CN102898524A - Human-mouse chimeric antibody of anti-human tumor necrosis factor related apoptosis-inducing ligand receptor DR5, preparation method and uses thereof - Google Patents

Abstract

The invention provides a human-mouse chimeric antibody against human tumor necrosis factor-related apoptosis-inducing ligand receptor DR5 and its preparation method and application. Specifically, the present invention relates to a human-mouse chimeric antibody against the extracellular region of the human tumor necrosis factor-related apoptosis-inducing ligand receptor, which consists of the variable region of the mouse antibody IgG and the constant region of the human antibody IgG. district composition. The present invention also relates to a stable cell line that stably expresses a human-mouse chimeric antibody against the extracellular region of human tumor necrosis factor-related apoptosis-inducing ligand receptor, and a recombinant eukaryotic expression vector that stably expresses the human-mouse chimeric antibody , the method for preparing the human-mouse chimeric antibody, the use of the human-mouse chimeric antibody and the composition thereof. The chimeric antibody of the present invention has good affinity to the extracellular region of DR5, has specific apoptosis-inducing activity on tumor cells, and can be used alone or in combination with other drugs for the treatment of various tumors and other DR5-related diseases .

Description

Human-mouse chimeric antibody against human tumor necrosis factor-related apoptosis-inducing ligand receptor DR5 and its preparation method and application

technical field

The present invention relates to a human-mouse chimeric antibody against human tumor necrosis factor-related apoptosis-inducing ligand (tumor necrosis factor-related apoptosis-inducing ligand, TRAIL) receptor (death receptor 5, DR5), and highly efficient expression of the antibody The eukaryotic expression vector, the stable cell line producing the antibody, the method for preparing the human-mouse chimeric antibody and the application of the human-mouse chimeric antibody and its composition. the

Background technique

Death receptor 5 (death receptor 5, DR5) is a receptor for tumor necrosis factor related apoptosis-inducing ligand (TRAIL), and its intracellular region contains a death domain. DR5 combined with the natural ligand TRAIL can effectively activate the apoptosis signal transduction pathway, resulting in cell death. Previous studies have shown that mouse-derived functional anti-DR5 monoclonal antibodies can induce the death of various tumor cells cultured in vitro, effectively inhibit tumor growth in vivo, and have no toxicity to normal tissues and cells (Guo Y et al., A novel anti-human DR5 monoclonal antibody with tumoricidal activity induces caspase-dependent and caspase-independent cell death J Biol Chem 2005;280(51):41940-52). However, the monoclonal antibody prepared by hybridoma technology is a heterologous immunoglobulin of mouse origin, which may cause human anti-mouse antibody (HAMA) reaction and weaken its therapeutic effect when used directly in the treatment of human tumors. and have harmful effects on the human body. the

Therefore, modifying the murine antibody to reduce its immunogenicity to the human body is a necessary way to successfully apply the antibody to pharmaceutical use. Common murine antibody engineering approaches include chimerization and humanization. Human-mouse chimeric antibody is the use of genetic engineering technology to link the variable region of murine antibody with the constant region of human antibody to construct a complete chimeric antibody, which can maintain the specificity and affinity of the original monoclonal antibody , reduce human anti-mouse antibody response, prolong its half-life and improve pharmacokinetic properties. the

The mammalian cell expression system can efficiently produce complete antibody molecules with correct folding, assembly, spatial conformation and good biological effects, and is the most ideal expression system for chimeric antibodies. The expression level of the chimeric antibody is very important. An ideal eukaryotic expression vector requires a strong promoter and an appropriate selection marker, which is not only conducive to the amplification of the gene itself, but also can be used as a screening of positive cells. the

Therefore, the technical problem of the present invention is to prepare anti-DR5 human-mouse chimeric antibody using chimeric antibody technology. the

Contents of the invention

Therefore, the technical purpose of the present invention is to provide a human-mouse chimeric antibody against human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor (DR5). the

Therefore, the first aspect of the present invention relates to a human-mouse chimeric antibody against the extracellular region of human tumor necrosis factor-related apoptosis-inducing ligand receptor, characterized in that the light chain and heavy chain of the chimeric antibody can be The amino acid sequence of the variable region includes the light chain variable region (VL) amino acid sequence shown in SEQ ID NO: 1 and the heavy chain variable region (VH) amino acid sequence shown in SEQ ID NO: 2, the chimeric antibody The constant region is that of a human antibody. the

