CN110357960A

CN110357960A - Antibody and antibody remodeling method

Info

Publication number: CN110357960A
Application number: CN201810319413.4A
Authority: CN
Inventors: 张文军; 张兵; 刘嘉熙; 黄引娣; 邱小林
Original assignee: Guangzhou Esme Biomedical Technology Co Ltd
Current assignee: Guangzhou Esme Biomedical Technology Co Ltd
Priority date: 2018-04-10
Filing date: 2018-04-10
Publication date: 2019-10-22
Also published as: WO2019196522A1

Abstract

The present invention provides the methods that the area Fab to antibody is transformed.Specifically, the present invention provides the specific sites of CH1- hinge area and the area CL to antibody to be mutated the method to form non-native disulfide bond.The present invention also provides the antibody generated by above-mentioned remodeling method and its applications.

Description

Antibody and antibody remodeling method

Technical field:

The invention belongs to protein engineering field, it is related in protein molecule or intermolecular disulfide bond remodeling method.Specifically For, the present invention relates to the disulfide bond between the heavy chain and light chain to antibody to be transformed to change or improve its binding specificity Method.The invention further relates to the antibody such as bispecific antibodies, preparation method and use by disulfide bond transformation.

Technical background:

In biomedicine field, antibody drug has been widely used for various diseases such as malignant tumour, autoimmunity disease The major diseases such as disease, inflammation infection, cardiovascular disease etc. treatment.Wherein bispecific antibody (Bispecific Antibody, BsAb) it is a kind of antibody that can combine at least two epitopes, the antibody relative to the single target spot of tradition comes It says, safety and validity are greatly improved, and are one of antibody engineering field and immunotherapy of tumors field Research and development focus.

Bispecific antibody and the naturally-produced antibody of acellular can only be carried out by cell fusion or DNA recombinant technique Artificial preparation.It when preparing bispecific antibody, needs to express two different heavy chains and two different light chains simultaneously, leads to The pairing for crossing two couples of HC-LC forms the Fab of two kinds of antigen of identification.In the process, homologous heavy chain combination and heterologous light chain knot The mismatch problems generated are closed, ten kinds of different products can be generated, wherein only one is required correct products.Thus prepare The main problem solved required for this kind of antibody be for example homologous heavy chain mispairing of random assembling of different chains, light chain mispairing and other The problems such as pollution of non-purpose product.

Although having had some solutions for the mismatch problems of bispecific antibody, there are still stability and molten The various problems such as solution property low, complex process, low output.Therefore, there is still a need for being transformed bispecific antibody to reduce it Mispairing between heavy chain and light chain, and improve the new method of the yield of purpose product.

Summary of the invention

The present inventor has carried out deep analysis to the interaction of the heavy chain and light chain of antibody and space structure, and discovery will Specific site in the heavy chain constant region 1 (CH1) of antibody-interface constant region of light chain (CL) introduces non-native disulfide bond, is introducing Few site mutation and on the basis of not influencing antibody structure and function, can change or improve antibody heavy chain and light chain it Between interaction.The above method can be used for being transformed antibody or antibody fragment.

Accordingly, on the one hand, the present invention relates to the methods that the area Fab to antibody is transformed, and the method includes selecting The step of introducing the area Fab from one or more mutation below:

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL, wherein according to EU number pair The heavy chain of antibody and light chain are numbered.

In some embodiments of the above method, mutation a)-e is introduced into the area Fab) in 1,2,3,4 or 5 ?.In preferred embodiments, mutation a)-d is introduced into the area Fab) in 1 or 2 mutation and optional mutation e).

In some embodiments, the above method further include destroy hinge area-CL between natural disulphide bonds, that is, destroy by Two formed between the 214th cysteine (C214) in the 220th cysteine (C220) and CL in hinge area Sulfide linkage.Therefore, in some embodiments, method of the invention further includes sporting the C220 in hinge area except cysteine Other amino acid in addition or missing C220, and/or the C214 in CL is sported to other amino acid in addition to cysteine Or missing C214.In preferred embodiments, other amino acid in addition to cysteine are selected from serine, alanine Or glycine.

In any embodiment of the above method, the area Fab derives from IgG, IgA, IgM, IgE or IgD, such as IgG1, IgG2, IgG3 or IgG4 isotype.

On the other hand, there is the method for the antibody or antibody fragment in at least two different areas Fab the present invention relates to production, It the described method comprises the following steps:

1) one or more mutation selected from the following are introduced in the first area Fab of the antibody or antibody fragment:

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and CL in E213R or E213K mutation, wherein according to EU number into Row number,

2) under conditions of expressing the antibody or antibody fragment, the core containing encoding said antibody or antibody fragment is cultivated The host cell of acid, and

3) antibody or antibody fragment are recycled from the host cell cultures.

In some embodiments, the above method further includes introducing in the 2nd area Fab of the antibody or antibody fragment One or more mutation selected from the following:

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL, wherein introducing described first The mutation in the area Fab and the mutation for introducing the 2nd area Fab are not exactly the same.

In some embodiments of the above method, mutation a)-e is introduced into the first area Fab) in 1,2, 3,4 or 5, mutation a)-e is introduced into the 2nd area Fab) in 1,2,3,4 or 5, and wherein draw The mutation of the mutation and introducing the 2nd area Fab that enter the first area Fab is not exactly the same.In preferred embodiments, Mutation a)-d is introduced into the first area Fab and/or the 2nd area Fab) in 1 or 2 and optional mutation e).

In some embodiments, the above method further includes destroying hinge in the first area Fab and/or the 2nd area Fab C220 in the hinge area in the first area Fab and/or the 2nd area Fab is sported and is removed by the natural disulphide bonds between area CL Other amino acid other than cysteine or missing C220, and/or by the first area Fab and/or the 2nd area Fab and/or the C214 in the CL in two areas Fab sports other amino acid or missing C214 in addition to cysteine.In preferred embodiment party In case, the amino acid in addition to cysteine is selected from serine, alanine and glycine.

In some embodiments of the above method, the first Fab and the 2nd area Fab combine different antigen.

In some embodiments of the above method, the first area Fab and the 2nd area Fab combine two of same antigen Different epitopes.

In some embodiments of the above-mentioned method being transformed to the area Fab and the production method of antibody or antibody fragment In, the area Fab, the first area Fab and the 2nd area Fab combine antigen selected from the group below: CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32a, CD32b, CD33 (p67 albumen), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3, IL-1 α, IL-1 β, IL- 1R, IL-2, IL-2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-12, IL-13, IL-15, IL-17, IL-17R, IL-18, IL-23, interferon-' alpha ', interferon beta, interferon gamma；TNF-α, TNF β, TNF-R1, TNF-RII, FasL, Receptor -1 CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL, Adenosine receptor, lymphotoxin-beta-receptor, TACI, BAFF-R, EPO；LFA-3, ICAM-1, ICAM-3, EpCAM, integrin β 1, Integrin β 2, integrin alpha-4/β 7, beta 2 integrin alpha 2, beta 2 integrin alpha 3, integrin alpha-4, beta 2 integrin alpha 5, integrin α 6, beta 2 integrin alpha v, beta 2 integrin alpha V β 3, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, Anti- Id, E-Selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGF β 1, TGF β 2, Eosinophil chemokine 1 (eotaxin1), Blys (bone-marrow-derived lymphocyte stimulating factor), complement C5, IgE, factor Ⅴ II, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), Tissue factor, endothelin receptor, VLA-4, haptens NP-cap or NIP-cap, E-Selectin, digoxin, human placental alkaline phosphoric acid Enzyme (PLAP) and testis PLAP sample alkaline phosphatase, TfR, carcinomebryonic antigen (CEA), CEACAM5, HMFG1, PEM, Mucin1, MUC18, Heparinase I, human heart myosin, tumor-associated glycoprotein -72 (TAG-72), tumour correlation are anti- Former CA 125, prostate-specific membrane antigen (PSM-A), high molecular weight melanoma related antigen (HMW-MAA), cancer (carcinoma) related antigen, Gco protein I ib/IIIa (GPIIb/IIIa), expression Lewis Y related carbohydrate swell Tumor related antigen, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV breathe syncytial virus RSV F, RSVF Fgp, cytokeratin tumor associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 ring, Respiratory Syncytial Virus(RSV) (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB coating sugar egg White and C.perfringens (Clostridium perfringens) toxin, CD133, CD138, OX40, GITR, PD-1, PD- L1, PD-L2, CTLA-4, KIR, LAG-3, TCR α, TCR β, TCR γ, TCR δ, VEGF, EGF, VEGFR, EGFR, EpCAM, mesothelium Element, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, Fucosido-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM-CSFR, ADAMS, flesh generate inhibin, PCSK9, CXCR4, MIF, PEG2.In preferred embodiments, the area Fab, the first area Fab and the 2nd area Fab combine choosing From the antigen of CD3 and Her2.

In some embodiments, aforementioned production method is selected from Fab piece for producing antibody fragment, the antibody fragment Section, Fab' segment and F (ab')₂Segment.

In some embodiments, aforementioned production method is for producing bispecific antibody, the bispecific antibody tool There are the first heavy chain and the first light chain and the second heavy chain and the second light chain, wherein first heavy chain and the first light chain form institute The first area Fab is stated, second heavy chain and the second light chain form the 2nd area Fab.

In some embodiments, the above method further includes introducing P395K, P396K and V397K to first heavy chain, And to second heavy chain introduce T394D, P395D and P396D, or to first heavy chain introduce T394D, P395D and P396D, and P395K, P396K and V397K are introduced to second heavy chain.Above-mentioned mutation can increase the first heavy chain and the second weight Binding specificity between chain, to avoid heavy chain homodimer is formed.

In some embodiments of the above method, the antibody or antibody fragment from IgG, IgA, IgM, IgE or IgD, such as IgG1, IgG2, IgG3 or IgG4 isotype.

The invention further relates to the antibody or antibody fragment that are generated by the above method.

In another aspect, the present invention relates to at least two different areas Fab antibody or antibody fragment, the antibody or First area Fab of antibody fragment has one or more mutation selected from the following:

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL,

Wherein it is numbered according to EU number.

In some embodiments of above-mentioned antibody or antibody fragment, the 2nd area Fab of the antibody or antibody fragment has There are one or more mutation selected from the following:

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL,

Wherein the mutation in the area second Fab and the mutation in the first area Fab are not exactly the same.

In some embodiments of above-mentioned antibody or antibody fragment, the first area Fab has mutation a)-e) in 1 , 2,3,4 or 5, the 2nd area Fab has mutation a)-e) in 1,2,3,4 or 5, and wherein The mutation in the first area Fab and the mutation in the 2nd area Fab are not exactly the same.In preferred embodiments, described One area Fab and/or the 2nd area Fab have mutation a)-d) in one or two and optional mutation e).

In some embodiments of above-mentioned antibody or antibody fragment, the first area Fab and/or the 2nd area Fab also have The C214 for having the C220 in hinge area to sport other amino acid in addition to cysteine or lack in C220 and/or CL is prominent Become the other amino acid or missing C214 in addition to cysteine.Wherein, other amino acid in addition to cysteine It is preferably selected from serine, alanine and glycine.

In some embodiments of above-mentioned antibody and antibody fragment, the first area Fab and the 2nd area Fab are combined Different antigen.

In some embodiments of above-mentioned antibody and antibody fragment, the first area Fab and the 2nd area Fab are in conjunction with same Two different epitopes on antigen.

In some embodiments of above-mentioned antibody and antibody fragment, the antigen is selected from the group: CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32a, CD32b, CD33 (p67 albumen), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3, IL-1 α, IL-1 β, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-12, IL-13, IL- 15, IL-17, IL-17R, IL-18, IL-23, interferon-' alpha ', interferon beta, interferon gamma；TNF-α, TNF β, TNF-R1, TNF- RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, Receptor -1 TRAIL, adenosine receptor, lymphotoxin-beta-receptor, TACI, BAFF-R, EPO；LFA-3, ICAM-1, ICAM-3, EpCAM, Integrin β 1, integrin β 2, integrin alpha-4/β 7, beta 2 integrin alpha 2, beta 2 integrin alpha 3, integrin alpha-4, integrin α 5, beta 2 integrin alpha 6, beta 2 integrin alpha v, beta 2 integrin alpha V β 3, FGFR-3, keratinocyte growth factor, VLA-1, VLA- 4, L-selectin, anti-Id, E-Selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGF β 1, TGF β 2, eosinophil chemokine 1 (eotaxin1), Blys (bone-marrow-derived lymphocyte stimulating factor), complement C5, IgE, factor Ⅴ II, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, endothelin receptor, VLA-4, haptens NP-cap or NIP-cap, E-Selectin, digoxin, P-ALP (PLAP) and testis PLAP sample alkaline phosphatase, TfR, carcinomebryonic antigen (CEA), CEACAM5, HMFG1, PEM, Mucin1, MUC18, Heparinase I, human heart myosin, tumor-associated glycoprotein -72 (TAG-72), tumor associated antigen CA 125, prostate-specific membrane antigen (PSM-A), high molecular weight melanoma correlation are anti- Former (HMW-MAA), cancer (carcinoma) related antigen, Gco protein I ib/IIIa (GPIIb/IIIa), expression Lewis Y are related The tumor associated antigen of carbohydrate, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV, breathing are closed Cellular virus RSV F, RSVF Fgp, cytokeratin tumor associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 ring, Respiratory Syncytial Virus(RSV) (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and C.perfringens (Clostridium perfringens) toxin, CD133, CD138, OX40, GITR, PD-1, PD-L1, PD-L2, CTLA-4, KIR, LAG-3, TCR α, TCR β, TCR γ, TCR δ, VEGF, EGF, VEGFR, EGFR, EpCAM, mesothelin, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, fucosido-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM- CSFR, ADAMS, flesh generate inhibin, PCSK9, CXCR4, MIF, PEG2.In preferred embodiments, the antigen is selected from CD3 and HER2.

In some embodiments, the antibody fragment is selected from Fab segment, Fab' segment and F (ab')₂Segment.

In some embodiments, the antibody is with the first heavy chain and the first light chain and the second heavy chain and second The bispecific antibody of light chain, wherein first heavy chain and the first light chain form the first area Fab, second heavy chain and Second light chain forms the 2nd area Fab.In a further embodiment, the first heavy chain has P395K, P396K and V397K Mutation, and second heavy chain have T394D, P395D and P396D be mutated or first heavy chain have T394D, P395D and P396D and mutation, and second heavy chain is mutated with P395K, P396K and V397K.

In some embodiments, the antibody or antibody fragment derive from IgG, IgA, IgM, IgE or IgD, such as IgG1, IgG2, IgG3 or IgG4.

On the other hand, the present invention relates to the methods being transformed to bispecific antibody, wherein the bispecific is anti- Body has the first heavy chain and the first light chain in conjunction with CD3, and combines the second heavy chain and the second light chain of HER2, second heavy chain Amino acid sequence with SEQ ID NO:7, and second light chain has the amino acid sequence of SEQ ID NO:5, the side Method includes that 1 or 2 mutation selected from following a)-d) is introduced to second heavy chain and the second light chain:

A) S162C in the F170C and constant region of light chain (CL) in heavy chain constant region 1 (CH1)；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；With

D) Q160C in the V173C and CL in CH1,

Wherein the heavy chain of antibody and light chain are numbered according to EU number.

It further include that the C220 in the hinge area of second heavy chain dashes forward in some embodiments of above-mentioned remodeling method Become the other amino acid or missing C220 in addition to cysteine, and/or the C214 in the CL of second light chain is mutated For in addition to cysteine other amino acid or missing C214 the step of.

It further include introducing K218E or K218D to first heavy chain to dash forward in some embodiments of above-mentioned remodeling method Become, and introduces E213R or E213K mutation to first light chain；Or K218E or K218D is introduced to all second heavy chains and is dashed forward Become, and introduces E213R or E213K mutation to second light chain.

In some embodiments of above-mentioned remodeling method, first heavy chain has the amino acid sequence of SEQ ID NO:3 Column, first light chain have the amino acid sequence of SEQ ID NO:1.

The invention further relates to the bispecific antibodies obtained by above-mentioned remodeling method.

On the one hand, the present invention relates to antibody or antibody fragments, with the area CL selected from the group below: SEQ ID NO:11, SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19、SEQ ID NO:21、SEQ ID NO: 23, SEQ ID NO:25 and SEQ ID NO:27.

On the one hand, the present invention relates to antibody or antibody fragments, with the area CH1 selected from the group below or hinge area: SEQ ID NO:29、SEQ ID NO:31、SEQ ID NO:33、SEQ ID NO:35、SEQ ID NO:37、SEQ ID NO:39、SEQ ID NO:41 and SEQ ID NO:43.

