CN110563841A

CN110563841A - Humanized anti-Grb 2 monoclonal antibody, and preparation method and application thereof

Info

Publication number: CN110563841A
Application number: CN201910796374.1A
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: 2019-12-13

Abstract

The invention discloses a humanized anti-Grb 2 monoclonal antibody, and a preparation method and application thereof, and belongs to the technical field of medicines. The humanized anti-Grb 2 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-Grb 2 monoclonal antibody. The humanized anti-Grb 2 monoclonal antibody provided by the invention not only has the specificity of combining with Grb2 protein and Grb2 positive tumor cells, but also has human source and can reduce the toxic and side effect for human use.

Description

humanized anti-Grb 2 monoclonal antibody, and preparation method and application thereof

Technical Field

The invention relates to a humanized anti-Grb 2 monoclonal antibody, and a preparation method and application thereof, and belongs 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.

Growth factor receptor bound protein2 (Grb 2) is an adaptor protein widely expressed in many cells, and is composed of 217 amino acids and has a molecular weight of about 25 kDa. The greatest feature of the structure of the Grb2 protein is that the Grb2 protein contains a SH2 domain in the center and two SH3 domains on both sides. SH2 and SH3 are connected with each other by two flexible arm chains. The two flexible arm chains give greater spatial structural variation to Grb 2. Grb2 regulates cellular function by modulating Receptor Tyrosine Kinase (RTK) signaling pathways. It is clear that Grb2 is not only used as a main signal conduction molecule to be highly expressed in various tumor tissues such as gastric cancer, colorectal cancer, esophageal squamous cell carcinoma, cervical cancer and the like, but also the expression level is closely related to lymph node metastasis and prognosis of tumors. Abnormal expression of Grb2 or abnormal activation of its signaling pathway will decrease adhesion between tumor cells or between tumor cells and extracellular matrix, decrease stability of extracellular matrix structure, increase flexibility of cytoskeleton and migration ability of cells, and promote tumor angiogenesis. Considering that Grb2 has important pathophysiological functions, according to the structural characteristics of Grb2, screening of specific compounds and polypeptides aiming at Grb2 is always a hotspot of tumor research. However, the anti-Grb 2 small molecule drugs developed at present are far from meeting the clinical treatment requirements.

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 expression specificity of Grb2 in tumor tissues and the inhibition efficiency of corresponding murine monoclonal antibodies on a nude mouse transplantation tumor model, the development of antibody drugs aiming at Grb2 will probably provide new targeted therapeutic drugs of new targets for tumor clinical treatment.

Disclosure of Invention

One of the objects of the present invention is to provide a humanized anti-Grb 2 monoclonal antibody. The inventors of the present application determined the CDR region sequences of the humanized anti-Grb 2 monoclonal antibody by cloning, identification and analysis of the gene structure. The humanized anti-Grb 2 monoclonal antibody provided by the invention not only has the specificity of combining with Grb2 protein and Grb2 positive tumor cells, but also has human source and can reduce the toxic and side effect for human use.

The technical scheme for solving the technical problems is as follows: a humanized anti-Grb 2 monoclonal antibody, comprising 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 specific sequences of SEQ ID NO.1-6 are as follows:

SEQ ID NO.1：Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu Ala；

SEQ ID NO.2：Trp Ala Ser Thr Arg Asp Ser；

SEQ ID NO.3：Lys Gln Ser Tyr Asn Leu Arg Thr；

SEQ ID NO.4：Ser Tyr Trp Ile Glu；

SEQ ID NO.5：Glu Ile Leu Pro Gly Ile Gln Ser Thr Asn Tyr Asn Glu Lys Phe Lys Val Arg；

SEQ ID NO.6：Arg Tyr Tyr Gly Ser Ser Tyr Gly Phe Asp Tyr。

the invention has the beneficial effects that:

