CN108299557A

CN108299557A - IgG classes antibody is improved to the binding affinity of FcRn and extends the method for its serum half-life

Info

Publication number: CN108299557A
Application number: CN201710024748.9A
Authority: CN
Inventors: 张发明; 席甘; 黄莺
Original assignee: Hangzhou Hice Biological Medicine Co Ltd
Current assignee: Hangzhou Hice Biological Medicine Co Ltd
Priority date: 2017-01-13
Filing date: 2017-01-13
Publication date: 2018-07-20

Abstract

The invention discloses improving IgG classes antibody to the binding affinity of FcRn and extend the method for its serum half-life, this method be the heavy chain constant region for making the IgG classes antibody FcRn binding sites region the 254th, 308,434 amino acids mutate.Thereby, it is possible to effectively improve IgG classes antibody to the binding affinity of FcRn and extend its serum half-life, also, by the IgG class antibody of modification, will not be reduced with the binding affinity of corresponding antigens.

Description

IgG classes antibody is improved to the binding affinity of FcRn and extends its serum half-life Method

Technical field

The present invention relates to immunologys and antibody engineering technical field, and in particular to improves combination of the IgG classes antibody to FcRn Affinity and the method for extending its serum half-life, and modification IgG class antibody.

Background technology

Antibody is also immunoglobulin (Ig), is a kind of protein of energy molecule of the antigen binding.Antibody is by 4 peptide chains Composition, larger two heavy chains (H chains, 50kD) of molecular weight and the smaller two light chains (L chains, 23kD) of molecular weight, H chains and L chains It is connected by disulfide bond.The hydroxyl terminal of polypeptide chain, the amino acid classes that this region includes relatively are stablized, without apparent number difference The variation of different and order, referred to as constant region or stable region, the i.e. areas C, this end account for the 3/4 of the 1/2 and H chains of L chains.It is not of the same race in aminoterminal The characteristics of amino acid, which puts in order, to be changed, this region is known as variable region, the i.e. areas V, and variable region height becomes determines antibody Diversity also determines antibody and specific antigen binding.

The mankind there are five types of different classes of antibody, including IgA (including subclass IgA1 and IgA2), IgD, IgE, IgG (including Subclass IgG1, IgG2, IgG3 and IgG4) and IgM.IgG is most important immune globulin classes in the mankind, is usually used in treating In.But natural IgG resists its one of critical issue in therapeutical uses to be that their persistence in blood circulation are low, blood Clear half-life short, so that the effect of speed of cleaning antibody directly affects treatment, and therefore influence to cause side effect in patients And also increase the frequency and amount of the medicament administration of medical expense.Therefore, extend IgG classes antibody (especially natural IgG classes antibody) Serum half-life and improve its binding affinity to FcRn, it is significant.

However, extending the serum half-life of IgG classes antibody (especially natural IgG classes antibody) at present and improving it to FcRn The method of binding affinity still need further to be studied.

Invention content

The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention It is to propose a kind of means improving IgG classes antibody to the binding affinity of FcRn and extend its serum half-life.

It should be noted that the present invention is following discovery based on inventor and work and completes：

With papain hydrolysis IgG antibody, it can be cut off from the N-terminal position of hinge area disulfide bond, obtain three pieces Section：Two identical to be known as Fab segments with the segment of antigen binding；One crystallizable segment is known as Fc segments.Antibody The areas Fc interact with a large amount of Fc receptors and ligand, assign the performance of some important effector functions of antibody.Effector functions Including starting the cytotoxicity (CDC) of Complement Dependent, starting the cell-mediated cytotoxicity of phagocytosis and antibody-dependant (ADCC) and antibody is transported by transcytosis and passes through barrier cell.In addition, the regions FC are to maintaining partly declining for IgG class antibody serums Phase is extremely important.

Neonatal Fc receptor (FcRn) is responsible for the receptor of epithelial cell active transport Immunoglobulin IgG, be by α chains and The heterodimer that two subunits of β chains are formed in the form of non-covalent bond.The parts Fc of IgG molecules include 2 identical polypeptides Chain, wherein every polypeptide chain combines single FcRn molecules by its FcRn binding site.In Adult Mammals, IgG passes through The parts Fc are combined with FcRn, to protect IgG antibody from degradation, are played a crucial role in maintaining antibody level of serum.IgG points Son, if they are combined with FcRn, is re-circulated into cycle by after endothelial cell endocytosis.In contrast, not with FcRn In conjunction with IgG molecules enter cell, by lysosomal degradation.Thus, the regions FC for IgG antibody to the binding affinity of FcRn It is strong and weak also most important.