On the one hand, the present invention relates to antibody or antibody fragments, with the area CL selected from the group below and the area CH1:

A) area CH1 in the area CL of SEQ ID NO:11 and SEQ ID NO:33；

B) area CH1 in the area CL of SEQ ID NO:13 and SEQ ID NO:29；

C) area CH1 in the area CL of SEQ ID NO:15 and SEQ ID NO:35；

D) area CH1 in the area CL of SEQ ID NO:17 and SEQ ID NO:33；With

E) area CH1 in the area CL of SEQ ID NO:27 and SEQ ID NO:43.

On the other hand, the present invention relates to antibody coupling matter, it includes antibody of the invention or antibody fragments or double special Property antibody, and the part being coupled with the antibody or antibody fragment or bispecific antibody, wherein the part is selected from cell Toxin, radioactive isotope, fluorescent marker, shiner, substance that show color or enzyme.

In some embodiments, anti-to be formed with antibody or antibody fragment of the invention or bispecific antibody coupling The part of body conjugate is cytotoxin.In some embodiments, the cytotoxin is selected from: colchicine, Emtansine, maytansinoid, auristatin, vindesine, tubulysin etc..

In some embodiments, anti-to be formed with antibody or antibody fragment of the invention or bispecific antibody coupling The part of body conjugate is radioactive isotope.In some embodiments, the radioactive isotope is selected from: At²¹¹,I¹³¹, I¹²⁵,Y⁹⁰,Re¹⁸⁶,Re¹⁸⁸,Sm¹⁵³,Bi²¹²,P³²Radioactive isotope etc..

In some embodiments, anti-to be formed with antibody or antibody fragment of the invention or bispecific antibody coupling The part of body conjugate is selected from fluorescent marker, shiner and substance that show color, such as: FITC, luciferase, HRP etc..

In some embodiments, anti-to be formed with antibody or antibody fragment of the invention or bispecific antibody coupling The part of body conjugate is enzyme, for example, bacterium, fungi, plant or animal origin enzyme activity toxin, including its active fragment and/ Or variant.

In another aspect, the present invention relates to pharmaceutical composition, it includes antibody of the invention or antibody fragments, double special Property antibody or antibody coupling matter, and optionally one or more pharmaceutically acceptable carriers, surfactant and/or dilution Agent.

In one aspect, the present invention relates to antibody of the invention or antibody fragments, bispecific antibody or antibody coupling matter Preparing the purposes in the pharmaceutical composition for treating disease.In some embodiments, the disease is cancer.

On the other hand, the present invention relates to the methods for the treatment of disease comprising uses antibody of the invention or antibody piece The step of section, bispecific antibody, antibody coupling matter or pharmaceutical composition.In some embodiments, the disease is cancer.

In one aspect, the present invention includes nucleic acid molecules, and it includes nucleotide sequences selected from the group below: SEQ ID NO: 12、SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44.

On the other hand, the present invention includes carrier, and it includes nucleic acid molecules of the invention.

In another aspect, the present invention includes host cell, and it includes nucleic acid molecules of the invention or carriers.

Detailed description of the invention

Fig. 1 shows the schematic diagram that the structure for the antibody that method of the invention is transformed or produces can be used.It is described Antibody can have different structures, and can be the antibody of bispecific, tri-specific or four specificity.Wherein, Figure 1A It shows the antibody of common " Y " font, can be bispecific antibody.Figure 1B -1H shows three spy as derived from Figure 1A Anisotropic and four specific antibody forms.

Fig. 2 shows protein A affinity chromatography after purification, and the anti-Her2 × AntiCD3 McAb being transformed by means of the present invention is double special Heterogenetic antibody MutB, MutC, MutD and MutE and the SDS-PAGE electrophoresis result figure for compareing bispecific antibody WT.WT in figure Refer to by plasmid pFUSE-Her2-HC-OB-6His, pFUSE-C31-HC-OA, pCDNA3.1-C31-LC and pCDNA3.1- The bispecific antibody of Her2-LC coding, wherein WT (1:1:1:1) indicates that above-mentioned four kinds of plasmids are transfected with the ratio of 1:1:1:1； WT (2:1:1:1) indicates plasmid pFUSE-Her2-HC-OB-6His, pFUSE-C31-HC-OA, pCDNA3.1-C31-LC, The transfection ratio of pCDNA3.1-Her2-LC is 2:1:1:1.Fig. 2A shows irreducibility SDS-PAGE electrophoresis result, and Fig. 2 B is aobvious Reproducibility SDS-PAGE electrophoresis result is shown.

Fig. 3 shows protein A affinity chromatography after purification, the bispecific antibody being further transformed on the basis of MutC The SDS-PAGE electrophoresis result figure of MutC-ER, MutC+ER and MutC-DeC.Fig. 3 A shows irreducibility SDS-PAGE electrophoresis As a result, Fig. 3 B shows reproducibility SDS-PAGE electrophoresis result.

Fig. 4 shows the peak shape that bispecific antibody MutC is further purified by cation-exchange chromatography (CIEX) method Figure and SDS-PAGE electrophoresis result figure after purification.Wherein Fig. 4 A is the CIEX peak shape figure of bispecific antibody MutC sample；Figure 4B is irreducibility SDS-PAGE electrophoresis, and swimming lane 1 is albumen Marker from left to right；Swimming lane 2 is by protein A affinity chromatography The sample for not yet carrying out CIEX method after purification, is labeled as Input；Swimming lane 3-4 is the sample at the peak A of corresponding CIEX；Swimming lane 5-10 is the sample at the peak B of corresponding CIEX.Fig. 4 C is the reproducibility SDS-PAGE electrophoresis of corresponding diagram 4B.

Fig. 5 shows protein A affinity chromatography after purification, bispecific antibody MutC-D+ER and MutC-D- of the invention The SDS-PAGE electrophoresis result figure of DeC.Wherein 5A is irreducibility SDS-PAGE electrophoresis, and Fig. 5 B is reproducibility SDS-PAGE electricity Swimming figure.

Fig. 6 shows anti-Her2 × AntiCD3 McAb bispecific antibody combination source of people Her2 antigen ELISA result.Fig. 6 A is aobvious The result of bispecific antibody MutC, MutC+ER and MutC-ER are shown.Fig. 6 B show MutC-DeC, MutC-D+ER and The result of MutC-D-DeC.

Fig. 7 shows Her2 × AntiCD3 McAb bispecific antibody combination source of people CD3 antigen ELISA result.Fig. 7 A is shown The result of bispecific antibody MutC, MutC+ER and MutC-ER.Fig. 7 B shows MutC-D+ER, MutC-D-DeC and MutC- The result of DeC.

Fig. 8 shows the source of people Her2 antigen using HRP label and is adsorbed in the dual anti-original of the source of people CD3 antigen of ELISA Plate Result of the ELISA experiment detection antibody to the combination of two kinds of antigen.Fig. 8 A is shown compared to control WT and hIgG, of the invention Bispecific antibody MutB, MutC and MutD are in the ELISA result for combining two kinds of antigen.Fig. 8 B shows bispecific antibody The result of MutE.

Fig. 9 shows the T cell activation of the Mediated by Bi-specific Antibodies of various concentration and to tumor cytotoxicity (CTL) Experimental result.Fig. 9 A shows the result of bispecific antibody MutB, MutC and MutD compared to control WT and hIgG.Fig. 9 B is aobvious The result of bispecific antibody MutC and MutE is shown.

Figure 10 shows the T cell activation of the Mediated by Bi-specific Antibodies of various concentration and to tumor cytotoxicity (CTL) Experimental result.Figure 10 A shows the knot of bispecific antibody MutC, MutC+ER and MutC-ER compared to control WT and hIgG Fruit.Figure 10 B shows the result of bispecific antibody MutC-D+ER and MutC-D-DeC and MutC-Dec.

Detailed description of the invention

Term and abbreviation

Unless otherwise defined herein, the scientific and technical terms and its abbreviation being used in combination with the application should have this public affairs Open the normally understood meaning of those of ordinary skill in the art.Part term and breviary used herein is listed below Language.

BsAb: bispecific antibody (bispecific antibody)

HC: heavy chain (heavy chain)

LC: light chain (light chain)

VH: heavy chain variable region (variable region of heavy chain)

VL: light chain variable region (variable region of light chain)

CH: heavy chain constant region (constant region of heavy chain)

CL: constant region of light chain (constant region of light chain)

CDR: antigen complementary determining region (complementarity determining region)

ScFv: Single chain antibody segment (single-chain variable fragment)

ADCC: the cytotoxic effect (antibody dependent cellular cytotoxicity) of antibody-dependant

ELISA: enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay)

Molecular cloning of the present invention, cell culture, protein purification, immunological experiment operating procedure be quilt in the field Widely applied conventional steps.Unless otherwise specified, amino acid sequence of the present invention is according to from aminoterminal to c-terminus Direction arrange and write.The abbreviation of amino acid trigram mentioned by the present invention and nucleotide one-letter abbreviations are the technical field Generally accepted form, amino acid one-letter abbreviations are the biochemical nomenclature commission IUPAC-IUB (IUPAC-IUB Biochemical Nomenclature Commission) recommend form.

Term " amino acid " refers to one of 20 kinds of naturally occurring amino acid or can reside in any non-of specific position Natural analog." amino acid mutation " of the present invention refers to amino acid substitution in polypeptide sequence, addition, insertion and/or lacks It loses.Amino acid mutation preferred herein is to replace." amino acid substitution " or " substitution " refers to parental polypeptide sequence in the present invention The amino acid of specific position is replaced by another amino acid in column.For example, C220S is replaced to refer to variant polypeptide, wherein polypeptide Cysteine at position 220 is replaced by serine.

Term " antibody " refers to the immunoglobulin point comprising at least one antigen recognition site and energy molecule of the antigen binding Son.Here, term " antigen " is to induce immune response and the substance in conjunction with antibody specificity in body, as protein, The combination of polypeptide, peptide, carbohydrate, polynucleotide, lipid, haptens or above-mentioned substance.The combination of antibody and antigen according to The interaction formed between the two mediates, including hydrogen bond, Van der Waals force, ionic bond and hydrophobic bond.Antigenic surface and anti- The region that body combines is " antigenic determinant " or " epitope ", and in general, each antigen has multiple determinants.

Term mentioned by the present invention " antibody " includes that monoclonal antibody is (including complete with the understanding of its broadest sense Long monoclonal antibody), polyclonal antibody, antibody fragment, the polyspecific for containing at least two different antigen-binding domains Antibody (for example, bispecific antibody).Antibody further includes source of mouse antibody, humanized antibody, chimeric antibody, human antibody and other The antibody in source.Antibody of the invention can derive from any animal, including but not limited to people, non-human primate, mouse, Rat, ox, horse, chicken, camel, Llama (Llama), alpaca (Alpaca), llama (Guanaco), vigone (Vicunas) Or immunoglobulin molecules of shark etc..Antibody can contain other change, such as unnatural amino acid, and Fc effector function is dashed forward Change and glycosylation site mutation.Antibody further includes the fusion protein of the antibody of posttranslational modification, antigenic determinant comprising antibody, And the immunoglobulin molecules comprising any other modification to antigen recognition site, as long as these antibody show it is desired Bioactivity.In other words, antibody includes the immunoreactive fragments of immunoglobulin molecules and immunoglobulin molecules, i.e., extremely Few molecule containing an antigen-binding domains.

According to the amino acid sequence of heavy chain constant region, human immunoglobulin(HIg) can be fallen into 5 types: IgA, IgD, IgE, IgG and IgM can also be further separated into different subclass (isotype), such as IgG1, IgG2, IgG3, IgG4, IGA1, IGA2 Deng.According to light-chain amino acid sequence, light chain can be classified as to λ chain or κ chain.Antibody of the invention can be any type (such as IgA, IgD, IgE, IgG and IgM) or subclass (such as IgG1, IgG2, IgG3, IgG4, IGA1 or IGA2).

Term " humanized antibody " refers to the antibody of the generations such as non-human species such as rat, mouse, after modification transformation Retain the affinity of parental antibody and antigen binding, while reducing heterologous antibody in the intracorporal immunogenicity of people.In general, people The CDR region amino acid sequence of source antibody and parental antibody (non-human source antibodies) are almost the same, and framework region and constant region ammonia Base acid sequence is the sequence of human immunoglobulin(HIg).Humanized antibody can be any type of the immunoglobulin of any classification (such as IgA, IgD, IgE, IgG and IgM) or subclass (such as IgG1, IgG2, IgG3, IgG4, IGA1 or IGA2).Humanized antibody CDR region or framework region and the sequence of parental antibody are not fully corresponding, can carry out according to actual needs residue insertion, missing or Replace to make antibody generate desired characteristic.To non-human source antibodies carry out it is humanization modified can be by as known in the art Prepared by method, including CDR grafting, surface recombination method, molecule modeling method etc..

Term " bispecific antibody " is to refer to combine two independent antigens or have to different epitopes in same antigen The antibody molecule of binding specificity.Such as an arm combination tumor associated antigen of bi-specific antibody molecule, another arm, which combines, exempts from Epidemic disease cell-associated antigens, can activate at tumour cell in this way and related mechanism is immunized in active cell." polyspecific is anti-for term Body " refers to bispecific, tri-specific or four specific antibodies.Multi-specificity antibody includes two or more different antigens Binding structural domain, therefore can be from two kinds, three kinds, four kinds or more different antigen bindings.

Term " antibody " further includes antibody fragment." antibody fragment " or " antigen-binding fragment " includes but is not limited to: (i) Fab segment, with V_L、C_L、V_HAnd C_H1 domain；(ii) Fab' segment is in C_HThe C-terminal in 1 domain has one or more half Guang ammonia The Fab segment of sour residue；(iii) there is V_HAnd C_HThe Fd segment in 1 domain；(iv) Fd' segment, with V_HAnd C_H1 domain and in CH1 One or more cysteine residues of the C-terminal in domain；(v) Fv segment, the V of the single arm with antibody_LAnd V_HDomain；(vi)dAb Segment is made of the domain VH or the domain VL；(vii) 2 segment of F (ab'), a kind of two comprising the disulfide bond connection by hinge area The bivalent fragment of Fab' segment；(viii) single chain variable fragment (scFv)." antibody fragment " used herein is not only comprising above-mentioned anti- Body segment further includes the antibody from whole antibody transformation and the new antibody that is synthesized using recombinant DNA technology.

" variable region " of antibody refers to the variable region of heavy chain of antibody or light chain, comprising each variable region individually and combination Form.The variable region of heavy chain and light chain is respectively by three complementary determining region (complementarity determining Region, CDR, and be called hypervariable region) and positioned at CDR flank four framework regions (framework region, FR) Composition.Framework region plays a supporting role to CDR, and defines the spatial relationship between each CDR.The CDR of heavy chain or light chain is by ammonia Cardinal extremity starts to be expressed as CDR1, CDR2, CDR3.Heavy chain and light chain variable region are combined by non-covalent bond, and 3 of heavy chain 3 CDR of CDR and light chain together constitute antigen recognition site, which is that antibody participates in antigen binding Main body constitutes the specificity of antibody identification antigen.

Term " area Fc of antibody " or " area human immunoglobulin(HIg) Fc " include antibody in addition to heavy chain constant region 1 (CH1) Constant region polypeptides, i.e. two constant region domain CH2 of human immunoglobulin(HIg) IgA, IgD, IgG heavy chain constant region c-terminus and Three constant region domains CH2, CH3 and CH4 of CH3 and human immunoglobulin(HIg) IgE and IgM heavy chain constant region c-terminus, and It and further include the flexible hinge sequence of these structural domain aminoterminals.Although the boundary in the area Fc can change, the human IgG area heavy chain Fc is logical It is often defined as including since A231 to the residue of its carboxyl terminal.

Immunoglobulin fc region is the functional domain that antibody plays immunological effect.The Fc of IgG antibody can be with a variety of receptor phases Interaction, most important of which is that Fc γ receptor family.This receptor family includes 5 kinds of activation receptors: Fc γ RI, Fc γ RIIa, Fc γ RIIc, Fc γ RIIIa and Fc γ RIIIb and a kind of inhibition receptor: Fc γ RIIb.Fc and Fc γ R's is extracellular Area combines and forms Fc/Fc γ R compound, leads to Fc γ R intracellular region ITAM (immunoreceptor tyrosine-based Activation motifs, immunity receptor Tyrosine Activating Motifs) or ITIM (immunoreceptor tyrosine-based Inhibitory motifs, immunity receptor Tyrosine Inhibitory Motifs) phosphorylation activation downstream signal transduction access, it generates immune Response such as endocytosis, phagocytosis, killing functions of immunocytes etc..In addition to this, Fc can also be generated in conjunction with complement protein C1q Cytotoxicity (complement dependent cytotoxicity, CDC) effect of Complement Dependent.