The humanized anti-Grb 2 monoclonal antibody provided by the invention not only has the specificity of combining with Grb2 protein and Grb2 positive tumor cells, but also has human source and can reduce the toxic and side effect 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 humanized anti-Grb 2 antibody gene has CDR region from mouse monoclonal antibody 3B8 prepared from human breast cancer cell immunized mouse, and the target antigen of the mouse monoclonal antibody is Grb2 through mass spectrum identification. Early-stage research proves that the mouse monoclonal antibody can be combined with Grb2, and an in-vivo animal tumor suppression experiment proves that the antibody can block the in-vivo function of Grb2 and obviously inhibit the growth of in-vivo Grb2 positive lung cancer nude mouse transplanted tumor, and the tumor suppression rate is as high as 81.27%; the 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 treatment for treating the malignant tumor with strong positive Grb2 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-Grb 2 monoclonal antibody. The preparation method provided by the invention can be used for obtaining the humanized anti-Grb 2 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-Grb 2 monoclonal antibody comprises the following steps:

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

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 on Pst II and BstE II, directionally cloning into a corresponding site of a cloning vector pRGWH, screening out positive recombinant clones, carrying out nucleotide sequence analysis, and constructing an expression vector for expressing the anti-Grb 2 humanized monoclonal antibody, wherein the expression vector is used for transfecting Chinese hamster ovary cells in pSRNC-Ck-Grb 2 or pSRDC-Cgamma 1-Grb2 to express, so as to obtain the humanized anti-Grb 2 monoclonal antibody.

The invention has the beneficial effects that:

The preparation method provided by the invention can be used for obtaining the humanized anti-Grb 2 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 step 1, the method for extracting total RNA of mouse hybridoma LC-5A1 comprises the following steps: collecting 1X 10⁷Centrifuging mouse monoclonal antibody hybridoma cells at 10,000rpm for 1min, discarding the supernatant, adding 1mL of Trizol to fully dissolve the cells, standing at room temperature for 3min-5min, adding 0.2mL of chloroform, and reversing and mixing; centrifuging at 12,000rpm for 10min at 4 deg.C, transferring the supernatant into a clean 1.5mL centrifuge tube, adding 0.5mL isopropanol, and mixing by inversion; standing at room temperature for 20 min; centrifuging at 4 deg.C and 12,000rpm for 10min, and removing supernatant; washed 2 times with 75% vol ethanol, air dried, and added 50. mu.L of ddH₂And dissolving the precipitate with O to obtain the final product.

Total RNA of murine hybridoma LC-5A1 was extracted as described above, according to TRIzol of Gibco^TMThe Reagent kit (Invitrogen) was operated under the cat # 15596026.

the mouse hybridoma cell LC-5A1 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: mu.L of a total reaction system comprising 4. mu.L of 5 Xbuffer, 10mM DDT, 10g of total RNA extracted in step 1, dNTP at a final concentration of 0.5mM, Oligo (dT) at a final concentration of 10. mu.g/mL₁₅40U of RNAsin and 200U of MMLV-reverse transcriptase are evenly mixed, and are subjected to water bath at 37 ℃ for 1h and 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) as described above₁₅Purchased from Promega corporation.

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-Grb 2 antibody. The humanized anti-Grb 2 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-Grb 2 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-Grb 2 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 the particular embodiment of the invention described, the humanized anti-Grb 2 antibody is a recombinant monoclonal antibody having specific light and heavy chain amino acid sequences, also called recombinant humanized antibody, since the framework and constant regions of the specific antibody are derived from human.

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

SEQ ID NO.7：

DIVMSQSPSS LAVSVGEKVT MSCKSSQSLL YSSNQKNYLA WYQQKPGQSP KLLIYWASTR DSGVPDRFTG SGSGTDFTLT ISSVQAEDLA VYYCKQSYNL RTFGGGTKLE IKSGSGSGQD YSLTISSLESDDTATYYCLQ HGESPYTFGG GTNHPPCMHR WERESLSLAR RVRTSWQMGS HQDSVAVDLG KIILGLCSLLLCKILLWWGH GLL。