Thus, inventor attempts the sequence by changing IgG antibody heavy chain constant region (i.e. the areas FC), finds and extends IgG classes The serum half-life of antibody (especially natural IgG classes antibody) simultaneously improves its method to the binding affinity of FcRn.That is, hair A person of good sense is intended to the ends Fc by being mutated IgG antibody, reaches and changes its serum half-life, and the mesh of binding affinity to FcRn 's.By a series of experimental design and exploration, inventor surprisingly has found, several to be introduced in human IgG molecule constant region Based on a mutation, these mutation change affinity of the IgG molecules to FcRn, and the serum to change antibody partly declines Phase.Specifically, it is introduced in the amino acid residue 254,308,434 of the heavy chain constant region of IgG class antibody and is different from unmodified resist Amino acid residue in body, this mutation is so that the antibody of optimization has the serum half-life longer than wild-type antibodies.Change speech It, inventor find, make the heavy chain constant region of IgG class antibody FcRn binding sites region the 254th, 308,434 amino acids Mutate, can effectively improve the IgG antibody to the binding affinity of FcRn, extend its serum half-life.

In turn, in one aspect of the invention, the present invention provides a kind of raising IgG classes antibody is affine to the combination of FcRn Power and the method for extending its serum half-life.According to an embodiment of the invention, this method is to keep the heavy chain of the IgG classes antibody permanent Determine area FcRn binding sites region the 254th, 308,434 amino acids mutate.It is anti-thereby, it is possible to effectively improve IgG classes Body is to the binding affinity of FcRn and extends its serum half-life, also, by the IgG class antibody of modification, with corresponding antigens Binding affinity will not reduce.

Wherein, it should be noted that make the heavy chain constant region of the IgG classes antibody FcRn binding sites region the 254th, 308, the method that 434 amino acids mutate is not particularly limited, such as chemical derivatization, fixed-point mutation method etc., as long as It can effectively realize purpose site mutation.Under normal circumstances, it can be carried out from gene level, that is, be based on amino acid mutation pair The encoding gene of pending IgG class antibody is carried out corresponding rite-directed mutagenesis, then, base by the encoding gene mutated site answered It can be readily available IgG classes antibody variants (alternatively referred to as " the IgG classes antibody of modification ") in the antibody-encoding genes of mutation.From For this respect, IgG classes antibody is improved to the binding affinity of FcRn and extends the method for its serum half-life, can also be considered as It is to be based on known IgG classes antibody, obtains a kind of new, to the binding affinity enhancing of FcRn, extended serum half lives IgG classes The method of antibody (i.e. IgG classes antibody variants).

According to an embodiment of the invention, make the FcRn binding sites region of the heavy chain constant region of the IgG classes antibody 254,308,434 amino acids sport threonine, proline and alanine respectively.The serum of the IgG class antibody partly declines as a result, Phase extends, to the enhancing of the binding affinity of FcRn significantly.

In still another aspect of the invention, the present invention provides a kind of IgG class antibody of modification.Implementation according to the present invention Example, relative to wild type IgG class antibody, the FcRn binding sites region of the heavy chain constant region of the IgG class antibody of the modification the 254,308,434 amino acids are mutated.It is surprisingly found by the inventors that the IgG class antibody of the modification, relative to wild type IgG class antibody, to the binding affinity enhancing of FcRn and extended serum half lives, also, the combination of itself and corresponding antigens is affine Power will not reduce.

According to an embodiment of the invention, relative to wild type IgG class antibody, the heavy chain of the IgG class antibody of the modification is permanent Determine area FcRn binding sites region the 254th, 308,434 amino acids be respectively threonine, proline and alanine.As a result, Relative to wild type IgG class antibody, the extended serum half lives of the IgG class antibody enhance more the binding affinity of FcRn Significantly.

In another aspect of this invention, the present invention provides a kind of sides for the IgG class antibody preparing foregoing modification Method.According to an embodiment of the invention, this approach includes the following steps：

According to the amino acid sequence of purpose IgG class antibody, the nucleic acid sequence of purpose IgG class antibody described in full genome composite coding Row；

Structure encodes the expression vector of the nucleic acid sequence of the purpose IgG class antibody；And

Antibody preparation vehicles cells are transfected using the expression vector, to make the Antibody preparation vehicles cells expression simultaneously Secrete the IgG class antibody of the modification.

Object here IgG class antibody is the IgG class antibody of the modification.Thereby, it is possible to quickly and easily prepare to obtain The IgG classes antibody (i.e. purpose IgG classes antibody) that must be modified, and relative to wild type IgG class antibody, pair of the IgG class antibody The binding affinity of FcRn enhances and extended serum half lives.In other words, according to an embodiment of the invention, the side of the present invention is utilized Method, can prepare that a kind of binding affinity to FcRn is strong, the new IgG class antibody of serum half-life length.Furthermore, it is necessary to Illustrate, the IgG class antibody of modification of the invention, relative to wild type IgG class antibody, the binding affinity with corresponding antigens It will not reduce.

The type for the Antibody preparation vehicles cells that may be used is not particularly limited, as long as can make corresponding IgG classes antibody Effective expression and secretion, such as mouse cell, rat cell, rabbit cell, common people's vehicles cells may be used etc.. According to the embodiment of the present invention, the Antibody preparation vehicles cells are 293 cells.More convenient, product yield higher is prepared as a result, Effect is more preferable.

Also, the type of expression vector, specific cell transfecting method are also not particularly limited, as long as disclosure satisfy that antibody table The requirement reached.

