CN109810194B - anti-DR 5 antibody and preparation method and application thereof - Google Patents

Abstract

The invention particularly relates to an anti-DR 5 antibody and a preparation method and application thereof. An anti-DR 5 antibody comprises a constant region and a variable region comprising a heavy chain variable region comprising the following 3 CDRs: CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3; the light chain variable region comprises the following 3 CDRs: CDR1 ' shown in SEQ ID NO. 4, CDR2 ' shown in SEQ ID NO. 5, and CDR3 ' shown in SEQ ID NO. 6. The antibody has unique antibody variable region sequence, can be specifically combined with an antigen DR5, has no cross reaction with DR4, has high affinity and biological activity, can induce apoptosis without adding a cross-linking agent, and can be combined with TRAIL to synergistically induce apoptosis.

Description

anti-DR 5 antibody and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an anti-DR 5 antibody and a preparation method and application thereof.

Background

According to the research of the world health organization, the cancer mortality rate is increased from 12% to 15% from 1990 to 2013, and the cancer mortality rate is high mortality disease second to cardiovascular disease, and the worldwide new cancer cases are predicted to reach 2000 ten thousand in 2020 and die for 1200 ten thousand people due to cancer; therefore, there is a great need for the development of new cancer therapeutic drugs.

Induction of cancer cell apoptosis is one of the many cancer therapies, and Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can bind to Death Receptor (DR) to induce cancer cell apoptosis without damaging normal tissues, so TRAIL-DR becomes a hot target for development of anti-Tumor antibody drugs. TRAIL, also known as Apo2L, was first discovered by Wiley in 1995 and is an apoptotic molecule of the third TNF family following TNF, FasL. TRAIL is a type II transmembrane protein, the extracellular region of which can be cut by metalloprotease to form soluble TRAIL, which is widely expressed in various tissues of normal human body and acts by specifically binding with the receptor on the surface of target cell. Currently, 5 TRAIL receptors have been found in humans: (ii) 2 death receptors DR4(death receptor 4) and DR5(death receptor 5); 2 decoy receptors DcR1(decoy receptor) and DcR2(decoy receptor 2); (iii) 1 soluble receptor OPG (osteoprotegerin). Among them, the intracellular domains of DR4 and DR5 possess complete death domains and are capable of inducing apoptosis, while DcR1 has no death domain, DcR2 has only a truncated death domain and is unable to transmit apoptosis signals, and OPG is the only soluble protein in these five receptors and is mainly involved in regulation of bone density. TRAIL binds to DR5 with higher affinity than other membrane-expressed TRAIL receptors at 37 ℃; thus, TRAIL is more likely to bind to DR5 under physiological conditions, especially when endogenous TRAIL is limited. DR5 is highly expressed in human cancers including colon, stomach, pancreas, ovary, breast, and non-small cell lung cancers, and is not or is not low expressed in normal cells. After the death receptor DR4 or DR5 is combined with TRAIL, a ligand/receptor trimerization complex is formed, the death receptor cytoplasmic Death Domain (DD) is combined with the C terminal DD of a Fas-associated death domain protein (FADD), and FADD is combined with procaspase-8 by an N-terminal death effector domain (DEDD) to form a DR4/DR5/FADD/procaspase-8 Death Inducing Signal Complex (DISC), so that procaspase-8 is promoted to be catalyzed into active caspase-8, and after the caspase-8 is activated, an apoptosis signal is transmitted through 2 signal pathways (a mitochondrion-independent pathway and a mitochondrion-dependent pathway).

TRAIL can selectively kill tumor cells, so that a plurality of TRAIL receptor antagonists enter clinical development, and the TRAIL receptor antagonists are mainly classified into 2 types: 1) a recombinant TRAIL protein; 2) an antibody that antagonizes DR4 or DR 5. However, none of these drugs currently benefits clinical cancer patients. The main drawback of recombinant TRAIL proteins is poor drug stability and targeting, and other forms of recombinant TRAIL protein show hepatotoxicity at high doses. The main reasons for the failure of TRAIL receptor antagonists in clinical trials are: 1) insufficient antagonistic activity of the drug candidate; 2) many primary cancer cells are resistant to single antibody therapy; 3) lack of suitable biomarkers to identify susceptibility of patients to TRAIL receptor antagonists. Monoclonal antibodies targeting DR5 that have currently entered clinical studies are: lexatumumab, Drozitumab, Conatumumab, Tigatuzumab, LBY135, etc., whereas many DR5 antibodies (e.g., Drozitumab and Tigatuzumab) require the addition of additional cross-linking agents (cross-linking agents) to achieve optimal activity in vitro.

Disclosure of Invention

The invention aims to overcome the defects in the prior art, provides an anti-DR 5 antibody, and a preparation method and application thereof, and aims to solve the technical problems that the antagonistic activity of the existing TRAIL receptor antagonist anti-tumor drugs is insufficient, and a cross-linking agent needs to be added to induce apoptosis.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the present invention provides an antibody variable region comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following 3 complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3;

the light chain variable region comprises the following 3 complementarity determining regions CDRs:

CDR1 ' shown in SEQ ID NO. 4, CDR2 ' shown in SEQ ID NO. 5, and CDR3 ' shown in SEQ ID NO. 6.