Preferably, the heavy chain constant region of the chimeric antibody is a human IgG1 constant region, and the light chain constant region is a human κ chain. the

Preferably, the amino acid sequences of the light chain variable regions CDR1, CDR2 and CDR3 of the chimeric antibody are respectively:

Amino acids from position 24 to position 39 of CDR1: RSSQSLVHSNGNTYLH (SEQ ID NO: 3);

Amino acids from position 55 to position 61 of CDR2: KVSNRFS (SEQ ID NO: 4);

94 to 102 amino acids of CDR3: FQSTHVPHT (SEQ ID NO: 5); and/or

The amino acid sequences of the heavy chain variable regions CDR11, CDR22 and CDR33 of the chimeric antibody are respectively:

The 31st to 35th amino acids of CDR11: DFSMN (SEQ ID NO: 6);

Amino acids from position 55 to position 66 of CDR22: WINTETGEPTYADDFKG (SEQ ID NO: 7);

Amino acids 99 to 101 of CDR33: IDY (SEQ ID NO: 8). the

Most preferably, the chimeric antibody is expressed by a stable cell line with the preservation number CGMCC No. 4938 deposited in the General Microorganism Center of China Microbiology Culture Collection Management Committee on June 15, 2011, and named Zaptuximab. the

The second aspect of the present invention relates to a stable cell line stably expressing a human-mouse chimeric antibody against the extracellular region of the human tumor necrosis factor-related apoptosis-inducing ligand receptor, and the classification name is stable expression of anti-DR5 human-mouse chimeric antibody Antibody CHO cell strain was preserved in the General Microbiology Center (CGMCC) of China Microbiological Culture Collection Management Committee (CGMCC) on June 15, 2011, and the preservation number is CGMCC No.4938. the

The third aspect of the present invention relates to a recombinant eukaryotic expression vector that stably expresses the human-mouse chimeric antibody against the extracellular region of human tumor necrosis factor-related apoptosis-inducing ligand receptor as described in the first aspect of the present invention, It is characterized in that the recombinant eukaryotic expression vector has a CAG strong promoter and a dihydrofolate reductase screening marker, and the coding sequence of the heavy chain variable region and the light chain variable region of the monoclonal antibody from the mouse anti-human DR5, human The sequences of IgG1 heavy chain constant region and light chain constant region κ chain and the Furin/2A sequence with self-cleavage function added between the heavy chain and the light chain are operatively linked to efficiently express the human-mouse chimeric antibody , preferably, the starting plasmid of the recombinant eukaryotic expression vector is pCDNA3, preferably, the mouse monoclonal antibody against human DR5 is AD5-10 (Guo Y et al., A novel anti-human DR5 monoclonal antibody with tumoricidal activity induces caspase-dependent and caspase-independent cell death J Biol Chem 2005;280(51):41940-52). the

Preferably, the coding sequence of the heavy chain variable region and the light chain variable region of the monoclonal antibody derived from murine anti-human DR5, the sequence of the human IgG1 heavy chain constant region and the light chain constant region kappa chain and between the heavy chain and the light chain The concatenated sequence of the Furin/2A sequence with self-cleavage function added between them is shown in SEQ ID NO: 9. the

The fourth aspect of the present invention relates to a method for preparing the human-mouse chimeric antibody against the extracellular region of human tumor necrosis factor-related apoptosis-inducing ligand receptor as described in the first aspect of the present invention, which is characterized in that it comprises the following steps:

A) using the recombinant eukaryotic recombinant human-mouse chimeric antibody stably expressing the anti-human tumor necrosis factor-related apoptosis-inducing ligand receptor ectoregion as described in the third aspect of the present invention as described in the first aspect of the present invention The expression vector was transfected into CHO- ^dhfr- cells;

B) A cell line stably expressing the chimeric antibody was obtained by pressurized screening with methotrexate. the

A fifth aspect of the present invention relates to a stable cell line obtained by a method as described in the fourth aspect of the present invention. the

The sixth aspect of the present invention relates to a composition, the active ingredient of which is the human-mouse chimeric antibody against the extracellular region of human tumor necrosis factor-related apoptosis-inducing ligand receptor and chemotherapy as described in the first aspect of the present invention. Drug composition, preferably, the chemotherapy drug is TRAIL or epirubicin. the

