CN112279914A

CN112279914A - IL4RA antibodies, genes, vectors, host cells and IL4RA antagonists

Info

Publication number: CN112279914A
Application number: CN201910676472.1A
Authority: CN
Inventors: 张欣; 费烨琼; 宁姗姗; 赵猛; 马树立; 傅士龙
Original assignee: Dingfu Biotarget Co ltd
Current assignee: Dingfu Biotarget Co ltd
Priority date: 2019-07-25
Filing date: 2019-07-25
Publication date: 2021-01-29

Abstract

The invention relates to the technical field of antibodies, in particular to an IL4RA antibody, a gene, a vector, a host cell and an IL4RA antagonist. The heavy chain of the IL4RA antibody comprises SEQ ID NO: 1, CDR1 of the sequence shown in SEQ ID NO: 2, CDR2 of the sequence shown in SEQ ID NO: 3 CDR3 of the sequence shown in seq id no; the light chain of IL4RA antibody comprises SEQ ID NO: 4, CDR4 of the sequence shown in SEQ ID NO: 5, CDR5 of the sequence shown in SEQ ID NO: 6, CDR6 of the sequence shown in seq id no. The IL4RA antibody of the invention can be specifically combined with cell surface hIL4RA, and can be used as an antagonist of IL4RA signal transduction, so the antibody can be used for treating IL4RA related diseases, such as asthma.

Description

IL4RA antibodies, genes, vectors, host cells and IL4RA antagonists

Technical Field

The invention relates to the technical field of antibodies, in particular to an IL4RA antibody, a gene, a vector, a host cell and an IL4RA antagonist.

Background

IL-4 (also known as B cell stimulating factor or BSF-1) has a wide range of biological functions that can stimulate the activation of T cells, mast cells, granulocytes and megakaryocytes, while inducing expression of MHC-II in resting B cells and enhancing IgE secretion by stimulating B cells (Reddy et al (2000) J.Immunol.164: 1925-.

Asthma is a chronic inflammatory allergic disease of respiratory tract, the incidence rate of the asthma is high, and no medicament with particularly good curative effect exists at present. It is well known that IL-4 is a Th2 type cytokine, the biological activity of which is mediated by its specific cell surface IL-4 receptor. IL-4 is a cytokine secreted by Th2 cells, which plays an important role with IL-13 in the development of allergic asthma (Zura Wski G.et al, Immunol.today, Vol.15:19-26 (1994)). The two cytokines, IL-13 and IL-4, share significant structural similarities in cells, and share certain receptor components while having separate receptors. Such as IL-4 and IL-13 dual receptors IL-4R/IL-13Ra1, the receptor contains IL-4Ra chain and IL-13Ra1 chain, its main expression in hematopoietic and non-hematopoietic cells, including lung epithelial and smooth muscle cells. In addition, IL-4 and IL-13 each have a receptor that recognizes themselves but not the other. The type I receptor IL-4R, such as IL-4, comprises an IL-4Ra chain and a common gamma chain, which specifically bind IL-4, and the type I receptor IL-4R for IL-4 is exclusively expressed on cells of hematopoietic origin. The receptor specific for IL-13 is IL-13Ra2, which has a specific high affinity for IL-13, but which is not capable of downstream signaling (Caput D.et al, J.biol.Chern., Vol.271: 16965 (1996)). Antagonists of IL4RA signaling would therefore be useful in the treatment of IL4RA related diseases, such as asthma.

Disclosure of Invention

In view of the above, the present invention provides IL4RA antibodies, genes, vectors, host cells and IL4RA antagonists. The IL4RA antibody can be specifically combined with cell surface hIL4RA, and can be used as IL4RA signaling antagonist.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides an IL4RA antibody, the heavy chain of which IL4RA antibody comprises SEQ ID NO: 1, CDR1 of the sequence shown in SEQ ID NO: 2, CDR2 of the sequence shown in SEQ ID NO: 3 CDR3 of the sequence shown in seq id no;

the light chain of IL4RA antibody comprises SEQ ID NO: 4, CDR4 of the sequence shown in SEQ ID NO: 5, CDR5 of the sequence shown in SEQ ID NO: 6, CDR6 of the sequence shown in seq id no.

