CN112279916A

CN112279916A - IL13RA1 antibodies, genes, vectors, host cells and IL13RA1 antagonists

Info

Publication number: CN112279916A
Application number: CN201910676953.2A
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 IL13RA1 antibody, a gene, a vector, a host cell and an IL13RA1 antagonist. The heavy chain of the IL13RA1 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 IL13RA1 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 IL13RA1 antibody can be specifically combined with cell surface hIL13RA1 and can be used as an antagonist of IL13RA1 signaling, so that the antibody can be used for treating IL13RA1 related diseases, such as asthma.

Description

IL13RA1 antibodies, genes, vectors, host cells and IL13RA1 antagonists

Technical Field

The invention relates to the technical field of antibodies, in particular to an IL13RA1 antibody, a gene, a vector, a host cell and an IL13RA1 antagonist.

Background

IL-13 can induce monocyte differentiation and enhance the expression of MHC class II molecules; inhibiting the secretion of mononuclear factors induced by lps and controlling inflammatory reaction; inducing B cell proliferation and IgE antibody synthesis, and enhancing expression of MHC class II molecules, CD23 and CD72 on the surface of B cell; in conjunction with IL-2, stimulates the production of IFN by NK cells, thereby promoting monocyte-macrophage activation and TH1 type cellular immune responses. Recently, IL-13 has been found to have various functions of inhibiting HIV-1 replication in macrophages, inducing IL-1RA gene expression and protein synthesis in neutrophils, and the like.

IL-13 is produced by TH2 cells and has a molecular weight of about 10 KD; the gene is located on chromosome 5 and is closely linked with the IL-4 gene. The amino acid sequence of the IL-13 molecule is 20% to 25% homologous to IL-4, and also bears many similarities to IL-4 in function. 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 IL13RA1 signaling would therefore be useful in the treatment of IL13RA1 related diseases, such as asthma.

Disclosure of Invention

In view of the above, the present invention provides IL13RA1 antibodies, genes, vectors, host cells and IL13RA1 antagonists. The IL13RA1 antibody can be specifically combined with cell surface hIL13RA1, and can be used as an antagonist of IL13RA1 signaling.

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

the invention provides an IL13RA1 antibody, the heavy chain of which IL13RA1 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 IL13RA1 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 IL13RA1 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 IL13RA1 antibody, wherein the amino acid sequence of the IL13RA1 antibody is shown in SEQ ID NO: shown in fig. 8.

Preferably, the IL13RA1 antibody is a humanized antibody.

The invention also provides a gene for coding the IL13RA1 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 IL13RA1 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 IL13RA1 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 IL13RA1 antagonist, comprising the IL13RA1 antibody provided by the invention.

The invention provides IL13RA1 antibodies, genes, vectors, host cells and IL13RA1 antagonists. The heavy chain of the IL13RA1 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 IL13RA1 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 IL13RA1 antibody can be specifically combined with cell surface hIL13RA1 and can be used as an antagonist of IL13RA1 signaling, so that the antibody can be used for treating IL13RA1 related diseases, such as asthma.

Drawings

FIG. 1 shows the characterization results of anti-IL13RA1 antibody, where 1-1 is blank control, 1-2 is negative control, and 1-3 is flow cytogram of anti-hIL13RA1scFv antibody combined with cell surface hIL13RA 1.

Detailed Description

The invention discloses IL13RA1 antibody, gene, vector, host cell and IL13RA1 antagonist, and the technical personnel can use the content and appropriately modify the technological parameters for realization. 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 IL13RA1 antibodies, genes, vectors, host cells and IL13RA1 antagonists provided by the invention are all commercially available.

