WO2020047705A1 - Dr5单域抗体及其用途 - Google Patents

Dr5单域抗体及其用途 Download PDF

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WO2020047705A1
WO2020047705A1 PCT/CN2018/103799 CN2018103799W WO2020047705A1 WO 2020047705 A1 WO2020047705 A1 WO 2020047705A1 CN 2018103799 W CN2018103799 W CN 2018103799W WO 2020047705 A1 WO2020047705 A1 WO 2020047705A1
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seq
sequence
domain antibody
cancer
single domain
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PCT/CN2018/103799
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English (en)
French (fr)
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查丽莎
禾立春
陈宏权
邓婕
周霆
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安菲尼生命科技有限公司
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    • 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 [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants

Definitions

  • the present application relates to the field of genetic engineering technology, in particular to a human-derived anti-DR5 single domain antibody and use thereof.
  • Apoptosis is a type of programmed cell death found in animals (Kerr et al., 1972). Apoptosis is a highly regulated and controlled process that plays an important role in the life cycle of multicellular organisms. Normal cell apoptosis is an essential way to maintain the stability of the body, but disorders of the apoptotic system can promote various diseases, including cancer, neurodegenerative diseases and acquired immunodeficiency syndrome (Agostini et al., 2011) . Apoptosis is the most important and common type of programmed cell death. There are two main apoptotic pathways: intrinsic and extrinsic. The intrinsic pathway, also known as mitochondrial apoptosis, is essential for cell survival.
  • TNF Tumor Necrosis Factor
  • TRAIL Tumor Necrosis-inducing ligand
  • TRAIL receptor 1 and TRAIL receptor 3 (DcRl, TR5, TRIDD, LIT) and TRAIL receptor 4 (DcR2, TRUNDD), among which TRAIL receptor 4 lacks a cytoplasmic death domain and cannot mediate apoptosis.
  • DR4 or DR5 expression is often detected in human cancers, including non-small cell lung cancer, gastric cancer, breast cancer, colon cancer, ovarian cancer, and pancreatic cancer, while there is no DR4 or DR5 expression or low expression in normal tissues.
  • DR5 The expression of DR5 is regulated by a variety of stimulating factors in the cells, and the accumulation of ROS in tumor cells can induce the significant expression of DR5 in cells (Chang et al., 2015; Jayasooriya et al., 2014). Kimihisa et al. In 2001 synthesized a foreign sequence of human DR5 and an Fc segment of human Ig1 (DR5-Ig) into a fusion protein that can be used to immunize female mice with BALB / c. Research has enabled the synthesis of a specific monoclonal antibody against DR5, which is TRA-8.
  • TRA-8 works only in primary and metastatic hepatocellular carcinoma, and that TRA-8 does not play a pro-apoptotic role in normal hepatocytes (Ichikawa et al., 2001).
  • IgG1 TRAIL-R2, Conatumumab
  • AMG-655 a fully human agonistic monoclonal antibody against TRAIL-R2, Conatumumab
  • the agonism-specific antibody shows higher stability and extended half-life (Kaplan-Lefko et al., 2010; Rosevear et al., 2010).
  • Previous single-domain antibodies (nanobodies) on DR5 have also achieved significant results in preclinical experiments.
  • the anti-DR5sdAb tetramer (TAS266) has shown excellent pharmacokinetics and efficacy in preclinical models, inducing a powerful anti-tumor response and continuous caspase activation in the body.
  • TAS266 The anti-DR5sdAb tetramer
  • Bifunctional single-domain antibodies that target EGFR and TRAIL simultaneously inhibit the growth of different tumor cell types that do not respond to EGFR antagonists or death receptor agonists monotherapy, which is a significant advance in the clinical application of single-domain antibodies (Zhu et al. , 2017).
  • tumor cells are able to avoid programmed cell death and have the ability to invade and metastasize simultaneously with unlimited cell division (Hanahan and Weinberg, 2000).
  • the escape of tumor cells from apoptosis is key to the pathogenesis and progression of cancer, and advances in our understanding of the regulation of programmed cell death pathways have facilitated the development of new agents that re-stimulate tumor cell apoptosis.
  • agonistic antibodies against DR4 or DR5 have been produced, and this agonistic antibody against DR4 or DR5 represents a new generation of cancer therapy.
  • the present invention provides a humanized single domain antibody against DR5, which has a simple structure, a small relative molecular weight, strong penetrating power, and immunogenicity. Low characteristics, and has cytotoxic effect on a variety of tumor cells, can be used as an ideal targeted drug and carrier.
  • the present invention provides a single domain antibody against DR5, the single domain antibody comprising a monomer having a sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or an amino acid sequence containing at least one CDR region or a dimer or a multimer.
  • the CDR sequence comprises SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8.
  • the present invention also provides a single domain antibody against DR5, the single domain antibody comprising the full sequence of the first antibody sequence (SEQ ID NO: 1) or the second antibody sequence (SEQ ID NO: 2).
  • a long sequence or an amino acid sequence of at least one CDR region thereof has an amino acid sequence monomer, dimer or multimer with at least 95% homology.