SEQ ID NO.8：

QVQLQQPGAE LVKPGASVKL SCKASGYTFT SYLMHWVKQR PGRGLEWIGR IDPNSGGTKY NEKFKSKATL TVDKPSSTAY MQLSSLTSED SAVYFCARYP YGRAMDYWGQ GTSVTVSSGS PDSGEEAGVGRNHRWWWVSH LLSRHCEGPI HHLQRQCQEQ PVPANEQSEV LGHGLVSLCK TGLGRVLLRL LGPRHHSHSLLSQNDTPICL STGPWICCPN WLHGDPGMPG QGLFPGASDS DLELWIPVQR CAHLPSCPAV TPLHSEQLSDCPLQPSAQRD RHLQRCPPGQ QHQGGQENCA QGLWLPALHM YSPRSIICLH LPPKAQGCPH HYSDSCGHVCCGRHQQGESR GPVQLVCRQC GGAHSSDATP GGAVQQHFPL SQATSHHAPG LAQWQGVQMQ GQQCSFPCPHRENHIQNQYL HPSLYKHSTQ HCLGTL。

The invention has the beneficial effects that:

The humanized anti-Grb 2 antibody provided by the invention has humanized antibody, and can greatly reduce the side immune reaction of heterologous antibody to human body.

It should be noted that, in the present application, the term "antibody of the invention" refers in particular to the "humanized anti-Grb 2 monoclonal antibody" according to the invention, the light chain complementarity determining region of which comprises the CDR regions shown in SEQ ID Nos. 1-3 and the heavy chain complementarity determining region of which comprises the CDR regions shown in SEQ ID Nos. 4-6.

the "humanized anti-Grb 2 antibody" is an anti-Grb 2 humanized monoclonal antibody having a specific complete sequence consisting of the amino acid sequence of the light chain protein contained in or consisting of SEQ ID NO.7 and the amino acid sequence of the heavy chain protein contained in or consisting of SEQ ID NO.8, wherein the complete sequence of the specific anti-Grb 2 humanized monoclonal antibody is composed of the CDR region sequence provided by the present invention embedded and spliced into the FR region sequence and the constant region sequence of a specific 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-Grb 2 monoclonal antibodies described above.

The technical scheme for solving the technical problems is as follows: an isolated polynucleotide encoding the humanized anti-Grb 2 monoclonal antibody described above.

The invention has the beneficial effects that:

The isolated polynucleotides provided by the present invention may encode the humanized anti-Grb 2 monoclonal antibodies described above.

According to a consensus in the art, even if the nucleotide sequences coding for the antibody proteins of SEQ ID Nos. 1-3 and 4-6 are altered, said nucleic acids are DNA, as long as the translated amino acid sequences three positions after the triplet codon are retained, both being polynucleotides coding for the anti-Grb 2 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-Grb 2 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, the culture supernatant of the host cells contains a large amount of the anti-Grb 2 humanized antibody protein secreted by the host cells, the anti-Grb 2 humanized antibody protein can be conveniently extracted and prepared therefrom according to art-recognized techniques, in the examples, the expression vectors were designated pSRNC-Ck-Grb 2-1 and pSRDC-Cy 1-Grb2-1, which contain the humanized antibody gene against Grb2 and methotrexate pressure-amplified expression selection marker gene (dhfr) and were expressed in Chinese Hamster Ovary (CHO) cells, respectively.

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.

Such host cells include, but are not limited to, cells 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 an embodiment of the invention, the host cell is a chinese hamster ovary Cell (CHO). pSRNC-Ck-Grb 2-1 and pSRDC-Cy 1-Grb2-1 are introduced into eukaryotic CHO cells, eukaryotic host cells expressing the antibody of the invention in high yield are obtained by screening, and the anti-Grb 2 humanized antibody protein secreted in a large amount is contained in the culture supernatant of the host cells, from which the anti-Grb 2 humanized antibody protein can be conveniently extracted and prepared according to the common knowledge in the art.

The seventh object of the present invention is to provide the use of the above humanized anti-Grb 2 monoclonal antibody or the above polynucleotide or the above expression vector or the above host cell in the preparation of humanized anti-Grb 2 positive tumor drugs. The humanized anti-Grb 2 monoclonal antibody, the polynucleotide or the expression vector or the host cell provided by the invention can be used for preparing humanized anti-Grb 2 positive tumor medicaments and treating malignant tumors with Grb2 expression positive on the cell surface.

