CN101928345B - Human source antibody and humanization remolding method thereof - Google Patents

Human source antibody and humanization remolding method thereof Download PDF

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CN101928345B
CN101928345B CN2010102115548A CN201010211554A CN101928345B CN 101928345 B CN101928345 B CN 101928345B CN 2010102115548 A CN2010102115548 A CN 2010102115548A CN 201010211554 A CN201010211554 A CN 201010211554A CN 101928345 B CN101928345 B CN 101928345B
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CN101928345A (en
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肖卫华
常亮
郭雨刚
胡思怡
刘兢
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University of Science and Technology of China USTC
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
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    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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Abstract

The invention discloses a human source antibody and a humanization remolding method thereof. The antibody is one of the following proteins: a) protein consisting of amino acid sequences from 1st to 253rd locus from the N end in a sequence 3 in a sequence table; and b) protein consisting of amino acid sequences shown in the sequence 3 in the sequence table. When the remolded antibody structure is compared with the initial chimeric antibody, the affinity of the remolded antibody with antigen is approximate, and the remolded antibody reaches complete humanization, and has better clinical application prospect.

Description

A kind of humanized antibody and humanization modified method thereof
Technical field
The present invention relates to a kind of humanized antibody and humanization modified method thereof.
Background technology
P185 is by the important receptor tyrosine kinase of a kind of cell surface of oncogene Her2/erbB-2 coding, belongs to Epidermal Growth Factor Receptor Family.P185/Her2 acceptor and other members of this family are similar; By extracellular region; Striding three structural domains of film district and intracellular region forms; Wherein extracellular region (ECD) is divided into four subprovinces again, and I wherein, III subprovince are that leucine is rich in the district, and II, IV subprovince are that halfcystine is rich in district (Carpenter G. (1987) Receptors of epidermal growth factor and other polypeptide mitogens.Annu.Rev.Biochem.56:881-914).The epidermal growth factor family acceptor is distributed in multiple tissue widely, especially in epithelium, stroma and neural organizing, express, propagation, the differentiation of adjusting cell, grow, stick and move in play an important role.P185/Her2 is as most important a member in the signal transduction network of Epidermal Growth Factor Receptor Family; Unusual and the cell signalling disorder of its expression level, the generation and the malignant development of tumour (the Nancy EH that is closely related; Heidi A, Lane N. (2005) ErbB receptors and cancer:the complexity of targeted inhibitors Nat.Rev.Cancer 5:341-354).
In recent years P185/Her2 always as the focus in the oncotherapy field by broad research.1985; Greene M.I. study group finds that the monoclonal antibody of anti-P185/Her2 extracellular region can make the NIH3T3 phenotype of the Her2 high expression level that has transformed reverse, and makes the phenotype of tumour cell be tending towards normalizing; Simultaneously can reduce Her2 receptor expression level (Drebin JA; Link VC, Stern DF, Weinberg RA; Greene, MI. (1985) Down-modulation of an oncogene protein product and reversion of the transformed phenotype by monoclonal antibodies.Cell 41:695-706).This laboratory is found again subsequently; The monoclonal anti physical efficiency inhibition of anti-Her2 is implanted into the growth of the NIH3T3 tumour cell of the intravital Her2 conversion of nude mice; Thereby proved that through experiment in the body anti-Her2 monoclonal antibody possibly have value (the Drebin JA of clinical treatment to Her2 high expression level tumour for the first time; Link VC; Weinberg RA, Greene MI. (1986) Inhibition of tumor growth by a monoclonal antibody reactive with an oncogene-encoded tumor antigen.Proc Natl Acad Sci USA 83:9129-9133).This discovery has promoted the Antitumor Effects of anti-Her2 monoclonal antibody greatly, has also disclosed antibody Application Prospect in oncotherapy.But,, can produce HAMA (HAMA) reaction when being applied to human body, so can not directly be used for the intravital disease treatment of people because the monoclonal antibody of hybridoma technology preparation at present all is a mouse source antibody.Therefore must carry out monoclonal antibody humanization modified, to reduce and to eliminate the immunogenicity of mouse source protein.
The method that is used for the transformation of monoclonal antibody human source the earliest is to make up chimeric antibody (Vaughan TJ, Osbourn JK, Tempest PR. (1998) Human antibodies by design.Nat.Biotechnol 6:535-539).This method is that the variable region of mouse source antibody and people's antibody constant region are coupled together through genetic engineering technique, and the constant region gene replacement that is about to mouse source antibody remains mouse source antibody for people's antibody constant region gene with antigen bonded variable region part.The through engineering approaches chimeric antibody that this method constructs has kept the mouse source antibody variable region of identification target antigen ability to greatest extent; The immunology lethal effect that has simultaneously had all constant regions of people's antibody again; And since the immunogenicity of this chimeric antibody compare primary mouse source antibody and reduced by 2/3, so can be applied to human body therapy (Davis TA. (1999) Final report on the safety and efficacy of retreatment with rituximab for patients with non-Hodgkin ' s lymphoma.Blood 94:88a).But, be used for long-term clinical treatment the HAMA reaction also might take place owing to also kept 1/3 mouse source part in its molecule.
Therefore,, need carry out further humanization modifiedly, obtain full humanized antibody the variable region, mouse source of chimeric antibody in order further to reduce the immunogenicity of chimeric antibody.Use at present more full humanization method and be the framework region of participating in antigen bonded part in the antibody variable region of mouse source and selecting to be used to do people's antibody variable region of template is spliced again; Make that the complementary determining region (CDR) except that alterable height derives from the murine antibody in the antibody variable region, rest part all changes the people source into.Because participating in the antigen bonded in the antibody variable region generally is its 6 complementary determining regions (CDR); So process also is called as " CDR transplanting ", descend in order to overcome the avidity that possibly cause then owing to " CDR transplanting ", need through a series of sudden changes the framework region residue; Progressively recover or improve the antigen binding capacity of antibody; Thereby obtain the higher reshaped antibody of humanization degree (Jones PT, Dear PH, Foote J. (1986) Replacing the complementarity-determining regions in a human antibody with those from a mouse; Riechmann L; Clark M, Waldmann H. (1988) Reshaping human antibodies for therapy, Chothia C; Lesk AM, Tramontano. (1989) A Conformat ions of immunoglobulin hypervariable regions).The method of the structure reshaped antibody of having reported mainly can be divided into two kinds again according to the selection of the template of its humanization modified use and the method for answer antibody binding capacity.One of which; Contrast through sequence; Be that to select the human antibody of the highest light chain of antibody of similarity and variable region of heavy chain respectively be template sequence to standard with the sequence similarity; On light, sequence of heavy chain after transplanting through CDR is basic, make up a series of two mutants respectively, differentiates that suitable sudden change makes up to the framework residue that possibly influence binding ability.These two mutants need independently to prepare separately and test its antigen binding capacity, and will suddenly change preferably then combines, promptly need be to the sudden change of each possible residue, the recirculation experiment of screening, so step is complicated, efficient is low, and effect is undesirable.Its two, also be a kind of universal method of present antibody humanization.Concrete way is; The CDR of inhuman source (for example mouse source) antibody is transplanted on the framework of general human antibody variable region template VL κ I-VHIII; Through one group of selected crucial framework region residue is carried out random mutagenesis; Make up the phage library of monoclonal antibody, identify best framework sequence through the screening of phage avidity again.This method is compared first kind can obtain the framework sequence optimized more quickly, and can further improve the antigen avidity of antibody on this basis; But because it selects for use is common template, though universality is arranged, possibly not be optimum to different antibodies, and also more complicated of the method through making up phage library screening high-affinity antibody wastes time and energy in addition.
Summary of the invention
An object of the present invention is to provide a kind of albumen and encoding sox thereof.
Albumen provided by the present invention is following arbitrary said protein:
A) by the protein of forming from N-terminal 1-253 amino acids sequence in the sequence in the sequence table 3; (H1-2:V L+ Linker+V H)
B) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 3; (H1-2:V L+ Linker+V H+ Fc)
C) with amino acid passes through the replacement of one or several amino-acid residue and/or the protein with identical function of disappearance and/or interpolation from its N-terminal 115-134 position (linker) in the albumen that a) limits;
D) with b) in the albumen that limits from its N-terminal 115-134 position (linker) amino acid and/or 254-489 position (Fc fragment) amino acid through the replacement of one or several amino-acid residue and/or the protein with identical function of disappearance and/or interpolation;
E) by the protein of forming from N-terminal 1-253 amino acids sequence in the sequence in the sequence table 1; (H1-1:V L+ Linker+V H)
F) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1; (H1-1:V L+ Linker+V H+ Fc)
G) with e) in the albumen that limits from its N-terminal 115-134 position (linker) amino acid through the replacement of one or several amino-acid residue and/or the protein with identical function of disappearance and/or interpolation;
H) with f) in the albumen that limits from its N-terminal 115-134 position (linker) amino acid and/or 254-489 position (Fc fragment) amino acid through the replacement of one or several amino-acid residue and/or the protein with identical function of disappearance and/or interpolation;
I) by the protein of forming from N-terminal 1-253 amino acids sequence in the sequence in the sequence table 5; (H1-V1:V L+ Linker+V H)
J) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 5; (H1-V1:V L+ Linker+V H+ Fc)
K) with i) in the albumen that limits from its N-terminal 115-134 position (linker) amino acid through the replacement of one or several amino-acid residue and/or the protein with identical function of disappearance and/or interpolation;
L) with j) in the albumen that limits from its N-terminal 115-134 position (linker) amino acid and/or 254-489 position (Fc fragment) amino acid through the replacement of one or several amino-acid residue and/or the protein with identical function of disappearance and/or interpolation.