  • Another object of the present invention is to provide the use of an anti-DR5 single domain antibody in the preparation of a tumor or cancer targeting drug, which is lung cancer, liver cancer or breast cancer.
  • Another object of the present invention is to provide a method for treating a tumor or cancer, which comprises administering an effective amount or a sufficient amount of a single domain antibody against DR5 provided by the present invention, wherein the tumor or cancer is lung cancer, liver cancer or breast cancer.
  • the advantages of the embodiments of the present invention are as follows: 1.
  • the humanized anti-DR5 single domain antibody provided has a simple structure, is composed only of the variable region VH of the antibody heavy chain, contains a complete antigen-binding site, and its trimer can be composed of A linker peptide (SEQ ID NO: 9) is connected, and the trimer has a stronger affinity and a longer half-life than a single molecule; 2.
  • the trimers of two single-domain antibodies have the ability to tumor cells Universal killing effect.
  • the experimental results show that the trimers linked by these two single-domain antibodies through linker peptides have cytotoxic effects on lung cancer (A549), liver cancer (HepG2) and breast cancer (MDA-MB-231) cell lines. 3.
  • the two single-domain antibodies of the present invention have small molecular weights, both below 12.5 KDa, have the characteristics of strong penetrating power and low immunogenicity, and can be used as drugs or drug carriers to transport drugs or toxin molecules for targeted treatment of tumors. It can be coupled with fluorescent or isotope markers for contrast imaging of tumor lesions in vivo.
  • Figure 1 shows the results of the binding activities of two DR5 single domain antibodies screened by ELISA.
  • Figure 2 is a sequence analysis diagram of two DR5 single domain antibodies.
  • FIG. 3 shows two DR5 single domain antibodies in E. coli Electrophoresis of expression and purification results in T7 Express.
  • FIG. 4 is a graph of the affinity measurement data of DR5 single domain antibody binding to DR5 using an ITC titration experiment.
  • Figure 5 is an electrophoresis diagram of the construction and expression purification of a trimer of a DR5 single domain antibody.
  • FIG. 6 is a diagram showing detection of endotoxin in two DR5 single domain antibody trimers expressed and purified by anion exchange chromatography.
  • FIG. 7 is a detection diagram of specific inhibition of trimers of two DR5 single domain antibodies against lung cancer (A549), liver cancer (HepG2), and breast cancer (MDA-MB-231) cell lines.
  • cancer and “tumor” are used herein with the same meaning.
  • gene as used herein includes not only DNA, but also its mRNA, its cDNA and cRNA.
  • nucleic acid as used herein includes DNA, RNA, probes, oligonucleotides and primers.
  • polypeptide As used herein, the terms “polypeptide” and “protein” are used without distinction.
  • cell as used herein also includes cells in individual animals and cultured cells.
  • cytotoxic activity refers to a state that causes some form of pathological change in the cell.
  • Cell damage is not limited to direct damage, but also includes various types of damage to cell structure and function, such as DNA cutting, bases Dimer formation, chromosome cutting, impaired cell division machinery, and reduced various enzyme activities.
  • receptor containing a death domain refers to a receptor molecule having an apoptosis signal transduction region (called a "death domain”) in the intracellular domain, which shows homology with the fruit fly suicide gene reaper .
  • the term "functional fragment of an antibody” as used herein refers to a partial fragment of an antibody having antigen-binding activity, and includes all fragments having binding affinity for an antigen.
  • these functional fragments include not only fragments obtained by treating a full-length molecule of an antibody protein with a suitable enzyme, but also proteins produced in a suitable host cell using a genetically modified antibody gene.
  • CDR complementarity determining region
  • CDRs complementarity determining regions
  • DR5 death receptor 5
  • DR5 also includes one to several amino acid substitutions, deletions or additions of amino acid sequences and also has a biological activity equivalent to DR5 protein.
  • apoptosis refers to an ordered or controlled form of cell death in mammals, usually accompanied by one or more specific cellular changes, including cytoplasmic condensation, loss of plasma membrane microvilli, fragmentation of the nucleus, and chromosomal DNA. Degradation or loss of mitochondrial function.
  • DR5 antibody refers to the antibody that has similar activity to DR5 ligand, which is known as Apo-2 ligand (TRAIL); or refers to the ability to activate the DR5 receptor, thereby generating the activation of another intracellular signaling pathway
  • TRAIL Apo-2 ligand
  • the intracellular signaling pathway may include caspase 3, caspase 8, caspase 10, or FADD activation.
  • variable refers to the variable domain used for the binding and specificity of each particular antibody to its particular antigen. However, variability is not evenly distributed across the variable domains of antibodies. It concentrates in three fragments called complementary determining regions (CDRs) or hypervariable regions in the variable domains of the light and heavy chains.
  • CDRs complementary determining regions
  • single domain antibody refers to an antibody containing only one heavy chain variable region (VHH), also known as a nanobody, and the corresponding English language is Single Domain Antibody or Nanobody, respectively.
  • VHH heavy chain variable region
  • the embodiment of the present invention provides a single domain antibody against DR5.
  • the single domain antibody comprises an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 or at least one CDR region comprising the two sequences.
