CN103374071B - A kind of preparation of the humanized antibodies of anti-DERLIN 1 - Google Patents

Abstract

The present invention relates to the preparation of an anti-DERLIN-1 humanized antibody, and further, the present invention relates to a humanized antibody against DERLIN-1 protein expressed on the surface of cancer cells, a polynucleotide encoding the antibody, comprising the polynuclear Expression vectors of nucleotides, host cells containing the expression vectors, and their use in the preparation of drugs for treating tumors.

Description

Preparation of an anti-DERLIN-1 humanized antibody

technical field

What the present invention relates to is biopharmaceutical technical field. The present invention relates to a humanized antibody against DERLIN-1 protein expressed on the surface of cancer cells, a polynucleotide encoding the antibody, an expression vector containing the polynucleotide, and a host cell containing the expression vector, and their expression in Use in the preparation of medicines for treating tumors.

Background technique

Tumor is one of the most threatening malignant diseases to human beings. Its death rate ranks second and third among various diseases in the world and our country respectively, seriously affecting human health. The main methods of clinical treatment of cancer are surgical resection, radiotherapy and chemotherapy, but these three conventional treatment methods do not significantly reduce the mortality of tumors, and the 5-year survival rate is only 10-30%. In recent years, targeted therapy of tumors has achieved encouraging results in clinical practice and has become the most promising and active field. For therapeutic target proteins that are specifically expressed in tumors and play an important role in tumor growth or metastasis, a variety of targeted therapeutic agents are used to specifically block the biological functions that interfere with therapeutic targets, thereby exerting the effect of affecting tumor growth and metastasis. Therapeutic effect, it will be possible to effectively treat malignant tumors. Among the various targeted therapy drugs for tumors, the most successful ones are antibody drugs. Due to their good specificity, strong targeting, and high efficiency in blocking target proteins, antibody drugs have made breakthroughs in the research of treating tumors in recent years. Rituxan is the first anti-tumor antibody drug approved by the FDA in 1997 for the treatment of B-cell non-Hodgkin's lymphoma. Herceptin was approved in 1998, mainly for HER-2/neu positive breast cancer. Mylotarg (2000) for the treatment of acute relapsed myeloid leukemia. In recent years, antibody drugs that have been approved for the treatment of tumors include Mylotarg, Campath-1H, Zevalin, Bexxer, Erbitux, and Avastin. At present, Rituxan and Herceptin have achieved great success in clinical tumor treatment.

Although there are dozens of tumor-specific protein targets for anti-tumor monoclonal antibody drugs, for example: CD20 (Grillo-Lopez AJ. Anti-CD20 monoclonal antibody: strategies and results of leukemia treatment. Anti-tumor therapy Experience Review. 2002; 2: 323-329; Grillo-Lopez AJ. AntiCD20 mAbs: modifying therapeutic strategies and outcomes in the treatment of lymphoma patients. Expert Rev Anticancer Ther. 2002; 2: 323-329.), Her2-neo (Mendelsohn J , Baselga J. The EGF receptor family as targets for cancer therapy. Oncogene. 2000; 19: 6550-6565; The EGF receptor family atargets for cancer therapy. Oncogene. 2000; 19: 6550-6565.), VEGF (Schwartzberg LS. Clinical experience with edrecolomab: a monoclonal antibody therapy for colorectal carcinoma. Review of Antitumor Hematology. 2001; 40: 17-24; Schwartzberg LS. Clinical experience with edrecolomab: a monoclonal antibody therapy for colorectal carcinoma. Crit Rev Oncol Hematol.2001; 40:17-24.), CD33, CD52, EGRFR, CD25, CO17-1A, CEA, CD22, GD2, EGFR, EGF, CD6, CD11a, etc., but for the clinical treatment of various malignant tumors However, targeted therapy drugs against new tumor targets are still far from enough. Since the occurrence and development of tumors is a process of multiple factors and multiple gene mutations, targeted therapy drugs targeting only a small number of targets cannot effectively treat tumors, but often can only delay this process; in addition, different tumors have different incidences The mechanism has different therapeutic targets and requires different targeted therapeutic drugs. Therefore, the clinical treatment of tumors requires more new drugs targeting different therapeutic targets in order to work synergistically and treat tumors more effectively.

The target protein recognized by the monoclonal antibody involved in the present invention is cell membrane protein Derlin-1. Derlin-1 is a protein located on the membrane of the endoplasmic reticulum. Current studies have shown that it is involved in the transport and degradation of misfolded proteins in the endoplasmic reticulum to the cytoplasm (Yihong Ye1, Yoko Shibata1, Chi Yun2, David Ron2&TomA .Rapoport1. A Membrane Protein Complex Involved in Mediating Protein Translocation from the Endoplasmic Reticulum to the Cytoplasm. Nature. 2004;(429):841-847; Yihong Ye1, Yoko Shibata1, Chi Yun2, David Ron2&Tom A.Rapoport1 .Amembrane protein complex mediates retro-translocation from the ER lumen into the cytosol.NATURE.2004;(429):841-847), whose interaction with mutant SOD1 is associated with familial amyotrophic lateral sclerosis (Nishitoh, H ., Kadowaki, H., Nagai, A., Maruyama, T., Yokota, T., Fukutomi, H., Noguchi, T., Matsuzawa, A., Takeda, K., Ichijo, H. ALS-linked mutant SOD1 induces ER stress-and ASK1-dependent motor neuron death by targeting Derlin-1. Genes Dev. 22:1451-1464, 2008.). However, although there are few reports on the expression and role of Derlin-1 in tumors, there are still reports that it is up-regulated by vascular endothelial growth factor VEGF in hepatocellular carcinoma endothelial cells. Apoptosis, growth inhibited (Ran Y, Jiang Y, Zhong X, Zhou Z, Liu H, Hu H, Lou JN, Yang Z. Identification of Derlin-1 as a novel growth factor-responsive endothelial antigen by suppression subtractive hybridization[J] . Biochem Biophys Res Commun. 2006, 348(4): 1272-1278.). Derlin-1 can be expressed on the surface of various tumor cells and cancer cells in human cancer tissues through some unknown mechanism, and it is expressed in polarity, that is, it is only expressed on the matrix surface of the cell membrane, while it is not expressed on the cell membrane of normal tissues or Low expression, Derlin-1 is specifically highly expressed in nine common malignant tumors such as breast cancer, colon cancer, esophageal cancer, lung cancer, gastric cancer, and liver cancer, with a positive rate of about 60-90%, especially on the cancer cell membrane. No expression or low expression in 21 kinds of normal tissues, and basically no normal cell membrane expression, with excellent tumor specificity; Derlin-1 extracellular domain epitope peptide was used to prepare rabbit polyclonal antibody, and 125I-labeled Derlin-1 antibody was used for detection In vivo distribution and imaging studies, the results showed that Derlin-1 antibody specifically aggregated in tumor tissues, and the radiation intensity in tumor tissues was significantly higher than that in other normal tissues. Competed by non-labeled antibodies, it shows that anti-Derlin-1 antibodies can specifically recognize tumors in vivo, gather at tumor sites and stay for a long time. On the one hand, it further proves that Derlin-1 can be expressed on the surface of tumor cells in vivo. 1 The homology of human and mouse protein sequences is as high as 99%, which fully proves that the expression of Derlin-1 has excellent tumor targeting in vivo and has the potential as a target for tumor therapy; using anti-Derlin-1 extracellular region polypeptide antibody, Antitumor experiments in animals have shown that blocking the function of Derlin-1 in vivo can significantly inhibit the growth of human colon cancer in vivo, with a tumor inhibition rate of 54.84% (Ran Y, Hu H, Hu D, Zhou Z, Sun Y, Yu L , Sun L, Pan J, Liu J, Liu I, Yang Z. Derlin-1 is overexpressed on the tumor cell surface and enables antibody-mediated tumor targeting therapy. Clin Cancer Res. 2008, 14(20): 6538-45.) . In addition, studies using immunohistochemistry and Western Blot simultaneous detection have confirmed that the positive rate of Derlin-1 expression in breast cancer is high, and the positive rate and expression intensity of Derlin-1 are related to the grade and metastasis of breast cancer, and in HER2 positive The frequency of expression is higher in cases of breast cancer. Chemotherapeutic drugs can cause endoplasmic reticulum pressure in breast cancer cells and up-regulate the expression of Derlin-1. Blocking Derlin-1 can make breast cancer cells resistant to apoptosis induced by various chemotherapeutic drugs. Derlin-1 is overexpressed in human breast cancer and protects cancer cells from endoplasmic reticulum stress -induced apoptosis. Breast Cancer Res. 2008, 10(1): R7.). Therefore, these existing reports suggest that Derlin-1 is a new target with important functions and tumor-specific tumor therapy. Tumor-specific high expression, good tumor-specific targeting in vivo, interference blocking its function can significantly inhibit tumor growth, the development of antibody drugs against Derlin-1 may provide new targets for clinical treatment of tumors Treatment with new drugs.