Term " mini antibody (minibody) " refers to the artificial antibody's segment being made of antibody fragment VL-VH-CH3.

Term " nano antibody (nanobody) ", which refers to, can be changed the Camelidae antibodies segment that antibody district forms by single monomer.

Term " original antibody (probody) " is that antigen-binding site is masked, until activating the artificial of competence exertion function Antibody molecule.

" epitope " used herein or " antigenic determinant ", which refer to, to be combined by immunoglobulin or antibody specificity on antigen Position.Antigenic determinant is present in the surface of antigenic substance mostly, some are present in the inside of antigenic substance, must through enzyme or other Mode is just exposed after handling.Epitope or antigenic determinant are usually by the chemically active surface group of molecule, such as amino acid, carbon Hydrate or carbohydrate side chain composition, and usually there is specific three-dimensional structural feature and specific point and feature.Antigen table Position can be " linear " or " conformation ".In linear epitope, institute between protein and interacting molecule (such as antibody) There is the point of interaction linearly to exist along the primary amino acid sequences of protein；In comformational epitope, the point of interaction across Gal4 amino acid residue more separated from each other and exist.One native antigen substance can there are many and multiple determinants.Generally For, antigen molecule is bigger, and the number of determinant is more.

" specific binding " used herein refers to, two kinds of intermolecular nonrandom association reactions, such as antibody and its institute's needle Pair antigen between reaction.In certain embodiments, the antibody for specifically binding certain antigen (or has certain antigen special Property antibody) refer to, antibody be less than about 10^-5M is, for example, less than about 10^-6M、10^-7M、10^-8M、10^-9Or 10 M,^-10M or Smaller affinity (K_D) combine the antigen.In some embodiments of the present invention, term " targeting " refers to specific binding.

" K used herein_D" refer to, specific antibodies-antigen interactions Dissociation equilibrium constant, for describing antibody and resisting Binding affinity between original.Equilibrium dissociation constant is smaller, and antibody-antigen binding is closer, the affinity between antibody and antigen It is higher.In general, antibody is to be less than about 10^-5M is, for example, less than about 10^-6M、10^-7M、10^-8M、10^-9Or 10 M,^-10M or smaller Balance dissociate often (K_D) combine antigen.

Terms used herein " EC50 " i.e. half-maximal effect concentration (concentration for 50%of maximal Effect), refer to and cause antibody concentration corresponding to 50% ceiling effect.

" carrier (Vector) " used herein refers to a kind of nucleic acid delivery vehicle that can be inserted polynucleotide. And when carrier can make the albumen of the polynucleotide encoding of insertion obtain expression, which is known as expression vector.Carrier can lead to It crosses the methods of conversion, transduction or transfection and imports host cell, the inhereditary material element for then carrying it is in host cell It is expressed.Carrier be skilled artisans recognize that, including but not limited to: (1) plasmid；(2) phasmid；(3) Ke Si Plasmid；(4) artificial chromosome, such as the artificial dye of yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or the source P1 Colour solid (PAC)；(5) bacteriophage such as λ bacteriophage or M13 bacteriophage and (6) animal virus, such as retrovirus (including slow disease Poison), adenovirus, adeno-associated virus, spore exanthema virus (such as gebitalis virus), poxvirus, baculoviral.A kind of carrier can contain There are many elements of control expression, including but not limited to, promoter sequence, transcriptional initiation sequence, enhancer sequence, selection member Part and reporter gene；In addition, carrier can also contain replication origin.

Antibody of the present invention such as bispecific antibody can be extracted from host cell with the experimental method of standard Purifying.For example, albumin A or protein g affinity chromatography method antibody purification can be used.Means of purification includes but is not limited to affinity chromatography Chromatography, ion-exchange, size exclusion chromatography and albumen ultrafiltration.The separation of bispecific antibody of the present invention is pure Change method also includes the combination of the above method." purifying " as used herein refers to from cell, cell culture or other natural groups Separation and/or recycling purpose component in point.If not specified, antibody of the present invention is purified antibody.Term " isolated antibody " refers to the antibody for being substantially free of other molecules of different structure or antigentic specificity, " separation it is double special Property antibody " is the antibody for being substantially free of other kinds of antibody molecule.

In the present invention, the numbering of residue is such as Kabat et al., Sequences in heavy chain immunoglobulin of Proteins of Immunological Interest,5th Ed.Public Health Service,National The numbering of EU index in Institutes of Health, Bethesda, Md. (1991), can also be in WWW Upper acquisition, and be clearly completely incorporated herein by reference." the EU index in such as Kabat " refers to the residual of human IgG1's EU antibody Base numbering.As used herein, " Kabat sequence number mode " or " Kabat label " refers to that the EU index in Kabat such as is compiled The sequence of number encoding variable regions.It for heavy chain variable region, is numbered according to Kabat, hypervariable region range is the amino acid position of CDR1 The amino acid position 50 to 65 of 31 to 35, CDR2 and the amino acid position 95 to 102 of CDR3.For light chain variable region, according to Kabat number, hypervariable region range are the amino acid position 24 to 34 of CDR1, the amino acid position 50 to 56 of CDR2, the ammonia of CDR3 Base acid position 89 to 97.

Antibody remodeling method

Antibody is after antigenic stimulus body generates immune response, by capable of exempting from conjunction with antigentic specificity for plasma cell secretion Epidemic disease globulin (Immunoglobulin, Ig).Natural antibody is generally bivalent antibody (in addition to IgM classification), i.e. an antibody Molecule includes two antigen binding sites, and each Fab arm contains an antigen binding site.Antibody light chain passes through two disulfide bond It is covalently attached with heavy chain, heavy chain-light chain dimer constitutes Y-shaped antibody molecule by the disulfide bond formed between heavy chain again.Inhomogeneity Disulfide bond quantity between other heavy chain of antibody can be variant.Region between Y-shaped two arms and trunk is hinge area, is had Certain flexibility.Every antibody polypeptides chain includes variable region and constant region, and different structural domain lists is formed by space folding Member.Wherein, the heavy chain aminoterminal of antibody is variable region, is followed by three constant regions CH1, CH2, CH3；Light chain aminoterminal is variable Area is followed by a constant region CL.Heavy chain constitutes the region in conjunction with antigen recognizing with the interaction of the variable region of light chain, in addition CL and CH1 interaction and the part hinge area between CH1 and CH2 constitute monoclonal antibody region；CH2, CH3 of two heavy chains Form the region Fc that homodimer is IgG antibody；The disulfide bond that hinge area between CH1, CH2 is formed further stabilizes anti- The structure of body.

The area Fab of antibody includes the Variable domain of heavy chain of antibody and light chain aminoterminal and one after it Constant region domain and part hinge area are constituted.Wherein, the constant region knot of first constant region domain (CH1) and light chain of heavy chain Structure domain (CL) combines, and the Variable domain (VH) of heavy chain and the Variable domain (VL) of light chain combine.

Compared to classic chemotherapy drug, antibody have toxic side effect it is smaller, it is specific it is high, that Cell killing efficacy is good etc. is excellent Point is one of the research and development focus of current pharmaceutical field.It is anti-from first monoclonal antibody drug for clinical treatment in 1986 CD3 monoclonal antibody Mo Luomona (Muromonab OKT3) through FDA ratify list, monoclonal antibody medicine malignant tumour, autoimmune disease, It is quickly grown in the treatment of the major diseases such as inflammation infection, cardiovascular disease, is biomedicine field compound growth rate highest one Class product.By the existing 63 kinds of antibody class drugs listing in 2016 end of the year whole world, global antibody drug sales volume is more than 100,000,000,000 beauty Member, 20% or more of Zhan Quanqiu medicine sales volume.And in the drug of global marketing top 10,6 are antibody class drugs, In it is more than half be used for field of cancer treatment.The mechanism of action of monoclonal antibody medicine includes blocking growth signals, blocking tumor vessel raw At, induce cell apoptosis, immune cell activated generate immunological effect, wherein monoclonal antibody play critical function mainly pass through Fc and exempt from The Fc γ R (Fc γ receptor) on epidemic disease cell (such as NK cell, monocyte) surface combines active cell to enhance ADCC, antibody-dependant Cytotoxic effect), ADCP (antibody dependent cellular phagocytosis) or pass through Fc and complement PROTEIN C 1q plays killing functions of immunocytes in conjunction with enhancing CDC.

Although antibody drug achieves breakthrough development in the nearly more than ten years, it has also become the main side of the following pharmaceutical industry development To, but monoclonal antibody medicine still suffers from that collective effectiveness is low, is easy to produce the problems such as drug resistance in actual application.FcγR There is polymorphism in crowd, the Fc γ RIIIa-V158 and Fc binding ability such as NK cell surface is higher, and Fc γ RIIIa- F158 binding ability is weaker, this directly affects response and monoclonal antibody medicine validity lower original of some patients to drug One of because.In addition when treating malignant tumour especially entity tumor, monoclonal antibody is ineffective, Tumor Heterogeneity, tumor stem cell And the multi signal access of tumour cell itself adjusts so that the immunization therapy for single target spot is easy to produce drug resistance (Beck A,et al.Nat Rev Immunol.2010；10(5):345-52.).

Since T cell does not express Fc γ R, traditional monoclonal antibody can not directly activate killer T cell, and then people attempt By the way that traditional monoclonal antibody is transformed to achieve the purpose that T cell is activated to kill tumour, wherein bispecific antibody (BsAb) is at present most There is one of the method for application prospect.BsAb is a kind of antibody that can combine at least two epitopes, relative to the single target of tradition For the antibody of point, safety and validity are greatly improved, and are antibody engineering field and immunotherapy of tumors The research and development focus in field.Treatment malignant tumour practical application in, BsAb usually in combination with tumor cell surface antigen with And immune cell surface antigenic, by activating self immune system killing tumor cell (Chames P, et al.Curr Opin Drug Discov Devel 2009,12:276-283.)。

Bispecific antibody and the naturally-produced antibody of acellular can only be carried out by cell fusion or DNA recombinant technique Artificial preparation, there are many preparation methods at present.The bispecific antibody of early stage is handed over by purified monoclonal antibody chemistry Connection or two different hybridoma cell fusions generate, but the product of these methods production can there are many problem such as product is unstable Under fixed, low output, antibody modification is improper, immunogenicity, production purification difficult etc..With technique for gene engineering in recent years into Step, BsAb are largely prepared by technique for gene engineering, have been had more than 50 kinds of different bispecific antibody forms at present, totally may be used To be divided into two classes: the bispecific antibody not comprising Fc and comprising the region Fc (Brinkmann U, et al.MAbs2017,9: 182-212.).The former is usually the lesser antibody fragment of molecular weight, cannot mediate the relevant biological function of Fc.And the latter is logical Often be IgG form, this kind of antibody is similar with natural antibody form, the two-arm of Y-shaped can respectively in connection with two different antigens, The region Fc can mediate ADCC, ADCP and CDC, increase half-life period, stability and the dissolubility of antibody in blood, be easy logical Existing method is crossed largely to prepare.

It when preparing the antibody of IgG structure, needs to express two different heavy chains and two different light chains simultaneously, leads to The pairing for crossing two couples of HC-LC forms the Fab of two kinds of antigen of identification, and in this process, homologous heavy chain combines and heterologous light chain In conjunction with the mismatch problems of generation, ten kinds of different products can be generated, and only one of them is required correct product.Thus Prepare for example homologous heavy chain mispairing of random assembling that the main problem solved required for this kind of antibody is different chains, light chain mispairing and The problems such as pollution of other non-purpose products.

For homologous heavy chain mismatch problems, a solution is the IgG antibody " Knob invented by Ridgeway et al. Into Hole " is fitted into technology (referred to as " KIH "), the basic principle is that introducing in the region Fc of two heavy chains of IgG antibody different Amino acid mutation, be introduced into first heavy chain with bulky side chain group amino acid (T366Y) and in Article 2 heavy chain Introduce the amino acid (Y407T) with small side-chain radical.When this two chains are expressed simultaneously in the cell, since space structure is mutual It mends, they tend to be combined together to form heterodimer, rather than homodimer, in order to overcome brought by mutation not Stability, researcher further screen random mutation by display technique of bacteriophage, construct more stable structure, it may be assumed that Bulge-structure-T366W, sunk structure-T366S, L368A, Y407V.Theoretically, any two can be made not by this method Synantibody forms heterodimer, but wherein still has 5% homodimer, and can not solve the problems, such as light chain mispairing (US 5731168A, US5731168A；Ridgway JB,et al.Protein Eng1996,9:617-621.；Atwell S,et al.J Mol Biol 1997,270:26-35.；Merchant AM,et al.Nat Biotechnol1998,16:677- 681.)。

In addition to the hydrophobic effect formed using steric hindrance overcomes the formation of homodimer, another solution is benefit With ionic bond, it is artificially introduced the opposite amino acid of charge property in two heavy chains, passes through the repulsive interaction of like charges in this way Inhibit the formation of homodimer, correlation technique has a detailed description (US8592562B2, US in patent and document 20170058054A1, WO 2014084607A1；Gunasekaran K,et al.J Biol Chem2010,285:19637- 19646；Strop P,et al.J Mol Biol 2012,420:204-219；Choi HJ,et al.Mol Immunol 2015,65:377-383)。

In the earlier patent application (publication number: WO 2017034770A1) of the present inventor, the heavy chain to antibody is disclosed Part is transformed to increase the method for combination activity and specificity between two heavy chains.Specifically, this method is by changing Become the charge property of the amino acid at CH3 regional interaction interface, the formation for reducing homodimer, promoting heterodimer, Successfully solves the problems, such as heavy chain mispairing in bispecific antibody preparation process.

For the mismatch problems between heavy chain and light chain, there are several types of solutions at present: 1) screening and using common Light chain；2) light chain is fused to heavy chain；3) being transformed to light chain and heavy chain can specific pairs.

Light chain mispairing is solved, most straightforward approach is using common light chain, i.e. 2 heavy chains share identical light chain.Generally In the case of, the Variable Area of antibody light chain is different, and various Variable Area also determines antibody to the special of antigen recognizing Property.In practical application, the acquisition of common light chain is relatively difficult, needs that a large amount of manpowers and time is spent to go to screen, while can not Guarantee screens suitable common light chain；In addition, the use of common light chain may be decreased the specificity and affinity to antigen, because This common light chain is not particularly suited for all bispecific antibodies.

Other solutions are to make light chain and heavy chain fusion by transformation, such as pass through one section of non-natural linking sequence It connects heavy chain variable domain and light chain variable region generates single chain variable fragment (single chain variable Fragment, scFv), or the Variable Area that light chain is connected to heavy chain is formed into single chain Fab antibody fragment (single chain Fab antibody fragment, scFab), but the problems such as the linking sequence of this introducing can bring stability, dissolubility, hold Easily formed antibody polymerization object, may cause Immunogenicity (Gunasekaran K, et al.J Biol Chem 2010, 285:19637-19646.；Muda M, et al. albumen Eng Des Sel2011,24:447-454.；Wranik BJ,et al.J Biol Chem 2012,287:43331-43339.).It, can be by two for the bispecific antibody with 2 light chains Kind of antibody separately express to avoid light chain mispairing, such as controllable Fab arm exchange (controlled Fab-arm exchange, CFAE), chemical crosslinking etc..CFAE method is by two species specificity monoclonal antibody of expression and purification respectively, in vitro using redox Method make two strain specific antibodies be assembled together to form the bispecific antibody of similar native IgG structure.Its principle is used for reference The physiological processes of the Fab arm exchange of 4 molecule of human IgG in vivo, at the 409th of this process kind IgG4CH3 region Arginine (R409) and hinge area the 228th serine (S228) have played key effect.Current this assembly method technique It is complicated, need to carry out secondarily purified, low output, can greatly increase production cost (WO 2008119353, WO2011131746A2；Labrijn AF,et al.Proc Natl Acad Sci USA 2013,110:5145-5150.； Cook AG,et al.J Immunol Methods 1994,171:227-237.)。

Lindhofer et al. (Lindhofer H, et al.J Immunol 1995,155:219-225) describes another The characteristics of kind solution, this method can be combined naturally using rat heavy with murine heavy chain, will produce rat source monoclonal antibody The thin of bispecific antibody is able to produce by the technology formation of Somatic Fusion with two kinds of hybridomas of mouse source monoclonal antibody Born of the same parents.The antibody that this method generates overcomes light chain mispairing by kind limitation, and mismatch rate can be reduced to 4-10%.Listing earliest Bispecific antibody Catumaxomab (trade name: Removab^TM) it is to be produced by the technology, the antibody is for treating Malignant ascite is swollen.But the problem of being source of mouse, immunogenicity can be brought due to antibody.