Further, the heavy chain variable region comprises an amino acid sequence shown as SEQ ID NO.7, or an amino acid sequence with the same function obtained by deletion, insertion or substitution of the amino acid sequence shown as SEQ ID NO. 7; and/or

The light chain variable region contains an amino acid sequence shown as SEQ ID NO.8, or an amino acid sequence with the same function obtained by deletion, insertion or substitution of the amino acid sequence shown as SEQ ID NO. 8.

In another aspect, the invention also provides an antibody comprising a constant region as described above, said constant region comprising a heavy chain constant region and a light chain constant region.

In another aspect, the present invention also provides a recombinant protein comprising a sequence having the above-described variable region; and/or

Sequences comprising the above antibodies, and tag sequences to facilitate expression and/or purification.

In another aspect, the present invention also provides a nucleic acid molecule encoding the variable region described above; and/or

Encoding the above antibody; and/or

Encoding the recombinant protein.

Further, the nucleotide sequence encoding the heavy chain variable region is shown as SEQ ID NO.9, or the nucleotide sequence with the same function is obtained by deletion, insertion or substitution of the nucleotide sequence shown as SEQ ID NO. 9; and/or

The nucleotide sequence for coding the light chain variable region is shown as SEQ ID NO.10, or the nucleotide sequence with the same function is obtained by deletion, insertion or substitution of the nucleotide sequence shown as SEQ ID NO. 10.

In another aspect, the present invention also provides a vector having the above-described nucleic acid molecule.

In another aspect, the present invention also provides a genetically engineered host cell comprising the vector described above; and/or

The host cell has integrated into its genome the nucleic acid molecule described above.

In another aspect, the invention also provides an immunoconjugate comprising the variable region described above; and/or

Comprises the above-mentioned antibody; and/or

Contains the recombinant protein and at least one of detectable marker, medicine, toxin, cytokine, radionuclide and enzyme.

In another aspect, the present invention also provides a pharmaceutical composition comprising the above variable region; and/or

Comprises the above-mentioned antibody; and/or

Contains the recombinant protein; and/or

Comprises the immune conjugate and a pharmaceutically acceptable carrier.

In another aspect, the invention further provides an application of the variable region, the antibody, the recombinant protein and the immunoconjugate in preparing a tumor drug for resisting DR5 protein, or preparing a reagent and/or a kit for detecting DR5 protein.

Finally, the invention relates to a preparation method of the antibody or the recombinant protein, which comprises the steps of culturing host cells or producing the antibody or the recombinant protein by using ascites of mice by a hybridoma method.

The variable region of the antibody provided by the invention contains six special Complementarity Determining Regions (CDRs) (shown in SEQ ID NO: 1-6), can specifically recognize the antigen DR5, has high affinity and biological activity, and can induce apoptosis.

The antibody provided by the invention has the unique antibody variable region sequence, can be combined with an antigen DR5, has high affinity and biological activity, and can induce apoptosis without adding a cross-linking agent; and the specific recognition epitope of the polypeptide and the antigen DR5 is different from the binding site of TRAIL and DR5, and the polypeptide can be combined with the TRAIL to synergistically induce apoptosis.

The antibody provided by the invention has wide application, can be used as an antitumor drug (the tumor expresses DR5 protein) alone, can be used together with TRAIL, and can also be used together with common tumor radiotherapy drugs or chemotherapy drugs, or a gene engineering antibody generated on the basis is used as a targeting part and is coupled with radionuclide or chemical drugs or toxins. In addition, the antibody is used for preparing a reagent and/or a kit for detecting DR5 protein, such as a related kit in the fields of ELISA detection, Western blot detection, flow cytometry detection and the like, and the sensitivity of the antibody is obviously superior to that of the prior art.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis of the 3E73F11 antibody in example 1 of the present invention;

FIG. 2 is a diagram showing the electrophoresis of the amplified heavy and light chain variable regions of the 3E73F11 antibody in example 1; wherein, Y is a negative control, M is Marker, 1 is the heavy chain of the 3E73F11 antibody, and 2 is the light chain of the 3E73F11 antibody;

FIG. 3 is a diagram showing the subtype identification of the 3E73F11 antibody in example 2 of the present invention;

FIG. 4 is an affinity assay graph for the 3E73F11 antibody in example 3 of the invention;

FIG. 5 is a graph showing the results of the 3E73F11 antibody inducing apoptosis of HepaRG cells in the absence of cross-linking agent in example 4 of the present invention;

FIG. 6 is a graph showing the results of the 3E73F11 antibody inducing apoptosis of HepaRG cells in the presence of a cross-linking agent in example 4 of the present invention;

FIG. 7 is a graph showing the results of using the 3E73F11 antibody in flow detection of jurkat cells in example 5 of the present invention;

FIG. 8 is a graph showing the results of ELISA detection using the 3E73F11 antibody in example 6 of the present invention;

FIG. 9 is a graph showing the results of Western blot detection using the 3E73F11 antibody in example 7 of the present invention;

FIG. 10 is a graph showing the results of the synergistic induction of HepaRG apoptosis by the 3E73F11 antibody and TRAIL in example 8 of the present invention;

FIG. 11 is a graph showing the results of detecting the concentration of DR5 in human serum using the 3E73F11 antibody in example 9 of the present invention.