In the invention, the heavy chain and the light chain of the IL4RA antibody are connected by a linker, and the amino acid sequence of the linker is shown as SEQ ID NO: shown at 7.

The invention also provides an IL4RA antibody, wherein the amino acid sequence of the IL4RA antibody is shown in SEQ ID NO: shown in fig. 8.

Preferably, the IL4RA antibody is a humanized antibody.

The invention also provides a gene for coding the IL4RA antibody, and the base sequence of the gene is shown as SEQ ID NO: shown at 9.

The invention also provides a vector comprising a gene encoding an IL4RA antibody.

Preferably, the plasmid used for the vector is pcDNA 4/myc-HisA.

The invention also provides a host cell comprising the vector.

Preferably, the host cell is a HEK293 cell.

The invention also provides a preparation method of the IL4RA antibody, which comprises the following steps: after fusion of scFv gene (shown in SEQ ID NO: 9) and IgG1-Fc gene, the single-chain antibody was cloned into vector pcDNA4/myc-HisA by HindIII and EcoRI double digestion, cloning and plasmid miniextraction were carried out, the extracted plasmid was expressed in HEK293 cells and purified by protein A column.

The invention also provides an IL4RA antagonist, including the IL4RA antibody provided by the invention.

The invention provides IL4RA antibodies, genes, vectors, host cells and IL4RA antagonists. The heavy chain of the IL4RA antibody comprises SEQ ID NO: 1, CDR1 of the sequence shown in SEQ ID NO: 2, CDR2 of the sequence shown in SEQ ID NO: 3 CDR3 of the sequence shown in seq id no; the light chain of IL4RA antibody comprises SEQ ID NO: 4, CDR4 of the sequence shown in SEQ ID NO: 5, CDR5 of the sequence shown in SEQ ID NO: 6, CDR6 of the sequence shown in seq id no. The invention has the technical effects that:

the IL4RA antibody of the invention can be specifically combined with cell surface hIL4RA, and can be used as an antagonist of IL4RA signal transduction, so the antibody can be used for treating IL4RA related diseases, such as asthma.

Drawings

FIG. 1 shows the characterization results of anti-IL4RA antibody, where 1-1 is blank control, 1-2 is negative control, and 1-3 is flow cytometry diagram of the combination of anti-hIL4RA scFv antibody and cell surface hIL4 RA.

Detailed Description

IL4RA antibodies, genes, vectors, host cells and IL4RA antagonists are disclosed and can be achieved by one skilled in the art with appropriate modification of the process parameters in view of the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

Reagents, instruments or materials used in the IL4RA antibodies, genes, vectors, host cells and IL4RA antagonists provided by the invention are commercially available.

The invention is further illustrated by the following examples:

example 1: expression of recombinant human IL4RA and EGFP cell preparation

Obtaining the amino acid sequence of the extracellular domain of human IL-4RA (i.e. residues 1 to 232 in P24394) according to the amino acid sequence of human IL-4RA on Uniprot (P24394) of a protein database; the amino acid sequence of IL4RA is as follows (wherein the underlined part is the extracellular domain):