  • the two amino acid sequences are as follows:
  • the total length of the sequence is 111 amino acids, the relative molecular weight is 12034.50 Da, and the theoretical isoelectric point is 5.02.
  • the total length of the sequence is 112 amino acids, the relative molecular weight is 12271.68 Da, and the theoretical isoelectric point is 6.85.
  • amino acid sequences corresponding to the above two sequences are relatively small in molecular weight, have strong penetrating power and low immunogenicity. They can be used as drugs or drug carriers to transport drugs or toxin molecules to target tumors, or they can be coupled to fluorescence or isotopes. The markers are used for imaging of tumor lesions in vivo.
  • the two antibody sequences of the single-domain antibody each contain three CDR sequences, and the three CDR sequences in the first antibody sequence (SEQ ID NO: 1) are SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5, the specific sequence is as follows:
  • the sequence of the CDR1 region is SEQ ID NO: 3: GSLGGIFA;
  • the sequence of the CDR2 region is SEQ ID NO: 4: ILGTGVT;
  • the sequence of the CDR3 region is SEQ ID NO: 5: NLRDY.
  • the three CDR sequences in the above second antibody sequence are SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and the specific sequences are as follows:
  • the sequence of the CDR1 region is SEQ ID NO: 6: GNIDSIET;
  • the sequence of the CDR2 region is SEQ ID NO: 7: IHRDGTT;
  • the sequence of the CDR3 region is SEQ ID NO: 8: TADQRD.
  • the present application also provides a single-domain antibody comprising the full-length sequence of the first antibody sequence (SEQ ID NO: 1) or the second antibody sequence (SEQ ID NO: 2) or Any of its functional fragments (CDR1, CDR2, and CDR3) has an amino acid sequence monomer, dimer, or multimer with at least 95% homology.
  • the "multimer” described herein refers to an amino acid sequence formed by combining three or more identical monomers.
  • the present application relates to a form of a dimer and a multimer using the above-mentioned single domain antibody, for example, a trimer formed by a DR5 single domain antibody by linking a peptide.
  • the dimer or multimer includes two or more polypeptide or antibody amino acid sequences each having the ability to bind to DR5.
  • the dimer or multimer is connected by a linker peptide GGGGSGGGGSGGGGS (SEQ ID NO: 9).
  • the present application provides the use of a humanized anti-DR5 single domain antibody in the preparation of a tumor-targeting drug.
  • the tumor is lung cancer (A549), liver cancer (HepG2), or breast cancer (MDA-MB -231), wherein the anti-DR5 single domain antibody is a monomer, dimer or multimer.
  • the concentration of the anti-DR5 single domain antibody used for treating tumor or cancer is greater than or equal to 10 ng / ml.
  • the anti-DR5 single domain antibody is in a trimer form when used for treating tumors or cancers or for inhibiting cancer cells.
  • the present application also relates to a method for treating a tumor.
  • the method further comprises administering an effective amount or a sufficient amount of an anti-DR5 single domain antibody provided by the present application.
  • the single domain antibody is a monomer, dimer, or multimer.
  • the tumor is specifically lung cancer (A549), liver cancer (HepG2) or breast cancer (MDA-MB-231).
  • Example 1 ELISA to identify the antigen-binding activity of DR5 single domain antibodies
  • 10X PBS 80g NaCl, 2g KCl, 14.4g Na 2 HPO 4 and 2.4g KH 2 PO 4 , add 1L of pure water and adjust the pH to 7.4;
  • Blocking solution 10mg / ml BSA (1% BSA, 1X PBS configuration);
  • Antibody dilution primary antibody (screening for enriched potential single domain antibodies), secondary antibody are diluted with 1% BSA-PBS;
  • Dissolve the antigen DR5 in the coating solution at the set concentration then add 100 ⁇ l of antigen to the corresponding wells of the 96-well plate, and incubate at 37 ° C for 2 hours (or 4 ° C overnight); empty the liquid and pat dry (96-well plate Tap toward the absorbent paper) Residual liquid, rinse the washing solution 5 times; add 200 ⁇ l of blocking solution to each well, and incubate for 1 hour at 37 ° C; immunize alpaca, select and obtain monoclonal clones obtained after two rounds, prepare monoclonal recombinant expression vectors, and wait until The monoclonal antibody to be tested was added to a 96-well plate and incubated at 37 ° C for 1 hour; the liquid was emptied and the residual liquid was patted dry, and the washing solution was washed 7 times; PBS was used as a negative control; 100 ⁇ l of secondary antibody was added to each well, and incubated at 37 ° C for 1 hour Empty the
  • the strong positive clone screened by ELISA in Example 1 was sequenced to identify the sequence of the single domain antibody screened. Sequencing results showed that most of the antibody sequences had high homology or were the same.
  • the binding sequences of the CDR regions of the two antibodies disclosed in the present invention have significant differences, corresponding to the sequences SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • CDR sequences can be based on Or ordinary numbering system, which is reserved with Common sequence in.