FIG. 12 is a graph showing the results of no cross-reaction between the 3E73F11 antibody and human DR4 in example 10 of the present invention.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

In one aspect, the present embodiments provide a variable region of an antibody, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the following 3 complementarity determining regions CDR: CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3;

the light chain variable region comprises the following 3 complementarity determining regions CDRs: CDR1 ' shown in SEQ ID NO. 4, CDR2 ' shown in SEQ ID NO. 5, and CDR3 ' shown in SEQ ID NO. 6.

That is, the CDR amino acid sequence of the heavy chain variable region is:

CDR1(SEQ ID NO.1)：GFDFSTCW；

CDR2(SEQ ID NO.2)：INPDSSRI；

CDR3(SEQ ID NO.3)：ARGGTFYAMDY；

the CDR amino acid sequence of the light chain variable region is:

CDR1’(SEQ ID NO.4)：QSLLNSRTRKNY；

CDR2’(SEQ ID NO.5)：WAS；

CDR3’(SEQ ID NO.6)：KQSYNLPFT。

the CDRs, also known as complementary determining regions (complementary determining regions), are spatially structurally closely complementary to the antigenic determinants, and are the regions of the antibody that recognize and bind to the antigen, directly determining the specificity of the antibody. The variable region of the antibody provided by the invention contains six special Complementarity Determining Regions (CDRs), can specifically recognize an antigen DR5, has high affinity and biological activity, and can induce apoptosis.

Further, in one embodiment of the present invention, the heavy chain variable region comprises an amino acid sequence shown in SEQ ID NO.7, or an amino acid sequence with the same function obtained by deletion, insertion or substitution of the amino acid sequence shown in SEQ ID NO. 7; and/or the light chain variable region contains an amino acid sequence shown as SEQ ID NO.8, or an amino acid sequence with the same function obtained by deletion, insertion or substitution of the amino acid sequence shown as SEQ ID NO. 8.

Heavy chain variable region amino acid sequence (SEQ ID NO. 7):

EVKLLESGGGLVQPGGSLKLSCAASGFDFSTCWMNWVRQAPGKGLEWIGEINPDSSRINYMPSLKEKFIISRDNAKNTLYLQMSKVRSEDTALYYCARGGTFYAMDYWGQGTSVTVSS。

light chain variable region amino acid sequence (SEQ ID NO. 8):

DIVMTQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLPFTFGAGTKLELK。

in particular, there may be mentioned as alternatives amino acid sequences which are at least 80% homologous to the published sequences of SEQ ID NO.7 or SEQ ID NO. 8.

In another aspect, the embodiments also provide an antibody comprising a constant region comprising a heavy chain constant region and a light chain constant region, and the variable region described above.

In another aspect, the embodiments of the present invention also provide a recombinant protein, wherein the recombinant protein comprises a sequence having the above-mentioned variable region; and/or sequences comprising the above antibodies, and tag sequences to facilitate expression and/or purification.

The antibody or the recombinant protein provided by the embodiment of the invention has the unique antibody variable region sequence, can be combined with an antigen DR5, has high affinity and biological activity, and can induce apoptosis without adding a cross-linking agent; and the specific recognition epitope of the polypeptide and the antigen DR5 is different from the binding site of TRAIL and DR5, and the polypeptide can be combined with the TRAIL to synergistically induce apoptosis.

In another aspect, the embodiments provide a nucleic acid molecule encoding the above variable region of the embodiments; and/or encodes the above antibody; and/or encode the above recombinant proteins.

Further, in a preferred embodiment, the nucleotide sequence encoding the amino acid in the heavy chain variable region is shown in SEQ ID NO.9, or the nucleotide sequence with the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown in SEQ ID NO. 9; and/or a nucleotide sequence with the same function obtained by deleting, inserting or replacing the nucleotide sequence shown in SEQ ID NO.10 or the nucleotide sequence shown in SEQ ID NO. 10.

A nucleotide sequence encoding the heavy chain variable region (SEQ ID NO. 9):

GAAGTGAAGCTTCTCGAGTCTGGAGGTGGCCTGGTGCAGCCTGGAGGATCCCTGAAACTCTCCTGTGCAGCCTCAGGATTCGATTTTAGTACATGCTGGATGAATTGGGTCCGGCAGGCTCCAGGGAAAGGACTAGAATGGATTGGAGAAATTAATCCAGATAGTAGTAGGATAAATTATATGCCATCTCTAAAGGAAAAATTCATCATCTCCAGAGACAACGCCAAAAATACACTGTACCTGCAAATGAGCAAAGTGAGATCTGAAGACACAGCCCTTTATTACTGTGCAAGGGGAGGAACTTTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA。

the nucleotide sequence encoding the heavy chain CDR1 is (SEQ ID NO. 11):

GGATTCGATTTTAGTACATGCTGG；

the nucleotide sequence encoding the heavy chain CDR2 is (SEQ ID NO. 12):

ATTAATCCAGATAGTAGTAGGATA；

the nucleotide sequence encoding the heavy chain CDR3 is (SEQ ID NO. 13):

