polypeptide, can use appointing in the multiple methods availalbe A kind of generation comprises the polypeptide of the change of this sudden change. For example, can prepare this multinuclear by recombination method Thuja acid. And, because the degeneracy of genetic code, can use the multiple nucleic acids sequential coding each The expectation polypeptide.

For the nucleic acid molecules of the amino acid sequence variant for preparing the initial polypeptide of coding, exemplary ability The territory known method includes, but not limited to decide by the early DNA of preparation to this polypeptide of encoding Point (or oligonucleotide mediated) mutagenesis, PCR mutagenesis and cassette mutagenesis prepare.

Direct mutagenesis is the method for optimizing of preparation alternative variations. This technology be well known in the art (for example see, The Proc.Natl. such as the Nucleic Acids Res.13:4431-4443 (1985) such as Carter and Kunkel Acad.Sci.USA 82:488 (1987)). In brief, when DNA is implemented direct mutagenesis, by as Lower mode changes parent DNA: one of the oligonucleotides that at first making encodes expects sudden change and parent DNA The hybridization of bar strand. After the hybridization, use the oligonucleotides of hybridization as primer, and use parent DNA's This strand utilizes archaeal dna polymerase to synthesize the second complete chain as template. Like this, coding expectation The oligonucleotides of sudden change is merged in the double-stranded DNA of gained.

PCR mutagenesis also is applicable to the amino acid sequence variant of the initial polypeptide of preparation. See Higuchi, " PCR Protocols ", pp.177-183 (Academic Press, 1990); With Vallette etc., Nuc.Acids Res. 17:723-733 (1989). In brief, when in PCR, using a small amount of template DNA as parent material The time, can use with template DNA in respective regions slightly discrepant primer on sequence, produce phase To a large amount of specific DNA fragments, this dna fragmentation will only be different from template at primer with template sequence The position on there are differences.

The other method of preparation variant, cassette mutagenesis, based on Wells etc., Gene 34:315-323 (1985) The technology of describing. Parent material be the plasmid that comprises initial polynucleotide dna to be suddenlyd change (or other Carrier). Identify the codon among the parent DNA to be suddenlyd change. In each of the mutational site of identifying Must there be the Single restriction restriction enzyme site in side. If do not have this type of restriction site to exist, then can So that with above-mentioned oligonucleotide mediated method of mutagenesis, by they being introduced the suitable of initial polypeptid DNA Produce this site when the position. At these sites cutting DNAs with it linearisation. Application standard Method, the dna sequence dna between the composite coding restriction site but contain the double-stranded few nucleosides of expectation sudden change Acid, Application standard technical point two chains of synthetic oligonucleotide of turning up the soil wherein, and then with they hybridization Together. This double chain oligonucleotide is called box. This box design has the terminal phase with linearization plasmid 5 ' and the 3 ' end that holds, this box can directly be connected with plasmid like this. This plasmid contains sudden change thus Dna sequence dna.

Alternatively, perhaps additionally, can determine the expectation amino acid sequence of coded polypeptide variant, and Can be by the nucleotide sequence of this amino acid sequence variant of synthetic method preparation coding.

Those of ordinary skills understand that the polypeptide that the present invention has the FcRn combination of change can advance One step was modified, and expected activity thereby change their amino acid sequence but do not change. For example, can To carry out other nucleotide substitution at protein, to cause amino acid at " nonessential " amino acid residue position Substitute. For example, the nonessential amino acid residue in the immunoglobulin polypeptides can be used from the same side chain Other amino acid residue of family substitutes. In another embodiment, can be with one section amino acid chain with tying Chain (difference is side chain family member's kind and/or composition) similar on the structure is replaced, that is, can advance Row is conservative to be substituted, the radical amino acid replacement of one of them amino acid residue with similar side chain.

Have the in the art existing definition of family of the amino acid residue of similar side chain, comprise alkaline side Chain (for example, lysine, arginine, histidine), acid side-chain (for example, aspartic acid, glutamic acid), Uncharged polar side chain (for example, glycine, asparagine, glutamine, serine, Soviet Union's ammonia Acid, tyrosine, cysteine), non-polar sidechain (for example, alanine, valine, leucine, different Leucine, proline, phenylalanine, methionine, tryptophan), the side chain of β branch (for example, Soviet Union Propylhomoserin, valine, isoleucine) and aromatic side chains (for example, tyrosine, phenylalanine, tryptophan, Histidine).

Except amino acid replacement, the present invention also considers other modification to initial Fc region amino acid sequence The Fc region variants that has the effector function of change with generation. For example, can lack of Fc district Or a plurality of amino acid residues, with the combination of reduction or enhancing and FcR. In the embodiment, can Modify one or more residues in Fc district to produce this Fc region variants. Usually, according to the present invention this Embodiment, disappearance are no more than 1 to about 10 Fc district residues. Comprise one or more herein Initial Fc district at least about that the Fc district of amino acid deletions is preferred to keep having people Fc district native sequences 80%, preferably at least about 90%, most preferably at least about 95%.

The amino acid that also can prepare the effector function with change inserts the Fc region variants. For example, can So that (for example, one to two amino acid residue usually is no more than 10 with at least one amino acid residue Individual residue) introduces one or more juxtapositions that affect the Fc of FcR combination zone position that are accredited as herein Put. One of the Fc district residue that " vicinity " span is identified from herein is to the scope of two amino acid residues. This Fc region variants can demonstrate FcRn combination enhancing or that weaken.

This Fc region variants comprises in the Fc district usually that at least one is amino acid modified. An enforcement side In the case, amino acid modified can the combination. For example, variant Fc district can comprise therein to for example this paper In two, three, four, five of the specific Fc zone position identified etc. substitute. Another embodiment In, the polypeptide of change can have a change with FcRn and with the combination of another Fc acceptor.

The Fc district is made up of two same protein chains. Therefore, in the embodiment, sudden change occurs in Article two, on the protein chain. In another embodiment, sudden change occurs over just in the protein chain.

V. preferably change

The polypeptide of change of the present invention contains at least one sudden change in its Fc district (for example, amino acid replaces Generation). In the embodiment, the amino acid that substitutes is arranged in the CH2 domain in Fc district. Another is real Execute in the scheme, the amino acid that substitutes is arranged in the CH3 domain in Fc district. In another embodiment, replace The amino acid in generation is arranged in CH2 and two domains of CH3 in Fc district.

In the embodiment, the polypeptide of change of the present invention comprises at least one amino acid in the Fc district Sudden change, the effect of this sudden change is to strengthen the half-life of molecule in blood. Half-life with increase Molecule have the cyclical administration that reduces simultaneously medicine or the advantage that reduces alternatively drug dose, and Keep identical pharmacokinetics spectrum.

In another embodiment, the polypeptide of change of the present invention comprises at least one amino acid in the Fc district Sudden change, the effect of this sudden change is to reduce the half-life of this antibody in blood. Have partly declining of reduction The molecule of phase has patient's open-assembly time of minimizing and the advantage of dosage. When the antibody that changes is puted together toxicity Or during radiopharmaceutical (for example anticancer therapeutic agent) diagnostic flag since must balance antibody non-specific In conjunction with and specific binding, so the half-life that reduces be even more important.

The change of half-life or other antigen dependent/non-dependent effector function can be from initial antibody and change The difference of antibody on the FcRn binding affinity predict.

In another exemplary, by regulating the FcRn binding affinity, regulate the tissue of polypeptide Distribute or bioavilability. In the embodiment, the polypeptide that the present invention changes in the Fc district, comprise to Few one causes polypeptide to particular target tissue (for example mucomembranous surface or disease site, for example tumour or other Special disease take pathology as feature) amino acid mutation that location increases.

In another embodiment, the polypeptide that the present invention changes comprises at least one in the Fc district and causes described changing The polypeptide that becomes is at the amino acid mutation that reduces such as the location in the undertissue, and wherein said tissue is not to changing The effect sensitivity of initial polypeptide. In the exemplary, the polypeptide of change demonstrates from female Systemic circulatory system shifts minimizing to the placenta of fetus. Its of (or increasing) location that may have benefited from reducing Its sensitive organization comprises brain, kidney and liver. In the exemplary, change of the present invention Polypeptide demonstrates the transhipment minimizing of striding glomerular epithelium from vascular system. In another embodiment, this The polypeptide of bright change demonstrates the transhipment minimizing of striding blood-brain barrier (BBB) from brain.

In another embodiment, the polypeptide of change of the present invention comprises at least one amino acid in the Fc district Sudden change, this amino acid mutation cause the IgG of the accumulation that mediated by FcRn and glomerular epithelium cell In conjunction with minimizing, and cause comparing less membranous nephropathy with the initial polypeptide that lacks this sudden change. (the 2004.Nephron Exp.Nephrol.90:e13-e21 such as Haymann)

The increase of the tissue location of Fc (or contain Fc polypeptide) or reduce can be respectively right by this Fc district The increase of the affinity of FcRn (neonatal Fc receptor) or minimizing reflect. Similarly, Fc polypeptide The Tissue distribution of FcRn binding affinity and Fc polypeptide or the correlation of bioavilability also meet FcRn Enhancing antibody is by the biological action of transcytosis transepithelial barrier transhipment.

In some embodiments, the polypeptide of change of the present invention demonstrates the antigen dependent/non-dependent effect of change Answer subfunction but do not change antigen dependence effector function (for example ADCC or CDC). Other enforcement In the scheme, the polypeptide of change of the present invention has antigen dependent/non-dependent effector function and the change of change Antigen dependence effector function. In the embodiment, disclosed herein one or more prominent Change can be given the antigen dependence effector function of increase and the half-life of reduction.

In another embodiment, one or more sudden changes disclosed herein can be given the antigen of increase The half-life of dependence effector function and increase. In another embodiment, one disclosed herein Or a plurality of sudden changes can be given the antigen dependence effector function of reduction and the half-life of reduction. Another In the embodiment, one or more sudden changes disclosed herein can be given the antigen dependence effect of reduction Answer the half-life of subfunction and increase.

In specific embodiments, the polypeptide of change of the present invention is included in corresponding to being selected from following EU Substituting of the amino acid position of amino acid position: 248,249,250,251,252,255,256,257,258, 260,277,279,280,281,282,283,284,285,286,287,288,289,290,304,305, 306,307,309,310,311,312,313,314,315,316,317,340,343, 344,345,374,376,378,383,386,387,389,426,428,430,431,432,434,436, or 438.

In an exemplary, the polypeptide of described change is at 15  FcRn contact zones or " band " Amino acid (for example, from the EU position 243 to 261,275 to the EU positions 280 from the EU position, from 282 to EU positions 293, EU position, 302 to the EU positions 319 from the EU position are from the EU position 336 to EU positions 348, EU position 367, EU position 369,372 to the EU positions from the EU position 389, EU position 391, EU position 393, EU position 408 and 424 to the EU positions from the EU position 440) comprise alternative in.

In another exemplary, the polypeptide of change is comprising EU amino acid position 280,281, 282,283,284,285,286,287,288,289,290,291,292,293,294,295,296,297, Comprise alternative in 298 and 299 the FcRn contact ring.

The site, exemplary Fc district (or its FcRn bound fraction) that is used for sudden change comprises EU position 280, 281,282,283,285,286,288,289,290,305,307,308,309,315,340,344 and 378.

In another embodiment, the polypeptide of change can comprise that arbitrary or any combination of following sudden change is (straight To all): EU position 248 is replaced by aspartic acid; With EU position 249 be replaced by arginine or Lysine; EU position 250 is replaced by arginine or lysine; EU position 251 is replaced by essence Propylhomoserin, lysine, or asparagine; EU position 252 is replaced by glutamine, asparagine, silk Propylhomoserin or threonine; EU position 255 is replaced by methionine, aspartic acid, or leucine; With EU Position 256 is replaced by arginine, glutamic acid, or lysine; EU position 257 is replaced by arginine, Aspartic acid, glutamic acid, or lysine; EU position 258 is replaced by arginine, glutamine, or rely Propylhomoserin; EU position 260 is replaced by lysine; EU position 277 is replaced by arginine, asparagus fern ammonia Acid, glutamine, or lysine; EU position 279 is replaced by arginine, aspartic acid, glutamic acid, Or lysine; EU position 280 is replaced by asparagine; EU position 281 is replaced by asparagus fern ammonia Acid, glutamic acid, or glutamine; EU position 282 is replaced by arginine, aspartic acid, paddy ammonia Acid, or lysine; EU position 283 is replaced by glutamine; EU position 284 is replaced by smart ammonia Acid, lysine, aspartic acid or glutamic acid; EU position 285 is replaced by arginine, aspartic acid, Glutamic acid, lysine, proline, threonine, or phenylalanine; EU position 286 is replaced by asparagus fern Propylhomoserin, glutamic acid, threonine, or methionine; EU position 287 is replaced by aspartic acid, paddy ammonia Acid, lysine, proline, or threonine; EU position 288 is replaced by methionine, arginine, day Winter propylhomoserin or glutamic acid; EU position 289 is replaced by proline; EU position 290 is replaced by dried meat Propylhomoserin, aspartic acid or glutamic acid; EU position 304 is replaced by aspartic acid or glutamic acid; With EU Position 305 is replaced by arginine or threonine; EU position 306 is replaced by arginine, aspartic acid, Glutamic acid, or lysine; EU position 307 is replaced by arginine, proline, aspartic acid, or paddy ammonia Acid; EU position 308 is replaced by arginine, aspartic acid, glutamic acid, lysine, or isoleucine; Position 309 is replaced by threonine, arginine, aspartic acid, lysine or glutamic acid; With the EU position 310 are replaced by arginine, leucine, lysine or asparagine; EU position 311 is replaced by smart ammonia Acid or lysine; EU position 312 is replaced by arginine, asparagine, leucine, or lysine; EU position 313 is replaced by aspartic acid, arginine, or lysine; EU position 314 is replaced by Arginine, lysine, or asparagine; EU position 315 is replaced by arginine, aspartic acid or paddy Propylhomoserin; EU position 316 is replaced by aspartic acid or lysine; EU position 317 is replaced by the sky Winter propylhomoserin or glutamic acid; EU position 340 is replaced by arginine; EU position 343 is replaced by paddy Glutamine or lysine; EU position 344 is replaced by leucine; EU position 345 is replaced by essence Propylhomoserin, lysine, or glutamine; EU position 374 is replaced by arginine, lysine, or leucine; EU position 376 is replaced by asparagine, arginine, leucine, or lysine; With EU position 378 Be replaced by serine; EU position 383 is replaced by lysine; In EU position 385 glycine is substituted Be lysine; In EU position 386 glutamine is replaced by alanine or lysine; With the EU position 387 are replaced by alanine; EU position 389 is replaced by aspartic acid; EU position 426 is replaced by Arginine, aspartic acid, or glutamic acid; EU position 428 is replaced by aspartic acid, glutamic acid, essence Propylhomoserin, glutamine, or lysine; EU position 430 is replaced by arginine, glutamine, first sulphur Propylhomoserin, or lysine; EU position 431 is replaced by lysine or proline; EU position 432 is replaced On behalf of arginine and phenylalanine; EU position 434 is replaced by leucine, arginine, or lysine; EU position 436 is replaced by arginine or glutamic acid; Or EU position 438 is replaced by glutamic acid.

More particularly, the polypeptide of change can comprise arbitrary or any associating (until owning) of following sudden change: Lysine is replaced by aspartic acid in EU position 248; Aspartic acid 249 is substituted in the EU position Be arginine or lysine; Threonine is replaced by arginine or lysine in EU position 250; With bright Propylhomoserin is replaced by arginine in EU position 251, lysine, or asparagine; With methionine at EU Position 252 is replaced by glutamine, asparagine, serine or threonine; With arginine in the EU position Put 255 and be replaced by methionine, aspartic acid, or leucine; Threonine 256 is substituted in the EU position Be arginine, glutamic acid, or lysine; Proline is replaced by arginine in EU position 257, asparagus fern Propylhomoserin, glutamic acid, or lysine; Glutamic acid is replaced by arginine in EU position 258, glutamine, Or lysine; Threonine is replaced by lysine in EU position 260; With tryptophan in EU position 277 Be replaced by arginine, aspartic acid, glutamic acid, glutamine, or lysine; With valine in the EU position Put 279 and be replaced by arginine, aspartic acid, glutamic acid, or lysine; With aspartic acid in the EU position 280 are replaced by asparagine; Glycine is replaced by aspartic acid in EU position 281, glutamic acid, or Glutamine; Valine is replaced by arginine in EU position 282, aspartic acid, glutamic acid, or rely Propylhomoserin; Glutamic acid is replaced by glutamine in EU position 283; With valine in EU position 284 Be replaced by arginine, lysine, aspartic acid or glutamic acid; With histidine or alanine in the EU position 285 are replaced by arginine, aspartic acid, glutamic acid, lysine, proline, threonine, or phenylpropyl alcohol ammonia Acid; Asparagine or lysine are replaced by aspartic acid in EU position 286, glutamic acid, threonine, Or methionine; Alanine is replaced by aspartic acid in EU position 287, glutamic acid, lysine, dried meat Propylhomoserin, or threonine; Lysine is replaced by methionine in EU position 288, arginine, asparagus fern ammonia Acid or glutamic acid; Threonine is replaced by proline in EU position 289; With lysine in the EU position 290 are replaced by proline, aspartic acid or glutamic acid; Serine is replaced by the sky in EU position 304 Winter propylhomoserin or glutamic acid; Valine is replaced by arginine or threonine in EU position 305; With bright ammonia Acid or valine are replaced by arginine in EU position 306, aspartic acid, glutamic acid, or lysine; Will Threonine or valine are replaced by arginine in EU position 307, proline, aspartic acid, or glutamic acid; Valine is replaced by arginine in EU position 308, aspartic acid, glutamic acid, lysine, or different bright Propylhomoserin; Leucine is replaced by threonine in EU position 309, arginine, aspartic acid, lysine or Glutamic acid; Histidine is replaced by arginine in EU position 310, leucine, lysine or asparagus fern acyl Amine; Glutamine is replaced by arginine or lysine in EU position 311; With aspartic acid or bright ammonia Acid is replaced by arginine in EU position 312, asparagine, leucine, or lysine; With asparagine Be replaced by aspartic acid in EU position 313, arginine, or lysine; With leucine in EU position 314 Be replaced by arginine, lysine, or asparagine; Asparagine is replaced by smart ammonia in EU position 315 Acid, aspartic acid or glutamic acid; Asparagine or glycine are replaced by asparagus fern ammonia in EU position 316 Acid or lysine; Lysine is replaced by aspartic acid or glutamic acid in EU position 317; With lysine Be replaced by arginine in EU position 340; With proline be replaced by in EU position 343 glutamine or Lysine; Arginine is replaced by leucine in EU position 344; With glutamic acid in EU position 345 Be replaced by arginine, lysine, or glutamine; Proline is replaced by arginine in EU position 374, Lysine, or leucine; Aspartic acid is replaced by asparagine in EU position 376, arginine, bright Propylhomoserin, or lysine; Alanine is replaced by serine in EU position 378; With serine in the EU position Put 383 and be replaced by lysine; Glycine is replaced by lysine in EU position 385; Glutamine is existed EU position 386 is replaced by alanine or lysine; Proline is replaced by third ammonia in EU position 387 Acid; Asparagine is replaced by aspartic acid in EU position 389; With serine in EU position 426 Be replaced by arginine, aspartic acid, or glutamic acid; Methionine is replaced by asparagus fern in EU position 428 Propylhomoserin, glutamic acid, arginine, glutamine, or lysine; Glutamic acid 430 is substituted in the EU position Be arginine, glutamine, methionine, or lysine; Leucine is replaced by in EU position 431 Lysine or proline; Histidine is replaced by arginine in EU position 432 is replaced by phenylalanine; Asparagine is replaced by leucine in EU position 434, arginine, or lysine; Tyrosine is existed EU position 436 is replaced by arginine or glutamic acid; Or glutamine is replaced by paddy in EU position 438 Propylhomoserin.

In another embodiment, it is prominent that polypeptide of the present invention can contain at least one amino acid that is selected from lower group Become: according to the EU numbering system, EU position 281 is replaced by E, EU position 282 is replaced by E is replaced by Q with EU position 283, and EU position 285 is replaced by R, and EU position 286 is replaced On behalf of T, EU position 288 is replaced by R, EU position 289 is replaced by P, with EU position 290 Be replaced by P, EU position 305 is replaced by T, EU position 307 is replaced by P, with the EU position 308 are replaced by I, and EU position 309 is replaced by T, and EU position 315 is replaced by R, with the EU position Put 340 and be replaced by R, EU position 344 is replaced by L, EU position 378 is replaced by S, with EU Position 383 is replaced by K, and EU position 385 is replaced by K, with EU position 386 be replaced by A and EU position 389 is replaced by D.

More particularly, polypeptide of the present invention can contain the amino acid mutation that at least one is selected from lower group: root According to the EU numbering system, (wherein the D indication is at described EU position (280) ammonia to be suddenlyd change for Asp280Asn The sour position of base, and N indication for the polypeptide that reaches described change at this position amino acid to be substituted), Gly281Glu, Val282Glu, Glu283Gln, His285Arg, Asn286Thr, Lys288Arg, Thr289Pro, Lys290Pro, Val305Thr, Thr307Pro, Val308Ile, Leu309Thr, Asn315Arg, Lys340Arg, Arg344Leu, Ala378Ser, Ser383Lys, Glu386Lys, Pro387Ala, and Asn389Asp.

In a particular, the polypeptide of change is corresponding to the ammonia that is selected from lower group EU position The sour position of base comprises alternative: 248,249,250,251,252,, 256,255,260,257,277,281,282, 287,284,285,286,288,290; 304,305,306,307,309,310,312,313,315,343, 374,426,428,430,431,432,434, or 438.

