WO2019100193A1 - Anticorps anti-dr5, son procédé de préparation et son utilisation - Google Patents

Anticorps anti-dr5, son procédé de préparation et son utilisation Download PDF

Info

Publication number
WO2019100193A1
WO2019100193A1 PCT/CN2017/112072 CN2017112072W WO2019100193A1 WO 2019100193 A1 WO2019100193 A1 WO 2019100193A1 CN 2017112072 W CN2017112072 W CN 2017112072W WO 2019100193 A1 WO2019100193 A1 WO 2019100193A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
seq
variable region
chain variable
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/112072
Other languages
English (en)
Chinese (zh)
Inventor
万晓春
陈倩
夏蒙
刘绿艳
李俊鑫
张青梅
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shenzhen Institute of Advanced Technology of CAS
Original Assignee
Shenzhen Institute of Advanced Technology of CAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Shenzhen Institute of Advanced Technology of CAS filed Critical Shenzhen Institute of Advanced Technology of CAS
Priority to PCT/CN2017/112072 priority Critical patent/WO2019100193A1/fr
Publication of WO2019100193A1 publication Critical patent/WO2019100193A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the invention belongs to the technical field of biomedicine, and particularly relates to an antibody against DR5 and a preparation method and application thereof.
  • cancer mortality increased from 12% to 15%, which has become a high-mortality disease after cardiovascular disease. It is predicted that new global cancer cases will reach 2020. 20 million people have died of cancers of 12 million people; therefore, humans need to develop new cancer treatment drugs.
  • TRAIL Tumor necrosis factor-related apoptosis-inducing ligands
  • TRAIL-DR death receptor
  • TRAIL The extracellular domain can be excised by metalloproteinases to form soluble TRAIL.
  • TRAIL is widely expressed in various tissues of normal human body and is specifically bound by receptors on the surface of target cells. Effect.
  • DR4 death receptors
  • DR5 decoy receptor 5
  • DcR1 decoy Receptor l
  • DcR2 decoy receptor 2
  • 31 soluble receptor OPG osteoprotegerin
  • the intracellular region of DR4 and DR5 possesses a complete death domain, which can induce apoptosis, while DcR1 has no death domain, and DcR2 has only a truncated death domain, which can not transmit apoptosis signals.
  • OPG is The only soluble protein of these five receptors, which is primarily involved in the regulation of bone density.
  • TRAIL binds to DR5 with higher affinity than other membrane-expressed TRAIL receptors; therefore, under physiological conditions, TRAIL is more likely to bind to DR5, especially when endogenous TRAIL is limited.
  • DR5 is highly expressed in human cancer, including colon cancer, gastric cancer, pancreatic cancer, ovarian cancer, breast cancer, non-small cell lung cancer, and has no or low expression in normal cells.
  • the death receptor DR4 or DR5 binds to TRAIL to form a ligand/receptor trimeric complex that induces death cytoplasmic segment death domain (death Domain, DD) Fas-associated death domain Protein, FADD) C-terminal DD binding, while FADD uses its N-terminal death effector Domain,DED) binds to procaspase-8 to form a DR4/DR5/FADD/procaspase-8 death-inducing signaling complex (death-inducing)
  • the signaling complex (DISC) promotes the activation of procaspase-8 itself into active caspase-8, which is activated by two signaling pathways (non-mitochondria-dependent pathway and mitochondria-dependent pathway).
  • TRAIL selectively kills tumor cells, allowing multiple TRAIL receptor antagonists to enter clinical development. It is mainly divided into two categories: 1) recombinant TRAIL protein; 2) antibodies that antagonize DR4 or DR5. However, none of these drugs currently benefit patients with clinical cancer.
  • the main drawback of recombinant TRAIL protein 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 antagonist clinical trials are: 1) insufficient antagonism of candidate drugs; 2) many primary cancer cells are resistant to single antibody therapy; 3) lack of suitable biomarkers to identify patients to TRAIL Susceptibility to receptor antagonists.