Said encoding sox is following arbitrary said:
1) encode said a) shown in proteic encoding sox be: nucleotide sequence be in the sequence table sequence 4 from 5 ' terminal 1-759 position Nucleotide shown in dna molecular;
2) proteic encoding sox is the said b that encodes): nucleotide sequence is a dna molecular shown in the sequence 4 in the sequence table;
3) proteic encoding sox is the said e that encodes): nucleotide sequence be in the sequence table sequence 2 from dna molecular shown in the Nucleotide of 5 ' terminal 1-759 position;
4) proteic encoding sox is the said f that encodes): nucleotide sequence is a dna molecular shown in the sequence 2 in the sequence table;
5) proteic encoding sox is the said i that encodes): nucleotide sequence be in the sequence table sequence 6 from dna molecular shown in the Nucleotide of 5 ' terminal 1-759 position;
6) proteic encoding sox is the said j that encodes): nucleotide sequence is a dna molecular shown in the sequence 6 in the sequence table;
7) under stringent condition with 1)-6) the dna sequence dna hybridization of arbitrary qualification and the dna molecular of encoding said proteins;
8) with 1)-6) dna sequence dna of arbitrary qualification has homology and the dna molecular of encoding said proteins more than 90%.
The recombinant vectors, reorganization bacterium, transgenic cell line, recombinant virus or the expression cassette that contain above-mentioned arbitrary said encoding sox also belong to protection scope of the present invention.
Said recombinant vectors is that the MCS at the pSectag2A carrier inserts said encoding sox and obtains.
Another object of the present invention provides a kind of antibody.
Antibody provided by the present invention is made up of two identical above-mentioned arbitrary said albumen; Said two identical albumen connect through disulfide linkage.
Above-mentioned arbitrary said albumen also belongs to protection scope of the present invention in the application that preparation prevents and/or treats in the medicine of tumour.
Above-mentioned arbitrary said antibody also belongs to protection scope of the present invention in the application that preparation prevents and/or treats in the medicine of tumour.
Another object of the present invention provides a kind of humanization modified method of antibody.
The humanization modified method of antibody provided by the invention comprises the steps:
(1), select to obtain the humanized antibody template according to following I or the said method of II:
I, select variable region of heavy chain and variable region of light chain respectively with the amino acid sequence similarity of the variable region of heavy chain of treating engineered antibody and variable region of light chain all at the humanized antibody 50% or more or more than 60%, with it as preparatory humanized antibody template;
From said preparatory humanized antibody template, select with the highest humanized antibody of the variable region space structure similarity of treating engineered antibody as the humanized antibody template;
II, select to satisfy following i) with ii) shown in the humanized antibody of condition, with it as preparatory humanized antibody template;
I) variable region of heavy chain and variable region of light chain respectively with the amino acid sequence similarity of the variable region of heavy chain of treating engineered antibody and variable region of light chain all more than 50% or more than 60%;
Ii) the framework region of the framework region of variable region of heavy chain and variable region of light chain respectively with the amino acid sequence similarity of the framework region of the framework region of the variable region of heavy chain of treating engineered antibody and variable region of light chain all more than 50% or more than 60%;
From said preparatory humanized antibody template, select with the highest humanized antibody of the variable region space structure similarity of treating engineered antibody as the humanized antibody template;
Said variable region space structure be following a) and/or b) shown in: a) space structure that constitutes together of variable region of heavy chain and variable region of light chain; B) space structure that constitutes together of framework region in the variable region of heavy chain and the framework region in the variable region of light chain;
The antibody of unknown space structure can go out its space structure through software simulation, comprises the space structure that space structure that variable region of heavy chain and variable region of light chain constitute together and the framework region in the variable region of heavy chain and the framework region in the variable region of light chain constitute together.
(2), obtain by the humanized antibody template after the displacement according to method shown in following III or the IV:
III, replace in the said humanized antibody template corresponding cdr amino acid sequence, obtain by the humanized antibody template after the displacement with the said variable region of heavy chain of engineered antibody and at least one cdr amino acid sequence in the variable region of light chain treated;
(antibody in unknown CDR district can be identified through people's such as people such as comprehensive Kabat and Chothia sequence definition, and the integrated value of two kinds of method qualification results as end value, is promptly got minimum value to peak section in two qualification results as the CDR district).
IV, replace antigen binding domain aminoacid sequence corresponding in the said humanized antibody template, obtain by the humanized antibody template after the displacement with the said antigen binding domain aminoacid sequence of treating engineered antibody;
(3), with in the said step (2) by the displacement after the humanized antibody template as improved purpose humanized antibody;
(4) obtain said improved purpose humanized antibody with the gene engineering method expression.
In the aforesaid method; After said step (2); Said step (3) is preceding; Comprise as becoming step next time: at least one amino-acid residue of the framework region of the antibody that said step (2) is obtained replaces with the said amino-acid residue of treating corresponding position in the engineered antibody, and is stable to the antibody space structure that obtains, with the antibody that obtains as improved purpose humanized antibody.
(return to become and satisfy following two conditions: 1) the humanization degree is high, 2) makes the antibody structure that obtains stable; Said variable region is variable region of heavy chain and/or variable region of light chain; )
In the aforesaid method, become in the step for said time, the amino-acid residue that is replaced is stable or influence the amino-acid residue of the antigen binding domain structural stability of the antibody that step (2) obtains for the CDR plot structure of the antibody that influence step (2) and obtain;
In the aforesaid method; Become for said time in the step, the amino-acid residue that is replaced is positioned at the vernier district of the antibody that said step (2) obtains and/or the amino-acid residue that is replaced is positioned in said cdr amino acid residue 3 dusts of side chain middle distance of the antibody that said step (2) obtains;
In the aforesaid method; After said time change; Said step (3) is preceding; Comprise like servant source sudden change step: in the framework region of the antibody that will become for said time inhuman source and be different from and saidly treat that the sudden change of corresponding position amino group of amino acids acid residue is a people source amino-acid residue in the engineered antibody, with the antibody that obtains as improved purpose humanized antibody.
The humanized antibody that is obtained by above-mentioned arbitrary said method also belongs to protection scope of the present invention.
Antibody of the present invention is specific recognition TSA P185 Hor2/erbB-2The humanized antibody varient of chimeric antibody ChA21.
The humanized antibody varient of ChA21 provided by the present invention comprises that the single-chain antibody in mouse source among the former chimeric antibody ChA21 is by the Fc district in the scFv of full-length human, hinge area and people source.Wherein be by the aminoacid sequence of humanized scFv:
DIVLTQSPDSLAVSLGERVTINC KSSQTLLYSNNQKNYLA WYQQKPGQSPKLLIS WAFTRKSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYC QQYSNYPWT FGAGTKLEIKR GGGGSGGGGSGGGGSGGGGS QVQLVQSGAEVVKPGASVKISCKAS GYSFTGYFIN WVKQNPGQRLEWIG HISSSYATSTYNQKFKG KATLTVDTSASTAYMELSSLRSEDTAVYYCVR SGNYEEYAMDYWGQGTLVTVS.
Underscore partly is respectively 3 light chains and 3 totally 6 complementary determining regions (CDRs) that heavy chain sequence is arranged of antibody, and black matrix partly is the joining region (Linker) between the weight chain variable region.
The unaltered initial chimeric antibody of the improved antibody framework of the present invention is compared, and is close with antigenic avidity, and reached humanization completely, has better potential applicability in clinical practice.
Humanized antibody remodeling method of the present invention comprises the steps:
The selection of the human antibody template of the variable region sequences of based target antibody and structural information
At first; To need that humanized target antibody (ChA21) is light, the sequence of variable region of heavy chain is carried out the sequence similarity search through IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/) respectively, will be gently, heavy chain all has higher similarity (greater than 50%) and the humanization template of several human antibodies of crystalline structure as the candidate arranged; Again through the superimposed structural similarity that compares candidate template antibody and target antibody framework region of molecule, a kind of template that the choice structure similarity is the highest as the antibody humanization.If target antibody does not have the crystalline structure data, can obtain the model molecule structure through the homology simulation and compare again.
The transplanting of antigen binding domain
At first, the antigen binding domain section of select target antibody.For the antibody that does not have the crystalline structure data, generally be to select its complementary determining region (CDR); (the CDR sequence of antibody can be identified through people's such as people such as comprehensive Kabat and Chothia sequence definition) for the antibody that antibody and antigenic compound crystalline structure are arranged, comprehensively CDR sequence and structured data obtain its antigen binding domain territory.
Then, with the residue on the section residue replacement people From Template antibody corresponding position of selecting, the anti-P185/Her2 antibody sequence of the humanization called after that obtains initial release is H1, and obtains the mimic space structure of H1 through the simulation of computingmachine homology.