In another embodiment, the polypeptide of change can comprise that arbitrary or any combination of following sudden change is (straight To all): EU position 248 is replaced by aspartic acid; With EU position 249 be replaced by arginine or Lysine; EU position 250 is replaced by arginine or lysine; EU position 251 is replaced by essence Propylhomoserin, lysine, or asparagine; EU position 252 is replaced by serine or threonine; With EU Position 256 is replaced by arginine, glutamic acid, or lysine; EU position 255 is replaced by leucine, Aspartic acid or methionine; EU position 260 is replaced by lysine; With EU position 257 smart ammonia Acid, aspartic acid, glutamic acid, or lysine; EU position 277 is replaced by arginine, aspartic acid, Glutamine, or lysine; EU position 279 is replaced by glutamic acid; EU position 281 is replaced by Glutamine; EU position 282 is replaced by arginine, aspartic acid, glutamic acid, or lysine; Will EU position 287 is replaced by aspartic acid, glutamic acid, lysine, proline, or threonine; With the EU position Put 284 and be replaced by aspartic acid or glutamic acid; EU position 285 is replaced by aspartic acid, glutamic acid Or phenylalanine; EU position 286 is replaced by aspartic acid, glutamic acid, or methionine; With EU Position 288 is replaced by aspartic acid or glutamic acid; EU position 290 is replaced by aspartic acid or paddy ammonia Acid; EU position 304 is replaced by aspartic acid or glutamic acid; EU position 305 is replaced by smart ammonia Acid; EU position 306 is replaced by arginine, aspartic acid, glutamic acid, or lysine; With the EU position 307 are replaced by arginine, aspartic acid, or glutamic acid; 309 positions are replaced by arginine, asparagus fern ammonia Acid, lysine or glutamic acid; EU position 310 is replaced by arginine, leucine, lysine or asparagus fern Acid amides; EU position 312 is replaced by arginine, asparagine, or lysine; With EU position 313 Be replaced by aspartic acid, arginine, or lysine; EU position 315 is replaced by aspartic acid or paddy ammonia Acid; EU position 343 is replaced by glutamine or lysine; EU position 345 is replaced by smart ammonia Acid or glutamine; EU position 374 is replaced by arginine, lysine, or leucine; With the EU position Put 376 and be replaced by asparagine; EU position 426 is replaced by arginine, aspartic acid, or glutamic acid; EU position 428 is replaced by arginine, glutamine, or lysine; EU position 430 is replaced by Lysine; EU position 431 is replaced by proline; EU position 432 is replaced by arginine; Will EU position 434 is replaced by leucine or lysine; Or EU position 438 is replaced by glutamic acid.

In another embodiment, introduce substituting in the Fc district of IgG1, described substituting is selected from: K248D, D249R, D249K, T250R, T250K, L251R, L251K, L251N, M252S, M252T, M252Q, M252N, R255D, R255L, R255M, T256R, T256E, T256K, P257R, P257D, P257E, P257K, E258R.E258Q.E258K, T260K, T260R, T260K, T260Q, W277R, W277D, W277Q, W277E, W277K, V279R, V279D, V279E, V279K, D280N, G281D, G281E, G281Q, V282R, V282D, V282E, V282K, E283Q, V284R, V284D, V284E, V284K, H285R, H285D, H285E, H285K, H285P, H285T, H285F, A285D, A285E, N286D, N286E, N286T, N286M, A286M, A286E, A286D, A287D, A287E, A287K, A287P, A287T, K288D, K288E, K288M, K288R, T289P, K290D, K290E, K290P, S304D, S304E, V305T, V305R, V306D, V306E, L306R, L306D, L306E, L306K, V307E, T307R, T307D, T307P, V308R, V308D, V308E, V308K, V308I, L309R, L309D, L309E, L309K, L309T, H310R, H310N, H310L, H310K, L312K, Q311R, Q311K, D312R, D312N, D312L, D312K, L312K, N313R, N313K, W313D, N313K, W313K, L314R, L314N, L314K, N315R, N315D, N315E, N316D, N316K, K317D, K317E, K340R, P343Q, P343K, R344L, E345R, E345Q, E345R, P374R, P374L, P374K, D376R, D376L, D376K, D376N, A378S, S383K, G385K, Q386A, Q386A, P387A, N389D, S426R, S426D, S426E, M428R, M428D, M428E, E430R, E430Q, E430K, E430M, A431P, L431K, H432F, L432R, N434R, N434K, N434L, Y436R, Y436E or Q438E.

As mentioned above, can understand that the present composition can comprise one or more described herein prominent Become. In the embodiment, the polypeptide of change of the present invention comprises only listed sudden change of this paper. One In the individual embodiment, the polypeptide of change of the present invention comprises only two listed sudden changes of this paper. An enforcement In the scheme, the polypeptide of change of the present invention comprises only three sudden changes that this paper is listed. An embodiment In, the polypeptide of change of the present invention comprises only four sudden changes that this paper is listed.

A. the polypeptide of change that has the FcRn binding affinity of enhancing

In the embodiment, the invention provides with corresponding initial polypeptide and compare the neonate with enhancing The polypeptide of the change of Fc receptor affinity. Preferably, the polypeptide of this change demonstrates than not containing sudden change The circulating half-life that corresponding polypeptide is long.

In the embodiment, have enhancing the FcRn binding affinity change polypeptide can with One of lower EU position comprises at least one amino acid replacement: 284,285,286,288,290 and 304.

In another embodiment, the polypeptide of change with FcRn binding affinity of enhancing can comprise At least one following amino acid replacement: EU position 284 is replaced by glutamic acid; With EU position 285 Be replaced by glutamic acid; EU position 286 is replaced by aspartic acid; EU position 288 is replaced by paddy Propylhomoserin; EU position 290 is replaced by glutamic acid.

In an exemplary, the polypeptide of described change comprises the Fc district of IgG1 molecule. Preferably, this molecule contains at least one following sudden change: V282E, V284E, and H285E, N286D, N286E, K288E, K290E, and S304D.

In a preferred embodiment of the invention, compare the FcRn of modified polypeptide with initial polypeptide At least about 30%, 50%, 80%, 2 times of binding affinity increase, 3 times, 4 times, 5 times, 10 times, 15 times, 20 times, 25 times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or 100 times.

When being administered to the patient, the polypeptide of change of the present invention can have little above 1 in patient body The time, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 10 days, 12 days, the circulating half-life in 2 weeks, 3 weeks or 1 month. In the exemplary, the polypeptide of change of the present invention has in patient body and surpasses 21 days Circulating half-life.

In the embodiment, the binding molecule of described change can be organized in particular target, and is for example ill The location or the bioavilability that have increase in the tissue. Exemplary illing tissue comprise the neoplasia tissue or Tumour or other tissue or organ take the pathology of any illness described herein as feature, comprise brain or CNS, lung, the heart, pancreas, liver, kidney, bladder, stomach, large intestine or small intestine, respiratory tract, lymph node, flesh Meat, epidermis, bone, cartilage, joint, blood vessel, marrow, prostate, ovary or uterus.

B. the polypeptide of change that has the FcRn binding affinity of reduction

In another embodiment, the invention provides neonatal Fc receptor than corresponding initial many Peptide has the polypeptide of change of the affinity of reduction. Preferably, the polypeptide of described change show with not The corresponding polypeptide that the contains sudden change short circulating half-life of comparing.

The polypeptide of change with FcRn binding affinity of reduction can comprise in the EU position at least one Individual amino acid replacement, wherein said EU position is selected from one of lower area: a) from 248 to 260; B) from 277 to 315; C) from 343 to 374; And d) from 426 to 438.

In the embodiment, the polypeptide of change with FcRn binding affinity of reduction can comprise the amino acid replacement that at least one is positioned at one of following EU position: 248,249,250,251,252,256,255,260,257,277,281,282,287,284,285,286,288,290; 304,305,306,307,309,310,312,313,315,343,374,426,428,430,431,432,434, or 438.

In another embodiment, the Fc polypeptide of change with FcRn binding affinity of reduction can comprise at least one following amino acid replacement: EU position 248 is replaced by aspartic acid; EU position 249 is replaced by arginine or Methionin; EU position 250 is replaced by arginine or Methionin; EU position 251 is replaced by arginine, Methionin, or l-asparagine; EU position 252 is replaced by Serine or Threonine; EU position 256 is replaced by arginine, L-glutamic acid, or Methionin; EU position 255 is replaced by leucine, aspartic acid or methionine(Met); EU position 260 is replaced by Methionin; EU position 257 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin; EU position 277 is replaced by arginine, aspartic acid, glutamine, or Methionin; EU position 279 is replaced by L-glutamic acid; EU position 281 is replaced by glutamine; EU position 282 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin; EU position 287 is replaced by aspartic acid, L-glutamic acid, Methionin, proline(Pro), or Threonine; EU position 284 is replaced by aspartic acid; EU position 285 is replaced by aspartic acid or phenylalanine; EU position 286 is replaced by L-glutamic acid or methionine(Met); EU position 288 is replaced by aspartic acid; EU position 290 is replaced by aspartic acid or L-glutamic acid; EU position 304 is replaced by aspartic acid or L-glutamic acid; EU position 305 is replaced by arginine; EU position 306 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin; EU position 307 is replaced by arginine, aspartic acid, or L-glutamic acid; Position 309 is replaced by arginine, aspartic acid, Methionin or L-glutamic acid; EU position 310 is replaced by arginine, leucine, Methionin or l-asparagine; EU position 312 is replaced by arginine, l-asparagine, or Methionin; EU position 313 is replaced by aspartic acid, arginine, or Methionin; EU position 315 is replaced by aspartic acid or L-glutamic acid; EU position 343 is replaced by glutamine or Methionin; EU position 345 is replaced by arginine or glutamine; EU position 374 is replaced by arginine, Methionin, or leucine; EU position 376 is replaced by l-asparagine; EU position 426 is replaced by arginine, aspartic acid, or L-glutamic acid; EU position 428 is replaced by arginine, glutamine, or Methionin; EU position 430 is replaced by Methionin; EU position 431 is replaced by proline(Pro); EU position 432 is replaced by arginine; EU position 434 is replaced by leucine (leucine) or Methionin; Or EU position 438 is replaced by L-glutamic acid.

In another embodiment, the polypeptide of change can comprise arbitrary or any combination (up to all) of following sudden change: K248D, D249R, D249K, T250R, T250K, L251R, L251K, L251N, M255S, M255T, T256R, T256E, T256K, R255D, R255L, R255M, T260K, T260R, T260K, T260Q, P257R, P257D, P257E, P257K, W277R, W277D, W277Q, W277K, V279E, G281Q, V282R, V282D, V282E, V282K, V282E, A287D, A285E, A287K, A287P, A287T, V284D,, H285D, H285F, N286E, N286M, K288D, K290D, K290E, S304D, S304E, V305R, V306E, L306R, L306D, L306E, L306K, V307E, T307R, T307D, L309R, L309D, L309E, L309K, H310R, H310N, H310L, H310K, L312K, D312R, D312N, D312K, N313R, W313D, N313K, W313K, N315D, N315E, P343Q, P343K, E345R, P374R, P374L, P374K, D376N, S426R, S426D, S426E, E430K, A431P, L432R, N434K, N434L, or Q438E.

In some preferred embodiment, the Fc of change with FcRn binding affinity of reduction can comprise at least one sudden change: M252S, M252T, V282E, K288E; V308E, V308D, L314K, N434L, or Q438E.In a more preferred embodiment, the polypeptide of change with FcRn binding affinity of reduction can comprise at least one sudden change: M252S, M252T, N434L or Q438E.In preferred embodiment again, have the Fc polypeptide that the FcRn bonded of reduction changes and comprise sudden change N434L or Q438.In another embodiment, the polypeptide of change with FcRn binding affinity of reduction can comprise at least one following amino acid replacement: EU position 252 is replaced by Threonine; EU position 255 is replaced by aspartic acid; EU position 282 is replaced by arginine, aspartic acid, L-glutamic acid or Methionin; Position 309 is replaced by arginine, aspartic acid, Methionin or L-glutamic acid; With position 434 is replaced by leucine..

In an exemplary, the polypeptide of described change comprises the Fc district of IgG1 molecule.Preferably, this molecule comprises at least one following sudden change: M252T, R255D, V282R, V282D, V282E, V282K, L309R, L309D, L309K, L309E, or N434L.

In a preferred embodiment of the invention, to compare with initial polypeptide, modified polypeptide is reduced by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% to the binding affinity of FcRn, or 99%.

When being administered to the patient, the polypeptide of change of the present invention can have in patient's body and is less than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 10 days, 12 days, 2 weeks, the circulating half-life in 3 weeks or 1 month.In an exemplary, the polypeptide of change of the present invention has in patient's body and is less than about 21 days circulating half-life.

In the embodiment, the polypeptide of change can be in particular organization, for example is vulnerable to have in the tissue of toxicity injury of unaltered binding molecule the location or the bioavailability of reduction.The example organization that typically is vulnerable to the toxic action injury of therapeutical agent comprises brain or CNS, heart, liver and kidney.In another embodiment, the location of going to fetus or placenta that the polypeptide of change can have reduction shift.In another embodiment, the polypeptide of change when by milk or colostrum picked-up, can have newborn infant's recycle system location of reduction.

V. the polypeptide expression of Gai Bianing

Polypeptide of the present invention, for example initial polypeptide and modified polypeptide can pass through recombinant methods.

For example, the polynucleotide of coded polypeptide can insert be used in the suitable expression vector recombinant expressed.When polypeptide was antibody, the polynucleotide of encode extra light chain and variable region of heavy chain (randomly being connected with constant region) can insert in the same or different expression vectors.Affinity tag sequence (for example His (6) label) can randomly be attached to initial peptide sequence or be included in the initial peptide sequence and be beneficial to downstream purification.Guarantee that the control sequence of the expression of immunoglobulin polypeptides is operably connected in dna fragmentation and the expression vector of coding immunoglobulin chain.Expression control sequenc includes but not limited to promotor (for example, natural bonded or allogenic promotor), signal sequence, enhancer element and transcription termination sequence.Preferably, expression control sequenc can transform or the carrier of transfection eukaryotic host cell in be the eukaryotic promoter system.In case after mixing carrier among the suitable host, under the condition that is suitable for collection of this nucleotide sequence high level expression and polypeptide and purifying, keep the host.

These expression vectors typically can duplicate in host organisms as episome or as an integral part of host chromosome DNA.Usually, expression vector contains selective marker (for example, amicillin resistance, hygromycin resistance, tetracyclin resistance or neomycin resistance) to allow to detect those cells (for example seeing U.S. Patent number 4,703,362) of the dna sequence dna conversion that is supposed to.

Intestinal bacteria (E.coli) are the prokaryotic hosts of a kind of clone who is particularly useful polynucleotide of the present invention (for example dna sequence dna).Other microorganism host that is suitable for comprises genus bacillus (bacilli), for example subtilis (Bacillus subtilus) and other enterobacteriaceae (Enterobacteriaceae), for example Salmonellas (Salmonella), Serratia (Serratia) and various Rhodopseudomonas (Pseudomonas) species.

Other microorganism for example yeast also can be used for expressing.Yeast belong (Saccharomyces) and Pichia (Pichia) are exemplary yeast hosts, and suitable carrier has expression control sequenc (for example promotor), replication orgin, terminator sequence and according to the sequence of expectation or the like sequence.Typical promotor comprises glycerol 3-phosphate acid kinase and other glycolytic ferment promotor.The induction type Yeast promoter for example comprises the promotor from the enzyme of aldehyde dehydrogenase, isocytochrome C and responsible methyl alcohol, maltose and galactose utilization.

Except microorganism, also can use the mammalian tissues culture to express and produce polypeptide of the present invention (for example encode immunoglobulin (Ig) or its segmental polynucleotide).See Winnacker, From Genes toClones, VCH Publishers, N.Y., N.Y. (1987).It is preferred that eukaryotic cell is actually, because this area developed in a large number can secretion heterogenous protein (for example complete immunoglobulin (Ig)) suitable host cell system, comprise the B cell and the hybridoma of Chinese hamster ovary celI system, various Cos clone, HeLa cell, 293 cells, myeloma cell line, conversion.The expression vector that is used for these cells comprises expression control sequenc, for example replication orgin, promotor and enhanser (Queen etc., Immunol.Rev.89:49 (1986)) and essential machining information site for example ribosome bind site, RNA splice site, polyadenylation site and transcription termination sequence.Preferred expression control sequenc is to derive from immunoglobulin gene, SV40, adenovirus, bovine papilloma virus.The promotor of cytomegalovirus etc.See Co etc., J.Immunol.148:1149 (1992).

The carrier that contains polynucleotide of interest sequence (for example heavy chain and light chain encoding sequence and expression control sequenc) can be transferred in the host cell by well-known process, and described method can be different according to the type of cell host.For example, the calcium chloride transfection is generally used for prokaryotic cell prokaryocyte, and calcium phosphate processing, electroporation, fat transfection, biological bombardment or can be used for other cell host based on the transfection of virus.(generally seeing Sambrook etc., Molecular Cloning:A Laboratory Manual (Cold Spring HarborPress), second edition, 1989).Other method that is used for transformed mammalian cell comprises uses Polybrene, protoplastis fusion, liposome, electroporation and microinjection (generally seeing Sambrook etc., the same quoted passage).For the preparation of transgenic animal, can with the transgenosis microinjection to the ovocyte of fertilization, perhaps it can be mixed the genome of embryonic stem cell, and the nucleus of this cell is transferred in the enucleation oocyte.

Also polypeptide of the present invention can be mixed and be used to import the genome of transgenic animal in the transgenosis and express subsequently, for example, in the milk of transgenic animal, express and (for example see Deboer etc., 5,741,957; Rosen5,304,489; With Meade 5,849,992).Suitable transgenosis comprises operationally the light chain that is connected with enhanser with promotor from mammary gland specific gene such as casein or beta lactoglobulin and/or the encoding sequence of heavy chain.

The polypeptide (for example polypeptide) that changes can use single carrier or two vector expressions.For example, heavy chain of antibody and light chain can be cloned on the different expression vectors, and cotransfection is to cell.

In the embodiment, can use signal sequence to be beneficial to polypeptide expression of the present invention.

In case after expressing, polypeptide can be according to the ability standard method, (for example comprise ammonium sulfate precipitation, affinity column, albumin A or Protein G), purifying such as column chromatography, HPLC purifying, gel electrophoresis (generally sees Scopes, Protein Purification (Springer-Verlag, N.Y., (1982)).In the preferred embodiment, purification process can use following polymer Fc acceptor of the present invention.

VI. analyze binding affinity

Can measure binding affinity in many ways.Usually, and regardless of being used for determining or measuring avidity butt formula really, the inventive method is regulated the binding affinity with FcRn, and when the inventive method produces polypeptide, this polypeptide aspect its clinical application any, be better than its from initial polypeptide (for example, when modified polypeptides can with low dosage more or more low frequency or so that route of administration is used more easily or when modified polypeptides had the bio distribution of the side effect of reduction or change, it is effective or successful that the inventive method is considered to).

The change of polypeptide on effector function that changes can be determined by measuring its binding affinity to specific Fc acceptor.In the embodiment, can pass through to measure the binding affinity of the polypeptide of change, determine the change of antigen dependency effector function Fc γ acceptor.In another embodiment, can determine antigen dependent/non-dependent effector function (for example, transformation period or bio distribution) by measuring binding affinity to other Fc acceptor, especially neonatal Fc receptor (for example people FcRn).

The change of polypeptide on binding affinity that the present invention changes can be determined with the binding affinity of the contrast polypeptide (for example corresponding initial polypeptide) that suits by the polypeptide that relatively changes.In the embodiment, can pass through binding affinity and contrast polypeptide the binding affinity in second combination test of polypeptide in first test of relatively change, determine the change of binding affinity.In alternative embodiment, can determine the variation of binding affinity by polypeptide and the binding affinity of contrast polypeptide in identical test that relatively changes.For example, described test can be a CBA, wherein uses the contrast polypeptide of progressive concentration, estimates the binding affinity of the polypeptide of described change.In a particular, can under a pH (for example acid pH) and the 2nd pH (for example alkaline pH), measure binding affinity to Fc acceptor (for example FcRn).

More particularly, can be by various tests, for example comprise surface plasma body resonant vibration (for example BiaCore test), analytical super centrifugal, gel-filtration, FRET and ELISAL or KinExA ^TM3000 tests (can be measured the avidity between polypeptide and its bonded acceptor from Sapidyne Instruments (Boise ID) obtains).Exemplary test is at following detailed description.

I) Cell free assay

Several external Cell free assay that are used to check effector function (for example FcR binding affinity) have been described in this area.Preferably, can estimate the antibody and the solubility Fc acceptor form of change, for example combination of monomer Fc acceptor or polymer Fc acceptor of the present invention based on the test of cell.Automatically the HTS technology can be used for this screening procedure.Screening can be used the marker (for example isotopic label, chromophore, fluorophore, luminophor (lumiphore) or epi-position) that allows to implement detection.Marker can adhere to polymer Fc acceptor to be analyzed or the polypeptide that contains Fc.

Exemplary Cell free assay includes but not limited to FRET (fluorescence resonance energy transmission), BRET (transmission of noclilucence resonance energy), based on the test of Alphascreen (Amplied Luminescent ProximityHomogeneous), flicker near detection method (scintillation proximity assays), ELISA (enzyme linked immunosorbent assay), SPR (surface plasma body resonant vibration, BIACORE  for example), constant temperature titration calorimetry, differential scanning calorimetry, gel electrophoresis, analytical super centrifugal and chromatography, comprise gel permeation chromatography.

Ii) based on the test of cell

The testing based on cells in vitro of effector function (for example, FcR binding affinity) of several polypeptide that are used to check change described in this area.Preferably, the Fc acceptor of the antibody that can estimate change based on the test of cell and format surface combines.Exemplary test based on cell comprises bridge joint test (bridging assay) and flow cytometry.