  • Monoclonal antibodies targeting DR5 that have entered clinical research are: Lexatumumab, Drozitumab, Conatumumab, Tigatuzumab, LBY135, etc., while many DR5 antibodies (such as Drozitumab and Tigatuzumab) require an additional cross-linking agent to The best activity is achieved in vitro.
  • the object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide an anti-DR5 antibody, a preparation method and application thereof, and aim to solve the problem that the antagonistic activity of the existing TRAIL receptor antagonist antitumor drugs is insufficient, and cross-linking needs to be added.
  • the technical problem of the agent to induce apoptosis is to overcome the above-mentioned deficiencies of the prior art, and to provide an anti-DR5 antibody, a preparation method and application thereof, and aim to solve the problem that the antagonistic activity of the existing TRAIL receptor antagonist antitumor drugs is insufficient, and cross-linking needs to be added.
  • the invention provides a variable region of an antibody, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the following three complementarity determining region CDRs:
  • the light chain variable region comprises the following three complementarity determining region CDRs:
  • the heavy chain variable region contains an amino acid sequence as shown in SEQ ID NO: 7, or an amino acid sequence having the same function obtained by deletion, insertion or substitution of the amino acid sequence shown in SEQ ID NO: ;and / or
  • the light chain variable region contains an amino acid sequence as shown in SEQ ID NO: 8, or an amino acid sequence having the same function obtained by deletion, insertion or substitution of the amino acid sequence shown in SEQ ID NO: 8.
  • the invention provides an antibody comprising a constant region comprising the variable region, the constant region comprising a heavy chain constant region and a light chain constant region.
  • the present invention provides a recombinant protein, the recombinant protein comprising a sequence having the above variable region; and/or
  • the invention provides a nucleic acid molecule encoding the variable region; and/or
  • the above recombinant protein is encoded.
  • nucleotide sequence encoding the heavy chain variable region is as shown in SEQ ID NO: 9, or the nucleotide sequence shown in SEQ ID NO: 9 is obtained by deletion, insertion or substitution.
  • a nucleotide sequence encoding the light chain variable region is represented by SEQ ID NO: 10, or a nucleotide having the same function obtained by deletion, insertion or substitution of the nucleotide sequence shown in SEQ ID NO: Glycosidic acid sequence.
  • the invention also provides a vector having the above nucleic acid molecule.
  • the invention provides a genetically engineered host cell, the host cell comprising the above vector; and/or
  • the nucleic acid molecule described above is integrated into the genome of the host cell.
  • the present invention provides an immunoconjugate comprising the above variable region; and/or
  • the recombinant protein described above and at least one of a detectable label, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.
  • the present invention provides a pharmaceutical composition comprising the above variable region; and/or
  • the present invention provides a variable region, the above antibody, the above recombinant protein, the above immunoconjugate in the preparation of an anti-DR5 protein tumor drug, or a reagent and/or a kit for preparing a DR5 protein. application.
  • the present invention provides a method for producing the above antibody or the above recombinant protein, which comprises culturing a host cell or using a hybridoma method to produce mouse ascites.
