CN110551214A

CN110551214A - Humanized anti-Periostin monoclonal antibody, and preparation method and application thereof

Info

Publication number: CN110551214A
Application number: CN201910796392.XA
Authority: CN
Inventors: 杨澜; 舒雄
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-08-27
Filing date: 2019-08-27
Publication date: 2019-12-10

Abstract

The invention discloses a humanized Periostin-resistant monoclonal antibody, and a preparation method and application thereof, and belongs to the technical field of medicines. The humanized anti-Periostin monoclonal antibody comprises a light chain complementarity determining region and a heavy chain complementarity determining region, wherein the amino acid sequence of the light chain complementarity determining region is shown in SEQ ID NO.1-3, and the amino acid sequence of the heavy chain complementarity determining region is shown in SEQ ID NO. 4-6. The invention also discloses a preparation method and application of the humanized anti-Periostin monoclonal antibody. The humanized anti-Periostin monoclonal antibody provided by the invention not only has the specificity of being combined with Periostin protein and Periostin positive tumor cells, but also has humanity, and can reduce the toxic and side effects for human use.

Description

Humanized anti-Periostin monoclonal antibody, and preparation method and application thereof

Technical Field

The invention relates to a humanized Periostin-resistant monoclonal antibody, and a preparation method and application thereof, belonging to the technical field of medicines.

Background

Since the 80 s of the last century, tumors have gradually become the most serious disease threatening human health and life globally. In the last two decades, the incidence of malignant tumor in China has been on a continuous rising trend along with the development of the economic society of China and the improvement of the living standard of people. Chemotherapy, neoadjuvant chemotherapy and surgery remain the major strategies for clinical treatment of most solid tumors at present. Since 2001, tumor molecular targeted therapy opened a completely new field of view for the treatment of solid tumors. Molecular targeted cancer drugs (molecular targeted cancer therapy) usually employ monoclonal antibodies or small molecular substances to block specific signal transduction pathways or molecules in tumor pathogenesis, reduce side effects caused by treatment, and improve safety. At present, drugs which are specifically targeted at receptor tyrosine kinases (EGFR, HER2, VEGF, PDGF and the like) have achieved considerable curative effects in clinic. For example, the Epidermal Growth Factor Receptor (EGFR) blocker gefitinib (iressa) and the receptor Tyrosine Kinase Inhibitor (TKI) erlotinib (tarceva) have been widely used in clinical treatment of lung cancer; the anti-EGFR monoclonal antibody Cetuximab (Cetuximab, trade name: erbitux) and Panitumumab (Panitumumab, trade name: Vectibix) have also been used as important targeted therapeutic drugs for patients with advanced metastatic colorectal cancer.

Although the targeted drug brings hope for the long-term survival of the colorectal cancer patients, the response rate of the single drug therapy of Cetuximab and Panitumumab is only 8-13%, and compared with the combined drug of FOLFOX and FOLFIRI which are main chemotherapy schemes for colorectal cancer, the response rate is only improved by about 16% compared with the conventional chemotherapy scheme. Even patients who are initially highly sensitive to the anti-EGFR mAb are almost refractory to evasion of acquired drug resistance after median non-progressive treatment at 12-18 m. Targeted therapeutic drug resistance is the biggest obstacle preventing the continued benefit of colorectal cancer patients. Therefore, how to help patients who target EGFR therapy resistance to benefit from the targeted drug again is a key problem to be solved urgently in the field of colorectal cancer therapy at present.

The human Periostin protein contains 836 amino acid residues, has a relative molecular weight of about 93.3KDa, has a gene sequence positioned on chromosome 13q13.3, has about 89.2 percent of homology with a mouse amino acid sequence, and has high conservation. Periostin in human tissues found so far shares 8 isoforms, and its different expression patterns are closely related to the development of human tissues and the development of certain tumors. It has been found that the expression of Periostin in various normal tissues of the human body is positively correlated with the number of fibroblasts in the tissues (e.g., Periostin is very low in peripheral blood and spleen, low in salivary glands and thymus, fluctuating in skin, breast and ovary, and uniform in solid tissues such as liver, pancreas, lung and colon. recent studies have found that cytokines such as BMP-2, TGF-beta, PDGF-alpha, PDGF-beta, alpha-FGF, beta-FGF, AngII, etc. are all capable of up-regulating the expression of Periostin in cells. The expression level of the polypeptide has obvious correlation with the stages of tumors, metastasis, postoperative recurrence rate and survival rate.

At present, the industry is commonly recognized: the binding specificity and affinity of a monoclonal antibody is determined in large part by the amino acid sequences of the light and heavy chain variable regions (CDRs) of the antibody, and any monoclonal antibody having identical CDR sequences recognizes the target antigen with substantially the same specificity and affinity and therapeutic effect. The U.S. Food and Drug Administration (FDA) recognizes in its guidelines that all antibodies of the same type having the same complementarity determining regions belong to the same antibody and are treated with the same antibody in clinical treatment and drug approval. Therefore, after obtaining the CDR sequences of the same antibody with clinical therapeutic value, the industry can easily change the amino acid sequences of the non-CDR regions thereof according to the mature and well-known prior art to obtain other monoclonal antibodies with the same biological activity.

In view of the specificity of the expression of Periostin in tumor tissues and the inhibition efficiency of corresponding murine mAbs on nude mouse transplanted tumor models, the development of antibody drugs against Periostin will likely provide new targeted therapeutic drugs with new targets for clinical tumor therapy.

Disclosure of Invention

An object of the present invention is to provide a humanized anti-Periostin monoclonal antibody. The inventor of the present application determined the CDR region sequences of the humanized anti-Periostin monoclonal antibody by cloning, identification and analysis of the gene structure. The humanized anti-Periostin monoclonal antibody provided by the invention not only has the specificity of being combined with Periostin protein and Periostin positive tumor cells, but also has humanity, and can reduce the toxic and side effects for human use.

The technical scheme for solving the technical problems is as follows: a humanized anti-Periostin monoclonal antibody, which comprises a light chain complementarity determining region and a heavy chain complementarity determining region, wherein the amino acid sequence of the light chain complementarity determining region is shown in SEQ ID Nos. 1-3, and the amino acid sequence of the heavy chain complementarity determining region is shown in SEQ ID Nos. 4-6.

The specific sequence of SEQ ID NO.1-6 is as follows:

SEQ ID NO.1：Lys Ala Ser Gln Asp Val Ser Ala Ala Val Ala。

SEQ ID NO.2：Trp Thr Ser Thr Arg His Thr。

SEQ ID NO.3：Gln Gln His Tyr Tyr Thr Pro Arg Thr。

SEQ ID NO.4：Ala Glu Leu Val Lys。

SEQ ID NO.5：Asp Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly。

SEQ ID NO.6：Asn Thr Ala Tyr Leu Gln Leu Ser Ser。

the invention has the beneficial effects that:

The humanized anti-Periostin monoclonal antibody provided by the invention not only has the specificity of being combined with Periostin protein and Periostin positive tumor cells, but also has humanity, and can reduce the toxic and side effects for human use.

It is well recognized in the art that the binding specificity and affinity of an antibody is primarily determined by the CDR sequences, and that the amino acid sequence of the non-CDR regions can be readily altered to obtain variants with similar biological activity, according to the presently known art. The monoclonal antibody variant of the invention, which has the CDR sequence completely identical with the CDR sequence, has similar biological activity.

The invention provides a CDR region sequence of a monoclonal antibody, which has a specific sequence, can be specifically combined with a Periostin protein and can specifically recognize tumor cells over-expressed by the Periostin protein in cells. The CDR sequence is derived by amplifying, cloning and analyzing the variable region gene of a Periostin mouse monoclonal antibody with tumor targeting and in-vivo tumor treatment effects. The specificity and affinity of the monoclonal antibody and antigen combination are determined by the 6 CDR sequences of the light and heavy chains, on the basis of obtaining the complete CDR sequence, various monoclonal antibody variants with completely identical CDR sequences can be easily constructed and produced according to the industry consensus and the prior art, and the modified variants have similar biological activity and therapeutic action.