  • the anti-DR5 single domain antibody of the present invention whose full-length sequence is SEQ ID NO: 1 includes the following CDRs: CDR1, the corresponding sequence of SEQ 3; CDR2, the corresponding sequence of SEQ 4;
  • the single-domain anti-DR5 antibody of ID NO: 2 includes the following CDRs: CDR1, corresponding to SEQ 6; CDR2, corresponding to SEQ 7; CDR3, corresponding to SEQ 8; CDR region of the domain, the binding domain includes any one or more of SEQ 3, SEQ 4, SEQ 5, SEQ 6, SEQ 7, and SEQ 8. Also included is one or more having a homology of at least 90% with SEQ 3, SEQ 4, SEQ 5, SEQ 6, SEQ 7, and SEQ 8.
  • the required solution configuration method is:
  • LB liquid culture medium Trytone 10g, yeast extract 5g, NaCl 10g, and distilled water to 1000mL;
  • IPTG 1M IPTG (isopropylthio- ⁇ -D-galactoside): 2.38 g of IPTG was dissolved in 10 ml of ddH 2 O, filtered through a 0.22 ⁇ m filter membrane, and stored at -20 ° C;
  • Loading buffer 50 mM Tris, 150 mM Nacl, pH 8.0;
  • Washing buffer 50 mM Tris, 150 mM Nacl, pH 8.0, 30 mM Imidazole;
  • Elution buffer 50 mM Tris, 150 mM Nacl, 500 mM Imidazole, pH 8.0.
  • the single-domain antibody DNA sequence containing the full-length sequences of the first and second sequences was ligated into an expression vector, transformed into T7Shuffle competent cells, and then picked.
  • the clone was taken in 50 mL of LB liquid medium (containing 100 ug / mL ampicillin), and cultured with shaking at 37 ° C for 5 h. After that, they were transferred to 1 L of LB liquid medium (containing 100 ug / mL ampicillin) to expand the culture. When the OD 600 reached 0.7, 0.4 mM ITPG was added to induce expression for 4 h.
  • Example 4 Affinity determination data for DR5 single domain antibody binding to DR5
  • ITC Isothermal Titration Calorimetry
  • FIG. 4 shows that the single-domain antibodies of the first sequence and the second sequence bind to the DR5 antigen and emit heat, and the molar ratio (Stoichiometry) of the binding between the two is about 1.
  • the dimer and more of DR5 have better cytotoxic activity. Therefore, in the present invention, the first sequence and the second sequence are linked into a homotrimeric form through a linker peptide.
  • the method is shown in Figure 5.
  • the linker sequence is: GGGGSGGGGSGGGGS (SEQ ID NO: 9).
  • Ligation is to synthesize the gene sequence encoding the protein of interest according to the designed method, then ligate it into an expression vector and express it in E. coli of T7shuffle.
  • the solution configuration methods required in expression and purification are:
  • LB liquid culture medium Trytone 10g, yeast extract 5g, NaCl 10g, and distilled water to 1000mL;
  • IPTG 1M IPTG (isopropylthio- ⁇ -D-galactoside): 2.38 g of IPTG is dissolved in 10 ml of ddH 2 O, 0.22 ⁇ m filter membrane is suction filtered, and stored at -20 ° C;
  • Loading buffer 50 mM Tris, 150 mM Nacl, pH 8.0;
  • Washing buffer 50 mM Tris, 150 mM Nacl, pH 8.0, 30 mM Imidazole;
  • Elution buffer 50 mM Tris, 150 mM Nacl, 500 mM Imidazole, pH 8.0.
  • the experimental steps are as follows: the trimer sequence containing the first sequence (SEQ ID NO: 1), the second sequence (SEQ ID NO: 2) and the linker peptide (SEQ ID NO: 9) is ligated into an expression vector, respectively, and transformed To T7SHuffle competent cells, clones were then picked up in 50mL LB liquid medium (containing 100ug / mL ampicillin), and cultured with shaking at 37 ° C for 5h. Thereafter, they were transferred to 1 L of LB liquid medium (containing 100 ug / mL ampicillin) to expand the culture, and 0.4 mM ITPG was added to induce expression for 4 h when the OD 600 reached 0.7.
  • LB liquid medium containing 100ug / mL ampicillin
  • the two DR5 single domain antibodies disclosed in the present invention can be expressed in E. coli after being fused into a trimer form by a linker peptide, and can be purified using a Ni column to prepare high-purity single domain antibodies.
  • the two single-domain antibodies of DR5 disclosed in the present invention are expressed by E. coli (Example 5 above), so the endotoxin needs to be removed before the cell viability test.
  • the method used is: using an anion exchange column Q column to bind negatively charged endotoxin to remove endotoxin.
  • the buffer needed in the experiment is:
  • a buffer solution 50mM Tris, 50mM Nacl, pH8.0;
  • B buffer 50 mM Tris, 2M Nacl, pH 8.0.
  • the specific experimental steps are: in the case of low salt ion concentration, the diluted single domain antibody is loaded as a trimer and eluted with different concentration gradients of elution buffer to separate endotoxin and single domain antibody. . All equipment and tubing used during the experiment were sterile, pyrogen-free equipment. Single-domain antibodies in the form of trimers before and after purification using a Q column were measured for endotoxin content using a perylene tripeptide kit (commercially available).
  • FIG. 6A The results of Q column chromatography are shown in Figures 6A and 6B.