The selection in framework region residue reverse mutation site
Based on mimic H1 structure, adopt the calculation biology method, to the suddenly change screening of direction of some important framework region residues.These residues comprise all vernier residues (Vernier) and all side chain distance C DR residue 3 dusts with interior framework region residue, select more to meet the direction of the site of antibody structure stability for the needs sudden change.Specific in this example; The the 4th and 49 residue of the variable region of light chain VL of initial human antibody H1 and the 71st and 93 residue of variable region of heavy chain VH; Be selected the corresponding residue that need sport on the target antibody; Be that VL the 4th residue methionine(Met) is replaced by leucine, the 49th residue tyrosine is replaced by Serine; The 71st residue L-Ala of VH is replaced by Xie Ansuan, and the 93rd residue Threonine replaced by Xie Ansuan.Obtain humanized anti-P185/Her2 antibody sequence H1-1 thus.According to transforming back avidity analytical results and the needs that further improve the humanization degree; Also can combine residue to advance the pedestrianization to the non-antigen beyond the number of C DR district modifies; As specific to this example being the residue 5,21 and 106 and the residue 61,62,80 and 91 in VH district in VL district among the H1-1; Then select in the DB on the human antibody corresponding position the higher residue of the frequency of occurrences that they are replaced respectively, finally obtain the anti-P185/Her2 antibody sequence H1-2 higher than initial release humanization degree.
Method provided by the present invention is compared with existing antibody humanization's remodeling method, and following advantage and specificity are arranged:
At first; Present method is in the selection of humanization template antibody; Method different from the past respectively selection and light chain similar with heavy chain two people's From Template sequences, but select a kind of human antibody that the weight chain all has higher similarity with target antibody, and the selection of trying one's best of candidate template antibody has the crystalline structure data as masterplate; Through comparing the integrally-built similarity of candidate's masterplate antibody and target antibody, final decision goes out template antibody.This weight chain with antibody is done as a wholely to consider; Selected weight chain template comes from same antibody; Itself be exactly from natural framework; Compare the method that in the past artificially makes up the weight chain and more help weight chain framework region stability of structure, and in the Selection Framework district residue when sudden change, the weight chain interface residue of can considering again whether need suddenly change makes the weight chain more stable problem that interacts.
Secondly, select template to compare the simple in the past sequence similarity screening template of passing through by structural similarity and have more reasonable structure.Because antibody and the antigenic conformation that combines directly to depend on the complementary antibody determining area; And complementary determining region is to build on the constructed platform of framework region; Be beneficial to keeping of complementary determining region conformation after the transplanting more so the space structure similarity of people's From Template and target antibody is high more, promptly be beneficial to the antigenic ability of antibodies that keeps more.Though sequence similarity to a certain extent can the representative structure similarity, and not really accurate.For example in the embodiment of the invention one, the some templates with highest serial similarity but are not that structural similarity is best.
Once more, when residue suddenlyd change in the Selection Framework district, the binding antibody structured data was analyzed, and can intuitively make the selection of answering the mutational site rapidly.Select in the vernier residue those might influence the residue of CDR plot structure and determine some other direction that is positioned at the antibody molecule surface and can further humanized residue whether suddenlys change and suddenly change according to constitutionally stable needs; With needed many wheel screen mutations or simplified greatly in the past through the mode of phage library screening sudden change; All sudden changes are selected to concentrate in together to carry out structural analysis and feasibility thereof, and a direct step obtains final sudden change scheme.Therefore compare previous methods, present method is simpler and more direct clear and definite.
At last, than existing humanization method, the correctional effect of present method is also higher.The ChA21 antibody humanization's who obtains through present method version H1-2 has the antigen-binding activity suitable with original antibody, and relative affinity is about about 93% of original antibody.It is low that the avidity of passing through the improved antibody of universal antibody peopleization method of report has height to have, level very unstable (Carter P, Presta L, Gorman CM; Ridgway JB, Henner D, Wong WL, Rowland AM; Kotts C, Carver ME, Shepard HM. (1992) Humanization of an anti-p185HER2 antibody for human cancer therapy.Proc Natl Acad Sci USA 89:4285-4289, Presta LG; Chen H, O Connor SJ, Chisholm V; Meng YG, Krummen L, Winkler M; Ferrara N, (1997) Humanization of an anti-VEGFmonoclonal antibody for the therapy of solid tumors and other disorders, Cancer Res.57:4593-4599).For directly comparing the correctional effect of present method and universal method; The general people's From Template VL κ I-VHIII that uses simultaneously in embodiments of the present invention in the general humanization method carries out humanization modified to ChA21; Obtain another humanization version H2-1 of ChA21; Compare original antibody through the avidity of avidity mensuration discovery H2-1 and descended more than one times, explain that with regard to the antibody ChA21 in this example, the correctional effect of the peopleization method among the present invention obviously is better than existing additive method.
Known in the art have various different methods to can be used for expressing and the humanized antibody of purifying.Like escherichia expression system, simple to operate quick, cost is low; But soluble antibody expression amount low (100ug/L), expression product can not correctly be modified, and forms inclusion body easily; Purification difficult; Expression amount is widely different between the different mutants clone, makes the mensuration of next step affinity of antibody influenced by AC, is difficult for judging; Yeast expression system is like pPIC9K carrier, secreting, expressing; Available His-tag column purification, purifying is more convenient, but needs to make up stable cell line; Cycle is long, though expression amount than intestinal bacteria system high (generally in the mg/L rank), expression amount difference is also very big between the different mutants clone; And the 293T eucaryon transient expression system that adopts among the present invention is a carrier with pSectag2A, but secreting, expressing; Product can be used His-tag post or albumin A column purification; Therefore with the recombinant plasmid dna of the humanized antibody that designs through liposome transient transfection 293T cell, expression product can correctly be modified, purifying is simple; Expression amount higher (3-10mg/L); And expression amount difference is little between the different mutants clone, and is though cost is higher relatively, clear and definite, stable with this system expression antibody purified property testing result.In addition, use the ELISA method and measure antibody expression amount and relative affinity, the result is accurate, good reproducibility, and need not to carry out purifying to expressing supernatant, be convenient to Rapid identification and filter out people source two mutants.
Description of drawings
Fig. 1 is the variable region amino acid sequence of antibody ChA21 light chain and heavy chain, and sequence numbering is according to the Kabat rule compositor.Underscore partly is the complementary determining region (CDRs) that the sequence definition of comprehensive Kabat and Chothia marks off.
Fig. 2 is strand variable region (scFv, PDB number: the people's From Template antibody huCC49 that 2GJJ) selectes together (PDB number: the space structure comparison result of variable region 1ZA6) of antibody ChA21.
Fig. 3 is the composite structure of strand variable region and its identification antigen part subprovince of antibody ChA21.
Fig. 4 is used to explain the aminoacid sequence of transplanting the humanized antibody H1 of the initial release that obtains through the antigen binding domain of ChA21, and compares with the variable region primary sequence of original mouse source antibody and people's From Template antibody.
Fig. 5 is the molecular model of simulating the humanized antibody H1-1 that obtains through the computingmachine homology.
Fig. 6 is used to explain the selection result of on humanized antibody H1 sequence and architecture basics, carrying out the sudden change of a series of framework region residue, and with the comparison of the sequence of new humanized antibody varient H1-1 that obtains and H1-2.Complementary determining region marks with square frame.
Fig. 7 is used to explain that (PDB number: it is humanization modified 1FVC) be that people's From Template carries out ChA21, and the aminoacid sequence of the humanized antibody H2-1 of acquisition with the humanized antibody hu4D5 that obtains based on common template VL κ I-VHIII.
Fig. 8 is the structure synoptic diagram of humanized antibody H1-1 and H1-2 recombinant expression vector.
Fig. 9 is the antigen binding curve of antibody ChA21 and its various humanization two mutants.The initial concentration of the antibody in the supernatant of the 293T cell of various transfections all is mixed with 1ug/ml, detects various two mutants and antigenic binding curve through ELISA again.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
Embodiment 1, chimeric antibody chA21's is humanization modified
The sequence of chimeric antibody chA21 is described (Chen L.S. by people such as L.S.Cheng already; Liu A.P.; Liu.J.Construction, expression and characterization of the engineered antibody against tumor surface antigen P185 C-erbb-2.Cell Res.2003 is 13:35-48), as shown in Figure 1.The structure of chimeric antibody chA21: the disulfide-bonded of two identical protein chains through forming in the Fc district.Every protein chain is successively by variable region of light chain (V L), linker, variable region of heavy chain (V H) and the Fc district connect to form successively, its aminoacid sequence is shown in SEQ ID NO:9, wherein, the 1-114 position is V L, the 115-134 position is linker, the 135-253 position is V H, the 254-489 position is Fc.
One, the selection of human antibody template
Sequence similarity comparison: the variable region of light chain of chA21 and the variable region of light chain of humanized antibody are carried out the amino acid sequence similarity comparison, the variable region of heavy chain of chA21 and the variable region of heavy chain of humanized antibody are carried out the amino acid sequence similarity comparison.In albumen database (PDB, Protein Data Bank), carry out the sequence similarity search respectively through IgBLAST (http://www.ncbi.nlm.nih.gov/igblast/).The concrete search parameter of selecting is following: program: blastp; Submission sequence source: mouse; DB: pdb; Aligned sequences source: human.The quantity that shows comparison result can be come concrete the selection according to comparison result, and other parameters can be selected default parameters.