The CDR region of the humanized anti-Periostin antibody gene is derived from a mouse monoclonal antibody 3B8 (the research is not published) prepared from a previous human breast cancer cell immune mouse, and the target antigen of the mouse monoclonal antibody is Periostin through mass spectrum identification. Early-stage research proves that the mouse monoclonal antibody can be combined with Periostin, and an in-animal tumor suppression experiment proves that the antibody can block the in-vivo function of Periostin by interference and obviously inhibit the growth of in-vivo Periostin positive lung cancer nude mouse transplanted tumor, and the tumor suppression rate is up to 82.91%; the body weight of an animal is not obviously reduced in treatment, no pathological change is observed under a mirror after the tissue section of an experimental animal is stained by H & E, the research result preliminarily indicates that the toxic and side effects of the antibody treatment are not obvious or low, the safety of the antibody used for treating the malignant tumor with strong positive human Periostin expression is reliable, and the antibody is expected to be used for clinical tumor treatment after the immunogenicity toxicity of the mouse monoclonal antibody is eliminated by humanized modification.

The second object of the present invention is to provide a method for producing the above humanized anti-Periostin monoclonal antibody. The preparation method provided by the invention can be used for obtaining the humanized Periostin-resistant monoclonal antibody, the monoclonal antibody can inhibit the growth and recurrence of human lung cancer in the body of an animal model, and the preparation method is simple, easy to operate and suitable for large-scale popularization and application.

The technical scheme for solving the technical problems is as follows: the preparation method of the humanized anti-Periostin monoclonal antibody comprises the following steps:

Step 1: extracting total RNA of mouse hybridoma LC-5A 6;

Step 2: and (2) synthesizing a first strand of the mouse hybridoma cDNA by using the total RNA extracted in the step (1) as a template:

And step 3: amplifying a light chain complementary determining region by using the cDNA obtained in the step 2 as a template and adopting a light chain upstream primer PVL5 and a light chain downstream primer PVL 3; amplifying a heavy-light chain complementarity determining region by adopting a heavy chain upstream primer PVH5 and a heavy chain downstream primer PVH 3;

And 4, step 4: separating, recovering, purifying and transfecting the target fragment obtained in the step 3, carrying out double enzyme digestion by using Pst II and BstE II, directionally cloning into a corresponding site of a cloning vector pRGWH, screening out a positive recombinant clone, carrying out nucleotide sequence analysis, and constructing an expression vector for expressing the anti-Periostin humanized monoclonal antibody, wherein the expression vector is expressed in pSRNC-Ck-Periostin or pSRDC-Cgamma 1-Periostin transfected Chinese hamster ovary cells, so as to obtain the humanized anti-Periostin monoclonal antibody.

The invention has the beneficial effects that:

The preparation method provided by the invention can be used for obtaining the humanized Periostin-resistant monoclonal antibody, the monoclonal antibody can inhibit the growth and recurrence of human lung cancer in the body of an animal model, and the preparation method is simple, easy to operate and suitable for large-scale popularization and application.

On the basis of the technical scheme, the invention can be further improved as follows.

Further, in the step 1, the method for extracting the total RNA of the mouse hybridoma LC-5A6 comprises the steps of collecting 1 × 10 ⁷ mouse monoclonal antibody hybridoma cells, centrifuging at 10,000rpm for 1min, discarding the supernatant, adding 1mL of Trizol to fully dissolve the cells, standing at room temperature for 3min to 5min, adding 0.2mL of chloroform, reversing and mixing uniformly, centrifuging at 4 ℃, at 12,000rpm for 10min, transferring the supernatant to a clean 1.5mL centrifuge tube, adding 0.5mL of isopropanol, reversing and mixing uniformly, standing at room temperature for 20min, centrifuging at 4 ℃, at 12,000rpm for 10min, discarding the supernatant, washing for 2 times with 75% ethanol, drying in air, and adding 50 μ L of ddH ₂ O to dissolve precipitates to obtain the mouse hybridoma cells.

Total RNA from murine hybridoma LC-5A6 was extracted as described above using the kit TRIZOL ^TM reagent (Invitrogen) from Gibco, cat # 15596026.

The mouse hybridoma cell LC-5A6 is purchased from China general microbiological culture Collection center with the preservation number of CGMCC No. 10898.

Further, in step 2, the reaction system for synthesizing the first strand of the mouse hybridoma cDNA is 20. mu.L of a total reaction system comprising 4. mu.L of 5 Xbuffer, 10mM DDT, 10g of the total RNA extracted in step 1, dNTP at a final concentration of 0.5mM, oligo (dT) ₁₅ at a final concentration of 10. mu.g/mL, 40U of RNAsin and 200U of MMLV-reverse transcriptase, and the mixture is uniformly mixed, subjected to water bath at 37 ℃ for 1h, and subjected to metal bath inactivation at 100 ℃.

among them, DDT, D-Dopachrome Tautomerase, a Chinese name of D-DOPACHROME Tautomerase, was purchased from Promega corporation.

the dNTPs were purchased from Promega corporation.

Oligo (dT) ₁₅, available from Promega corporation, supra.

The above RNAsin was purchased from Promega corporation.

The MMLV-reverse transcriptase described above was purchased from Gibco.

Further, in step 3, the nucleotide sequence of the light chain upstream primer PVL5 is shown as SEQ ID No.9, the nucleotide sequence of the light chain downstream primer PVL3 is shown as SEQ ID No.10, the nucleotide sequence of the heavy chain upstream primer PVH5 is shown as SEQ ID No.11, and the nucleotide sequence of the heavy chain downstream primer PVH3 is shown as SEQ ID No. 12.

the sequences of SEQ ID NO.9-12 are specifically as follows:

SEQ ID NO. 9: 5'-gacattcagctgacccagtctcca-3' (light chain upstream primer PVL 5).

SEQ ID NO. 10: 5'-gttagatctccagcttggtccc-3' (light chain downstream primer PVL 3).

SEQ ID NO. 11: 5 '-aggtsmartgcagsaggcwgg-3' (s ═ c or g, m ═ a or c, r ═ a or g, w ═ a or t) (heavy chain upstream primer PVH 5).

SEQ ID NO. 12: 5'-tgaggagacggtgaccgtcctcccttggccccag-3' (heavy chain downstream primer PVH 3).

Further, in step 3, the method for amplifying the light chain complementarity determining region comprises: in a total reaction system of 100. mu.L, 10. mu.L of 10 Xbuffer, 2. mu.L of dNTP with a concentration of 10mM, 20. mu.L of cDNA obtained in step 2, 50pmol of light chain upstream primer PVL5 and 50pmol of light chain downstream primer PVL3 were mixed, the surface was covered with paraffin oil, after a water bath at 95 ℃ for 5min, 2U of Taq and Pfu DNA polymerase were added through paraffin oil, and a PCR amplification procedure was performed in the following cycle: 94 ℃ for 1 min; 55 deg.C, 1min, 72 deg.C, 1min, 30 cycles in total, and 72 deg.C, 10min in the last cycle.

Further, in step 3, the method for amplifying the heavy chain complementarity determining region comprises: in a total reaction system of 100. mu.L, 10. mu.L of 10 XTbuffer, 2. mu.L of 10mM dNTP, 20. mu.L of the cDNA obtained in step 2, 50pmol of heavy chain upstream primer PVH5 and 50pmol of heavy chain downstream primer PVH3, after mixing, the surface is covered with paraffin oil, after a water bath at 95 ℃ for 5min, 2U of Taq and Pfu DNA polymerase are added through the paraffin oil, and a PCR amplification procedure is performed according to the following cycle: 94 ℃ for 1 min; 55 deg.C, 1min, 72 deg.C, 1min, 30 cycles in total, and 72 deg.C, 10min in the last cycle.