  • variable region of the antibody provided by the present invention comprising six unique complementarity determining region CDRs (see 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 above-mentioned unique antibody variable region sequence, can bind to the antigen DR5, has high affinity and biological activity, can induce apoptosis without adding a cross-linking agent; and it is associated with the antigen DR5
  • the specific recognition epitope is different from the binding site of TRAIL and DR5, and can cooperate with TRAIL to induce apoptosis.
  • the antibody provided by the invention has wide use and can be used as an antitumor drug (the tumor expresses DR5 protein) alone or in combination with TRAIL, and can also be used in combination with a commonly used tumor radiotherapy or chemotherapy drug, or a gene generated based on the tumor.
  • the engineered antibody acts as a targeting moiety and is coupled to a radionuclide or chemical drug or toxin.
  • the antibody is used for preparing reagents and/or kits for detecting DR5 protein, such as ELISA detection, Western blot detection and flow cytometry, and the sensitivity is significantly superior to the prior art.
  • Example 1 is an SDS-PAGE electrophoresis pattern of the 3E73F11 antibody in Example 1 of the present invention
  • 2 is an electrophoresis pattern of the heavy and light chain variable regions of the 3E73F11 antibody in Example 1 of the present invention; wherein Y is a negative control, M is a Marker, 1 is a heavy chain of 3E73F11 antibody, and 2 is a 3E73F11 antibody.
  • Figure 3 is a subtype identification diagram of the 3E73F11 antibody in Example 2 of the present invention.
  • Figure 4 is a diagram showing the affinity detection of the 3E73F11 antibody in Example 3 of the present invention.
  • Figure 5 is a graph showing the results of apoptosis of HepaRG cells induced by 3E73F11 antibody in the absence of a cross-linking agent in Example 4 of the present invention
  • Figure 6 is a graph showing the results of apoptosis of HepaRG cells induced by 3E73F11 antibody in the presence of a cross-linking agent in Example 4 of the present invention
  • Figure 7 is a diagram showing the results of flow cytometric detection of jurkat cells by the 3E73F11 antibody of Example 5;
  • Figure 8 is a diagram showing the results of the 3E73F11 antibody used in the ELISA assay in Example 6 of the present invention.
  • Figure 9 is a diagram showing the results of Western blot analysis of the 3E73F11 antibody in Example 7 of the present invention.
  • Figure 10 is a graph showing the results of synergistic induction of apoptosis of HepaRG cells by the 3E73F11 antibody and TRAIL in Example 8;
  • Figure 11 is a graph showing the results of detecting the concentration of DR5 in human serum by the 3E73F11 antibody in Example 9 of the present invention.
  • Figure 12 is a graph showing the results of no cross-reaction of 3E73F11 antibody with human DR4 in Example 10 of the present invention.
  • the embodiments of the invention provide a variable region of an antibody, comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the following three complementarity determining regions CDR: SEQ CDR1 shown by ID NO: 1, CDR2 shown by SEQ ID NO: 2, and CDR3 shown by SEQ ID NO: 3;
  • the light chain variable region comprises the following three complementarity determining region CDRs: CDR1', SEQ ID NO: 4, SEQ ID NO: CDR2' shown by 5, and CDR3' shown by SEQ ID NO: 6.
  • 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 CDR also known as the complementarity determining region, forms a precise complement to the antigenic determinant in the spatial structure, and is a region in which the antibody recognizes and binds to the antigen, directly determining the specificity of the antibody.
  • the variable region of the antibody provided by the invention contains six unique complementarity determining region CDRs, which can specifically recognize the antigen DR5, has high affinity and biological activity, and can induce apoptosis.
  • the heavy chain variable region comprises SEQ ID NO: the amino acid sequence shown in 7 or SEQ ID An amino acid sequence having the same function obtained by deleting, inserting or replacing the amino acid sequence shown by NO: 7; and/or the light chain variable region containing SEQ.