In the result of search, the antibody of selecting with light, heavy chain the higher sequence similarity to be arranged all generally selects similarity to select threshold value in conduct more than 50% as candidate's humanized antibody template, also can select different threshold values according to concrete Search Results.In the comparison result of ChA21, because that sequence similarity is higher than 50% candidate template is too many, select threshold value so improve, select two chain similaritys all to be higher than 60% antibody as candidate's humanized antibody template;
The structural similarity comparison: Using P yMOL software compares the variable region (scFv of candidate's humanized antibody template and ChA21 through molecule superimposed (align) again; PDB number: overall space structural similarity 2GJJ) (being the space structure that variable region of heavy chain and variable region of light chain are formed together in the albumen) and framework region space structure similarity (being the space structure that variable region of heavy chain middle frame district and variable region of light chain middle frame district form together in the albumen) (size of being represented its structure differences by RMS), the sequence alignment of each candidate template and space structure comparison result are seen table 1 (sequence numbering in all tables all adopts the Kabat sequence numbering).
The huCC49 that the choice structure similarity is the highest (PDB:1ZA6) (seeing it is minimum in the table) is as the humanized template of chimeric antibody ChA21, and Fig. 2 is seen in the comparison of two antibody space structures.
Wherein template 1FVC is that antibody 4D5 carries out the transformation of people source and the humanized antibody hu4D5 of acquisition based on general human antibody template VL κ I-VHIII; Its framework region sequence and VL κ I-VHIII basically identical; Based on this common template structure ChA21 is carried out simultaneously humanization modified, in order to the peopleization correctional effect of the inventive method and universal method relatively.
Table 1, ChA21 variable region and each candidate template sequence and structural similarity comparison
Candidate template structure (PDB number) Variable region of light chain sequence similarity degree % Weight chain variabl area sequence similarity % Light chain framework region sequence similarity degree % Heavy chain framework region sequence similarity degree % Overall space textural difference (RMS) Framework region space structure difference (RMS)
1ZA6 78.1 61.7 79 73.6 0.666 0.646
1B4J 64 60.5 76.5 73.6 0.679 0.662
1I9R 62.3 61.7 76.5 74.7 0.705 0.675
3DIF 65.8 66.7 79 79.3 0.778 0.691
3FCT 63.2 60.8 71.6 73.6 0.841 0.765
1FVC 58.8 47.5 71.6 54 0.881 0.866
Two, the transplanting of antigen binding domain
Select the antigen binding domain section of ChA21.The CDR sequence of antibody ChA21 can identify that CDR sequence results is seen table 2 through the sequence definition that combines people such as people such as Kabat and Chothia.Final each CDR district that delimit is the section of band underscore among Fig. 1.
The complementary determining region of table 2, ChA21 (CDR) sequence results
CDR LOOP Kabat Chothia Combine
L1 L24-L34 L24-L34 L24-L34
L2 L50-L56 L50-L56 L50-L56
L3 L89-L97 L89-L97 L89-L97
H1 H31-H35B H26-H32..34 H26-H35B
H2 H50-H65 H52-H56 H50-H65
H3 H95-H102 H95-H102 H95-H102
Because the strand variable region of antibody ChA21 and the compound crystal structured data of its identification antigen part subprovince have obtained to resolve, its antigen binding domain section can obtain from composite structure intuitively, and is as shown in Figure 3.Can find out that the second half section of heavy chain CDR2 is positioned at the surface of antibody molecule; Neither participating in antigen combines; To other CDR plot structures yet not influence, do not participate in the interaction between the antibody weight chain, so consider from the requirement that improves sequence humanization degree yet; It as non-antigen binding domain, and is not transplanted this section sequence on the heavy chain CDR2 of ChA21.Residue with the CDR zone on residue replacement antibody huCC49 (1ZA6) corresponding position in all the other CDR zones; Obtain the humanized antibody sequence H1 (the sequence comparative result is seen Fig. 4) of initial release, and application protein structure simulation software MODELLER obtains its mimic space structure through the homology simulation.(H3 that provides in the table 3 is H95-H102; This sequence numbering is the Kabat sequence number; But not sequence PS numbering is H95-H102 (SGNYEEYAMDY) like the CDR district H3 sequence that is identified among Fig. 1, and its position is this section of 101-111 (SGNYEEYAMDY) among Fig. 4 relatively.)
Three, (return and become) selected in the sudden change of H1 framework region residue
Mimic space structure based on initial release humanized antibody H1; To the suddenly change screening of direction of some important framework region residues: one type of residue at first considering is the residue that possibly influence complementary determining region conformation or structure; Or possibly directly influence antigen bonded residue; For the research of this class framework residue, list the inventory (seeing table 3) of this type of residue according to before, these residues comprise nearly more than 30 kinds of vernier residues (Vernier) that the CDR structure had potential contribution; In addition, in mimic H1 structure, select all side chain distance C DR residue 3 dusts, consider that from the structure angle these residues also possibly directly influence the conformation of CDR district residue, and they are included in all just among the listed vernier residue of table 3 with interior residue.Usually need these residues all be sported the residue in the corresponding site of original antibody ChA21; Be called " return and become "; But do the danger that might cause immunogenicity to increase like this because of the element that has added inhuman source sequence; Therefore, in that returning of vernier residue become in the chosen process, can keep the vernier residue that some side chains are positioned at the antibody molecule surface; Also should select not return change, for example H69Leu-＞Phe for returning the residue that may cause back becoming after the change with the residue that residue recurring structure around it conflicts simultaneously.Through selecting back the humanization sequence H1-1 that becomes, its model configuration is as shown in Figure 5.
Table 3, the sudden change of vernier residue are selected
The vernier residue People's From Template residue Mouse source ChA21 residue Humanization H1-1 sequence
L2 Ile Ile Ile
L4 Met Leu Leu
L35 Trp Trp Trp
L36 Tyr Tyr Tyr
L46 Leu Leu Leu
L47 Leu Leu Leu
L48 Ile Ile Ile
L49 Tyr Ser Ser
L64 Gly Gly Gly
L66 Gly Gly Gly
L68 Gly Gly Gly
L69 Thr Thr Thr
L71 Phe Phe Phe
L98 Phe Phe Phe
H2 Val Val Val
H47 Trp Trp Trp
H48 Ile Ile Ile
H49 Gly Gly Gly
H67 Ala Ala Ala
H69 Leu Phe Leu
H71 Ala Val Val
H73 Thr Thr Thr
H78 Ala Ala Ala
H93 Thr Val Val
H94 Arg Arg Arg
H103 Trp Trp Trp
Four, people source sudden change
Combining residue to advance the pedestrianization to some non-antigens again modifies.Because the From Template antibody huCC49 that chooses of institute is humanized antibody; Also exist on its framework sequence and return the residue that becomes parental generation mouse source antibody in some its humanization processes; These residues comprise total residue of some mouse source antibody and the distinctive residue of some huCC49 parental generation mouse source antibody mCC49; And the latter is dispensable for other mouse source antibody; Therefore see from the angle that improves the humanization degree, do not influencing on the constitutionally stable basis, need these residues be sported corresponding people source residue.In the sequence specific to H1-1; Pass through sequence alignment; Find the site of the people's From Template sequence (huCC49 light chain template sequence LEN and huCC49 heavy chain template sequence 21/28 ' CL) that both had been different from ChA21 in the framework sequence and also has been different from humanized antibody huCC49; They be among the VL residue 5,21 and 106 and VH in residue 61,62,80 and 91, select sudden change to be the residue (seeing table 4) on people's From Template sequence LEN and 21/28 ' the CL corresponding position.The final humanization sequence H1-2 that obtains, the comparison result of all sequences is seen Fig. 6.
Table 4, the transformation of non-antigen binding sequence peopleization
In addition; With humanized antibody hu4D5 (PDB:1FVC; Usually be regarded as common template) be template; Obtain humanization sequence H2-1 (Fig. 7) through same procedure, this humanized antibody is as contrast, the human antibody relatively influence of antagonist avidity each other that obtains with humanization template system of selection among the present invention.Among Fig. 7; Hu4D5 is humanization template (being common template); H2 is the antibody that obtains after the CDR displacement (method of replacing with above-mentioned steps two in consistent); HumanVL-KappaI is the corresponding people's source sequence of variable region of light chain of antibody hu4D5, and HumanVH-III is the corresponding people's source sequence of variable region of heavy chain of antibody hu4D5, and H2-1 is the sequence behind the final humanization.
The expression vector establishment of embodiment 2, various antibody
One, pSectag2A-dFc vector construction
The pSectag2A carrier is available from Invitrogen company.Utilize pcr amplification Fc fragment, the design primer adds EcoRI and XhoI restriction enzyme site respectively at Fc fragment N end and C end, then with the Fc fragment with EcoRI and XhoI double digestion, fragment behind the recovery double digestion; Simultaneously the pSectag2A plasmid is carried out double digestion with EcoRI and XhoI, fragment behind the recovery double digestion; Fc fragment behind the double digestion is connected with the T4-DNA ligase enzyme with the pSectag2A carrier; To connect product and be transformed into the Top10 competent cell, the extracting plasmid carries out double digestion and identifies and sequence verification the result; The gene order of inserting between the EcoRI of pSectag2A and XhoI restriction enzyme site is (being the Fc fragment) shown in 760-1470 position Nucleotide among the SEQ ID NO:2; The recombinant vectors that shows structure is correct, this carrier note is made pSectag2A-dFc, as the expression vector of humanized antibody.