The above Taq was purchased from Promega corporation.

The Pfu DNA polymerase was purchased from Promega corporation.

It is a further object of the present invention to provide a humanized anti-Periostin antibody. The humanized anti-Periostin antibody provided by the invention has humanized antibody, and can greatly reduce the side immune reaction of heterologous antibody to human body.

The technical scheme for solving the technical problems is as follows: a humanized anti-Periostin antibody comprising a light chain complementarity determining region having an amino acid sequence shown in SEQ ID No.7 and a heavy chain complementarity determining region having an amino acid sequence shown in SEQ ID No. 8.

The humanized anti-Periostin antibody of the invention has a light chain protein amino acid sequence comprising or consisting of SEQ ID No.7 and a heavy chain protein amino acid sequence comprising or consisting of SEQ ID No. 8. In a particular embodiment of the invention, the humanized anti-Periostin antibody is a recombinant monoclonal antibody having the amino acid sequences of the light and heavy chains specified therein, which is also referred to as a recombinant humanized antibody since the framework and constant regions of the specified antibody are derived from human.

the specific sequences of SEQ ID NO.7-8 are as follows:

SEQ ID NO.7：

IQAFVFVFLWLSGARVSIPCKASQDVSAAVAWYLQKPGHSPTLLIYWTSTRHTGVPDRFTGSGSGTDYTLTISSVQAEGPGTLLLSTTLLHSSDVRWRHQAGNQTVGTKLQTGVCCTNCIRHLPTIQTAVNIWRCLSRVLLEQLLPQRHQCQVEDKWQGTTKWRPEQLDPSGQQRQHLQHEQHPHVDQGRVLTTHQLYLYGHSQDINFTHCQELQQERV。

SEQ ID NO.8：

EVQLQQSGAELVKPGSSVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARDYYGSRFIYVPETLLCSLWIHFQALWHVLGSPDSGEEAGVGRNHEWWWYSHLLSRHCEGPIHHLQRQCQEQPVPANEQSEVSGHGLVSLCKTGLGRVLLGLLGPRHHSHSLLSQNDTPICLSTGPWICCPNSLHGDPGMPGQGLFPLASDSDLELWIPVQRCAHLPSCPAVIPLHSEQLSDCPLQPSAQRDRHLQRCPPGQQHQGGQENCAQGLWLAALHMYSPRSIICLHLPPKAQGCPHHYSDSQGHVCCGRHQQGESRGPVQLVCRIICGGAHSSDATPGGAVQQHFPLSQPTSHHAPGLAQWQGVQMQGQQCSFPCPHRENHIQNQRQTEGSTGVHHSTSQGADGQGKSQSDLHDNRLLPQRHNSGVAVEWAASGELQEHSAHHEH。

The invention has the beneficial effects that:

The humanized anti-Periostin antibody provided by the invention has humanized antibody, and can greatly reduce the side immune reaction of heterologous antibody to human body.

It is to be noted that, in the present application, the term "antibody of the present invention" specifically means a "humanized anti-Periostin monoclonal antibody" according to the present invention, wherein the light chain complementarity determining region comprises CDR regions shown in SEQ ID Nos. 1 to 3, and the heavy chain complementarity determining region comprises CDR regions shown in SEQ ID Nos. 4 to 6.

"humanized anti-Periostin antibody" is an anti-Periostin humanized monoclonal antibody comprising the amino acid sequence of the specified light chain protein comprised by or consisting of SEQ ID NO.7 and the amino acid sequence of the heavy chain protein comprised by or consisting of the specified complete sequence of SEQ ID NO.8, the complete sequence of the specified anti-Periostin humanized monoclonal antibody being composed of CDR sequences provided by the present invention embedded and spliced to the FR region sequences and constant region sequences of the specified human antibody.

the fourth object of the present invention is to provide an isolated polynucleotide. The isolated polynucleotides provided by the present invention may encode the humanized anti-Periostin monoclonal antibodies described above.

The technical scheme for solving the technical problems is as follows: an isolated polynucleotide encoding the humanized anti-Periostin monoclonal antibody described above.

The invention has the beneficial effects that:

The isolated polynucleotides provided by the present invention may encode the humanized anti-Periostin monoclonal antibodies described above.

According to a consensus in the art, even if the nucleotide sequences encoding the antibody proteins of SEQ ID Nos. 1-3 and 4-6 are altered, the nucleic acid is DNA, as long as the translated amino acid sequences three positions after the triplet codon are retained, and are all polynucleotides encoding the anti-Periostin humanized antibody.

The fifth purpose of the invention is to provide an expression vector. The expression vector of the present invention comprises the above-mentioned polynucleotide, and specifically, the present invention recombines and clones the polynucleotide encoding the light chain and the heavy chain of the humanized chimeric antibody into two vectors containing eukaryotic promoter, respectively, and introduces the obtained expression vector into eukaryotic host cells, and obtains the eukaryotic host cells with high-yield expression by screening, and the culture supernatant of the host cells contains a large amount of the humanized antibody protein secreted therefrom, and can be conveniently extracted and prepared therefrom according to the technical method known in the art.

The technical scheme for solving the technical problems is as follows: an expression vector comprising the polynucleotide.

The invention has the beneficial effects that:

The expression vector of the present invention comprises the above-mentioned polynucleotide, and specifically, the present invention recombines and clones the polynucleotide encoding the light chain and the heavy chain of the humanized chimeric antibody into two vectors containing eukaryotic promoter, respectively, and introduces the obtained expression vector into eukaryotic host cells, and obtains the eukaryotic host cells with high-yield expression by screening, and the culture supernatant of the host cells contains a large amount of the humanized antibody protein secreted therefrom, and can be conveniently extracted and prepared therefrom according to the technical method known in the art.

One skilled in the art can readily construct expression vectors in a variety of forms that can express the antibody, provided that the amino acid sequence or nucleotide sequence of the expressed antibody is known, based on common knowledge and general techniques in the art.

As is well known in the art, many forms of expression vectors for antibodies can be readily constructed using techniques common in the art, provided that the amino acid or nucleotide sequence of the antibody desired to be expressed is known. Vectors may be derived from plasmids such as F, R1, RP1, Co1, pBR322, TOL, Ti, and the like; sticking particles; bacteriophages such as lambda, Iambdoid, M13, Mu, P1, P22, Q, T-even, T-odd, T2, T4, T7; plant or animal viruses, and the like. The vectors may be used for cloning and/or expression purposes and for gene therapy purposes. Vectors comprising one or more nucleic acid molecules encoding an antibody of the invention operably linked to one or more expression-modulating nucleic acid molecules may also be included in the invention. The choice of vector will depend on the recombination procedure and the host used. Introduction of the vector into the host cell can be achieved by calcium phosphate transfection, viral infection, DEAE-dextran mediated transfection, lipofectamin transfection or electroporation. The vector may be autonomously replicating or may be replicating together with the chromosome(s) into which it has been integrated. The vector typically contains one or more selectable markers, the selection of which may be dependent on the host cell selected, including but not limited to kanamycin, neomycin, puromycin, hygromycin, Zeocin, the thymidine kinase gene of herpes simplex virus (HSV-TK), the mouse dihydrofolate reductase gene (dhfr). Specifically, the invention recombines and clones the polynucleotides for encoding the light chain and the heavy chain of the anti-Periostin humanized chimeric antibody into two vectors containing eukaryotic promoters respectively, and the obtained expression vector is introduced into eukaryotic host cells, and the eukaryotic host cells which express the antibody of the invention with high yield are obtained by screening, containing in the culture supernatant of the host cell a plurality of the anti-Periostin humanized antibody proteins secreted therefrom, the anti-Periostin humanized antibody protein can be conveniently extracted and prepared therefrom according to a technical method commonly recognized in the art, in the examples, the expression vectors were designated pSRNC-Ck-Periostin-1 and pSRDC-Cy 1-Periostin-1, respectively, which contain the anti-Periostin humanized antibody gene and methotrexate pressor-amplified expression selection marker gene (dhfr) and are expressed in Chinese Hamster Ovary (CHO) cells.