At present, it has become known in the industry that the binding specificity and affinity of a monoclonal antibody are largely determined by the hypervariable region (or complementarity determining region, complementary determinant region, CDR) of the light chain and heavy chain of the antibody. ) determined by the amino acid sequence, any monoclonal antibody with the same CDR sequence has basically the same specificity, affinity and therapeutic effect on the target antigen. The U.S. Food and Drug Administration (FDA) has determined in its guidelines that all antibodies of the same type with the same complementarity-determining region belong to the same antibody and should be treated as the same antibody in the approval of clinical therapeutic drugs. Therefore, after obtaining the CDR sequence of an antibody with clinical therapeutic value, it is easy for the industry to change the amino acid sequence of its non-CDR region according to mature and well-known existing technologies to obtain other monoclonal antibodies with the same biological activity .

Contents of the invention

The present invention is based on an obtained parental anti-Derlin-1 mouse monoclonal antibody with specific binding to Derlin-1 protein, through cloning, identification and analysis of gene structure, the sequence of its CDR region is determined, and the corresponding The humanized antibody and its eukaryotic cell expression vector were obtained, and the cell line expressing and secreting the anti-Derlin-1 humanized antibody was obtained, and the humanized antibody was produced and purified.

The present invention further proves that the humanized antibody not only has the specificity of binding to Derlin-1 protein and Derlin-1 positive tumor cells, but also has human origin, which can reduce the toxic and side effects when applied to human body. The present invention further uses animal models to prove that the humanized antibody has the ability to inhibit the growth of Derlin-1 positive tumors, and therefore can be used to prepare targeted therapeutic drugs for Derlin-1 positive malignant tumors.

Therefore, the present invention mainly relates to the following aspects:

In the first aspect, the present invention relates to a humanized monoclonal antibody, wherein the monoclonal antibody consists of a light chain containing three light chain complementarity determining regions and a heavy chain containing three heavy chain complementarity determining regions, wherein the The amino acid sequences of the three light chain complementarity determining regions are shown in SEQ ID NO: 1-3, and the amino acid sequences of the three heavy chain complementarity determining regions are shown in SEQ ID NO: 4-6.

It is well known in the art that the binding specificity and affinity of an antibody are mainly determined by the CDR sequence, and the amino acid sequence of the non-CDR region can be easily changed according to mature and well-known existing technologies to obtain variants with similar biological activities. body. The monoclonal antibody variants of the present invention having completely identical CDR sequences to the above-mentioned CDR sequences have similar biological activities because they have completely identical CDR sequences to the humanized antibodies of the present invention.

In a second aspect, the present invention relates to a specific anti-Derlin-1 humanized monoclonal antibody, wherein the light chain amino acid sequence of the monoclonal antibody is shown in SEQ ID NO: 7, and the heavy chain amino acid sequence is shown in SEQ ID NO: 8 shown. The light chain of the monoclonal antibody includes the CDR regions shown in SEQ ID NO: 1-3, and the heavy chain includes the CDR regions shown in SEQ ID NO: 4-6.

In the third aspect, the present invention relates to an isolated polynucleotide encoding the monoclonal antibody described in the first aspect or the second aspect. It is well known in the art that even if the nucleotide sequence is changed, as long as the antibody protein containing the amino acid sequence of SEQ ID NO: 1-3 and the amino acid sequence of SEQ ID NO: 4-6 can be translated in accordance with the genetic rules of triplet codons, they are all encoded The polynucleotide of the anti-Derlin-1 humanized antibody. The nucleic acid is DNA.

In a fourth aspect, the present invention relates to an expression vector containing the polynucleotide described in the third aspect, and the expression vector is expressed in eukaryotic cells. According to the well-known and common techniques in the art, as long as the amino acid sequence or nucleotide sequence of the antibody to be expressed is known, the practitioners in the field can easily construct expression vectors capable of expressing the antibody in various forms.

In the fifth aspect, the present invention relates to a specific expression vector capable of expressing anti-Derlin-1 humanized monoclonal antibody, the expression vector is pSRNC-Cκ-Derlin-1 as shown in Figure 5 or as shown in Figure 6 pSRDC-Cγ1-Derlin-1. In the sixth aspect, the present invention relates to a host cell comprising the expression vector described in the fifth aspect or the fourth aspect.

In the seventh aspect, the present invention relates to a therapeutically effective amount of the antibody described in the first or second aspect, or the nucleic acid described in the third aspect, or the carrier described in the fourth aspect or the fifth aspect, or the sixth aspect Use of the host cell in the preparation of antitumor drugs against Derlin-1 positive tumors. The tumor is selected from the group of malignant tumors with positive expression of Derlin-1 on the cell surface. According to the nucleic acid described in the third aspect, the vectors described in the fourth aspect and the fifth aspect can be prepared, and after the cells for expression are transfected, the host cells described in the sixth aspect can be prepared for the final production of the first or second The antibody according to one aspect, the antibody can be directly used in the treatment of malignant tumors with positive expression of Derlin-1 on the cell surface.

Description of drawings

Figure 1. Analysis of PCR products of VL and VH genes of anti-Derlin-1 mouse monoclonal antibody 8D3 by agarose gel electrophoresis.

Lane 1. Molecular weight standard, λDNA/Hind III;

Swimming lane 2. Mouse monoclonal antibody VL gene PCR product;

Swimming lane 3. Mouse monoclonal antibody VH gene PCR product;

Swimming lane 4. Mouse monoclonal antibody VL gene PCR amplification blank control product;

Swimming lane 5. Mouse monoclonal antibody VH gene PCR amplification blank control product.