MGWLCSGLLFPVSCLVLLQVASSGNMKVLQEPTCVSDYMSISTCEWKMNGPTNCSTELRLLYQLVFLLS EAHTCIPENNGGAGCVCHLLMDDVVSADNYTLDLWAGQQLLWKGSFKPSEHVKPRAPGNLTVHTNVSDTLLLTWSNP YPPDNYLYNHLTYAVNIWSENDPADFRIYNVTYLEPSLRIAASTLKSGISYRARVRAWAQCYNTTWSEWSPSTKWHN SYREPFEQHLLLGVSVSCIVILAVCLLCYVSITKIKKEWWDQIPNPARSRLVAIIIQDAQGSQWEKRSRGQEPAKCPHWKNCLTKLLPCFLEHNMKRDEDPHKAAKEMPFQGSGKSAWCPVEISKTVLWPESISVVRCVELFEAPVECEEEEEVEEEKGSFCASPESSRDDFQEGREGIVARLTESLFLDLLGEENGGFCQQDMGESCLLPPSGSTSAHMPWDEFPSAGPKEAPPWGKEQPLHLEPSPPASPTQSPDNLTCTETPLVIAGNPAYRSFSNSLSQSPCPRELGPDPLLARHLEEVEPEMPCVPQLSEPTTVPQPEPETWEQILRRNVLQHGAAAAPVSAPTSGYQEFVHAVEQGGTQASAVVGLGPPGEAGYKAFSSLLASSAVSPEKCGFGASSGEEGYKPFQDLIPGCPGDPAPVPVPLFTFGLDREPPRSPQSSHLPSSSPEHLGLEPGEKVEDMPKPPLPQEQATDPLVDSLGSGIVYSALTCHLCGHLKQCHGQEDGGQTPVMASPCCGCCCGDRSSPPTTPLRAPDPSPGGVPLEASLCPASLAPSGISEKSKSSSSFHPAPGNAQSSSQTPKIVNFVSVGPTYMRVS

the base sequence is as follows:

atggggtggctttgctctgggctcctgttccctgtgagctgcctggtcctgctgcaggtggcaagctc tgggaacatgaaggtcttgcaggagcccacctgcgtctccgactacatgagcatctctacttgcgagtggaagatg aatggtcccaccaattgcagcaccgagctccgcctgttgtaccagctggtttttctgctctccgaagcccacacgt gtatccctgagaacaacggaggcgcggggtgcgtgtgccacctgctcatggatgacgtggtcagtgcggataacta tacactggacctgtgggctgggcagcagctgctgtggaagggctccttcaagcccagcgagcatgtgaaacccagg gccccaggaaacctgacagttcacaccaatgtctccgacactctgctgctgacctggagcaacccgtatccccctg acaattacctgtataatcatctcacctatgcagtcaacatttggagtgaaaacgacccggcagatttcagaatcta taacgtgacctacctagaaccctccctccgcatcgcagccagcaccctgaagtctgggatttcctacagggcacgg gtgagggcctgggctcagtgctataacaccacctggagtgagtggagccccagcaccaagtggcacaactcctaca gggagcccttcgagcagcacctcctgctgggcgtcagcgtttcctgcattgtcatcctggccgtctgcctgttgtgctatgtcagcatcaccaagattaagaaagaatggtgggatcagattcccaacccagcccgcagccgcctcgtggctataataatccaggatgctcaggggtcacagtgggagaagcggtcccgaggccaggaaccagccaagtgcccacactggaagaattgtcttaccaagctcttgccctgttttctggagcacaacatgaaaagggatgaagatcctcacaaggctgccaaagagatgcctttccagggctctggaaaatcagcatggtgcccagtggagatcagcaagacagtcctctggccagagagcatcagcgtggtgcgatgtgtggagttgtttgaggccccggtggagtgtgaggaggaggaggaggtagaggaagaaaaagggagcttctgtgcatcgcctgagagcagcagggatgacttccaggagggaagggagggcattgtggcccggctaacagagagcctgttcctggacctgctcggagaggagaatgggggcttttgccagcaggacatgggggagtcatgccttcttccaccttcgggaagtacgagtgctcacatgccctgggatgagttcccaagtgcagggcccaaggaggcacctccctggggcaaggagcagcctctccacctggagccaagtcctcctgccagcccgacccagagtccagacaacctgacttgcacagagacgcccctcgtcatcgcaggcaaccctgcttaccgcagcttcagcaactccctgagccagtcaccgtgtcccagagagctgggtccagacccactgctggccagacacctggaggaagtagaacccgagatgccctgtgtcccccagctctctgagccaaccactgtgccccaacctgagccagaaacctgggagcagatcctccgccgaaatgtcctccagcatggggcagctgcagcccccgtctcggcccccaccagtggctatcaggagtttgtacatgcggtggagcagggtggcacccaggccagtgcggtggtgggcttgggtcccccaggagaggctggttacaaggccttctcaagcctgcttgccagcagtgctgtgtccccagagaaatgtgggtttggggctagcagtggggaagaggggtataagcctttccaagacctcattcctggctgccctggggaccctgccccagtccctgtccccttgttcacctttggactggacagggagccacctcgcagtccgcagagctcacatctcccaagcagctccccagagcacctgggtctggagccgggggaaaaggtagaggacatgccaaagcccccacttccccaggagcaggccacagacccccttgtggacagcctgggcagtggcattgtctactcagcccttacctgccacctgtgcggccacctgaaacagtgtcatggccaggaggatggtggccagacccctgtcatggccagtccttgctgtggctgctgctgtggagacaggtcctcgccccctacaacccccctgagggccccagacccctctccaggtggggttccactggaggccagtctgtgtccggcctccctggcaccctcgggcatctcagagaagagtaaatcctcatcatccttccatcctgcccctggcaatgctcagagctcaagccagacccccaaaatcgtgaactttgtctccgtgggacccacatacatgagggtctcttag