Although having existed the technical solution of mismatch problems between the heavy chain and light chain of above-mentioned solution bispecific antibody, still So there are complex process, antigenicity, the low output of target antibody and the various problems such as be not suitable for all antibody.Therefore, right Needs are still had in the new and effective method for solving mispairing between heavy chain and light chain.

The present invention has analysed in depth the space structure of humanized IgG 1CH1-CL and hinge area-CL interaction, and discovery exists CH1-CL interface introduces non-native disulfide bond, and the optional removal region hinge area-CL natural disulphide bonds (HC-C220S, LC-C214S), can be on the basis of introducing few site mutation and not influencing antibody structure and function, utilization is artificial reconstructed Disulfide bond formation pairing heavy chain-light chain Fab or whole antibody.

Specifically, the present invention identifies 4 amino acid between heavy chain of antibody CH1 and light chain CL to (see following table 1), It is suitable for sporting cysteine to form non-native disulfide bond between heavy chain CH1 and light chain CL.

Table 1. can form the amino acid pair that two sulphur are strong between heavy chain CH1- light chain CL

In addition, the present inventor analyzes conservative of the above-mentioned site between different antibody isotypes.Such as table 2 Shown, above-mentioned 4 amino acid is guarded in IgG1, IgG2, IgG3 and IgG4 isotype.

2. amino acid of table is to the conservative in IgG1, IgG2, IgG3 and IgG4

The present invention also provides a kind of charge remodeling methods, i.e., introduce K218E or K218D to the hinge area of antibody and be mutated, And E213R or E213K is introduced to CL and is mutated, increase the binding specificity between heavy chain and light chain.This can be with to charge transformation It is used alone or is combined with disulfide bond remodeling method of the invention for antibody such as bispecific antibody or multi-specificity antibody Building design in, promote the heavy chain-light chain of antibody correctly to match.

Therefore, on the one hand, the present invention relates to the methods that the area Fab to antibody is transformed, and the method includes selecting The step of introducing the area Fab from one or more mutation below:

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL,

Can the area Fab to antibody be introduced into above-mentioned mutation combination a)-e) in it is one or more, such as 1,2,3, 4 or 5.In preferred embodiments, above-mentioned mutation combination a)-d is introduced into the area Fab of antibody) in 1 or 2, with 1 pair or 2 pairs of non-native disulfide bonds are formed between the heavy chain and light chain or heavy chain of antibody and the segment of light chain, and optional are drawn Enter mutation combination e) to further increase the binding specificity between heavy chain of antibody and light chain.

In some embodiments, remodeling method of the invention further includes natural two sulphur destroyed between CH1-CL Key, that is, destroy by the 214th cysteine (C214) in the 220th in hinge area cysteine (C220) and CL it Between the disulfide bond that is formed.It can be realized by the following method: the cysteine in 214 site of the region mutated light chain CL is any non- Cysteine, preferably serine, alanine and glycine, or delete the cysteine in 214 sites；Or mutagenized heavy chain hinge The cysteine that sequence is 220 is any non-cysteine；Or the cysteine in 214, the region mutated light chain CL is any non-half Cystine, the cysteine that simultaneous mutation heavy chain hinge region is 220 are any non-cysteine；Or delete the region light chain CL 214 The cysteine of position, the cysteine that simultaneous mutation heavy chain hinge region is 220 are any non-cysteine.

In any embodiment of the above method, the area Fab derives from IgG, IgA, IgM, IgE or IgD, such as IgG1, IgG2, IgG3 or IgG4 isotype.As shown in table 2, the amino acid that the present invention identifies in IgG1, IgG2, IgG3 and It is guarded in IgG4 isotype.Therefore method of the invention can be used for above-mentioned antibody isotype.

It should be appreciated that the method that the area Dui Fab of the present invention is transformed can be applied to it is any have at least one area Fab Antibody or antibody fragment, including such as monoclonal antibody, bispecific antibody or multi-specificity antibody；Source of mouse antibody, source of people Change antibody, chimeric antibody, human antibody and the antibody in other sources；And any antibody fragment with the area Fab, such as Fab Segment, Fab' segment and F (ab')₂Segment.

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；With

D) Q160C in the V173C and CL in CH1,

Antibody production method

On the one hand, there is the method for the antibody or antibody fragment in at least two different areas Fab, institute the present invention relates to production State method the following steps are included:

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

3) antibody or antibody fragment are recycled from the host cell cultures.

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

In some embodiments, the above method further includes destroying hinge in the first area Fab and/or the 2nd area Fab Natural disulphide bonds between the area-CL, area sport the C220 in the hinge area in the first area Fab and/or the 2nd area Fab Other amino acid in addition to cysteine or missing C220, and/or by the first area Fab and/or the 2nd area Fab and/or C214 in the CL in the 2nd area Fab sports other amino acid or missing C214 in addition to cysteine.Preferably implementing In scheme, the amino acid in addition to cysteine is selected from serine, alanine and glycine.

In some embodiments of the above method, the first area Fab and the 2nd area Fab are combined on identical antigen Different epitopes.

In some embodiments of the above method, the first area Fab combines the first antigen, the 2nd area the Fab knot The second antigen is closed, and first antigen and the second antigen are different.

It should be appreciated that can be used for as described herein for the method for producing antibody or antibody fragment any at least two The antibody or antibody fragment in a different area Fab, including bispecific antibody and multi-specificity antibody.Furthermore, it is possible to according to need Select the different areas Fab to construct bispecific antibody or multi-specificity antibody.

The antibody of transformation

On the other hand, the present invention relates to at least two different areas Fab antibody or antibody fragment, the antibody or First area Fab of antibody fragment has one or more mutation selected from the following:

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and CL in E213R or E213K mutation, wherein according to EU number into Row number.

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL, wherein second Fab The mutation in area and the mutation in the first area Fab are not exactly the same.

In some embodiments of above-mentioned antibody and antibody fragment, the first area Fab and the 2nd area Fab are in conjunction with identical Different epitopes on antigen.

In some embodiments of above-mentioned antibody and antibody fragment, the first area Fab combines the first antigen, and described the In conjunction with second antigen, and wherein, first antigen and the second antigen are different in two areas Fab.

In any embodiment, the area Fab, antibody or the antibody fragment combination cancer antigen that the present invention is transformed, for cancer Disease development or the relevant non-cancer protein of invasion or viral associated proteins.In fact, the present invention be transformed the area Fab, antibody Or antibody fragment can combine any antigen, including but not limited to following albumen, subunit, structural domain, motif and epitope: CD2； CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32, CD33 (p67 albumen), CD38, CD40, CD40L, CD52, CD54, CD56, CD80, CD147, GD3, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-6, IL-6R, IL-8, IL-12, IL-15, IL-18, IL-23, interferon-' alpha ' are done Disturb plain β, interferon gamma；TNF-α, TNF β, TNF-R1, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, Receptor -1 TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL, adenosine receptor, lymphotoxin-beta-receptor, TACI, BAFF-R, EPO；LFA-3, ICAM-1, ICAM-3, EpCAM, integrin β 1, integrin β 2, integrin alpha-4/β 7, integrin Protein alpha 2, beta 2 integrin alpha 3, integrin alpha-4, beta 2 integrin alpha 5, beta 2 integrin alpha 6, beta 2 integrin alpha v, beta 2 integrin alpha V β 3, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, anti-Id, E-Selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGF β 1, TGF β 2, eosinophil chemokine 1 (eotaxin1), Blys (bone-marrow-derived lymphocyte stimulating factor), complement C5, IgE, factor Ⅴ II, CD64, CBL, NCA 90, EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, VEGF, VEGFR, Endothelin Receptor, VLA-4, haptens NP-cap or NIP-cap, T cell receptor α/β, E-Selectin, digoxin, P-ALP (PLAP) it is glued with testis PLAP sample alkaline phosphatase, TfR, carcinomebryonic antigen (CEA), CEACAM5, HMFG1, PEM Albumen MUC1, MUC18, Heparinase I, human heart myosin, tumor-associated glycoprotein -72 (TAG-72), tumor associated antigen CA 125, prostate-specific membrane antigen (PSMA), high molecular weight melanoma related antigen (HMW-MAA), cancer (carcinoma) related antigen, Gco protein I ib/IIIa (GPIIb/IIIa), expression Lewis Y related carbohydrate swell Tumor related antigen, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV breathe syncytial virus RSV F, RSVF Fgp, VNR integrin, cytokeratin tumor associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 ring, Respiratory Syncytial Virus(RSV) (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and C.perfringens (Clostridium perfringens) toxin.

In some embodiments, the area Fab, antibody or the antibody fragment that the present invention is transformed combine antigen selected from the group below: PSMA, CD133, CD138, CD20, CD19, OX40, GITR, PD-1, PD-L1 or PD-L2, CTLA-4, KIR, LAG-3, CD3, TCR α, TCR β, TCR γ, TCR δ, CD40, CD40L, VEGF, EGF, VEGFR, EGFR, Her1, Her2, Her3, EpCAM, mesothelium Element, Glypicans, CD28, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, fucosido-GM1, IGF1, IGF2, IGF1R, IGF2R, CD64, CD32a, CD32b, CD16, integrin, RANK Ligand, CEA, DLL-4, GM-CSFR, ADAMS, flesh generate inhibin, PCSK9, CXCR4, IL-1 α, IL-1 β, IL-12, IL- 18, TNF α, IL-23, IL-13, MIF, IL-17, IL-17R, IL-15, IL-9, IL-5, IL-5R, IL-6, IL-25, PEG2 etc.. Antibody of the invention can be specific, such as HIV albumen, HPV albumen, CMV albumen, influenza disease for viral associated target Toxalbumin or prion protein.

In some aspects, the area Fab, antibody or the antibody fragment that the present invention is transformed can be used for diagnosing and treating application.More Body, method of the invention can be used for the bispecific or polyspecific that generate can in conjunction with two kinds or more than two kinds of target antigens Antibody, the target antigen can be selected from, but be not limited to following target: IL-1 α, IL-1 β, IL-12, IL-18, TNF α, IL-23, IL-13, MIF, IL-17, IL-17R, IL-15；VEGF, VEGFR, EGFR；IL-9, IL-5, IL-5R, IL-6, IL-25, IL- 13, ADAMS, PEG2, Her1, Her2 and Her3.In addition, those skilled in the art will identify other possible targets.

Method of the invention can be used for that bispecific antibody is transformed, and can be used for but be not limited to activation T cell to kill Carry the target cell of tumour antigen, one of antigen binding regions are made of the region Fab of monoclonal antibody, C-terminal and its In the N-terminal of hinge area of a heavy chain be connected, it is logical in combination with the signal on tumor-cell antigen target spot or immune effector cell Road target spot；Another antigen binding regions is made of the region Fab of monoclonal antibody, the hinge area of C-terminal and Article 2 heavy chain N-terminal be connected；Similarly, it can also specifically bind the signal on the Antigenic Target or immune effector cell of tumour cell Access target spot；Equally, the present invention can be used for blocking according to the bispecific antibody that above-mentioned technical proposal mutation generates, antagonism Or activation target antigen, such as antagonism cell factor or cytokine receptor.

A) area CH1 in the area CL of SEQ ID NO:11 and SEQ ID NO:33；

B) area CH1 in the area CL of SEQ ID NO:13 and SEQ ID NO:29；

C) area CH1 in the area CL of SEQ ID NO:15 and SEQ ID NO:35；

D) area CH1 in the area CL of SEQ ID NO:17 and SEQ ID NO:33；With

E) area CH1 in the area CL of SEQ ID NO:27 and SEQ ID NO:43.

Nucleic acid sequence and host cell

The invention further relates to the nucleotide sequences for encoding antibody of the invention, and the load comprising these nucleotide sequences Body.During expressing antibody, the nucleotide sequence is inserted into suitable carrier, carrier includes but is not limited to: plasmid, Phagocytosis expression vector, coemid, artificial chromosome, bacteriophage and animal virus.Comprising for regulating and controlling table in expression vector The element reached, including but not limited to promoter, transcriptional initiation sequence, enhancer, signal peptide sequence etc..Promoter includes but is not limited to T7 promoter, T3 promoter, SP6 promoter, β-actin promoter, EF-1 α promoter, CMV promoter and SV40 starting Son.Expression vector is transferred to, appropriate method known in the art can be used in host cell, including but not limited to: calcium phosphate is heavy Shallow lake method, polyethyleneimine infection protocol, lipofection, electroporation, PEI (polyethyleneimine) infection protocol.

In another aspect, the present invention includes host cell, and it includes nucleic acid molecules of the invention or carriers.The place Chief cell include but is not limited to Chinese hamster ovary celI (Chinese hamster ovary cells, Chinese hamster ovary cell), HEK293 cell (Human embryonic kidney cells 293, human embryonic kidney cells 293), myeloma cell, yeast or Prokaryotic cell such as Escherichia coli (Escherichia coli).

Antibody coupling matter

Pharmaceutical composition and disease treatment method

On the other hand, the present invention relates to the methods for the treatment of disease comprising uses antibody of the invention or antibody piece The step of section, bispecific antibody, antibody coupling matter or pharmaceutical composition.

In some embodiments, the disease is cancer.

In one aspect, the present invention relates to pharmaceutical compositions, and it includes antibody of the invention or antibody fragments, bispecific Antibody or antibody coupling matter, and optionally pharmaceutically acceptable carrier, surfactant and/or diluent.

Phrase " pharmaceutically acceptable carrier " refers to pharmaceutically acceptable material, composition or medium, such as liquid Or solid-filling agent, diluent, excipient, solvent, medium, encapsulating material, manufacture auxiliary agent (such as lubricant, talcum magnesium, tristearin Sour calcium or zinc or stearic acid) or solvent encapsulating material, it is related to maintaining stability, solubility or the activity of LAP bonding agent.With In the sense that other ingredients of preparaton are compatible and unharmful to patient, every kind of carrier must be " acceptable ".It may act as Some examples of the material of pharmaceutically acceptable carrier include: (1) sugar, such as lactose, dextrose and saccharose；(2) starch, it is such as beautiful Rice starch and potato starch；(3) cellulose and its derivates, such as sodium carboxymethylcellulose, methylcellulose, ethyl cellulose Element, microcrystalline cellulose and cellulose acetate；(4) powdered tragacanth；(5) malt；(6) gelatin；(7) excipient, such as cocoa butter and Suppository wax；(8) oily, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil；(9) glycol, such as third Glycol；(10) polyalcohol, such as glycerol, D-sorbite, mannitol and polyethylene glycol (PEG)；(11) ester, such as ethyl oleate and laurel Acetoacetic ester；(12) agar；(13) buffer, such as magnesium hydroxide and aluminium hydroxide；(14) alginic acid；(15) apirogen water；(16) Isotonic salt water；(17) Ringer's solution；(19) pH buffer solution；(20) polyester, polycarbonate and/or polyanhydride；(21) filler, Such as polypeptide and amino acid (22) serum component, such as seralbumin, HDL and LDL；(23) C2-C12 alcohol, such as ethyl alcohol；(24) medicine Other non-toxic compatible substances used in object preparaton.Releasing agent, coating agent, preservative and antioxidant can also exist on medicine In object preparaton.The term of " excipient ", " carrier ", " pharmaceutically acceptable carrier " etc. is interchangeable herein to be made With.

In some embodiments, except antibody of the invention, bispecific antibody or antibody coupling beyond the region of objective existence, the medicine group Closing object also includes one or more other therapeutic agents.

In some embodiments, the other therapeutic agent includes but is not limited to chemotherapeutics, growth inhibitor, cell toxicant Property agent, the examination of the reagent for radiotherapy, anti-angiogenic agent, apoptosis agent, antitublin and other treating cancers Agent, such as anti-CD 20 antibodies, EGF-R ELISA (EGFR) antagonist (such as tyrosine kinase inhibitor), HER1/EGFR Inhibitor (such as Tarceva), platelet derived growth factor inhibitor is (for example, GLEEVEC^TM(she Imatinib mesylate (Imatinib Mesylate))), cox 2 inhibitor (such as celecoxib (celecoxib)), interference Element, cell factor, antagonist (such as neutralizing antibody), in conjunction with one or more following target PD-1, PD-L1, PD-L2 (examples Such as, pyridine aldoxime methyliodide (PAM) monoclonal antibody (pembrolizumab)；nivolumab；MK-3475；AMP-224；MPDL3280A；MEDI0680； MSB0010718C；And/or MEDI4736)；CTLA-4 (for example, tremelimumab (PFIZER) and ipilimumab))； LAG-3 (such as BMS-986016)；CD103；TIM-3 and/or other TIM series members；CEACAM-1 and/or other CEACAM Family member, ErbB2, ErbB3, ErbB4, PDGFR- β, BlyS, APRIL, BCMA or vegf receptor, TRAIL/Apo2 and other Bioactivity and organic chemistry agent etc..Also special consideration should be given to a combination thereof for method described herein.