Figure 2. Nucleotide sequence, amino acid sequence and antibody variable region (complementarity determining regions (CDRs) are underlined) of the amplified 8D3 mouse monoclonal antibody VL and VH genes.

SEQ ID NO: 1Ser Ala Ser Ser Ser Ser Val Ser Tyr Met His;

SEQ ID NO: 2Leu Thr Ser Lys Leu Ala Ser;

SEQ ID NO: 3Gln His Ser Arg Asn Asn Pro Tyr Thr;

SEQ ID NO: 4His Tyr Tyr Met Ser;

SEQ ID NO: 5Trp Ile Asn Pro Glu Ala Asn Gly Tyr Thr Glu Tyr Ala SerAla SerVal Lys Gly;

SEQ ID NO: 6Leu Tyr Tyr Tyr Trp Gly Gln Gly Met Asp Val.

Figure 3. Amino acid sequence of the humanized antibody variable region gene of the designed anti-Derlin-1 murine monoclonal antibody (complementarity determining regions (CDRs) are underlined).

Figure 4. Complete amino acid sequence of anti-Derlin-1 humanized monoclonal antibody.

SEQ ID NO: 7, light chain, constant region is human antibody CK;

SEQ ID NO: 8, heavy chain, constant region is human antibody Cγ1.

Figure 5. Schematic diagram of the structure of the eukaryotic expression vector pSRNC-Cκ-Derlin-1 for the light chain of the anti-Derlin-1 humanized antibody.

The abbreviations in the figure are as follows, Pw, weakened eukaryotic promoter; Neo, aminoglycoside phosphotransferase (neo) gene; PhCMV-IE, human cytomegalovirus immediate early promoter and enhancer; VLgene, with leader peptide sequence and The light chain variable region gene fragment of the 5' intron end splice site sequence; Cκgene, human antibody light chain κ chain constant region gene fragment; BGHpolyA, bovine growth hormone polyA tailing site; Ap, ampicillin resistance gene.

Fig. 6. Schematic diagram of the structure of the heavy chain eukaryotic expression vector pSRDC-Cγ1-Derlin-1 of the humanized anti-Derlin-1 antibody.

The abbreviations in the figure are as follows, Pw, weakened eukaryotic promoter; dhfr, dihydrofolate reductase (dhfr) gene; PhCMV-IE, human cytomegalovirus immediate promoter and enhancer; VHgene, with leader peptide sequence and Heavy chain variable region gene fragment of 5' intron end splice site sequence; Cγ1gene, human antibody heavy chain γ1 chain constant region gene fragment; BGHpolyA, bovine growth hormone polyA tailing site; Ap, ampicillin resistance gene.

Figure 7. Antigen specificity of anti-Derlin-1 humanized antibodies analyzed by immunofluorescence.

detailed description

The antibody CDR region of the anti-Derlin-1 humanized antibody gene involved in the present invention comes from an anti-Derlin-1 mouse monoclonal antibody 8D3 prepared by us in the past by immunizing mice with Derlin-1 polypeptide (others prepared in the same batch) For the experimental data of monoclonal antibodies, see Ran Y, Hu H, Hu D, Zhou Z, Sun Y, Yu L, Sun L, Pan J, Liu J, Liu T, YangZ. Derlin-1 is overexpressed on the tumor cell surface and enables Antibody-mediated tumor targeting therapy. Clin Cancer Res. 2008, 14(20): 6538-45.). The results of live cell immunofluorescence experiments in previous studies showed that the mouse monoclonal antibody can bind to Derlin-1 expressed on the plasma membrane of various tumor cells, which was further confirmed by immunohistochemical chip and section studies involving more than 1,000 cases. The antibody recognizes Derlin-1, which is specifically and highly expressed in nine common malignant tumors, including breast cancer, colon cancer, esophageal cancer, lung cancer, gastric cancer, and liver cancer, with a positive rate of about 60-90%, while 21 normal tissues do not stain or Weak staining, with excellent tumor specificity; the results of in vivo distribution and imaging studies show that the anti-Derlin-1 mouse monoclonal antibody specifically accumulates in tumor tissue, and the radiation intensity in tumor tissue is significantly higher than that in other normal tissues. T/NT distribution It shows remarkable tumor specificity, and the tumor is clearly visualized. It can specifically recognize the tumor in vivo, gather at the tumor site and stay for a long time. Since the homology of Derlin-1 human and mouse protein sequence is as high as 99%, it fully proves that the antibody can be used in the human body. It will also have excellent tumor targeting; in vivo tumor inhibition experiments in animals have proved that the use of this antibody to interfere with and block the function of Derlin-1 in vivo can significantly inhibit the growth of human colon cancer xenografts in nude mice, and the tumor inhibition rate reaches 54.84%; The body weight of the animals did not decrease significantly during the treatment, which preliminarily suggested that the toxic and side effects of the treatment were not obvious or very low. All the organs of the experimental animals were stained with HE staining in histological sections, and observed under a microscope whether there was any toxic or side effect on each organ. No pathological changes were seen in normal organs, directly proving that the anti-Derlin-1 antibody has high safety in treating human colon cancer. In summary, it proves that the anti-Derlin-1 mouse monoclonal antibody has important value for further development as an anti-tumor targeted therapy drug, and its humanized transformation eliminates the immunogenic toxicity of its mouse monoclonal antibody, which is expected to be used in the clinical and effective treatment of tumors .

definition

Monoclonal antibodies

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homologous antibodies, ie, comprising individual antibodies that are identical except for naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific antibodies directed against a single target site. Furthermore, each monoclonal antibody is directed against a single determinant on the target, in contrast to conventional (polyclonal) antibody preparations, which typically comprise different antibodies directed against different determinants (epitopes). In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture without contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that an antibody is obtained from a substantially homogeneous population of antibodies and does not imply that the antibody was produced by any particular method. For example, monoclonal antibodies used in the present invention can be isolated from phage antibody libraries by using well-known techniques. The parental monoclonal antibodies used according to the invention can be produced by the hybridoma method first described by Kohler and Milstein, Nature 256, 495 (1975), or can be produced by recombinant methods.