The invention is further illustrated by the following examples:

example 1: expression of recombinant human IL13RA1 and EGFP cell preparation

Obtaining the amino acid sequence of the extracellular domain of human IL13RA1 (namely residues 1 to 343) according to the amino acid sequence of human IL13RA1 on a protein database Unit (P78552); the amino acid sequence of IL13RA1 is as follows (wherein the underlined part is the extracellular domain):

MEWPARLCGLWALLLCAGGGGGGGGAAPTETQPPVTNLSVSVENLCTVIWTWNPPEGASSNCSLWYFSH FGDKQDKKIAPETRRSIEVPLNERICLQVGSQCSTNESEKPSILVEKCISPPEGDPESAVTELQCIWHNLSYMKCSW LPGRNTSPDTNYTLYYWHRSLEKIHQCENIFREGQYFGCSFDLTKVKDSSFEQHSVQIMVKDNAGKIKPSFNIVPLT SRVKPDPPHIKNLSFHNDDLYVQWENPQNFISRCLFYEVEVNNSQTETHNVFYVQEAKCENPEFERNVENTSCFMVP GVLPDTLNTVRIRVKTNKLCYEDDKLWSNWSQEMSIGKKRNSTLYITMLLIVPVIVAGAIIVLLLYLKRLKIIIFPPIPDPGKIFKEMFGDQNDDTLHWKKYDIYEKQTKEETDSVVLIENLKKASQ

the base sequence is as follows:

atggagtggccggcgcggctctgcgggctgtgggcgctgctgctctgcgccggcggcgggggcggggg cgggggcgccgcgcctacggaaactcagccacctgtgacaaatttgagtgtctctgttgaaaacctctgcacagta atatggacatggaatccacccgagggagccagctcaaattgtagtctatggtattttagtcattttggcgacaaac aagataagaaaatagctccggaaactcgtcgttcaatagaagtacccctgaatgagaggatttgtctgcaagtggg gtcccagtgtagcaccaatgagagtgagaagcctagcattttggttgaaaaatgcatctcacccccagaaggtgat cctgagtctgctgtgactgagcttcaatgcatttggcacaacctgagctacatgaagtgttcttggctccctggaa ggaataccagtcccgacactaactatactctctactattggcacagaagcctggaaaaaattcatcaatgtgaaaa catctttagagaaggccaatactttggttgttcctttgatctgaccaaagtgaaggattccagttttgaacaacac agtgtccaaataatggtcaaggataatgcaggaaaaattaaaccatccttcaatatagtgcctttaacttcccgtg tgaaacctgatcctccacatattaaaaacctctccttccacaatgatgacctatatgtgcaatgggagaatccaca gaattttattagcagatgcctattttatgaagtagaagtcaataacagccaaactgagacacataatgttttctac gtccaagaggctaaatgtgagaatccagaatttgagagaaatgtggagaatacatcttgtttcatggtccctggtg ttcttcctgatactttgaacacagtcagaataagagtcaaaacaaataagttatgctatgaggatgacaaactctg gagtaattggagccaagaaatgagtataggtaagaagcgcaattccacactctacataaccatgttactcattgttccagtcatcgtcgcaggtgcaatcatagtactcctgctttacctaaaaaggctcaagattattatattccctccaattcctgatcctggcaagatttttaaagaaatgtttggagaccagaatgatgatactctgcactggaagaagtacgacatctatgagaagcaaaccaaggaggaaaccgactctgtagtgctgatagaaaacctgaagaaagcctctcag

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 hIL13RA1-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 (P78552) of human IL13RA1 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 hIL13RA1 and EGFP fusion protein, and a gene of hIL13RA 1-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-hIL13RA1-Fc and pcDNA4-hIL13RA 1-EGFP.

The relevant EGFP recombinant plasmid was transfected into HEK293(ATCC, CRL-1573)^TM) In cells, expression of hCSF1R was confirmed 48h after transfection by fluorescence activated signal sorting (FACS).

pcDNA4-hIL13RA1-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 culturing for 5-6 days, transient expression culture supernatant is collected and subjected to primary purification by a protein affinity chromatography method to obtain a hIL13RA1-Fc protein sample which is 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-IL13RA1 antibody from yeast display library, cloning and expressing

The yeast display technology is adopted to screen and select a fully human antibody of human IL13RA 1. 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 capacity, inducing yeast surface expression antibodies, enriching twice by using 100nM biotinylated hIL13RA1-Fc antigen in a magnetic bead sorting mode, and then enriching twice by using biotinylated hIL13RA1-Fc as flow sorting. The resulting yeast was plated and single clones were picked. After amplification and induction of expression, the monoclonal yeast were analyzed by staining with biotinylated hIL13RA1-Fc 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-hIL13RA1scFv antibody is shown as SEQ ID NO: 8 (VH-Linker-VL, CDR in the wavy line portion and Linker in the underline):