  • A is the result of the endotoxin removal experiment in the form of a single domain antibody trimer of the first sequence (SEQ ID NO: 1).
  • the test result of the ⁇ tripeptide kit shows that the endotoxin content will be 5EU after Q column separation. / ml or less (see Fig. 6C).
  • Fig. 6B is the result of the endotoxin removal experiment in the form of a single domain antibody trimer of the second sequence (SEQ ID NO: 2).
  • the test result of the triseptide kit shows that the endotoxin content will be 5EU / ml or less (see Figure 6D).
  • Example 7 Cancer cell suppression detection of two DR5 single domain antibody trimers
  • the concentration is 2-1250ng / ml (divided into 5 concentration gradients, as shown in Figure 7).
  • the single-domain antibody in the form of a trimer of the first sequence (SEQ ID NO: 1) and the second sequence (SEQ ID NO: 2) disclosed in the present invention is a concentration-dependent manner for all three tumor cell lines. It shows a cytocidal effect, but has almost no effect on normal vascular endothelial cells, proving that it has a specific tumor cell killing effect.
  • the two DR5 single domain antibodies disclosed by the present invention can be used as antitumor drugs or drug carriers, such as conjugated drugs, toxins or radioisotopes, for targeted therapy of tumors, either after modification by genetic engineering or other methods.
  • these two DR5 single-domain antibodies also have the ability to couple small molecules such as fluorescent labels to develop tumor cells and tumor lesions, and play a role in tumor diagnosis and localization.
  • Tanshinone IIA Facilitates TRIL Sensitization Up-regulating DR5 through the ROS-JNK-CHOP Signaling, Axis, Human, Ovarian, Carcinoma Cell Lines. Chem. Res. Toxicol. 28, 1574-1583.
  • Conatumumab a fully human, human body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, body, human, body, body, body, body, body, body, body, body, body, body, body, human, body, body, body, body, body, body, body, human, body, body, body, body, body, human, body, body, body, body, body, human, body, body, body, body, body, body, human, body, body, body, body, body, body, human, body, body, body, body, body, body, body, human, body, body, body, body, body,
  • Apoptosis a basic biological phenomenon with wide-ranging imimplications in kintissues. Br. J. Cancer 26, 239-257.

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Abstract