Complementarity Determining Regions (CDRs)

As used herein, the term "complementarity-determining region" refers to a sequence in the variable region of a binding molecule such as an immunoglobulin, which typically primarily provides an antigen-binding site that is complementary in shape and charge distribution to an epitope recognized on the antigen . The CDR regions may be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments that are present on the protein in its native conformation or in some cases as denatured epitopes, e.g. by solubilization in SDS. forms exist on proteins. Epitopes can also consist of post-translationally modified proteins.

polynucleotide

As used herein, "polynucleotide" includes deoxyribose polynucleotides, ribopolynucleotides, or analogs thereof that have the essential properties of natural ribonucleotides, as long as they are identical to natural nucleotides under stringent hybridization conditions. Nucleotide sequences that are substantially identical hybridize and/or can be translated to the same amino acids as native nucleotides. A polynucleotide may be the full length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes the specific sequence as well as its complement. Thus, the term "polynucleotide" herein includes DNA with a backbone modified for stability or for other reasons.

polypeptide

As used herein, the term "polypeptide" is used interchangeably 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 a man-made chemical analog of the corresponding natural amino acid, as well as for natural amino acid polymers. An essential property of such analogs of natural amino acids is that when incorporated into a protein, the protein specifically elicits an antibody reaction with a protein consisting of the same but consisting entirely of natural amino acids. The terms "polypeptide", "peptide" and "protein" also include modifications including, but not limited to, phosphorylation, glycosylation, lipid attachment, sulfuration, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribose Basicization.

specific binding

As used herein, the term "specific binding" in reference to the interaction between an antibody and its binding partner, such as an antigen, means that the interaction is dependent on the presence of a specific structure on the binding partner, such as an antigenic determinant or presence of the epitope. In other words, the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms. The association may be mediated by covalent or non-covalent interactions or both. In other words, the term "specifically binds" refers to immunospecific binding to an antigen or a fragment thereof and non-immunospecific binding to other antigens. A binding molecule that immunospecifically binds an antigen may bind other peptides or polypeptides with lower affinity as determined, for example, 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 an antigen may cross-react with a related antigen. Preferably, a binding molecule or fragment thereof that immunospecifically binds an antigen does not cross-react with other antigens.

Variants

As used herein, the term "variant" refers to a nucleotide sequence comprising altered nucleotides and/or amino acids outside one or more CDR regions compared to the nucleotide and/or amino acid sequence of the parental binding molecule and Binding molecules of binding partners such as Derlin-1 that have an amino acid sequence and/or that are still able to compete for binding to the parental binding molecule. In other words, modifications in the amino acid and/or nucleotide sequence of the parental binding molecule do not significantly affect or alter the binding properties of the binding molecule encoded by said nucleotide sequence or comprising said amino acid sequence, i.e. said binding The molecule is still able to recognize and bind its target. The functional variants may have conservative sequence modifications, including nucleotide and amino acid substitutions, additions and deletions. These 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 as well as unnatural nucleotides and amino acids.

Conservative amino acid substitutions involve the replacement of an amino acid residue with one having similar structural or chemical properties. Families of amino acid residues having similar side chains have been defined in the art. These families include those 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, aspartic acid) Amide, glutamine, serine, threonine, tyrosine, cystine, tryptophan), non-polar side chains (e.g. alanine, valine, leucine, isoleucine, pro amino acid, phenylalanine, methionine), β-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine , tryptophan, histidine) amino acids. It will be clear to those skilled in the art that other amino acid residue family classifications than those described above may also be applied. In addition, a variant may have non-conservative amino acid substitutions, eg, replacing an amino acid residue with an amino acid residue having different structural or chemical properties. Similar minor changes may also include amino acid deletions or insertions or both. Guidance for determining which amino acid residues may be substituted, inserted, or deleted without abrogating their immunological activity can be found using computer programs well known in the art.

A mutation in a nucleotide sequence may be a single change at a locus (point mutation), such as a transition or transversion mutation, or may insert, delete or change multiple nucleotides at a single locus. Additionally, one or more alterations may be made at any number of loci within the nucleotide sequence. Mutations can be performed by suitable methods known in the art.

Humanized Antibody

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv, Fab, Fab ', F(ab') ₂ fragments, or other target-binding subsequences). In general, a humanized antibody will comprise substantially all of at least one, and usually two, variable domains, wherein all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin FR regions are those of the human immunoglobulin consensus sequence. The humanized antibody will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin template of choice.

carrier

The term "vector" refers to a nucleic acid molecule into which another nucleic acid molecule can be inserted for introduction into a host for its replication, and in some cases expression. In other words, a vector is capable of transporting a nucleic acid molecule to which it is attached. Cloning as well as expression vectors are encompassed by the term "vector" as used herein. Vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs), and vectors derived from bacteriophage or plant or animal (including human) viruses. A vector contains an origin of replication recognized by a given host and, in the case of an expression vector, a promoter and other regulatory regions recognized by the host. A vector containing another nucleic acid molecule is introduced into a cell by transformation, transfection, or by utilizing a viral entry mechanism. Certain vectors are capable of autonomous replication in the host into which they are introduced (eg, vectors with a bacterial origin of replication can replicate in bacteria). Other vectors can be integrated into the host genome when introduced into the host, thereby replicating together with the host genome.

Host

As used herein, the term "host" 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 organism or cell. It is to be understood that this term refers not only to a particular subject cell, but also to the progeny of such cells. Certain modifications may occur in subsequent generations as a result of mutations or environmental influences, so that such progeny are not substantially identical to the parental cells, but are still included within the term "host" as used herein.

Pharmaceutically acceptable excipients

A "pharmaceutically acceptable excipient" refers to any inert substance that is combined with an active molecule such as a drug, an agent or a binding molecule to produce a suitable or convenient dosage form. A "pharmaceutically acceptable excipient" is an excipient that is nontoxic to the recipient at the dosages and concentrations used and is compatible with the other ingredients of the formulation comprising the drug, agent or binding molecule.

therapeutically effective amount

The term "therapeutically effective amount" refers to the amount of the antibody of the present invention that is effective in preventing, improving and/or treating cancer.

treat

The term "treatment" refers to both therapeutic treatment and prophylactic measures to cure a disease or to arrest or at least delay the progression of a disease. Those in need of treatment include those already with cancer as well as those in which cancer is to be prevented. Subjects who have partially or fully recovered from cancer are also in need of treatment. Prevention includes inhibiting or slowing cancer progression or inhibiting or reducing the occurrence, development or progression of one or more symptoms associated with cancer.

In this specification, the term "comprising" means including the stated elements, integers or steps, or groups of elements, integers or steps, but not excluding any other elements, integers or steps, or other groups of elements, integers or steps.

First, the present invention provides a CDR region sequence of a monoclonal antibody with a specific sequence that can specifically bind to Derlin-1 protein and specifically recognize tumor cells expressing Derlin-1 protein on the cell membrane surface. The CDR sequence is obtained by amplifying, cloning, and analyzing the variable region gene of an anti-Derlin-1 mouse monoclonal antibody with tumor targeting and in vivo treatment of tumors. It has become well known in the art that the specificity and affinity of monoclonal antibodies binding to antigens are mainly determined by the 6 CDR sequences of their light and heavy chains. On the basis of obtaining the complete CDR sequences, it is easy to obtain Known existing technologies construct and produce various monoclonal antibody variants with identical CDR sequences, and these modified variants have similar biological activities and therapeutic effects. Therefore, according to the specific and complete CDR amino acid sequence provided by the present invention, the content of the invention involved in the present invention can be achieved in the following ways.

In one aspect, the present invention provides an anti-Derlin-1 humanized monoclonal antibody, which is characterized in that the heavy chain of the monoclonal antibody comprises the CDR region shown in SEQ ID NO 1-3, and the monoclonal antibody The light chain of Derlin-1 contains the CDR region shown in SEQ ID NO: 4-6, any monoclonal antibody with the above specific and complete CDR sequence will be directed against the same epitope of Derlin-1, and have similar biological activity and treatment effect.