The technical scheme for solving the technical problems is as follows: the humanized anti-Grb 2 monoclonal antibody or humanized anti-Grb 2 antibody or polynucleotide or expression vector or host cell is used for preparing anti-human anti-Grb 2 positive tumor drugs.

The invention has the beneficial effects that:

The humanized anti-Grb 2 monoclonal antibody or humanized anti-Grb 2 antibody or polynucleotide or expression vector or the host cell can be used for preparing humanized anti-Grb 2 positive tumor drugs and treating cell surface Grb2 positive malignant tumors.

The humanized anti-Grb 2 monoclonal antibody of the invention can be specifically combined with Grb2 protein in tumor cells, can directly block Grb 2-mediated over-activation of RTKs related pathways in vivo and inhibit growth of Grb2 over-expressed tumors in vivo, so that the antibody can be directly used for preparing antibody medicines for targeted therapy of anti-Grb 2 expression strong positive tumors or synergistic RTKs targeted therapy. According to the consensus in the art, when obtaining humanized anti-Grb 2 antibody protein, the antibody drug can be prepared by adding proper stabilizers, protective agents, pH regulators, salt balancing agents, excipients and the like, and can be directly used for human body treatment. In the examples of the invention, the antibodies of the invention can be used for treating nude mice inhibitive tumors that express Grb2 strongly positive. In a particular colorectal cancer model, the humanized anti-Grb 2 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 humanized antibody against Grb2 significantly inhibited the growth of Grb 2-overexpressing human colorectal cancer cells in a dose-dependent manner.

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 Grb2) that comprises an altered nucleotide and/or amino acid sequence of one or more nucleotides and/or amino acids outside of the CDR regions as 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

"humanized" forms of non-human antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab' of an antibody)₂Fragments or other subsequences that bind to the target). In general, humanized antibodies are generally two variable domains (comprising at least one), ideally all the CDRs correspond to those of a non-human immunoglobulin, and almost all the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody may comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a selected human immunoglobulin template.

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-Grb 2 monoclonal antibody light chain eukaryotic expression vector pSRNC-Ck-Grb 2-1.

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-Grb 2 monoclonal antibody heavy chain eukaryotic expression vector pSRDC-Cgamma 1-Grb 2-1.

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; VH gene represents a heavy chain variable region gene fragment with a leader peptide sequence and a 5' intron end cleavage site sequence; c gamma 1gene represents human antibody heavy chain gamma 1 chain constant region gene segment; BGHpolyA 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-Grb 2 monoclonal antibody

the preparation method of the humanized anti-Grb 2 monoclonal antibody of the embodiment comprises the following steps:

Step 1: total RNA of mouse hybridoma LC-5A1 was extracted

The mouse hybridoma cell LC-5A1 is purchased from China general microbiological culture Collection center with the preservation number of CGMCC No.10898 and preserved by liquid nitrogen. Total RNA of murine hybridoma LC-5A1 was extracted according to the instructions of Trizol (Gibco Co.), specifically: collecting 1X 10⁷Murine monoclonal antibody hybridoma cells were centrifuged at 10,000rpm for 1min and the supernatant discarded. Adding 1mL of Trizol to fully dissolve cells, standing at room temperature for 3-5min, adding 0.2mL of chloroform, and reversing and uniformly mixing; centrifuging at 12,000rpm for 10min at 4 deg.C, transferring the supernatant into a clean 1.5mL centrifuge tube, adding 0.5mL isopropanol, and mixing by inversion; standing at room temperature for 20 min; centrifuging at 4 deg.C and 12,000rpm for 10min, and removing supernatant; washing with 75% ethanol for 2 times, air drying, and adding 50 μ L ddH₂O dissolves the precipitate.

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-Grb 2 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, Oligo d (T)15 at a final concentration of 10. mu.g/mL, 40U of RNAsin and 200U of MMLV-reverse transcriptase was mixed homogeneously, incubated in a water bath at 37 ℃ for 1 hour, and inactivated in a metal bath at 100 ℃.

The dNTPs were purchased from Promega corporation.

Oligo (dT) as described above₁₅Purchased from Promega corporation.

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 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.