GCAAGGGGAGGAACTTTCTATGCTATGGACTAC。

nucleotide sequence encoding the variable region of the light chain (SEQ ID NO. 10):

GACATTGTGATGACACAATCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGGAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTTCCGTTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA。

the nucleotide sequence encoding the light chain CDR 1' is (SEQ ID NO. 14):

CAGAGTCTGCTCAACAGTAGAACCCGGAAGAACTAC；

the nucleotide sequence encoding the light chain CDR 2' is (SEQ ID NO. 15):

TGGGCATCC；

the nucleotide sequence encoding the light chain CDR 3' is (SEQ ID NO. 16):

AAGCAATCTTATAATCTTCCGTTCACG。

the nucleic acid molecules provided in the embodiments of the present invention may be sequences that are different from the nucleotide sequences disclosed in the embodiments of the present invention but encode the same protein due to the complementary sequence of the nucleotide sequences or due to the degeneracy of the genetic code; alternatively, the nucleotide sequence disclosed in the examples of the present invention may be at least 80% homologous thereto.

In another aspect, the embodiments of the present invention provide a vector having the above-described nucleic acid molecule. The recombinant vector can be used for expressing an antibody or a recombinant protein containing the heavy chain variable region and the light chain variable region.

In another aspect, the embodiments of the present invention also provide an immunoconjugate comprising the variable region described above; and/or comprising the above antibody; and/or contains the recombinant protein and at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide and an enzyme.

In another aspect, the embodiments of the present invention further provide a pharmaceutical composition, which contains the above-mentioned variable region; and/or comprising the above antibody; and/or comprising the above recombinant protein; and/or comprises the immunoconjugate and a pharmaceutically acceptable carrier.

On the other hand, the embodiment of the invention also provides an application of the variable region, the antibody, the recombinant protein and the immunoconjugate in the embodiment of the invention in preparing a tumor medicament for resisting the DR5 protein or preparing a reagent and/or a kit for detecting the DR5 protein.

The antibody or recombinant protein provided by the embodiment of the invention has wide application, can be used as an antitumor drug (the tumor expresses DR5 protein) alone, can be combined with TRAIL for use, and can also be combined with common tumor radiotherapy drugs or chemotherapy drugs, or can be used as a targeting part for coupling with radionuclide or chemical drugs or toxins by using a gene engineering antibody generated on the basis. Further, in a preferred embodiment, the invention discloses the killing effect of the monoclonal antibody against human DR5 on liver cancer cell lines, and can be used for treating various other tumor types with DR5 expression. In addition, the reagent and/or the kit for detecting the DR5 protein is a related kit in the fields of ELISA detection, Western blot detection, flow cytometry detection and the like, and the sensitivity of the reagent and/or the kit is obviously superior to that of the prior art.

The embodiment of the invention provides the variable region amino acid sequence and the nucleotide sequence of the anti-human DR5 monoclonal antibody, on the basis, the recombinant vector or the host cell can be used for producing the genetic engineering antibody, and the heavy chain and light chain variable region sequences contained in the monoclonal antibody are consistent with the variable region sequences disclosed by the embodiment of the invention. Such engineered antibodies include, but are not limited to: the functional fragment Fab of the antibody is a single-chain antibody, or an antibody functional fragment VH-L formed by fusing a heavy chain variable region and a complete light chain, or an antibody functional fragment formed by arranging, connecting in series or combining one or more CDRs of a heavy chain and a light chain, or an antibody-like functional fusion protein formed by connecting, splicing and fusing the antibody, the antibody functional fragment and other various proteins or polypeptides.

Finally, the invention relates to a preparation method of the antibody or the recombinant protein, which comprises the steps of culturing host cells or producing the antibody or the recombinant protein by using ascites of mice by a hybridoma method.

In a preferred embodiment, the preparation method uses mouse ascites to produce monoclonal antibody; or the monoclonal antibody is produced by culturing hybridoma cells, CHO cells or 293F cells. In a preferred embodiment of the present invention, the variable region amino acid sequence and nucleotide sequence of mouse anti-human DR5 monoclonal antibody are provided, and a human mouse chimeric antibody and a humanized antibody of anti-human DR5 can be obtained by antibody humanization modification. The human-mouse chimeric antibody of anti-human DR5 is produced by connecting the variable region of the mouse monoclonal antibody gene and the human constant region, and then expressing them in mammalian cells; in addition to the constant region of the antibody being converted to human origin, the FR region of the variable region is further converted to human origin, thereby reducing immunogenicity of the humanized antibody against human DR 5. The human-mouse chimeric antibody and humanized antibody of anti-human DR5 generated by the method also have the effect of binding human DR5 molecule to induce cancer cell apoptosis so as to treat tumors.

The invention is described in further detail with reference to a part of the test results, which are described in detail below with reference to specific examples.

Example 1

Preparation of monoclonal antibody against DR5 in this example, monoclonal antibody against DR5 was produced using mouse ascites by hybridoma method.