The seventh aspect of the present invention relates to the human-mouse chimeric antibody against the extracellular region of human tumor necrosis factor-related apoptosis-inducing ligand receptor as described in the first aspect of the present invention, and the stable antibody as described in the third aspect of the present invention. A recombinant eukaryotic expression vector expressing the human-mouse chimeric antibody against the extracellular region of human tumor necrosis factor-related apoptosis-inducing ligand receptor according to the first aspect of the present invention or the composition according to the sixth aspect of the present invention Use in the preparation of medicaments for treating tumors, preferably, the tumors are leukemia, liver cancer, colon cancer or lung cancer. the

In other words, in order to accomplish the object of the present invention, the present invention provides a human-mouse chimeric antibody whose light chain and heavy chain variable region amino acid sequences include the amino acid of the light chain variable region (VL) of SEQ ID NO: 1 sequence and the amino acid sequence of the heavy chain variable region (VH) of SEQ ID NO:2. the

The amino acid sequences of the light chain variable regions CDRL1, CDRL2 and CDRL3 of the chimeric antibody are respectively:

CDRL1 (24 to 39 amino acids): RSSQSLVHSNGNTYLH (SEQ ID NO: 3),

CDRL2 (55 to 61 amino acids): KVSNRFS (SEQ ID NO: 4),

CDRL3 (94 to 102 amino acids): FQSTHVPHT (SEQ ID NO: 5),

The amino acid sequences of the heavy chain variable regions CDRH1, CDRH2 and CDRH3 of the chimeric antibody are respectively:

CDRH1 (31 to 35 amino acids): DFSMN (SEQ ID NO: 6),

CDRH2 (50 to 66 amino acids): WINTETGEPTYADDFKG (SEQ ID NO: 7),

CDRH3 (amino acids 99 to 101): IDY (SEQ ID NO: 8). the

The present invention also provides a preparation method of a human-mouse chimeric antibody against the extracellular region of DR5, the amino acid sequences of the light chain and heavy chain variable regions of the chimeric antibody include the light chain variable region of SEQ ID NO: 1 (VL) and the amino acid sequence of the heavy chain variable region (VH) of SEQ ID NO:2. The constant region of the light chain is human κ chain, and the subtype of the constant region of the heavy chain is human IgG1. The method includes the following steps:

Using the anti-human DR5 single-chain antibody (scFv) prokaryotic expression vector pET15b-AD5scFvDNA as a template, the heavy chain variable region and light chain variable region gene fragments of the anti-DR5 chimeric antibody were amplified by polymerase chain reaction (PCR) , respectively linked to the human IgG1 heavy chain constant region and κ chain, and then overlapped PCR to link the heavy chain and light chain of the human-mouse chimeric antibody, and added a self-cleaving function between the heavy chain and the light chain The Furin/2A sequence. the

Using pCDNA3 as the original vector, a new eukaryotic expression vector containing CAG promoter and dihydrofolate reductase (dhfr) selection marker was constructed, and the gene of the above human-mouse chimeric antibody was cloned into this vector to construct anti-DR5 human - A eukaryotic expression vector for a murine chimeric antibody. The new expression vector obtained by the present invention connects the heavy chain and light chain of the chimeric antibody in an open reading frame with the Furin/2A sequence with self-cutting function. the

The eukaryotic expression vector of anti-DR5 human-mouse chimeric antibody was transfected into CHO-dhfr ^- cells, and the cell line with stable and high expression of chimeric antibody was obtained by methotrexate (MTX) gradient pressure selection. It can express the heavy chain and light chain of the chimeric antibody in a balanced and matched manner at the same time, and automatically assemble into a functionally complete human-mouse chimeric antibody against human DR5, named Zaptuximab.