In some embodiments, the other therapeutic agent is chemotherapeutics.The non-limiting example of chemotherapeutics can wrap Include alkylating agents (alkylating agents), such as phosphinothioylidynetrisaziridine (thiotepa) andCyclophosphamide (cyclophosphamide), Temozolomide (temozolomide)；Alkylsulfonates (alkyl sulfonates), such as Busulfan (busulfan), Improsulfan (improsulfan) and piposulfan (piposulfan)；Aziridines (aziridines), such as Benzodepa (benzodepa), carboquone (carboquone), meturedepa (meturedepa) With uredepa (uredepa)；Ethylenimines (ethylenimines) and methylamelamines (methylamelamines), Including hemel (altretamine), triethylenemelamine (triethylenemelamine), triethylphosphoramide (trietylenephosphoramide, triethylenephosphoramide), triethylene thiophosphamide (triethiylenethiophosphoramide, triethylenethiophosphoramide) and trimethylolmelamine (trimethylolomelamine)；Annonaceousacetogenicompounds (acetogenin) (especially bullatacin (bullatacin) and Bullatacinone (bullatacinone))；Camptothecine (camptothecin) (including synthetic analogues Hycamtin (topotecan)；Bryostatin (bryostatin)；callystatin；CC-1065 (including its Adozelesin (adozelesin), Carzelesin (carzelesin) and Bizelesin (bizelesin) synthetic analogues)；Cryptophycins (cryptophycins) (especially cryptophycin 1 and cryptophycin 8)；Dolastatin (dolastatin)；Duocarmycin (packet Include synthetic analogues, KW-2189 and CB1-TM1)；Eleutherobin (eleutherobin)；pancratistatin； sarcodictyin；Spongistatin (spongistatin)；Nitrogen mustards (nitrogen mustards), such as Chlorambucil (chlorambucil), Chlornaphazine (chlornaphazine), cholophosphamide (cholophosphamide), Estramustine (estramustine), ifosfamide (ifosfamide), chlormethine (mechlorethamine), hydrochloric acid oxygen nitrogen Mustard (mechlorethamine oxide hydrochloride), melphalan (melphalan), novoembichin (novembichin), phenesterin (phenesterine), prednimustine (prednimustine), Trofosfamide (trofosfamide), uracil mastard (uracil mustard)；Nitrosourea (nitrosoureas), such as Carmustine (carmustine), chlorozotocin (chlorozotocin), Fotemustine (fotemustine), lomustine (lomustine), Nimustine (nimustine) and Ranimustine (ranimustine)；Antibiotics, such as Enediyne Antibiotic (enediyne) (such as Calicheamicin (calicheamicin), especially Calicheamicin γ 1I and Calicheamicin ω I1 (see, for example, Agnew.Chem Intl.Ed.Engl., 33:183-186 (1994))；Anthracycline antibiotic Including dynemicin A (dynemicin),；Ai sibo mycin (esperamicin)；And Neocarzinostatin (neocarzinostatin) chromophore and related chromoprotein Enediyne Antibiotic chromophore), aclacinomycin (aclacinomycin), D actinomycin D (actinomycin), anthramycin (anthramycin), azaserine (azaserine), bleomycin (bleomycin), act-C (cactinomycin), carabicin, carminomycin (caminomycin, carminomycin), cardinophyllin (carzinophilin), chromomycin (chromomycinis, Chromomycin), actinomycin D (dactinomycin), daunorubicin (daunorubicin), Detorubicin (detorubicin), 6- phenodiazine -5- oxygen-L- nor-leucine,Doxorubicin (doxorubicin) (including morpholino Doxorubicin, Cyanomorpholino Doxorubicin, 2- pyrroles are for Doxorubicin and deoxydoxorubicin), table are soft Than star (epirubicin), esorubicin (esorubicin), idarubicin (idarubicin), marcellomycin (marcellomycin), mitomycin (mitomycins) such as mitomycin C, mycophenolic acid (mycophenolic Acid), nogalamycin (nogalamycin), olivomycin (olivomycin), Peplomycin (peplomycin), pool are non-mould Element (potfiromycin, potfiromycin), puromycin (puromycin), triferricdoxorubicin (quelamycin), Luo Duo Than star (rodorubicin), streptonigrin (streptonigrin), streptozotocin (streptozocin), tubercidin (tubercidin), ubenimex (ubenimex), Zinostatin (zinostatin), zorubicin (zorubicin)；Anti- generation Thank species, such as methotrexate (MTX) and 5 FU 5 fluorouracil (5-FU)；Folacin, such as denopterin (denopterin), first Aminopterin, pteroyltriglutamic acid (pteropterin), Trimetrexate (trimetrexate)；Purine analogue, such as fluorine, which reach, to be drawn Shore (fludarabine), Ismipur (mercaptopurine), thiapurine (thiamiprine), thioguanine (thioguanine)；Pyrimidine analogue, such as ancitabine (ancitabine), azacitidine (azacitidine), 6- nitrogen Uridine, Carmofur (carmofur), cytarabine (cytarabine), dideoxyuridine (dideoxyuridine), deoxidation fluorine Uridine (doxifluridine), enocitabine (enocitabine), floxuridine (floxuridine)；Androgens such as block Shandong testosterone (calusterone), dromostanolone propionate (dromostanolone propionate), epitiostanol (epitiostanol), Mepitiostane (mepitiostane), Testolactone (testolactone)；Anti- adrenal gland class, such as ammonia Shandong Meter Te (aminoglutethimide), mitotane (mitotane), Trilostane (trilostane)；Folic acid supplement, such as Folinic acid (folinic acid)；Aceglatone (aceglatone)；Aldophosphamideglycoside (aldophosphamide glycoside)；Amino-laevulic acid (aminolevulinic acid)；Eniluracil (eniluracil)；Amsacrine (amsacrine)；bestrabucil；Bisantrene (bisantrene)；Edatrexate (edatraxate)；Defosfamide (defosfamide)；Demecolcine (demecolcine)；Diaziquone (diaziquone)；elformithine；Elliptinium Acetate (elliptinium acetate)；epothilone；Ethoglucid (etoglucid)；Gallium nitrate；Hydroxyurea (hydroxyurea)；Lentinan (lentinan)；Lonidamine (lonidamine)；Maytansinoids (maytansinoids), such as maytansine (maytansine) and ansamitocin (ansamitocin)；Mitoguazone (mitoguazone)；Mitoxantrone (mitoxantrone)；Mopidamol (mopidamol)；C-283 (nitracrine)；Pentostatin (pentostatin)；Phenamet (phenamet)；Pirarubicin (pirarubicin)； Losoxantrone (losoxantrone)；Podophyllic acid (podophyllinic acid)；2- ethylhydrazide (ethylhydrazide)； Procarbazine (procarbazine)；Polysaccharide compound (JHS Natural Products, Eugene, Oreg.)；Thunder Help raw (razoxane)；Rhizomycin (rhizoxin)；Sizofiran (sizofuran, sizofiran)；Spirogermanium (spirogermanium)；Tenuazonic acid (tenuazonic acid)；Triethyleneiminobenzoquinone (triaziquone)；2,2', 2 "-trichlorotriethylamines；Trichothecin class (trichothecenes) (especially T-2 toxin, verrucarine (verrucarin) A, roridin (roridin) A and snake rhzomorph (anguidin))；Urethane (urethan)；Eldisine (vindesine)；Dacarbazine (dacarbazine)；Mannomustin (mannomustine)；Dibromannitol (mitobronitol)；Mitolactol (mitolactol)；Pipobroman (pipobroman)；gacytosine；Arabinose born of the same parents Glycosides (arabinoside) (" Ara-C ")；Cyclophosphamide；Phosphinothioylidynetrisaziridine (thiotepa)；Taxoid (taxoids), such asTaxol (paclitaxel) (Bristol-Myers Squibb Oncology, Princeton, N.J.),The nano particle dosage form Taxol that no cremophor (Cremophor-free), albumin are transformed (American Pharmaceutical Partners, Schaumberg, Ill.) andTaxotere (doxetaxel)(Rhone-Poulenc Rorer,Antony,France)；Chlorambucil (chlorambucil)；Gemcitabine；6- thioguanine (thioguanine)；Purinethol (mercaptopurine)；First ammonia butterfly Purine (methotrexate)；Platinum analogs, such as cis-platinum (cisplatin), oxaliplatin (oxaliplatin) and carboplatin (carboplatin)；Vincaleukoblastinum (vinblastine)；Platinum；Etoposide (etoposide) (VP-16)；Ifosfamide (ifosfamide)；Mitoxantrone (mitoxantrone)；Vincristine；NAVELBINE, vinorelbine (vinorelbine)； Can destroy tumors (novantrone)；Teniposide (teniposide)；Edatrexate (edatrexate)；Daunomycin (daunomycin)；Aminopterin (aminopterin)；Xeloda (xeloda)；Ibandronate (ibandronate)；She It is vertical for health (irinotecan) (Camptosar, CPT-11) (including Irinotecan and 5-FU and formyl tetrahydrofolic acid (leucovorin) therapeutic scheme)；Topoisomerase enzyme inhibitor RFS 2000；Difluoromethylornithine (DMFO)；Retinoids (retinoids), such as Tretinoin (retinoic acid), capecitabine (capecitabine)；Combretastatin (combretastatin)；Formyl tetrahydrofolic acid (LV)；Oxaliplatin (oxaliplatin), including oxaliplatin therapeutic regimen (FOLFOX)；lapatinib(TYKERB.)；The inhibitor of PKC-alpha, Raf, H-RasVEGF-A；With above-mentioned any substance Pharmaceutically acceptable salt, acid or derivative.

Pharmaceutical composition as described herein can especially be formulated for applying with solid, liquid or gel form to subject Compound, including be suitable for those of following: (1) parenteral administration, such as pass through subcutaneous, intramuscular, intravenous or Epidural cavity Injection, as such as sterile solution or suspension or sustained release preparaton；(2) surface is applied, such as is used as and is applied to skin Creme, ointment or controlled release patch or spray；(3) in intravaginal or rectum, such as pessary, emulsifiable paste or bubble Foam；(4) eye；(5) percutaneous；(6) transmucosal；Or (7) nose.

On the other hand, the present invention relates to antibody of the invention or antibody fragment, bispecific antibody, antibody coupling matters Or pharmaceutical composition, it is used to treat disease.In some embodiments, the disease is cancer.

In another aspect, the present invention relates to the methods for the treatment of disease comprising antibody of the invention is applied to subject Or the step of antibody fragment, bispecific antibody, antibody coupling matter or pharmaceutical composition.In some embodiments, the disease Disease is cancer.

The example of cancer includes but is not limited to basal-cell carcinoma, cancer of bile ducts；Bladder cancer；Osteocarcinoma；Brain and CNS cancer；Breast cancer； Peritoneal cancer；Cervical carcinoma；Cholangiocarcinoma；Choriocarcinoma；Colon and the carcinoma of the rectum；Connective tissue cancer；Cancer in digestive system；Endometrium Cancer；Cancer of the esophagus；Cancer eye；Head and neck cancer；Gastric cancer (including human primary gastrointestinal cancers)；Glioblastoma；Liver cancer；Liver cancer；Upper intradermal neoformation；Kidney Cancer；Laryngocarcinoma；Leukaemia；Liver cancer；Lung cancer (for example, Small Cell Lung Cancer, non-small cell lung cancer, adenocarcinoma of lung and squamous cell lung carcinoma)； Lymthoma, including Hodgkin lymphoma and non-Hodgkin lymphoma；Melanoma；Myeloma；Neuroblastoma；Carcinoma of mouth (example Such as lip, tongue, mouth and pharynx)；Oophoroma；Cancer of pancreas；Prostate cancer；Retinoblastoma；Rhabdomyosarcoma；The carcinoma of the rectum；Breathing System cancer；Salivary-gland carcinoma；Sarcoma；Cutaneum carcinoma；Squamous cell carcinoma；Gastric cancer；Teratocarcinoma；Carcinoma of testis；Thyroid cancer；Uterus or uterus Endometrial carcinomas；Urinary system cancer；Carcinoma of vulva；And other cancers and sarcoma；With lymphoproliferative disorders after transplanting (PTLD), and Abnormal angiogenesis relevant to phakomatoss, oedema (oedema such as relevant to brain tumor), original origin tumour and Plum Gus (Meigs) Cotard.

Antibody or antibody fragment of the invention, bispecific antibody, antibody coupling matter or pharmaceutical composition can be applied In subject in need or patient.Term " subject ", " patient " and " individual " is used interchangeably herein, and refers to Animal, such as the mankind.Term " non-human animal " and " non-human mammal " are used interchangeably herein, and dynamic including lactation Object such as rat, mouse, rabbit, sheep, cat, dog, ox, pig and non-human primate.Term " subject " further includes any vertebra Animal, including but not limited to mammal, reptile, amphibian and fish.Advantageously however, subject is mammal Such as people or other mammals, such as the mammal raised and train, such as dog, cat, horse etc..Produce mammal, as ox, sheep, Pig etc. is also included in term subject.

Specific implementation method

The contents of the present invention are further illustrated below with reference to embodiment.It should be understood that following embodiment is only illustrative , and it is not considered as limitation of the scope of the invention.

Embodiment 1: the design and expression vector establishment of bispecific antibody

(1) design of bispecific antibody

The Exemplary bispecific antibodies of design and building in this implementation are " Y " font antibody (see Figure 1A) comprising 2 The complete heavy chain of item and 2 complete light chains, are collectively formed Fab the and Fc structural domain of antibody.Fab two-arm is specifically bound respectively People CD3 and people Her2.The part for wherein specifically binding people CD3 is named as C31 herein, it includes a heavy chain of antibody and One antibody light chain, sequence is from heavy chain of antibody and light chain from source of mouse monoclonal antibody (referring to patent No. US 9587021).The part for specifically binding people Her2 includes a heavy chain of antibody and an antibody light chain, and sequence derives from people The sequence of source monoclonal antibody Herceptin (referring to patent No. US 5821337).The part Fc of antibody is according to inventor elder generation The transformation that method in preceding patent application publication WO2017034770A1 carries out, wherein being done to the heavy chain for combining the part people CD3 Following mutation: P395K, P396K, V397K, the mutation markers are OA；Following mutation is done to the heavy chain for combining the part people Her2: T394D, P395D, P396D, the mutation markers are OB, form it into heavy chain heterodimer.The bispecific antibody is herein In be known as bispecific antibody WT.

AntiCD3 McAb (C31) light-chain amino acid sequence (SEQ ID NO:1)

QTVVTQEPSLTVSPGGTVTLTCGSSTGAVTTSNYANWVQEKPGQAPRGLIGGTNKRAPWTPARFSGSLLGGKAALTI TGAQAEDEADYYCALWYSNLWVFGGGTKLTVLGKLVTMEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYTCEVTHQ GLSSPVTKSFNRGEC AntiCD3 McAb (C31) heavy chain amino acid sequence (SEQ ID NO:3)

EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWVRQAPGKGLEWVARIRSKYNNYATYYADSVKDR FTISRDDSKNSLYLQMNSLKTEDTAVYYCARHGNFGNSYVSWFAYWGQGTLVTVSSISSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVD KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTKKKLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

Anti- Her2 (Herceptin) light-chain amino acid sequence (SEQ ID NO:5)

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGT DFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKLVTMEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYTCEVTHQGLSSPVTKSFNRGEC

Anti- Her2 (Herceptin) heavy chain amino acid sequence (SEQ ID NO:7)

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFT ISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSISSASTKGPSVFPLAPSSKSTSGGTA ALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP KSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

(2) molecular cloning of expression plasmid

The nucleotide sequence (SEQ ID NO:2) of artificial synthesized coding C31 light chain and the nucleotide sequence for encoding C31 heavy chain (SEQ ID NO:4).Utilize method (Sambrook JF, the E.F.et al.Molecular of general molecular cloning cloning:a laboratory manual.4th ed.Cold Spring Harbor Laboratory Press,Cold Spring Harbor, New York:2012), the nucleotide sequence for encoding C31 light chain is cloned into the plasmid being transformed On pCDNA3.1 (+) (Invitrogen, article No. V790-20), which joined one section in multiple cloning sites N-terminal by transformation The signal peptide sequence (SEQ ID NO:9 and SEQ ID NO:10) of human interleukin-2 (Interleukin-2, IL-2), can Enough that secretory antibody is expressed in cell (such as HEK293 cell), resulting expression plasmid is named as pCDNA3.1-C31-LC.It will The nucleotide sequence of coding C31 heavy chain is cloned on plasmid pFUSE-hIgG1-Fc2 (InvivoGene), resulting expression plasmid It is named as pFUSE-C31-HC-OA.