The sixth object of the present invention is to provide a host cell. The host cell provided by the invention contains the polynucleotide and provides a vector for secreting antibody protein.

The technical scheme for solving the technical problems is as follows: a host cell comprising the polynucleotide or the expression vector.

The invention has the beneficial effects that:

The host cell provided by the invention contains the polynucleotide and provides a vector for secreting antibody protein.

The host is a host cell including, but not limited to, a cell of mammalian, plant, insect, fungal or bacterial origin. Expression systems using mammals such as chinese hamster ovary Cells (CHO) are often preferred for use in the present invention. In embodiments, the host cell is a chinese hamster ovary Cell (CHO). pSRNC-Ck-Periostin-1 and pSRDC-Cy 1-Periostin-1 were introduced into eukaryotic CHO cells, eukaryotic host cells expressing the antibody of the present invention in high yield were obtained by selection, containing a large amount of the secreted anti-Periostin humanized antibody protein in the culture supernatant of the host cells, from which the anti-Periostin humanized antibody protein could be conveniently extracted and prepared according to consensus in the art.

The seventh object of the present invention is to provide the use of the above humanized anti-Periostin monoclonal antibody or the above polynucleotide or the above expression vector or the above host cell in the preparation of a humanized anti-Periostin positive tumor medicament. The humanized anti-Periostin monoclonal antibody or polynucleotide or expression vector or the host cell can be used for preparing humanized anti-Periostin positive tumor medicaments and treating malignant tumors with positive Periostin expression on the cell surface.

The technical scheme for solving the technical problems is as follows: use of a humanized anti-Periostin monoclonal antibody as described above or a humanized anti-Periostin antibody as described above or a polynucleotide as described above or an expression vector as described above or a host cell as described above for the preparation of a medicament for combating human-derived anti-Periostin positive tumors.

The invention has the beneficial effects that:

The humanized anti-Periostin monoclonal antibody or humanized anti-Periostin antibody or polynucleotide or expression vector or the host cell can be used for preparing humanized anti-Periostin positive tumor medicaments and treating malignant tumors with positive Periostin expression on the cell surface.

The humanized anti-Periostin antibody can be specifically combined with Periostin protein secreted by tumor cells, can directly play a role in blocking the biological function of Periostin in vivo and inhibiting the growth of a Periostin over-expression tumor in vivo, so that the antibody can be directly used for preparing a targeted therapeutic antibody medicine for resisting the Periostin expression positive tumor. According to the consensus in the art, when obtaining humanized anti-Periostin antibody protein, the antibody drug can be prepared for direct use in human therapy by adding appropriate stabilizers, protectants, pH regulators, salt balancers, excipients, and the like. In embodiments, the antibodies of the invention are useful for treating a nude mouse melanoma positive for Periostin expression. In a particular breast cancer model, the humanized anti-periodin chimeric antibody may be administered as a drug to a patient by conventional routes of administration, such as, but not limited to, parenteral administration, e.g., intravenous infusion; topical administration, and the like. The appropriate dosage depends on several parameters, including the method of administration and the degree of tolerance of the subject being treated. It is clear that the anti-Periostin humanized antibody can obviously inhibit the growth of human breast cancer cells overexpressed by Periostin and is in a dose-dependent relationship.

Definition of

Monoclonal antibodies

The term "monoclonal antibody" as used herein refers to an antibody from a population of substantially homologous antibodies, i.e., comprising the same collection of individual antibodies except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific antibodies directed against a single target site, each monoclonal antibody being directed against a single determinant on the target, in contrast to conventional (polyclonal) antibody preparations, which comprise different antibodies directed against different determinants (epitopes). In addition to its specificity, the advantages of monoclonal antibodies can be synthesized by hybridoma culture, thereby avoiding immunoglobulin contamination. The modifier "monoclonal" indicates the character of the antibody as being from a substantially homogeneous population of antibodies, and does not require production of the antibody by any particular method.

Complementarity Determining Region (CDR)

The term "complementarity determining region" as used herein refers to sequences in binding molecules such as immunoglobulin variable regions. It generally provides primarily an antigen binding site that is complementary in shape and charge distribution to the epitope recognized on the antigen. The CDR regions may be specific for linear epitopes, discontinuous epitopes or conformational epitopes of proteins, or protein fragments, which epitopes are present on the protein in its native conformation or in some cases in denatured form, for example by solubilization in SDS, or may consist of post-translationally modified proteins.

Polynucleotide

As used herein, the term "polynucleotide" includes deoxyribonucleotides, ribonucleotides, or analogs that have the requisite properties for a natural ribonucleic acid to hybridize under stringent hybridization conditions to the same and substantially the same nucleotide sequence as a natural nucleotide and/or to be translated into the same amino acid as a natural nucleotide. The polynucleotide may be the full length or a subsequence of a native or heterologous structure or regulatory gene. Unless otherwise indicated, the term includes the specific sequence and its complement. Thus, the term "polynucleotide" in this application encompasses DNA having a backbone modified for stability or other reasons.

Polypeptides

the term "polypeptide" is used interchangeably herein with "peptide" and "protein" and refers to a polymer of amino acid residues. The term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding natural amino acid, as well as for natural amino acid polymers. An essential property of such analogues of natural amino acids is that when incorporated into a protein, the protein specifically reacts with antibodies raised against the same protein but consisting entirely of natural amino acids. The terms "polypeptide", "peptide" and "protein" also include modifications, but are not limited to, phosphorylation, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

Specific binding

The term "specific binding" as used herein in reference to an interaction between an antibody and its binding partner, e.g., an antigen, means that the interaction is dependent on the presence of a particular structure on the binding partner, e.g., the presence of an antigenic determinant or epitope. In other words, the antibody also preferentially binds or recognizes the binding partner when the binding partner is present in a mixture of other molecules or organisms. The binding may be mediated by covalent or non-covalent interactions, or both. "specifically binds" also refers to immunospecific binding to an antigen or fragment thereof and non-immunospecific binding to other antigens. Binding molecules that immunospecifically bind to an antigen can bind other peptides or polypeptides with lower affinity as determined by Radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), BIACORE, or other assays known in the art. A binding molecule or fragment thereof that immunospecifically binds to an antigen can cross-react with the relevant antigen. Preferably, the binding molecule or fragment thereof that immunospecifically binds an antigen does not cross-react with other antigens.

Variants

The term "variant" as used herein refers to a binding partner (e.g., a binding molecule of periodinan) that comprises a nucleotide sequence and/or amino acid sequence that is altered from the nucleotide and/or amino acid sequence of one or more CDR regions compared to the nucleotide and/or amino acid sequence of the parent binding molecule, but that is still capable of competitively binding to the parent binding molecule. In other words, modifications in the amino acid and/or nucleotide sequence of the parent binding molecule do not significantly affect or alter the binding properties of the binding molecule encoded by or comprising the nucleotide sequence, i.e. the binding molecule is still able to recognize and bind its target. The functional variants have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. Such modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can comprise natural and non-natural nucleotides and amino acids.

Conservative amino acid substitutions include the replacement of an amino acid residue with an amino acid residue having similar structural or chemical properties. The present field has identified a family of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cystine, tryptophan), nonpolar side chains (e.g., alanine, leucine, isoleucine, proline, phenylalanine, methionine, valine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine). In addition to this, other amino acid residue family classes than those described above may also be applied. In addition, variants may also be substituted with non-conservative amino acids, such as replacing an amino acid residue with an amino acid residue having a different structure or chemical property. Similar minor changes may also include amino acid deletions or insertions. Techniques for determining whether an amino acid residue can be substituted, inserted, or deleted without abolishing its immunological activity can be explored using computer software common in the art.