The stable CHO cell strain obtained by the method of the present invention has been preserved in the General Microorganism Center (CGMCC) of China Microbiological Culture Collection Management Committee (CGMCC) on June 15, 2011, and its classification name is stably expressing anti-DR5 human-mouse chimeric antibody CHO cell line, its number is: CGMCC No.4938. the

The stable CHO cell line of the present invention can produce human-mouse chimeric antibody, and the subtype of the chimeric antibody antibody immunoglobulin is IgG1. the

Both the anti-DR5 human-mouse chimeric antibody and the natural ligand TRAIL of DR5 can bind to DR5, but their binding sites are different. In vitro studies have shown that the anti-DR5 human-mouse chimeric antibody can kill various tumor cells such as leukemia, liver cancer, colon cancer and lung cancer, and the lethality is time- and dose-dependent. the

In vivo studies have shown that the anti-DR5 human-mouse chimeric antibody can strongly inhibit the growth of tumor cells such as human colon cancer, liver cancer and lung cancer in nude mice, and can be combined with epirubicin and other chemotherapy drugs to achieve better the therapeutic effect. There was no obvious rebound phenomenon after drug withdrawal, and histological analysis did not cause pathological changes in the liver, kidney and other organs of mice. the

The stable CHO cell line of the present invention produces the human-mouse chimeric antibody Zaptuximab which has a strong effect of killing tumor cells in vitro. The chimeric antibody has the synergistic effect of killing tumor cells with epirubicin or other chemotherapeutic drugs, and can inhibit the formation and growth of various human xenograft tumors in nude mice. the

The human-mouse chimeric antibody Zaptuximab produced by the stable cell line induces two different forms of cell death, apoptosis and autophagic death. the

The present invention also provides the use of the human-mouse chimeric antibody Zaptuximab in the preparation of drugs for treating tumors. the

The present invention further provides a human-mouse chimeric antibody and a humanized modified antibody thereof. the

Finally, the present invention also provides a human-mouse chimeric antibody or its at least 80% homology, preferably at least 85%, 90%, 95%, 96%, 97%, 98% or 99% homology The application of the recombinant expression vector obtained by recombining the homologous sequence of the gene and the pharmaceutically acceptable carrier in the preparation of gene therapy drugs for treating tumors. the

It can be seen that in the present invention, a eukaryotic expression vector stably expressing a human-mouse chimeric antibody against human DR5 was constructed by using modern biological techniques and methods such as genetic engineering, and then the vector was stably transfected into CHO-dhfr ^-The cells were pressurized and screened with methotrexate to obtain a stable cell line capable of expressing a new type of anti-DR5 human-mouse chimeric antibody Zaptuximab. In vitro, the chimeric antibody has the ability to strongly induce the apoptosis of various tumor cells; in nude mice, the chimeric antibody has the biological activity of significantly inhibiting human tumor cells from forming tumors and inhibiting the growth of tumor cells; the chimeric antibody It can be used in combination with other anticancer drugs to induce tumor cell death in vivo and in vitro, enhance the curative effect of chemotherapy drugs, and reduce the dose and side effects of chemotherapy drugs. Therefore, the human-mouse chimeric antibody is of great significance in the development of new antitumor drugs.

The advantages and effects of the present invention are to obtain a strain of anti-DR5 human-mouse chimeric antibody and its eukaryotic expression vector and stable expression cell which have not been reported before. The gene expression regulatory elements of the eukaryotic expression vector include strong promoter CAG promoter, dhfr selection marker and Furin/2A self-cleavage sequence. In the eukaryotic expression vector, the heavy chain and light chain of the chimeric antibody are Linked in an open reading frame, the heavy chain and light chain of the chimeric antibody can be expressed in a balanced and matched manner at the same time, and can be automatically assembled into a functionally complete chimeric antibody molecule. After stably transfecting CHO- ^dhfr- cells with the eukaryotic expression vector, a CHO cell strain stably expressing anti-DR5 chimeric antibody was obtained through MTX pressure selection.

The binding site of the human-mouse chimeric antibody to DR5 is different from that of TRAIL, and it can be used in combination with chemotherapy drugs such as TRAIL and epirubicin to synergistically enhance their anti-tumor effects such as liver cancer, lung cancer and colon cancer. It can induce apoptosis of various tumor cells in vitro, but has no toxic side effects on normal cells. It has a strong biological activity of inhibiting the growth of human liver cancer, lung cancer and colon cancer cells in vivo, and has no toxic side effects on the liver, kidney and other organs of normal mice. It shows that the chimeric antibody can be developed into a safe and effective anti-tumor drug. The chimeric antibody sequence or further humanized modified antibody sequence is recombined with a pharmaceutically acceptable carrier, and the obtained recombinant expression vector can be used for Preparation of gene therapy drugs for treating tumors. the