The nucleotide sequence (SEQ ID NO:6) and coding of artificial synthesized anti-Her2 (Herceptin) light chain of coding are anti- The nucleotide sequence (SEQ ID NO:8) of Her2 (Herceptin) heavy chain.Using the method for general molecular cloning, will encode The nucleotide sequence of anti-Her2 light chain is cloned on the above-mentioned plasmid pCDNA3.1 (+) being transformed, can cell (such as HEK293 cell) in express secretory antibody, resulting expression plasmid is named as pCDNA3.1-Her2-LC；Anti- Her2 weight will be encoded The nucleotide sequence of chain is cloned on plasmid pFUSE-hIgG1-Fc2 (InvivoGene), and resulting expression plasmid is named as pFUSE-Her2-HC-OB。

In order to make four chains of above-mentioned bispecific antibody form differentiable 4 band on PAGE gel, anti- Her2 heavy chain C-terminal adds 6His label, and the expression plasmid of building is named as pFUSE-Her2-HC-OB-6His.

(3) to the transformation of bispecific antibody

According to the inventors have disclosed patent application WO2017034770A1 in method to antibody sequence carry out ammonia Base acid point mutation.It is residual that 2 non-cysteine residues of amino acid pair shown in B, C, D or E in table 2 are sported into cysteine Base makes to form disulfide bond between mutational site between heavy chain CH1 and light chain CL.Meanwhile destroying original heavy chain hinge region and light The natural disulphide bonds of chain CL interchain, can be realized by the following method: by the 214th cysteine mutation in the region light chain CL For any non-cysteine or the 214th cysteine is deleted, and/or by the cysteine of heavy chain hinge region the 220th Sport the cysteine of any non-cysteine or deletion the 220th.It is being non-cysteine by cysteine mutation In the case of, preferably sported serine, alanine or glycine.By above-mentioned mutation method, present invention produces 4 kinds to show The anti-Her2 of example property × AntiCD3 McAb bispecific antibody, is respectively designated as MutE, MutB, MutC and MutD (referring to table 3).Wherein, MutE introduces C220S and V173C mutation on the basis of bispecific antibody WT in anti-Her2 sequence of heavy chain, and anti- C214S and Q160C mutation is introduced in Her2 sequence of light chain；It is prominent that MutB introduces C220S and F170C in anti-Her2 sequence of heavy chain Become, and introduces C214S and S162C mutation in anti-Her2 sequence of light chain；MutC introduces C220S in anti-Her2 sequence of heavy chain It is mutated with L128C, and introduces C214S and F118C mutation in anti-Her2 sequence of light chain；MutD is in anti-Her2 sequence of heavy chain Middle introducing C220S and F126C mutation, and C214S and Q124C mutation is introduced in anti-Her2 sequence of light chain.

The anti-Her2 of table 3 × AntiCD3 McAb bispecific antibody mutation combination I

To further increase light chain to the selectivity of heavy chain, it is prominent that E213R is introduced on a light chain of bispecific antibody Become, and introduce K218E mutation on corresponding heavy chain, obtains anti-Her2 shown in table 4 × AntiCD3 McAb bispecific antibody mutation group Close MutC+ER and MutC-ER.Wherein, MutC+ER is to introduce in AntiCD3 McAb (C31) sequence of heavy chain on the basis of MutC K218E mutation, and E213R mutation is introduced in AntiCD3 McAb light chain；MutC-ER is on the basis of MutC, in anti-Her2 heavy chain K218E mutation is introduced, and introduces E213R mutation in anti-Her2 light chain.In addition, MutC-Dec construct is also constructed, wherein 214th cysteine of anti-Her light chain is deleted into (Del-C214).

The anti-Her2 of table 4 × AntiCD3 McAb bispecific antibody mutation combination II

It is strong in order to be formed simultaneously 2 two sulphur of non-natural in bispecific antibody, construct anti-Her2 shown in table 5 × AntiCD3 McAb bispecific antibody mutation combination MutC-D+ER and MutC-D-DeC.Wherein, MutC-D+ER is in bispecific antibody WT On the basis of, C220S, L128C and F126C mutation are introduced in anti-Her2 sequence of heavy chain, are introduced in anti-Her sequence of light chain C214S, F118C and Q124C mutation, while K218E mutation is introduced in AntiCD3 McAb sequence of heavy chain, and in AntiCD3 McAb sequence of light chain Introduce E213R mutation.MutC-D-DeC is mutated to introduce C220S, L128C and F126C in anti-Her2 sequence of heavy chain, and The 214th cysteine is deleted in anti-Her2 sequence of light chain, and introduces F118C and Q124C mutation.

The anti-Her2 of table 5 × AntiCD3 McAb bispecific antibody mutation combination III

Embodiment 2: bispecific antibody expression and purification

(1) anti-Her2 × AntiCD3 McAb bispecific antibody transient expression

Using the big extraction reagent kit of endotoxin-free plasmid, (Endo-Free-Plasmid Maxi Kit (100) is purchased from OMEGA Company, cat. no D6926-04) carry out plasmid largely extract, operating procedure according to specification provided by kit into Row.It is 2.0~3.0 × 10 by HEK293 cell culture to cell density⁶Cell suspension is centrifuged 5min by a/mL, and revolving speed is 1000rpm abandons old culture supernatant, with fresh culture medium (OPM-291CD03Medium, purchased from Shanghai Ao Pumai biology Science and Technology Ltd., article No. 81070-001) cell is resuspended, make its density 1.0 × 10⁶/mL.Use institute's structure in embodiment 1 Mutation combination MutB, MutC listed by the expression vector the built i.e. expression vector of bispecific antibody WT and table 4,5 and 6, MutD, MutE, MutC-DeC, MutC+ER, MutC-ER, MutC-D+ER and MutC-D-DeC carry out cotransfection to cell respectively.It will turn Cell suspending liquid after dye is placed on 37 DEG C, 5%CO₂, 120rpm culture shaking table in be protected from light culture 4-5 days.

(2) anti-Her2 × AntiCD3 McAb bispecific antibody purifying

Expression supernatant is collected by centrifugation and with 0.22 μm of membrane filtration cell conditioned medium.It is affine using the albumin A balanced Chromatographic stuffing (MabSelectSuRe^TM, it is purchased from GE Healthcare company, article No. 17-5438-02) and it captures in supernatant Bispecific antibody albumen uses equilibration buffer (137mMNaCl, 2.7mMKCl, 10mM Na₂HPO₄, 1.8mM KH₂PO₄) It washes away (about 10 column volumes) after the albumen of non-specific binding.Then, using elution buffer (100mM glycine, pH 3.5) 5 column volumes are eluted, collect eluent, and adjust pH to neutrality with neutralization buffer (1M Tris-HCl, pH9.0).It will Sample after elution is analyzed by SDS-PAGE.

For bispecific antibody MutB, MutC, MutD and MutE, the purity through step destination protein after purification is shown in In Fig. 2.Wherein, control WT (1:1:1:1) refers to plasmid pFUSE-Her2-HC-OB-6His, pFUSE-C31-HC-OA, The product that pCDNA3.1-C31-LC, pCDNA3.1-Her2-LC are obtained with the ratio transformed cells of 1:1:1:1.In Fig. 2 B Shown in reproducibility SDS-PAGE result, it is found that the content of anti-Her2 heavy chain in WT (1:1:1:1) bispecific antibody is relatively low, therefore Control WT (2:1:1:1) is devised, wherein the plasmid pFUSE-Her2-HC-OB-6His and other three that anti-Her2 heavy chain will be encoded The transfection ratio setting of kind plasmid is 2:1:1:1.However, from Fig. 2 B as it can be seen that compareing the ratio of Her2 heavy chain in WT (2:1:1:1) Content is equally relatively low.The result illustrates that the proportional amount for compareing Her2 heavy chain in bispecific antibody WT is not due to Her2 heavy chain Caused by low expression, but as caused by the incorrect pairing of interchain of not modified 2 heavy chains and 2 light chains.And through changing Bispecific antibody MutB, MutC, MutD and the MutE made, close to 1:1, the ratio of two light chains also connects the ratio of two heavy chains Nearly 1:1.

Bispecific antibody MutC+ER, MutC-ER and MutC-DeC through protein A affinity chromatography after purification, destination protein Purity is as shown in figure 3, the ratio of two heavy chains is close to 1:1, and the ratios of two light chains is close to 1:1.

Further purifying has been carried out to Mut C eluent using AKTA pure 25L1 protein purification system.Wherein, will Elution samples loading is to cation exchange column (the prepacked column Resource balanced^TMS, 1mL, GE Healthcare, goods Number GE17-1178-01), the albumen of non-specific binding is washed away to ultraviolet using equilibration buffer A (50mM sodium phosphate, pH6.0) Absorption Line is gentle, then uses elution buffer B (50mM sodium phosphate, 1M NaCl, pH 6.0) from 5%-45% linear elution 30- 50 column volumes, collect eluting peak, and Fig. 4 A shows elution curve.Albumen size and purity, bispecific are analyzed with SDS-PAGE After purification through the step, the purity of destination protein is as shown in Figure 4 B and 4C by antibody MutC.

In further experiment, to be formed to bispecific antibody the transformation (being shown in Table 6) of 2 pairs of disulfide bond.It is double special Property antibody MutC-D+ER, MutC-D-DeC expressing and analyzing destination protein by after Protein A purification, passing through SDS-PAGE Purity.As a result see Fig. 5 A and 5B, wherein Fig. 5 A shows irreducibility SDS-PAGE's as a result, Fig. 5 B shows reproducibility SDS The result of PAGE.

Embodiment 3: the antigen binding detection of antibody

The bispecific antibody antigen binding energy to be formed is transformed in order to be verified disulfide bond and the charge of present disclosure Power, the bispecific antibody for detecting purifying respectively by ELISA detect the combination of antigen CD3 and antigen Her2.Specific steps It is as follows.

1) the antigen behaviour Her2 antigen (purchased from Acrobiosystems company) and people's CD3 antigen that experiment uses (are purchased from Sino Biological Inc. company)

2) it is coated with: with 1 × PBS by antigen diluent to 100ng/mL.The antigen diluted is added in ELISA Plate, every hole adds 200 μ L seal reacting hole with sealing plate film, and room temperature is coated with 1.5 hours or 4 DEG C and is coated with 16 hours.Use 0.05%PBST board-washing 5 It is secondary.

3) it closes: preparing 3%M-PBS with skimmed milk power and PBS Buffer.Add 300 μ L 3%M- to the every hole of ELISA Plate PBS seals reacting hole with sealing plate film, is incubated at room temperature 1 hour.It uses 0.05%PBST board-washing 5 times.

4) antibody combines: being added with 100 holes μ L/ according to the diluted antibody of certain gradient concentration, each antibody concentration is with 3 Multiple holes are tested.After adding antibody, reacting hole is sealed with sealing plate film, is incubated at room temperature 1.5 hours.Use 0.05%PBST board-washing 5 times.

5) it secondary antibody: (is purchased from 3%M-PBS solution according to 1:2000 dilution secondary antibody, that is, HRP label Goat anti-Human IgG Peprotech company, cat. no SA0001-17), the secondary antibody after dilution is added to hole with 50 holes μ L/.With sealing plate film Reacting hole is sealed, is incubated at room temperature 1 hour.It uses 0.05%PBST board-washing 5 times.

6) it develops the color: 100 hole μ L/ of TMB developing solution is added, room temperature is protected from light 10~20min of incubation.

7) it terminates: 100 hole μ L/ terminate liquid (2M HCl) is added, measures the OD value at 450nm after mixing immediately.

8) data are analyzed: carrying out data processing using 5 software of GraphPad Prism.It generates with log (sample concentration) and is Abscissa, OD value are the curve of ordinate, and obtain kd, R²Etc. data.

As shown in fig. 6, anti-Her2 × AntiCD3 McAb bispecific antibody MutC, MutC+ER, MutC-ER, MutC-D+ of purifying ER, MutC-D-DeC and MutC-DeC are shown with the strong combination activity to Her2 antigen.By calculating, anti-Her2 × anti- CD3 bispecific antibody is 0.8nM to the kd value of Her2 antigen binding.In addition, anti-Her2 × AntiCD3 McAb is double special as Fig. 7 is shown Property antibody MutC, MutC+ER, MutC-ER, MutC-D+ER, MutC-D-DeC and MutC-DeC are maintained to CD3 antigen binding Ability.The above results show that the bispecific antibody being transformed through method of the invention maintains the ability to antigen binding.

Embodiment 4: dual anti-original is in combination with detection

During antibody producing, if mismatching phenomenon, bispecific antibody occur for the heavy chain of bispecific antibody and light chain Lose the binding ability and biological function to 2 kinds of not synantigens.In order to which the remodeling method for further verifying of the invention solves The above problem, the bispecific for having used the dual anti-former ELISA method verifying of bispecific to assemble through remodeling method of the invention are anti- Body can be and double special with being assembled by not modified two native heavies and two natural light chains in combination with 2 kinds of antigens Heterogenetic antibody (WT) is compared.Theoretically, this not modified two native heavies and two natural light chains assemble to be formed Bispecific antibody only have about 25% its two heavy chains correctly to match with two light chains, therefore can be in combination with 2 kinds of antigens Antibody ratios be 25%.

This experiment is using the dual anti-former ELISA method of bispecific, by corresponding first Antigen adsorption in surface of solid phase carriers, Bispecific antibody to be tested is added, makes its antigen-reactive with surface of solid phase carriers.Then, it washs and is formed on solid phase carrier Antigen-antibody complex, add enzyme label the second antigen, formed first the-the second antigenic compound of Ag-Ab.So Afterwards, the reaction substrate of the enzyme is added, by enzymatic at color products.The amount of the product and two kinds of antigens can be specifically bound Bispecific antibody amount it is directly proportional.

Specific experiment step are as follows: be coated in antigens c D3 ELISA Plate (NUNC), overnight, degreasing is added in 4 DEG C of coatings after board-washing Milk is closed, and bispecific antibody and corresponding control antibodies (WT and hIgG) are added after board-washing, is incubated at room temperature 1.5 hours, The people Her2 antigen (HRP-Her2) of HRP label is added in board-washing, is incubated at room temperature 2 hours, board-washing, and luminous substrate is added, uses enzyme It marks instrument (Synergy HTX, BioTeck) and detects luminous value.As a result referring to Fig. 8.Wherein, Fig. 8 A shows bispecific antibody MutB, MutC and MutD are compared to control as a result, Fig. 8 B shows the result of MutE.The double antigen ELISAs of above-mentioned bispecific Binding assay shows that, compared to the WT not being transformed, bispecific antibody MutB, MutC, MutD, MutE are in combination with Her2 antigen About 2 times, 4 times, 3 times, 2 times have been respectively increased with the amount of CD3 antigen.The above results demonstrate through the present invention design and transformation MutB, MutC, MutD, MutE improve the correct pairing between bispecific antibody heavy chain and light chain.

Embodiment 5:T cell tumour killing detection (CTL)

The bispecific antibody to be formed is transformed in cellular level in order to further be verified disulfide bond and charge of the present invention Antigen binding capacity and killing activity to tumour, the lethal detection of CTL is carried out to the bispecific antibody of purifying.Specific step It is rapid as follows.