With scFv-Fc/pEE14 (Chinese patent CN1613873) is template, uses following primer, and pcr amplification obtains the Fc fragment.The perhaps Fc fragment shown in the Nucleotide of 760-1470 position among the synthetic SEQ ID NO:2.DFc-EcoRI:5 '-AAT GAATTCGCGGCCGCAGAGCCCAAATCTTGTGACAAA-3 ' (underscore is EcoRI and NotI site)
DFc-XhoI:5 '-TTT CTCGAGTCATTTACCCGGAGACAGGGAGAG-3 ' (underscore is the XhoI site)
Two, the structure of humanized antibody H1-1 recombinant expression vector
Gene shown in the Nucleotide of 1-759 position (is V among the synthetic SEQ ID NO:2 L+ Linker+V H), import carrier pMD-19T (by the precious biosynthesizing in Dalian), called after pMD-19T-H1-1.With pMD-19T-H1-1 is template; At primer P0 (PH1-Sfi1): carry out pcr amplification under the guiding of 5 '-ATATGGCCCAGCCGGCCGACATCGTTTTGTCTCAATCTCCAGACTCTTTG-3 ' and P7 (H1-Rv-Not1): 5 '-ATATGCGGCCGCAGAAACAGTAACCAAAGTACC-3 '; Pcr amplification product is carried out 1% agarose gel electrophoresis detect and reclaim purpose band, called after " H1-1 ".With restriction enzyme SfiI and Not I the H1-1 gene fragment that obtains is carried out single endonuclease digestion respectively; Again endonuclease bamhi is connected with the T4DNA ligase enzyme with the plasmid pSectag2A-dFc that cuts through the same enzyme enzyme respectively; To connect product transformed into escherichia coli Top10 or DH5 α competent cell, screening positive recombinant, extracting DNA; Order-checking, the gene order that the result inserts between the SfiI of pSectag2A-dFc and Not I site is shown in 1-759 position Nucleotide among the SEQ IDNO:2.With positive recombinant plasmid called after pSectag2A-dFc-H1-1.Contain gene shown in the SEQ ID NO:2 in this recombinant vectors, the 1-342 position is V in this gene L, the 343-402 position is Linker, the 403-759 position is V H, the 760-1470 position is Fc; The albumen of this genes encoding is shown in SEQ ID NO:1, and the 1-114 position is V in this albumen L, the 115-134 position is Linker, the 135-253 position is V H, the 254-489 position is Fc.
Three, the structure of humanized antibody H1-2 recombinant expression vector
(1) construction of recombinant vector of expression H1-V1
Method through point mutation on the gene order basis of the H1-1 that obtains obtains the encoding sequence of H1-2.Concrete grammar is as shown in Figure 8, divides the two-wheeled point mutation to carry out, and earlier H1-1 is carried out first round point mutation and obtains the middle two mutants of H1-V1, again H1-V1 is carried out second and takes turns the sequence that point mutation obtains H1-2.
Mutational site to humanized antibody H1-1 is as shown in table 5:
The mutational site table of table 5, humanized antibody H1-V1 gene
The site L5 L21 L106 H80 H91
H1-1 S(TCT) L(TTG) L(TTG) V(GTT) F(TTC)
H1-V1 T(ACT) I(ATT) I(ATT) M(ATG) Y(TAC)
Adopt fixed-point mutation method to divide three sections amplifications to have the sequence in said mutation site, use bridging PCR method then, amplification obtains humanized antibody H1-V1 coding gene sequence, and concrete grammar may further comprise the steps:
1) amplification of 5 ' terminal sequence of humanized antibody H1-V1 encoding sox
With pMD-19T-H1-1 is template; At primer P2 (P5T-21I-Fw): carry out pcr amplification under the guiding of 5 '-ACTCAATCTCCAGACTCTTTGGCTGTTTCTTTGGGTGAAAGAGTTACTATTAACTG T-3 ' and P3 (P112I-Rv): 5 '-CTAAGTTGGAAATTAAGAGAGGTGGTGG-3 '; Reclaim the purpose band, called after " H1-V1-5 ' ".
2) amplification of the interlude sequence of humanized antibody H1-V1 encoding sox
With pMD-19T-H1 is template; At primer P4 (P112I-Fw): carry out pcr amplification under the guiding of 5 '-CTAAGTTGGAAATTAAGAGAGGTGGTGG-3 ' and P5 (P215M-RV): 5 '-GTCTTCCGATCTCAAAGAAGACAATTCCATGTAAGCAG-3 '; Reclaim the purpose band, called after " H1-V1-m ".
3) amplification of 3 ' terminal sequence of humanized antibody H1-V1 encoding sox
With pMD-19T-H1 is template; At primer P6 (P229Y-Fw): carry out pcr amplification under the guiding of 5 '-GTCTTCTTTGAGATCGGAAGACACTGCTGTTTACTACTGTG-3 ' and P7 (H1-Rv-Not1): 5 '-ATATGCGGCCGCAGAAACAGTAACCAAAGTACC-3 '; Reclaim the purpose band, called after " H1-V1-3 ' ".
4) acquisition of humanized antibody H1-V1 encoding sox
H1-V1-5 ', step 2 with step 1) amplification) H1-V1-m and these three segments of H1-V1-3 ' gene that step 3) increases of amplification are that template is at primer P1 (P5T-Sfi1): ATATGGCCCAGCCGGCCGAC
The pcr amplification of putting up a bridge under the guiding of ATCGTTTTGACTCAATCTCCAGACTCTTTG and P7 reclaims the purpose band, called after " H1-V1 "
5) acquisition of the expression vector of humanized antibody H1-V1 encoding sox
With restriction enzyme SfiI and NotI the H1-V1 gene fragment that step 4) obtains is carried out single endonuclease digestion respectively; Again endonuclease bamhi is connected with plasmid pSectag2A-dFc through the same enzyme double digestion respectively; To connect product transformed into escherichia coli Top10 competent cell, screening positive recombinant, extracting DNA; Order-checking, the gene order that the result inserts between the SfiI of pSectag2A-dFc and NotI site is shown in 1-759 position Nucleotide among the SEQ ID NO:6.With positive recombinant plasmid called after pSectag2A-dFc-H1-V1.Contain gene shown in the SEQ ID NO:6 in this recombinant vectors, the 1-342 position is V in this gene L, the 343-402 position is Linker, the 403-759 position is V H, the 760-1470 position is Fc; The albumen of this genes encoding is shown in SEQ ID NO:5, and the 1-114 position is V in this albumen L, the 115-134 position is Linker, the 135-253 position is V H, the 254-489 position is Fc.
(2) structure of the recombinant expression vector of expression H1-2
The following site of humanized antibody H1-V1 suddenlyd change obtains H1-2, and the mutational site is shown in (table 6):
The mutational site of table 6 humanized antibody H1-2
The site H61 H62
H1-V1 E(GAA) R(AGA)
H1-2 Q(CAA) K(AAA)
Adopt the fixed-point mutation method amplification to have the humanized antibody H1-2 encoding sox in said mutation site, concrete grammar may further comprise the steps:
1) amplification of 5 ' terminal sequence of humanized antibody H1-2 encoding sox
With pSectag2A-dFc-H1-V1 is template, at primer P1 and P8 (H1-2-Rv): and carry out pcr amplification under the guiding of 5 '-GCCTTACCCTTGAATTTTTGGTTGTAAGTAGAAG-3 ', reclaim the purpose band, called after " H1-2-5 ' ".
2) amplification of 3 ' terminal sequence of humanized antibody H1-2 encoding sox
With pSectag2A-dFc-H1-V1 is template, at primer P9 (H1-2-Fw): 5 '-CTTCTACTTACAACCAAAAATTCAAGGGTAAGGC-3 ' and P7: guiding under carry out pcr amplification, reclaim the purpose band, called after " H1-2-3 ' ".
3) acquisition of humanized antibody H1-2 encoding sox
H1-2-5 ' and step 2 with step 1) amplification) H1-2-3 ' gene segment of amplification is that template is carried out pcr amplification under the guiding of primer P1 and P7, recovery purpose band, called after " H1-2 ".
4) acquisition of the expression vector of humanized antibody H1-2 encoding sox
With restriction enzyme SfiI and NotI the H1-2 gene fragment that step 3) obtains is carried out single endonuclease digestion respectively; Again endonuclease bamhi is connected with plasmid pSectag2A-dFc through the same enzyme double digestion respectively; To connect product transformed into escherichia coli Top10 competent cell, screening positive recombinant, extracting DNA; Order-checking, the gene order that the result inserts between the SfiI of pSectag2A-dFc and NotI site is shown in 1-759 position Nucleotide among the SEQ ID NO:4.With positive recombinant plasmid called after pSectag2A-dFc-H1-2.Contain gene shown in the SEQ ID NO:4 in this recombinant vectors, the 1-342 position is V in this gene L, the 343-402 position is Linker, the 403-759 position is V H, the 760-1470 position is Fc; The albumen of this genes encoding is shown in SEQ ID NO:3, and the 1-114 position is V in this albumen L, the 115-134 position is Linker, the 135-253 position is V H, the 254-489 position is Fc.