The anti-DR5 human-mouse chimeric antibody is a complete chimeric antibody constructed by linking the variable region of the murine anti-DR5 antibody with the constant region of the human antibody by genetic engineering technology, which can maintain the specificity of the original monoclonal antibody and On the basis of affinity, it can reduce human anti-mouse antibody response, prolong its half-life and improve pharmacokinetic properties, and can effectively activate complement and Fc receptor-related effector systems. the

The present invention connects the variable region of a murine functional anti-human DR5 monoclonal antibody prepared by us with the constant region of the human antibody to establish a human-mouse chimeric antibody, and stably express it in mammalian cells to obtain A cell line stably expressing the novel human-mouse chimeric antibody. The antibody has specific apoptosis-inducing activity on a variety of tumor cells in vivo and in vitro, and has no toxic and side effects on normal cells and tissues. It can be used alone or in combination with TRAIL or other chemotherapy drugs for a variety of tumors and other tumors with DR5 expression Treatment of disorders associated with abnormalities. the

Description of drawings

Figure 1: It is a new eukaryotic expression vector for highly expressing anti-DR5 human-mouse chimeric antibody with CAG promoter and dhfr selection marker. the

Figure 2: It is the detection result of the expression level of the chimeric antibody after the CHO cell line stably expressing the chimeric antibody was screened by MTX pressure. the

Figure 3: shows the binding of the chimeric antibody to its antigen human DR5. The figure shows that the chimeric antibody binds to DR5 in a typical single-site binding mode, K _d =1.1nM, and the data processing uses GraphPad software.

Figure 4: shows the activity of the chimeric antibody in killing tumor cells in vitro. Panel A shows the cell survival rate of human T lymphocytic leukemia Jurkat cells treated with different concentrations of the chimeric antibody for 24 hours; panel B shows the cell survival rate of human liver cancer SMMC-7721 cells treated with the chimeric antibody of different concentrations for 24 hours ; Figure C shows the cell survival rate of human colon cancer HCT116 cells treated with different concentrations of the chimeric antibody for 24 hours; Figure D shows the cell survival rate of the chimeric antibody of different concentrations treated human liver cancer BEL-7402 cells for 24 hours; Panel E shows the cell viability of human non-small cell lung cancer H460 cells treated with different concentrations of the chimeric antibody for 24 hours. Cell viability was determined using MTS kit (Promega, G1112). the

Figure 5: shows the activity of the chimeric antibody and its combination with chemotherapy drugs in inhibiting the formation and growth of human transplanted tumors in nude mice. Figure A shows that the chimeric antibody and its combined chemotherapy drugs inhibit the growth of human liver cancer BEL-7402 cells in nude mice; Figure B shows that the chimeric antibody and its combined chemotherapy drugs inhibit the formation of human colon cancer HCT116 cells in nude mice Tumor growth; Panel C shows that the chimeric antibody and its combination with chemotherapy drugs inhibit the growth of human non-small cell lung cancer H460 cells forming tumors in nude mice. EPB stands for the chemotherapy drug epirubicin. the

Figure 6: It shows the activity of the chimeric antibody to activate the caspase cascade and lead to the degradation of the caspase substrate PARP (polyADP-ribose polymerase) in human liver cancer, colon cancer and non-small cell lung cancer cells. the

Detailed ways

The present invention will be further illustrated by the following non-limiting examples below. It is well known to those skilled in the art that many modifications can be made to the present invention without departing from the spirit of the present invention, and such modifications also fall within the scope of the present invention. the

The following experimental methods are conventional methods unless otherwise specified, and the experimental materials used can be easily obtained from commercial companies unless otherwise specified. the

Example

Experimental Example 1 Construction of a new eukaryotic expression vector for highly expressing anti-DR5 human-mouse chimeric antibody with CAG promoter and dhfr selection marker