the amino acid sequence of the domain of human IgG1-Fc (i.e., residues 104 to 330 in P01857) was obtained from the amino acid sequence of the constant region of human immunoglobulin gamma (gamma) 1(IgG1) on Unit protein database (P01857). The IgG1-Fc amino acid sequence is as follows:

DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

the base sequence is as follows:

gacaaaactcacacatgcccaccgtgcccagctccggaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa

the gene of hIL4RA-Fc fusion protein is obtained by designing a corresponding coding DNA sequence by using a DNAworks online tool (http:// helix web. nih. gov/DNAworks /).

Obtaining an enhanced green fluorescent protein EGFP amino acid sequence (C5MKY7) and an amino acid sequence (P24394) of human IL4RA according to information on a protein database Unit, wherein the EGFP amino acid sequence is as follows: MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK

The base sequence is as follows:

atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag

a DNAworks online tool (http:// helix web. nih. gov/DNAworks /) is used for designing a corresponding coding DNA sequence to obtain a gene of the hIL4RA and EGFP fusion protein, and a gene of hIL4 RA-EGFP. The DNA fragment is obtained by artificial synthesis. The synthesized gene sequence is subcloned into a commercial vector pcDNA4/myc-HisA (Invitrogen, V863-20) through HindIII and EcoRI double enzyme digestion of Fermentas company, and the accuracy of the constructed plasmid is verified by sequencing to obtain recombinant plasmid DNA, namely: pcDNA4-hIL4RA-Fc and pcDNA4-hIL4 RA-EGFP.

The relevant EGFP recombinant plasmid was transfected into HEK293(ATCC, CRL-1573)^TM) In cells, expression of hIL4RA was confirmed 48h after transfection by fluorescence activated signal sorting (FACS).

pcDNA4-hIL4RA-Fc was transiently transfected into HEK293 cells for protein production. Diluting the recombinant expression plasmid with Freestyle293 medium and adding PEI (polyethyleneimine) solution required for transformation, adding each group of plasmid/PEI mixture into the cell suspension respectively, and placing at 37 ℃ and 10% CO₂Culturing at 90 rpm; after 5-6 days of culture, transient expression culture supernatant was collected and subjected to primary purification by protein affinity chromatography to obtain a hIL4RA-Fc protein sample, which was used in the following examples. The obtained protein sample is subjected to primary detection by SDS-PAGE, and a target band can be clearly seen.