In an exemplary, can use the test of FcR bridge joint, measure the FcR binding affinity of the polypeptide that changes.Can with based on antibody at antigen with carry the test that forms the ability of " bridge " between the cell of FcR, measure FcR (for example, FcRn or Fc γ R) binding affinity.

Ii) animal pattern test

For the veterinary science purpose or as the animal model of human diseases (for example above-mentioned Immunological diseases or illness), also the polypeptide that the present invention changes can be administered to animal pattern, so that by transformation period or the bio distribution of for example checking antibody, the potentiality that the polypeptide that inspection the present invention changes is used for the treatment of.About the latter, this animal model can be used to estimate the curative effect (for example check effector function, dosage and use time-histories) of antibody of the present invention.

In the embodiment, can check the improvement of polypeptide aspect circulating half-life that the present invention changes.The polypeptide of change with enhanced transformation period when being administered to animal pattern can or expection will be retained in the circulation more longways than the corresponding polypeptide that does not contain sudden change (the same type antibody (for example IgG such as IgG1) that for example, on the polypeptide that changes comprises the position of sudden change, does not contain sudden change).For example, owing to be the typical β phase transformation period of human antibodies in 21 days, the antibody that the present invention changes can be those antibody of being longer than cycling time 21 days.In another embodiment, the polypeptide of change has the transformation period of reduction with respect to target antibody.For example, can select to have the antibody of change of the transformation period (for example, being less than 21 days) of reduction with respect to target antibody.

The animal model example that can be used to estimate the polypeptide prevention of the present invention's change or treat the curative effect of tumour formation comprises the tumour heteroplastic transplantation model.

The animal model example that can be used to estimate the curative effect of polypeptide prevention that the present invention changes or treatment rheumatoid arthritis (RA) comprises adjuvant inductive RA, collagen-induced RA and collagen mAb inductive RA (Holmdahl etc., Immunol.Rev.184:184,2001; Holmdahl etc., Ageing Res.Rev.1:135,2002; Van den Berg, Curr.Rheumatol.Rep.4:232,2002).

The animal model example that can be used to estimate the polypeptide prevention of the present invention's change or treat the curative effect of inflammatory bowel (IBD) comprises TNBS inductive IBD, DSS inductive IBD (Padol etc., Eur.J.Gastrolenterol.Hepatol.12:257,2000; Murthy etc., Dig.Dis.Sci.38:1722,1993).

The animal model example that can be used to estimate the polypeptide prevention of the present invention's change or treat brightic curative effect comprises anti-GBM inductive glomerulonephritis (Wada etc., Kidney Int.49:761-767,1996) and anti-thy1 inductive glomerulonephritis (Schneider etc., Kidney Int.56:135-144,1999).

The animal model example that can be used to estimate antibody of the present invention or Fab prevention or treat the curative effect of multiple sclerosis comprises experimental autoimmune encephalomyelitis (EAE) (Link and Xiao, Immunol.Rev.184:117-128,2001).

The VII.FcRn fusion rotein

On the other hand, the invention provides novel agent, the binding affinity of the polypeptide that contains Fc that for example be used for purifying, is used for determining that the present invention changes and be used for other Fc sudden change of elutriation influence and FcRn bonded.

Reagent of the present invention can be the polymer Fc conjugated protein that contains first polypeptide and at least one other polypeptide, and wherein said first respectively contains Fc district or its part that at least one operationally is connected in conjunction with the territory with at least one monomer Fc with other polypeptide.

In the embodiment, the technology that the Fc district of each polypeptide or its part and Fc use this area to be easy to obtain respectively in conjunction with territory or its part merges by mode of inheritance.In certain embodiments, polymer by described first and the non-covalent combination of other polypeptide form.In another embodiment, polymer by described first and other polypeptide between covalent attachment (for example, disulfide-bonded) and form.Preferably, first and second polypeptide at its corresponding Fc district covalent bonding (for example passing through disulfide linkage) to form " dimerization " (or bidendate) Fc in conjunction with the territory.In the embodiment, the Fc district can be changed (for example sudden change) so that its effector function (especially with Fc acceptor combine) is abolished or inactivation.Described Fc district and Fc can derive from any organism of expressing the corresponding construction territory in conjunction with the territory.Preferred species comprise people, monkey, mouse, rat and rabbit.Polymer Fc acceptor can be chimeric, and for example the Fc district can derive from rodent (for example mouse or rat), and Fc derives from primate (for example, people or monkey) in conjunction with the territory.

In some preferred embodiment, reagent of the present invention can comprise derive from Fc acceptor (for example Fc newborn infant's acceptor) or its extracellular part Fc in conjunction with the territory.In the embodiment, FcRn:Fc molecule of the present invention is a dimer molecule; Constitute dimeric each monomer and comprise first polypeptide and the second beta-2 microglobulin (β that contains newborn infant's receptor alpha chain extracellular domain (for example, the α 1 of this receptor and α 2 territories) and Fc district or its part respectively ₂M) polypeptide.In one embodiment, the Fc district derives from IgG1.The dna sequence dna of preferred people FcRn α-Fc fusions is presented among the SEQ ID NO:9, and the aminoacid sequence of prediction is presented among the SEQID NO:10.At least one bonded that reduces itself and monomer or polymer Fc acceptor sudden change is preferably contained in the Fc district.For example, can suddenly change, for example describe among the embodiment 4 in amino acid position 310,311,433 and 434 (EU numbering).

Surprisingly, polymer Fc acceptor can be than the much higher horizontal expression of monomer Fc acceptor.For example, using coding FcRn:Fc fusion rotein and β ₂Behind the expression vector stable transfection mammal cell line (for example CHO) of m (together on the identical carrier or), can obtain the nearly productive rate of 100mg/L on the different carriers.

Reagent of the present invention provides many advantages that are better than available reagent.The inventor is unexpected astoundingly to find that polymer Fc acceptor of the present invention has greatly enhanced binding interactions with the polypeptide that contains Fc.By the binding affinity of Biacore measuring h FcRnFc to immobilization hIgG, demonstrate 25 nmole binding affinities, relatively the binding affinity of monomer hFcRn is 2,000 nmoles.For example, reagent of the present invention can be in conjunction with at least two FcR binding sites that separate on the polypeptide that contains Fc (for example antibody Fc district).Especially, reagent of the present invention can be in conjunction with the FcR binding site on two heavy chains in Fc polypeptide (for example antibody) Fc district.These character make to have the enhanced binding interactions with the polypeptide that contains Fc.Especially, reagent of the present invention can be with enhanced stability and/or avidity in conjunction with the polypeptide that contain Fc.Available reagent typically comprises monomer solubility Fc acceptor or its fragment, and it has number of drawbacks.For example, monomer Fc acceptor, especially people's monomer FcRn, owing to have poor avidity and unstable at coated, mark or when being fixed on solid (for example plastics) surface, so can not be used for many high flux screenings (HTS) test form (for example HTS ELISA).And, the Fc-polypeptide relative with the combination of monomer Fc acceptor a little less than, this makes and to be difficult to measure binding signal.Compare with monomer Fc acceptor, polymer Fc acceptor of the present invention because have enhanced stability and (for example＞100 times) of improving at the specific binding affinity that contains the Fc polypeptide, so more strong test is provided.

Therefore, polymer Fc acceptor of the present invention can be used for multiple screening method and changes with the FcR binding affinity of the polypeptide of checking any Fc of containing.Polymer Fc acceptor of the present invention can be used for known in the art can the evaluation and contain Fc polypeptide (for example antibody) and any acellular test that combines of the bonded of polymer Fc acceptor.According to the requirement of described test, can be fixed on polymer Fc acceptor of the present invention on the solid-phase matrix or polymer Fc acceptor of the present invention can freely float in solution.Suitable acellular combination test has been described in this article.In the embodiment, polymer Fc acceptor of the present invention can be used to the variation of polypeptide on effector function of checking that the present invention changes.

In another embodiment, polymer Fc acceptor of the present invention can be used for the polypeptide that purifying contains Fc.For example, polymer Fc acceptor of the present invention can be fixed on the post, so as can be from mixture based on the bigger avidity of polypeptide that contains Fc and polymer Fc acceptor and be purified into the polypeptide that contains Fc.These methods are compared with the existing affinity purification method of label (for example with His or the epi-position label) labelled protein that is beneficial to purifying with requirement some advantage are provided.This type of label may disturb the desired use (for example using as therapeutical agent) of purifying protein, and if remove this type of label, the method for removing them needs a large amount of purification steps, thereby causes a large amount of losses of productive rate.

In an exemplary, the polypeptide that polymer neonatal Fc receptor of the present invention can be beneficial to contain Fc a pH (for example acid pH) combine with pillar down and under the 2nd pH (for example neutral pH) from the pillar wash-out.This purification process is used for the method that purifying contains the polypeptide of Fc and has extra advantage than existing.For example, the albumin A affinity column is used for antibody purification at large and other contains the polypeptide of Fc.Yet this polypeptide of wash-out requires harsh relatively processing (for example low pH) from the pillar, and these processing may destroy described polypeptide, cause a large amount of losses of productive rate.On the contrary, the polypeptide that wash-out contains Fc from the polymer neonatal Fc receptor is affine only requires slightly to rise to the 2nd pH (for example pH7.0-7.5) from a pH (for example pH5.8-6.5).

In another embodiment, (for example, in addition, reagent of the present invention can be used to check the security based on proteinic treatment, the especially mab treatment of any Fc of containing to polymer Fc acceptor of the present invention.For example, when being administered to the patient, some polypeptide (for example, the polypeptide that contains Fc of oxidation) that contains Fc can show the endocytosis of the not expecting transhipment (for example, shifting from the placenta of parent to fetus) to pregnable tissue.Expect this polypeptide that contains Fc with lower avidity and polymer Fc receptors bind, and will be before not oxidized polypeptide wash-out.Methionine(Met) (for example, EU position 253 and 429) residue especially is easy to be subjected to oxidation.Therefore, can based on polymer Fc acceptor combine screening Fc polypeptide, and if the binding affinity of polymer Fc acceptor is in acceptable threshold value, can be based on treatment application choice Fc polypeptide.For example, in the embodiment, can use and the combining of polymer Fc acceptor, consistence or the implementation quality control measured between polypeptide product batch are checked.

Polymer Fc acceptor of the present invention can use any method described herein to produce.

VIII. further modify the polypeptide that contains Fc that changes

Can further modify the polypeptide that contains Fc of change, so that desired effects to be provided.For example, in certain embodiments, can by with the extra section of the desired function that is used for improving polypeptide (for example curative effect), promptly, functional part, for example but PEGization part, blocking part test section, diagnosis part and/or treatment part are puted together (that is, physical connection), modify the polypeptide of described change.Chemically conjugated can the realization randomly or by the specific residue in the polypeptide of the described change of site-specific ground modification.Below at first describe the exemplary functions part, described the useful chemical process and the material of the different aminoacids side chain chemistry of the polypeptide that is used to connect this functional part and change afterwards.

A) functional part

But the example of useful functional part includes but not limited to PEGization part, blocking part test section, diagnosis part and treatment part.

Exemplary PEGization part comprises polyalkylene glycol part, for example peg moiety, preferably PEG-maleimide amine moiety.Preferred PEGization part (or related polymer) can for example be polyoxyethylene glycol (" PEG "), polypropylene glycol (" PPG "), polyoxy ethylization glycerine (" POG ") and other polyoxy ethylization polyvalent alcohol, polyvinyl alcohol (" PVA ") and other polyalkylene oxide, polyoxy ethylization Sorbitol Powder or polyoxy ethylization glucose.Polymkeric substance can be homopolymer, at random or segmented copolymer, based on above-mentioned monomeric terpolymers, straight or branched, that replace or unsubstituted, condition is that it has at least one active sulfone part.Polymeric part can be any length or molecular weight, but these features may influence biological property.Clearance rate during for the reduction medicinal application, the polymkeric substance molecular-weight average that is particularly useful is 2,000 to 35,000 dalton.In addition, if two groups are connected with polymkeric substance one first ground, the length of polymkeric substance may influence two operating ranges between the group and other spatial relation.Therefore, those skilled in the art's length that can change polymkeric substance is to optimize or to give desired biological activity.PEG is because several reasons can be used for biological applications.PEG transparent typically, colourless, tasteless, water soluble, have thermostability, many chemical reagent had inertia, not hydrolysis and nontoxic.

Preferably, the PEGization part is adhered to the polypeptide that contains Fc with the present invention's change that increases the life-span.PEGization part can be by increasing described change the apparent molecular weight of peptide molecule, be used for further increasing transformation period of the polypeptide of described change.The apparent molecular weight that increases can reduce the speed of removing behind subcutaneous or the systemic administration from health.In many cases, PEGization also can play and reduce antigenicity and immunogenic effect.In addition, PEGization can increase the solubility of the polypeptide of change.

Exemplary blocking part (blocking moiety) comprises that halfcystine adducts, Gelucystine, mixed disulfide adducts or other have enough spatial volumes and/or electric charge so that can reduce the antigen dependency effector function compound of (for example, by suppressing the ability of Fc district in conjunction with Fc acceptor or complement proteins).Preferably, the conjugation of polypeptides that the present invention of described blocking part and the effector function with reduction changes is with further reduction effector function.

But the exemplary test section that can be used for the conjugation of polypeptides that changes with the present invention comprises fluorescence part, radio isotope part, radiopaque part etc., detectable for example is as vitamin H, fluorophore, chromophore, spin resonance probe or radioactively labelled substance.Exemplary fluorophore comprises fluorescence dye (for example fluorescein, rhodamine etc.) and other light emitting molecule (for example versomnal).Fluorophore can be an environment sensitive, if its position is near the one or more residues that recurring structure changes when bound substrates (for example, the dansyl probe) in the modified protein like this, then its fluorescence will change.Exemplary radioactively labelled substance comprise contain atoms with one or more low responsive nuclears ( ¹³C, ¹⁵N, ²H, ¹²⁵I, ¹²³I, ⁹⁹Tc, ⁴³K, ⁵²Fe, ⁶⁷Ga, ⁶⁸Ga, ¹¹¹In etc.) small molecules.Other useful part is known in the art.

But the example that can be used for the diagnosis part of the conjugation of polypeptides that changes with the present invention comprises the test section that is applicable to the existence that discloses disease or illness.Typically, diagnose the part permission to determine the existence of the molecule (for example target peptide, protein or a plurality of protein) relevant, do not exist or level with disease or illness.This diagnosis also is applicable to prognosis and/or diagnoses the illness or illness and process thereof.

The exemplary treatment that can be used for the conjugation of polypeptides that changes with the present invention for example partly comprises anti-inflammatory agent, carcinostatic agent, anti-neurodegeneration agent and anti-infection agent.This functional part also can have one or more functions in the above-mentioned functions.

The exemplary treatment agent comprises and can cause nuclear DNA many places splitting of chain and be applicable to inducing cell (for example, cancer) radionuclide dead, that have high-energy ionization radiation thus.Exemplary high energy radionuclide comprises: ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re.These isotropic substances typically produce high energy α or the beta-particle with short path length.These radionuclides kill and wound its contiguous cell, for example this conjugate neoplastic cell accompanying or that entered.They almost do not have the non-cell that is positioned or do not have effect fully, and are non-immunogenicity basically.

The exemplary treatment agent also comprises cytotoxic agent, for example cytostatics (for example alkylating agent, DNA synthetic inhibitor, DNA intercalator or linking agent or the agent of DNA-RNA transcriptional regulatory), enzyme inhibitors, generegulation agent, cytotoxicity nucleosides, tubulin wedding agent, hormone and hormone antagonist, anti-angiogenic agent etc.

The exemplary treatment agent also comprises alkylating agent, for example the medicine of anthracycline antibiotic (anthracycline) family (for example, Zorubicin (adriamycin), carminomycin (carminomycin), cyclosporin A (cyclosporin-A), chloroquine (chloroquine), methylpterin (methopterin), mithramycin (mithramycin), porphyromycin (porfiromycin), streptonigrin, porphyromycin,, amerantrone class (anthracenediones) and aziridines (aziridines)).In another embodiment, chemotherapy partly is a cytostatics, for example the DNA synthetic inhibitor.The example of DNA synthetic inhibitor includes but not limited to methotrexate (methotrexate) and dichloro-methotrexate (dichloromethotrexate), 3-amino-1,2,4-phentriazine-1,4-dioxide (3-amino-1,2,4-benzotriazine 1,4-dioxide), aminopterin (aminopterin), cytosine(Cyt) β-D-arbinofuranose glycosides (cytosine β-D-arabinofuranoside), 5-fluoro-5 '-deoxyuridine (5-fluoro-5 '-deoxyuridine), 5 FU 5 fluorouracil (5-fluorouracil), 9-[1,3-dihydroxy-2-third oxygen methyl] guanine (ganciclovir), hydroxyurea (hydroxyurea), dactinomycin (actinomycin-D) and ametycin (mitomycin C).Exemplary DNA intercalator or linking agent include but not limited to bleomycin (bleomycin), carboplatin (carboplatin), Carmustine (carmustine), Chlorambucil (chlorambucil), endoxan (cyclophosphamide), along diamino platinum (II) dichloride (Platinol) (cis-diammineplatinum (II) dichloride (cisplatin)), melphalan (melphalan), mitoxantrone (mitoxantrone) and oxaliplatin (oxaliplatin).

The exemplary treatment agent also comprises the transcriptional regulatory agent, for example dactinomycin, daunoblastin (daunorubicin), Zorubicin (doxorubicin), homoharringtonine (homoharringtonine) and darubicin (idarubicin).Other exemplary cells inhibitor compatible with the present invention comprises ansamycin (Ansamycin), benzene a kind of jade class (benzoquinones), the quinoid derivative (for example, quinolone (quinolones), young fustic brass (genistein), bactacyclin), busulfan (busulfan), ifosfamide (Ifosfamide), mustargen (mechlorethamine), triaziquone (Triaziquone), diaziquone (Diaziquone), carbaxilquinone (carbazilquinone), indoloquinone EO9, two aziridinyls-benzoquinone methyl DZQ (diaziridinyl-benzoquinone methyl DZQ), triethylenephosphoramide (triethylenephosphoramide), and nitroso-urea compounds (for example, carmustine (carmustine), lomustine (lomustine), Semustine (semustine)).

The exemplary treatment agent also comprises the cytotoxicity nucleosides, for example adenosine Arabinoside, cytosine arabinoside (cytarabine), cytarabin, 5 FU 5 fluorouracil, fludarabine (fludarabine), 5-fluorine dechlorination uridine (floxuridine), fluorofur (ftorafur) and Ismipur; For example Japanese yew class medicine (Taxoids) is (for example for the tubulin wedding agent, Paclitaxel (Paclitaxel), polyenoid taxol (Docetaxel), taxane), R 17934 (nocodazole), agile new (Rhizoxin), the dolastatin class (for example, dolastatin-10,-11 or-15), colchicine (colchicine) and class colchicine (colchicinoid) (for example ZD6126), combretastatin class (combretastatin A-4 for example, AVE-6032), and vinca alkaloids (vinca alkaloids) (for example, vincaleucoblastine (vinblastine), vincristin (vincristine), vindesine (vindesine) and Vinorelbine (Vinorelbine) are (navelbine)); Anti-angiogenic compounds, for example angiostatin (Angiostatin) K1-3, DL-alpha-difluoromethyl-ornithine, interior chalone (endostatin), fumidil (fumagillin), young fustic brass, MINOCYCLINE HCL (minocycline), Staurosporine (staurosporine) and (±) thalidomide (Thalidomide).

The exemplary treatment agent also comprises hormone and hormone antagonist, as reflunomide (for example prednisone (prednisone)), progesterone (for example, hydroxyprogesterone (hydroxyprogesterone) or medroprogesterone, oestrogenic hormon (for example, diethyl diethylstilbestrol (diethylstilbestrol)), estrogen antagonist (for example, tamoxifen (tamoxifen)), male sex hormone (for example, testosterone (testosterone)), aromatase inhibitor (aromatase inhibition) (for example, aminoglutethimide (aminogluthetimide)), 17-(allyl amino)-17-demethoxylation geldanamycin (Geldanamycin), 4-amino-1, the 8-naphthalimide, apigenin (apigenin), brefeldin A (brefeldin A), Cimetidine Type AB (cimetidine), dichloro methylene radical-bisphosphate, Leuprolide (Leuprolide) (Leuprorelin), the lutein releasing hormone, pifithrin-α, rapamycin (rapamycin), sex hormone binding globulin and thapsigargin (thapsigargin).

The exemplary treatment agent also comprises enzyme inhibitors, S (+)-camptothecine (camptothecin) for example, turmeric yellow (curcumin), (-)-deguelin (deguelin), 5,6-dichloro benzo-imidazoles 1-β-D-ribofuranoside, etoposide (etoposide), Formestane (Formestane), phosphorus alkene rhzomorph (fostriecin), hispidin, 2-imino--1-imidazolidine acetate (cyclocreatine), nervinolin (Mevinolin), Atrichostatin A (trichostatin A), tyrphostin (tyrphostin) AG34 and tyrphostin AG879.

The exemplary treatment agent also comprises the generegulation agent, for example 5-azepine-2 '-Deoxyribose cytidine, 5-azacytidine, cholecalciferol (cholecalciferol) (Vitamin D3 500,000 I.U/GM), 4-hydroxy tamoxifen (4-hydroxytamoxifen), melatonin (melatonin), Mifepristone (Mifepristone), raloxifene (Raloxifene), trans-retinal (axerophthal), vitamin A acid (retinoic acid), retinoic acid, 9-cis-vitamin A acid, 13-cis-vitamin A acid, Vogan-Neu (vitamin A), tamoxifen and troglitazone.