The lethal detection of CTL: SKBR-3 cell is a kind of highly expressed breast cancer cell line of Her2, and is made in this experiment For target cell.Using trypsin digestion cell SKBR-3 cell, single cell suspension is prepared.With without phenol red 5%FBS-1640 culture Cell density is adjusted to 0.20 × 10 by base⁶/ mL, and with the hole of 50 holes μ L/ addition, 96 orifice plates, so that final concentration of cells is 1.0 ×10⁴A/hole.20 times (E:T=20:1) are added in the effector cell PBMC of target cell number in this experiment, i.e., and 1.50 × 10⁵A/hole (1.50×10⁶/ mL, 100 holes μ L/).Antibody is diluted to 4 μ g/mL with without phenol red 1640 culture medium of 5%FBS-RPMI, so Doubling dilution is carried out in the ratio of 1:4 afterwards, obtaining concentration is respectively 4000ng/mL, 1000ng/mL, 2500ng/mL, 625ng/ The antibody of mL, 156.25ng/mL, 39.06ng/mL, 9.77ng/mL, 2.44ng/mL, 0.61ng/mL, 0.15ng/mL.According to Antibody is added into corresponding aperture with 50 holes μ L/ in experimental design.In 37 DEG C after cell and antibody are mixed, 5%CO2 incubator is trained It supports, with killing of lactic dehydrogenase cytotoxic reagent box (the being purchased from Beyotime company) detection to cell, reflection after about 20h The killing activity of bispecific antibody.Killing rate is calculated according to following formula:

Killing rate (%)=(OD_Sample-S_{It is spontaneous})/(Max-S_{It is spontaneous}) × 100%

Wherein, S_{It is spontaneous}=OD_{Spontaneous relief hole (target cell+effector cell)}, Max=OD_{Maximum relief hole (target cell)}；

As illustrated in figures 9a and 9b, the bispecific without two native heavies of transformation and two natural light chain assemblings is anti- Body WT (1:1:1:1) and WT (2:1:1:1) because containing large scale heavy chain-light chain mispairing (theoretically only have 25% it is double special Property heavy chain of antibody and light chain correctly match) so that the ratio of the bispecific antibody with normal bioactivity correctly matched Example is lower in total protein, and the EC50 concentration of tumour cell SKBR-3 killing highly expressed to Her2 is respectively 21.16ng/ml And 41.65ng/ml.As a comparison, the anti-Her2 of purifying × AntiCD3 McAb bispecific antibody MutB, MutC, MutD and MutE couple The highly expressed tumour cell SKBR3 fragmentation effect of Her2 is significant, EC50 concentration be respectively 0.95ng/ml, 1.28ng/ml, 0.71ng/ml and 0.13ng/ml.

In addition, Figure 10 shows bispecific antibody MutC, MutC+ER, MutC-ER, MutC-DeC, MutC-D of purifying + ER and MutC-D-DeC is significant to the fragmentation effect of SKBR3 cell, and EC50 concentration is respectively 0.045ng/ml, 0.055ng/ Ml, 0.034ng/ml, 0.078ng/ml, 0.071ng/ml, 0.063ng/ml.

These statistics indicate that: disulfide bond through the invention and the bispecific antibody of charge remodeling method assembling are in vitro In cytotoxicity experiment, the CD3 antigen on immunocyte surface can be identified and be combined, while identifying and combining tumour cell The Her2 antigen on the surface SKBR3, and activate T cell killing tumor cell SKBR3.That is transformed by means of the present invention is double special Property antibody maintains antigen-binding activity in cellular level, and the cytotoxicity of enhancing is shown compared to not modified antibody Active (CTL effect).

Sequence table

<110>Guangzhou Ai Simai biological medicine Science and Technology Ltd.

<120>antibody and antibody remodeling method

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<170> PatentIn version 3.5

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Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val Thr Thr Ser

20 25 30

Asn Tyr Ala Asn Trp Val Gln Glu Lys Pro Gly Gln Ala Pro Arg Gly

35 40 45

Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Trp Thr Pro Ala Arg Phe

50 55 60

Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Leu Trp Tyr Ser Asn

85 90 95

Leu Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Lys Leu

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Val Thr Met Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile

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Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys

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Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu

165 170 175

Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu

180 185 190

Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Thr Cys Glu Val Thr

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His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu

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ggcgggggaa caaaactgac tgtgctggga aagcttgtca ccatggaaat caaacgtacg 360

gtggctgcac catctgtctt catcttcccg ccatctgatg agcagttgaa atctggaact 420

gcctctgttg tgtgcctgct gaataacttc tatcccagag aggccaaagt acagtggaag 480

gtggataacg ccctccaatc gggtaactcc caggagagtg tcacagagca ggacagcaag 540

gacagcacct acagcctcag cagcaccctg acgctgagca aagcagacta cgagaaacac 600

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Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr

20 25 30

Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp

50 55 60

Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser

65 70 75 80

Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr

85 90 95

Tyr Cys Ala Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe

100 105 110

Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ile Ser Ser

115 120 125

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

130 135 140

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

145 150 155 160

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

165 170 175

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

180 185 190

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

195 200 205

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

210 215 220

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

225 230 235 240

Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro

245 250 255

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

260 265 270

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

275 280 285

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

290 295 300

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

305 310 315 320

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

325 330 335

Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly

340 345 350

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu

355 360 365

Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

370 375 380

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

385 390 395 400

Asn Tyr Lys Thr Thr Lys Lys Lys Leu Asp Ser Asp Gly Ser Phe Phe

405 410 415

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

420 425 430

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

435 440 445

Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

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cacggcaact tcggcaattc ttacgtgagc tggtttgcct attggggcca gggcacactg 360

gtgaccgtga gctccatctc gagtgctagc accaagggcc catcggtctt ccccctggca 420

ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 480

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ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 600

tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc 660

aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacatg cccaccgtgc 720

ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 780

accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 840

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 900

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 960

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1020

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1080

accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1140

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1200

aactacaaga ccacgaagaa gaagctggac tccgacggct ccttcttcct ctacagcaag 1260

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcat 1320

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaa 1374

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<400> 5

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Val Thr Met Glu Ile Lys Arg Thr

100 105 110

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

115 120 125

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

130 135 140

Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly

145 150 155 160

Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr

165 170 175

Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His

180 185 190

Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val

195 200 205

Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 6

<211> 651

<212> DNA

<213>artificial sequence

<400> 6

gacatccaga tgacacagtc ccctagcagc ctgagcgcta gcgtgggcga cagggtcaca 60

atcacctgca gggccagcca ggatgtgaac accgccgtgg cctggtacca acagaagccc 120

ggcaaggccc ccaaactgct gatctacagc gccagcttcc tgtactccgg cgtgccctcc 180

agattcagcg gcagcaggag cggcaccgac ttcaccctga ccatcagcag cctgcagccc 240

gaggacttcg ccacctacta ttgccagcag cactacacaa cccctcccac cttcggccag 300

ggcaccaagc ttgtcaccat ggaaatcaaa cgtacggtgg ctgcaccatc tgtcttcatc 360

ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420

aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 480

aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 540

accctgacgc tgagcaaagc agactacgag aaacacaaag tctacacctg cgaagtcacc 600

catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg t 651

<210> 7

<211> 453

<212> PRT

<213>artificial sequence

<220>

<221> PEPTIDE

<222> (1)..(453)

<223>anti-Her2 heavy chain amino acid sequence

<400> 7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ile Ser Ser Ala Ser Thr Lys Gly

115 120 125

Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly

130 135 140

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

145 150 155 160

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

165 170 175

Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

180 185 190

Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val

195 200 205

Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys

210 215 220

Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu

225 230 235 240

Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

245 250 255

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

260 265 270

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

275 280 285

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

290 295 300

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

305 310 315 320

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

325 330 335

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

340 345 350

Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln

355 360 365

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

370 375 380

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

385 390 395 400

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

405 410 415

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

420 425 430

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

435 440 445

Leu Ser Pro Gly Lys

450

<210> 8

<211> 1359

<212> DNA

<213>artificial sequence

<400> 8

gaggttcagc tggtggaatc cggaggcgga ctggtgcagc ctggaggaag cctgagactg 60

agctgcgccg ccagcggctt caacatcaag gacacctata tccattgggt gaggcaggct 120

cccggaaaag gcctggagtg ggtggccagg atctacccta ccaacggcta caccaggtac 180

gccgacagcg tgaagggcag gttcaccatc agcgccgaca ccagcaagaa caccgcctac 240

ctgcagatga acagcctcag agccgaggac accgccgtgt attactgcag cagatggggc 300

ggcgacggct tctacgctat ggattactgg ggacagggca cactggtgac cgtgagctcc 360

atctcgagtg ctagcaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 420

acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 480

acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 540

cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 600

acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 660

gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 720

ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 780

cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 840

ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 900

cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 960

aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1020

accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1080

cgggaggaga tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1140

agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1200

cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1260

agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1320

cactacacgc agaagagcct ctccctgtct ccgggtaaa 1359

<210> 9

<211> 20

<212> PRT

<213>artificial sequence

<220>

<221> SIGNAL

<222> (1)..(20)

<223>human interleukin-2 signal peptide amino acid sequence

<400> 9

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser

20

<210> 10

<211> 60

<212> DNA

<213>artificial sequence

<220>

<221> sig_peptide

<222> (1)..(60)

<223>human interleukin-2 signal peptide nucleic acid sequence

<400> 10

atgtacagga tgcaactcct gtcttgcatt gcactaagtc ttgcacttgt cacgaattcg 60

<210> 11

<211> 107

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (11)..(11)

<223>MutC F118C light chain CL sequence

<400> 11

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Cys Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 12

<211> 321

<212> DNA

<213>artificial sequence

<400> 12

cgtacggtgg ctgcaccatc tgtcttcatc tgcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacacctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg t 321

<210> 13

<211> 107

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (17)..(17)

<223>MutD Q124C light chain CL sequence

<400> 13

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Cys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 14

<211> 321

<212> DNA

<213>artificial sequence

<400> 14

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagtg cttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacacctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg t 321

<210> 15

<211> 107

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (53)..(53)

<223>MutE Q160C light chain CL sequence

<400> 15

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Cys Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 16

<211> 321

<212> DNA

<213>artificial sequence

<400> 16

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcctgtg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacacctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg t 321

<210> 17

<211> 107

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (55)..(55)

<223>MutB S162C light chain CL sequence

<400> 17

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Cys Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 18

<211> 321

<212> DNA

<213>artificial sequence

<400> 18

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agtgtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacacctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg t 321

<210> 19

<211> 107

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (106)..(106)

<223>E213R light chain CL sequence

<400> 19

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Arg Cys

100 105

<210> 20

<211> 321

<212> DNA

<213>artificial sequence

<400> 20

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacacctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggaaggtg t 321

<210> 21

<211> 107

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (107)..(107)

<223>C214S light chain CL sequence

<400> 21

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser

100 105

<210> 22

<211> 321

<212> DNA

<213>artificial sequence

<400> 22

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacacctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtc t 321

<210> 23

<211> 106

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (1)..(106)

<223>C214 site cysteine deletion mutation light chain CL sequence

<400> 23

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu

100 105

<210> 24

<211> 318

<212> DNA

<213>artificial sequence

<400> 24

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacacctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagag 318

<210> 25

<211> 107

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (106)..(107)

<223>E213R-C214S light chain CL sequence

<400> 25

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

1 5 10 15

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Arg Ser

100 105

<210> 26

<211> 321

<212> DNA

<213>artificial sequence

<400> 26

cgtacggtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacacctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggaaggtc t 321

<210> 27

<211> 107

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (11)..(17)

<223>the light chain CL sequence of MutC-D F118C-Q124C

<400> 27

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Cys Pro Pro Ser Asp Glu

1 5 10 15

Cys Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

20 25 30

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

35 40 45

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

50 55 60

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

65 70 75 80

Lys His Lys Val Tyr Thr Cys Glu Val Thr His Gln Gly Leu Ser Ser

85 90 95

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

100 105

<210> 28

<211> 321

<212> DNA

<213>artificial sequence

<400> 28

cgtacggtgg ctgcaccatc tgtcttcatc tgcccgccat ctgatgagtg cttgaaatct 60

ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120

tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180

agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240

aaacacaaag tctacacctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300

agcttcaaca ggggagagtg t 321

<210> 29

<211> 98

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (9)..(9)

<223>MutD F126C heavy chain CH1 sequence

<400> 29

Ala Ser Thr Lys Gly Pro Ser Val Cys Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val

<210> 30

<211> 294

<212> DNA

<213>artificial sequence

<400> 30

gctagcacca agggcccatc ggtctgcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294

<210> 31

<211> 98

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (11)..(11)

<223>MutC L128C heavy chain CH1 sequence

<400> 31

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Cys Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val

<210> 32

<211> 294

<212> DNA

<213>artificial sequence

<400> 32

gctagcacca agggcccatc ggtcttcccc tgcgcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294

<210> 33

<211> 98

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (53)..(53)

<223>MutB F170C heavy chain CH1 sequence

<400> 33

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Cys Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val

<210> 34

<211> 294

<212> DNA

<213>artificial sequence

<400> 34

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacacctgcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294

<210> 35

<211> 98

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (56)..(56)

<223>MutE V173C heavy chain CH1 sequence

<400> 35

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Cys Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val

<210> 36

<211> 294

<212> DNA

<213>artificial sequence

<400> 36

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggcttgcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294

<210> 37

<211> 113

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (101)..(101)

<223>K218E heavy chain CH1 sequence

<400> 37

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Glu Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro

<210> 38

<211> 339

<212> DNA

<213>artificial sequence

<400> 38

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

gaatcttgtg acaaaactca cacatgccca ccgtgccca 339

<210> 39

<211> 113

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (103)..(103)

<223>C220S heavy chain CH1 sequence

<400> 39

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro

<210> 40

<211> 339

<212> DNA

<213>artificial sequence

<400> 40

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

aaatctagcg acaaaactca cacatgccca ccgtgccca 339

<210> 41

<211> 113

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (101)..(103)

<223>K218E-C220S heavy chain CH1 sequence

<400> 41

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Glu Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro

<210> 42

<211> 339

<212> DNA

<213>artificial sequence

<400> 42

gctagcacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300

gaatctagcg acaaaactca cacatgccca ccgtgccca 339

<210> 43

<211> 98

<212> PRT

<213>artificial sequence

<220>

<221> MUTAGEN

<222> (9)..(11)

<223>MutC-D F126C-L128C heavy chain CH1 sequence

<400> 43

Ala Ser Thr Lys Gly Pro Ser Val Cys Pro Cys Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val

<210> 44

<211> 294

<212> DNA

<213>artificial sequence

<400> 44

gctagcacca agggcccatc ggtctgcccc tgcgcaccct cctccaagag cacctctggg 60

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agtt 294

Claims

1. the method that the area Fab of pair antibody is transformed, the method includes introducing one or more mutation selected from the following The step of area Fab:

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL, wherein being numbered according to EU to described Heavy chain of antibody and light chain are numbered.

2. according to the method described in claim 1, be wherein introduced into a)-e to the area Fab) in 1,2,3,4 or 5 ?.

3. according to the method described in claim 1, be wherein introduced into a)-d to the area Fab) in 1 or 2, and it is optional e)。

4. method according to any one of claim 1-3, wherein the method also includes the C220 in hinge area dashes forward Become other amino acid in addition to cysteine or missing C220, and/or by the C214 in CL sport except cysteine with Outer other amino acid or missing C214.

5. method according to claim 4, wherein other amino acid in addition to cysteine are selected from serine, alanine And glycine.

6. method according to any one of claims 1-5, wherein the area Fab combines the anti-of antigen selected from the following Body: CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32a, CD32b, CD33 (p67 albumen), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3, IL-1 α, IL-1 β, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-12, IL-13, IL-15, IL-17, IL-17R, IL-18, IL-23, interferon-' alpha ', interferon beta, interferon gamma, TNF-α, TNF β, TNF-R1, TNF-RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, Receptor -1 VEG1, OX40L, TRAIL, adenosine receptor, lymphotoxin-beta-receptor, TACI, BAFF-R, EPO；LFA-3, ICAM-1, ICAM-3, EpCAM, integrin β 1, integrin β 2, integrin alpha-4/β 7, beta 2 integrin alpha 2, beta 2 integrin alpha 3, integrin egg White α 4, beta 2 integrin alpha 5, beta 2 integrin alpha 6, beta 2 integrin alpha v, beta 2 integrin alpha V β 3, FGFR-3, keratinocyte growth because Son, VLA-1, VLA-4, L-selectin, anti-Id, E-Selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGF β 1, TGF β 2, eosinophil chemokine 1 (eotaxin1), Blys (bone-marrow-derived lymphocyte stimulation because Son), complement C5, IgE, factor Ⅴ II, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, endothelin receptor, VLA-4, haptens NP-cap or NIP-cap, E- choosing Select element, digoxin, P-ALP (PLAP) and testis PLAP sample alkaline phosphatase, TfR, carcinomebryonic antigen (CEA), CEACAM5, HMFG1, PEM, Mucin1, MUC18, Heparinase I, human heart myosin, tumour associated sugars egg White -72 (TAG-72), tumor associated antigen CA 125, prostate-specific membrane antigen (PSM-A), high molecular weight melanoma phase It closes antigen (HMW-MAA), cancer (carcinoma) related antigen, Gco protein I ib/IIIa (GPIIb/IIIa), expresses Lewis Y The tumor associated antigen of related carbohydrate, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV are exhaled It is attracted cellular virus RSV F, RSVF Fgp, cytokeratin tumor associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 ring, Respiratory Syncytial Virus(RSV) (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and C.perfringens (Clostridium perfringens) toxin, CD133, CD138, OX40, GITR, PD-1, PD-L1, PD-L2, CTLA-4, KIR, LAG-3, TCR α, TCR β, TCR γ, TCR δ, VEGF, EGF, VEGFR, EGFR, EpCAM, mesothelin, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, fucosido-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM- CSFR, ADAMS, flesh generate inhibin, PCSK9, CXCR4, MIF, PEG2.