Example 2: screening anti-IL4RA antibody from yeast display library, cloning and expressing

The yeast display technology is adopted to screen the whole human antibody of the human IL4 RA. Construction of scFV yeast display library by cloning VH and VL genes in IgM and IgG cDNAs from PBMCs of 50 healthy humans (the linker sequence between VH and VL is GILGSGGGGSGGGGSGGGGS linker peptide, the library content is 5X 10⁸. Recovering the yeast library with 10 times of library volume, inducing yeast surface expression antibody, and using 100nM biotinylated hIL4RA-Fc antigenEnrichment was done twice using magnetic bead sorting and then twice again using biotinylated hIL4RA-Fc as flow sort. The resulting yeast was plated and single clones were picked. After amplification and induction of expression, the monoclonal yeast were analyzed by biotinylated hIL4RA-Fc staining to identify positive yeast. Yeast clones confirmed by FACS were subjected to yeast colony PCR and sequencing with PCR primers:

sequence-F：CGTAGAATCGAGACCGAGGAGA；

sequence-R：CTGGTGGTGGTGGTTCTGCTAGC；

the sequencing primer is sequence-R. And after a sequencing result is obtained, the sequences are compared and analyzed by using BioEdit software.

The scFv gene of the single-chain antibody obtained above and the human IgG1-Fc gene were fused and cloned into a commercial vector pcDNA4/myc-HisA by means of double digestion with HindIII and EcoRI from Fermentas corporation, and cloning and plasmid extraction were carried out according to standard procedures for molecular cloning. The extracted plasmid was transiently expressed in HEK293 cells and purified by protein a column. The amino acid sequence of the anti-hIL4RAscFv antibody is shown in SEQ ID NO: 8 (VH-Linker-VL, CDR in the wavy line portion and Linker in the underline):

QVQLVESEAEVRKPGASVKVSCKASGYTFT

WVRQAPGQGLEWMG

RVTMTRDTSTSIDYMELSSLRFEDTAVYYCVR

WGQGTLVTVSSGILGSGGGGSGGGGSGGGGSQSVLIQPASVSGSPGQSITISC

WYQQHPGKAPKLMIY

GVPDRFSGSKSGNTASLTISGLQPEDEADYYC

FGGGTKVTVL

the base sequence is shown as SEQ ID NO: 9 is as follows:

caggtgcagcttgtggagtctgaggctgaggtgaggaagcctggggcctcagtgaaggtttcctgcaaggcatctggatacaccttcaccacctactatatgaactgggtgcgacaggcccctggacaagggcttgagtggatgggaataatcagccctaggggtgacaggactgtctacgcaccgaagttccagggcagagtcaccatgaccagggacacgtccacgagtatagattacatggaactgagcagcctgagatttgaggacacggccgtgtattactgtgtgagaggtgatactggtaaccggataaagggagagtttgacctttggggccagggaaccctggtcaccgtctcctcaggaattctaggatccggtggcggtggcagcggcggtggtggttccggaggcggcggttctcagtctgttctgattcagcctgcctccgtgtctgggtctcctggacagtcgatcaccatctcctgcactggaacgagcggtgacgttggtggttataactatgtctcctggtaccaacagcacccaggcaaagcccccaaactcatgatttatgaggtcagtaagcggccctcaggggtccctgatcgcttctctggctccaagtcgggaaacacggcctccctgaccatctctgggctgcagcctgaggacgaggctgattattactgcttctcctacagacatactattagttctttcgtcttcggcggagggaccaaggtcaccgtccta

example 3: characterization of anti-IL4R antibodies (characterization by binding to IL 4R-FACS method)

Taking hIL4RA-EGFP cells, suspending the cells in 0.5% PBS-BSABuffer, adding 2 μ g of anti-hIL4RAscFv antibody after purification, and setting a relevant control at the same time, wherein the negative control is 2 μ g of hIgG1 protein. The secondary antibody is anti-hIg-PE of eBioscience. And detecting by a flow cytometer after dyeing. Antibodies that bind cell surface hIL4RA antigen were identified in this manner. As shown in FIG. 1, anti-IL4RA11# can bind to cell surface hIL4RA, while the negative control is unable to bind to cell surface hIL4 RA.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Suzhou Dingfu target biotech Co., Ltd

<120> IL4RA antibodies, genes, vectors, host cells and IL4RA antagonists

<130> MP1917792

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Thr Tyr Tyr Met Asn

1 5

<210> 2

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Ile Ile Ser Pro Arg Gly Asp Arg Thr Val Tyr Ala Pro Lys Phe Gln