Female BALB/c mice (6 weeks old) are immunized by using an sDR5-Fc protein antigen (provided by Shenzhen Zhongke El medicine Co., Ltd., preparation method reference patent 201610067931.2), the first immunization uses Freund's complete adjuvant to emulsify the antigen, the second immunization starts, uses Freund's incomplete adjuvant to emulsify the antigen, the subcutaneous injection is carried out at 5-6 points, and the amount of the antigen injected by each mouse is about 100 ug. 10 days after the 3 rd immunization, a small amount of blood is collected from the tail of the mice for serum titer ELISA detection, and the mice with high antibody titer (>1:100000) are selected for the 4 th immunization, and about 100ug of antigen protein is injected into each mouse by intraperitoneal injection. 3-5 days after 4 th immunization, the splenocytes of the killed mice are taken to be fused with SP2/0 cells, and the stable hybridoma cells are obtained by HAT culture medium culture. Hybridoma capable of secreting DR5 antibody is obtained by screening through ELISA method, subcloning is carried out through limiting dilution method, monoclonal hybridoma cell strain capable of secreting DR5 antibody (in the specification, the anti-DR 5 monoclonal antibody is named as 3E73F11 antibody all together) is obtained by screening through ELISA method, and the strain is preserved through stage-by-stage amplification culture and liquid nitrogen freezing.

Preparing an ascites antibody: female BALB/c mice (8 weeks old) were intraperitoneally injected with Freund's incomplete adjuvant, 0.5ml of each mouse was injected, 3-5 days later, the hybridoma cells in logarithmic phase were intraperitoneally injected, and 1-5 × 10 cells were injected into each mouse⁵Individual cells (0.5ml), and mice were sacrificed about 11 days after injection of hybridoma cells to obtain ascites. Centrifuging at 3000rpm and 4 deg.C for 10min, removing precipitate, diluting ascites with 10 times volume of 1 XPB solution, mixing, and filtering with 0.45 μm filter membrane. Ascites fluid was affinity-purified by Protein G (Protein GSsepharose 4Fast Flow, GE Healthcare) to obtain a purified DR5 antibody Protein, and the antibody concentration was measured by BCA method. Purified antibody was run on SDS-PAGE (5.4 ug loading), and stained with Coomassie Brilliant blueColor, results are shown in FIG. 1: the 3E73F11 antibody contained two bands, one heavy and one light chain.

Antibody gene variable region sequencing of DR5 monoclonal antibody hybridoma cells: harvesting monoclonal antibody hybridoma cells in logarithmic growth phase, carrying out TRIZOL lysis to carry out RNA extraction, carrying out reverse transcription to obtain cDNA, amplifying and obtaining DNA sequences of heavy chain and light chain variable regions (an electrophoresis chart is shown in figure 2), removing non-functional VK genes, cloning to a pMD18-T vector, carrying out sequencing, and carrying out sequencing result comparison by using an IMGT/V-QUEST database; the sequencing results and analysis are as follows.

The nucleotide sequence for coding the heavy chain variable region is SEQ ID NO. 9;

the nucleotide sequence of the coding light chain variable region is SEQ ID NO. 10;

heavy chain variable region amino acid sequence SEQ ID No. 7: the variable region amino acid sequence of light chain is SEQ ID NO. 8.

By comparison with the IMGT/V-QUEST database, the CDR amino acid sequence of the heavy chain variable region is:

CDR1-IMGT：SEQ ID NO.1；

CDR2-IMGT：SEQ ID NO.2；

CDR3-IMGT：SEQ ID NO.3；

the amino acid sequence of the FR region of the heavy chain is:

FR1-IMGT：EVKLLESGGGLVQPGGSLKLSCAAS；

FR2-IMGT：MNWVRQAPGKGLEWIGE；

FR3-IMGT：NYMPSLKEKFIISRDNAKNTLYLQMSKVRSEDTALYYC；

FR4-IMGT：WGQGTSVTVSS。

the CDR amino acid sequence of the light chain variable region is:

CDR1’-IMGT：SEQ ID NO.4；

CDR2’-IMGT：SEQ ID NO.5；

CDR3’-IMGT：SEQ ID NO.6；

the amino acid sequence of the FR region of the light chain is:

FR1’-IMGT：DIVMTQSPSSLAVSAGEKVTMSCKSS；

FR2’-IMGT：LAWYQQKPGQSPKLLIY；

FR3’-IMGT：TRESGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYC；

FR4’-IMGT：FGAGTKLELK。

example 2

Subtype identification of anti-DR 5 monoclonal antibodies.

The monoclonal antibody subtype identification was performed using a commercial mouse monoclonal antibody Isotyping kit (sino biological, SEK003), and the results are shown in fig. 3, from which: the 3E73F11 antibody is an IgG1 type antibody.

Example 3

Affinity assay for monoclonal antibodies against DR 5.

The sensor was immobilized using 10ug/ml of sDR5-Fc protein (available from Shenzhen, Zhongke El deep medicine Co., Ltd.), 3E73F11 antibodies (566.7nM, 283.3nM, 141.7nM, 70.8nM, 35.4nM, 17.7nM, 8.84nM) were formulated at different concentrations using SD buffer (PBS + 0.02% Tween 20+ 0.1% BSA) as mobile phase, and affinity assays were performed using an intermolecular interactor (OCTET K2, PALL life science) programmed as follows: baseline 240s, Loading 360s, Baseline 2180 s, Association 480s, Dissociation 480s, using AHC (Anti-hIgG Fc Capture) sensors. The results are shown in FIG. 4, which shows: the 3E73F11 antibody had an affinity for DR5 of 3.55E-09(M), i.e., the 3E73F11 antibody had a high affinity for DR 5.