Four, the structure of humanized antibody H2-1 recombinant expression vector
Among the synthetic SEQ ID NO:8 from gene shown in the Nucleotide of 5 ' terminal 1-759 position, and importing carrier pMD-19T, sequence verification is with positive recombinant called after pMD-19T-H2-1.With pMD-19T-H2-1 is template; At primer P10 (PH2-Sfi1): 5 '-ATAT GGCC CAGCC GGCC GACATCCAATTGACTCAATCTCCATCTTCTTTG-3 ' and P7 (H1-Rv-Not1): carry out pcr amplification under the guiding of 5 '-ATATGCGGCCGCAGAAACAGTAACCAAAGTACC-3 '; Reclaim the purpose band, called after " H2-1 ".With restriction enzyme SfiI and NotI the H2-1 gene fragment that obtains is carried out single endonuclease digestion respectively; Again endonuclease bamhi is connected with plasmid pSectag2A-dFc through the same enzyme double digestion respectively; The positive recombinant plasmid that obtains; Sequence verification, the result the gene order of inserting between the SfiI of pSectag2A-dFc and NotI site as among the SEQ ID NO:8 from shown in the Nucleotide of 5 ' terminal 1-759 position, with positive recombinant plasmid called after pSectag2A-dFc-H2-1.Contain gene shown in the SEQ ID NO:8 in this recombinant vectors, the 1-342 position is V in this gene L, the 343-402 position is Linker, the 403-759 position is V H, the 760-1470 position is Fc; The albumen of this genes encoding is shown in SEQ ID NO:7, and the 1-114 position is V in this albumen L, the 115-134 position is Linker, the 135-253 position is V H, the 254-489 position is Fc.
Five, the structure of chimeric antibody ChA21 recombinant expression vector
The construction process of the expression vector of chimeric antibody ChA21 is with consistent described in the Chinese patent CN1613873.
Embodiment 3, wink are changeed the purifying of 293T cell expressing humanized antibody and expression product
(1) expression of antibody H1-1, H1-2, H2-1, H1-V1, ChA21 and purifying
The 293T cell is available from ATCC, and catalog number is CRL-11268.
The DMEM substratum is available from GIBCO, and catalog number is 11960.
Cultivate the 293T cell (DMEM/10% serum/1% pair anti-) (two anti-be penicillium mould and Streptomycin sulphates are given birth to worker, article No. BS732 available from Shanghai) of q.s in advance,, be used to transfection recombinant antibodies DNA when being in logarithmic phase when cell reaches 80% left and right sides full scale.
The transfection step is following: (1) adds 20ul Lipofectamine 2000 in 1ml DMEM, the even or rifle head mixing with the finger bullet, and room temperature left standstill 5 minutes.(2) in 1ml DMEM, add 10ug then and be ready to the DNA that transfection is used, mixing.(3) (need forward a 4ml centrifuge tube to) mixed in (1) and (2), mixing, room temperature leaves standstill 20-30 minute (mixture need be finished transfection in 1 hour); (4) 20-30 minute waiting time, the 293T cell culture medium is sopped up, add 5-8ml PBS washed cell one time (as far as possible removing remaining PBS), add 8ml DMEM then, subsequent use; (5) mixed solution with (3) adds in (4), rocks the culturing bottle mixing gently, and places the incubator overnight cultures.The transfection time was generally 8-16 hour.
Transfection renews bright DMEM substratum (adds 1% pair resist prevent microbiological contamination) after 16 hours.Collected a culture supernatant in per two days, supernatant centrifugal 10 minutes with 3000rpm is collected supernatant, places 4 ℃ (using in the week) perhaps-80 ℃ (for a long time) preservation.
Contain antibody H1-1, H1-2, H2-1, H1-V1 or ChA21 in the supernatant.
(2) CHO expresses the also method of the ChA21 chimeric antibody (ChA21-CHO) of purifying:
The method of the ChA21 chimeric antibody (ChA21-CHO) of CHO expression and purifying is with consistent described in the Chinese patent CN1613873.
The sequence indifference of antibody ChA21 and antibody ChA21-CHO, different is that antibody ChA21 is come out by the 293T cell expressing, antibody ChA21-CHO is expressed by the CHO-GS engineering cell strain.
The affine active mensuration of the humanization two mutants of embodiment 4, ChA21 antibody
One, affine relatively determination of activity
Select the ELISA method to survey the affine relatively activity of antibody.It is following that the ELISA method is surveyed the affine relatively active step of antibody:
How anti-available from Sigma the anti-people Fc of rabbit is, and catalog number is A9544.
The preparation method of antigen GST-EPI is following:
The I.GST-EPI plasmid construction:
The outer I-II district gene (SEQ ID NO:10) of synthetic Her2 born of the same parents.Use EcoR I (available from Promega synthetic gene; Article No. R6011) and Xho I (available from Promega; Article No. R6161) carries out double digestion; After being connected to pGEX/4T-1 (available from GE Healthcare, the article No. 27-4580-01) carrier of cutting through same enzyme, choosing and identify with order-checking after clone's double digestion is identified; The gene order that the result inserts between the EcoR of pGEX/4T-1 I and Xho I restriction enzyme site shown in 1-603 position Nucleotide among the SEQ ID NO:10, the clone's called after pGEX/4T-1-EPI that checks order correct.
The prokaryotic expression of II.GST-EP I and purifying
PGEX/4T-1-EPI is converted into Origami B competence (available from novagen, article No. 71408-3), selects the positive expression clone.Positive colony is carried out enlarged culturing, treat that bacterium liquid OD600 value in the culturing bottle reaches about about 0.6 after, adding IPTG (available from Merch, article No. 420291-5GM) is that 1mM carries out abduction delivering to final concentration, 16 ℃, 220rpm continues to cultivate 20h; 5000g, 10min, 4 ℃ of centrifugal receipts bacterium, behind the hydraulic crushing with 16000g, 20min, 4 ℃ of centrifugal collection lysate supernatants; With GST-EP I in GSTrap FF (available from GE Healthcare, article No. 17-5131-02) the purifying cracking supernatant, be put in after the desalination-80 ℃ frozen subsequent use.
1) confirm the expression amount of the antibody mutation body of transient transfection: the anti-people Fc of rabbit is the anti-NaHCO that are diluted in how 3(the about 1ug/ml of final concentration) shop elisa plate in the aqueous solution, 4 ℃ of refrigerator overnight or 37 ℃ baking oven 2-3 hour; 5% skim-milk is dissolved in TPBS, and 37 ℃ were sealed 1 hour; The standard antibody that adds the different concns dilution is expressed supernatant with each 293T, room temperature concussion incubation 1 hour.To standard antibody (ChA21-CHO): CHO is expressed and the ChA21 chimeric antibody (ChA21-CHO) of purifying, be diluted to 1ug/ml with the DMEM substratum earlier, be diluted to 100 successively with encapsulant then; 50,25,12.5; 6.25,3.13,1.56; 0.78ng/ml 8 concentration are used as typical curve altogether; To expressing supernatant: directly with 10,40,160,640 times of encapsulant dilutions.Goat-anti people/the HRP two that adds dilution in 1: 2000 is anti-, room temperature concussion incubation 1 hour, and the colour developing of OPD substrate, sulfuric acid stops.According to typical curve, calculate each two mutants antibody expression amount.
2) relative affinity of mensuration humanized antibody two mutants: antigen used herein is GST-EPI; EPI is the outer I/II districts of membrane antigen ErbB2 born of the same parents; It has comprised the identification epi-position of ChA21 and humanization two mutants thereof, measures with the avidity that the EPI of gst fusion protein formal representation can replace holoantigen ErbB2 to be used for the antibody variation body.GST-EPI (1mg/ml liquid storage) to be diluted in NaHCO at 1: 4000 3(the about 0.25ug/ml of final concentration) shop elisa plate in the aqueous solution, 4 ℃ of refrigerator overnight or 37 ℃ baking oven 2-3 hour; 5% skim-milk is dissolved in TPBS, and 37 ℃ were sealed 1 hour; Add the standard antibody (ChA21-CHO) of different concns dilution and the expression supernatant of transfection with mutant 293T, room temperature concussion incubation 1 hour.Standard antibody (ChA21-CHO) and two mutants are expressed supernatant: be diluted to 1000,333,111,37,12.33,4.11,1.37 with encapsulant, 0.46ng/ml.Goat-anti people/the HRP two that adds dilution in 1: 2000 is anti-, room temperature concussion incubation 1 hour.The colour developing of OPD substrate, sulfuric acid stops.According to each two mutants EC50 value of curve calculation, the ratio of the EC50 value of primary standard antibody and two mutants is the relative affinity of two mutants.
Original antibody is as shown in Figure 9 with each one the antigen binding curve of source two mutants; Original ChA21 chimeric antibody (antibody ChA21) so that 293T expresses is active in contrast, and the relative affinity that calculates humanization two mutants H1-1, H1-V1, H1-2 and H2-1 is respectively 0.95,0.92,0.93,0.4.The result shows that with antibody huCC49 be the antigen binding capacity that humanization two mutants H1-1, H1-V1, H1-2 that template is carried out humanization modified acquisition have kept ChA21 basically, and has reduced more than one times based on the antigen binding capacity that general peopleization template is carried out the humanization two mutants H2-1 of humanization modified acquisition.The ChA21 chimeric antibody (antibody ChA21) that this experiment also is illustrated in the 293T cell expressing has suitable avidity with the antibody (antibody ChA21-CHO) of expressing at the CHO-GS engineering cell strain.