In one aspect, specifically, the present invention also specifically provides a specific anti-Derlin-1 humanized antibody, wherein the light chain protein amino acid sequence of the anti-antibody comprises or consists of SEQ ID NO: 7, and the heavy chain protein amino acid sequence Comprising or consisting of SEQ ID NO:8. In the specific embodiment described in the present invention, the anti-Derlin-1 humanized antibody is a recombinant monoclonal antibody with specific light and heavy chain amino acid sequences, because the framework region and constant region of the specific antibody are derived from In humans, it can also be called recombinant humanized antibody.

In this application, the term "antibody of the present invention" refers to the anti-Derlin-1 humanized monoclonal antibody of the present invention, wherein the heavy chain of the monoclonal antibody comprises SEQ ID NO: 1-3 The light chain of the monoclonal antibody comprises the CDR regions shown in SEQ ID NO: 4-6. In a specific embodiment, the anti-Derlin-1 humanized antibody is contained in the above-mentioned antibody concept, specific, the amino acid sequence of the light chain protein contains or consists of SEQ ID NO: 7, and the amino acid sequence of the heavy chain protein An anti-Derlin-1 humanized monoclonal antibody comprising or consisting of a specific complete sequence of SEQ ID NO: 8, the complete sequence of the specific anti-Derlin-1 humanized monoclonal antibody is the CDR region provided by the present invention Sequence embedding is spliced into specific human antibody FR region sequences and constant region sequences.

In another aspect, the invention relates to a nucleic acid encoding an antibody of the invention comprising a polynucleotide encoding an antibody polypeptide of the invention or a complementary sequence thereof. The nucleic acid can be DNA. It is well known in the art that even if the nucleotide sequence is changed, as long as the antibody protein containing the amino acid sequence SEQID NO: 7 and the amino acid sequence SEQID NO: 8 can be translated according to the genetic rules of triplet codons, they all encode the anti-Derlin-1 human Polynucleotides for derivatized antibodies.

In another aspect, the present invention provides a recombinant expression vector that can be used to prepare the anti-Derlin-1 humanized antibody, said vector comprising nucleic acid encoding the antibody of the present invention. According to the well-known and common techniques in the art, as long as the amino acid sequence or nucleotide sequence of the antibody to be expressed is known, the practitioners in the field can easily construct expression vectors capable of expressing the antibody in various forms. Vectors can be derived from plasmids such as F, R1, RP1, Col, pBR322, TOL, Ti, etc.; cosmids; bacteriophages such as λ, lambdoid, M13, Mu, P1, P22, Qβ, T-even, T-odd, T2, T4, T7, etc.; plant viruses; or animal viruses. Vectors can be used for cloning and/or expression purposes as well as for gene therapy purposes. Vectors comprising one or more nucleic acid molecules encoding the antibodies of the invention operably linked to one or more expression-modulating nucleic acid molecules are also encompassed by the invention. The choice of vector depends on the recombination procedure and the host used. The vector can be introduced into host cells by calcium phosphate transfection, virus infection, DEAE-dextran mediated transfection, lipofectamin transfection or electroporation. A vector can replicate autonomously or can replicate with the chromosome into which it has integrated. Preferably, the vector contains one or more selectable markers. The choice of said marker may depend on the chosen host cell, which is not critical to the invention and is already well known to those skilled in the art. Said markers include but are not limited to kanamycin, neomycin, puromycin, hygromycin, zeocin, thymidine kinase gene (HSV-TK) of herpes simplex virus, mouse dihydrofolate reductase gene (dhfr ). Specifically, the present invention recombinantly clones polynucleotides encoding the light chain and heavy chain of the anti-Derlin-1 humanized chimeric antibody into two vectors containing eukaryotic promoters, and introduces the resulting expression vectors into Eukaryotic host cells, obtained by screening eukaryotic host cells that express the antibody of the present invention in high yield, contain a large amount of the anti-Derlin-1 humanized antibody protein secreted by the host cell in the culture supernatant, according to the art Known technical methods can conveniently extract and prepare the anti-Derlin-1 humanized antibody protein. In an embodiment, the expression vectors are the specific vector pSRNC- CK-Derlin-1 and pSRDC-Cγ1-Derlin-1.

The present invention also provides hosts containing one or more copies of the above-mentioned vectors. The host is a host cell. Host cells include, but are not limited to, cells of mammalian, plant, insect, fungal, or bacterial origin. Expression systems using mammalian cells such as Chinese hamster ovary (CHO) cells are generally preferred for the present invention. In an embodiment, the host cell is Chinese hamster ovary cell CHO. Specifically, the present invention introduces pSRNC-Cκ-Derlin-1 and pSRDC-Cγ1-Derlin-1 into the eukaryotic host cell CHO, and obtains a eukaryotic host cell expressing the antibody of the present invention in a high yield through screening, and in the host cell The culture supernatant contains a large amount of the anti-Derlin-1 humanized antibody protein secreted by it, from which the anti-Derlin-1 humanized antibody protein can be conveniently extracted and prepared according to technical methods known in the art.

According to one aspect of the present invention, the anti-Derlin-1 humanized antibody protein can be used to prepare a drug for treating human Derlin-1 positive tumors. The anti-Derlin-1 humanized antibody of the present invention can specifically bind to the Derlin-1 protein on the surface of the tumor cell membrane, and can directly block the biological function of Derlin-1 in vivo, thereby inhibiting the expression of Derlin-1 positive tumors Growth in the body, so the antibody can be used to directly prepare anti-Derlin-1 expression positive tumor targeted therapeutic antibody medicine. It has become known in the field that when obtaining anti-Derlin-1 humanized antibody protein, it can be prepared by adding general appropriate stabilizers, protective agents, pH regulators, salt balancers, excipients, etc. Antibody drugs that can be directly used for human treatment. In an embodiment, the antibody of the present invention can be used to treat Derlin-1-positive tumor transplanted in nude mice, and in a specific embodiment, a colorectal cancer model is used. The anti-Derlin-1 humanized chimeric antibody can be administered to patients as a drug through conventional administration routes, such as but not limited to parenteral administration, such as intravenous, infusion, local administration medicine etc. Suitable dosages will depend on several parameters, including the method of administration and the subject being treated and how well tolerated. It is clear that the anti-Derlin-1 humanized antibody can significantly inhibit the growth of Derlin-1 positive human colorectal cancer in a dose-dependent manner.

The present invention is further illustrated by the following examples, but any example or combination thereof should not be construed as limiting the scope or implementation of the present invention. The scope of the present invention is defined by the appended claims, and those skilled in the art can clearly understand the scope defined by the claims in combination with the description and common knowledge in the field.

Example

Cloning, sequencing and analysis of the anti-Derlin-1 monoclonal antibody gene of embodiment 1

The light and heavy chain variable region genes of the anti-Derlin-1 mouse monoclonal antibody were cloned by gene cloning method, and the nucleotide sequence analysis was carried out.