Anti-human DR5 single-chain antibody (scFv) prokaryotic expression vector pET15b-AD5scFv DNA (Guo Y et al., A novel anti-human DR5 monoclonal antibody with tumoricidal activity induces caspase-dependent and caspase-independent cell death J Biol Chem 2005; 280 (51): 41940-52) as a template, PCR amplification of the heavy chain variable region and light chain variable region gene fragments of the anti-DR5 chimeric antibody (VH primer, heavy chain: upstream primer VHE: 5'CGGAATTCCAGATCCAGTTG3'( SEQ ID NO: 10), downstream primer VHS: 5' ATGTGTCGACGCTGAGGAGACTGT3' (SEQ ID NO: 11). VL primer, light chain: upstream primer VLE: 5' GCGGAATTCGATGTTGTGATG3' (SEQ ID NO: 12), downstream primer VLS: 5 'ACGCGTCGACCCGTTTTTATTTC3' (SEQ ID NO: 13), was inserted into the vector (pCI-neo, Promega Co., E1841) with human IgG1 heavy chain constant region and κ chain (general sequence), and human-mouse chimeric Antibody heavy and light chains linked (primer 1:

5'CCGCTCGAGG ATCCACCATGGAGACAGACACA3' (SEQ ID NO: 14),

Primer 2:

5'CTTGAGAAGGTCAAAGTTCAGTGTTTGCTTTACAGGTGCTCGCCTCTTCCTTTTACCCGGAGACAGG3' (SEQ ID NO: 15),

Primer 3:

5'CTTTGACCTTTCCAAGTTGGCTGGAGACGTTGAGTCCAATCCTGGGCCCGAGACAGACACACTC3' (SEQ ID NO: 16),

Primer 4:

5'CCGAAGCTTCTAGATATCTTACTAGCACTTCCCCTGTTG3'(SEQ ID NO: 17)), thereby adding a Furin/2A sequence with self-cleavage function between the heavy chain and the light chain. With pCDNA3 (Invitrogen.A-150228) as the original vector, with the pAM-CAG vector (provided by the University of Hong Kong) as the template, with the upstream primer 5'GGCGCAGATCTATTGACGTCAAT3'((SEQ ID NO: 18) and the downstream primer 5'GGCAAGCTTAATTCTTTGCCAAAATG 3'( SEQ ID NO: 19) amplifies chicken actin promoter CAG, using pSV2-dhfr vector (purchased from ATCC, 67110) as template, with upstream primer 5'AATCCCGGGACAGCTCAGGGCTGCG3' (SEQ ID NO: 20) and downstream primer 5' GGCGGCGCTTCGAAAAAGCCAGCAAAAGCTC3' (SEQ ID NO: 21) amplifies the gene fragment of dihydrofolate reductase dhfr. The CAG, the complete antibody gene HF2AL (SEQ ID NO: 9) and the dhfr gene fragment are inserted into the original vector in sequence, and are identified correctly by sequencing, and obtained The eukaryotic expression vector pcDNA3-CAG-HF2AL-dhfr of the new anti-DR5 human-mouse chimeric antibody. The map of the resulting recombinant plasmid is shown in Figure 1.

Experimental Example 2 Establishment of CHO cell lines that efficiently and stably express chimeric antibodies

The constructed eukaryotic expression vector pcDNA3-CAG-HF2AL-dhfr was transfected into CHO- ^dhfr- cells (purchased from ATCC, CRL-9096) by liposome transfection method, and cultured without hypoxanthine and thymine ^IMDM (HyClone, SH30228.01B) was used to screen positive monoclonals ^to obtain cell lines stably expressing chimeric antibodies. Press screening to obtain a cell line stably expressing the chimeric antibody with significantly increased expression yield. The results are shown in Figure 2. The cell line was preserved in the General Microorganism Center (CGMCC) of the China Committee for Culture Collection of Microorganisms on June 15, 2011. Its classification name is CHO cell line stably expressing anti-DR5 human-mouse chimeric antibody, and its number is: CGMCC No. 4938. The chimeric antibody was named Zaptuximab.

Experimental example 3 Determination of specific binding of chimeric antibody to its antigen by enzyme-linked immunosorbent assay (ELISA)

Coat 96-well ELISA plates with recombinant human DR5 expressed in Escherichia coli, block with 5% skimmed milk powder, and add different concentrations (0, 7.8, 15.6, 31.2, 62.5, 125, 250, 500, 1000, 2000, 4000ng/ml) chimeric antibody or mouse-derived anti-DR5 antibody, incubated at 37°C for 2 hours. Horseradish peroxidase (HRP)-labeled goat anti-human/mouse IgG secondary antibody (Zhongshan Jinqiao) was added and incubated at 37°C for 2 hours. Chromogenic substrate was added, and after 30 minutes, 2 M sulfuric acid was added to stop the reaction, and the OD value was measured. The results are shown in Figure 3. The results showed that the binding of the chimeric antibody Zaptuximab to DR5 showed a typical single-site binding mode, Kd=1.1nM, and the affinity was not significantly lower than that of the mouse-derived anti-DR5 antibody AD5-10. the