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 variable region gene fragment of the light chain of the anti-Grb 2 mouse monoclonal antibody in a large scale, separating and recovering by adopting a glass milk adsorption method, performing double enzyme digestion by Pvu II and Bg1 II, directionally cloning into the corresponding site of a cloning vector pRGWL, and collecting 221 transformed clones in total. Randomly selecting 15 clones, and screening to obtain 4 recombinant clones. The screened positive recombinant clones were subjected to nucleotide sequence analysis, and the sequences of 4 clones were completely identical, confirming that the light chain variable region gene of the anti-Grb 2 murine monoclonal antibody existed in the cloned antibody light chain variable region gene. One of the 4 clones was randomly selected and named pRGWL-Grb 2-1. Comparison of Kabat data revealed that the VL of the murine anti-Grb 2 monoclonal antibody belongs to subgroup III of the light chains of mouse k, the light chain CDR1-3 sequences (SEQ ID NO. 1-3).

Amplifying heavy chain variable region gene segments of anti-Grb 2 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 346 transformation clones in total. Randomly selecting 25 clones, and screening to obtain 11 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 that the anti-Grb 2 murine monoclonal antibody heavy chain variable region gene existed in the cloned antibody heavy chain variable region gene. One of the 5 clones was randomly selected and named pRGWH-Grb 2-1. Comparison of Kabat data revealed that the VH of the anti-Grb 2 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.2：Trp Ala Ser Thr Arg Asp Ser；

SEQ ID NO.3：Lys Gln Ser Tyr Asn Leu Arg Thr；

SEQ ID NO.4：Ser Tyr Trp Ile Glu；

SEQ ID NO.6：Arg Tyr Tyr Gly Ser Ser Tyr Gly Phe Asp Tyr。

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

The method comprises the steps of replacing a framework region of a variable region gene of the anti-Grb 2 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-Grb 2 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-Grb 2 humanized antibody and a eukaryotic expression vector containing the gene.

Synthesis of the humanized antibody variable region genes of the anti-Grb 2 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 designed humanized antibody variable region gene of the anti-Grb 2 mouse monoclonal antibody has translated amino acid sequence, and the light chain gene and the heavy chain gene are synthesized directly by commercial company through a segmented synthesis and overlapping PCR connection method. The humanized antibody light and heavy chain variable region genes of the anti-Grb 2 mouse monoclonal antibody were amplified by PCR in the same manner as in example 1, the products were digested by restriction enzymes, and were 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-Grb2-H and pRGWH-Grb2-H, respectively, and used as amplification templates for constructing the expression vectors described below.

Amplifying variable region gene fragments with regulatory sequences by PCR: using the recombinant clone plasmids pRGWL-Grb2-H and pRGWH-Grb2-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-Grb2-H and pRGWH-Grb2-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), after mixing, surface-coated with paraffin oil, after water bath at 95 ℃ for 5min, 2UTaq and Pfu DNA polymerase were added through paraffin oil, 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-Grb 2 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-Grb 2 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 4 light chain recombinant clones and 5 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-Grb 2 humanized monoclonal antibody gene and eukaryotic expression vector can be successfully constructed. The 3-time repeated nucleotide sequences prove that the variable region gene sequences of the anti-Grb 2 humanized monoclonal antibody eukaryotic expression pSRNC-Ck-Grb 2 and pSRDC-Cgamma 1-Grb2 are completely identical to the originally designed humanized variable gene sequences respectively. The amino acid sequences of the anti-Grb 2 humanized monoclonal antibodies produced by 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:

IQAFVFVFLWLSGARVSIPCKASQDVSAAVAWYLQKPGHSPTLLIYWTSTRHTGVPDRFTGSGSGTDYTLTISSVQAEGPGTLLLSTTLLHSSDVRWRHQAGNQTVGTKLQTGVCCTNCIRHLPTIQTAVNIWRCLSRVLLEQLLPQRHQCQVEDKWQGTTKWRPEQLDPSGQQRQHLQHEQHPHVDQGRVLTTHQLYLYGHSQDINFTHCQELQQERV。