7. according to the method described in claim 6, wherein the antigen is CD3 or HER2.

8. method according to any one of claims 1-7, wherein the antibody is IgG, IgA, IgM, IgE or IgD same Kind type.

9. method as claimed in one of claims 1-8, wherein the antibody is IgG1, IgG2, IgG3 or IgG4 isotype.

10. producing the method with the antibody or antibody fragment in at least two different areas Fab, the method includes following steps It is rapid:

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL, wherein being compiled according to EU Number,

2) under conditions of expressing the antibody or antibody fragment, nucleic acid of the culture containing encoding said antibody or antibody fragment Host cell, and

3) antibody or antibody fragment are recycled from the host cell cultures.

11. method as claimed in claim 10, wherein the method also includes the 2nd Fab in the antibody or antibody fragment Area introduces one or more mutation selected from the following:

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL, wherein introducing the first Fab The mutation in area and the mutation for introducing the 2nd area Fab are not exactly the same.

12. method as described in claim 10 or 11, wherein being introduced into mutation a)-e in the first area Fab) in 1,2 , 3,4 or 5, is introduced into the 2nd area Fab and is mutated a)-e) in 1,2,3,4 or 5, and wherein The mutation of the mutation and introducing the 2nd area Fab that introduce the first area Fab is not exactly the same.

13. the method as described in any one of claim 10-12, wherein the method also includes by the first area Fab C220 in hinge area sports other amino acid in addition to cysteine or missing C220, and/or by the first area Fab CL in C214 sport other amino acid in addition to cysteine or missing C214.

14. the method as described in any one of claim 10-13, wherein the method also includes by the 2nd area Fab C220 in hinge area sports other amino acid in addition to cysteine or missing C220, and/or by the 2nd area Fab CL in C214 sport other amino acid in addition to cysteine or missing C214.

15. 3 or 14 method according to claim 1, wherein other amino acid in addition to cysteine be selected from serine, Alanine and glycine.

16. method described in any one of 0-15 according to claim 1, wherein the area first Fab and the 2nd area Fab combine not Two different epitopes of same antigen or same antigen.

17. according to the method for claim 16, wherein the antigen is selected from the group: CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32a, CD32b, CD33 (p67 albumen), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3, IL-1 α, IL-1 β, IL- 1R, IL-2, IL-2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-12, IL-13, IL-15, IL-17, IL-17R, IL-18, IL-23, interferon-' alpha ', interferon beta, interferon gamma, TNF-α, TNF β, TNF-R1, TNF-RII, FasL, Receptor -1 CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, TRAIL, Adenosine receptor, lymphotoxin-beta-receptor, TACI, BAFF-R, EPO；LFA-3, ICAM-1, ICAM-3, EpCAM, integrin β 1, Integrin β 2, integrin alpha-4/β 7, beta 2 integrin alpha 2, beta 2 integrin alpha 3, integrin alpha-4, beta 2 integrin alpha 5, integrin α 6, beta 2 integrin alpha v, beta 2 integrin alpha V β 3, FGFR-3, keratinocyte growth factor, VLA-1, VLA-4, L-selectin, Anti- Id, E-Selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGF β 1, TGF β 2, Eosinophil chemokine 1 (eotaxin1), Blys (bone-marrow-derived lymphocyte stimulating factor), complement C5, IgE, factor Ⅴ II, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), Tissue factor, endothelin receptor, VLA-4, haptens NP-cap or NIP-cap, E-Selectin, digoxin, human placental alkaline phosphoric acid Enzyme (PLAP) and testis PLAP sample alkaline phosphatase, TfR, carcinomebryonic antigen (CEA), CEACAM5, HMFG1, PEM, Mucin1, MUC18, Heparinase I, human heart myosin, tumor-associated glycoprotein -72 (TAG-72), tumour correlation are anti- Former CA 125, prostate-specific membrane antigen (PSM-A), high molecular weight melanoma related antigen (HMW-MAA), cancer (carcinoma) related antigen, Gco protein I ib/IIIa (GPIIb/IIIa), expression Lewis Y related carbohydrate swell Tumor related antigen, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV breathe syncytial virus RSV F, RSVF Fgp, cytokeratin tumor associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 ring, Respiratory Syncytial Virus(RSV) (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB coating sugar egg White and C.perfringens (Clostridium perfringens) toxin, CD133, CD138, OX40, GITR, PD-1, PD- L1, PD-L2, CTLA-4, KIR, LAG-3, TCR α, TCR β, TCR γ, TCR δ, VEGF, EGF, VEGFR, EGFR, EpCAM, mesothelium Element, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, Fucosido-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM-CSFR, ADAMS, flesh generate inhibin, PCSK9, CXCR4, MIF, PEG2.

18. according to the method for claim 17, wherein first antigen and the second antigen are selected from CD3 and HER2.

19. method described in any one of 0-18 according to claim 1, wherein the antibody fragment is selected from Fab segment, Fab' piece Section and F (ab')₂Segment.

20. method described in any one of 0-18 according to claim 1, wherein the antibody is light with the first heavy chain and first The bispecific antibody of chain and the second heavy chain and the second light chain, wherein first heavy chain and the first light chain form described One area Fab, second heavy chain and the second light chain form the 2nd area Fab.

21. according to the method for claim 20, wherein the method also includes to first heavy chain introduce P395K, P396K and V397K, and T394D, P395D and P396D are introduced to second heavy chain, or introduce to first heavy chain T394D, P395D and P396D, and P395K, P396K and V397K are introduced to second heavy chain.

22. method described in any one of 0-21 according to claim 1, wherein the antibody or antibody fragment from IgG, IgA, IgM, IgE or IgD.

23. method described in any one of 0-22 according to claim 1, wherein the antibody or antibody fragment from IgG1, IgG2, IgG3 or IgG4.

24. antibody or antibody fragment that method described in any one of 0-23 generates according to claim 1.

25. antibody or antibody fragment at least two different areas Fab, the first area Fab of the antibody or antibody fragment With one or more mutation selected from the following:

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL, wherein being compiled according to EU Number.

26. antibody according to claim 25 or antibody fragment, wherein the 2nd area Fab of the antibody or antibody fragment has There are one or more mutation selected from the following:

A) S162C in the F170C and CL in CH1；

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；

D) Q160C in the V173C and CL in CH1；With

E) K218E in hinge area or K218D mutation and E213R or E213K mutation in CL, wherein the area second Fab Mutation and the mutation in the first area Fab are not exactly the same.

27. antibody or antibody fragment as described in claim 25 or 26, wherein the area first Fab has mutation a)-e) in 1,2,3,4 or 5, the 2nd area Fab has mutation a)-e) in 1,2,3,4 or 5, and Wherein the mutation in the area first Fab and the mutation in the 2nd area Fab are not exactly the same.

28. antibody or antibody fragment as described in any one of claim 25-27, wherein the area first Fab also has hinge The C214 that C220 in sequence is sported in other amino acid or missing C220 and/or CL in addition to cysteine is sported Other amino acid or missing C214 in addition to cysteine.

29. antibody or antibody fragment as described in any one of claim 25-28, wherein the area second Fab also has hinge The C214 that C220 in sequence is sported in other amino acid or missing C220 and/or CL in addition to cysteine is sported Other amino acid or missing C214 in addition to cysteine.

30. the antibody according to claim 28 or 29 or antibody fragment, wherein other ammonia in addition to cysteine Base acid is selected from serine, alanine and glycine.

31. antibody or antibody fragment according to any one of claim 25-30, wherein the area first Fab and second The area Fab combines two different epitopes of different antigen or same antigen.

32. antibody according to claim 31 or antibody fragment, wherein the antigen is selected from the group: CD2, CD3, CD3E, CD4, CD11, CD11a, CD14, CD16, CD18, CD19, CD20, CD22, CD23, CD25, CD28, CD29, CD30, CD32a, CD32b, CD33 (p67 albumen), CD38, CD40, CD40L, CD52, CD54, CD56, CD64, CD80, CD147, GD3, IL-1 α, IL-1 β, IL-1R, IL-2, IL-2R, IL-4, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-12, IL-13, IL- 15, IL-17, IL-17R, IL-18, IL-23, interferon-' alpha ', interferon beta, interferon gamma, TNF-α, TNF β, TNF-R1, TNF- RII, FasL, CD27L, CD30L, 4-1BBL, TRAIL, RANKL, TWEAK, APRIL, BAFF, LIGHT, VEG1, OX40L, Receptor -1 TRAIL, adenosine receptor, lymphotoxin-beta-receptor, TACI, BAFF-R, EPO；LFA-3, ICAM-1, ICAM-3, EpCAM, Integrin β 1, integrin β 2, integrin alpha-4/β 7, beta 2 integrin alpha 2, beta 2 integrin alpha 3, integrin alpha-4, integrin α 5, beta 2 integrin alpha 6, beta 2 integrin alpha v, beta 2 integrin alpha V β 3, FGFR-3, keratinocyte growth factor, VLA-1, VLA- 4, L-selectin, anti-Id, E-Selectin, HLA, HLA-DR, CTLA-4, T cell receptor, B7-1, B7-2, VNR integrin, TGF β 1, TGF β 2, eosinophil chemokine 1 (eotaxin1), Blys (bone-marrow-derived lymphocyte stimulating factor), complement C5, IgE, factor Ⅴ II, CD64, CBL, NCA 90, EGFR (ErbB-1), Her1, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB4), tissue factor, endothelin receptor, VLA-4, haptens NP-cap or NIP-cap, E-Selectin, digoxin, P-ALP (PLAP) and testis PLAP sample alkaline phosphatase, TfR, carcinomebryonic antigen (CEA), CEACAM5, HMFG1, PEM, Mucin1, MUC18, Heparinase I, human heart myosin, tumor-associated glycoprotein -72 (TAG-72), tumor associated antigen CA 125, prostate-specific membrane antigen (PSM-A), high molecular weight melanoma correlation are anti- Former (HMW-MAA), cancer (carcinoma) related antigen, Gco protein I ib/IIIa (GPIIb/IIIa), expression Lewis Y are related The tumor associated antigen of carbohydrate, human cytomegalovirus (HCMV) gH envelope glycoprotein, HIV gp120, HCMV, breathing are closed Cellular virus RSV F, RSVF Fgp, cytokeratin tumor associated antigen, Hep B gp120, CMV, gpIIbIIIa, HIV IIIB gp120V3 ring, Respiratory Syncytial Virus(RSV) (RSV) Fgp, herpes simplex virus (HSV) gD glycoprotein, HSV gB glycoprotein, HCMV gB envelope glycoprotein and C.perfringens (Clostridium perfringens) toxin, CD133, CD138, OX40, GITR, PD-1, PD-L1, PD-L2, CTLA-4, KIR, LAG-3, TCR α, TCR β, TCR γ, TCR δ, VEGF, EGF, VEGFR, EGFR, EpCAM, mesothelin, Glypicans, Erbl, Erb2, B7-H3, ICOS, BMP1, BMP2, BMP3B, BMP4, CSF1, GM-CSF, FGF1, FGF2, FGF3, FGF4, PDGFR, TIGIT, CS1, TWEAK, CCL1, CCL2, CCL3, CCL13, CXCL1, CXCL2, CXCL3, IP-10, fucosido-GM1, IGF1, IGF2, IGF1R, IGF2R, RANK ligand, DLL-4, GM- CSFR, ADAMS, flesh generate inhibin, PCSK9, CXCR4, MIF, PEG2.

33. antibody according to claim 29 or antibody fragment, wherein the antigen is selected from CD3 and HER2.

34. antibody or antibody fragment according to any one of claim 25-33, wherein the antibody fragment is selected from Fab Segment, Fab' segment and F (ab')₂Segment.

35. antibody or antibody fragment according to any one of claim 25-33, wherein the antibody is that have the first weight The bispecific antibody of chain and the first light chain and the second heavy chain and the second light chain, wherein first heavy chain and the first light chain The first area Fab is formed, second heavy chain and the second light chain form the 2nd area Fab.

36. antibody according to claim 35 or antibody fragment, wherein first heavy chain have P395K, P396K and V397K mutation, and second heavy chain have T394D, P395D and P396D be mutated or first heavy chain have T394D, P395D and P396D and mutation, and second heavy chain is mutated with P395K, P396K and V397K.

37. antibody or antibody fragment according to any one of claim 25-36, wherein the antibody or antibody fragment come Derived from IgG, IgA, IgM, IgE or IgD.

38. antibody or antibody fragment according to any one of claim 25-37, wherein the antibody or antibody fragment come Derived from IgG1, IgG2, IgG3 or IgG4.

39. the method that pair bispecific antibody is transformed, wherein the bispecific antibody has the first heavy chain in conjunction with CD3 There is the amino acid of SEQ ID NO:7 with the first light chain, and the second heavy chain and the second light chain of combination HER2, second heavy chain Sequence, and second light chain has the amino acid sequence of SEQ ID NO:5, the method includes to second heavy chain and the Two light chains introduce 1 or 2 mutation selected from following a)-d):

B) F118C in the L128C and CL in CH1；

C) Q124C in the F126C and CL in CH1；With

D) Q160C in the V173C and CL in CH1,

40. further including according to the method for claim 39, sporting the C220 in the hinge area of second heavy chain Other amino acid or missing C220 in addition to cysteine, and/or the C214 in the CL of second light chain is sported and is removed Other amino acid or missing C214 other than cysteine.

41. the method according to claim 38 or 39 further includes introducing K218E or K218D to first heavy chain to dash forward Become, and introduces E213R or E213K mutation to first light chain, or introduce K218E or K218D to all second heavy chains and dash forward Become, and introduces E213R or E213K mutation to second light chain.

42. the method according to any one of claim 39-41, wherein first heavy chain is with SEQ ID NO:3's Amino acid sequence, first light chain have the amino acid sequence of SEQ ID NO:1.

43. the bispecific antibody that the method according to any one of claim 39-42 generates.

44. antibody or antibody fragment, with the area CL selected from the group below: SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25 and SEQ ID NO:27。

45. antibody or antibody fragment, with the area CH1 selected from the group below or hinge area: SEQ ID NO:29, SEQ ID NO: 31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 and SEQ ID NO:43。

46. antibody or antibody fragment, with the area CL selected from the group below and the area CH1:

A) area CH1 in the area CL of SEQ ID NO:11 and SEQ ID NO:33；

B) area CH1 in the area CL of SEQ ID NO:13 and SEQ ID NO:29；

C) area CH1 in the area CL of SEQ ID NO:15 and SEQ ID NO:35；

D) area CH1 in the area CL of SEQ ID NO:17 and SEQ ID NO:33；With

E) area CH1 in the area CL of SEQ ID NO:27 and SEQ ID NO:43.

47. antibody coupling matter, it includes the antibody or antibody fragment or root according to any one of claim 24-38 and 44-46 According to the bispecific antibody described in claim 43, and be coupled with the antibody or antibody fragment or bispecific antibody Part, wherein the part is selected from cytotoxin, radioactive isotope, fluorescent marker, shiner, substance that show color or enzyme.

48. pharmaceutical composition, described pharmaceutical composition include according to the antibody of any one of claim 24-38 and 44-46 or Antibody fragment, bispecific antibody according to claim 43 or antibody coupling matter according to claim 47, with And one or more pharmaceutically acceptable carriers, surfactant and/or diluent.

49. according to claim 43 according to the antibody or antibody fragment of any one of claim 24-38 and 44-46 Bispecific antibody or antibody coupling matter according to claim 47 are in preparing the pharmaceutical composition for treating disease Purposes.

50. purposes according to claim 49, wherein the disease is cancer.

51. the method for treating disease, including subject in need is applied according to any in claim 24-38 and 44-46 The antibody or antibody fragment of item, bispecific antibody according to claim 43, antibody according to claim 47 Conjugate or pharmaceutical composition according to claim 48.

52. method according to claim 51, wherein the disease is cancer.

53. nucleic acid molecules, it includes nucleotide sequences selected from the group below: SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24、SEQ ID NO:26、SEQ ID NO:28、SEQ ID NO:30、SEQ ID NO:32、SEQ ID NO:34、SEQ ID NO:36、SEQ ID NO:38、SEQ ID NO:40, SEQ ID NO:42 and SEQ ID NO:44.

54. carrier, the carrier includes nucleic acid molecules according to claim 53.

55. host cell, the host cell includes nucleic acid molecules or claim 54 described in claims require 53 The carrier.