Two, the surface plasmon resonance method is surveyed the binding constant Kd of two mutants H1-2
Antigen Her2 is Recombinant Human ErbB2/Fc Chimera, available from R and D Systems, article No. 1129-ER
Utilize Biacore 3000 (Malmqvist M. (1999) BIACORE:an affinity biosensor system for characterization of biomolecular interactions. Biochem Soc Trans.Feb; 27 (2): 335-40) survey humanized antibody H1-2 and antigenic absolute avidity.Select the CM-5 vane for use, the surface adsorption Sensor Chip CM 5.Two flow-through cells (pathway) are all with the flow velocity activation 6min of 15 μ l/min; 2 μ g antigen Her2 are dissolved in the phosphoric acid solution of 50 μ l pH7.0; Flow velocity with 10 μ l/min injects chip circulation pond 1, and its surface density is about 10K Ru, with the 1M thanomin chip surface is sealed behind the antigenic mark again.In conjunction with condition: each detects the antibody original liquid concentration is 10mg/ml, is diluted to 200 μ g/ml successively, 100 μ g/ml, and 50 μ g/ml, 25 μ g/ml, 12 μ g/ml, 0 μ g/ml injects two flow-through cells of chip, last kind of 3min with the speed of 10 μ l/min; The condition of dissociating: flow velocity 10 μ l/min, 3min dissociates.Chip regeneration condition: 10mM NaOH, 1M NaCl.With Biacore evaluation software analysis antibody affinity costant KD.
Through calculating H1-2 antibody affinity costant KD=10 -8M.
Sequence table
< 110>China Science & Technology University
< 120>a kind of humanized antibody and humanization modified method thereof
<160>10
<210>1
<211>489
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>1
Asp Ile Val Leu Ser Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Leu Asn Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser
20 25 30
Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Trp Ala Phe Thr Arg Lys Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Ser Asn Tyr Pro Trp Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu
100 105 110
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
130 135 140
Val Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
145 150 155 160
Tyr Ser Phe Thr Gly Tyr Phe Ile Asn Trp Val Lys Gln Asn Pro Gly
165 170 175
Gln Arg Leu Glu Trp Ile Gly His Ile Ser Ser Ser Tyr Ala Thr Ser
180 185 190
Thr Tyr Asn Glu Arg Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr
195 200 205
Ser Ala Ser Thr Ala Tyr Val Glu Leu Ser Ser Leu Arg Ser Glu Asp
210 215 220
Thr Ala Val Tyr Phe Cys Val Arg Ser Gly Asn Tyr Glu Glu Tyr Ala
225 230 235 240
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala
245 250 255
Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
260 265 270
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
275 280 285
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
290 295 300
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
305 310 315 320
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
325 330 335
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
340 345 350
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
355 360 365
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
370 375 380
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
385 390 395 400
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
405 410 415
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
420 425 430
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
435 440 445
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
450 455 460
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
465 470 475 480
Lys Ser Leu Ser Leu Ser Pro Gly Lys
485
<210>2
<211>1470
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
gacatcgttt tgtctcaatc tccagactct ttggctgttt ctttgggtga aagagttact 60
ttgaactgta agtcttctca aactttgttg tactctaaca accaaaagaa ctacttggct 120
tggtaccaac aaaagccagg tcaatctcca aagttgttga tctcttgggc tttcactaga 180
aagtctggtg ttccagacag attctctggt tctggttctg gtactgactt cactttgact 240
atctcttctg ttcaagctga agacgttgct gtttactact gtcaacaata ctctaactac 300
ccatggactt tcggtgctgg tactaagttg gaattgaaga gaggtggtgg tggttctggt 360
ggtggtggtt ctggtggtgg tggttccggt ggtggtggtt cccaagttca attggttcaa 420
tctggtgctg aagttgttaa gccaggtgct tctgttaaga tcagttgtaa ggcttctggt 480
tactctttca ctggttactt catcaactgg gttaagcaaa acccaggtca aagattggaa 540
tggatcggtc acatctcttc ttcttacgct acttctactt acaacgaaag attcaagggt 600
aaggctactt tgactgttga cacttctgct tctactgctt acgttgaatt gtcttctttg 660
agatcggaag acactgctgt ttacttctgt gttagatcgg gtaactacga agaatacgct 720
atggactact ggggtcaagg tactttggtt actgtttctt cagcggccgc agagcccaaa 780
tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 840
tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 900
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 960
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 1020
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1080
tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1140
gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1200
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1260
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1320
gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1380
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1440
aagagcctct ccctgtctcc gggtaaatga 1470
<210>3
<211>489
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>3
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Ile Asn Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser
20 25 30
Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Trp Ala Phe Thr Arg Lys Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Ser Asn Tyr Pro Trp Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
130 135 140
Val Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
145 150 155 160
Tyr Ser Phe Thr Gly Tyr Phe Ile Asn Trp Val Lys Gln Asn Pro Gly
165 170 175
Gln Arg Leu Glu Trp Ile Gly His Ile Ser Ser Ser Tyr Ala Thr Ser
180 185 190
Thr Tyr Asn Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr
195 200 205
Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
210 215 220
Thr Ala Val Tyr Tyr Cys Val Arg Ser Gly Asn Tyr Glu Glu Tyr Ala
225 230 235 240
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala
245 250 255
Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
260 265 270
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
275 280 285
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
290 295 300
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
305 310 315 320
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
325 330 335
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
340 345 350
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
355 360 365
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
370 375 380
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
385 390 395 400
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
405 410 415
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
420 425 430
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
435 440 445
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
450 455 460
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
465 470 475 480
Lys Ser Leu Ser Leu Ser Pro Gly Lys
485
<210>4
<211>1470
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>4
gacatcgttt tgactcaatc tccagactct ttggctgttt ctttgggtga aagagttact 60
attaactgta agtcttctca aactttgttg tactctaaca accaaaagaa ctacttggct 120
tggtaccaac aaaagccagg tcaatctcca aagttgttga tctcttgggc tttcactaga 180
aagtctggtg ttccagacag attctctggt tctggttctg gtactgactt cactttgact 240
atctcttctg ttcaagctga agacgttgct gtttactact gtcaacaata ctctaactac 300
ccatggactt tcggtgctgg tactaagttg gaaattaaga gaggtggtgg tggttctggt 360
ggtggtggtt ctggtggtgg tggttccggt ggtggtggtt cccaagttca attggttcaa 420
tctggtgctg aagttgttaa gccaggtgct tctgttaaga tcagttgtaa ggcttctggt 480
tactctttca ctggttactt catcaactgg gttaagcaaa acccaggtca aagattggaa 540
tggatcggtc acatctcttc ttcttacgct acttctactt acaaccaaaa attcaagggt 600
aaggctactt tgactgttga cacttctgct tctactgctt acatggaatt gtcttctttg 660
agatcggaag acactgctgt ttactactgt gttagatcgg gtaactacga agaatacgct 720
atggactact ggggtcaagg tactttggtt actgtttctt cagcggccgc agagcccaaa 780
tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 840
tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 900
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 960
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 1020
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1080
tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1140
gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1200
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1260
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1320
gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1380
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1440
aagagcctct ccctgtctcc gggtaaatga 1470
<210>5
<211>489
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>5
Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly
1 5 10 15
Glu Arg Val Thr Ile Asn Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser
20 25 30
Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Trp Ala Phe Thr Arg Lys Ser Gly Val
50 55 60
Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Ser Asn Tyr Pro Trp Thr Phe Gly Ala Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu
130 135 140
Val Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
145 150 155 160
Tyr Ser Phe Thr Gly Tyr Phe Ile Asn Trp Val Lys Gln Asn Pro Gly
165 170 175
Gln Arg Leu Glu Trp Ile Gly His Ile Ser Ser Ser Tyr Ala Thr Ser
180 185 190
Thr Tyr Asn Glu Arg Phe Lys Gly Lys Ala Thr Leu Thr Val Asp Thr
195 200 205
Ser Ala Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
210 215 220
Thr Ala Val Tyr Tyr Cys Val Arg Ser Gly Asn Tyr Glu Glu Tyr Ala
225 230 235 240
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala
245 250 255
Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
260 265 270
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
275 280 285
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
290 295 300
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
305 310 315 320
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
325 330 335
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
340 345 350
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
355 360 365
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
370 375 380
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
385 390 395 400
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
405 410 415
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
420 425 430
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
435 440 445
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
450 455 460
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
465 470 475 480
Lys Ser Leu Ser Leu Ser Pro Gly Lys
485
<210>6
<211>1470
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>6
gacatcgttt tgactcaatc tccagactct ttggctgttt ctttgggtga aagagttact 60
attaactgta agtcttctca aactttgttg tactctaaca accaaaagaa ctacttggct 120
tggtaccaac aaaagccagg tcaatctcca aagttgttga tctcttgggc tttcactaga 180
aagtctggtg ttccagacag attctctggt tctggttctg gtactgactt cactttgact 240
atctcttctg ttcaagctga agacgttgct gtttactact gtcaacaata ctctaactac 300
ccatggactt tcggtgctgg tactaagttg gaaattaaga gaggtggtgg tggttctggt 360
ggtggtggtt ctggtggtgg tggttccggt ggtggtggtt cccaagttca attggttcaa 420
tctggtgctg aagttgttaa gccaggtgct tctgttaaga tcagttgtaa ggcttctggt 480