  • Heavy chain variable region amino acid sequence (SEQ ID NO. 7):
  • it may be an amino acid sequence having at least 80% homology to the sequence disclosed by SEQ ID NO: 7 or SEQ ID NO: 8 as an alternative.
  • embodiments of the invention also provide an antibody comprising a constant region and a variable region as described above, the constant region comprising a heavy chain constant region and a light chain constant region.
  • the present invention provides a recombinant protein comprising a sequence having the above variable region; and/or a sequence comprising the above antibody, and a tag sequence which facilitates expression and/or purification.
  • the above antibody or recombinant protein provided by the embodiment of the invention has the above-mentioned unique antibody variable region sequence, can bind to the antigen DR5, has high affinity and biological activity, and can induce apoptosis without adding a crosslinking agent; Moreover, its specific recognition epitope with antigen DR5 is different from the binding site of TRAIL and DR5, and can cooperate with TRAIL to induce apoptosis.
  • embodiments of the invention provide a nucleic acid molecule encoding the variable region of the embodiments of the invention; and/or encoding the antibody; and/or encoding the recombinant protein.
  • the nucleotide sequence encoding the heavy chain variable region ammonia is SEQ ID NO: 9 or as SEQ ID a nucleotide sequence having the same function obtained by deletion, insertion or substitution of a nucleotide sequence represented by NO: 9; and/or a nucleotide sequence encoding a variable region of a light chain such as SEQ ID NO: 10, or a nucleotide sequence having the same function obtained by deleting, inserting or replacing the nucleotide sequence shown by SEQ ID NO: 10.
  • Nucleotide sequence encoding the heavy chain variable region (SEQ ID NO. 9):
  • the nucleotide sequence encoding the heavy chain CDR1 is (SEQ ID NO. 11):
  • the nucleotide sequence encoding the heavy chain CDR2 is (SEQ ID NO. 12):
  • the nucleotide sequence encoding the heavy chain CDR3 is (SEQ ID NO. 13):
  • the nucleotide sequence encoding the light chain CDR1' is (SEQ ID NO. 14):
  • the nucleotide sequence encoding the light chain CDR2' is (SEQ ID NO. 15):
  • the nucleotide sequence encoding the light chain CDR3' is (SEQ ID NO. 16):
  • the nucleic acid molecule provided by the embodiment of the present invention may be a sequence different from the nucleotide sequence disclosed in the embodiment of the present invention but encoding the same protein due to the complementary sequence of the nucleotide sequence or the degeneracy of the genetic code; Is a sequence which is at least 80% homologous to the nucleotide sequence disclosed in the examples of the present invention as an alternative.
  • embodiments of the invention provide a vector having the nucleic acid molecule described above.
  • the recombinant vector can be used to express an antibody or recombinant protein comprising the above-described heavy chain variable region and light chain variable region.
  • embodiments of the present invention also provide an immunoconjugate comprising the above variable region; and/or comprising the above antibody; and/or comprising the above recombinant protein, and a detectable label, At least one of a drug, a toxin, a cytokine, a radionuclide, and an enzyme.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the above variable region; and/or containing the above antibody; and/or containing the above recombinant protein; and/or containing the above immunoconjugate And a pharmaceutically acceptable carrier.
  • the embodiment of the present invention further provides a tumor drug for preparing an anti-DR5 protein, or a reagent for detecting DR5 protein, wherein the variable region, the antibody, the recombinant protein, and the immunoconjugate are used in the embodiment of the present invention. And/or the application in the kit.