Wherein, term " amino acid " used herein means to can reside in 20 kinds on position that is specific, determining One of natural amino acid or arbitrary non-natural analogs.Can abridge native amino with three-letter code or with one-letter code Acid：

Expression way " the n-th amino acids " used herein (such as the 254th, 308,434 amino acids) be finger protein Position in the sequence of matter.It, can be according to the EU index numbers position in Kabat for the areas Fc being directed in the present invention.

The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description Obviously, or practice through the invention is recognized.

Description of the drawings

The above-mentioned and/or additional aspect and advantage of the present invention will become in the description from combination following accompanying drawings to embodiment Obviously and it is readily appreciated that, wherein：

Fig. 1 is the result figure of H8L2 and H2L2 to the combination ELISA of PD1 according to an embodiment of the invention；

Fig. 2 is result figures of the H8L2 and H2L2 to the Pd1 and PdL1 competition Elisa inhibited according to an embodiment of the invention；

Fig. 3 is result figures of the H8L2 and H2L2 to the Pd1 and PdL2 competition Elisa inhibited according to an embodiment of the invention；

Fig. 4 is H8L2 and H2L2 hemodynamic characteristics testing result figures according to an embodiment of the invention；

Fig. 5 is that according to an embodiment of the invention, antibody H8L2 and H2L2 is thin by blocking PD-1 protein functions activation stimulation T Intracrine IL-2 level views；

Fig. 6 is that according to an embodiment of the invention, antibody H8L2 and H2L2 is thin by blocking PD-1 protein functions activation stimulation T Intracrine IFNgamma level views；

Fig. 7 be according to an embodiment of the invention, in the research of machin serum-concentration, H8L2 and H2L2 that ELISA is measured Time front of blood concentration；

Fig. 8 be according to an embodiment of the invention, in machin pharmacokinetic, the 1mg/kg individual blood of variant H8L2 Concentration data；

Fig. 9 be according to an embodiment of the invention, in machin pharmacokinetic, the 3mg/kg individual blood of variant H8L2 Concentration data；

Figure 10 is that according to an embodiment of the invention, in machin pharmacokinetic, the 10mg/kg of variant H8L2 is individual Plasma drug concentration data

Figure 11 is that according to an embodiment of the invention, in machin pharmacokinetic, the 10mg/kg of wild type H2L2 is a Body plasma drug concentration data；

Figure 12 be according to an embodiment of the invention, in machin pharmacokinetic, variant H8L2 and wild type H2L2 Average effective half-life period.

Specific implementation mode

The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment Part, according to technology described in document in the art or condition, (such as with reference to works such as J. Pehanorm Brookers, Huang Peitang etc. is translated 's《Molecular Cloning:A Laboratory guide》, the third edition, Science Press) or carry out according to product description.Agents useful for same or instrument Production firm person is not specified, being can be with conventional products that are commercially available, such as can purchase from Illumina companies.

The protein expression of embodiment 1H8L2 mutant

On the basis of humanized antibody H2L2 (the IgG classes antibody of anti-PD-1), the FcRn of its heavy chain constant region is made to tie Conjunction site areas the 254th, 308,434 amino acids sport threonine, proline and alanine respectively, and it is (anti-to be named as H8L2 The IgG classes antibody variants of PD-1).

That is, purpose antibody H8L2 mutant, relative to humanized antibody H2L2, the FcRn of heavy chain constant region is tied Conjunction site areas the 254th, 308,434 amino acids sport threonine, proline and alanine respectively, other regional sequences are not Become.

In practical operation, the nucleic acid sequence of full genome composite coding humanized antibody H8L2, and it is building up to expression vector.It carries Take expression vector dna, 293 cell of transfection mammalian cell.After cell transfecting, antibody is expressed in mammalian cell, and It is secreted into extracellular.Then, by antibody A affinity column, the antibody of expression is purified, that is, obtains humanized antibody H8L2 eggs In vain.

As previously mentioned, humanized antibody H2L2 and H8L2 antibody, only the FcRn binding sites region of heavy chain constant region the 254,308,434 amino acids are different, thus the H8L2 sequences for only providing H8L2 mutant below are for reference.

H8L2 heavy chain amino acid sequences：

EVQLVQSGGGLVQPGGSLKLSCAASGFTFSSYGMSWVRQAPGKGLDWVATISGGGRDTYYPDSVKGRFTISRDNSKN NLYLQMNSLRAEDTALYYCARQKGEAWFAYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPE FLGGPSVFLFPPKPKDTLMIRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT LHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHHYTQKSLSLSLGK(SEQ ID NO：1), Wherein, the part of underscore mark is antibody variable region, and box label is mutational sites of the H8L2 relative to antibody H2L2, i.e., The FcRn binding sites region of heavy chain constant region the 254th, 308,434 amino acids.

Wherein, relative to humanized antibody H2L2, the FcRn binding sites area of the heavy chain constant region of H8L2 mutant 254th amino acids in domain by mutant serine be threonine, the 308th amino acids by valine mutation be proline, the 254th Amino acids are alanine by asparagine mutation.