tactctttca ctggttactt catcaactgg gttaagcaaa acccaggtca aagattggaa 540
tggatcggtc acatctcttc ttcttacgct acttctactt acaacgaaag attcaagggt 600
aaggctactt tgactgttga cacttctgct tctactgctt acatggaatt gtcttctttg 660
agatcggaag acactgctgt ttactactgt gttagatcgg gtaactacga agaatacgct 720
atggactact ggggtcaagg tactttggtt actgtttctt cagcggccgc agagcccaaa 780
tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 840
tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 900
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 960
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 1020
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1080
tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1140
gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1200
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1260
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1320
gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1380
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1440
aagagcctct ccctgtctcc gggtaaatga 1470
<210>7
<211>489
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>7
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ser Ser Gln Thr Leu Leu Tyr Ser
20 25 30
Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys
35 40 45
Ala Pro Lys Leu Leu Ile Ser Trp Ala Phe Thr Arg Lys Ser Gly Val
50 55 60
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
85 90 95
Tyr Ser Asn Tyr Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
130 135 140
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
145 150 155 160
Tyr Ser Phe Thr Gly Tyr Phe Ile Asn Trp Val Arg Gln Ala Pro Gly
165 170 175
Lys Gly Leu Glu Trp Val Ala His Ile Ser Ser Ser Tyr Ala Thr Ser
180 185 190
Thr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Val Asp Thr
195 200 205
Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
210 215 220
Thr Ala Val Tyr Tyr Cys Val Arg Ser Gly Asn Tyr Glu Glu Tyr Ala
225 230 235 240
Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ala
245 250 255
Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
260 265 270
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
275 280 285
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
290 295 300
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
305 310 315 320
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
325 330 335
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
340 345 350
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
355 360 365
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
370 375 380
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
385 390 395 400
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
405 410 415
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
420 425 430
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
435 440 445
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
450 455 460
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
465 470 475 480
Lys Ser Leu Ser Leu Ser Pro Gly Lys
485
<210>8
<211>1470
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>8
gacatccaat tgactcaatc tccatcttct ttgtctgctt ctgttggtga cagagttact 60
atcacttgta gatcctctca aactttgttg tactctaaca accaaaagaa ctacttggct 120
tggtaccaac aaaagccagg taaggctcca aagttgttga tctcttgggc tttcactaga 180
aagtctggtg ttccatctag attctctggt tctggttctg gtactgactt cactttgact 240
atctcttctt tgcaaccaga agacttcgct acttactact gtcaacaata ctctaactac 300
ccatggactt tcggtcaagg tactaaggtt gaaatcaaga gaggtggtgg tggttctggt 360
ggtggtggtt ctggtggtgg tggttccggt ggtggtggtt ccgaagttca attggttgaa 420
tctggtggtg gtttggttca accaggtggt tctttgagat tgtcttgtgc tgcttctggt 480
tactctttca ctggttactt catcaactgg gttagacaag ctccaggtaa gggtttggaa 540
tgggttgctc acatctcttc ttcttacgct acttctactt acgctgactc tgttaagggt 600
agattcacta tctctgttga cacttctaag aacactgctt acttgcaaat gaactctttg 660
agagctgaag acactgctgt ttactactgt gttagatccg gtaactacga agaatacgct 720
atggactact ggggtcaagg tactttggtt actgtttctt cagcggccgc agagcccaaa 780
tcttgtgaca aaactcacac atgcccaccg tgcccagcac ctgaactcct ggggggaccg 840
tcagtcttcc tcttcccccc aaaacccaag gacaccctca tgatctcccg gacccctgag 900
gtcacatgcg tggtggtgga cgtgagccac gaagaccctg aggtcaagtt caactggtac 960
gtggacggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gtacaacagc 1020
acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa tggcaaggag 1080
tacaagtgca aggtctccaa caaagccctc ccagccccca tcgagaaaac catctccaaa 1140
gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggaggagatg 1200
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 1260
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1320
gactccgacg gctccttctt cctctatagc aagctcaccg tggacaagag caggtggcag 1380
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1440
aagagcctct ccctgtctcc gggtaaatga 1470
<210>9
<211>489
<212>PRT
< 213>artificial sequence
<220>
<223>
<400>9
Asp Ile Val Leu Thr Gln Thr Pro Ser Ser Leu Pro Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Lys Ser Ser Gln Thr Leu Leu Tyr Ser
20 25 30
Asn Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Ser Trp Ala Phe Thr Arg Lys Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Ile Gly Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Tyr Ser Asn Tyr Pro Trp Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile
100 105 110
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu
130 135 140
Val Val Lys Thr Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
145 150 155 160
Tyr Ser Phe Thr Gly Tyr Phe Ile Asn Trp Val Lys Lys Asn Ser Gly
165 170 175
Lys Ser Pro Glu Trp Ile Gly His Ile Ser Ser Ser Tyr Ala Thr Ser
180 185 190
Thr Tyr Asn Gln Lys Phe Lys Asn Lys Ala Ala Phe Thr Val Asp Thr
195 200 205
Ser Ser Ser Thr Ala Phe Met Gln Leu Asn Ser Leu Thr Ser Glu Asp
210 215 220
Ser Ala Val Tyr Tyr Cys Val Arg Ser Gly Asn Tyr Glu Glu Tyr Ala
225 230 235 240
Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala
245 250 255
Ala Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
260 265 270
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
275 280 285
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
290 295 300
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
305 310 315 320
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
325 330 335
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
340 345 350
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
355 360 365
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
370 375 380
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
385 390 395 400
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
405 410 415
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
420 425 430
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
435 440 445
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
450 455 460
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
465 470 475 480
Lys Ser Leu Ser Leu Ser Pro Gly Lys
485
<210>10
<211>603
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>10
gaggctgaag cttacgtaga attcagcacc caagtgtgca ccggcacaga catgaagctg 60
cggctccctg ccagtcccga gacccacctg gacatgctcc gccacctcta ccagggctgc 120
caggtggtgc agggaaacct ggaactcacc tacctgccca ccaatgccag cctgtccttc 180
ctgcaggata tccaggaggt gcagggctac gtgctcatcg ctcacaacca agtgaggcag 240
gtcccactgc agaggctgcg gattgtgcga ggcacccagc tctttgagga caactatgcc 300
ctggccgtgc tagacaatgg agacccgctg aacaatacca cccctgtcac aggggcctcc 360
ccaggaggcc tgcgggagct gcagcttcga agcctcacag agatcttgaa aggaggggtc 420
ttgatccagc ggaaccccca gctctgctac caggacacga ttttgtggaa ggacatcttc 480
cacaagaaca accagctggc tctcacactg atagacacca accgctctcg ggcctgccac 540
ccctgttctc cgatgtgtaa gggctcccgc tgctggggag agagttctga ggattgtcag 600
agc 603

Claims (10)

1. albumen is following arbitrary said protein:
A) by the protein of forming from N-terminal 1-253 amino acids sequence in the sequence in the sequence table 3;
B) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 3;
E) by the protein of forming from N-terminal 1-253 amino acids sequence in the sequence in the sequence table 1;
F) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
I) by the protein of forming from N-terminal 1-253 amino acids sequence in the sequence in the sequence table 5;
J) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 5.
2. the said proteic encoding sox of claim 1.
3. encoding sox according to claim 2 is characterized in that: said encoding sox is for following arbitrary said:
1) encode said a) shown in proteic encoding sox be: nucleotide sequence be in the sequence table sequence 4 from 5 ' terminal 1-759 position Nucleotide shown in dna molecular;
2) proteic encoding sox is the said b that encodes): nucleotide sequence is a dna molecular shown in the sequence 4 in the sequence table;
3) proteic encoding sox is the said e that encodes): nucleotide sequence be in the sequence table sequence 2 from dna molecular shown in the Nucleotide of 5 ' terminal 1-759 position;
4) proteic encoding sox is the said f that encodes): nucleotide sequence is a dna molecular shown in the sequence 2 in the sequence table;
5) proteic encoding sox is the said i that encodes): nucleotide sequence be in the sequence table sequence 6 from dna molecular shown in the Nucleotide of 5 ' terminal 1-759 position;
6) proteic encoding sox is the said j that encodes): nucleotide sequence is a dna molecular shown in the sequence 6 in the sequence table.
4. the recombinant vectors that contains claim 2 or 3 said encoding soxs.
5. the reorganization bacterium that contains claim 2 or 3 said encoding soxs.
6. the transgenic cell line that contains claim 2 or 3 said encoding soxs.
7. the recombinant virus that contains claim 2 or 3 said encoding soxs.
8. the expression cassette that contains claim 2 or 3 said encoding soxs.
9. an antibody is made up of albumen described in two identical claims 1; Said two identical albumen connect through disulfide linkage.
10. antibody described in said albumen of claim 1 or claim 2 or 3 said encoding soxs or the claim 9 suppresses the application in the Her2 high expression level tumor growth medicine in preparation.
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CN103145834B (en) * 2013-01-17 2015-03-18 广州泰诺迪生物科技有限公司 Antibody humanization transformation method
NL2011406C2 (en) 2013-09-06 2015-03-10 Bionovion Holding B V Method for obtaining april-binding peptides, process for producing the peptides, april-binding peptides obtainable with said method/process and use of the april-binding peptides.
CN104225594B (en) * 2014-09-23 2016-03-23 合肥瀚科迈博生物技术有限公司 Anti-HER 2 humanized antibody and relevant anti-tumor compositions thereof
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CN105131117B (en) * 2015-09-25 2018-10-12 中国人民解放军第四军医大学 The humanization modified of targeting HER2 is internalized by single-chain antibody P1h2 and application
CN105131116B (en) * 2015-09-25 2018-10-09 中国人民解放军第四军医大学 The humanization modified of targeting HER2 is internalized by single-chain antibody P1h3 and preparation method and application
CN107446047B (en) * 2017-09-04 2020-08-11 山西省生物研究院有限公司 Bifunctional anti-tissue factor humanized antibody and preparation method thereof
TW202128748A (en) * 2020-01-24 2021-08-01 日商西斯美股份有限公司 Method for improving affinity of antibody for antigen and use thereof
CN111635457A (en) * 2020-05-22 2020-09-08 上海药明生物医药有限公司 Method for humanizing nano antibody
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