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

caggtacagctgcagcagtcaggcccaggactggtgaagccctcgcagaccctcttaatcacctgtgccatctccggggacagtgtctctagtaacagtgctgcttggaactggatcaggcagtcgccctcgggaagccttgagtggctgggaaggacatactacaggtccaagtggtatagtcattatgcagtatctgtgacaggtcgaatgaccatcaattcagactcatccaagaaccagttctccctgcagctggattctgtgactcccgaggacatggctatttattattgtgcaagagaggaagtggctggtagggggtcgtttgacttctggggccagggaaccctggtcaccgtctcctcaggaattctaggatccggtggcggtggcagcggcggtggtggttccggaggcggcggttctgacatccagttgacccagtctccgtccttcctgtctgcatctgtaggagacagagtcaccatcacttgccgggccagtcagagtattagtaagaatttggtctggtatcagcagaaaccagggaaagcccctaagctcctgatctattctgcatccactttgcaaagtggggtcccatcaaggttcagcggcagtggatctgggacagatttcactctcactatcagcagcctgcagcctgaagattgtgcactttacttttgtcaacaggctcacagtttcccggtgaccttcggccaagggacacgactggagattaaa

example 3: characterization of anti-IL13RA1 antibody (characterization by binding to IL13RA 1-FACS method)

Taking hIL13RA1-EGFP cells, suspending the cells in 0.5% PBS-BSABuffer, adding 2 μ g of anti-hIL13RA1scFv 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 hIL13RA1 antigen were identified in this manner. As shown in FIG. 1, anti-IL13RA 135 # can bind to cell surface hIL13RA1, while the negative control is not capable of binding to cell surface hIL13RA 1.

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> IL13RA1 antibodies, genes, vectors, host cells and IL13RA1 antagonists

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<170> SIPOSequenceListing 1.0

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Ser Asn Ser Ala Ala Trp Asn

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Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Ser His Tyr Ala Val Ser Val

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Gln Val Gln Leu Gln Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp

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

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Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Lys Asn Leu

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

180 185 190

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195 200 205

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225 230 235 240

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caggtacagc tgcagcagtc aggcccagga ctggtgaagc cctcgcagac cctcttaatc 60

acctgtgcca tctccgggga cagtgtctct agtaacagtg ctgcttggaa ctggatcagg 120

cagtcgccct cgggaagcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

agtcattatg cagtatctgt gacaggtcga atgaccatca attcagactc atccaagaac 240

cagttctccc tgcagctgga ttctgtgact cccgaggaca tggctattta ttattgtgca 300

agagaggaag tggctggtag ggggtcgttt gacttctggg gccagggaac cctggtcacc 360

gtctcctcag gaattctagg atccggtggc ggtggcagcg gcggtggtgg ttccggaggc 420

ggcggttctg acatccagtt gacccagtct ccgtccttcc tgtctgcatc tgtaggagac 480

agagtcacca tcacttgccg ggccagtcag agtattagta agaatttggt ctggtatcag 540

cagaaaccag ggaaagcccc taagctcctg atctattctg catccacttt gcaaagtggg 600

gtcccatcaa ggttcagcgg cagtggatct gggacagatt tcactctcac tatcagcagc 660

ctgcagcctg aagattgtgc actttacttt tgtcaacagg ctcacagttt cccggtgacc 720

ttcggccaag ggacacgact ggagattaaa 750

Claims

1. An IL13RA1 antibody, wherein the heavy chain of the IL13RA1 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 IL13RA1 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 IL13RA1 antibody of claim 1, wherein the heavy chain and the light chain of the IL13RA1 antibody are linked by a linker having an amino acid sequence as set forth in SEQ ID NO: shown at 7.

3. An IL13RA1 antibody, wherein the amino acid sequence of the IL13RA1 antibody is as set forth in SEQ ID NO: shown in fig. 8.

4. The IL13RA1 antibody of claim 3, wherein the IL13RA1 antibody is a humanized antibody.

5. A gene encoding the IL13RA1 antibody of claim 3 or 4, wherein 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 IL13RA1 antagonist, comprising the IL13RA1 antibody of any one of claims 1 to 4.