The exemplary treatment agent also comprises cytotoxic agent, for example the medicine of pteridine family, diyenes and podophillotoxines (podophyllotoxins).The member who is particularly useful in these classifications for example comprises, methylpterin, podophyllotoxin (podophyllotoxin) or podophyllotoxin derivative be etoposide or phosphoric acid etoposide, leurosidine (leurosidine), vindesine, leurosine (leurosine) etc. for example.

Again other cytotoxin compatible with the instruction of this paper comprises auristatin (for example auristatin E and monomethylauristan E), calicheamicin (calicheamicin), linear gramicidins (gramicidin) D, Caulis Mayteni tree alkaloid (maytansanoids) (for example maytenin (maytansine)), neocarzinostatin (neocarzinostatin), topotecan (topotecan), taxanes, cytochalasin B, the pyridine of bromination second, ipecamine (emetine), tenoposide, colchicine, dihydroxy anthracindione, mitoxantrone (mitoxantrone), PROCAINE HCL, PHARMA GRADE, tetracaine (tetracaine), lignocaine (lidocaine), Proprasylyte (Propranolol), tetracycline (puromycin) and their analogue or homologue.

The functional part of other type is known in the art, can easily be used for the inventive method and composition according to the instruction of this paper.

B) be used for the chemistry of linkage function part and amino acid side chain

The chemistry that is used to connect aforementioned functional part (they can be the molecules of small molecules, nucleic acid, polymkeric substance, peptide, protein, chemotherapeutics or other type) and specific amino acids side chain is known in the art (for the detailed summary of given joint, for example see, Hermanson, G.T., BioconjugateTechniques, Academic Press (1996)).

The example that is used for the linking group of sulfydryl part (for example halfcystine or sulfenyl side chain chemistry) known in the art includes but not limited to; activatory carboxyl groups (for example, alpha-halogen acetic ester, Mono Chloro Acetic Acid or chlor(o)acetamide), activatory alkyl group, Michael acceptor such as maleimide or acrylic acid groups, group and activatory disulphide group by redox reaction and sulfydryl partial reaction.The sulfydryl part also can be by disliking-1, the 3-diazole with bromine trifluoroacetone, α-bromo-β-(5-imidazolyl (imidozoyl))-propionic acid, chloracetyl phosphoric acid ester, N-alkyl maleimide, 3-nitro-2-pyridyl disulfide, methyl-2-pyridyl disulfide, parachloro-mercuri-benzoate, 2-chlorine mercury-4-nitrophenols or chloro-7-nitro benzo-2-.

In a preferred embodiment, halfcystine or other amino acid with sulfydryl side chain chemistry in the preparation process of the polypeptide that contains Fc or after connect.For example, use that the cell culture preparation modifies contain the polypeptide of Fc the time, provide condition so that the free cysteine in the solution can form the halfcystine adducts with the sulfydryl side chain of the polypeptide that contains Fc.The adducts of Xing Chenging can be used to suppress glycosylation and/or effector function thus, perhaps, and with being placed under the reductive condition to remove adducts and to allow to use one of above-mentioned sulfydryl chemical substance thus.

Known in the artly (for example be used for hydroxylic moiety; Serine, Threonine or tyrosine side chain chemistry) the example of linking group comprise above at the sulfydryl part described those, comprise activatory carboxyl groups, activatory alkyl group and Michael acceptor.

Known in the artly (for example be used for amino part; l-asparagine or arginine side chain chemistry) the example of linking group include but not limited to the N-succinimido; N-sulfosuccinic acylimino; N-phthaloyl imino (phthalimidyl); N-sulfo group phthaloyl imino; the 2-nitrophenyl; the 4-nitrophenyl; 2; the 4-dinitrophenyl; 3-alkylsulfonyl-4-nitrophenyl; 3-carboxyl-4-nitrophenyl; imido-ester (for example; pyridine formyl imido acid methyl ester); pyridoxal phosphate; pyridoxal; chlorine borohydride (chloroborohydride); trinitro-benzene-sulfonic acid; O-methyl-isourea and 2, the 4-diacetylmethane.

The example that is used for the linking group of acidic moiety (for example, aspartic acid or L-glutamic acid side chain chemistry) known in the art comprises activatory ester and activatory carbonyl.Acidic moiety also can be optionally by with carbodiimide (R ' N-C-N-R ') 1-cyclohexyl-3-[2-morpholinyl-(4-ethyl) for example] carbodiimide or 1-ethyl-3-(4-nitrogen drone assorted-4,4-dimethyl amyl group) carbodiimide.

When the functional part of expectation is the PEGization part, can use PEGization reaction well known in the art.For example, in the method, by with the acylation reaction or the alkylated reaction of reactive polyethylene glycol molecule (or similarly reaction water-soluble polymkeric substance), realize PEGization.The water-soluble polymers that is used for PEGization antibody of the present invention and antibody fragment is polyoxyethylene glycol (PEG).In another embodiment, the polymkeric substance that is used for PEGization is polyoxyethylene glycol-maleimide (that is a PEG-maleimide).

Prepare the antibody of PEGization of the present invention or the method for antibody fragment and generally comprise step: a) make antibody or antibody fragment and polyoxyethylene glycol, for example reactive ester or the aldehyde derivatives of PEG, under the condition that can cause antibody or antibody fragment, react in conjunction with one or more PEG groups, and b) the acquisition reaction product.Those skilled in the art will understand how to select optimum reaction condition or acylation reaction based on known parameters and expected result.In the embodiment, can the target particular amino acid residue, for example change first amino-acid residue so that suppress the glycosylation (preferably be halfcystine and have under the situation of sulfydryl chemistry) of second amino-acid residue at first amino acid.

IX. prevent, diagnosis and methods of treatment

The polypeptide (for example antibody or fusion rotein) that changes as the present invention combines with cell-surface antigens and this combination evokes essential effector when replying, and the present invention has conventional application.An example of replying of effector mediation is the basic reason (for example, the antigen of elimination tumour cell or participation immunity or inflammatory reaction carries cell) that reduces illness.In another embodiment, can reduce one or more symptoms of illness.In another embodiment, composition described herein can be used for diagnostic reagent (antibody that for example, is used for tumor imaging) to change replying of effector mediation.Method as herein described can be used to that the experimenter of the danger that disease takes place or the experimenter who shows the disease illness are at present arranged.

A. antineoplaston

Therefore, in some embodiment, the polypeptide that the present invention changes can be used for prevention or treatment cancer.In the embodiment, the polypeptide of change blocking-up autocrine or paracrine growth (for example, by transduction signal with receptors bind and not, or by and somatomedin in conjunction with).In preferred embodiments, the polypeptide of change can combine with tumor associated antigen.

In the embodiment, the polypeptide of change can reduce the survival time that the animal of tumour is carried in gross tumor volume, inhibition tumor growth and/or prolongation.Usually, the present invention can be used to prevent or treat any vegetation that comprises following antigen sign, and described antigen sign allows the antibody target cancer cells of modification.The exemplary cancer that can prevent or treat or tumorigenesis include but not limited to bladder cancer, mammary cancer, head and neck cancer, prostate cancer, colon-rectum cancer, melanoma or skin carcinoma, mammary cancer, ovarian cancer, cervical cancer, carcinoma of endometrium, kidney, lung cancer (for example minicell and non-squamous cell carcinoma), carcinoma of the pancreas and multiple myeloma.More particularly, the antibody modified of the present invention can be used for the treatment of sarcoma, rhabdosarcoma, Glioblastoma (preferably glioblastoma multiforme), leiomyosarcoma, the retinoblastoma of Kaposi sarcoma, CNS tumorigenesis (capillary vessel hemangioblastoma, meningioma and brain metastatic carcinoma), melanoma, GI and kidney, papillary cystic adenocarcinoma, WilmShi tumour or the small cell lung cancer of ovary.Be appreciated that in view of the present invention openly, need not undo experimentation and promptly can derive suitable initial polypeptide at the tumor associated antigen relevant with aforementioned tumorigenesis.

Be suitable for using the example of the hematologic cancers that the present invention treats to comprise He Jiejinshi and non_hodgkin lymphoma and leukemia, comprise ALL-L3 (Hugh Burkitt type leukemia (Burkitt ' s type leukemia)), lymphocytic leukemia (CLL) and monocytic leukemia.Be appreciated that, polypeptide and method that the present invention changes especially can be treated various B cell lymphomas effectively, comprise rudimentary/folliculus non_hodgkin lymphoma (NHL), cell lymphoma (FCC), lymphoma mantle cell (MCL), diffustivity large celllymphoma (DLCL), small lymphocyte (SL) type NHL, middle rank/folliculus NHL, intermediate diffustivity NHL, senior immunoblast NHL, senior lymphocytoblast NHL, senior little no schistocyte NHL, huge lump (bulky disease) NHL and WaldenstromShi macroglobulinemia.It should be understood by those skilled in the art that these lymphomas usually have different titles owing to the categorizing system that is changing, and, suffer from the lymphadenomatous patient who classifies under different titles and also can have benefited from combined treatment of the present invention.Except above-mentioned tumorigenesis illness, be appreciated that the present invention can also be advantageously used in other malignant tumour that treatment has compatible tumor associated antigen.

B. Immunological diseases treatment

Except the tumour illness, the polypeptide that the present invention changes is also especially effective aspect replying at treatment autoimmune disease or abnormal immune.In this regard, can understand that the polypeptide that the present invention changes can be used to control, prevent, regulate or eliminate the immunne response of not expecting to outside antigen and autoantigen.For example, in the embodiment, antigen is autoantigen.In another embodiment, antigen is allergen.In other embodiments, antigen is isoantigen or heterologous antigen.Modified polypeptide disclosed by the invention especially can be used for transplanting reducing at the purposes in the immunne response of isoantigen and heterologous antigen, for example to suppress transplant recipient to for example repulsion of tissue or organ graft or bone marrow graft of donor graft.In addition, the donor T cell in inhibition or the elimination bone marrow graft also can be used for the inhibition of transplant versus-host disease.

In other embodiments, the polypeptide that the present invention changes can be used for the treatment of immune disorders, includes but not limited to the allergy bronchopneumonic aspergillosis; Rhinallergosis; Autoimmune hemolytic anemia; Acanthosis; Allergic contact dermatitis; The AddisonShi disease; Atopic dermatitis; Alopecia areata (alopeciaareata); General alopecia (alopecia universalis); Amyloidosis (amyloidosis); Anaphylactoid purpura (anaphylactoid purpura); Anaphylactoid reaction (Anaphylactoid reaction); Aplastic anemia (aplastic anemia); Hereditary angiodysplasia (angioedema); The special property sent out angioedema; Ankylosing spondylitis (Ankylosing Spondylitis); Cranial arteritis (cranial arteritis); Giant cell arteritis (giant cell arteritis); Takyayasu heubner's artery Heubner inflammation; Temporal arteritis (temporalarteritis); Asthma; Telangiectasis-locomotor ataxia disease (ataxia-telangiectasia); Autoimmune oophoritis (autoimmune oophoritis); Autoimmunity orchitis (autoimmuneorchitis); The many endocrinasthenias of autoimmunity (autoimmune polyendocrine failure); The BehcetShi disease; The BergerShi disease; The BuergerShi disease; Bronchitis; BP (bullouspemphigus); Chronic mucocutaneous candidiasis (chronic mucocutaneous candidiasis); The Caplan syndrome; Postmyocardial infarction syndrome (post-myocardial infarction syndrome); Postpericardiotomy syndrome (post-pericardiotomy syndrome); Carditis (carditis); Sprue (celiac sprue); The ChagasShi disease; Chediak-Higashi syndrome; The Churg-Strauss disease; Liver cirrhosis (Cirrhosis); The Cogan Cotard; Cold agglutinin disease (cold agglutinin disease); CREST syndrome; The CrohnShi disease; Cryoglobulinemia (cryoglobulinemia); Idiopathic pulmonary fibrosis (Cryptogenic Fibrosing Alveolitis), dermatitis herpetiformis (dermatitisherpetifomis); Dermatomyositis (dermatomyositis); Diabetes; Diamond-Blackfan syndrome; DiGeorge syndrome; Discoid lupus erythematosus (discoid lupus erythematosus); Eosinophilic fasciitis (eosinophilic fasciitis); Episcleritis (episcleritis); Erythema elevatum diutinum (Drythema elevatum diutinum); Erythema marginatum (erythema marginatum); Erythema multiforme (erythema multiforme); Erythema nodosum (erythema nodosum); Familial Mediterranean fever (Familial Mediterranean fever); The Felty Cotard; Pulmonary fibrosis (fibrosispulmonary); Irritated sample glomerulonephritis (glomerulonephritis, anaphylactoid); The autoimmunity glomerulonephritis; Post-streptococcal glomerulonephritis (post-streptococcalglomerulonephritis); Transplant the back glomerulonephritis; Membranous glomerulopathy (glomerulopathy, membranous); The Goodpasture Cotard; Immune-mediated granulocytopenia (granulocytopenia, immune-mediated); Annular granuloma (granuloma annulare); Allergic granulomatosis (granulomatosis, allergic); Granulomatous myositis (granulomatous myositis); The GraveShi disease; Hashimoto thyroiditis (Hashimoto ' s Thyroiditis); Hemolytic disease of newborn; Idiopathic hemochromatosis (hemochromatosis, idiopathic); The Henoch-Schoenlein purpura; Chronic active and chronic progressive external hepatitis; Histiocytosis X (histiocytosis X); Hypereosinophilic syndrome (hypereosinophilic syndrome); Idiopathic thrombocytopenic purpura (idiopathicthrombocytopenic purpura); The Job Cotard; Juvenile form dermatomyositis (juveniledermatomyositis); Juvenile rheumatoid arthritis (juvenile rheumatoid arthritis) (juvenile chronic arthritis); The KawasakiShi disease; Keratitis (keratitis); Keratoconjunctivitis sicca (keratoconjunctivitis sicca); Landry-Guilain-Barre-Strohl syndrome; Lepromatous leprosy (Leprosy, lepromatous); The Loeffler Cotard; Lupus; Lupus nephritis (lupus nephritis); The Lyell Cotard; Lyme borrelia burgdorferi disease (Lyme disease); Lymphomatoid granulomatosis (lymphomatoid granulomatosis); The general mastocytosis (mastocytosis, systemic); Mixed connective tissue disease (mixed connective tissue disease); Multiple single neuritis (Mononeuritis multiplex); Muckle-Wells syndrome; Kawasaki disease (mucocutaneous lymph node syndrome); Kawasaki disease; Multicentricity reticulohistiocytosis (multicentric reticulohistiocytosis); Multiple sclerosis (Multiple sclerosis); Myasthenia gravis (myasthenia gravis); Mycosis fungoides (mycosisfungoides); The general necrotizing vasculitis (necrotizing vasculitis, systemic); Nephrotic syndrome (nephrotic syndrome); Overlap syndrome (overlap syndrome); Pimelitis (panniculitis); Paroxysmal cold hemoglobinuria (paroxysmal cold hemoglobinuria); Paroxysmal nocturnal hemoglobinuria (paroxysmal nocturnal hemoglobinuria); Pemphigoid (pemphigoid); Pemphigus (pemphigus); Pemphigus erythematosus (pemphigus erythematosus); Pemphigus foliaceus (pemphigus foliaceus); Pemphigus vulgaris (pemphigus vulgaris); Raise dove person disease (pigeonbreeder ' s disease); Hypersensitivity pneumonitis (Hypersensitivity Pneumonitis); Polyarteritis nodosa (polyarteritis nodosa); Polymyalgia rheumatica (Polymyalgia rheumatica); Polymyositis (polymyositis); The special property sent out polyneuritis (polyneuritis, idiopathic); Hereditary amyloid polyneuropathy Portuguese type familial polyneuropathy (portuguese familial polyneuropathy); Preeclampsia/eclampsia (pre-eclampsia/eclampsia); Primary biliary cirrhosis (Primary biliary cirrhosis); Progressive systemic sclerosis (scleroderma); Psoriatic; Psoriasis arthropathica; Pulmonary alveolar proteinosis (pulmonary alveolar proteinosis); Pulmonary fibrosis (pulmonary fibrosis), RaynaudShi phenomenon/syndrome; The ReidelShi thyroiditis; The Reiter Cotard, relapsing polychondritis (relapsingpolychrondritis); Rheumatic fever (rheumatic fever); Rheumatoid arthritis (rheumatoidarthritis); Sarcoidosis (Sarcoidosis); Scleritis (scleritis); Sclerosing cholangitis (sclerosingcholangitis); Scleroderma (scleroderma), serum sickness (serum sickness); Sezary syndrome; The Sjogren Cotard; Stevens-Johnson syndrome; The StillShi disease; Subacute sclerosing panencephalitis (subacute sclerosing panencephalitis); Sympathetic ophthalmia (sympathetic ophthalmia); Systemic lupus erythematosus; Transplant rejection; Ulcerative colitis (ulcerative colitis); Undifferentiated connective tissue disease (undifferentiated connective tissue disease); Chronic nettle disease (urticaria, chronic); Cold urticaria (urticaria, cold); Uveitis (uveitise); Vitiligo (vitiligo); The Weber-Christian disease; The WegenerShi granuloma; With Wiskott-Aldrich syndrome.

The C anti-inflammatory treatment

In other embodiment again, the polypeptide of change of the present invention can be used for the treatment of (at least in part) that cause because of inflammation or the inflammatory conditions of deterioration, for example activation of the blood flow of Zeng Jiaing, oedema, immunocyte (for example hyperplasia, cytokine produce or the enhanced phagolysis).Exemplary inflammatory conditions comprises and wherein relates to inflammatory factor (for example, matrix metalloproteinase (MMP), nitrogen protoxide (NO), TNF, interleukin, plasma proteins; Cell defense system, cytokine, lipid metabolism thing, proteolytic enzyme, toxicity free radical, plastosome, apoptosis, adhesion molecule etc.) or inflammatory factor be present in those illnesss in given area or the tissue with abnormal quantity (for example, advantageously Change Example as being of value to experimenter's amount).Inflammatory process is living tissue replying damage.The reason of inflammation can be because physical damnification, chemical substance, microorganism, tissue necrosis, cancer or other factors.The acute inflammation time length is short, only continues a couple of days.Yet, if its lasting long period can be called chronic inflammatory diseases.

Inflammatory conditions comprises acute inflammation illness, chronic inflammatory illness and recurrent inflammatory conditions.The acute inflammation illness generally has the relatively short extended period, and continues big approximate number minute to about 1 to 2 day, but they also can continue several weeks.The principal character of acute inflammation illness comprises that blood flow, body fluid and the plasma proteins of increase ooze out (oedema) and white corpuscle such as neutrophilic granulocyte and ooze out.The chronic inflammatory illness generally has the long extended period, for example several weeks to several months to the several years or even longer, and its hyperplasia with the existence of lymphocyte and scavenger cell and blood vessel and reticular tissue is relevant on histology.The recurrent inflammatory conditions comprises recurrence or periodical attack over time illness.The example of recurrent inflammatory conditions comprises asthma and multiple sclerosis.Some illnesss can fall into one or more classification.

Inflammatory conditions is general to be characterised in that heating, flush, enlargement, pain and loss of function.The exemplary reason of inflammatory conditions (for example includes but not limited to infected by microbes (for example bacterium, virus and fungi infestation), physical factor, burn, radiation and wound), chemical factor (for example, toxin and corrosives), tissue necrosis and various types of immune response.The example of inflammatory conditions includes but not limited to Alzheimer; Serious asthma, atherosclerosis, cachexy, CHF-ischemic and coronary restenosis; Osteoarthritis, rheumatoid arthritis, fibrosis/radiation-induced or juvenile arthritis; Acute and chronic infection (bacterium, virus and fungi); Acute and chronic bronchitis, sinusitis paranasal sinusitis (sinusitis) and other respiratory infection comprise flu; Acute and chronic enterogastritis and colitis and CrohnShi disease; Acute and chronic cystitis and urethritis; Adult respiratory distress syndrome; Cystic fibrosis; Acute and chronic dermatitis; Psoriatic; Acute and chronic conjunctivitis; Acute and chronic serositis (pericarditis, peritonitis, synovitis, pleuritis and tendonitis (tendinitis)); Uremic pericarditis; Acute and chronic cholecystitis; Acute and chronic vaginitis (vaginitis); Apoplexy, the brain that causes by wound or the inflammation of central nervous system, and ulcerative colitis; Acute and chronic eye uveitis; Drug reaction; Diabetic nephropathy, and burn (heat, chemistry with electricity).Can comprise the inflammation that causes owing to corneal transplantation, chronic obstructive pulmonary disease, hepatitis C, lymphoma, multiple myeloma and osteoarthritis with other inflammatory conditions or the situation of antibody of the present invention or Fab treatment.

In another embodiment, polypeptide of the present invention can be used for prevention or treatment neurodegenerative disease, includes but not limited to Alzheimer, apoplexy and traumatic brain or central nervous system injury.Other neurodegenerative disease comprises ALS/ motor neuron, diabetic peripheral neurophaty, diabetic retinopathy, Huntington Chorea, macular degeneration and Parkinson's disease.In the preferred embodiment, the polypeptide of change that FcRn is had the binding affinity of reduction is used for the treatment of nervous system disorders, because they can not stride across hemato encephalic barrier effectively as the polypeptide with higher FcRn binding affinity.For example, in the embodiment, the polypeptide that the present invention is changed is injected in the cerebrospinal fluid with the treatment neurodegenerative disease.

In prophylactic application, to comprise the pharmaceutical composition of polypeptide of the present invention or medicament administration and give disease danger is arranged the experimenter of (or suffer from disease but do not demonstrate symptom yet), wherein said disease is can be with the disease of the polypeptide treatment with Fc district, disorder of immune system for example, the wherein said amount of using employing will be enough to eliminate or reduce described danger, reduce severity of disease, or (wherein said disease comprises the biological chemistry of disease the time opening that postpones disease, histology and/or behavior symptom, its complication and the middle pathology phenotype that in the disease progression process, occurs).