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

EVQLQQSGAELVKPGSSVKLSCTASGFDIKDTYIHWVKQRPEQGLEWIGRIDPANGDTRYDPKFQGKATITADTSSNTAYLQLSSLTSEDTAVYYCARDYYGSRFIYVPETLLCSLWIHFQALWHVLGSPDSGEEAGVGRNHEWWWYSHLLSRHCEGPIHHLQRQCQEQPVPANEQSEVSGHGLVSLCKTGLGRVLLGLLGPRHHSHSLLSQNDTPICLSTGPWICCPNSLHGDPGMPGQGLFPLASDSDLELWIPVQRCAHLPSCPAVIPLHSEQLSDCPLQPSAQRDRHLQRCPPGQQHQGGQENCAQGLWLAALHMYSPRSIICLHLPPKAQGCPHHYSDSQGHVCCGRHQQGESRGPVQLVCRIICGGAHSSDATPGGAVQQHFPLSQPTSHHAPGLAQWQGVQMQGQQCSFPCPHRENHIQNQRQTEGSTGVHHSTSQGADGQGKSQSDLHDNRLLPQRHNSGVAVEWAASGELQEHSAHHEH。

Structure of eukaryotic expression vector of humanized anti-Grb 2 monoclonal antibody: the eukaryotic expression vector of the anti-Grb 2 humanized monoclonal antibody adopts a light chain eukaryotic expression vector (pSRNC-Ck-Grb 2) and a heavy chain expression vector (pSRDC-Cgamma 1-Grb2), and the structural schematic diagrams are shown in figure 1 and figure 2.

example 3: induction of expression in cells (CHO cells) of humanized anti-Grb 2 antibody expression vectors

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 5% CO₂Recovering and culturing CHO-dhfr in 37 deg.c environment^-A cell. Subculturing once for 3-4 days according to the proportion of 1: 10. The gene transfection method is adopted, Lipofect AMINE reagent (Gibco company) is used for transfection, light and heavy chain expression vectors of anti-Grb 2 humanized antibody are co-transfected into cells, H, T, Gly-free culture medium is used for screening, and after the clone is formed, a selective culture medium containing 200 mu G/mL G418(Gibco company) is used for screening. As a result, 4. mu.g each of the light and heavy chain antibody gene expression vectors was transfected into CHO-dhfr^-Cells, clonal growth visible to the naked eye within 2 weeks. The OD of the culture supernatant of the resistant clone mixture is measured by an indirect ELISA method using goat anti-human k-chain polyclonal antibody as coating and goat anti-human IgG-HRP as a secondary antibody₄₉₀1.645 as a negative control, CHO-dhfr^-Supernatant OD₄₉₀Only 0.073, suggesting that the supernatant of the transfected cells contained the expression of the humanized antibody against Grb 2.

Example 4: screening of anti-Grb 2 humanized chimeric antibody high-expression strain

DMEM medium containing 10% FBS, 100U/mL streptomycin, 2mmol/L glutamine at 5% CO₂The transfected CHO cells were recovered and cultured at 37 ℃. Subculturing once for 3-4 days according to the proportion of 1: 10. And screening the anti-Grb 2 antibody high-expression strain by adopting a Methotrexate (MTX) pressure amplification expression screening method. The cell clones containing the expression of the humanized anti-Grb 2 antibody in the supernatant were subsequently subjected to a method containing 3X 10^-8M and 10^-7And (3) performing pressurized culture on the M MTX complete culture medium, performing subcloning by adopting a limiting dilution method after each round of pressurized amplification expression, and screening the clone strain with the highest yield.

Transfection of humanized anti-Grb 2 antibody expression vector into CHO-dhfr^-Clone 7B4 (clone yield can reach 28.9 mu g/mL) which is obtained by primary screening after cells and can efficiently express humanized anti-Grb 2 antibody is added into the mixture containing 3X 10^-8Culturing continuously in M MTX culture medium for 30 days, when the cells are adapted to 3X 10^-8After the M MTX pressure concentration (the cell morphology and the growth speed are recovered to be normal), the antibody yield can reach 41.27 mu g/mL, and the clone 8E5 is continuously replaced by 1X 10 after 1:5 subculture^-7M MTX was continuously pressure-cultured. After cell adaptation, the antibody yield can reach 55.27 mu g/mL, and the antibody yield of the clone strain with the highest 5F9 antibody yield after subcloning can reach 112.71 mu g/mL through detection.