  • the antibody or the recombinant protein provided by the embodiment of the invention can be widely used as an antitumor drug (the tumor expresses the DR5 protein), can also be used in combination with TRAIL, and can also be used in combination with a commonly used tumor radiotherapy or chemotherapy drug, or
  • the genetically engineered antibody produced as a basis is coupled to a radionuclide or a chemical drug or toxin.
  • the present invention discloses the killing effect of the monoclonal antibody against human DR5 on a liver cancer cell line, and can be used for the treatment of other tumor types having DR5 expression.
  • the reagent and/or kit for detecting DR5 protein is a related kit in the fields of ELISA detection, Western blot detection and flow cytometry, and its sensitivity is significantly superior to the prior art.
  • the present invention provides a variable region amino acid sequence and a nucleotide sequence of a monoclonal antibody against human DR5, and on the basis of the above recombinant vector or host cell, a genetically engineered antibody can be produced, which comprises a heavy chain and a light chain.
  • the chain variable region sequence is identical to the variable region sequences disclosed in the examples of the present invention.
  • the genetically engineered antibody includes, but is not limited to, a functional fragment of the antibody, Fab, or a single-chain antibody, or an antibody functional fragment VH-L, which is a heavy chain variable region and a full light chain fusion, or a heavy chain or a light chain.
  • the present invention provides a method for producing the above antibody or the above recombinant protein, which comprises culturing a host cell or using a hybridoma method to produce mouse ascites.
  • the preparation method uses mouse ascites to produce monoclonal antibodies; or the cultured hybridoma cells, CHO cells or 293F cells are used to produce the monoclonal antibodies.
  • a variable region amino acid sequence and a nucleotide sequence of a mouse anti-human DR5 monoclonal antibody are provided, and a human mouse chimeric antibody and human against human DR5 can be obtained by humanized modification of the antibody. Sourced antibodies.
  • the anti-human DR5 human-mouse chimeric antibody ligates the variable region of the murine monoclonal antibody gene to the human constant region and then expresses it in mammalian cells; whereas the humanized antibody against human DR5 is In addition to changing the constant region of the antibody to an adult source, the FR region of the variable region is further converted to an adult source, thereby reducing immunogenicity.
  • the human-human chimeric antibody and humanized antibody against human DR5 thus produced will also have the effect of binding to human DR5 molecules to induce apoptosis of cancer cells, thereby treating tumors.
  • this example uses a hybridoma method to produce a monoclonal antibody against DR5 using mouse ascites.
  • mice Female BALB/c mice (6 weeks old) were immunized with sDR5-Fc protein antigen (provided by Shenzhen Zhongke Ai Shen Pharmaceutical Co., Ltd., preparation method reference patent 201610067931.2), and the first immunization was performed using Freund's complete adjuvant for emulsification antigen. At the beginning of the second immunization, the antigen was emulsified using Freund's incomplete adjuvant, subcutaneously injected at 5-6 points, and the amount of antigen injected per mouse was about 100 ug.
  • sDR5-Fc protein antigen provided by Shenzhen Zhongke Ai Shen Pharmaceutical Co., Ltd., preparation method reference patent 201610067931.2
  • the antigen was emulsified using Freund's incomplete adjuvant, subcutaneously injected at 5-6 points, and the amount of antigen injected per mouse was about 100 ug.
  • mice with high antibody titer >1:100000 were selected for the fourth immunization, and the antigenic protein was injected intraperitoneally. Each mouse was injected about 100 ug.