Experimental example 4 MTS method to determine the lethality of chimeric antibody Zaptuximab on tumor cells in vitro

Human T lymphocytic leukemia Jurkat cells in logarithmic growth phase (ATCC, TIB-152), liver cancer SMMC-7721 cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, TCHu 52), colon cancer HCT116 cells (ATCC, CCL -247), liver cancer BEL-7402 cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, TCHu 10) and non-small cell lung cancer H460 cells (ATCC, HTB-177) and other tumor cells were added to 96-well cell culture plates, and added Chimeric antibodies with different concentrations (0, 0.13, 0.25, 0.5, 1, 2, 4, 8 μg/ml) were treated for 24 hours, then added with MTS reagent, reacted for 2-4 hours, and measured the OD value (wavelength 492nm). Take the OD value of the well without cells as the blank control "0". Cell viability = the ratio of the OD value of the treated wells to the OD value of the untreated wells. The results are shown in Figure 4. The results showed that the cell viability decreased significantly, indicating that the chimeric antibody Zaptuximab had a strong tumor-inhibiting effect. the

Experimental example 5 Anti-tumor activity of chimeric antibody Zaptuximab in vivo

Nude mice (BALB/c, Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.) were subcutaneously injected with 5×10 ⁶ cells of BEL-7402. The treatment group (n=6) was divided into a chimeric antibody treatment group with different doses, a chemotherapy drug epirubicin (EPB) treatment group and a chimeric antibody and chemotherapy drug combination treatment group, and the administration began on the 5th day after tumor cell inoculation. , to detect the activity of the chimeric antibody in inhibiting tumor growth. The negative control group (n=6) was given PBS and normal saline. The route of administration was intraperitoneal injection. After 29 days of administration, the nude mice were sacrificed, and the tumors were weighed.

The results are shown in Figure 5. The results showed that the chimeric antibody Zaptuximab could significantly inhibit the growth of transplanted tumors of human liver cancer BEL-7402 cells, colon cancer HCT116 cells and non-small cell lung cancer H460 cells in a dose-dependent manner. The anti-tumor activity of the chimeric antibody Zaptuximab combined with the chemotherapy drug Epirubicin was further enhanced. the

Experimental example 6 Western blot detection chimeric antibody Zaptuximab activates caspase cascade and causes degradation of its substrate PARP (poly ADP-ribose polymerase)

BEL-7402, HCT116 and H460 cells in the logarithmic growth phase were treated with 100 ng/ml chimeric antibody Zaptuximab for 0, 4 and 8 hours, the cells were collected, lysed, and subjected to 12% SDS-polyacrylamide gel electrophoresis. The protein on the gel was transferred to PVDF membrane, and the primary antibodies of caspase-3, caspase-8 and PARP (Cell Signaling Technology Co.) were added for hybridization. Add the corresponding horseradish peroxidase-labeled secondary antibody (HRP-goat anti-rabbit or mouse IgG, Zhongshan Jinqiao Company), and add a luminescent substrate for development. the

Western blot results showed that caspase-8, caspase-3, and PARP were all degraded in a time-dependent manner, proving that the chimeric antibody Zaptuximab can activate the caspase cascade and cause the degradation of its substrate PARP (Figure 6). the

Claims (11)

1. people-the mouse chimeric antibody of an anti-human tumor necrosin relative death inducing ligand acceptor extracellular region, it is characterized in that the light chain of described chimeric antibody and the aminoacid sequence of variable region of heavy chain comprise the weight chain variable region amino acid sequence shown in the light chain variable region amino acid sequence shown in the SEQ ID NO:1 and the SEQ ID NO:2, the constant region of the constant region behaviour antibody of described chimeric antibody.

2. people-the mouse chimeric antibody of anti-human tumor necrosin relative death inducing ligand acceptor extracellular region according to claim 1 is characterized in that the CH of described chimeric antibody is human IgG1's constant region, constant region of light chain behaviour κ chain.