Example 4

In vitro cell function assay of monoclonal antibodies against DR 5.

1. Collecting HepaRG cells in logarithmic growth phase, digesting with pancreatin, stopping with 10% FBS-containing culture medium, blowing off, counting, and adjusting density to 8 × 10⁴Perml, add to a 96 well cell culture plate at 100 ul/well, i.e., 8000 cells per well. The cells were co-inoculated in 6X 10-60 wells, and the surrounding 36 wells were liquid-sealed with 200ul PBS per well to prevent evaporation of the medium. And placing the cells in an incubator for culturing for 22-24 h.

2. 10ml of culture solution is taken and added with 2ul of actinomycete D with the concentration of actinomycin D being 1 ug/ml.

3. Antibody dilution:

case without addition of cross-linking agent: the 3E73F11 antibody was diluted to 40ug/ml with a diluent, starting from this concentration, and diluted in 15 concentration gradients of 2 fold, 1 multiple well per gradient, 100ul per well, were added to the plate.

Crosslinking agent (anti-mouse lgG Fc antibody) dilution: 0.6ul anti-mouse lgG Fcanibody (1mg/ml) +3ml of the diluted solution, diluted to 0.2 ug/ml.

Addition of the crosslinking agent: taking 15 EP tubes, adding 125ul of 3E73F11 antibody gradient dilution solution into each tube, adding 125ul of 0.2ug/ml cross-linking agent dilution into each tube, and mixing uniformly. 100ul of each well was added to the plate, with one more well.

4. Placing the culture box for culturing for 18-22 hours.

5. MTS/PMS solution (Promega, G5430) is prepared according to the ratio of 20:1, 20ul of MTS/PMS solution is added to each hole, the incubator is placed for 3-4 h without a cover, and the final result is shown in the graph of figure 5 and figure 6 after A490-A630 detection by a microplate reader.

Figure 5 shows that 3E73F11 antibody induced hepadrg apoptosis in the absence of cross-linking agent, EC50 ═ 0.1856 μ g/ml. FIG. 6 shows that the EC50 of the 3E73F11 antibody induced apoptosis of HepaRG cells is reduced to 6ng/ml with 0.1ug/ml of cross-linking agent.

Example 5

Use of a monoclonal antibody against DR5 in flow cytometry.

1. Collecting jurkat cells (one of acute T cell leukemia cell lines), blowing, counting, and packaging into 5 centrifuge tubes (3 × 10 per tube)⁵Each cell was centrifuged at 500g for 5min, and the supernatant was removed, and 1ml of PBS was added thereto, and centrifuged at 500g for 5min, and the supernatant was removed.

2. Reagents were added as in table 1 below:

TABLE 1

Placing on ice, incubating for 30 min.

3. 1ml of PBS + 5% FBS was added, 500g was centrifuged for 5min, the supernatant was removed, 100ul of DMEM culture solution +1ul of PE goal anti-mouse lgG (minor x-reactivity) antibody (biolegend,405307) was added, and the mixture was incubated on ice for 20 min.

4. 1ml of PBS + 5% FBS was added to each, and the mixture was centrifuged at 500g for 5min to remove the supernatant.

5. 300ul PBS + 5% FBS was added to each tube, resuspended, and examined using a Beckman CytofleXm flow cytometer.

The results are shown in FIG. 7, which shows that: the 3E73F11 antibody can be combined with DR5 protein on the surface of a jurkat cell, is applied to flow cytometry detection, and has detection sensitivity superior to that of a commercial positive control. Therefore, it can be used for preparing a flow cytometry kit.

Example 6

Use of a monoclonal antibody against DR5 in an ELISA assay.

25ng of sDR5 protein is coated on each well of the ELISA plate, the plate is washed at 4 ℃ overnight, and 3E73F11 antibody diluted by 2 times is added after the plate is washed in a closed mode, the highest concentration is 40ug/ml, the lowest concentration is 19.5ng/ml, each well is 100ul, and incubation is carried out at 37 ℃ for 1 h. PBST washing plate 5 times, adding 1:5000 dilution of HRP-goat anti mouse IgG antibody, 37 degrees C were incubated for 45 min. And (3) washing the PBST for 5 times, adding 100ul of TMB into each hole, reacting for 3min in a dark place at room temperature, adding 50ul of stop solution into each hole, and detecting OD450 by using an enzyme-linked immunosorbent assay.

The results are shown in FIG. 8, which shows that: the 3E73F11 antibody can be used for ELISA detection, and can be used for preparing a kit for ELISA detection.

Example 7

Application of the anti-DR 5 monoclonal antibody in Western blot detection.