  • Hybridoma cells capable of secreting DR5 antibody were screened by ELISA, subcloned by limiting dilution, and monoclonal hybridoma cell lines capable of secreting DR5 antibody were screened by ELISA (in this specification, the monoclonal antibody against DR5) It was uniformly named as 3E73F11 antibody, and it was cultured by stepwise expansion, and stored in liquid nitrogen for storage.
  • mice Female BALB/c mice (8 weeks old) were injected intraperitoneally with Freund's incomplete adjuvant, 0.5 ml per mouse, and intraperitoneal injection of hybridoma cells in logarithmic growth phase after 3 to 5 days, each Mice were injected with 1 ⁇ 5 ⁇ 10 5 cells (0.5 ml), and the mice were sacrificed about 11 days after the injection of the hybridoma cells to obtain ascites. Centrifuge at 3000 rpm for 10 min at 4 ° C to remove the precipitate, dilute the ascites with 10 volumes of 1 ⁇ PB solution, mix and pass through a 0.45 ⁇ m filter.
  • the ascites was affinity-purified by Protein G (Protein G Sepharose 4 Fast Flow, GE Healthcare) to obtain a purified DR5 antibody protein, and the antibody concentration was measured by the BCA method.
  • the purified antibody was run on SDS-PAGE (loading amount 5.4 ug) and stained with Coomassie brilliant blue. The results are shown in Figure 1.
  • the 3E73F11 antibody contains two bands, one for the heavy chain and one for the light chain.
  • DR5 monoclonal antibody hybridoma cell antibody gene variable region sequencing harvesting monoclonal antibody hybridoma cells in logarithmic growth phase, TRIZOL cleavage for RNA extraction, reverse transcription to obtain cDNA, amplification and obtaining heavy chain and light
  • the DNA sequence of the variable region of the chain (electropherogram shown in Figure 2), the non-functional VK gene was removed, cloned into the pMD18-T vector, sequenced, and sequenced using the database; the sequencing results and analysis are as follows.
  • the nucleotide sequence encoding the heavy chain variable region is SEQ ID NO. 9;
  • the nucleotide sequence encoding the light chain variable region is SEQ ID NO. 10;
  • Heavy chain variable region amino acid sequence SEQ ID NO. 7 light chain variable region amino acid sequence SEQ ID NO.
  • CDR1-IMGT SEQ ID NO. 1;
  • CDR2-IMGT SEQ ID NO. 2;
  • CDR3-IMGT SEQ ID NO. 3;
  • 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;
  • amino acid sequence of the FR region of the light chain is:
  • FR1’-IMGT DIVMTQSPSSLAVSAGEKVTMSCKSS;
  • FR2’-IMGT LAWYQQKPGQSPKLLIY;
  • FR3’-IMGT TRESGVPDRFIGSGSGTDFTLTISSVQAEDLAVYYC;
  • FR4’-IMGT FGAGTKLELK.
  • the monoclonal antibody subtype was identified using a commercial mouse monoclonal antibody Isotyping kit (sino 003), and the results are shown in Fig. 3. As is clear from the figure, the 3E73F11 antibody is an IgG1 type antibody.
  • Dilution of anti-mouse lgG Fc antibody 0.6ul Anti-mouse lgG Fc antibody (1 mg/ml) + 3 ml dilution, diluted to 0.2 ug/ml.
  • Add cross-linking agent Take 15 EP tubes, add 125ul 3E73F11 antibody gradient dilution solution to each tube, add 125ul 0.2ug/ml cross-linker dilution to each tube and mix. 100 ul per well was added to the plate and a double hole was provided.
  • Figure 6 shows that the EC50 of apoptosis induced by 3E73F11 antibody in HepaRG cells was reduced to 6 ng/ml in the case of 0.1 ug/ml crosslinker.
  • Negative control DMEM 100ul DMEM medium
  • PBS 100ul PBS Isotype control PE
  • Mouse lgG1, ⁇ isotype control antibody (biolegend, 400111) 100ul DMEM medium + 2ul lgG1-PE sample 3E73F11 100ul DMEM medium + 3.5ul 3E73F11 Positive control PE anti-human CD262(DR5,TRAIL-R2)Antibody(biolegend,307405) 100ul DMEM medium + 4ul anti-DR5 antibody-PE
  • the 3E73F11 antibody can bind to the DR5 protein on the surface of jurkat cells, and is applied to flow cytometry, and the detection sensitivity is superior to the commercial positive control. Therefore, it can be used to prepare a flow cytometry kit.
  • the ELISA plate is coated with 25ng per well.