提供一种抗DR5的单域抗体，该单域抗体包含序列为SEQ ID NO:1或者SEQ ID NO:2或者含有至少一个CDR区域的氨基酸序列的单体或者二聚体或者多聚体，其中所述CDR序列包含SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6，SEQ ID NO:7，或SEQ ID NO:8，所述单域抗体可治疗肿瘤或制备癌症靶向药物。

Description

DR5单域抗体及其用途 技术领域
本申请涉及基因工程技术领域，具体涉及一种人源抗DR5单域抗体及其用途。
背景技术
细胞凋亡是动物中发现的一种细胞程序性死亡(Kerr et al.,1972)。细胞凋亡是一种高度调节和控制的过程，在多细胞生物体的生命周期中具有重要作用。细胞正常凋亡是维持机体稳定必不可少的途径，但是细胞凋亡系统的紊乱可促使各种疾病发生，包括癌症、神经退行性疾病和获得性免疫缺陷综合征(Agostini et al.,2011)。细胞凋亡是程序性细胞死亡的最重要和最常见的类型。有两种主要的凋亡途径：内在和外在。内在途径，也称为线粒体凋亡，线粒体对细胞生存至关重要。如果没有它们，细胞就会停止呼吸，并迅速死亡。这一事实构成了一些凋亡途径的基础。其特征是Bcl-2家族蛋白介导的线粒体破坏，导致细胞色素C从线粒体释放到细胞质中。细胞质细胞色素C与Apaf-1和caspase-9形成复合物，激活caspase-9，然后启动下游半胱天冬酶的活化，最终诱导细胞凋亡(Ola et al.,2011)。外源性细胞凋亡由死亡受体与其各自配体的结合介导。死亡受体DR5(death receptor 5)是一种重要类型的死亡受体，又名Trail受体2(TRAIL-R2、ApO-2)，其具有细胞质死亡结构域，可与TNF(Tumor Necrosis Factor)相关的凋亡诱导配体(TRAIL)结合(Yuan et al.,2018)导致caspase-8或caspase-10的募集和激活，从而诱导细胞凋亡通过直接激活caspase级联，或者切断Bid，Bcl-2家族的一个成员得到的截短的Bid易位至线粒体以引发内在的细胞凋亡(Lemke et al.,2014)。与DR5同属死亡受体的还有四种其他的TRAIL细胞表面受体。TRAIL受体1(DR4)和TRAIL受体3(DcRl、TR5、TRIDD、LIT)和TRAIL受体4(DcR2、TRUNDD)，其中TRAIL受体4缺乏细胞质死亡结构域，并且不能介导凋亡。经常在人类癌症中，包括非小细胞肺癌、胃癌、乳癌、结肠癌、卵巢癌和胰腺癌中检测到DR4或DR5的表达，而在正常组织中没有DR4或DR5的表达或者表达很低。
DR5的表达受细胞中多种刺激因子的调控，肿瘤细胞中ROS的聚集能诱导细胞DR5的显著表达(Chang et al.,2015；Jayasooriya et al.,2014)。Kimihisa等在2001年时将人体具有的DR5的外源序列和人体具有的Ig G1(DR5-Ig)中的Fc段合成一种可以用来免疫BALB/c的雌性小鼠的融合蛋白,这一研究使得DR5的特异单克隆抗体得以合成，这就是TRA-8。随后却发现TRA-8只对原发性和转移性肝细胞癌起作用，正常肝细胞中TRA-8并没有起到 促凋亡作用(Ichikawa et al.,2001)。此外另一种针对TRAIL-R2的完全人类激动性单克隆抗体(IgG1)Conatumumab(AMG-655)，与重组形式的TRAIL相比，激动性特异性抗体表现出更高的稳定性和延长的半衰期(Kaplan-Lefko et al.,2010；Rosevear et al.,2010)。此前关于DR5的单域抗体(纳米抗体)在临床前实验中也取得了显著效果。研究者们将单结构域抗体模块设计成多价结构以克服快速清除。抗DR5sdAb四聚体(TAS266)在临床前模型中显示出优异的药代动力学和功效，诱导强大的抗肿瘤反应和体内持续的胱天蛋白酶活化。同时靶向EGFR和TRAIL的双功能单域抗体抑制对EGFR拮抗剂或死亡受体激动剂单一疗法无反应的不同肿瘤细胞类型的生长，这是单域抗体临床应用的明显进步(Zhu et al.,2017)。
在很多肿瘤的发展或推进中，肿瘤细胞能够避免程序性死亡并且可以无限制的细胞分裂同时具有侵袭和转移的能力(Hanahan and Weinberg,2000)。肿瘤细胞对细胞凋亡的逃避是癌症发病机制和进展的关键，并且我们对细胞程序性死亡途径调节的理解的进步促进了重新刺激肿瘤细胞凋亡的新的药剂的开发。如上所述，已经生产出了针对DR4或DR5的激动性抗体，并且该针对DR4或DR5的激动性抗体代表了新一代的癌症疗法。目前涉及指向DR4，或DR5或DR4和DR5的激动性抗体的应用的专利，或者涉及针对DR5的抗体和另一化学治疗剂的组合应用的专利或专利申请包含：US20040147725；US20090022707；US20080248037；US20020155109；US20030036168；US20020072091；US20030190685S20040052788。这些抗体具有抗肿瘤作用的前提是肿瘤细胞表面表达DR5，但是目前已经揭示在临床前试验表明所述的抗肿瘤作用与DR5表达水平之间没有相关性，其中的原因可能包括细胞反应受到多种因素的调节。但是通过激活死亡受体诱导肿瘤细胞凋亡的机制为治疗癌症和免疫性疾病开辟了新的思路，其中促使癌细胞的凋亡诱导剂日益受到重视，并已经用于临床研究，是一类非常具有应用前景的药物。
发明内容