In therapeutic is used, give the experimenter who has suffered from this type of disease with composition or medicament administration, the wherein said amount of using employing will be enough to treat or stop at least in part the symptom (biochemical, histological and/or behavior) of disease, comprise its complication and the middle pathology phenotype that occurs in the disease progression process.Polypeptide of the present invention especially can be used for regulating the biologic activity that is present in the cell-surface antigens in the blood, and the disease of wherein treating or preventing causes owing to the unusual high or low biologic activity of described antigen at least in part.

In the certain methods, use medicament and can reduce or eliminate for example inflammation of immune disorders.The amount that is enough to finish treatment or prevents is defined as treatment or prevents effective dosage.In prevention and treatment plan, medicament divides several dosage to use up to realizing enough immunne responses usually.

Be appreciated that based on to the selection of modified polypeptide bonded target molecule of the present invention, polypeptide of the present invention can be used for the treatment of not specifically mentioned many illnesss herein.Can also recognize that any antibody known in the art or fusion rotein all can be modified and are used for the treatment of the illness that it is suitable for according to the inventive method.

D. application process

The polypeptide that the present invention changes can be used to prevent and/or treat by mode in startingeral, part, intravenously, oral, intra-arterial, encephalic, intraperitoneal or the nose.Term startingeral is used to comprise intravenously, intra-arterial, intraperitoneal, intramuscular, subcutaneous, rectum or vaginal application in this article.Most typical pharmaceutical grade protein route of administration is interior, the subcutaneous or intramuscular of blood vessel, but other approach also may be effective.In certain methods, medicament is injected directly into accumulated in the sedimental particular organization, for example intracranial injection.In the certain methods, antibody is with slow releasing composition or device (Medipad for example ^TMDevice) form is used.This pharmaceutical grade protein also can pass through respiratory tract, for example uses powder inhaler to use.

For above-mentioned treatment of conditions, the effective dose of the present composition changes along with many different factors, and described factor comprises that method of application, target site, experimenter's physiology situation, experimenter are that people or animal, the other medicines of using and treatment are preventative or curative.Usually, the experimenter is the people, but also can treat non-human mammal comprises transgene mammal.

For carrying out passive immunization with antibody, dosage range from about 0.0001 to 100mg/kg, be 0.01 to 20mg/kg host's body weight more generally.For example, dosage can be 1mg/kg body weight or 10mg/kg body weight or in the scope of 1-10mg/kg, preferably 1mg/kg at least.Can every day, the next day, weekly or according to any other by the scheme that empirical analysis is determined, use this dosage to the experimenter.An exemplary therapy needs for a long time, and for example at least 6 months, multiple doses was used.Other exemplary treatment scheme needs per two weeks to use once or used in every month once or used in per 3 to 6 months 1 time.The exemplary dose scheme comprise continuous every day 1-10mg/kg or 15mg/kg, the next day 30mg/kg or 60mg/kg weekly.In certain methods, two peptide species with different binding specificities are used simultaneously, and the application dosage of each polypeptide is respectively in specified scope in the case.

Usually repeatedly use polypeptide.Interval between two single doses can be weekly, every month or annual.In the certain methods, adjust dosage reaching the plasma antibody concentration of 1-1000 μ g/ml, and in the certain methods modulation to reach the plasma antibody concentration of 25-300 μ g/ml.Perhaps, can need lower frequency of administration in the case with the form administration of antibodies of sustained release preparation.Dosage and frequency along with polypeptide in the experimenter transformation period and change.Usually, people's antibody demonstrates the longest transformation period, is humanized antibody, chimeric antibody and non-human antibody afterwards.Discuss as this paper, the transformation period is also depended on the concrete sudden change that exists in the polypeptide of change.

Application dosage and frequency can be preventative or curative the variations according to treatment.In prophylactic application, the composition that contains antibody of the present invention or its cocktail type mixture is applied to the experimenter who is not in morbid state as yet so that strengthen experimenter's resistibility.This amount is defined as " prevention effective dose ".In this used, dosage depended on experimenter's healthy state and general immune state again accurately, but is generally 0.1 to 25mg/ agent, especially 0.5 to 2.5mg/ agent.Can be with the low relatively dosage of rare relatively interval long-term application.Some experimenters can remain at it and continue in lifetime to receive treatment.

In therapeutic is used, sometimes need use high relatively dosage (for example, 5-25mg dosage is more generally used in about 1 to 200mg antibody/agent) with short relatively interval, up to slowing down or stopping disease process, preferably the disease symptoms that demonstrates partially or completely up to the experimenter improves.Afterwards, the patient can accept preventative application program.

The dosage range of the nucleic acid of encoding antibody is the about 10ng to 1g of each experimenter, 100ng to 100mg, 1 μ g to 10mg or 30-300 μ g DNA.The dosage variation range of venereal infection poisonous carrier is 10 to 100, or more virosome/agent.

Those skilled in the art can determine that the polypeptide that changes is used for the treatment of the effective nontoxic amount of disease purpose by normal experiment.For example, the treatment significant quantity of modified polypeptide can be according to factor for example experimenter's disease stage (for example the I phase is to IV phase tumour), age, sex, medical science complication (for example, immunosuppression situation or disease) and body weight and change.Can modulate dosage so that best therapeutic response to be provided.For example, can divide several times every day and use, perhaps can reduce dosage in proportion along with the emergency of treatment situation.

E. the monitoring to treating

Can use standard method monitoring treatment to the experimenter that suffers from disease or illness.Certain methods need be measured baseline value before using drug dose, the baseline value of antibody horizontal or antibody repertoire among the experimenter for example, and with this with treatment after spectrum or level compare.The significance of this level value or spectrum value increases (promptly, model experiment limit of error (being expressed as a standard deviation of the mean value that departs from these measurements) the expression positive treatment result (that is, using of medicament realized the reaction of expecting) who is higher than the replicate measurement same sample.If significance variation or minimizing do not take place the value at immunne response, then indicate negative treatment result.

In other method,, determine control level value or spectrum value (that is, mean value adds standard deviation) at control population.Typically, the individuality in the control population was not accepted treatment formerly.Relatively experimenter's the level or the mean value and the control value of spectrum after the administering therapeutic agent then.Significance increase (standard deviation that for example, is higher than departure) with respect to control value shows the positive or sufficient treatment result.Lack the significance increase or reduction occurs and show negative or inadequate treatment result.When described level is increasing with respect to control value, generally continue using of medicament.As preceding, with respect to control value, the arrival of plateau indication can stop to treat uses or reduces dosage and/or the frequency that treatment is used.

In other method,, determine control level value or spectrum value (for example, mean value adds standard deviation) from by living through therapeutical agent treatment and level or spectrum has reached the control population that the individuality of platform is formed in response to treatment.Level value that will measure in the experimenter or spectrum value and this control value are relatively.If value that measures among the experimenter and control value do not have significant difference (for example greater than 1 standard deviation), then can stop treatment.If experimenter's level is markedly inferior to control value, continuing to use reasonable ground be provided then for medicament.If experimenter's level continues to be lower than control level, then may need to change therapy.

In other method, monitoring is not received treatment at present but the formerly experimenter's of the course of treatment polypeptide level or spectrum had been arranged, to determine whether restarting treatment.The value that the level that measures in the experimenter or spectrum and experimenter before can have been reached after the course of treatment formerly compares.Remarkable minimizing (that is the typical error scope that, is higher than the replicate measurement same sample) indication with respect to previous observed value can resume treatment.Perhaps, value and the following control value (mean value adds standard deviation) that measures among the experimenter can be compared, described control value is determined in the colony that is made up of the experimenter who lives through the course of treatment.Perhaps, the value that measures in the experimenter and the control value of determining in by colony that forms through preventative-therapeutic experimenter (keeping no disease symptoms) or the colony that is made up of the experimenter's (showing the improvement of genius morbi) who passes through therapeutic treatment can be compared.Under all these situations, indicate this experimenter should restart treatment with respect to the significance minimizing (that is, overgauge is poor) of control value.

Polypeptide spectrum typical earth surface after using reveal immediately the antibody concentration peak and exponential decay afterwards.No longer under the situation of administration, depend on the transformation period of used antibody, decay will be treated preceding level by convergence in a few days to several months.For example, the transformation period of some antibody is about 20 days.

In the certain methods, before using, in the experimenter, polypeptide is carried out base measurement, carry out second after using very soon and measure, and carry out one or many termly again and measure to monitor the decay of polypeptide level with definite polypeptide peak level at given antigen.When the polypeptide level drops to the percentage ratio of being scheduled to (for example, 50%, 25% or 10%) of baseline or deduction baseline postpeak, give a dosage again of polypeptide.In the certain methods, the peak after the background correction or measurement level subsequently and reference level are compared, described reference level are formerly determined to form useful prevention or treatment plan in other experimenter.If measured polypeptide level significantly is lower than reference level (for example, the mean value that is lower than the reference value in the population of subjects that has benefited from treating deducts a standard deviation), then explanation needs the polypeptide of a dosage to use again.

Other method be included in the course of treatment monitoring any this area approval, the person of being studied or doctor's routine be used to diagnose or the physiology symptom (for example, symptom health or spirit) of monitoring of diseases.

F. combination therapy

The polypeptide that the present invention changes can be randomly with known or to be determined other medicament (any medicament that comprises above VIII part) that can treat the illness that needs treatment (for example preventative or therapeutic) or disease effectively co-administered.In addition, polypeptide of the present invention can also and be puted together to the part (for example PEG, label, medicine or marker) that polypeptide adds function.

Be further appreciated that the polypeptide that the present invention changes can make up or unite use (for example, so that combined treatment to be provided) with any chemotherapeutics (one or more) that can digest, reduce, suppress or control the growth of tumorigenesis cell in the body.The exemplary chemotherapeutics compatible with the present invention comprises alkylating agent, vinca alkaloids (for example, vincristin, vincaleucoblastine), procarbazine (procarbazine), aminopterin and prednisone.Four medication combined MOPP (mechlethamine (mustargen), vincristin (Oncovin), procarbazine and prednisone) can treat various types of lymphomas very effectively, and comprise the preferred embodiments of the invention.In the resistance patient of MOPP, can use ABVD (for example, Zorubicin, bleomycin, vincristin and Dacarbazine (Dacarbazine)), ChlVPP (Chlorambucil, vincristin, procarbazine and prednisone), CABS (lomustine, Zorubicin (doxorubicin), bleomycin and U-9889 (Streptozotocin)), MOPP to add ABVD, MOPP and add ABV (Zorubicin, bleomycin and vincristin) or BCVPP (Carmustine, endoxan, vincristin, procarbazine and prednisone) associating.For standard dosage and progress, can be referring to ArnoldS.Freedman and Lee M.Nadler, Malignant Lymphomas, " Harrison ' s Principles ofInternal Medicine " 1774-1788 (Kurt J.Isselbacher etc., eds., 13th ed.1994) and V.T.DeVita etc., (1997) and the reference of wherein quoting.These therapies can be without changing or uniting use through changing the back with one or more modified polypeptides of the present invention described herein as required according to concrete patient.

Can be used for other treatment plan of the present invention and comprise the independent alkylating agent of use for example endoxan or Chlorambucil, or it is medication combined as CVP (endoxan, vincristin and prednisone), CHOP (CVP and Zorubicin), C-MOPP (endoxan, vincristin, prednisone and procarbazine), CAP-BOP (CHOP methylate benzyl hydrazine and bleomycin), (CHOP adds methotrexate to m-BACOD, bleomycin and formyl tetrahydrofolic acid (leucovorin)), ProMACE-MOPP (prednisone, methotrexate, Zorubicin, endoxan, etoposide and formyl tetrahydrofolic acid add standard MOPP), ProMACE-CytaBOM (prednisone, Zorubicin, endoxan, etoposide, cytosine arabinoside, bleomycin, vincristin, methotrexate and formyl tetrahydrofolic acid) and MACOP-B (methotrexate, Zorubicin, endoxan, vincristin, the prednisone of fixed dosage, bleomycin and formyl tetrahydrofolic acid).Those skilled in the art can easily be identified for each the standard dose and the progress of these schemes.CHOP also unites with bleomycin, methotrexate, procarbazine, mustargen, cytarabin and etoposide.Other compatible chemotherapeutics includes but not limited to 2-chlorodeoxyadenosine (2-CDA), 2 '-deoxycoformycin (Deoxycoformycin) and fludarabine.

For not realizing that disease is alleviated or the middle rank and the senior NHL patient of recurrence, use rescue treatment (salvage treatment).Medicine, for example cytarabin, carboplatin, cis-platinum, etoposide and ifosfamide (giving individually or jointly) are used in the rescue treatment.The recurrence or invasive some tumour illness in, usually use following scheme: IMVP-16 (ifosfamide, methotrexate and etoposide), MIME (methyl-gag, ifosfamide, methotrexate and etoposide), DHAP (dexamethasone (dexamethasone), high dosage cytosine arabinoside and cis-platinum), ESHAP (etoposide, methyl meticortelone (methylpredisolone), the HD cytosine arabinoside, cis-platinum), CEPP (B) (endoxan, etoposide, procarbazine, prednisone and bleomycin) and CAMP (lomustine, mitoxantrone, cytosine arabinoside and prednisone) (each all has medicine-feeding rate and the progress of knowing).

Can change along with the experimenter with the amount that modified polypeptide of the present invention is united the chemotherapeutics of use, perhaps can use according to method well known in the art.For example see Bruce A Chabner etc., Antineoplastic Agents, " Goodman﹠amp; Gilman ' s The Pharmacological Basis ofTherapeutics " 1233-1287 ((Joel G.Hardman etc., eds., 9th ed.1996).

Although can as described hereinly use modified polypeptides, what must emphasize is in other embodiments, modified polypeptides can be applied to healthy patients as first-line treatment.In this embodiment, can use modified polypeptide of the present invention to patient and/or to the patient who does not have, has now not have or do not having normal or average Red bone marrow deposit with normal or average Red bone marrow deposit.As used herein, the associating of modified polypeptide and adjuvant therapy or combined administration mean in succession (sequential), simultaneously (simutaneous), prolong and use or use described therapy and antibody disclosed by the invention with (contemporaneous) of (coextensive), simultaneous (concurrent) of time, (concomitant) that follow or contemporaneity.Those skilled in the art understand, can arrange each composition in the combined treatment use or the application time to strengthen the overall effectiveness of treatment.For example, can use chemotherapeutics the course of treatment, in several weeks, use radioimmunity conjugate of the present invention afterwards by knowing of standard.Conversely, can use and modified polypeptide bonded cytotoxin by intravenously, position outside beamed radiation afterwards in tumour.In other embodiment again, modified polypeptide can be used in an outpatient service simultaneously with one or more selected therapeutical agents.Those skilled in the art (for example experienced tumour scholar) need not undo experimentation and can easily differentiate effective combined treatment based on selected assisting therapy and the instruction of this explanation.

In this regard, the associating that is appreciated that modified polypeptide and chemotherapeutics can be used with any order and in any time framework that the treatment benefit is provided to the patient.That is, chemotherapeutics and modified polypeptide can or be used simultaneously with any order.In selected embodiment, modified polypeptide of the present invention is used to the patient who carried out chemotherapy before.In other embodiments, modified polypeptide and chemotherapy are basically simultaneously or use concurrently.For example, can give experiencing patient's modified antibodies of chemotherapy treatment.In preferred embodiments, in any chemotherapeutics or treatment 1 year, use modified antibodies.In other preferred embodiment, any chemotherapeutics or treatment 10,8,6,4 or 2 months in use modified polypeptide.In other preferred embodiment again, in 4,3,2 or 1 week of any chemotherapeutics or treatment, use modified polypeptide.In other preferred embodiment again, in selected chemotherapeutics or treatment 5,4,3,2 or 1 days, use modified polypeptide.Be further appreciated that two kinds of medicaments or treatment can (that is basically side by side) use in approximately a few hours or several minutes.

IX. pharmaceutical composition

Pharmaceutical composition of the present invention comprises at least a modified polypeptide that contains Fc by the methods described herein preparation in pharmaceutically acceptable carrier." pharmaceutically acceptable carrier " at least a composition in the pharmaceutical preparation that is usually used in delivery of active ingredients of making a comment or criticism.Similarly, carrier can contain any drug excipient and any type of remedium constituens that is used for administration that use this area.Composition can for example be injection solution, waterborne suspension or solution, non-aqueous suspension or solution, solid and liquid oral medicine, salve (salve), gel, ointment (ointment), transdermal patch (intradermal patch), creme, lotion, tablet, capsule, sustained release preparation etc.Other vehicle can comprise for example tinting material, taste masked agent, dissolution aids, suspension agent, compression agent (compressing agent), casing, slowly-releasing auxiliary agent etc.

Medicament of the present invention is usually used with the pharmaceutical compositions that comprises active therapeutic agent and various other pharmaceutically acceptable compositions.See Remington ' s Pharmaceutical Science (15th ed., Mack PublishingCompany, Easton, Pennsylvania (1980)).Preferred form depends on using of expectation and treats the mode of application.Composition can also comprise pharmaceutically useful non-toxic carrier or thinner (be defined as be generally used for preparing to the pharmaceutical composition of animal or human's class administration remedium constituens) according to the preparation of expectation.Can select thinner not influence the biologic activity of this combination.The example of this type of thinner is distilled water, physiology phosphate-buffered saline, RingerShi solution, glucose solution and HankShi solution.In addition, pharmaceutical composition or preparation can also comprise other carrier, adjuvant or nontoxicity non-therapeutic non-immunogenicity stablizer etc.

Can inject (depost injection) or the form of implantation preparation with depot and use polypeptide, described preparation can be prepared by the mode that allows slow-release.An exemplary composition comprises the 5mg/mL polypeptide that is formulated in the aqueous buffer solution (be made up of 50mM L-Histidine, 150mM NaCl, be adjusted to pH6.0 with HCl).An exemplary universal preparation damping fluid is the 20mM Trisodium Citrate, pH6.0,10% sucrose, 0.1%Tween 80.

Typically, preparation of compositions is become the injection of liquor or form of suspension; Also can prepare and be applicable to the solid form that before injection, is dissolved or suspended in the liquid remedium constituens.Preparation also can emulsification be encapsulated in liposome or particulate such as polylactide, polyglycolide or multipolymer in be used for enhanced adjuvant effect (seeing above discussion) and (see Langer, Science 249:1527,1990 and Hanes, Advanced Drug Delivery Reviews 28:97,1997).

The present invention further illustrates by some embodiment, and these embodiment should not be construed as restrictive.The content of the patent application of all reference, patent and the publication of using in whole the application and accompanying drawing and sequence table all is incorporated herein by reference hereby.

Embodiment

Example I-preparation has the change antibody in chimeric Fc district

For the combination of appraiser's rabbit igg chimeric construct body, at first may influence in 10  that bonded amino acid is defined as the interaction interface between these two interacting proteins.Based on the crystalline structure of rat IgG2a and rat FcRn, determine to be positioned on the rat IgG2a Fc amino acid within 10  at interface of these two molecules.The comparison of the homology in rabbit igg 1 Fc and rat IgG1 Fc district allows to determine which amino acid may be within 10  at interaction interface in the rabbit Fc district then.Determine the counterpart in rat and people Fc district then.Make up chimeric molecule then, be included in all mutant in the 10  interfaces.Then each single amino acids is substituted into separately among the hFc, analyzes with definite described each single composition amino acid bonded is contributed.Utilize then and demonstrate enhanced hFcRn bonded molecule, make up the combination of amino acid mutation body.In this way, only identify the positive contributor of bonded.

Use Biacore, analyze the avidity of each mutant, with demonstrate 25% or the molecule that increases of better avidity be assessed as positive findings.Test is carried out at pH6.

By the known standard technique of those of ordinary skills, transient expression sudden change antibody in 293 cells.Expressed protein contains λ type people F (ab), and it can easily use albumen L purifying.The epi-position of not expressing in this antibody and the animal has reactivity (pK has to be determined).By ELISA, use the antigen with responding property of variable region, and with anti-people H﹠amp; L HRP (horseradish peroxidase) two anti-detections are to be implemented in the detection of this mAb in the blood.These tests and animal model can easily be set up by those of ordinary skills.

On Biacore, measured the Kd of 1 couple of soluble and monomeric hFcRn of rabbit igg.The Kd that will be worth on identical chips and determine at the human IgG1 compares.In these experiments, IgG is fixed on the different quadrants of chip, and can determine the direct comparative result of Kd value.Buy protein and before using, verify lipidated protein by SDS PAGE and analytical gel-filtration.

Finish rabbit igg 1 and human IgG1's sequence alignment, and identified the sequence of determining from rat IgG2a/rFcRn crystalline structure that is positioned at people and rabbit igg interface 10 .

Determine to increase in the IgG constant region of people's antibody and IgG fusion rotein the concrete amino acid replacement of antibody half life.The present invention's sudden change specifically comes from the chimeric protein construct between rabbit igg 1 constant region (rFc) and the hIgG1 constant region (hFc).

Then, the binding affinity in the hFc district by measuring h FcRn and sudden change is determined the influence that these variations bring, and compares with natural hFc that the h/rFc mosaic demonstrates the avidity bigger to hFcRn.Then at monkey or other suitable model for example in gene wedging (knock-in) or the transgenosis hFcRn mouse, determine the transformation period in the body of these molecules, have the transformation period of increase with respect to natural hFc to confirm chimeric protein.

The result: obtained the Biacore data, wherein hFcRn demonstrates the Kd value for the tight twice of binding ratio hIgG of rabbit igg and hFcRn.The example that is used for the alternate residue comprises: EU position 280,281,282,283,285,286,288,289,290,305,307,308,309,315,340,344 and 378.More particularly, polypeptide of the present invention can contain at least one amino acid mutation that is selected from down group: Asp280Asn (wherein D is illustrated in described EU position (278) wait the to be suddenlyd change amino acid position of (by substituting), and N is expressed as and realizes that the polypeptide that changes treats the amino acid that enters this position by substituting), Gly281Glu, Val282Glu, Glu283Gln, His285Arg, Asn286Thr, Lys288Arg, Thr289Pro, Lys290Pro, Val305Thr, Thr307Pro, Val308Ile, Leu309Thr, Asn3153Arg, Lys340Arg, Arg344Leu, Ala378Ser, Ser383Lys, Glu386Lys, Pro387Ala, and Asn389Asp (according to the EU numbering system).