Encode the nucleic acid sequence of H8L2 heavy chains：

ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGCGCGCACTCCGAGGTGCAGCTGGTGCAGTC TGGCGGCGGACTGGTGCAGCCCGGCGGGTCACTGAAGCTGAGCTGCGCCGCTTCCGGCTTCACCTTTAGCTCCTACG GAATGTCCTGGGTGCGACAGGCACCCGGGAAGGGGCTGGACTGGGTCGCTACTATCTCAGGAGGCGGGAGAGACACC TACTATCCTGATAGCGTCAAGGGCCGGTTCACAATTAGCCGGGACAACAGCAAGAACAATCTGTACCTGCAGATGAA CAGCCTGAGGGCTGAGGATACTGCACTGTACTATTGTGCCCGCCAGAAGGGCGAAGCATGGTTTGCCTATTGGGGCC AGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGC ACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTC AGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGG TGACTGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTG GACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCACCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGT CTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCACCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACG TGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCG CGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCCCCCTGCACCAGGACTGGCTGAACGGCAA GGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGC CCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG GTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCAC GCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGG GGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACGCCCACTACACACAGAAGAGCCTCTCCCTGTCTCTG GGTAAA(SEQ ID NO：2), wherein the part of underscore mark is antibody variable region.

H8L2 light-chain amino acid sequences：

DIVLTQSPASLAVSPGQRATITCRASESVDNYGISFMNWFQQKPGQPPKLLIYAASNKGTGVPARFSGSGSGTDFTL NINPMEENDTAMYFCQQSKEVPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO： 3), wherein the part of underscore mark is antibody variable region.

Encode the nucleic acid sequence of H8L2 light chains：

ATGGGATGGAGCTGTATCATCCTCTTCTTGGTAGCAACAGCTACAGGCGTGCACTCCGATATTGTGCTGACTCAGAG CCCTGCTTCCCTGGCCGTGTCTCCAGGACAGCGAGCTACCATCACATGCAGAGCATCTGAGAGTGTGGACAACTACG GAATTAGTTTCATGAATTGGTTTCAGCAGAAGCCCGGCCAGCCCCCTAAACTGCTGATCTATGCCGCCAGCAACAAG GGCACCGGGGTGCCTGCTCGATTCTCAGGAAGCGGCTCCGGGACAGACTTTACTCTGAACATTAACCCAATGGAGGA AAATGATACAGCAATGTACTTCTGCCAGCAGAGCAAGGAGGTGCCCTGGACCTTTGGCGGGGGAACAAAGCTGGAAA TCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCT GTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGG TAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCA AAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGC TTCAACAGGGGAGAGTGT(SEQ ID NO：4), wherein the part of underscore mark is antibody variable region.

Embodiment 2H8L2 mutant recombinant humanized antibody ELISAs are tested

For the H8L2 antibody that H2L2 antibody and embodiment 1 prepare, ELISA Binding experiments and competitive ELISA are carried out Comparative experiments, it is specific as follows：

1,18A10 H8L2,18A10 H2L2 ELISA Binding experiments

It is as follows：

1) envelope antigen：0.25 μ g/ml of PD-1-his antigens, 100 holes μ l/, 4 DEG C of coatings are overnight；

2) 37 DEG C of 1%BSA (PBS dilutions) is closed 2 hours, and 1 × PBST (Tween-20,1%) is washed 3 times, is gently patted dry；

3) primary antibody：2 μ g/ml, 1:3 gradient concentrations of gradient dilution 7, blank control group PBS, 37 DEG C are incubated 1 hour；

4) secondary antibody：PBST is washed 3 times, is gently patted dry, and 1 is added per 100 μ l of hole:10000 diluted HRP enzymes mark goat-anti people IgG (H+L) secondary antibody, 37 DEG C are incubated 1 hour；

5) it develops the color：PBST is washed 3 times, is gently patted dry；TMB color developing agents are added per 100 μ l of hole, react at room temperature 5~10min；

6) colour developing terminates：2M H are added in 50 holes μ l/₂SO₄Solution color development stopping is reacted；

7) it reads：In microplate reader, the light absorption value in each hole is detected with absorbance 450nm.

The result is shown in Figure 1 can calculate the EC of H8L2 and H2L2 to PD-1₅₀Value is respectively 0.04nM and 0.05nM.It can by Fig. 1 Know, the mutation on FcRN calmodulin binding domain CaMs does not influence antibody and PD-1 affinity.