1 5 10 15

Gly

<210> 3

<211> 13

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Gly Asp Thr Gly Asn Arg Ile Lys Gly Glu Phe Asp Leu

1 5 10

<210> 4

<211> 14

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Thr Gly Thr Ser Gly Asp Val Gly Gly Tyr Asn Tyr Val Ser

1 5 10

<210> 5

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Glu Val Ser Lys Arg Pro Ser

1 5

<210> 6

<211> 11

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Phe Ser Tyr Arg His Thr Ile Ser Ser Phe Val

1 5 10

<210> 7

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Ile Leu Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 8

<211> 253

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Ile Asp Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Phe Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Arg Gly Asp Thr Gly Asn Arg Ile Lys Gly Glu Phe Asp Leu Trp

100 105 110

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Ile Leu Gly Ser Gly

115 120 125

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ser

130 135 140

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

145 150 155 160

Thr Ile Ser Cys Thr Gly Thr Ser Gly Asp Val Gly Gly Tyr Asn Tyr

165 170 175

Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu Met Ile

180 185 190

Tyr Glu Val Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser Gly

195 200 205

Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln Pro

210 215 220

Glu Asp Glu Ala Asp Tyr Tyr Cys Phe Ser Tyr Arg His Thr Ile Ser

225 230 235 240

Ser Phe Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu

245 250

<210> 9

<211> 759

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

caggtgcagc ttgtggagtc tgaggctgag gtgaggaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc acctactata tgaactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaata atcagcccta ggggtgacag gactgtctac 180

gcaccgaagt tccagggcag agtcaccatg accagggaca cgtccacgag tatagattac 240

atggaactga gcagcctgag atttgaggac acggccgtgt attactgtgt gagaggtgat 300

actggtaacc ggataaaggg agagtttgac ctttggggcc agggaaccct ggtcaccgtc 360

tcctcaggaa ttctaggatc cggtggcggt ggcagcggcg gtggtggttc cggaggcggc 420

ggttctcagt ctgttctgat tcagcctgcc tccgtgtctg ggtctcctgg acagtcgatc 480

accatctcct gcactggaac gagcggtgac gttggtggtt ataactatgt ctcctggtac 540

caacagcacc caggcaaagc ccccaaactc atgatttatg aggtcagtaa gcggccctca 600

ggggtccctg atcgcttctc tggctccaag tcgggaaaca cggcctccct gaccatctct 660

gggctgcagc ctgaggacga ggctgattat tactgcttct cctacagaca tactattagt 720

tctttcgtct tcggcggagg gaccaaggtc accgtccta 759

Claims

1. An IL4RA antibody, wherein the heavy chain of the IL4RA antibody comprises the amino acid sequence of SEQ ID NO: 1, CDR1 of the sequence shown in SEQ ID NO: 2, CDR2 of the sequence shown in SEQ ID NO: 3 CDR3 of the sequence shown in seq id no;

the light chain of the IL4RA antibody comprises SEQ ID NO: 4, CDR4 of the sequence shown in SEQ ID NO: 5, CDR5 of the sequence shown in SEQ ID NO: 6, CDR6 of the sequence shown in seq id no.

2. The IL4RA antibody of claim 1, wherein the heavy chain and the light chain of the IL4RA antibody are linked by a linker having an amino acid sequence as set forth in SEQ ID NO: shown at 7.

3. An IL4RA antibody, wherein the amino acid sequence of the IL4RA antibody is as set forth in SEQ ID NO: shown in fig. 8.

4. The IL4RA antibody according to claim 3, characterized in that the IL4RA antibody is a humanized antibody.

5. A gene encoding the IL4RA antibody of claim 3 or 4, characterized in that the base sequence is as set forth in SEQ ID NO: shown at 9.

6. A vector comprising the gene of claim 5.

7. The vector of claim 6, wherein the plasmid used in the vector is pcDNA 4/myc-HisA.

8. A host cell comprising the vector of claim 6.

9. The host cell of claim 8, wherein the host cell is a HEK293 cell.

10. An IL4RA antagonist, comprising the IL4RA antibody of any one of claims 1 to 4.