After mixing sDR5 protein with 5 × loading buffer, boiling for denaturation for 6min, running SDS-PAGE, loading 20ul of protein per well, 500ng of protein per well, transferring membrane for 1h with 400mA, sealing with 5% skimmed milk powder for 1h at room temperature, adding 3E73F11 antibody (adding 10ug of antibody to 3ml of 5% skimmed milk powder) for 4 ℃ incubation overnight, washing PBST for 3 times, adding 1:5000 diluted goat anti-mouse IgG secondary antibody, incubating for 45min at room temperature, washing PBST for 3 times, and adding Western HRP substrate (DenluR 0500) for development.

The result is shown in FIG. 9, the 3E73F11 antibody can be used for Western blot detection and can be used for preparing a kit for Westernblot detection. In addition, the DR5 antibody is applied in daily Western blot experiments, and the experimental effect of the 3E73F11 antibody is found to be better than that of the DR5 antibody commercialized by GeneTex company and Santa cruz company, wherein the cargo numbers are GTX21675 and sc166624 respectively.

Example 8

The relationship between the antigen binding epitope of the anti-DR 5 monoclonal antibody and the TRAIL binding site was verified.

1. Coating human TRAIL (sine biological, 10409-HANE, 250 ug/ml): 4ml coating solution +80ul human TRAIL to give human TRAIL at a concentration of 5ug/ml, which was used to coat ELISA plates at 100ul per well for 2h at 37 ℃.

2. The ELISA plate was removed, the well liquid was spun down, and washed 5 times 30 s/time with PBST (phosphate Tween buffer).

3. 300ul of blocking solution was added to each well and incubated at 37 ℃ for 2 h.

4. A mixture of 3E73F11 and sDR5-Fc-biotin was prepared.

60ul 3E73F11(2ug/ul) +2ml PBS (phosphate buffered saline) to give 2ml 60ug/ml 3E73F 11.

Dividing 32 EP tubes into 2 parts, each part comprises 16 EP tubes, and preparing sDR-Fc-biotin;

4ul of sDR5-Fc-biotin (1.5ug/ul) +200ul of PBS to obtain 200ul of 30ug/ml sDR 5-Fc-biotin; 100ul of sDR5-Fc-biotin (30ug/ml) +100ul of PBS to obtain 200ul of sDR5-Fc-biotin at 15 ug/ml; by analogy, 16 gradients were diluted. 100ul of 3E73F11 was added to each gradient and mixed well. Incubate at 37 ℃ for 1 h.

5. The ELISA plate was removed, the well was spun clean, and the PBST washed 5 times 30 s/time with shaking.

6. The incubated 3E73F11+ sDR5-Fc-biotin mixture was added to 100ul per well and incubated at 37 ℃ for 1 h.

7. The ELISA plate was removed, the well was spun clean, and the PBST washed 5 times 30 s/time with shaking.

8. 1:5000 dilution of Streptavidin-HRP was added to each well and incubated at 37 ℃ for 30 min.

9. The ELISA plate was removed, the well was spun clean, and the PBST washed 5 times 30 s/time with shaking.

10. 100ul TMB was added to each well and reacted for 10min at room temperature in the dark.

11. Stop adding 50ul stop solution to each well, and measuring OD450 by an enzyme-labeling instrument.

The working concentrations of the ELISA design and the corresponding OD450 results are shown in table 2.

TABLE 2

The data results of table 3 above show that: when excessive 3E73F11 antibody is combined with a small amount of antigen sDR5, the formed antigen-antibody complex can be combined with ligand TRAIL coated on an enzyme label plate, finally, a positive OD value is generated, and the size of the OD value is positively correlated with the amount of receptor DR 5. This demonstrates that the sites of binding of 3E73F11 antibody and DR5 are different from the sites of binding of TRAIL and DR5, that is binding of 3E73F11 antibody and DR5 does not affect the binding of TRAIL ligand to DR 5.

In addition, DR5 monoclonal antibody was also studied to induce apoptosis in conjunction with TRAIL: using the same method as in the in vitro cell function assay for the anti-DR 5 monoclonal antibody, HepaRG cell apoptosis was induced using a gradient dilution of human TRAIL in the presence of 1ug/ml 3E73F11 or 0.5ug/ml 3E73F11 or 0ug/ml 3E73F11 antibody, respectively, and the results are shown in figure 10. As can be seen from fig. 10: in the presence of 0.5ug/ml 3E73F11 antibody, the level of apoptosis induced by TRAIL at 0.013ng/ml was the same as that induced by TRAIL at 0.229ng/ml in the absence of 3E73F11 antibody, i.e., the TRAIL dosage was reduced by approximately 18-fold. Therefore, the 3E73F11 antibody can induce tumor cell apoptosis in cooperation with TRAIL.

Example 9

DR5 concentration in human serum was detected using 3E73F11 antibody.

1. 3E73F11 was diluted to 1ug/ml with coating solution, and 5 columns of FLUORO opaque enzyme plates (Thermo NUNC, 446473) were coated at 100ul per well for 2h at 37 ℃.

2. The plate was removed and the well was spun clean and washed 5 times 30 s/time with PBST shaking.

3. 250ul of blocking solution was added to each well and incubated at 37 ℃ for 1.5 h.

4. The plate was removed and the well was spun clean and washed 5 times 30 s/time with PBST shaking.

5. Human DR5(0.5mg/ml, Sino Biological Inc, 10465-H08H) was diluted with antibody dilutions, starting at 1000ng/ml, with 16 gradients, 1 replicate well per gradient, 100ul per well, incubated for 1H at 37 ℃.