2,18A10 H8L2,18A10 H2L2 and PDL1 competitive ELISAs are tested

It is as follows：

1) envelope antigen：0.5 μ g/ml of PD-1-hIgGFc antigens, 50 holes μ l/, 4 DEG C of coatings are coated in 96 hole elisa Plates Overnight；

2) PBST board-washings 3 times, gently pat dry, and 37 DEG C of 1%BSA (PBS dilutions) is added and closes 2 hours, 1 × PBST (Tween-20,1%) it washs 3 times；

3) primary antibody：6 μ g/ml, 1:3 gradient concentrations of gradient dilution 7, blank control group PBS, 50 holes μ l/ are added to packet By on good ELISA Plate, it is incubated at room temperature 10min；

4) ligand：0.6 μ g/ml of PDL1-mIgG2aFc solution are added, 50 holes μ l/, 37 DEG C are incubated 1 hour；

5) secondary antibody：PBST is washed 3 times, is gently patted dry；It is added 1 per 50 μ l of hole:5000 diluted HRP enzymes mark sheep anti-mouse iggs (H+L) secondary antibody, 37 DEG C are incubated 1 hour；

5) it develops the color：PBST is washed 3 times, is gently patted dry；TMB color developing agents are added per 50 μ l of hole, react at room temperature 5-10min；

6) it terminates：2M H are added in 50 holes μ l/₂SO₄Color development stopping is reacted；

As a result see Fig. 2, and H8L2 and H2L2 inhibits the EC of Pd-1 and PdL1₅₀Value is respectively 0.474nM and 0.783nM. It follows that the mutation on FcRN inhibits the combination of Pd-1 and PdL1 not influence antibody.

3,18A10 H2L2 and PDL2 competitive ELISAs are tested

It is as follows：

1) envelope antigen：PD-1-hIgGFc antigen 1 .0 μ g/ml, 100 holes μ l/, 4 DEG C of coatings are coated in 96 hole elisa Plates Overnight；

2) PBST board-washings 3 times, gently pat dry, and 37 DEG C of 1%BSA (PBS dilutions) is added and closes 2 hours, 1 × PBST (Tween-20,1%) it washs 4 times；

3) primary antibody：20 μ g/ml, 1:3 gradient concentrations of gradient dilution 7, blank control group PBS, 50 holes μ l/ are added to packet By on good ELISA Plate, it is incubated at room temperature 10min；

4) ligand：1.0 μ g/ml of PDL2-his tag solution are added, 50 holes μ l/, 37 DEG C are incubated 1 hour；

5) secondary antibody：PBST is washed 5 times, is gently patted dry；It is added 1 per 50 μ l of hole:The 750 diluted anti-his tag of HRP enzymes mark are small Mouse monoclonal antibody secondary antibody, 37 DEG C are incubated 1 hour；

5) it develops the color：PBST is washed 6 times, is gently patted dry；TMB color developing agents are added per 100 μ l of hole, react at room temperature 30min；

As a result see Fig. 3, and H8L2 and H2L2 inhibits the EC of Pd-1 and PdL2₅₀Value be respectively 1.83nM and 1.58nM. by This is it is found that the mutation on FcRN inhibits the combination of Pd-1 and PdL2 not influence antibody.

Embodiment 3 measures H8L2 and H2L2 kinetic parameters using Fortebio interaction of molecules instrument

The power of H8L2 (embodiment 1 prepares) and H2L2 is measured and compared using Fortebio interaction of molecules instrument Parameter is learned, it is specific as follows：

The antigen PD-1 of biotin labeling is fixed on SA sensor surfaces, after being balanced in PBST, is combined with antibody H8L2, H8L2 with PBST three times dilute, a concentration of 200,66.67,22.22,7.41,2.47,0.82,0.27,0nM, solved in PBST From.The detection method of H2L2 is identical as H8L2.H8L2, H2L2 kinetic parameter result figure are shown in Fig. 4.From fig. 4, it can be seen that on FcRN Mutation does not influence the kinetic parameter of antibody.

Embodiment 4 mixes lymph reaction detection PD1 antibody biological activities

It is tested using mixed lymphocyte reaction (MLP) (MLR) and detects and compare H8L2 (embodiment 1 prepares) and H2L2 thorns Swash T lymphocytic emiocytosis IL-2 and IFNgamma secretion capacities, it is specific as follows：

MLR experiments are mixed using the T cell (TC) and Dendritic Cells (DC) of different people sources, anti-using DC cells Body offers ability stimulation T cell and secretes IL-2 and IFNgamma.It uses first single in cell factor GM-CSF and IL-4 inducing blood Monocyte differentiation is then immature DC cell maturations with TNFa stimulations at Dendritic Cells.DC after maturation with it is of the same race different After the TC cells in source carry out mixing 5 days, the secretion level of the IL-2 and IFNgamma in cell conditioned medium are detected.It is mixed in 96 orifice plates TC and DC is closed, by the addition TC 1 × 10 per hole⁵With DC 1 × 10⁴, setting antibody concentration is from 10 μM to 0.09765625nM totally 8 Gradient, hybrid reaction quantitatively detect supernatant IL-2 contents after 5 days, with IL-2 detection kits.Setting antibody concentration 300nM is arrived 0.1nM totally 5 gradients.After hybrid reaction 5 days, IFNgamma contents are quantitatively detected with IFNgamm detection kits.

It is as shown in Figure 5 that antibody H8L2 and H2L2 stimulation T cell secretes IL-2 secretion levels.As seen from Figure 5, antibody H8L2 and H2L2 can effectively stimulate T cell to secrete IL-2, and the ability that the mutation in the sites FcRN secretes IL-2 to antibody stimulation T cell does not have It influences.