Anti-Derlin-1 mouse monoclonal antibody variable region gene amplification method: the extraction of total RNA from mouse monoclonal antibody hybridoma cells 8D3 was carried out according to the instructions of Trizol reagent (Gibco Company), and 1×10 ⁷ mouse monoclonal antibody hybridomas were collected Cells were centrifuged at 10,000 rpm for 1 min, and the supernatant was discarded. Add 1ml Trizol to fully dissolve the cells, let stand at room temperature for 3-5min, add 0.2ml chloroform, invert and mix well, centrifuge at 12000rpm at 4°C for 10min, transfer about 0.6ml supernatant to a new centrifuge tube. Add 0.5ml of isopropanol, mix by inversion, let stand at room temperature for 5-10min, centrifuge at 12000rpm at 4°C for 10min, discard the supernatant, wash once with 75% ethanol, air dry, add 50μ1ddH ₂ O to dissolve the precipitate. The synthesis of the first strand of mouse monoclonal antibody hybridoma cell cDNA was carried out using MMLV-reverse transcriptase (Gibco Company) according to the instructions provided by the manufacturer: 4 μl of 5× buffer and 10 mM DDT (Promega Company) were added to the 20 μl system , 10 μg total RNA, the dNTP (Promega company) of final concentration 0.5mM, the Oligo d (T) 15 (Promega company) of final concentration 10 μ g/ml, 40u RNasin (Promega company), 200u (U) MMLV-reverse transcriptase (Gibco), mix well. Water bath at 37°C for 1 hour, followed by boiling water bath for 5 minutes to inactivate reverse transcriptase. The mouse monoclonal antibody light and heavy chain variable region genes were amplified using high-precision DNA polymerase Taq (Promega) + Pfu DNA polymerase (Promega). The 100 μl reaction system contained: 10 μl of 10× buffer, 10 mM dNTP 2 μl, 20 μl of cDNA, 50 pmol of each amplification primer, mix well and cover the surface with paraffin oil. After bathing in water at 95°C for 5 minutes, add 1-2u of Taq+Pfu DNA polymerase through paraffin oil, and start the following cycle: 94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, a total of 30 cycles, and the last cycle is 72°C for 10 minutes. PCR primers: primers for amplifying the light chain variable region: PVL5: 5'-GACAT TCAGCTGACC CAGTC TCCA-3'; PVL3: 5'-GTTAG ATCTC CAGCT TGGTC CC-3'. Primers used to amplify the heavy chain variable region: PVH5: 5′-AGGTSMARCT GCAGS AGTCW GG-3′ (S=C/G, M=A/C, R=A/G, W=A/T); PVH3 : 5'-TGAGG AGACGGTGAC CGTGG TCCCTTGGCC CCAG-3'.

The total RNA extracted from the parental anti-Derlin-1 mouse monoclonal antibody hybridoma cell line was analyzed by 0.7% non-denaturing agarose gel electrophoresis. The size of 18S RNA and 28S RNA was correct, the brightness ratio was about 1:2, and the swimming lanes were clear. The first strand of cDNA was synthesized with Oligo d(T)15 as a primer, and the cDNA was used as a template to carry out PCR using light chain primers PVL5 and PVL3 to amplify a light chain variable region gene fragment with a size of about 320 bp; Chain primers PVH5 and PVH3 were used for PCR, and a heavy chain variable region gene fragment of about 360 bp in size was amplified; the blank control without template had no amplified band (Figure 1). The size of the amplified variable region gene fragment is consistent with the size of the general antibody variable region gene.

Cloning, sequencing, and gene structure analysis of light and heavy chain variable region genes of anti-Derlin-1 mouse monoclonal antibody: a large number of anti-Derlin-1 mouse monoclonal antibody light chain variable region gene fragments were amplified by glass milk adsorption method After separation and recovery, they were digested with Pvu II and Bgl II, and cloned into the corresponding site of the cloning vector pRGWL. A total of 203 transformed clones were obtained. 24 clones were randomly selected and screened, and 8 recombinant clones were obtained. Three VL gene recombinant clones were selected for nucleotide sequence analysis. The nucleotide sequence and deduced amino acid sequence are shown in Figure 2. The sequences of the three clones are identical, indicating that the cloned antibody light chain variable region gene is indeed It is the gene of the light chain variable region of the real anti-Derlin-1 mouse monoclonal antibody. A clone was randomly selected from these 3 clones and named pRGWL-D1. Compared with the data of Kabat, it can be seen that the VL of the anti-Derlin-1 mouse monoclonal antibody belongs to the III subgroup of the mouse κ light chain, and the light chain CDR1-3 sequence (SEQ ID NO: 1-3) is shown in FIG. 2 . A large number of anti-Derlin-1 mouse monoclonal antibody heavy chain variable region gene fragments were amplified, separated and recovered by the glass milk method, and then digested with Pst I and BstE II, and directional cloned into the corresponding site of the cloning vector pRGWH, a total of 321 Transformed clones, 24 clones were randomly selected for screening, and 15 recombinant clones were obtained. Select 3 VH gene recombinant clones for nucleotide sequence analysis. The nucleotide sequence and deduced amino acid sequence are shown in Figure 2. The sequences of the 3 clones are identical, which proves that the cloned antibody heavy chain variable region gene is indeed true. The heavy chain variable region gene of the anti-Derlin-1 mouse monoclonal antibody was randomly selected and a clone was named pRGWH-D1. Compared with the data of Kabat, it can be seen that the VH of the anti-Derlin-1 mouse monoclonal antibody belongs to the III (A) subgroup of the mouse IgG1 heavy chain, and the heavy chain CDR1-3 sequence is shown in Figure 2 (SEQ ID NO: 4- 6). Specifically, the sequence of SEQ ID NO: 1-6 is:

SEQ ID NO: 1Ser Ala Ser Ser Ser Ser Val Ser Tyr Met His;

SEQ ID NO: 2Leu Thr Ser Lys Leu Ala Ser;

SEQ ID NO: 3Gln His Ser Arg Asn Asn Pro Tyr Thr;

SEQ ID NO: 4His Tyr Tyr Met Ser;

SEQ ID NO: 5Trp Ile Asn Pro Glu Ala Asn Gly Tyr Thr Glu Tyr Ala SerAla Ser ValLys Gly;

SEQ ID NO: 6Leu Tyr Tyr Tyr Trp Gly Gln Gly Met Asp Val.

Example 2 Construction of humanized antibody gene and expression vector of anti-Derlin-1 mouse monoclonal antibody

Using the method of gene synthesis, the framework region of the variable region gene of the anti-Derlin-1 mouse monoclonal antibody is replaced by the framework region of the human antibody, and the humanized antibody of the anti-Derlin-1 mouse monoclonal antibody is synthesized by the total synthesis method Region gene fragments are recombined into vectors containing regulatory sequences and human antibody constant region genes to construct the complete gene of the anti-Derlin-1 humanized antibody and the eukaryotic expression vector containing the gene.

Synthesis of humanized antibody variable region gene of anti-Derlin-1 mouse monoclonal antibody: Using the variable region of the published humanized 4D5 monoclonal antibody as a template, the CDR region was replaced with the corresponding 8D3 by CDR grafting method In the CDR region of the monoclonal antibody, the enzyme-cleaved cloning site sequences at both ends are retained. The amino acid sequence translated by the humanized antibody variable region gene of the designed anti-Derlin-1 mouse monoclonal antibody is shown in Figure 3, light and heavy The chain gene was directly synthesized by a commercial company by segmented synthesis + overlapping PCR ligation method, and the light and heavy chain variable regions of the humanized antibody against Derlin-1 mouse monoclonal antibody were amplified by PCR using the same method as in Example 1 After the gene was recovered, the product was digested and re-cloned back to the pRGWL and pRGWH vectors. Sequencing confirmed that the cloned sequence was the same as the original designed sequence. One clone was selected and named pRGWL-hD1 and pRGWH-hD1, as the following amplification and construction of the expression vector. template.