Example 5: specificity, humanized identification of anti-Grb 2 humanized antibodies

1. Specific identification of the humanized antibody against Grb 2: using ELISA method, coating enzyme label plate with 1 microgram/mL recombinant Grb2 protein, adding sample for reaction, adding goat anti-human IgG Fc-HRP enzyme labeled antibody (Sigma company) (without cross reaction with mouse Ig) or goat anti-mouse IgG Fc-HRP enzyme labeled antibody (Sigma company), incubating, developing, determining OD₄₉₀. The anti-Grb 2 humanized antibody purified by a protein A affinity chromatographic column from the supernatant of the clone strain 5F9 can be combined with the coated recombinant Grb2 protein, and is identified by a goat anti-human IgG Fc fragment polyclonal antibody to present positive reaction; and not transfected CHO-dhfr^-The cell culture supernatant reacted negatively with the parent murine monoclonal antibody 5A 1. CHO-dhfr was not transfected when goat anti-mouse IgG Fc-HRP was used as the secondary antibody^-Cell culture supernatants and purified anti-Grb 2 humanized antibody were negative, while the parent murine monoclonal antibody 5a1 reacted positive. The irrelevant antibody human IgG1 was negative, confirming that the expressed anti-Grb 2 humanized antibody can be specifically combined with the recombinant Grb2 protein.

TABLE 1 ELISA analysis of antigen binding specificity of anti-Grb 2 humanized antibodies

sample (I)	OD490^a	OD490^b
			anti-Grb 2 humanized antibody (100ng/mL)	2.965±0.215	2.761±0.299
Human IgG1(100ng/mL)	0.073±0.002	0.073±0.001
			Parent mouse monoclonal antibody (100ng/mL)	0.073±0.001	2.461±0.153
PBS control	0.076±0.003	0.074±0.002
			CHO-dhfr cell supernatant	0.074±0.002	0.075±0.003

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.

The immobilized cell of the human colon cancer cell HT29 highly expressed by Grb2 is taken as a target cell, an anti-Grb 2 humanized antibody (10 mu g/mL) is added, a goat anti-human IgG-fluorescent secondary antibody (Sigma company) is added after incubation at 37 ℃, and the anti-Grb 2 humanized antibody is observed under a fluorescent microscope to recognize Grb2 protein expressed by the Grb2 high-surface human colon cancer cell HT 29.

2. Humanized identification of the humanized anti-Grb 2 antibody: in ELISA, the ELISA 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-Grb 2 humanized antibody reacted strongly positively, while the parent murine monoclonal antibody 3B8 of the anti-Grb 2 humanized antibody reacted negatively. It was demonstrated that the purified humanized antibody against Grb2 contained the light and heavy chain constant regions of human IgG.

TABLE 2 detection of humanity of anti-Grb 2 humanized antibodies by ELISA

sample (100ng/mL)	OD490^a	OD490^b
			anti-Grb 2 humanized antibody (5F9)	2.761±0.299	2.442±0.159
Parent murine monoclonal antibody	0.076±0.007	0.073±0.004
			Human IgG1	2.525±0.126	2.361±0.189
PBS control	0.074±0.003	0.074±0.006

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

Example 6: preparation of therapeutic drug by using humanized antibody against Grb2 for in vivo treatment of Grb2 overexpression human nude mouse transplantation tumor

In the Grb2 positive human colon cancer cell HT29 (the nude mice are injected with 10 right dorsal skin⁶Individual cells) was investigated for the effectiveness of the humanized antibody against Grb2 in vivo for the treatment of human colon cancer. Intraperitoneal administration treatment was started on day 3 after tumor cell inoculation, and the anti-Grb 2 humanized antibody in vivo treatment was divided into 3 dose groups: 3mg/kg body weight, 10mg/kg body weight, 30mg/kg body weight, PBS and 30mg/kg body weight human IgG control group were established simultaneously. The administration was 2 times per week. The results are shown in table 3, the anti-Grb 2 humanized antibody can significantly inhibit the growth of human Grb2 positive human colon cancer cell HT29 in vivo, and presents an obvious dose-dependent relationship, and preliminary studies on hematology and growth change of each experimental animal performed simultaneously with treatment indicate that, when the anti-Grb 2 humanized antibody is used for treating colon cancer in vivo, the hematology index and mouse growth change of the anti-Grb 2 humanized antibody have no significant difference between the experimental group and the control group, and the fact that the anti-Grb 2 humanized antibody has no obvious in vivo toxicity effect is preliminarily confirmed.