It is as shown in Figure 6 that antibody H8L2 and H2L2 stimulation T cell secretes IFNgamma secretion levels.As seen from Figure 6, antibody H8L2 and H2L2 can effectively stimulate T cell to secrete IFNgamma, and antibody stimulation T cell is secreted in the mutation in the sites FcRN The ability of IFNgamma does not influence.Wherein, " IgG " in Fig. 6 is isotype antibody controls.

5 machin serum-concentration of embodiment is studied

Machin serum-concentration comparative studies is carried out to H8L2 (embodiment 1 prepares) and H2L2, it is specific as follows：

4 machins are randomly divided into 2 groups according to weight, and respectively H8L2 (1mg/kg) is organized and H2L2 (1mg/kg) dosage Group, every group 2.Intravenous injection administration every time.Each dosage group is before administration, 5 minutes and 5,24,72,168,240 after administration Hour acquisition whole blood, centrifuges serum.The concentration that H8L2 and H2L2 in machin serum is measured using ELISA method (see the table below And Fig. 7).

6 machin pharmacokinetic of embodiment

Machin pharmacokinetics comparative studies is carried out to H8L2 (embodiment 1 prepares) and H2L2, it is specific as follows：

24 machins are randomly divided into 4 groups, respectively wild type H2L2 groups (10mg/kg) and variant according to weight (H8L2) basic, normal, high (1,3,10mg/kg) dosage group, every group 6, half male and half female.Intravenous injection administration every time.Each dosage group Before administration, 5,30 minutes and 1,2,4,8,24,48,144,216 hour acquisition whole bloods, centrifuge serum after administration. The concentration of wild type and variant in machin serum is measured using ELISA method, and uses PhoenixWinNonlin (Pharsight) 6.4 relevant pharmacokinetic parameter is calculated.

The serum drug level level of all individuals is below lower limit of quantitation before machin single-dose.Three agent of variant group Amount group, serum drug level level increases with dosage and is increased in cynomolgus monkey；Variant is basic, normal, high (1,3,10mg/kg) The average effective half-life period of dosage group is respectively 215.72 (see Fig. 8), 288.78 (see Fig. 9), 268.92h (see Figure 10), wild The average effective half-life period of type (10m g/kg) is 224h (see Figure 11).Under Isodose (10mg/kg), variant compares wild type With longer average effective half-life period (see Figure 12).

Antitumor actions of 7 H8L2 of embodiment in HCC827 Non-small cell lung carcinoma subcutaneous transplantation MiXeno models

Human tumour transplantation model is established using NSG mouse, research H8L2 (embodiment 1 prepares) is non-small in HCC827 people Cell lung carcinoma subcutaneous transplants the antitumor action in MiXeno models, specific as follows：

NSG mouse have NOD, Prkdc_scid, IL2rg_nullMissing/variation features, be current immune deficiency degree highest, It is most suitable for the tool mouse of human archeocyte transplanting, to human archeocyte and tissue almost without rejection.Therefore, inventor selects Adopt graft-versus-host reaction (GVHD) model transferred constructed by human peripheral blood mononuclear cell (PBMC) to NSG mouse, and Thus the internal pharmacodynamics of H8L2 is weighed.Inventor establishes human tumour transplantation model (Mixeno models) with NSG mouse, grinds Study carefully antitumor actions of the H8L2 in HCC827 Non-small cell lung carcinoma subcutaneous transplantation MiXeno models.

0th day (Day 0) is inoculated with 5 × 10 in right side dorsal sc⁶HCC827 cells are in 40 NCG mouse (32 mouse In addition 8 surplus capacities) in, 6 days (Day 6) reaches 66mm when mean tumor volume after inoculated tumour cell³, select gross tumor volume compared with 32 good mouse are uniformly divided into 4 groups, every group of 8 mouse.From tail vein transplanting PBMC, in 32 NCG mouse, (1-4 groups are small Mouse) in, cell is resuspended in PBS (0.1ml is inoculated with volume).Experiment is divided into test medicine H8L2 5mg/kg and 10mg/kg, the positive Compare Opdivo 5mg/kg groups and Isotype antibody Human IgG4 5mg/kg control groups.Tail vein injection is administered, respectively at connecing It is administered within the 6th, 9,13,16,19,22 day after kind tumour cell, is administered six times (is shown in Table 1) altogether.According to Relative tumor inhibiting rate (TGI_RTV) therapeutic evaluation is carried out, safety evaluatio is carried out according to the weight of animals variation and death condition.

Table 1 is tested antitumor action experiments of the medicine H8L2 in HCC827 Non-small cell lung carcinoma Mixeno tumor models and is set Meter

Note：Administered volume is 10ul/g；n：Animal number of elements；On the day of Day 0 is tumor cell inoculation；i.v.：Tail vein is given Medicine.