Amplify the variable region gene fragments with regulatory sequences by PCR: with the recombinant cloning plasmids pRGWL-hD1 and pRGWH-hD1 obtained in Example 1 identified through sequencing as templates, use the two-terminal cloning sites BamH I and Primers PVLS, PVNP (light chain) and PVHS, PVNP (heavy chain) of the Not I restriction site, use PCR technology to amplify humanized VL and VH with leader peptide and 5′-end splice site . A humanized VL fragment with a leader peptide and 5'-end splice site was amplified from the recombinant plasmid of the light chain by PCR reaction, with a size of about 500bp; amplified with a guide peptide from the recombinant plasmid of the heavy chain and 5'-end splice site humanized VH fragment, about 700bp in size. Specifically, use high-precision DNA polymerase Taq+Pfu DNA polymerase, which contains in 100 μl reaction system: 10 μl of 10× buffer, 2 μl of 10 mM dNTP, 1 μg of plasmid (pRGWL-hD1 or pRGWH-hD1), amplification primer (PVLS, PVNP (light chain) and PVHS, PVNP (heavy chain)) each 50pmol, after mixing, the surface is covered with paraffin oil. After 5 minutes in water bath at 95°C, add 1-2u Taq+Pfu DNA polymerase through paraffin oil, start the following cycle, 94°C for 1 minute, 55°C for 1 minute, 72°C for 1 minute, a total of 20 cycles, and the last cycle 72°C for 10 minutes. Amplification primers:

PVLS: 5′-CTCGGAATTCGGATCCATGGGATGGAGCTGTATCATCC-3′

PVHS: 5′-GGTCCAGCTTGCGGCCGCAACTGAGGAAGCAAAGTTTAAATTCTACTCACGTTTGATCACCA-3′

PVNP: 5′-GGTCCGAATTCGCGGCCGCTATAAATCCTCTGGCCATGAAG-3′

Construction and identification of anti-Derlin-1 humanized antibody eukaryotic expression vector: The above PCR products were separated and recovered by the glass milk method, and then digested with BamH I and Not I. Carry out routine DNA recombination operations as described in "Molecular Cloning", clone VL into pSRNC-Cκ (source, if it is a known antibody, please provide the literature prepared according to it), VH clone into pSRDC-Cγ1 (source, if it is known For the antibody, please provide the corresponding site in the prepared literature) to construct a complete eukaryotic expression vector of the anti-Derlin-1 humanized antibody gene. After linking VL and VH into expression vectors pSRNC-Cκ and pSRDC-Cγ1, respectively, 12 clones were selected for screening, and 8 light chain and 8 heavy chain recombinant clones were identified by enzyme digestion. After digestion with BamH I and Not I, the corresponding VL and VH fragments can be cut out, which proves that the complete anti-Derlin-1 humanized monoclonal antibody gene and its eukaryotic expression vector have been successfully constructed. The repeated nucleotide sequence determination proved that the variable region gene sequences in the anti-Derlin-1 humanized monoclonal antibody eukaryotic expression vectors pSRNC-Cκ-Derlin-1 and pSRDC-Cγ1-Derlin-1 were the same as The original designed humanized variable region gene sequence is exactly the same. The amino acid sequence of the anti-Derlin-1 humanized monoclonal antibody produced by this set of expression vectors is SEQ ID NO: 7 (light chain, constant region is human antibody CK) and SEQ ID NO: 8 (heavy chain, constant region) as shown in Figure 4. Region is indicated by human antibody Cγ1).

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

DIQLTQSPSS LSASVGDRVT ITCSASSSVS YMHWYQQKPG KAPKLLIYLT SKLASGVPSR

FSGSRSGTDF TLTISSLQPE DFATYYCQHS RNNPYTFGGG TKVEIKTVAA PSVFIFPPSD

EQLKSGTASV VCLLNNFYPR EAKVQWKVDN ALQSGNSQES VTEQDSKDST YSLSSTLTLS

KADYEKHKVY ACEVTHQGLS SPVTKSFNRG EC

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

QVQLQQSGGG LVQPGGSLRL SCAASGFNIK HYYMSWVRQA PGKGLEWVAW INPEANGYTE

YASASVKGRF TISADTSKNT AYLQMNSLRA EDTAVYYCSR LYYYWGQGMD VWGQGTSVTV

SSSSASTKGP SVFPLAPSSK STSGGTAALG CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV

LQSSGLYSLS SVVTVPSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTCPPCPAPE

LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE

EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP

SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD

KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

The structure of eukaryotic expression vector of anti-Derlin-1 humanized monoclonal antibody: The eukaryotic expression vector of anti-Derlin-1 humanized monoclonal antibody adopts light chain eukaryotic expression vector (pSRNC-Cκ-Derlin-1) and The structure diagrams of the heavy chain eukaryotic expression vector (pSRDC-Cγ1-Derlin-1), which are two light and heavy chain expression vectors, are shown in FIGS. 5 and 6 .

Example 3 Anti-Derlin-1 humanized antibody expression vector was transfected into CHO cells for expression

CHO-dhfr ^- cells (preserved in this laboratory) adopt 10% FBS, 0.03mmol/l hypoxanthine (H), 0.003mmol/l thymidine (T), 0.1mmol/l proline (Pro) , 0.1mmol/l glycine (Gly), 100u/ml penicillin, streptomycin, 2mmol/l glutamine DMEM complete culture solution cultured under the conditions of 5% CO ₂ and 37°C, routinely passed down at 1:10, approximately Passage to one generation in 3-4 days. The above cell culture reagents were purchased from Gibco. Using the method of gene transfection, transfect with LipofectAMINE reagent (Gibco company), co-transfect the cells with the light and heavy chain expression vectors of anti-Derlin-1 humanized antibody, and use the medium without H, T, and Gly Cultivate for screening, and then use selection medium containing 200 μg/ml G418 (Gibco Company) to culture for screening after the clones are formed. As a result, 4 μg of light and heavy chain antibody gene expression vectors were transfected into CHO ^- dhfr- cells, and clonal growth was observed after about 10 days, and a total of about 483 clones were counted. The OD490 of the culture supernatant of the resistant clone mixture was measured by indirect ELISA method coated with goat anti-human κ chain polyclonal antibody and goat anti-human IgG-HRP as the secondary antibody, while the negative control CHO ^- dhfr- supernatant OD490 was only is 0.073, indicating that there is expression of anti-Derlin-1 humanized antibody in the supernatant of transfected cells.

Example 4 Pressurized amplified expression, screening anti-Derlin-1 humanized chimeric antibody high expression strain

The transfected CHO cells were cultured in DMEM (Gibco company) containing 10% FBS, 100u/ml penicillin and streptomycin, 2mmol/l glutamine under the condition of 5% CO ₂ and 37°C. 1:10 subculture, about 3-4 days for one generation. High expression strains were screened by methotrexate (MTX) pressurized amplification expression screening method. The cell clones expressing the humanized anti-Derlin-1 antibody in the supernatant were cultured in the complete medium containing 3×10 ^-8 M and 10 ^-7 M methotrexate (MTX) (Sigma Company) sequentially, and then pressurized and expanded. After each round of pressurized amplification and expression, the limited dilution method was used for subcloning, and the clone with the highest yield was screened.