6. The plate was removed and the well was spun clean and washed 5 times 30 s/time with PBST shaking.

7. DR5 rabbit polyclonal antibody (1mg/ml, Nano Biological Inc, 10465-RP02) was diluted with antibody diluent to 1ug/ml, 100 ul/well, incubated at 37 ℃ for 1 h.

8. The plate was removed and the well was spun clean and washed 5 times 30 s/time with PBST shaking.

9. Donkey anti-rabbitIgG-HRP (Abcam, ab7803) was diluted at a ratio of 1:10000 with antibody dilutions, and 100ul was added to each gradient, incubated at 37 ℃ for 45 min.

10. The plate was removed and the well was spun clean and washed 5 times 30 s/time with PBST shaking.

11. 100ul of chemiluminescent color-developing solution (Thermo, 37075) was added, the mixture was shaken for 30s in the absence of light, and OD425nm was detected by SpectraMax M5.

TABLE 3

As a result, as shown in table 3 and fig. 11, it can be seen from table 3 that: the lower limit of detection of sDR5 concentration in human serum detected by the 3E73F11 antibody can reach 61 pg/ml; as can be seen from fig. 11: the linear interval is 244 pg/ml-125 ng/ml.

Example 10

It was tested whether the 3E73F11 antibody cross-reacted with human DR4 antigen.

1.7 concentrations of DR4 and DR5 were diluted to 2.5ug/ml, 1.25ug/ml, 625ng/ml, 312.5ng/ml, 156.25ng/ml, 78ng/ml, and 39ng/ml with coating solution, and 96-well plates were coated at 4 ℃ overnight at 100ul per well.

2. PBST was washed 3 times with shaking, 30 s/time.

3. 250ul of blocking solution was added to each well and incubated at 37 ℃ for 1.5 h.

4. PBST was washed 5 times with shaking, 30 s/time.

5. 3E73F11 was diluted to 2ug/ml in PBS, 100 ul/well and incubated for 1h at 37 ℃.

6. PBST was washed 5 times with shaking, 30 s/time.

7. Goat anti-mouse lgG-HRP was diluted 1:5000 with PBS at 100 ul/well and incubated at 37 ℃ for 45 min.

8. The plate was removed and the well was spun clean and washed 5 times 30 s/time with PBST shaking.

9. 100ul of TMB was added to each well and reacted at room temperature for 4min in the dark.

10. The addition of 50ul of stop solution per well was terminated and the OD450 was determined.

The results are shown in FIG. 12: the 3E73F11 antibody specifically recognizes DR5 antigen and does not cross-react with DR4 antigen.

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

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Claims (12)

1. An antibody targeting DR5, comprising a heavy chain variable region and a light chain variable region, wherein said heavy chain variable region comprises the following 3 complementarity determining regions CDRs:

CDR1 shown in SEQ ID NO.1, CDR2 shown in SEQ ID NO. 2, and CDR3 shown in SEQ ID NO. 3;

the light chain variable region comprises the following 3 complementarity determining regions CDRs:

CDR1 ' shown in SEQ ID NO. 4, CDR2 ' shown in SEQ ID NO. 5, and CDR3 ' shown in SEQ ID NO. 6.

2. The antibody of claim 1, wherein the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO 7; and/or

The light chain variable region comprises an amino acid sequence shown as SEQ ID NO. 8.

3. The antibody of claim 1 or 2, wherein the antibody comprises a constant region comprising a heavy chain constant region and a light chain constant region.

4. A recombinant protein comprising the sequence of the antibody of claim 1 or 2; and/or

A sequence comprising the antibody of claim 3, and a tag sequence to facilitate expression and/or purification.

5. A nucleic acid molecule encoding the antibody of claim 1 or 2; and/or

Encoding the antibody of claim 3; and/or

Encoding the recombinant protein of claim 4.

6. The nucleic acid molecule of claim 5, wherein the nucleotide sequence encoding said heavy chain variable region is set forth in SEQ ID NO 9; and/or

The nucleotide sequence for coding the light chain variable region is shown as SEQ ID NO. 10.

7. A vector having the nucleic acid molecule of claim 5 or 6.

8. A genetically engineered host cell comprising the vector of claim 7; and/or

The host cell having integrated into its genome the nucleic acid molecule of claim 5 or 6.

9. An immunoconjugate comprising the antibody of claim 1 or 2; and/or

Comprising the antibody of claim 3; and/or

Comprising the recombinant protein of claim 4 and at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme.

10. A pharmaceutical composition comprising the antibody of claim 1 or 2; and/or

Comprising the antibody of claim 3; and/or

Comprising the recombinant protein of claim 4; and/or

Comprising the immunoconjugate of claim 9, and a pharmaceutically acceptable carrier.

11. Use of the antibody of any one of claims 1 to 3, the recombinant protein of claim 4, the immunoconjugate of claim 9 for the preparation of a tumor drug against DR5 protein, or for the preparation of a reagent and/or kit for the detection of DR5 protein.

12. A method of producing the antibody of claim 3 or the recombinant protein of claim 4, comprising culturing the host cell of claim 8.

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