Embodiment 2-identifies candidate's residue by static optimization

In the present embodiment, description is used for the method for modified antibodies constant region to the avidity of people FcRn.In order to obtain when acid pH and the neutral pH Fc district FcRn is had the mutant of the binding affinity of change, we are applied to homologue model with people FcRn bonded people Fc with the static electric charge optimisation technique.The model of the people Fc/FcRn complex body when acid (6.0) pH and neutrality (7.4) pH uses MODELLER program (Accelrys, Inc., San Diego, CA) by crystalline structure (the PDB code: 1I1A) derive of rat Fc/FcRn complex body, and at CHARMM (Accelrys, Inc., San Diego has carried out energy minimization in CA).In the computational optimizing process, we use static charge optimization so that determine can adjustment of acidity pH and position (Lee and Tidor, J.Chem.Phys.106:8681-8690,1997 of the bonded Fc residue of Fc and FcRn during neutral pH; Kangas and Tidor, J.Chem.Phys.109:7522-7545,1998).Two pH values, finish these calculating when acid (6.0) pH and neutrality (7.4) pH, when acid pH in pinocytotic vacuole because known Fc discharges at ECS in conjunction with FcRn and when the neutral pH.Fc is combined with the transformation period that may damage Fc to the enhancing of FcRn during neutral pH.If IgG can not discharge from cell bonded FcRn in typical IgG purge mechanism, then the minimizing of transformation period will be an expected result.Therefore, the containing Fc albumen and will from blood, remove of avidity in that higher pH has increase to FcRn, cause protein to have than short-half-life.

These mutation forecastings can be divided into and relate to: (1) in conjunction with the time sudden change that related residue place, the hidden interaction interface of part carries out (form hydrogen bond by face transboundary, improve interacting) that becomes; (2) sudden change of the following polar residues on the antibody, described polar residues in conjunction with the time become hidden and pay a price for desolvation thus but not with FcRn form any direct electrostatic interaction (usually by be mutated into have the hydrophobic residue of similar shape with the wild-type residue or by adding the residue that can cause favourable electrostatic interaction, to be improved); (3) sudden change of the following surface residue on the Fc, described surface residue is present in the zone of incomplementarity electromotive force.Think these sudden changes can improve the long-range electrostatic interaction between Fc and FcRn and do not upset the accumulative facies mutual effect of bonding interface, and comprise two kinds of sudden changes that keep and change the branch charge of the electron.

When carrying out electric charge optimization, various restrictions are set to represent natural side chain feature.For example, for-1,0 and+1 clean side-chain charges, additional restrictions during optimization-none atomic charge surpasses the absolute value of 0.85 elementary charge unit.(CA) standard scheme of interpolation hydrogen has prepared model for Accelrys, Inc.SanDiego with program CHARMM in use.Except the N end of B2M chain, N-ethanamide and N-methane amide patchery thing (patch) are added to N end and C end respectively.Separately preparation is used for the model of acid pH and pH neutral, with the different protonated and rotational isomeric state of explanation histidine residues and the rotational isomeric state of l-asparagine and glutamine.Use continuous static model (continnum electrostatics model), we have carried out static electric charge optimization to each side chain that is positioned at the amino acid (candidate's residue) of 15  at FcRn interface among the Fc when acid pH and the pH neutral.Result based on electric charge is optimized has determined to be used for the sudden change of other computational analysis.In this process, our visual inspection best charge distribution and having designed than present residue more near the sudden change of optimum.Electric charge optimization provides electric charge at former subcenter, but does not produce true sudden change.Under adding that various restricted conditions are with the situation of representing natural side chain feature, carried out one and taken turns electric charge optimization.For example, utilizing additional restrictions---non-atomic electric charge surpasses the absolute value of 0.85 elementary charge unit, and at-1,0 and+1 clean side-chain charges is optimized.Only select as upper/lower positions, described desirable charge distribution place alternative can between neutral pH and acid pH, produce be no less than the 0.3kcal/ mole people FcRn in conjunction with difference.Analyze the desirable charge distribution on the existing side chain, and proposed a cover substituting from 20 kinds of natural amino acids.

Following example has shown the optimum result that Xie Ansuan 284, Histidine 285, l-asparagine 286 and the Methionin 290 (example of selected residue) at the Fc molecule obtains.Mut (sudden change energy) hurdle corresponding to from natural residue to complete uncharged side chain isostere (that is, have identical shaped but on atom, do not have electric charge or the residue of Partial charge is arranged) in conjunction with free energy difference (kcal/mol).The binding affinity of negative indication prediction increases.The Opt-1 hurdle corresponding to the clean side-chain charges with charge distribution best in the side chain and-1 can access in conjunction with free energy difference.Hurdle Opt0 and Opt1 respectively corresponding in net charge be 0 and had in+1 o'clock best electric charge in conjunction with free energy difference.

For acid pH, obtained following result:

Residue Mut Opt-1 Opt0 Opt1

V284 0.0 -0.9 -0.3 0.5

H285 -0.6 -2.0 -1.6 -1.1

N286 0.0 -1.1 -0.2 1.1

K290 -0.7 -0.8 -0.9 -0.8

For neutral pH, obtained following result:

Residue Mut Opt-1 Opt0 Opt1

V284 0.0 -0.2 -0.3 -0.3

H285 0.1 -0.2 -0.1 0.2

N286 0.1 -0.3 -0.3 -0.3

K290 -0.2 0.1 -0.3 -0.3

Based on these results with to the visual analysis of model, the transformations to L-glutamic acid that can utilize these to improve in conjunction with free energy have been proposed.In fact, for all positions, by wild-type amino acid is mutated into L-glutamic acid, all can between neutral pH and acid pH, obtain Δ Δ G greater than the people FcRn of 0.3kcal/mol in conjunction with difference.

By in CHARMM, being rotated the scanning of isomer interfacial angle, use the interfacial angle angle step of 30 or 60 degree, set up the sudden change side chain, to determine the optimal position of each side chain.Use Poisson-Boltzmann electrostatic energy and the extraneous term that is used for Fan Dewaershi energy and hidden surface area then, calculate the bound energy of wild-type and sudden change complex body.

Use the amino acid electric charge of PARSE or CHARMM to set, when acid pH and neutral pH, calculate.Use consistent marking scheme, mean that using two kinds of electric charges to set the result who obtains should have the indication that conforms to, so that produce significant prediction.

Following example illustrates the use of this method on Xie Ansuan 284, Histidine 285, l-asparagine 286 and the Methionin 288 of Fc molecule:

Residue numbering sudden change G _6.0parseG _7.4parseG _6.0charmmG _7.4charmm

V 284 E -1.7 0?.7 -1.4 -0.3

H 285 E -2.5 0.4 -2.3 -1.2

N 286 E -0.4 1?.2 -1.5 -0.7

K 290 E -0.9 -0.4 -1.8 0.7

When acid pH, the combination of people Fc and people FcRn is with respect to the combination of wild-type Fc and FcRn, and Δ Δ G all is lower than-0.3kcal/mol under two kinds of electric charges are set, and illustrates that avidity increases for wild-type people Fc.When neutral pH, Δ Δ G has different indications under two kinds of electric charges are set, and illustrates that mutant can not have higher people FcRn binding affinity (in the content of consistent marking scheme) than wild-type people Fc when neutral pH.Therefore, estimate that V284E, H285E, N286E and K290E mutant (example of selected mutant) have the transformation period of longer FcRn mediation than wild type molecule in organism.

In a word, we have carried out the aforementioned calculation method to predict the sudden change that can regulate the FcRn binding affinity of Fc molecule in neutral and acid ph value allowance below nominal size strange land.We propose, and have the FcRn avidity of increase in the time of can causing acid pH in people Fc but do not influence or the amino acid replacement of the interaction avidity when reducing neutral pH, will cause having the longer body Fc molecule of interior transformation period.Fc/FcRn combination that generation increases when neutral pH and/or the Fc/FcRn bonded amino acid replacement that generation weakens when acid pH will show and cause transformation period minimizing in the body.

Embodiment 4: the structure of the Fc polypeptide of change

To introduce in the initial polypeptide of coding humanization IgG1 monoclonal antibody huCBE11 heavy chain by the change of the inventive method prediction.Figure 1A and 1B have shown the nucleotide sequence (SEQ ID NO:3) of this heavy chain and the aminoacid sequence (SEQ ID NO:4) of prediction respectively.Utilize the standard recombinant dna technology,, introduce sudden change in the huCBE11 heavy chain that on the expression vector that is called pEAG1787, carries by site-directed mutagenesis.Antibody variable region is that 1-120 position residue, human IgG1's constant region are 121-449 position residues.The C end lysine residue heredity of huIgG1 is removed.For reference: the glycosylation site (EU residue N297) that N connects in above-mentioned sequence is the 300th residue.Fig. 2 shows the aminoacid sequence in the Fc district of huCBE11 according to the EU numeral index.

The HuCBE11 monoclonal antibody is humanization IgG1, κ recombinant antibodies, identification human lymphotoxin beta receptor.The clone of mAb CBE11, chimericization and humanization are described in the U.S. Patent application 2004/0058394.The HuCBE11 light chain is carried in the expression vector that is called pEAG1754.The variable region is a 1-107 position residue, and people κ constant region is a 108-214 position residue.Fig. 3 A and 3B show the nucleotide sequence (SEQ ID NO:5) of this light chain and the aminoacid sequence (SEQ IDNO:6) of prediction respectively.

The polypeptide of wild-type and change is by expressing with heavy chain carrier (pEAG1787) and light chain carrier (pEAG1754) transient cotransfection human embryonic kidney cell system (HEK293E).

The structure of embodiment 5:FcRn-Fc fusion rotein agent

Merge the extracellular domain of neonatal Fc receptor and the Fc district of IgG1 antibody by heredity, it is conjugated protein to make up dimerization Fc.

In brief, by RT-PCR from people's placenta polyA+RNA human cloning β2Wei Qiudanbai cDNA.The aminoacid sequence (SEQ ID NO:8) of the nucleotide sequence of people's β2Wei Qiudanbai cDNA (SEQ ID NO:7) and prediction is presented at respectively among Fig. 4 A and the 4B.From people's placenta polyA+RNA, clone cDNA by RT-PCR corresponding to the α chain of people FcRn, and order-checking.With extracellular domain (ECD) (the 1-297 position residue) subclone of people FcRn α chain, so that merge with the people Fc district that derives from IgG1 antibody.

Fc district (398-535 position residue) derives from human IgG1's antibody (seeing U.S. Patent number 5,928,643) of having described earlier.IgG1 Fc contains in the district hinge of brachymemma, but has kept the CH2 and the CH3 structural domain of this antibody, and the C end lysine residue of CH3 structural domain has been removed in heredity.Figure 1A and 1B have shown the nucleotide sequence (SEQ ID NO:3) of IgG1 heavy chain of antibody cDNA and the aminoacid sequence (SEQ ID NO:4) of prediction.

In order to eliminate the possibility of fused protein in conjunction with FcRn, pass through site-directed mutagenesis, the Fc district (is shown as the residue 388 of band underscore 4 positions in the sequence that Fig. 5 B shows, 389,511 and 512) locate to suddenly change from its wild-type sequence, to produce previous verified FcRn binding affinity and serum half-life (Kim etc., 1994, the Eur.J.Immunol.24:542-548 that can reduce significantly with FcRn; With Popov etc. 1996, people's Equivalent (" huM4Fc ") of H310A/Q311N/H433A/N434Q Mol.Immunol.33:521-530) (EU numbering) mutant.

Use the standard recombinant dna technology, the C end of this Fc district and FcRn ECD is merged to form FcRn-Fc fusion rotein cDNA.β2Wei Qiudanbai and FcRn-Fc fusion rotein cDNA are inserted in the expression vector (pEAG1761), and this expression vector carries this two kinds of cDNA in the tandem transcription units that is driven by the cytomegalovirus immediate early promoter.Secrete the solubility dimer stable cell lines of (assorted four poly-FcRn-Fc fusion roteins are made up of two B2Ms and two FcRn α-Fc fusion chains) with the pEAG1761 transfection CHO cell to produce.Fig. 5 A and 5B show that respectively the people FcRn α-Fc of pEAG1761 coding merges the nucleotide sequence (SEQ ID NO:9) of cDNA and the aminoacid sequence (SEQ ID NO:10) of prediction.

Use 10,000 MWCO poly (ether sulfone) film, in Amicon jar unit (stirred cell unit), the conditioned medium (from CHO or 293 cells) that will contain excretory FcRn-huM4Fc (α chain and B2M) fusion rotein concentrate about 6 times to 150ml.This enriched material of dialysis spends the night in the 20mM of 40 times of volumes MES (pH5.8), 150mM NaCl, and centrifugal 10 minutes 0 of 4 ℃ of 2060xg are with the sedimentation cell fragment afterwards.It is added to 6ml human IgG-Sepharose 6 Fast Flow (AmershamBiosciences) posts, this post in 20mM MES (pH5.8), 150mM NaCl in advance through overbalance.Damping fluid with one step changes (pH8 among 20mM Tris, the 150mM NaCl), the FcRnFc of elution of bound.The hIgG-Sepharose post is successfully used to catch rat, mouse and people FcRn-huM4Fc albumen.

By the fraction of irreducibility SDS-PAG (4%-20% gradient gel) analysis wash-out, the fraction that will contain FcRn-huM4Fc merges, to be further purified by anion-exchange chromatography.Use 1mg/ml people or rat protein matter solution (A ₂₈₀Value is 1.80), and 1mg/ml murine protein matter solution (A ₂₈₀Value is 1.84), spectrophotometry is determined protein concn.The about 70mg hFcRn-M4Fc of wash-out from the IgG-Sepharose post.This is merged thing in 20mM Tris pH8, dialyse, go up 5ml DEAE post afterwards.From the DEAE post use be up to 0.5M NaCl final concentration increase progressively salt linear gradient (20 times of column volumes), the protein of elution of bound.FcRn-huM4Fc albumen is located wash-out at about 150mM-175mM NaCl (30%-35%0.5M NaCl damping fluid).Utilize the same SDS-PAGE to analyze the purity of DEAE fraction, merge the fraction that contains FcRn-huM4Fc, the FcRnFc that obtains the 48mg homogeneous merges thing.

Concentrate this DEAE and merge thing, and it is centrifugal by Vivaspin concentrating unit (10,000 MWCO) buffer exchange is become 20mM MES (pH5.8), 150mM NaCl damping fluid so that-80 ℃ of storages, perhaps be used for size exclusion chromatography (Superdex200 16/60, Amersham Biosciences)---last purification step---to remove accumulations in a small amount.

Embodiment 6: the FcRn binding affinity of analyzing the polypeptide that changes

Use various Cell free assay to identify the human monoclonal antibodies that the present invention changes by its ability in conjunction with biotinylation FcRn.

A) the Fc polypeptide of purifying change

Use the FcRn-huM4Fc fusion rotein of embodiment 4, purifying contains the human monoclonal antibodies in the Fc district that changes.In order to be beneficial to purifying, the FcRn-huM4Fc fusion rotein is fixed on the Sepharose 4 FastFlow Media.In PBS (pH2.2), shake following 4 ℃ of couplings of spending the night by about 20mg hFcRn and NHS activatory Sepharose 4 Fast Flowmedia (Amersham Biosciences), preparation 2ml hFcRnFc post.Wash this post with PBS, be used for purifying afterwards.Everyone FcRnFc post 1mg.

Each 1mg of human IgG1 mAb of every kind of change is put on this post in sample-loading buffer 20mM MES (pH5.8), 150mM NaCl.With the sample-loading buffer washing of 10 times of column volumes, use the mAb of pH substep gradient (20mM Tris, the 150mM NaCl) elution of bound of rising pH to 8.0 after the pillar.

B) biotinylation of FcRn-huM4Fc (bhFcRnFc):

Vitamin H XX sulfosuccinimide base ester (two amino caproyl chains of XX=) be used for to FcRn-huM4Fc carry out vitamin H for use in following competitiveness, based on pearl, FRET and AlphaScreen test.This reagent can obtain from the Mini-Biotin-XX protein labeling test kit of Molecular Probes.FcRnFc dialyses among pH7.0 and the 150mMNaCl at PBS 150mM Na/K phosphoric acid salt.1M hydrogen-carbonate Na (50 μ L) carbonate (NaHCO3) liquid storage that adds 1/10 volume to 0.72mg/ml hFcRnFcIgG (500 μ l).Then add 10 μ l vitamin H-XX reagent (facing with before being dissolved in the water) by Molecular Probes is described.Reaction incubated at room 1 hour, 4 ℃ of thoroughly dialysis in PBS then.During at-80 ℃, this biotinylated fusion rotein (bhFcRnFc) is stable when frozen.

C) directly in conjunction with test:

Use the ELISA form, estimate unlabelled hFcRn-Fc and combine with the direct of Fc polypeptide that the present invention changes.

Unless otherwise indicated, otherwise all washings and the dilution all in pH6.0 PBS, carry out.Plank is spent the night by every hole 100 μ l bag at 4 ℃ of IgG antibody that change with 5 μ g/ml.Washing plank 3 times wraps by 2 hours with 1%BSA then.Wash plank then 3 times, and with the FcRnFc incubation of the suitable serial dilution of 100 μ L 90 minutes.Wash plank once more 3 times, and add suitable secondary mAb-HRP of 100 μ L or Streptavidin HRP, incubation 90 minutes washs plank once more, and according to for the colour developing of selected secondary mAb appropriate means, and read absorbancy.Use four parameter fittings of binding data shown in Figure 10, determine apparent dissociation constant (Kd).

D) CBA:

Variant antibody uses two kinds to determine near test (FRET and AlphaScreen) to the RA of the dimerization FcRn construction (bhFcRnFc) of mark, and the competition of estimating in described test between the polypeptide of fluorescently-labeled control antibodies (5C8) and the change of embodiment 4 combines.

I) FRET is in conjunction with test:

In black half hole elisa plate, carry out the FRET test with 30 μ L total reaction volume.(Biacore contains in pH5.8 damping fluid Tween-20): a) the 1nM control antibodies (h5C8) of europium mark each reaction mixture having 100mM NaCl and 0.005%P2O stain remover; B) 250nM is with the Streptavidin of second fluorophore (APC) mark; C) from suitable rival mAb (the 0-5 μ Molar of embodiment 4; 0-0.75mg/ml); And d) 200nM bhFcRnFc.Add bhFcRnFc to reaction mixture at last.

Be reflected at the room temperature incubation 30 minutes, and went up at LJL Analyst (Molecular Devices) then and use 615 excitation wavelengths and 665 fluorescent emission wavelength to carry out reading.LJL Analyst is set to 50 μ s time lags and 400 μ s readings.The signal of APC and Eu (665nm/615nm) is mapped with respect to the concentration of rival mAb than (Δ F), to determine IC50 (μ g/ml).The hAb and the rival IgG of negative control reaction lack europium mark, positive control does not contain rival IgG.

Determine the relative IC of the antibody of every kind of change ₅₀Value (mutant IC50/w.t. contrasts IC50), and mapping (Fig. 6).Fig. 6 shows, 285,286,290 and 304 antibody that contain sudden change especially contain sudden change H285E in the EU position, and N286D, the antibody of K290E and S304D cause dimerization FcRn/Fc fusion rotein enhanced RA (relative IC ₅₀＜1).On the contrary, the antibody of many changes is (that is, in the EU position 252,255,279,282,284,285,287,288,290,304,306,309,376,434, with 438 antibody that contain those changes of sudden change) dimerization FcRn/Fc fusion rotein is demonstrated apparent binding affinity reduce (relative IC ₅₀＞1).Especially, the binding affinity that demonstrates FcRn of the sudden change located of EU position 282 (V282E), 290 (K290D), 438 (Q438E) and 434 (N434L) significantly reduces.For example, sudden change N434L causes comparing according to the low about 400 times FcRn binding affinity of antibody.

Ii) ALPHA Screen is in conjunction with test:

AlphaScreen is based on the test of pearl, and it uses respectively the donor and the acceptor bead of puting together with binding partners (that is the Fc polypeptide of dimeric Fc binding proteins and change).Donor bead produces singlet oxygen with photosensitizers bag quilt, this photosensitizers in laser excitation (for example, at 680nm) back.The acceptor bead of chemoluminescence bag quilt singlet oxygen is therewith reacted, and discharges the light of 520-620nm.Because the transformation period that singlet oxygen is lacked in solution very much is so the degree of approach of the chemoluminescence of acceptor bead and donor bead is proportional.Therefore, the combination of donor and acceptor bead causes greatly enhanced chemiluminescence signal.

(Corning, Acton add biotinylated Fc/FcRn fusion rotein (bhFcRnFc) to the antibody of the change of the embodiment 4 of two times of serial dilutions in MA) at 384 holes white Costar plate.Behind 30 minutes room temperature incubations, the acceptor bead of puting together with 20 μ g/ml (final concentration) adding human IgG1 in 25 μ l end reaction volumes is (by PerkinElmer Biosignal Inc., Montreal, Canada puts together) and donor bead (the PerkinElmer Biosignal Inc. that puts together of Streptavidin, Montreal, Canada).Donor bead that this arrangement causes reaction mixture to contain puting together with Fc/FcRn fusion rotein bonded Streptavidin and the acceptor bead of puting together with the IgG1 of the antibodies that changes.