The mutation in the nucleotide sequence may be a single mutation (point mutation) generated at a locus, or a transition or transversion mutation, or may be insertion, deletion or alteration of multiple nucleotides at a single locus. In addition, one or more changes may be made at any number of loci within the nucleotide sequence. Mutations can be manipulated by suitable methods known in the art.

Humanized antibodies

₂In general, humanized antibodies are generally two variable regions (at least comprising one), ideally all CDRs corresponding to those of the non-human immunoglobulin, and substantially all FR regions being those of a human immunoglobulin consensus sequence.

Carrier

The term "vector" as used herein refers to a nucleic acid molecule that can be inserted into another nucleic acid molecule for introduction into a host for replication and induction of expression in a particular environment. In other words, the vector is capable of transporting the nucleic acid molecule to which it is linked. Both cloning and expression vectors are covered by the term "vector" as used herein, including but not limited to plasmids, cosmids, Bacterial Artificial Chromosomes (BACs) and Yeast Artificial Chromosomes (YACs) and vectors derived from phage, or plant (animal, human) viruses. The vector contains an origin of replication recognized by the intended host, and promoters and other regulatory regions recognized by the host are also encompassed in the expression vector. The vector containing the nucleic acid molecule of interest is introduced into the cell by transformation, transfection or by using viral entry mechanisms. Certain vectors are capable of autonomous replication in a host into which they are introduced (e.g., vectors having a bacterial origin of replication may replicate in bacteria). Other vectors may be integrated into the host genome upon introduction into the host, and thereby replicated together with the host genome.

Host computer

the term "host" as used herein refers to a cell into which a vector, such as a cloning vector or an expression vector, has been introduced. The cell may be a prokaryotic or eukaryotic cell or organism. The term refers not only to the particular subject cell, but also includes progeny of that cell. Such progeny may not be identical to the parent cell, but are still included within the scope of the "host" referred to herein, since certain modifications may occur in succeeding generations due to either mutation or environmental influences.

pharmaceutically acceptable excipient

The term "pharmaceutically acceptable excipient" as used herein refers to any inert substance used in combination with an active molecule, such as a drug, active substance or binding molecule, to prepare a suitable or convenient dosage form. A "pharmaceutically acceptable excipient" is an excipient that is non-toxic to the recipient at the dosages and concentrations employed and is compatible with the other ingredients of the formulation containing the drug, agent or binding molecule.

A therapeutically effective amount

The term "therapeutically effective amount" as used herein refers to the amount of an antibody of the invention that is effective in preventing, ameliorating and/or treating a tumor.

Treatment of

The term "treatment" as used herein refers to therapeutic treatment and prophylactic measures to cure a disease or to arrest or at least slow down the progression of a disease. Subjects in need of treatment include subjects already having a tumor or in need of prevention of tumorigenesis. Prevention includes inhibiting the development of a tumor or delaying the development of a tumor or inhibiting or reducing the development, progression and/or onset of one or more symptoms associated with a tumor.

As used herein, the term "comprising" means including the stated elements, integers or steps; or a group of elements, integers or steps, but does not exclude any other element, integer or step, or group of elements, integers or steps.

Drawings

FIG. 1 is a schematic structural diagram of a humanized anti-Periostin antibody light chain eukaryotic expression vector pSRNC-Ck-Periostin-1 of the present invention.

In the figure, Pw represents an attenuated eukaryotic promoter; neo represents an aminoglycoside phosphotransferase (Neo) gene; PhCMV-IE represents the promoter and enhancer of the human cytomegalovirus immediate early gene; VL gene represents a light chain variable region gene fragment with a leader peptide sequence and a 5' intron-end cleavage site sequence; c kappa gene represents a human antibody light chain K chain constant region gene fragment; BGHpolyA represents a bovine growth hormone polyA tailing site; ap represents an ampicillin resistance gene.

FIG. 2 is a schematic structural diagram of a humanized anti-Periostin antibody heavy chain eukaryotic expression vector pSRDC-Cgamma 1-Periostin-1 of the present invention.

In the figure, Pw represents an attenuated eukaryotic promoter; dhfr represents a dihydrofolate reductase gene; PhCMV-IE represents the promoter and enhancer of the human cytomegalovirus immediate early gene; VHgene represents a heavy chain variable region gene fragment with a leader peptide sequence and a 5' intron end cleavage site sequence; c gamma 1 gene represents human antibody heavy chain gamma 1 chain constant region gene segment; BGH polyA represents a bovine growth hormone polyA tailing site; ap represents an ampicillin resistance gene.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

Example 1: preparation method of humanized anti-Periostin monoclonal antibody

The method for preparing the humanized anti-Periostin monoclonal antibody comprises the following steps:

Step 1: total RNA of mouse hybridoma LC-5A6 was extracted

Mouse hybridoma cell LC-5A6 is purchased from China general microbiological culture collection center with the preservation number of CGMCC No.10898, stored in liquid nitrogen, total RNA of mouse hybridoma cell LC-5A6 is extracted and operated according to the instructions of Trizol (Gibco company), and the method specifically comprises the steps of collecting 1 × 10 ⁷ mouse monoclonal antibody hybridoma cells, centrifuging for 1min at 10,000rpm, discarding supernatant, adding 1mL Trizol to fully dissolve the cells, standing for 3-5min at room temperature, adding 0.2mL chloroform, reversing and mixing, centrifuging for 10min at 4 ℃, 12,000rpm, transferring supernatant to a clean 1.5mL centrifuge tube, adding 0.5mL isopropanol, reversing and mixing, standing for 20min at room temperature, centrifuging for 10min at 4 ℃, 12,000rpm, discarding supernatant, washing for 2 times with 75% ethanol, drying in air, and adding 50 μ L ddH ₂ O to dissolve precipitates.

Step 2: taking the total RNA extracted in the step 1 as a template to synthesize a first strand of the mouse hybridoma cell cDNA

0.7% non-denaturing agarose gel electrophoresis analysis Total RNA 18S and 28S RNA sizes were extracted from parental anti-Periostin murine monoclonal antibody hybridoma cell lines. First strand of murine monoclonal antibody hybridoma cell cDNA synthesized using MMLV-reverse transcriptase: mu.L of a total reaction system comprising 4. mu.L of 5 Xbuffer, 10mM DDT, 10. mu.g of total RNA extracted in step 1, dNTP at a final concentration of 0.5mM, Oligod (T)15 at a final concentration of 10. mu.g/mL, 40U of RNAsin and 200U of MMLV-reverse transcriptase was mixed homogeneously, incubated for 1h at 37 ℃ and inactivated at 100 ℃ in a metal bath.

The dNTPs were purchased from Promega corporation.

Oligo (dT) ₁₅, available from Promega corporation, supra.

The above RNAsin was purchased from Promega corporation.

The MMLV-reverse transcriptase described above was purchased from Gibco.

And step 3: amplifying a light chain complementary determining region by using the cDNA obtained in the step 2 as a template and adopting a light chain upstream primer PVL5 and a light chain downstream primer PVL 3; heavy and light chain complementarity determining regions were amplified using a heavy chain upstream primer PVH5 and a heavy chain downstream primer PVH 3.

the nucleotide sequence of the light chain upstream primer PVL5 is shown as SEQ ID NO.9, the nucleotide sequence of the light chain downstream primer PVL3 is shown as SEQ ID NO.10, the nucleotide sequence of the heavy chain upstream primer PVH5 is shown as SEQ ID NO.11, and the nucleotide sequence of the heavy chain downstream primer PVH3 is shown as SEQ ID NO. 12.