为解决上述问题，促进针对某些肿瘤的特异性药物的开发，本发明提供一种抗DR5的人源化单域抗体，该抗体具有结构简单、相对分子量小、穿透力强、免疫原性低的特点，并且对多种肿瘤细胞具有细胞毒作用，可以作为理想的靶向药物和运载载体。
本发明提供了一种抗DR5的单域抗体，该单域抗体包含序列为SEQ ID NO:1或者SEQ ID NO:2或者含有至少一个CDR区域的氨基酸序列的单体或者二聚体或者多聚体，其中所述CDR序列包含SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6，SEQ ID NO:7，或SEQ ID NO:8。
另一方面，本发明还提供一种抗DR5的单域抗体，该单域抗体包含与第一个抗体序列(SEQ ID NO:1)或第二个抗体序列(SEQ ID NO:2)的全长序列或其至少一个CDR区域的氨基酸序列具有至少95％同源性的氨基酸序列单体，二聚体或者多聚体。
本发明的另一目的在于提供抗DR5单域抗体在制备肿瘤或癌症靶向药物中的用途，所述肿瘤或癌症为肺癌、肝癌或乳腺癌。
本发明的另一目的在于提供一种肿瘤或癌症的治疗方法，该治疗方法包括给予有效量或足量的本发明提供的抗DR5的单域抗体，所述肿瘤或癌症为肺癌、肝癌或乳腺癌。
本发明的实施方案的优点在于：1.提供的人源化的抗DR5的单域抗体结构简单，仅由抗体重链可变区VH组成，含有完全的抗原结合部位，其三聚体可以由连接肽(SEQ ID NO:9)连接而成，该三聚体相对于单分子而言具有更强亲和力及更长半衰期的特性；2.两种单域抗体的三聚体均具有对肿瘤细胞普适的杀伤作用，实验结果显示这两种单域抗体通过连接肽连接成的三聚体对肺癌(A549)，肝癌(HepG2)和乳腺癌(MDA-MB-231)细胞株产生细胞毒作用；3.本发明的两种单域抗体分子量小，都在12.5KDa以下，具有穿透力强，免疫原性低的特点，可以作为药物或者药物载体运输药物或者毒素分子靶向治疗肿瘤，也可以偶联荧光或者同位素等标记物做体内肿瘤病灶的造影。
附图说明
图1为ELISA检测筛选出的两种DR5单域抗体的结合活性结果。
图2为两种DR5单域抗体的序列分析图。
图3为两种DR5单域抗体在大肠杆菌
Figure PCTCN2018103799-appb-000001
T7 Express中的表达和纯化结果电泳图。
图4为采用ITC滴定实验对DR5单域抗体与DR5结合的亲和力测定数据图。
图5为DR5单域抗体的三聚体的构建及表达纯化电泳图。
图6为利用阴离子交换层析的方法去除表达纯化的两种DR5单域抗体三聚体中的内毒素检测图。
图7为两种DR5单域抗体的三聚体对于肺癌(A549)，肝癌(HepG2)，乳腺癌(MDA-MB-231)细胞株的特异抑制检测图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
本文所用术语“癌症”和“肿瘤”以相同含义使用。
本文所用术语“基因”不仅包括DNA，而且还包括其mRNA、其cDNA和cRNA。
本文所用术语“核酸”相同的含义包括DNA、RNA、探针、寡核苷酸和引物。
本文所用术语“多肽”和“蛋白质”无差别地使用。
本文所用术语“细胞”还包括动物个体和培养细胞中的细胞。
本文所用术语“细胞毒性活性”是指在细胞中引起某种形式的病理变化的状态，细胞损伤不限于直接损伤，还包括对细胞结构和功能的各种类别的损害，例如DNA切割、碱基二聚体形成、染色体切割、细胞分裂机器受损和各种酶活性降低。
本文所用术语“含有死亡结构域的受体”是指胞内域中具有凋亡信号转导区(称为“死亡结构域”)的受体分子，其与果蝇自杀基因reaper显示同源性。
本文所用术语“抗体的功能性片段”是指具有抗原结合活性的抗体的部分片段，包括对抗原具有结合亲和力的所有片段。此外，这些功能性片段不仅包括通过用合适的酶处理抗体蛋白质的全长分子所得到的片段，而且还包括使用经遗传修饰的抗体基因在合适的宿主细胞中产生的蛋白质。
本文所用术语“CDR”是指互补决定区(complementarity determining region,CDR)，已知单域抗体分子的重链具有3个互补决定区(CDR)，该结构亦称为超变结构域，它是在其一级结构中具有极高可变性的部位，重链的CDR用自重链氨基酸序列的氨基末端侧开始的CDR H1、CDR H2和CDR H3表示，这些部位在三级结构中彼此接近，并决定抗体与之结合的抗原的特异性。
本文所用术语人DR5(死亡受体5)基因的核苷酸序列及其氨基酸序列在GenBank以GI:22547118(登录号:NM_147187)登记。其中DR5也包括一个至几个氨基酸的取代、缺失或添加的氨基酸序列并还具有相当于DR5的生物活性的蛋白质。
术语"凋亡"是指哺乳动物中有序或受控形式的细胞死亡，通常伴随有一种或多种特有的细胞变化，包括细胞质凝聚、质膜微绒毛的丢失、细胞核片段化、染色体DNA的降解或线粒体功能的丢失。
术语"激动性"指能够实质上直接或间接地诱导促进或增强DR5生物活性或活化的分子。DR5抗体是指该抗体具有DR5的配体类似的活性，DR5的配体已知为Apo-2配体(TRAIL)；或者指能够激活DR5受体，从而产生另一细胞内信号传导通路的激活，该细胞内信号传导通路可以包括caspase 3、caspase 8、caspase 10或FADD的激活。
术语"可变"指该可变结构域用于每一具体抗体对其具体抗原的结合和特异性。但是， 可变性在抗体的可变结构域上并不是均匀分布的。它集中在轻链和重链的可变结构域中的三个被称为互补决定区(CDR)的片段或高变区中。
术语"单域抗体"指仅含有一个重链可变区(VHH)的抗体，也称为纳米抗体，对应的英文分别是Single Domain Antibody或者Nanobody。