Reaction mixture room temperature incubation 1 hour, (PerkinElmerBiosignal Inc., Montreal Canada) go up reading at the Fusion-Aplha reader.Use PBS, 0.1% bovine serum albumin and 0.01Tween-20, pH6.0 is as the test damping fluid.End user IgG1 is as positive control.The test damping fluid that uses pH7.0 as negative control with identifier IgG1 and people FcRnFc debond.Use GraphPad Prism software analysis result.

At the definite IC of the antibody of every kind of change ₅₀Value (μ g/ml) is summarised among Fig. 7.Fig. 7 explanation, 284,285,286 and 288 contain sudden change in the EU position, the V284E that especially suddenlys change, H285E, N286D, the antibody of K290E causes the apparent binding affinity (IC of dimerization FcRn/Fc fusion rotein enhanced with respect to control antibodies ₅₀＜1).On the contrary, the antibody of many changes is (that is, in EU position 252,255,279,282,284,285,286,290,304,306,309,314,313,345,376,434 and 438 contain the antibody of those changes of sudden change) for control antibodies, dimerization FcRn/Fc fusion rotein is demonstrated the apparent binding affinity (IC of reduction ₅₀＞1).Especially, EU position 252 (M252S and M252T), 434 (N434L) and 438 (Q438E) demonstrate significantly reduced binding affinity to FcRn.For example, sudden change Q438E causes comparing according to the low about 1500 times FcRn binding affinity of antibody.

Equivalent

One of skill in the art will appreciate that or can only use normal experiment promptly to determine the many schemes that are equal to specific embodiments of the present invention as herein described.These equivalents all are intended to be included in the following claim.

Sequence table

SEQ?ID?NO：1

231 APELLGG

238 PSVFLFPPKP

248 KDTLMISRTP

258 EVTCVVVDVS

268 HEDPEVKFNW

278 YVDGVEVHNA

288 KTKPREEQYN

298 STYRVVSVLT

308 VLHQDWLNGK

318 EYKCKVSNKA

328 LPAPIEKTIS

338 KAK

SEQ?ID?NO：2

341 GQPREPQ

348 VYTLPPSRDE

358 LTKNQVSLTC

368 LVKGFYPSDI

378 AVEWESNGQP

388 ENNYKTTPPV

398 LDSDGSFFLY

408 SKLTVDKSRW

418 QQGNVFSCSV

428 MHEALHNHYT

438 QKSLSLSPG

Ripe huCBE11 heavy chain dna sequence dna (SEQ ID NO.3) by the pEAG1787 coding

1 GAGGTACAAC?TGGTGGAGTC?TGGGGGAGGC?TTAGTGAAGC?CTGGAGGGTC

51 CCTGAGGCTC?TCCTGTGCAG?CCTCTGGATT?CACTTTCAGT?GACTATTACA

101 TGTATTGGTT?TCGCCAGGCC?CCGGGAAAGG?GGCTGGAGTG?GGTCGCAACC

151 ATTAGTGATG?GTGGTAGTTA?CACCTACTAT?CCAGACAGTG?TGAAGGGGCG

201 ATTCACCATC?TCCAGAGACA?ATGCCAAGAA?CAGCCTCTAC?CTGCAGATGA

251 GCAGCCTGAG?GGCTGAGGAC?ACAGCTGTGT?ATTACTGCGC?AAGAGAGGAG

301 AATGGTAACT?TTTACTACTT?TGACTACTGG?GGCCAAGGGA?CCACGGTCAC

351 CGTCTCCTCA?GCCTCCACCA?AGGGCCCATC?GGTCTTCCCC?CTGGCACCCT

401 CCTCCAAGAG?CACCTCTGGG?GGCACAGCGG?CCCTGGGCTG?CCTGGTCAAG

451 GACTACTTCC?CCGAACCGGT?GACGGTGTCG?TGGAACTCAG?GCGCCCTGAC

501 CAGCGGCGTG?CACACCTTCC?CGGCTGTCCT?ACAGTCCTCA?GGACTCTACT

551 CCCTCAGCAG?CGTGGTGACC?GTGCCCTCCA?GCAGCTTGGG?CACCCAGACC

601 TACATCTGCA?ACGTGAATCA?CAAGCCCAGC?AACACCAAGG?TGGACAAGAA

651 AGTTGAGCCC?AAATCTTGTG?ACAAGACTCA?CACATGCCCA?CCGTGCCCAG

701 CACCTGAACT?CCTGGGGGGA?CCGTCAGTCT?TCCTCTTCCC?CCCAAAACCC

751 AAGGACACCC?TCATGATCTC?CCGGACCCCT?GAGGTCACAT?GCGTGGTGGT

801 GGACGTGAGC?CACGAAGACC?CTGAGGTCAA?GTTCAACTGG?TACGTGGACG

851 GCGTGGAGGT?GCATAATGCC?AAGACAAAGC?CGCGGGAGGA?GCAGTACAAC

901 AGCACGTACC?GTGTGGTCAG?CGTCCTCACC?GTCCTGCACC?AGGACTGGCT

951 GAATGGCAAG?GAGTACAAGT?GCAAGGTCTC?CAACAAAGCC?CTCCCAGCCC

1001 CCATCGAGAA?AACCATCTCC?AAAGCCAAAG?GGCAGCCCCG?AGAACCACAG

1051 GTGTACACCC?TGCCCCCATC?CCGGGATGAG?CTGACCAAGA?ACCAGGTCAG

1101 CCTGACCTGC?CTGGTCAAAG?GCTTCTATCC?CAGCGACATC?GCCGTGGAGT

1151 GGGAGAGCAA?TGGGCAGCCG?GAGAACAACT?ACAAGACCAC?GCCTCCCGTG

1201 TTGGACTCCG?ACGGCTCCTT?CTTCCTCTAC?AGCAAGCTCA?CCGTGGACAA

1251 GAGCAGGTGG?CAGCAGGGGA?ACGTCTTCTC?ATGCTCCGTG?ATGCATGAGG

1301 CTCTGCACAA?CCACTACACG?CAGAAGAGCC?TCTCCCTGTC?TCCCGGTTGA

The aminoacid sequence (SEQ ID NO.4) of the prediction of ripe huCBE11 heavy chain

1 EVQLVESGGG?LVKPGGSLRL?SCAASGFTFS?DYYMYWFRQA?PGKGLEWVAT

51 ISDGGSYTYY?PDSVKGRFTI?SRDNAKNSLY?LQMSSLRAED?TAVYYCAREE

101 NGNFYYFDYW?GQGTTVTVSS?ASTKGPSVFP?LAPSSKSTSG?GTAALGCLVK

151 DYFPEPVTVS?WNSGALTSGV?HTFPAVLQSS?GLYSLSSVVT?VPSSSLGTQT

201 YICNVNHKPS?NTKVDKKVEP?KSCDKTHTCP?PCPAPELLGG?PSVFLFPPKP

251 KDTLMISRTP?EVTCVVVDVS?HEDPEVKFNW?YVDGVEVHNA?KTKPREEQYN

301 STYRVVSVLT?VLHQDWLNGK?EYKCKVSNKA?LPAPIEKTIS?KAKGQPREPQ

351 VYTLPPSRDE?LTKNQVSLTC?LVKGFYPSDI?AVEWESNGQP?ENNYKTTPPV

401 LDSDGSFFLY?SKLTVDKSRW?QQGNVFSCSV?MHEALHNHYT?QKSLSLSPG*

Dna sequence dna (SEQ ID NO.5) by the ripe huCBE11 light chain of pEAG1754 coding

1?GATATCCAGA?TGACCCAGTC?TCCATCATCC?TTGTCTGCAT?CGGTGGGAGA

51?CAGGGTCACT?ATCACTTGCA?AGGCGGGTCA?GGACATTAAA?AGCTATTTAA

101?GCTGGTACCA?GCAGAAACCA?GGGAAAGCGC?CTAAGCTTCT?GATCTATTAT

151?GCAACAAGGT?TGGCAGATGG?GGTCCCATCA?AGATTCAGTG?GCAGTGGATC

201?TGGTACAGAT?TATACTCTAA?CCATCAGCAG?CCTGCAGCCT?GAGGATTTCG

251?CAACTTATTA?CTGTCTACAG?CATGGTGAGA?GCCCGTGGAC?GTTCGGTGGA

301?GGCACCAAGC?TGGAGATCAA?ACGAACTGTG?GCTGCACCAT?CTGTCTTCAT

351?CTTCCCGCCA?TCTGATGAGC?AGTTGAAATC?TGGAACTGCC?TCTGTTGTGT

401?GCCTGCTGAA?TAACTTCTAT?CCCAGAGAGG?CCAAAGTACA?GTGGAAGGTG

451?GATAACGCCC?TCCAATCGGG?TAACTCCCAG?GAGAGTGTCA?CAGAGCAGGA

501?CAGCAAGGAC?AGCACCTACA?GCCTCAGCAG?CACCCTGACG?CTGAGCAAAG

551?CAGACTACGA?GAAACACAAA?GTCTACGCCT?GCGAAGTCAC?CCATCAGGGC

601?CTGAGCTCGC?CCGTCACAAA?GAGCTTCAAC?AGGGGAGAGT?GTTAG

The aminoacid sequence (SEQ ID NO.6) of the prediction of ripe huCBE11 light chain

1 DIQMTQSPSS?LSASVGDRVT?ITCKAGQDIK?SYLSWYQQKP?GKAPKLLIYY

51 ATRLADGVPS?RFSGSGSGTD?YTLTISSLQP?EDFATYYCLQ?HGESPWTFGG

101 GTKLEIKRTV?AAPSVFIFPP?SDEQLKSGTA?SVVCLLNNFY?PREAKVQWKV

151 DNALQSGNSQ?ESVTEQDSKD?STYSLSSTLT?LSKADYEKHK?VYACEVTHQG

LSSPVTKSFN?RGEC*

Dna sequence dna (SEQ ID NO.7) by people's beta-2-microglobulin of pEAG1761 coding

1?ATGTCTCGCT?CCGTGGCCTT?AGCTGTGCTC?GCGCTACTCT?CTCTTTCTGG

51?CCTGGAGGCT?ATCCAGCGTA?CTCCAAAGAT?TCAGGTTTAC?TCACGTCATC

101?CAGCAGAGAA?TGGAAAGTCA?AATTTCCTGA?ATTGCTATGT?GTCTGGGTTT

151?CATCCATCCG?ACATTGAAGT?TGACTTACTG?AAGAATGGAG?AGAGAATTGA

201?AAAAGTGGAG?CATTCAGACT?TGTCTTTCAG?CAAGGACTGG?TCTTTCTATC

251?TCTTGTACTA?CACTGAATTC?ACCCCCACTG?AAAAAGATGA?GTATGCCTGC

301?CGTGTGAACC?ATGTGACTTT?GTCACAGCCC?AAGATAGTTA?AGTGGGATCG

351?AGACATGTAA

The aminoacid sequence of the prediction of people's beta-2-microglobulin (SEQ ID NO.8)

1?MSRSVALAVL?ALLSLSGLEA?IQRTPKIQVY?SRHPAENGKS?NFLNCYVSGF

51?HPSDIEVDLL?KNGERIEKVE?HSDLSFSKDW?SFYLLYYTEF?TPTEKDEYAC

101?RVNHVTLSQP?KIVKWDRDM*

Dna sequence dna (SEQ ID NO.9) by the people FcRn α-Fc fusion rotein of pEAG1761 coding

1?ATGGGGGTCC?CGCGGCCTCA?GCCCTGGGCG?CTGGGGCTCC?TGCTCTTTCT

51?CCTTCCTGGG?AGCCTGGGCG?CAGAAAGCCA?CCTCTCCCTC?CTGTACCACC

101?TTACCGCGGT?GTCCTCGCCT?GCCCCGGGGA?CTCCTGCCTT?CTGGGTGTCC

151?GGCTGGCTGG?GCCCGCAGCA?GTACCTGAGC?TACAATAGCC?TGCGGGGCGA

201?GGCGGAGCCC?TGTGGAGCTT?GGGTCTGGGA?AAACCAGGTG?TCCTGGTATT

251?GGGAGAAAGA?GACCACAGAT?CTGAGGATCA?AGGAGAAGCT?CTTTCTGGAA

301?GCTTTCAAAG?CTTTGGGGGG?AAAAGGTCCC?TACACTCTGC?AGGGCCTGCT

351?GGGCTGTGAA?CTGGGCCCTG?ACAACACCTC?GGTGCCCACC?GCCAAGTTCG

401?CCCTGAACGG?CGAGGAGTTC?ATGAATTTCG?ACCTCAAGCA?GGGCACCTGG

451?GGTGGGGACT?GGCCCGAGGC?CCTGGCTATC?AGTCAGCGGT?GGCAGCAGCA

501 GGACAAGGCG?GCCAACAAGG?AGCTCACCTT?CCTGCTATTC?TCCTGCCCGC

551 ACCGCCTGCG?GGAGCACCTG?GAGAGGGGCC?GCGGAAACCT?GGAGTGGAAG

601 GAGCCCCCCT?CCATGCGCCT?GAAGGCCCGA?CCCAGCAGCC?CTGGCTTTTC

651 CGTGCTTACC?TGCAGCGCCT?TCTCCTTCTA?CCCTCCGGAG?CTGCAACTTC

701 GGTTCCTGCG?GAATGGGCTG?GCCGCTGGCA?CCGGCCAGGG?TGACTTCGGC

751 CCCAACAGTG?ACGGATCCTT?CCACGCCTCG?TCGTCACTAA?CAGTCAAAAG

801 TGGCGATGAG?CACCACTACT?GCTGCATTGT?GCAGCACGCG?GGGCTGGCGC

851 AGCCCCTCAG?GGTGGAGCTG?GAATCTCCAG?CCAAGTCCTC?CGTCGACAAA

901 ACTCACACAT?GCCCACCGTG?CCCAGCACCT?GAACTCCTGG?GGGGACCGTC

951 AGTCTTCCTC?TTCCCCCCAA?AACCCAAGGA?CACCCTCATG?ATCTCCCGGA

1001 CCCCTGAGGT?CACATGCGTG?GTGGTGGACG?TGAGCCACGA?AGACCCTGAG

1051 GTCAAGTTCA?ACTGGTACGT?GGACGGCGTG?GAGGTGCATA?ATGCCAAGAC

1101 AAAGCCGCGG?GAGGAGCAGT?ACAACAGCAC?GTACCGTGTG?GTCAGCGTCC

1151 TCACCGTCCT?GGCTAACGAC?TGGCTGAATG?GCAAGGAGTA?CAAGTGCAAG

1201 GTCTCCAACA?AAGCCCTCCC?AGCCCCCATC?GAGAAAACCA?TCTCCAAAGC

1251 CAAAGGGCAG?CCCCGAGAAC?CACAGGTGTA?CACCCTGCCC?CCATCCCGGG

1301 ATGAGCTGAC?CAAGAACCAG?GTCAGCCTGA?CCTGCCTGGT?CAAAGGCTTC

1351 TATCCCAGCG?ACATCGCCGT?GGAGTGGGAG?AGCAATGGGC?AGCCGGAGAA

1401 CAACTACAAG?ACCACGCCTC?CCGTGTTGGA?CTCCGACGGC?TCCTTCTTCC

1451 TCTACAGCAA?GCTCACCGTG?GACAAGAGCA?GGTGGCAGCA?GGGGAACGTC

1501 TTCTCATGCT?CCGTGATGCA?TGAGGCTCTG?GCGCAGCACT?ACACGCAGAA

1551 GAGCCTCTCC?CTGTCTCCGG?GTTGA

The aminoacid sequence of the prediction of people FcRn α-Fc fusion rotein (SEQ ID NO.10)

1 MGVPRPQPWA?LGLLLFLLPG?SLGAESHLSL?LYHLTAVSSP?APGTPAFWVS

51 GWLGPQQYLS?YNSLRGEAEP?CGAWVWENQV?SWYWEKETTD?LRIKEKLFLE

101 AFKALGGKGP?YTLQGLLGCE?LGPDNTSVPT?AKFALNGEEF?MNFDLKQGTW

151 GGDWPEALAI?SQRWQQQDKA?ANKELTFLLF?SCPHRLREHL?ERGRGNLEWK

201 EPPSMRLKAR?PSSPGFSVLT?CSAFSFYPPE?LQLRFLRNGL?AAGTGQGDFG

251 PNSDGSFHAS?SSLTVKSGDE?HHYCCIVQHA?GLAQPLRVEL?ESPAKSSVDK

301 THTCPPCPAP?ELLGGPSVFL?FPPKPKDTLM?ISRTPEVTCV?VVDVSHEDPE

351 VKFNWYVDGV?EVHNAKTKPR?EEQYNSTYRV?VSVLTVL AND?WLNGKEYKCK

401 VSNKALPAPI?EKTISKAKGQ?PREPQVYTLP?PSRDELTKNQ?VSLTCLVKGF

451 YPSDIAVEWE?SNGQPENNYK?TTPPVLDSDG?SFFLYSKLTV?DKSRWQQGNV

501 FSCSVMHEAL? AQHYTQKSLS?LSPG*

Claims (70)

1. comprise the polypeptide of the change of the FcRn bound fraction in Fc district at least, wherein said polypeptide is compared with initial polypeptide and is comprised at least one sudden change, and wherein said at least one sudden change is selected from:

EU amino acid position 248 is replaced by charged amino acid;

EU amino acid position 249 is replaced by positively charged amino acid;

EU amino acid position 251 is replaced by polare Aminosaeren or Methionin;

EU amino acid position 252 is replaced by polare Aminosaeren;

EU amino acid position 255 is replaced by polare Aminosaeren;

EU amino acid position 256 is replaced by Methionin;

EU amino acid position 257 is replaced by charged amino acid;

EU amino acid position 258 is replaced by polare Aminosaeren or charged amino acid;

Substituting of EU amino acid position 277;

EU amino acid position 279 is replaced by charged amino acid;

EU amino acid position 280 is replaced by charged amino acid;

EU amino acid position 281 is replaced by charged amino acid or glutamine;

EU amino acid position 282 is replaced by charged amino acid;

EU amino acid position 284 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 285 is replaced by positively charged amino acid, polare Aminosaeren, or aspartic acid;

EU amino acid position 286 is replaced by L-glutamic acid, Threonine, or methionine(Met);

EU amino acid position 287 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 288 is replaced by charged amino acid;

Substituting of EU amino acid position 289;

EU amino acid position 304 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 305 is replaced by polare Aminosaeren or charged amino acid;

Substituting of EU amino acid position 306;

EU amino acid position 307 is replaced by polarity or charged amino acid;

EU amino acid position 308 is replaced by charged amino acid;

EU amino acid position 309 is replaced by charged amino acid;

EU amino acid position 310 is replaced by charged amino acid or polare Aminosaeren;

EU amino acid position 311 is replaced by positively charged amino acid;

EU amino acid position 312 is replaced by positively charged amino acid or polare Aminosaeren;

EU amino acid position 313 is replaced by charged amino acid;

EU amino acid position 315 is replaced by charged amino acid;

EU amino acid position 316 is replaced by positively charged amino acid;

EU amino acid position 317 is replaced by charged amino acid or polare Aminosaeren;

EU amino acid position 340 is replaced by charged amino acid;

EU amino acid position 343 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 344 is replaced by leucine;

EU amino acid position 345 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 376 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 378 is replaced by Serine;

EU amino acid position 383 is replaced by charged amino acid;

EU amino acid position 385 is replaced by charged amino acid;

EU amino acid position 389 is replaced by electronegative amino acid;

EU amino acid position 424 is replaced by charged amino acid;

EU amino acid position 426 is replaced by charged amino acid;

EU amino acid position 430 is replaced by polare Aminosaeren or charged amino acid;

EU amino acid position 431 is replaced by charged amino acid;

EU amino acid position 432 is replaced by polare Aminosaeren;

EU amino acid position 434 is replaced by Methionin, arginine, or leucine;

EU amino acid position 436 is replaced by electronegative amino acid; With

EU amino acid position 438 is replaced by charged amino acid.