3. people-the mouse chimeric antibody of anti-human tumor necrosin relative death inducing ligand acceptor extracellular region according to claim 1 and 2 is characterized in that variable region of light chain CDR1, the CDR2 of described chimeric antibody and the aminoacid sequence of CDR3 are respectively:

24 of CDR1 to 39 amino acids: shown in SEQ ID NO:3;

55 of CDR2 to 61 amino acids: shown in SEQ ID NO:4;

94 of CDR3 to 102 amino acids: shown in SEQ ID NO:5; And/or

Variable region of heavy chain CDR11, the CDR22 of described chimeric antibody and the aminoacid sequence of CDR33 are respectively:

31 of CDR11 to 35 amino acids: shown in SEQ ID NO:6;

55 of CDR22 to 66 amino acids: shown in SEQ ID NO:7;

99 of CDR33 to 101 amino acids: shown in SEQ ID NO:8.

4. according to claim 1 to the people-mouse chimeric antibody of 3 each described anti-human tumor necrosin relative death inducing ligand acceptor extracellular regions, it is characterized in that described chimeric antibody is the stable cell line expression of CGMCC No.4938 by the preserving number that was deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 15th, 2011, called after Zaptuximab.

5. the stable cell lines of the people-mouse chimeric antibody of the anti-human tumor necrosin relative death inducing ligand acceptor of strain stably express extracellular region, its specific name is the anti-DR5 people of stably express-mouse chimeric antibody Chinese hamster ovary celI strain, be deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on June 15th, 2011, preserving number is CGMCC No.4938.

6. the recombinant eukaryon expression vector of the people-mouse chimeric antibody of each described anti-human tumor necrosin relative death inducing ligand acceptor extracellular region of stably express claim 1 to 4, it is characterized in that described recombinant eukaryon expression vector is with CAG strong promoter and Tetrahydrofolate dehydrogenase selection markers, from the monoclonal antibody variable region of heavy chain of mouse-anti people DR5 and the encoding sequence of variable region of light chain, the sequence of human IgG1's CH and constant region of light chain κ chain and the Furin/2A sequence with self splicing function that adds between heavy chain and light chain are effectively connected to efficiently express described people-mouse chimeric antibody, preferably, the initial plasmid of described recombinant eukaryon expression vector is pCDNA3, preferably, the monoclonal antibody of described mouse-anti people DR5 is AD5-10.

7. the recombinant eukaryon expression vector of the people-mouse chimeric antibody of each described anti-human tumor necrosin relative death inducing ligand acceptor extracellular region of stably express claim 1 to 4 according to claim 6 is characterized in that the sequence that couples together from the sequence of encoding sequence, human IgG1's CH and the constant region of light chain κ chain of the monoclonal antibody variable region of heavy chain of mouse-anti people DR5 and variable region of light chain and the Furin/2A sequence with self splicing function that adds is shown in SEQ ID NO:9 between heavy chain and light chain.

8. method for preparing such as the people-mouse chimeric antibody of each described anti-human tumor necrosin relative death inducing ligand acceptor extracellular region of claim 1 to 4 is characterized in that may further comprise the steps:

A) usefulness is such as the recombinant eukaryon expression vector transfection CHO-dhfr of the people-mouse chimeric antibody of claim 6 or 7 each described anti-human tumor necrosin relative death inducing ligand acceptor extracellular regions of described stably express claim 1 to 4 ^-Cell;

B) utilize methotrexate pressurization screening to obtain the cell strain of stably express chimeric antibody.

9. stable cell line that method as claimed in claim 8 obtains.

10. composition, its activeconstituents is to form such as the people-mouse chimeric antibody of each described anti-human tumor necrosin relative death inducing ligand acceptor extracellular region of claim 1 to 4 and chemotherapeutics, preferably, described chemotherapeutics is TRAIL or epirubicin.

11. such as the people-mouse chimeric antibody of each described anti-human tumor necrosin relative death inducing ligand acceptor extracellular region of claim 1 to 4, such as recombinant eukaryon expression vector or the purposes of composition according to claim 10 in the medicine of preparation treatment tumour of the people-mouse chimeric antibody of claim 6 or 7 each described anti-human tumor necrosin relative death inducing ligand acceptor extracellular regions of described stably express claim 1 to 4, preferably, described tumour is leukemia, liver cancer, colorectal carcinoma or lung cancer.

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