TABLE 3 anti-Grb 2 humanized antibody against human Grb2 expressing positive colon carcinoma cell tumor re-inhibition rate

Group of	Number of animals	Average tumor weight (g)	Inhibition ratio (%)	P value
					PBS	10	1.27±0.29	/	/
Human IgG30mg/kg	10	1.19±0.32	/
					anti-Grb 2 humanized antibody 3mg/kg	10	0.84±0.11	33.1	<0.05
anti-Grb 2 humanized antibody 10mg/kg	10	0.62±0.27	52.7	<0.05
					anti-Grb 2 humanized antibody 30mg/kg	10	0.35±0.14	75.3	<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-Grb 2 monoclonal antibody, comprising 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 represented by SEQ ID NO.1-3, and the amino acid sequence of the heavy chain complementarity determining region is represented by SEQ ID NO. 4-6.

2. a method for preparing a humanized anti-Grb 2 monoclonal antibody, comprising the following steps:

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

And 4, step 4: separating, recovering, purifying and transfecting the target fragment obtained in the step 3, carrying out double enzyme digestion on Pst II and BstE II, directionally cloning into a corresponding site of a cloning vector pRGWH, screening out positive recombinant clones, carrying out nucleotide sequence analysis, and constructing an expression vector for expressing the anti-Grb 2 humanized monoclonal antibody, wherein the expression vector is used for transfecting Chinese hamster ovary CHO cells in pSRNC-Ck-Grb 2 or pSRDC-Cgamma 1-Grb2 to express, so as to obtain the humanized anti-Grb 2 monoclonal antibody.

3. The method for preparing humanized anti-Grb 2 monoclonal antibody of claim 2, wherein the method for extracting total RNA of murine hybridoma LC-5A1 in step 1 comprises: collecting 1X 10⁷Centrifuging mouse monoclonal antibody hybridoma cells at 10,000rpm for 1min, discarding the supernatant, adding 1mL of Trizol to fully dissolve the cells, standing at room temperature for 3min-5min, adding 0.2mL of chloroform, and reversing and mixing; centrifuging at 12,000rpm for 10min at 4 deg.C, transferring the supernatant into a clean 1.5mL centrifuge tube, adding 0.5mL isopropanol, and mixing by inversion;standing at room temperature for 20 min; centrifuging at 4 deg.C and 12,000rpm for 10min, and removing supernatant; washed 2 times with 75% vol ethanol, air dried, and added 50. mu.L of ddH₂And dissolving the precipitate with O to obtain the final product.

4. The method of claim 2, wherein the first strand reaction system for the synthesis of murine hybridoma cDNA is: mu.L of a total reaction system comprising 4. mu.L of 5 Xbuffer, 10mM DDT, 10g of total RNA extracted in step 1, dNTP at a final concentration of 0.5mM, Oligo (dT) at a final concentration of 10. mu.g/mL₁₅40U of RNAsin and 200U of MMLV-reverse transcriptase are evenly mixed, and are subjected to water bath at 37 ℃ for 1h and metal bath inactivation at 100 ℃.

5. The method for preparing humanized anti-Grb 2 monoclonal antibody according to claim 2, wherein 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 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-Grb 2 antibody, comprising a light chain complementarity determining region having an amino acid sequence represented by SEQ ID No.7 and a heavy chain complementarity determining region having an amino acid sequence represented by SEQ ID No. 8.

7. An isolated polynucleotide encoding the humanized anti-Grb 2 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-Grb 2 monoclonal antibody of claim 1 or the humanized anti-Grb 2 antibody of claim 6 or the polynucleotide of claim 7 or the expression vector of claim 8 or the host cell of claim 9 for the preparation of a medicament against humanized anti-Grb 2 positive tumors.