2. comprise the polypeptide of the change of the FcRn bound fraction in Fc district at least, wherein said polypeptide is compared with initial polypeptide and is comprised at least one sudden change, and wherein said at least one sudden change is selected from:

The Methionin of EU amino acid position 248 is replaced by charged amino acid;

The aspartic acid of EU amino acid position 249 is replaced by positively charged amino acid;

The leucine of EU amino acid position 251 is replaced by polare Aminosaeren or Methionin;

Substitute the methionine(Met) of EU amino acid position 252;

The arginine of EU amino acid position 255 is replaced by polare Aminosaeren;

The Threonine of EU amino acid position 256 is replaced by Methionin;

The proline(Pro) of EU amino acid position 257 is replaced by charged amino acid;

The L-glutamic acid of EU amino acid position 258 is replaced by polare Aminosaeren or charged amino acid;

Substitute the tryptophane of EU amino acid position 277;

The Xie Ansuan of EU amino acid position 279 is replaced by charged amino acid;

The aspartic acid of EU amino acid position 280 is replaced by charged amino acid;

The glycine of EU amino acid position 281 is replaced by charged amino acid or glutamine;

The Xie Ansuan of EU amino acid position 282 is replaced by charged amino acid;

The Xie Ansuan of EU amino acid position 284 is replaced by polare Aminosaeren or charged amino acid;

The Histidine or the L-Ala of EU amino acid position 285 are replaced by charged amino acid or polare Aminosaeren;

The Histidine or the L-Ala of EU amino acid position 285 are replaced by positively charged amino acid, polare Aminosaeren, or aspartic acid;

The l-asparagine or the Methionin of EU amino acid position 286 are replaced by L-glutamic acid, Threonine, or methionine(Met);

The L-Ala of EU amino acid position 287 is replaced by polare Aminosaeren or charged amino acid;

The Methionin of EU amino acid position 288 is replaced by charged amino acid;

Substitute the Threonine of EU amino acid position 289;

The Serine of EU amino acid position 304 is replaced by polare Aminosaeren or charged amino acid;

The Xie Ansuan of EU amino acid position 305 is replaced by polare Aminosaeren or charged amino acid;

Substitute the leucine or the Xie Ansuan of EU amino acid position 306;

The Threonine or the Xie Ansuan of EU amino acid position 307 are replaced by polarity or charged amino acid;

The Xie Ansuan of EU amino acid position 308 is replaced by charged amino acid;

The leucine of EU amino acid position 309 is replaced by charged amino acid;

The Histidine of EU amino acid position 310 is replaced by charged amino acid or polare Aminosaeren;

The glutamine of EU amino acid position 311 is replaced by positively charged amino acid;

The aspartic acid or the leucine of EU amino acid position 312 are replaced by positively charged amino acid or polare Aminosaeren;

The l-asparagine of EU amino acid position 313 is replaced by charged amino acid;

The l-asparagine of EU amino acid position 315 is replaced by charged amino acid;

The l-asparagine of EU amino acid position 316 is replaced by positively charged amino acid;

The Methionin of EU amino acid position 317 is replaced by charged amino acid or polare Aminosaeren;

The Methionin of EU amino acid position 340 is replaced by charged amino acid;

The proline(Pro) of EU amino acid position 343 is replaced by polare Aminosaeren or charged amino acid;

The arginine of EU amino acid position 344 is replaced by leucine;

The L-glutamic acid of EU amino acid position 345 is replaced by polare Aminosaeren or charged amino acid;

The aspartic acid of EU amino acid position 376 is replaced by polare Aminosaeren or charged amino acid;

The L-Ala of EU amino acid position 378 is replaced by Serine;

The Serine of EU amino acid position 383 is replaced by charged amino acid;

The glycine of EU amino acid position 385 is replaced by charged amino acid;

The l-asparagine of EU amino acid position 389 is replaced by electronegative amino acid;

The Serine of EU amino acid position 424 is replaced by charged amino acid;

The Serine of EU amino acid position 426 is replaced by charged amino acid;

The L-glutamic acid of EU amino acid position 430 is replaced by polare Aminosaeren or charged amino acid;

The leucine of EU amino acid position 431 is replaced by charged amino acid;

The Histidine of EU amino acid position 432 is replaced by polare Aminosaeren;

The l-asparagine of EU amino acid position 434 is replaced by Methionin, arginine, or leucine;

The tyrosine of EU amino acid position 436 is replaced by electronegative amino acid; With

The glutamine of EU amino acid position 438 is replaced by charged amino acid.

3. the polypeptide of claim 1 or 2 change wherein is positioned at EU amino acid position 277,289,306,344, or at least one the locational amino acid in 378 is by charged amino acid, polare Aminosaeren, or nonpolar amino acid substitutes.

4. the polypeptide of the change of claim 3, wherein said charged amino acid is electronegative amino acid.

5. the polypeptide of the change of claim 4, wherein said electronegative amino acid is selected from aspartic acid and L-glutamic acid.

6. the polypeptide of the change of claim 3, wherein said charged amino acid is positively charged amino acid.

7. the polypeptide of the change of claim 6, wherein said positively charged amino acid is selected from arginine, Histidine, and Methionin.

8. the polypeptide of the change of claim 7, wherein said positively charged amino acid is Methionin.

9. the polypeptide of the change of claim 3, wherein polare Aminosaeren is selected from methionine(Met), phenylalanine, tryptophane, Serine, Threonine, tyrosine, l-asparagine, glutamine and halfcystine.

10. the polypeptide of the change of claim 3, wherein nonpolar amino acid is selected from L-Ala, leucine, Isoleucine, Xie Ansuan, glycine and proline(Pro).

11. the polypeptide of the change of claim 1 or 2, the polypeptide of wherein said change are antibody or its fragment.

12. the polypeptide of the change of claim 1 or 2, the polypeptide of wherein said change is a fusion rotein.

13. the polypeptide of the change of claim 1 or 2, wherein Fc district or its FcRn bound fraction derive from people's antibody.

14. the polypeptide of the change of claim 13, it comprises complete Fc district.

15. the polypeptide of the change of claim 14, wherein initial polypeptide comprises the aminoacid sequence of SEQ ID NO:2.

16. each the polypeptide of change of claim 11-13, wherein Fc district or its Fc bound fraction are the IgG isotypes.

17. the polypeptide of the change of claim 16, wherein said IgG isotype is the IgG1 subclass.

18. each the polypeptide of change of claim 11-13, wherein said polypeptide comprises one or more inhuman amino-acid residues at the complementary determining region (CDR) of VL or VH.

19. the polypeptide of the change of claim 11 or 13, wherein said polypeptide can be in conjunction with (a) antigen and (b) FcR.

20. the polypeptide of the change of claim 19, wherein said antigen is tumor associated antigen.

21. the polypeptide of the change of claim 12, wherein said polypeptide can be in conjunction with (a) part and (b) FcR.

22. the polypeptide of the change of claim 19 or 21, wherein FcR is FcRn.

23. the polypeptide of the change of claim 19 or 21, wherein said polypeptide and the initial polypeptide that does not contain described sudden change with different binding affinities in conjunction with FcR.

24. the polypeptide of the change of claim 23, the binding affinity of the polypeptide of wherein said change is compared with high about 1.5 times to about 100 times of the beginning polypeptide.

25. the polypeptide of the change of claim 34, the binding affinity of the polypeptide of wherein said change is compared with low about 1.5 times to about 100 times of the beginning polypeptide.

26. the polypeptide of the change of claim 19 or 21, the polypeptide of wherein said change demonstrates a kind of binding affinity to FcR when a pH, and when the 2nd pH FcR is demonstrated different binding affinities.

27. the polypeptide of the change of claim 26, the binding affinity of the polypeptide of wherein said change when a pH than at the 2nd pH the time high about 1.5 times to about 100 times.

28. the polypeptide of the change of claim 27, the binding affinity of the polypeptide of wherein said change when a pH than at the 2nd pH the time low about 1.5 times to about 100 times.

29. each the polypeptide of change of claim 11-13, the polypeptide of wherein said change when being administered to the patient, demonstrates and the different circulating half-life of initial polypeptide that does not contain sudden change.

30. the polypeptide of the change of claim 29, the transformation period of the polypeptide of wherein said change is grown up about 1 hour to about 1 week than the initial polypeptide that does not contain sudden change.

31. the polypeptide of the change of claim 30, the transformation period of the polypeptide of wherein said change is lacked about 1 hour to about 1 week than the initial polypeptide that does not contain sudden change.

32. the polypeptide of the change of claim 1 or 2, the polypeptide of wherein said change can combine with albumin A or G.

33. comprise the pharmaceutical composition of polypeptide of the change of claim 1 or 2.

34. comprise the nucleic acid molecule of the nucleotide sequence of coding claim 1 or 2 polypeptide.

35. the nucleic acid molecule of claim 34, it is an expression vector.

36. comprise the host cell of the expression vector of claim 35.

37. be used for the treatment of the patient's who suffers from illness method, described method comprises using to the patient and comprises the polypeptide of the change of the FcRn bound fraction in Fc district at least that wherein said polypeptide comprises at least one and is selected from following sudden change:

EU amino acid position 284 is replaced by L-glutamic acid;

EU amino acid position 285 is replaced by L-glutamic acid;

EU amino acid position 286 is replaced by aspartic acid;

EU amino acid position 288 is replaced by L-glutamic acid or aspartic acid;

EU amino acid position 290 is replaced by L-glutamic acid; With

EU amino acid position 304 is replaced by aspartic acid,

The polypeptide of wherein said change is compared with the initial polypeptide that does not contain sudden change and is demonstrated longer circulating half-life.

38. be used for the treatment of the patient's who suffers from illness method, described method comprises using to the patient and comprises the polypeptide of the change of the FcRn bound fraction in Fc district at least that wherein said polypeptide comprises at least one and is selected from following sudden change:

The Xie Ansuan of EU amino acid position 284 is replaced by L-glutamic acid;

The Histidine of EU amino acid position 285 is replaced by L-glutamic acid;

The l-asparagine of EU amino acid position 286 is replaced by aspartic acid;

The Methionin of EU amino acid position 288 is replaced by L-glutamic acid or aspartic acid;

The Methionin of EU amino acid position 290 is replaced by L-glutamic acid; With

The Serine of EU amino acid position 304 is replaced by aspartic acid,

The polypeptide of wherein said change is compared with the initial polypeptide that does not contain sudden change and is demonstrated longer circulating half-life.

39. be used for the treatment of the patient's who suffers from illness method, described method comprises using to the patient and comprises the polypeptide of the change of the FcRn bound fraction in Fc district at least that wherein said polypeptide comprises at least one and is selected from following sudden change:

EU amino acid position 248 is replaced by aspartic acid;

EU amino acid position 249 is replaced by arginine or Methionin;

EU amino acid position 250 is replaced by arginine or Methionin;

EU amino acid position 251 is replaced by arginine, Methionin, or l-asparagine;

EU amino acid position 252 is replaced by Serine or Threonine;

EU amino acid position 254 is replaced by Serine or Threonine;

EU amino acid position 256 is replaced by arginine, L-glutamic acid, or Methionin;

EU amino acid position 255 is replaced by leucine, aspartic acid or methionine(Met);

EU amino acid position 260 is replaced by Methionin;

EU amino acid position 257 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

EU amino acid position 277 is replaced by arginine, aspartic acid, glutamine, or Methionin;

EU amino acid position 279 is replaced by L-glutamic acid;

EU amino acid position 281 is replaced by glutamine;

EU amino acid position 282 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

EU amino acid position 287 is replaced by aspartic acid, L-glutamic acid, Methionin, proline(Pro), or Threonine;

EU amino acid position 284 is replaced by aspartic acid;

EU amino acid position 285 is replaced by aspartic acid or phenylalanine;

EU amino acid position 286 is replaced by L-glutamic acid or methionine(Met);

EU amino acid position 288 is replaced by aspartic acid;

EU amino acid position 290 is replaced by aspartic acid;

EU amino acid position 304 is replaced by aspartic acid or L-glutamic acid;

EU amino acid position 305 is replaced by arginine;

EU amino acid position 306 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

EU amino acid position 307 is replaced by arginine, aspartic acid, or L-glutamic acid;

EU amino acid position 309 is replaced by arginine, aspartic acid, Methionin or L-glutamic acid;

EU amino acid position 310 is replaced by arginine, leucine, Methionin or l-asparagine;

EU amino acid position 312 is replaced by arginine, l-asparagine, or Methionin;

EU amino acid position 313 is replaced by aspartic acid, arginine, or Methionin;

EU amino acid position 315 is replaced by aspartic acid or L-glutamic acid;

EU amino acid position 343 is replaced by glutamine or Methionin;

EU amino acid position 345 is replaced by arginine or glutamine;

EU amino acid position 374 is replaced by arginine, Methionin, or leucine;

EU amino acid position 376 is replaced by l-asparagine;

EU amino acid position 426 is replaced by arginine, aspartic acid, or L-glutamic acid;

EU amino acid position 428 is replaced by arginine, glutamine, or Methionin;

EU amino acid position 430 is replaced by Methionin;

EU amino acid position 431 is replaced by proline(Pro);

EU amino acid position 432 is replaced by arginine;

EU amino acid position 434 is replaced by leucine or Methionin; With

EU amino acid position 438 is replaced by L-glutamic acid,

The polypeptide of wherein said change demonstrates short circulating half-life than the initial polypeptide that does not contain sudden change.

40. be used for the treatment of the patient's who suffers from illness method, described method comprises using to the patient and comprises the polypeptide of the change of the FcRn bound fraction in Fc district at least that described polypeptide comprises at least one and is selected from following sudden change:

The Methionin of EU amino acid position 248 is replaced by aspartic acid;

The aspartic acid of EU amino acid position 249 is replaced by arginine or Methionin;

The Threonine of EU amino acid position 250 is replaced by arginine or Methionin;

The leucine of EU amino acid position 251 is replaced by arginine, Methionin, or l-asparagine;

The methionine(Met) of EU amino acid position 252 is replaced by Serine or Threonine;

The methionine(Met) of EU amino acid position 254 is replaced by Serine or Threonine;

The Threonine of EU amino acid position 256 is replaced by arginine, L-glutamic acid, or Methionin;

The arginine of EU amino acid position 255 is replaced by leucine, aspartic acid or methionine(Met);

The Threonine of EU amino acid position 260 is replaced by Methionin;

The proline(Pro) of EU amino acid position 257 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

The tryptophane of EU amino acid position 277 is replaced by arginine, aspartic acid, glutamine, or Methionin;

The Xie Ansuan of EU amino acid position 279 is replaced by L-glutamic acid;

The glycine of EU amino acid position 281 is replaced by glutamine;

The Xie Ansuan of EU amino acid position 282 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

The L-Ala of EU amino acid position 287 is replaced by aspartic acid, L-glutamic acid, Methionin, proline(Pro), or Threonine;

The Xie Ansuan of EU amino acid position 284 is replaced by aspartic acid;

The Histidine of EU amino acid position 285 is replaced by aspartic acid or phenylalanine;

The l-asparagine of EU amino acid position 286 is replaced by L-glutamic acid or methionine(Met);

The Methionin of EU amino acid position 288 is replaced by aspartic acid;

The Methionin of EU amino acid position 290 is replaced by aspartic acid;

The Serine of EU amino acid position 304 is replaced by aspartic acid or L-glutamic acid;

The Xie Ansuan of EU amino acid position 305 is replaced by arginine;

The leucine of EU amino acid position 306 is replaced by arginine, aspartic acid, L-glutamic acid, or Methionin;

The Threonine of EU amino acid position 307 is replaced by arginine, aspartic acid, or L-glutamic acid;

The leucine of EU amino acid position 309 is replaced by arginine, aspartic acid, Methionin or L-glutamic acid;

The Histidine of EU amino acid position 310 is replaced by arginine, leucine, Methionin or l-asparagine;

The aspartic acid of EU amino acid position 312 is replaced by arginine, l-asparagine, or Methionin;

The tryptophane of EU amino acid position 313 is replaced by aspartic acid, arginine, or Methionin;

The l-asparagine of EU amino acid position 315 is replaced by aspartic acid or L-glutamic acid;

The proline(Pro) of EU amino acid position 343 is replaced by glutamine or Methionin;

The L-glutamic acid of EU amino acid position 345 is replaced by arginine or glutamine;

The proline(Pro) of EU amino acid position 374 is replaced by arginine, Methionin, or leucine;

The aspartic acid of EU amino acid position 376 is replaced by l-asparagine;

The Serine of EU amino acid position 426 is replaced by arginine, aspartic acid, or L-glutamic acid;

The methionine(Met) of EU amino acid position 428 is replaced by arginine, glutamine, or Methionin;

The L-glutamic acid of EU amino acid position 430 is replaced by Methionin;

The L-Ala of EU amino acid position 431 is replaced by proline(Pro);

The leucine of EU amino acid position 432 is replaced by arginine;

The l-asparagine of EU amino acid position 434 is replaced by leucine or Methionin; With

The glutamine of EU amino acid position 438 is replaced by L-glutamic acid,

The polypeptide of wherein said change demonstrates short circulating half-life than the initial polypeptide that does not contain sudden change.

41. the method for the polypeptide of the change of preparation claim 1 or 2, described method comprises:

(a) with the nucleic acid molecule transfectional cell of nucleotide sequence of the polypeptide that comprises the described change of encoding; With

(b) from the polypeptide of described cell or the described change of cell conditioned medium liquid purifying.

42. the method for the antibody of preparation claim 11 or 13, described method comprises:

(a) provide and comprise encoding said antibody variable region of light chain (V _L) and constant region (C _L) first nucleic acid molecule of nucleotide sequence;

(b) provide and comprise encoding said antibody variable region of heavy chain (V _H) and constant region (CH ₁, CH ₂, and CH ₃) second nucleic acid molecule of nucleotide sequence;

(c) with first and second nucleic acid molecule transfectional cell under the condition of the antibody of change that allow to express the light chain that comprises described coding and heavy chain; With

(d) from described cell or the described antibody of cell conditioned medium liquid purifying.

43. the method for claim 42, wherein said cell are 293 cells.

44. it is conjugated protein to comprise the dimerization Fc of first and second polypeptide chains, wherein first and second polypeptide chains respectively comprise the Fc plot structure territory that at least one operationally is connected in conjunction with the territory with Fc.

45. the dimerization Fc of claim 44 is conjugated protein, described Fc structural domain the combining with reduction or elimination and FcRn of wherein suddenling change.

46. the dimerization Fc of claim 44 is conjugated protein, wherein said first and second polypeptide chains are covalently bound.

47. the dimerization Fc of claim 44 is conjugated protein, wherein the Fc binding domains comprises the extracellular domain of FcRn.

48. the dimerization Fc of claim 44 is conjugated protein, wherein the Fc binding domains is attached to β2Wei Qiudanbai in conjunction with last.

49. the dimerization Fc of claim 44 is conjugated protein, wherein the Fc binding domains derives from people FcRn.

50. the dimerization Fc of claim 59 is conjugated protein, the wherein said conjugated protein aminoacid sequence shown in the SEQ IDNO:10 that comprises.

51. be used to measure the polypeptide that comprises Fc district FcRn bound fraction at least method at the binding affinity of FcR, this method comprises makes that the polypeptide that comprises the FcRn bound fraction in Fc district at least and the dimerization Fc of claim 44 are conjugated protein to be contacted and determine this interactional avidity.

52. be used in the library screening that comprises the polypeptide of the FcRn bound fraction in Fc district at least FcRn being had the method for those polypeptides of the binding affinity of change, wherein said method comprises:

(a) the library member is contacted with the dimerization Fc of claim 44 is conjugated protein; With

(b) measure described polypeptide to the protein-bonded binding affinity of dimerization Fc; With

(c) selection has the those polypeptides of the binding affinity of change to FcRn.

53. be used for containing the method for the polypeptide of the FcRn bound fraction in Fc district at least from the polypeptide mixture purifying, described method comprises the mixture application of sample to the protein-bonded affinity column of the dimerization Fc that contains claim 44, wash-out comprises the polypeptide of the FcRn bound fraction in Fc district at least, the described polypeptide of purifying thus.

54. the method for claim 53, wherein when a pH with described mixture application of sample to affinity column, and when the 2nd pH from the described polypeptide of affinity column wash-out.

55. the method for claim 54, wherein said polypeptide in the purifying engineering not by sex change.

56. be used to identify with initial polypeptide compare the method for polypeptide that FcRn has the binding affinity of change, described method comprises:

The associated change in conjunction with free energy of performance of the space of the best charge distribution of initial polypeptide amino acid and initial polypeptide when (a) determining in solvent in conjunction with FcRn;

Change at least one the candidate amino acid residue position in having of the initial polypeptide initial polypeptide to be finished when (b) being accredited as in conjunction with free energy in conjunction with FcRn; With

(c) identify the elected amino acid be positioned at this amino acid position, so that this sudden change mixing in initial polypeptide will cause FcRn is had the polypeptide of change of the binding affinity of change.

57. the method for claim 56 also comprises selected amino acid is mixed in the initial polypeptide.

58. the method for claim 56 comprises that also the polypeptide that calculate to change compares variation in conjunction with free energy with initial polypeptide in conjunction with FcRn the time in conjunction with FcRn the time..

59. the method for claim 58, wherein said calculation procedure comprise at first that on computers mould is built described sudden change, the variation of calculations incorporated free energy then in initial polypeptide.

60. the method for claim 58, wherein said calculation procedure is used the following at least a definite method that is selected from: use and determine the static bound energy based on the method for Poisson-Boltzmann formula; Determine the Fan Dewaershi bound energy; Determine bound energy with using based on the solvent-accessible surface Method for Area.

61. the method for claim 56, wherein said sudden change is an amino acid replacement.

62. the method for claim 61, wherein said amino acid replacement cause mixing the elected amino acid that has different electric charges with candidate amino acid.

63. the method for claim 56, wherein said sudden change increase the polypeptide that contains Fc that changes and FcRn in solvent in conjunction with the time between the two in conjunction with free energy, that reduces described change thus contains the binding affinity of Fc polypeptide to FcRn.

64. the method for claim 56, wherein said sudden change reduce the polypeptide that contains Fc that changes and FcRn in solvent in conjunction with the time between the two in conjunction with free energy, what increase described change thus contains the binding affinity of Fc polypeptide to FcRn.

65. identify the method for polypeptide of the change that contains Fc that FcRn is had the binding affinity of change two different pH levels, described method comprises:

(a) determine the space performance of the amino acid whose best charge distribution of initial polypeptide when a pH level combines with FcRn and the associated change in conjunction with free energy of initial polypeptide in solvent;

(b) determine the space performance of the amino acid whose best charge distribution of initial polypeptide when the 2nd pH level combines with FcRn and the associated change in conjunction with free energy of initial polypeptide in solvent;

(c) by the residue of charge distribution, the different charge distribution of the demonstration under the first and second pH levels relatively, identifying has at least one candidate amino acid residue position in conjunction with free energy when changing initial polypeptide and combine with FcRn to be finished in the initial polypeptide; With

(d) select to be positioned at the elected amino acid of described amino acid position, so as should elected amino acid to mix cause behind the initial polypeptide to FcRn have change in conjunction with polypeptide affinity, that contain Fc, that change.

66. the method for claim 65, wherein a pH is about 7.4.

67. the method for claim 65, the avidity of wherein said polypeptide when a pH than at the 2nd pH the time high about 1.5 times to about 100 times.

FcRn is demonstrated during at the 2nd pH the polypeptide of the change of different avidity 68. when a pH FcRn demonstrated a kind of avidity, wherein said polypeptide comprises the aminoacid sequence by the method prediction of claim 56.

69. comprise the pharmaceutical composition of the polypeptide of claim 68.

70. comprise the nucleic acid molecule of nucleotide sequence of the polypeptide of the claim 68 of encoding.

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