Relative comparison group is vaccinated with the Isotype antibody (Human lgG4) of PBMC, and test medicine H8L2 (10mg/kg) is being inoculated with All show within the 9th, 13 day after tumour cell significant Tumor growth inhibition effect, Relative tumor inhibiting rate TGI_RTV(%) is respectively 30% (p=0.007), 30% (p=0.039))；H8L2 (5mg/kg) all performances in the 9th, 13 day after inoculated tumour cell are notable Tumor growth inhibition effect, Relative tumor inhibiting rate TGI_RTV(%) is respectively 18% (p=0.049), 25% (p= 0.041).Corresponding, Opdivo does not show significant Tumor growth inhibition effect, the 9th, 13 day after inoculated tumour cell Relative tumor inhibiting rate TGI_RTV(%) is respectively 17% (p=0.084), 23% (p=0.073) (table 2).Experiment results proved H8L2 significantly inhibits tumour growth to HCC827 people source non-small cell lung cancer Mixeno tumor models, and drug effect is relatively positive Property reference substance Opdivo is more preferable.H8L2 (10mg/kg, 5mg/kg) and Opdivo (5mg/kg) each treatment group start 16 days in administration The relevant toxic reaction of drug therapy (such as severe weight decline or dead), explanation do not occur for interior (i.e. tumor inoculation 22 days in) Treat well-tolerated.

Toxicity Analysis of the table 2 in HCC827 Non-small cell lung carcinoma Mixeno tumor models

Note：P value¹It is compared with Isotype control group (G1 Human IgG4)

Anti- PD-1 monoclonal antibody injections liquid is at 10mg/kg, 5mg/kg dosage to HCC827 people source non-small cell lung cancer Mixeno tumor models significantly inhibit tumour growth, the anti-PD-1 monoclonal antibody injections liquid suppressions of wherein 10mg/kg Tumor growth effect processed is most apparent, and drug effect is more preferable with respect to the positive reference substance Opdivo of 5mg/kg.It is mono- that tumor-bearing mice fights PD-1 Clonal antibody injection well-tolerated under proof load.

Antitumor actions of 8 H8L2 of embodiment in MC38 mouse colorectal cancer HuGEMM models

Preclinical validation is swollen using H8L2 (embodiment 1 prepares) treatment mouse MC38 in PD-1 HuGEMM mouse The drug effect of tumor model, it is specific as follows：

MC38 cells are the mouse source colon cancer cell from C57BL/6 mouse.PD-1 HuGEMM mouse are by gene work Journey technology behaves the mouse source PD-1 protein molecular partial replacements with the interaction of PD-L1 protein moleculars in C57BL/6 Mice Bodies The mouse model of source protein.

MC38 tumour cells (1 × 10 are inoculated on the right side of test mice⁶/ only), when mean tumour volume reaches about 134mm³When, mouse is randomized by 4 experimental groups according to gross tumor volume, every group 8, per 4, cage.4 experimental groups are divided into survey Reagent H8L2 5mg/kg and 10mg/kg, control Keytruda 10mg/kg groups and Isotype antibody Human lgG4 5mg/kg couple According to group.Tail vein injection is administered, and is administered six times (is shown in Table 3) altogether.

3. pharmacodynamic experiment of table designs

The 13rd day after grouping, the mean tumour volume of the 1st group of mouse reached 1933.67mm³；At the time point, 2nd group (high dose group) of Keytruda treatments and the 3rd group (low dose group) of H8L2 treatments, the 4th group (high dose group) TGI (%) is respectively 85%, 93%, 90% (being shown in Table 4).It is respectively 8.72%, 0.94% that mice with tumor weight, which changes percentage ,- 2.07%, 1.68%.Each treatment group's mice with tumor has statistics without apparent weight loss or death, 2-4 groups compared with the 1st group Significant difference (P<0.05).

Tumor killing effects of the table 4.H8L2 in PD-1 HuGEMM MC38 mice with tumor

Annotation：A. data are indicated with " mean+/-standard error "；

B. the conspicuousness between each treatment group tumors volume is carried out using one-way analysis of variance (one-way ANOVA) method Variance analysis；2-4 groups statistically significant difference (P compared with the 1st group<0.05).

The T-C of 2-4 groups is (when gross tumor volume reaches 1000mm³When) be respectively 10 days, 14 days,>14 days.When this experiment exists At the end of 55th day, 2-4 groups have 3,5,5 mouse tumors to subside completely and continue one month or more (being shown in Table 5) respectively.

The initial data of table 5.MC38 gross tumor volumes

H8L2 (5mg/kg, 10mg/kg) shows statistically significant in PD-1 HuGEMM mouse MC38 tumor models Antitumor action；Compared with Keytruda, H8L2 more effectively can make mouse interior tumor subside completely.

In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any One or more embodiments or example in can be combined in any suitable manner.

Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that：Not In the case of being detached from the principle of the present invention and objective a variety of change, modification, replacement and modification can be carried out to these embodiments, this The range of invention is limited by claim and its equivalent.