  • the sDR5 protein was coated overnight at 4 °C. After blocking the plate, 2 times diluted 3E73F11 antibody was added. The highest concentration was 40 ug/ml, the lowest concentration was 19.5 ng/ml, 100 ul per well, and incubated at 37 degrees for 1 h.
  • the plate was washed 5 times with PBST, and a 1:5000 dilution of HRP-goat anti-mouse IgG antibody was added and incubated at 37 ° C for 45 min. Wash the plate 5 times with PBST and add 100ul per well TMB, room temperature and light-proof reaction for 3 min, 50 ul of stop solution was added to each well, and OD450 was measured by a microplate reader.
  • the 3E73F11 antibody can be used for ELISA assay, which can be used to prepare a kit for ELISA assay.
  • sDR5 protein plus 5 ⁇ loading After buffer mixing, boil and denature for 6 min, run SDS-PAGE, load 20ul per well, ⁇ 500ng protein/well, 400mA transfer membrane for 1h, 5% skim milk powder for 1h at room temperature, add 3E73F11 antibody (3ml 5% skim milk powder was added with ⁇ 10ug antibody) and incubated overnight at 4 degrees. After washing with PBST for 3 times, add 1:5000 diluted goat anti-mouse IgG secondary antibody, incubate for 45 min at room temperature, wash PBST for 3 times and add Western HRP substrate (Millipore, WBLUR0500) Development.
  • 3E73F11 antibody 3ml 5% skim milk powder was added with ⁇ 10ug antibody
  • the 3E73F11 antibody can be used in Western
  • the blot assay can be used to prepare a kit for Western blot detection. Also in everyday Western The DR5 antibody was used in the blot experiment and found that the 3E73F11 antibody was superior to the commercial DR5 antibody of GeneTex and Santa cruz, and the numbers were GTX21675 and sc166624, respectively.
  • TRAIL ino biological, 10409-HANE, 250 ug/ml: 4 ml of coating solution + 80 ul of human TRAIL to obtain human TRAIL at a concentration of 5 ug/ml, and coated with an ELISA plate, 100 ul per well, 37 °C is coated for 2h.
  • the ELISA plate was taken out, and the liquid in the well was cleaned, and PBST (phosphate tween buffer) was shaken and washed 5 times for 30 s/time.
  • PBST phosphate tween buffer
  • DR5 monoclonal antibody and TRAIL synergistically induced apoptosis were also studied: using the same method as the anti-DR5 monoclonal antibody in vitro cell function assay, respectively at 1 ug/ml 3E73F11 or 0.5ug/ml 3E73F11 or 0ug/ml Apoptosis of HepaRG cells was induced using a gradient dilution of human TRAIL in the presence of the 3E73F11 antibody, and the results are shown in FIG.
  • the concentration of DR5 in human serum was measured using the 3E73F11 antibody.
  • Sheep anti-mouse lgG-HRP was diluted 1:5000 with PBS, 100 ul/well, and incubated at 37 ° C for 45 min.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Public Health (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Wood Science & Technology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un anticorps anti-DR5, son procédé de préparation et son utilisation. L'anticorps comprend une région constante et une région variable, la région variable de chaîne lourde comprend CDR1 telle que représentée dans SEQ ID NO : 1, CDR2 telle que représentée dans SEQ ID NO : 2 et CDR3 telle que représentée dans SEQ ID NO : 3; la région variable de chaîne légère comprend CDR1' telle que représentée dans SEQ ID NO : 4, CDR2' telle que représentée dans SEQ ID NO : 5 et CDR3' telle que représentée dans SEQ ID NO : 6. L'anticorps peut se lier de manière spécifique à l'antigène DR5 mais n'a pas de réaction croisée avec DR4, présente une affinité et une activité biologique très élevées, peut induire l'apoptose sans ajout d'agents de réticulation, et peut induire de manière synergique l'apoptose en combinaison avec TRAIL.
PCT/CN2017/112072 2017-11-21 2017-11-21 Anticorps anti-dr5, son procédé de préparation et son utilisation Ceased WO2019100193A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/112072 WO2019100193A1 (fr) 2017-11-21 2017-11-21 Anticorps anti-dr5, son procédé de préparation et son utilisation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2017/112072 WO2019100193A1 (fr) 2017-11-21 2017-11-21 Anticorps anti-dr5, son procédé de préparation et son utilisation