After transfecting the anti-Derlin-1 humanized antibody expression vector into CHO-dhfr ^- cells, the clone 2B8 (antibody production up to 0.34 μg/ml) obtained by primary screening for highly expressing anti-Derlin-1 humanized antibody was used containing 3 Culture medium of ×10 ^-8 M methotrexate (MTX) (Sigma Company). After continuous culture for about 30 days, it was observed that the cell morphology and growth rate returned to normal, and had adapted to 3×10 ^-8 M MTX, and the antibody expression yield was 12.8 After subcloning, the clone 5B3 with the highest antibody production was screened out, and its antibody production could reach 18 μg/ml. Clone 5B3 continued to be cultured in the complete medium containing 10 ^-7 M MTX after subcloning at 1:5. After the cells adapted, the antibody production could reach 38 μg/ml. After subcloning, the clone 9D8 with the highest antibody production was screened out. The output can reach more than 90μg/ml after testing.

Example 5 Identification of Specificity and Humanity of Anti-Derlin-1 Humanized Antibody

(1) Specific identification of anti-Derlin-1 humanized antibody:

Using the ELISA method, the recombinant Derlin-1 protein expressed in the prokaryotic plate was coated with 1 μg/ml prokaryotic plate, and after the sample was added for reaction, the goat anti-human IgG Fc fragment-HRP enzyme-labeled antibody (Sigma Company) (not crossed with mouse Ig) was added. reaction) or goat anti-mouse IgG Fc fragment-HRP enzyme-labeled antibody (Sigma Company) (does not cross-react with human Ig) to incubate, develop color, and measure OD490 value. Anti-Derlin-1 humanized antibody purified from clone 9D8 cell supernatant using protein A affinity chromatography can bind to the coated recombinant Derlin-1 protein and be recognized by goat anti-human IgG Fc fragment polyclonal antibody , showed a strong positive reaction, while untransfected CHO-dhfr ^- cell culture supernatant and parental mouse monoclonal antibody 8D3 showed a negative reaction. When goat anti-mouse IgG Fc fragment-HRP was used as the secondary antibody, the culture supernatant of untransfected CHO ^- dhfr- cells and the purified anti-Derlin-1 humanized antibody were negative, while the parental mouse monoclonal antibody 8D3 was negative positive response. Unrelated antibody human IgG1 was negative. It is proved that the expressed anti-Derlin-1 humanized antibody can specifically combine with the recombinant Derlin-1 protein.

Table 1. Antigen-binding specificity of anti-Derlin-1 humanized antibodies analyzed by direct ELISA

a, using goat anti-human IgG Fc fragment-HRP as the secondary antibody b, using goat anti-mouse IgG Fc fragment-HRP as the secondary antibody.

Using immunofluorescence test, colon cancer cell line LS180 (purchased from ATCC) with high expression of Derlin-1 was used as the target cell, anti-Derlin-1 humanized antibody (10 μg/ml) was added, and goat anti-human IgG was added after incubation at 37°C -Fluorescent secondary antibody (Sigma Company), observed under a fluorescent microscope after the reaction, and an irrelevant antibody control was set at the same time. The results are shown in FIG. 7 , the anti-Derlin-1 humanized antibody can recognize the Derlin-1 protein on the cell membrane of colon cancer cell LS180 with high expression of Derlin-1.

(2) Identification of human origin of anti-Derlin-1 humanized antibody:

In the ELISA experiment, use goat anti-human κ chain (Sigma Company) or goat anti-human IgG polyclonal antibody (Sigma Company) to coat the microplate, and use goat anti-human IgG Fc fragment-HRP (Sigma Company) as the enzyme-labeled antibody The ELISA results (Table 2) showed that the purified anti-Derlin-1 humanized antibody showed a strong positive reaction, while the parental mouse monoclonal antibody 8D3 of the anti-Derlin-1 humanized antibody showed a negative reaction. It was proved that the purified anti-Derlin-1 humanized antibody contained the light and heavy chain constant regions of human IgG.

Table 2. Detection of Humanity of Anti-Derlin-1 Humanized Antibodies by ELISA

a, coated with goat anti-human IgG; b, coated with goat anti-human κ chain.

Example 6 Anti-Derlin-1 humanized antibody preparation of therapeutic drugs for in vivo treatment of Derlin-1-positive human nude mice transplanted tumors

Anti-Derlin-1 humanized antibody was used to treat human colon cancer in vivo in mice transplanted with human Derlin-1 positive colon cancer cell line LS180 (purchased from ATCC, USA) (1 million LS180 were subcutaneously injected into the right back of the nude mouse). For the study of the effectiveness of cancer tumors, intraperitoneal administration was started on the 3rd day after tumor inoculation, and anti-Derlin-1 humanized antibody treatment in vivo was divided into 3 dose groups, 3mg/kg body weight, 9mg/kg body weight, and 27mg/kg body weight , at the same time set up PBS and 27mg/kg body weight human IgG control group, administered 2 times a week. The results are shown in Table 3, the anti-Derlin-1 humanized antibody can significantly inhibit the growth of human Derlin-1 expression-positive colon cancer in vivo, and there is an obvious dose-dependent relationship. Preliminary toxicological studies on the blood phase analysis and body weight changes of experimental animals carried out at the same time as treatment showed that when the anti-Derlin-1 humanized antibody treats human colon cancer in vivo, the components of blood phase and the body weight of the mice were significantly different between the experimental group and the control group. There was no significant difference between them, proving that the anti-Derlin-1 humanized antibody has no obvious toxicity in vivo.

Table 3. Anti-Derlin-1 humanized antibody anti-tumor rate of human Derlin-1 expression positive colon cancer cell line LS180 tumor weight.

Claims (6)

1. A humanized monoclonal antibody, wherein the monoclonal antibody consists of a light chain containing three specific light chain complementarity determining regions and a heavy chain containing three heavy chain complementarity determining regions, wherein the three light chains The amino acid sequence of the complementarity determining region is shown in SEQ ID NO:1-3, the amino acid sequence of the three heavy chain complementarity determining regions is shown in SEQ ID NO:4-6, wherein the light chain amino acid sequence of the monoclonal antibody It is shown in SEQ ID NO:7, and the heavy chain amino acid sequence is shown in SEQ ID NO:8. 2. An isolated polynucleotide encoding the humanized monoclonal antibody of claim 1. 3. An expression vector comprising the polynucleotide according to claim 2. 4 . The expression vector according to claim 3 , which is pSRNC-Cκ-DERLIN-1 shown in FIG. 5 or pSRDC-Cγ1-DERLIN-1 shown in FIG. 6 . 5. A host cell containing the polynucleotide according to claim 2, or containing the expression vector according to claim 3 or 4. 6. The humanized monoclonal antibody described in claim 1 or the polynucleotide described in claim 2 or the expression vector described in claim 3 or 4 or the host cell described in claim 5 in the preparation of a therapeutically effective dose Use in anti-tumor drugs expressing DERLIN-1 positive on the cell surface.