SEQUENCE LISTING

<110>Hangzhou Han Si biological medicines Co., Ltd

<120>IgG classes antibody is improved to the binding affinity of FcRn and extends the method for its serum half-life

<130> PIDC1168053

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 445

<212> PRT

<213> Artificial

<220>

<223>H8L2 heavy chain amino acid sequences

<400> 1

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

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Ala Arg Gln Lys Gly Glu Ala Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

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

115 120 125

Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

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

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys

210 215 220

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

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Thr Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

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

290 295 300

Thr Pro Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

355 360 365

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

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Ala

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 2

<211> 1392

<212> DNA

<213> Artificial

<220>

<223>Encode the nucleic acid sequence of H8L2 heavy chains

<400> 2

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggcgc gcactccgag 60

gtgcagctgg tgcagtctgg cggcggactg gtgcagcccg gcgggtcact gaagctgagc 120

tgcgccgctt ccggcttcac ctttagctcc tacggaatgt cctgggtgcg acaggcaccc 180

gggaaggggc tggactgggt cgctactatc tcaggaggcg ggagagacac ctactatcct 240

gatagcgtca agggccggtt cacaattagc cgggacaaca gcaagaacaa tctgtacctg 300

cagatgaaca gcctgagggc tgaggatact gcactgtact attgtgcccg ccagaagggc 360

gaagcatggt ttgcctattg gggccaggga accctggtca ccgtctcctc agcttccacc 420

aagggcccat ccgtcttccc cctggcgccc tgctccagga gcacctccga gagcacagcc 480

gccctgggct gcctggtcaa ggactacttc cccgaaccgg tgacggtgtc gtggaactca 540

ggcgccctga ccagcggcgt gcacaccttc ccggctgtcc tacagtcctc aggactctac 600

tccctcagca gcgtggtgac tgtgccctcc agcagcttgg gcacgaagac ctacacctgc 660

aacgtagatc acaagcccag caacaccaag gtggacaaga gagttgagtc caaatatggt 720

cccccatgcc caccatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 780

cccccaaaac ccaaggacac tctcatgatc acccggaccc ctgaggtcac gtgcgtggtg 840

gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 900

gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 960

agcgtcctca cccccctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 1020

tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 1080

cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 1140

agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 1200

aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 1260

ttcttcctct acagcaggct aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 1320

tcatgctccg tgatgcatga ggctctgcac gcccactaca cacagaagag cctctccctg 1380

tctctgggta aa 1392

<210> 3

<211> 218

<212> PRT

<213> Artificial

<220>

<223>H8L2 light-chain amino acid sequences

<400> 3

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

1 5 10 15

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

20 25 30

Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Ala Ala Ser Asn Lys Gly Thr Gly Val Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile Asn

65 70 75 80

Pro Met Glu Glu Asn Asp Thr Ala Met Tyr Phe Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 4

<211> 711

<212> DNA

<213> Artificial

<220>

<223>Encode the nucleic acid sequence of H8L2 light chains

<400> 4

atgggatgga gctgtatcat cctcttcttg gtagcaacag ctacaggcgt gcactccgat 60

attgtgctga ctcagagccc tgcttccctg gccgtgtctc caggacagcg agctaccatc 120

acatgcagag catctgagag tgtggacaac tacggaatta gtttcatgaa ttggtttcag 180

cagaagcccg gccagccccc taaactgctg atctatgccg ccagcaacaa gggcaccggg 240

gtgcctgctc gattctcagg aagcggctcc gggacagact ttactctgaa cattaaccca 300

atggaggaaa atgatacagc aatgtacttc tgccagcaga gcaaggaggt gccctggacc 360

tttggcgggg gaacaaagct ggaaatcaaa cgaactgtgg ctgcaccatc tgtcttcatc 420

ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 480

aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 540

aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 600

accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 660

catcagggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg t 711

Claims

1. a kind of method improving IgG classes antibody to the binding affinity of FcRn and extend its serum half-life, it is characterised in that：

Make the heavy chain constant region of the IgG classes antibody FcRn binding sites region the 254th, 308,434 amino acids occur it is prominent Become.

2. according to the method described in claim 1, it is characterized in that, the FcRn of the heavy chain constant region of the IgG classes antibody is made to tie Conjunction site areas the 254th, 308,434 amino acids sport threonine, proline and alanine respectively.

3. a kind of IgG class antibody of modification, which is characterized in that relative to wild type IgG class antibody, the IgG classes of the modification are anti- The FcRn binding sites region the 254th of the heavy chain constant region of body, 308,434 amino acids are mutated.

4. the IgG class antibody of modification according to claim 3, which is characterized in that relative to wild type IgG class antibody, institute State the heavy chain constant region of the IgG class antibody of modification FcRn binding sites region the 254th, 308,434 amino acids be respectively revive Propylhomoserin, proline and alanine.

5. a kind of method for the IgG class antibody preparing the modification described in claim 3 or 4, which is characterized in that including following step Suddenly：

According to the amino acid sequence of purpose IgG class antibody, the nucleic acid sequence of purpose IgG class antibody described in full genome composite coding；

Antibody preparation vehicles cells are transfected using the expression vector, to make the Antibody preparation vehicles cells express and to secrete The IgG class antibody of the modification.

6. according to the method described in claim 5, it is characterized in that, the Antibody preparation vehicles cells are 293 cells.