Publications (1)

Publication Number Publication Date
WO2019100193A1 true WO2019100193A1 (fr) 2019-05-31

Family

ID=66630597

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/112072 Ceased WO2019100193A1 (fr) 2017-11-21 2017-11-21 Anticorps anti-dr5, son procédé de préparation et son utilisation

Country Status (1)

Country Link
WO (1) WO2019100193A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001083560A1 (fr) * 2000-05-02 2001-11-08 Uab Research Foundation Un anticorps selectif pour un recepteur de ligand induisant l'apoptose en relation avec le facteur de necrose tumorale et ses utilisations
CN101247825A (zh) * 2005-02-02 2008-08-20 健泰科生物技术公司 Dr5抗体及其用途
CN103282495A (zh) * 2010-10-29 2013-09-04 第一三共株式会社 新的抗dr5抗体
CN106397594A (zh) * 2016-10-25 2017-02-15 中国药科大学 一种全人源的抗人死亡受体5的激动剂单链抗体及应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001083560A1 (fr) * 2000-05-02 2001-11-08 Uab Research Foundation Un anticorps selectif pour un recepteur de ligand induisant l'apoptose en relation avec le facteur de necrose tumorale et ses utilisations
CN101247825A (zh) * 2005-02-02 2008-08-20 健泰科生物技术公司 Dr5抗体及其用途
CN103282495A (zh) * 2010-10-29 2013-09-04 第一三共株式会社 新的抗dr5抗体
CN106397594A (zh) * 2016-10-25 2017-02-15 中国药科大学 一种全人源的抗人死亡受体5的激动剂单链抗体及应用

Similar Documents

Publication Publication Date Title
AU2019280900B2 (en) DLL3/CD3 binding proteins for the treatment of cancer
CN110305210B (zh) 新型抗体分子、其制备方法及其用途
EP3604339B1 (fr) Anticorps thérapeutiques contre la protéine ror-1 et leurs procédés d'utilisation
CN109310756A (zh) 新型血管生成素2，vegf双特异性拮抗剂
WO2019184909A1 (fr) Nouvelle molécule d'anticorps, son procédé de préparation et son utilisation
BR112012028306A2 (pt) anticorpos para proteína 6 relacionada com lipoproteína de baixa densidade (lrp6) e seus fragmentos, seu uso, ácidos nucléicos, vetores, bem como composições farmacêuticas e combinações
WO2023193732A1 (fr) Anticorps anti-ccr8 ou fragment de liaison à l'antigène de celui-ci
WO2021110095A1 (fr) Anticorps anti-gpc3, fragment de liaison à l'antigène de celui-ci, et son utilisation médicale
CN105452297A (zh) mAb 2抗-Met抗体
CN109810194B (zh) 抗dr5的抗体及其制备方法和应用
WO2021143914A1 (fr) Anticorps anti-ox40, son procédé de production et son application
CN110172100B (zh) 抗人cd3e抗体及其用途
US9840560B2 (en) Monoclonal antibodies to EGFR, and uses therefor
CN104861068B (zh) 一种全人源抗her3抗体及其治疗相关疾病的用途
CN110950959B (zh) 靶向EpCAM的抗体及其制备和应用
CN114040924A (zh) 对lif具有特异性的结合分子及其用途
WO2019100193A1 (fr) Anticorps anti-dr5, son procédé de préparation et son utilisation
CN110964107B (zh) Met结合分子、其组合和用途
WO2019100194A1 (fr) Anticorps anti-dr5, son procédé de préparation et son utilisation
TW202128751A (zh) 抗gdf15抗體
CN109810193B (zh) 抗dr5的抗体及其制备方法和应用
KR102952495B1 (ko) 항-cxcr2 항체 및 이의 용도
US20230041071A1 (en) Antibodies against areg and its use
KR101996627B1 (ko) Vcam-1-d6에 결합하는 항체 및 그 용도
WO2025113639A1 (fr) Anticorps anti-gdf15, et méthode et utilisation associés

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17933127

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 17933127

Country of ref document: EP

Kind code of ref document: A1

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205 DATED 02/10/2020)

122 Ep: pct application non-entry in european phase

Ref document number: 17933127

Country of ref document: EP

Kind code of ref document: A1