The sequences of SEQ ID NO.9-12 are specifically as follows:

the method for amplifying the light chain complementarity determining region comprises the following steps: in a total reaction system of 100. mu.L, 10. mu.L of 10 Xbuffer, 2. mu.L of dNTP with a concentration of 10mM, 20. mu.L of cDNA obtained in step 2, 50pmol of light chain upstream primer PVL5 and 50pmol of light chain downstream primer PVL3 were mixed, the surface was covered with paraffin oil, after a water bath at 95 ℃ for 5min, 2U of Taq and PfuDNA polymerase were added through the paraffin oil, and the PCR amplification procedure was performed in the following cycle: 94 ℃ for 1 min; 55 deg.C, 1min, 72 deg.C, 1min, 30 cycles in total, and 72 deg.C, 10min in the last cycle.

The method for amplifying the heavy chain complementarity determining region comprises the following steps: in a total reaction system of 100. mu.L, 10. mu.L of 10 XPuffer, 2. mu.L of 10mM dNTP, 20. mu.L of the cDNA obtained in step 2, 50pmol of heavy chain upstream primer PVH5 and 50pmol of heavy chain downstream primer PVH3, after mixing, the surface is covered with paraffin oil, after a water bath at 95 ℃ for 5min, 2U of Taq and PfuDNA polymerase are added through the paraffin oil, and the PCR amplification procedure is performed according to the following cycle: 94 ℃ for 1 min; 55 deg.C, 1min, 72 deg.C, 1min, 30 cycles in total, and 72 deg.C, 10min in the last cycle.

The above Taq was purchased from Promega corporation.

The Pfu DNA polymerase was purchased from Promega corporation.

The results are shown in FIG. 1: the product at about 320bp is the gene segment of light chain variable region, the product at about 360bp is the gene segment of heavy chain variable region, and the amplified segment size is identical to that of common antibody variable region gene.

And 4, step 4: amplifying the light chain variable region gene fragment of the anti-Periostin mouse monoclonal antibody in a large scale, separating and recovering by adopting a glass milk adsorption method, performing double enzyme digestion by PvuII and Bg1 II, directionally cloning into corresponding sites of a cloning vector pRGWL, and collecting 358 transformed clones in total. Randomly selecting 25 clones, and screening to obtain 7 recombinant clones. The screened positive recombinant clones were subjected to nucleotide sequence analysis, and the sequences of 7 clones were completely identical, confirming that the light chain variable region gene of the anti-Periostin murine monoclonal antibody existed in the cloned antibody light chain variable region gene. One of the clones selected at random from 7 clones was designated pRGWL-Periostin-1. Comparison of Kabat data revealed that the VL of the anti-Periostin murine monoclonal antibody belongs to subgroup III of the light chain of mouse k, the light chain CDR1-3 sequence (SEQ ID NO. 1-3).

amplifying heavy chain variable region gene segments of the anti-Periostin mouse monoclonal antibody in a large scale, separating and recovering by adopting a glass milk adsorption method, performing double enzyme digestion by using Pst II and BstE II, directionally cloning into corresponding sites of a cloning vector pRGWH, and collecting 271 transformed clones in total. Randomly selecting 25 clones, and screening to obtain 9 recombinant clones. The nucleotide sequence analysis was performed on 5 of the positive recombinant clones, and the sequences of the 5 clones were completely identical, confirming the presence of the anti-Periostin murine monoclonal antibody heavy chain variable region gene in the cloned anti-heavy chain variable region genes. One of the 5 clones was randomly selected and named pRGWH-Periostin-1. Comparison of Kabat data revealed that the VH of the anti-Periostin murine monoclonal antibody belongs to subgroup III of the heavy chains of mouse IgG, the CDR1-3 sequences of the heavy chains (SEQ ID NO. 4-6).

The specific sequences of SEQ ID NO.1-6 are as follows:

SEQ ID NO.1.Lys Ala Ser Gln Asp Val Ser Ala Ala Val Ala；

SEQ ID NO.2.Trp Thr Ser Thr Arg His Thr；

SEQ ID NO.3.Gln Gln His Tyr Tyr Thr Pro Arg Thr；

SEQ ID NO.4.Ala Glu Leu Val Lys；

SEQ ID NO.5.Asp Ile Lys Asp Thr Tyr Ile His Trp Val Lys Gln Arg Pro Glu Gln Gly；

SEQ ID NO.6.Asn Thr Ala Tyr Leu Gln Leu Ser Ser。

Example 2: construction of humanized antibody gene and expression vector of anti-Periostin mouse monoclonal antibody

The method comprises the steps of replacing a framework region of a variable region gene of the anti-Periostin mouse monoclonal antibody with a framework region of a human antibody by adopting a gene synthesis method, synthesizing a humanized antibody variable region gene fragment of the anti-Periostin mouse monoclonal antibody by adopting a total synthesis method, recombining the humanized antibody variable region gene fragment into a vector containing a regulatory sequence and a human antibody constant region gene, and constructing a complete gene of the anti-Periostin humanized antibody and an eukaryotic expression vector containing the gene.

Synthesis of humanized antibody variable region genes of anti-Periostin murine monoclonal antibody: the humanized 4D5 monoclonal antibody variable region is used as a template, a CDR grafting method is adopted to replace the CDR region with the CDR region of the corresponding 3B8 monoclonal antibody, and the cloning enzyme cutting site sequences at the two ends are reserved. The light and heavy chain genes are directly synthesized by commercial companies by adopting a segmented synthesis + overlapping PCR connection method. The humanized antibody light and heavy chain variable region genes of the anti-Periostin murine monoclonal antibody were amplified by PCR in the same manner as in example 1, the products were digested, re-cloned into pRGWL and pRGWH vectors, the sequences of the clones were identified by sequencing to be identical to the originally designed sequences, and the positive clones were selected and named pRGWL-Periostin-H and pRGWH-Periostin-H, respectively, and used as amplification templates for constructing the following expression vectors.

amplifying variable region gene fragments with regulatory sequences by PCR: using the recombinant cloning plasmids pRGWL-Periostin-H and pRGWH-Periostin-H obtained in example 1 identified by sequencing as templates, humanized VL and VH with leader peptides and 5' -end cleavage sites were PCR-amplified using primers PVLS/PVNP (light chain) and PVHS/PVNP (heavy chain) containing BamH I and Not I cleavage sites at both ends. Amplifying a humanized VL fragment with a leader peptide and a 5' end shearing site from a recombinant plasmid of a light chain by PCR (polymerase chain reaction), wherein the size of the humanized VL fragment is about 500 bp; a humanized VH segment with a leader peptide and a 5' cleavage site was amplified from a recombinant plasmid for the heavy chain, approximately 700bp in size. Specifically, high-precision DNA polymerases Taq and Pfu DNA polymerase were used, which contained: 10 μ L of 10 Xbuffer, 2 μ L of 10mM dNTP, 1 μ g of plasmid (pRGWL-periodin-H and pRGWH-periodin-H), 50pmol each of amplification primers (light chain upstream primer PVLS, light chain downstream primer PVNP, heavy chain upstream primer PVHS, heavy chain downstream primer PVNP), mixing uniformly, covering the surface with paraffin oil, adding 2U Taq and Pfu DNA polymerase through the paraffin oil after 5min of water bath at 95 ℃, and heating at 94 ℃ for 1 min; 55 deg.C, 1min, 72 deg.C, 1min, 20 cycles; and performing PCR amplification at 72 ℃ for 10min in the last cycle.

The nucleotide sequence of the light chain upstream primer PVLS is shown as SEQ ID NO.13, the nucleotide sequence of the light chain downstream primer PVNP or the heavy chain downstream primer PVNP is shown as SEQ ID NO.14, and the nucleotide sequence of the heavy chain upstream primer PVHS is shown as SEQ ID NO. 15.

The sequence of SEQ ID NO.13-15 is specifically as follows:

SEQ ID NO. 13: 5'-ctcggaattcggatccatgggatggagctgtatcatcc-3' (light chain upstream primer PVLS).

SEQ ID No. 14: 5'-ggtccgaattcgcggccgctataaatctctggccatgaag-3' (light chain downstream primer PVNP or heavy chain downstream primer PVNP).

SEQ ID NO.15:

5'-ggtccagcttgcggccgcaactgaggaagcaaagtttaaattctactcacgtttgatcacca-3' (heavy chain upstream primer PVHS).

Construction and identification of humanized anti-Periostin antibody integration expression vector: the PCR product was separated and recovered by a glass milk adsorption method, and then digested with BamH I and Not I, followed by conventional DNA recombination as described in molecular cloning (fourth edition). VL is cloned into pSRNC-Ck, VH is cloned into corresponding sites in pSRDC-Cgamma 1, and a complete eukaryotic expression vector of the anti-Periostin humanized antibody gene is constructed. And respectively connecting VL and VH into expression vectors pSRNC-Ck and pSRDC-Cgamma 1, respectively selecting 5 clones for screening, and carrying out enzyme digestion identification to obtain 5 light chain recombinant clones and 3 heavy chain recombinant clones. After double enzyme digestion of BamH I and Not I, the corresponding VL fragment and VH fragment can be cut off, and the complete anti-Periostin humanized monoclonal antibody gene and eukaryotic expression vector can be successfully constructed. 3 times of repeated nucleotide sequences prove that the variable region gene sequences of the anti-Periostin humanized monoclonal antibody eukaryotic expression pSRNC-Ck-Periostin and pSRDC-Cgamma 1-Periostin are completely identical to the originally designed humanized variable gene sequences respectively. The amino acid sequences of the k-producing anti-Periostin humanized monoclonal antibody of the expression vector are shown in SEQ ID NO.7 (light chain, constant region is human antibody C kappa) and SEQ ID NO.8 (heavy chain, constant region is human antibody C gamma 1).

SEQ ID NO.7 (light chain sequence) is as follows:

SEQ ID NO 8 (heavy chain sequence) is as follows:

The eukaryotic expression vector structure of the humanized anti-Periostin monoclonal antibody comprises the following components: the eukaryotic expression vector of the anti-Periostin humanized monoclonal antibody adopts a light chain eukaryotic expression vector (pSRNC-Ck-Periostin) and a heavy chain expression vector (pSRDC-Cgamma 1-Periostin) respectively, and the structural schematic diagram of the eukaryotic expression vector is shown in figure 1 and figure 2.

Example 3: inducible expression in cells (CHO cells) of humanized anti-Periostin antibody expression vectors

A DMEM medium containing 10% FBS, 0.03mmol/L hypoxanthine (H), 0.003mmol/L thymidine (T), 0.1mmol/L proline (Pro), 0.1mmol/L glycine (Gly), 100U/mL streptomycin, 2mmol/L glutamine was placed in an environment of 5% CO ₂, 37 ℃ to resuscitate and culture CHO-dhfr ^- cells, passaged once in a 1:10 ratio for 3-4 days by the gene transfection method using Lipofect AMINE reagent (Gibco Co.), light and heavy chain expression vectors for anti-Periostin humanized antibody were CO-transfected into the cells, selected using a medium containing no H, T, Gly, selected using a selective medium containing 200. mu.g/mL G418(Gibco Co.) after the formation of clones, and as a result, 4. mu.g of each light and heavy chain expression vector was transfected into CHO-dhfr-cells, 2 weeks, a monoclonal antibody was grown in a visual culture medium containing polyclonal IgG 82 (Gibco K) and a monoclonal antibody was expressed as a control, and indirect monoclonal antibody was obtained by ELISA ₄₉₀.

Example 4: screening of high-expression strain of anti-Periostin humanized chimeric antibody

Recovering and culturing transfected CHO cells in DMEM medium containing 10% FBS, 100U/mL streptomycin and 2mmol/L glutamine at 37 ℃ in an environment of 5% CO ₂, subculturing once for 3-4 days according to a ratio of 1:10, screening the anti-Periostin antibody high-expression strain by adopting a Methotrexate (MTX) pressure amplification expression screening method, carrying out pressure culture on cell clones containing humanized anti-Periostin antibody expression in supernatant by adopting complete medium containing 3X 10 ^-8 M and 10 ^-7 M MTX in sequence, subcloning by adopting a limiting dilution method after each round of pressure amplification expression, and screening the highest-yield clone.

After a humanized anti-Periostin antibody expression vector is transfected into CHO-dhfr ^- cells, clone 7B4 (the clone yield can reach 28.9 mu g/mL) which is obtained by primary screening and highly efficiently expresses the humanized anti-Periostin antibody is added into a culture medium containing 3 × 10 ^-8 M MTX for continuous culture for 30 days, after the cells are adapted to 3 × 10 ^-8 M MTX pressure concentration (the cell morphology and the growth speed are recovered to be normal), the antibody yield can reach 37.51 mu g/mL, clone 9E6 is continuously subjected to 1:5 subculture, 1 × 10 ^-7 M MTX is replaced for continuous pressurized culture, after the cells are adapted, the antibody yield can reach 46.95 mu g/mL, and the antibody yield of the clone strain with the highest antibody yield of 2A5 screened after subcloning can reach 81.07 mu g/mL through detection.

Example 5: specificity, humanity identification of anti-Periostin humanized antibodies

1. The specificity of the anti-Periostin humanized antibody was determined by coating an ELISA plate with 1. mu.g/mL of recombinant Periostin protein by ELISA, adding a goat anti-human IgG Fc-HRP enzyme-labeled antibody (Sigma) which does not cross-react with mouse Ig or a goat anti-mouse IgG Fc-HRP enzyme-labeled antibody (Sigma) after the reaction, developing color, and determining OD ₄₉₀. the anti-Periostin humanized antibody purified from the supernatant of the clone 2A5 cell by a protein A affinity column was bound to the coated recombinant Periostin protein and recognized by the goat anti-human IgG Fc fragment polyclonal antibody to show a positive reaction, whereas the culture supernatant of the untransfected CHO-dhfr ^- cell and the parent mouse monoclonal antibody 3B8 showed a negative reaction, and the culture supernatant of the untransfected CHO-dhfr ^- cell and the purified anti-Periostin humanized antibody were both negative and the murine monoclonal antibody was found to show a negative reaction when the goat anti-mouse IgG Fc-HRP was used as a secondary antibody, while the monoclonal antibody was found to show a negative reaction, whereas the monoclonal antibody was found to show a negative reaction when the monoclonal antibody was found to bind to human IgG 1.

TABLE 1 ELISA analysis of antigen binding specificity of anti-Periostin humanized antibodies

sample (I)	OD₄₉₀ ^a	OD₄₉₀ ^b
			Humanized anti-Periostin antibody (100ng/mL)	2.471±0.200	2.339±0.176
human IgG1(100ng/mL)	0.073±0.001	0.073±0.001
			Parental mouse monoclonal antibody 3B8(100ng/mL)	0.073±0.000	2.197±0.242
PBS control	0.076±0.001	0.074±0.000
			CHO-dhfr^-Cell supernatant	0.074±0.003	0.074±0.001

a goat anti-human IgG Fc fragment-HRP is used as a secondary antibody; b goat anti-mouse IgG Fc fragment-HRP was used as the secondary antibody.

Taking an immobilized cell of a human breast cancer cell MDA-MB-231 with high expression of Periostin as a target cell, adding a humanized anti-Periostin antibody (10 mu g/mL), incubating at 37 ℃, adding a goat anti-human IgG-fluorescent secondary antibody (Sigma company), and observing that the anti-Periostin humanized antibody recognizes the Periostin protein expressed by the human breast cancer cell MDA-MB-23 with high expression of Periostin under a fluorescent microscope.

2. Humanized identification of humanized anti-periodin antibodies: in ELISA, the plate was coated with goat anti-human k chain (Sigma) or goat anti-human IgG polyclonal antibody (Sigma), and the results of ELISA using goat anti-IgG Fc-HRP (Sigma) as the enzyme-labeled antibody (Table 2) showed that the purified anti-Periostin humanized antibody reacted strongly positively, while the parent murine monoclonal antibody 3B8 of the anti-Periostin humanized antibody reacted negatively. The purified humanized anti-Periostin antibody was shown to contain the light and heavy chain constant regions of human IgG.

TABLE 2 detection of human origin of anti-Periostin humanized antibodies by ELISA

Sample (100ng/mL)	OD₄₉₀ ^a	OD₄₉₀ ^b
			Humanized anti-Periostin antibody (2A5)	2.915±0.232	2.532±0.171
Parental murine monoclonal antibody 3B8	0.074±0.003	0.073±0.001
			Human IgG1	2.961±0.087	2.083±0.102
PBS control	0.074±0.001	0.073±0.003

a coating with goat anti-human IgG; b coating with goat anti-human k-chain.

Example 6: preparation of therapeutic drug by using anti-Periostin humanized antibody for in vivo treatment of Periostin overexpression human nude mouse transplantation tumor

The research on the effectiveness of the in-vivo treatment of human breast cancer by the anti-Periostin humanized antibody is carried out in human breast cancer cells with high expression of human Periostin (10 ⁶ cells are injected into the skin of the right chest fat pad of the nude mouse), the intraperitoneal administration treatment is started 3 days after the inoculation of the tumor cells, the in-vivo treatment of the anti-Periostin humanized antibody is divided into 3 dose groups, namely 3mg/kg, 9mg/kg and 20mg/kg body weights, and a PBS and 30mg/kg body weight human IgG control group is simultaneously set up, the administration is carried out 2 times per week, the result is shown in Table 3, the anti-Periostin humanized antibody can obviously inhibit the growth of MDA-MB-231 in vivo and has obvious dose dependence, and the preliminary research on the hematology and growth change of each experimental animal simultaneously with the treatment shows that the hematology index and the growth change of the anti-Periostin humanized antibody have no obvious difference between the experimental group and the control group when the anti-Periostin humanized antibody is used for treating the breast cancer in vivo, and the preliminary verification of the obvious in-vivo toxicity.

TABLE 3 anti-Periostin humanized antibody tumor rebibition Rate against human Periostin expression positive breast cancer cell tumors

Group of	Number of animals	Average tumor weight (g)	Inhibition ratio (%)	P value
					PBS	10	1.35±0.21	/	/
Human IgG30mg/Kg	10	1.27±0.16	/
					humanized anti-Periostin antibody 3mg/kg	10	0.82±0.23	35.4	<0.05
anti-Periostin humanized antibody 9mg/kg	10	0.69±0.28	45.7	<0.05
					anti-Periostin humanized antibody 20mg/kg	10	0.47±0.13	63.0	<0.05

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A humanized anti-Periostin monoclonal antibody, comprising a light chain complementarity determining region having an amino acid sequence represented by SEQ ID nos. 1-3 and a heavy chain complementarity determining region having an amino acid sequence represented by SEQ ID nos. 4-6.

2. A method for preparing a humanized anti-Periostin monoclonal antibody, which comprises the following steps:

Step 1: extracting total RNA of mouse hybridoma LC-5A 6;

And 4, step 4: separating, recovering, purifying and transfecting the target fragment obtained in the step 3, carrying out double enzyme digestion by using Pst II and BstE II, directionally cloning into a corresponding site of a cloning vector pRGWH, screening out a positive recombinant clone, carrying out nucleotide sequence analysis, and constructing an expression vector for expressing the anti-Periostin humanized monoclonal antibody, wherein the expression vector is expressed by transfecting Chinese hamster ovary CHO cells in pSRNC-Ck-Periostin or pSRDC-Cgamma 1-Periostin, so as to obtain the humanized anti-Periostin monoclonal antibody.

3. The method for preparing a humanized anti-Periostin monoclonal antibody according to claim 2, wherein in step 1, the total RNA of mouse hybridoma LC-5A6 is extracted by collecting 1X 10 ⁷ mouse monoclonal antibody hybridoma, centrifuging at 10,000rpm for 1min, discarding the supernatant, adding 1mL Trizol to dissolve the cells sufficiently, standing at room temperature for 3min to 5min, adding 0.2mL chloroform, reversing and mixing, centrifuging at 4 ℃, 12,000rpm for 10min, transferring the supernatant to a clean 1.5mL centrifuge tube, adding 0.5mL isopropanol, reversing and mixing, standing at room temperature for 20min, centrifuging at 4 ℃, 12,000rpm for 10min, discarding the supernatant, washing with 75% ethanol for 2 times, air-drying, and adding 50 μ L ddH ₂ O to dissolve the precipitate.

4. the method of claim 2, wherein the first strand of cDNA from the murine hybridoma cells is synthesized in step 2 by a 20 μ L total reaction system comprising 4 μ L of 5 Xbuffer, 10mM DDT, 10g of total RNA extracted in step 1, dNTP at a final concentration of 0.5mM, oligo (dT) ₁₅ at a final concentration of 10 μ g/mL, 40U of RNAsin and 200U of MMLV-reverse transcriptase, mixing homogeneously, incubating in a 37 ℃ water bath for 1h, and inactivating in a 100 ℃ metal bath.

5. The method for preparing a humanized anti-Periostin monoclonal antibody according to claim 2, wherein in step 3, the nucleotide sequence of the light chain upstream primer PVL5 is represented by SEQ ID No.9, the nucleotide sequence of the light chain downstream primer PVL3 is represented by SEQ ID No.10, the nucleotide sequence of the heavy chain upstream primer PVH5 is represented by SEQ ID No.11, and the nucleotide sequence of the heavy chain downstream primer PVH3 is represented by SEQ ID No. 12; the method for amplifying the light chain complementarity determining region comprises the following steps: in a total reaction system of 100. mu.L, 10. mu.L of 10 Xbuffer, 2. mu.L of dNTP with a concentration of 10mM, 20. mu.L of cDNA obtained in step 2, 50pmol of light chain upstream primer PVL5 and 50pmol of light chain downstream primer PVL3 were mixed, the surface was covered with paraffin oil, after a water bath at 95 ℃ for 5min, 2U of Taq and Pfu DNA polymerase were added through paraffin oil, and a PCR amplification procedure was performed in the following cycle: 94 ℃ for 1 min; 30 cycles of 55 deg.C, 1min, 72 deg.C, 1min, and the last cycle of 72 deg.C, 10 min; the method for amplifying the heavy chain complementarity determining region comprises the following steps: in a total reaction system of 100. mu.L, 10. mu.L of 10 XTbuffer, 2. mu.L of 10mM dNTP, 20. mu.L of the cDNA obtained in step 2, 50pmol of heavy chain upstream primer PVH5 and 50pmol of heavy chain downstream primer PVH3, after mixing, the surface is covered with paraffin oil, after a water bath at 95 ℃ for 5min, 2U of Taq and Pfu DNA polymerase are added through the paraffin oil, and a PCR amplification procedure is performed according to the following cycle: 94 ℃ for 1 min; 55 deg.C, 1min, 72 deg.C, 1min, 30 cycles in total, and 72 deg.C, 10min in the last cycle.

6. a humanized anti-Periostin antibody comprising a light chain complementarity determining region having an amino acid sequence shown in SEQ ID No.7 and a heavy chain complementarity determining region having an amino acid sequence shown in SEQ ID No. 8.

7. An isolated polynucleotide encoding the humanized anti-Periostin monoclonal antibody of claim 1.

8. An expression vector comprising the polynucleotide of claim 7.

9. A host cell comprising the polynucleotide of claim 7 or comprising the expression vector of claim 8.

10. Use of the humanized anti-Periostin monoclonal antibody of claim 1 or the humanized anti-Periostin antibody of claim 6 or the polynucleotide of claim 7 or the expression vector of claim 8 or the host cell of claim 9 in the preparation of a medicament for anti-humanized anti-Periostin positive tumors.