本发明实施例提供一种抗DR5的单域抗体，具体地，该单域抗体包含序列为SEQ ID NO:1或者SEQ ID NO:2或者包含所述两个序列的至少一个CDR区域的氨基酸序列的单体或者二聚体或者多聚体。具体地，这两种氨基酸序列如下：
第一个抗体序列SEQ ID NO:1：
Figure PCTCN2018103799-appb-000002
该序列总长度为111个氨基酸，相对分子量为12034.50Da，理论等电点为5.02。
第二个抗体序列SEQ ID NO:2：
Figure PCTCN2018103799-appb-000003
该序列总长度为112个氨基酸，相对分子量为12271.68Da，理论等电点为6.85。
上述两种序列对应的氨基酸序列分子量都比较小，具有穿透力强，免疫原性低的特点，可以作为药物或者药物载体运输药物或者毒素分子靶向治疗肿瘤，也可以偶联荧光或者同位素等标记物做体内肿瘤病灶的造影。
进一步地，所述的单域抗体的两个抗体序列中均含有三个CDR序列，其中第一个抗体序列(SEQ ID NO:1)中的三个CDR序列分别为SEQ ID NO:3，SEQ ID NO:4，和SEQ ID NO:5，具体序列如下：
CDR1区域的序列为SEQ ID NO:3：GSLGGIFA；
CDR2区域的序列为SEQ ID NO:4：ILGTGVT；
CDR3区域的序列为SEQ ID NO:5：NLRDY。
另一方面，上述第二个抗体序列(SEQ ID NO:2)中的三个CDR序列分别为SEQ ID NO:6，SEQ ID NO:7，和SEQ ID NO:8，具体序列如下：
CDR1区域的序列为SEQ ID NO:6：GNIDSIET；
CDR2区域的序列为SEQ ID NO:7：IHRDGTT；
CDR3区域的序列为SEQ ID NO:8：TADQRD。
根据另一实施方案，本申请还提供一种单域抗体，其包含与上述第一个抗体序列(SEQ ID NO:1)或第二个抗体序列(SEQ ID NO:2)的全长序列或其任一功能性片段(CDR1， CDR2和CDR3)具有至少95％同源性的氨基酸序列单体，二聚体或者多聚体。
本申请所描述的“多聚体”是指由三个或更多个相同的单体结合而成的氨基酸序列。
根据具体实施例，本申请涉及使用上述单域抗体的二聚体及多聚体的形式，例如，DR5单域抗体通过链接肽形成的三聚体。该二聚体或多聚体包括都具有与DR5结合的能力的两条或更多条的多肽或抗体氨基酸序列。优选地，所述二聚体或多聚体由连接肽GGGGSGGGGSGGGGS(SEQ ID NO:9)连接而成。
根据另一实施方案，本申请提供人源化的抗DR5单域抗体在制备肿瘤靶向药物中的用途，具体地，该肿瘤为肺癌(A549)、肝癌(HepG2)或乳腺癌(MDA-MB-231)，其中该抗DR5单域抗体为单体、二聚体或者多聚体。
优选地，所述抗DR5单域抗体用于治疗肿瘤或者癌症时的浓度为大于或者等于10ng/ml。
进一步优选地，所述抗DR5单域抗体在用于治疗肿瘤或者癌症或者用于抑制癌细胞时为三聚体形式。
本申请还涉及一种肿瘤的治疗方法，该治疗方法还包括给予有效量或足量的本申请提供的抗DR5单域抗体，该单域抗体为单体、二聚体或者多聚体，该肿瘤具体为肺癌(A549)、肝癌(HepG2)或乳腺癌(MDA-MB-231)。
以下通过具体实施例对本发明的实验步骤进行详细描述。
实施例1:ELISA鉴定DR5单域抗体的抗原结合活性
实验方法:按照如下配方配置各种缓冲液：
1)10X PBS:80g NaCl，2g KCl,14.4g Na 2HPO 4和2.4g KH 2PO 4，加1L纯水，调pH至7.4；
2)包被液：1X PBS；
3)洗涤液：含0.05％Tween-20的1X PBS(1X PBST)或纯水；
4)封闭液：10mg/ml BSA(1％BSA，1X PBS配置)；
5)抗体稀释液：一抗(筛选富集的潜在单域抗体)、二抗均用1％BSA-PBS稀释；
6)2M H 2SO 4终止显色液：108ml 98％H 2SO 4，加入纯水中稀释到1L。
将抗原DR5按设定浓度溶解于包被液中，之后在96孔板对应的孔中加入100μl抗原，37℃孵育2小时(或4℃过夜)；倒空液体并拍干(将96孔板朝向吸水纸拍打)残留液体，洗涤液冲洗5次；每孔加200μl封闭液，37℃孵育1小时；免疫羊驼，挑选筛选二轮后获得的单 克隆，制备单克隆重组表达载体，将待测的单克隆抗体加入96孔板中，37℃孵育1小时；倒空液体并拍干残留液体，洗涤液冲洗7次；以PBS作为阴性对照；每孔加100μl二抗，37℃孵育1小时；倒空液体并拍干残留液体，洗涤液冲洗7次；拍干孔中残留液体，每孔加100μl TMB显色液，37℃避光显色10min；每孔加50μl 2M H 2SO 4终止显色，并用酶标仪测定OD 450值。
实验结果:结果参见图1，经过两轮富集后大部分克隆呈现阳性结合抗原的特性。有约一半左右的克隆显示OD 450值超过1.5，显示为强阳性结合。
结果说明:抗体筛选的方法效率很高，两轮之后富集的有效结合单克隆抗体的比率超过50％。
实施例2:DR5单域抗体的序列分析
将实施例1中ELISA筛选的强阳性克隆测序，以鉴定所筛选的单域抗体的序列。测序的结果显示大部分抗体序列同源度较高或者相同。其中本发明公开的两种抗体CDR区的结合序列具有显著差异，分别对应序列SEQ ID NO:1和SEQ ID NO:2。CDR序列可以根据
Figure PCTCN2018103799-appb-000004
或普通编号系统限定，该普通编号系统保留
Figure PCTCN2018103799-appb-000005
和
Figure PCTCN2018103799-appb-000006
中的常见序列。本发明的全长序列为SEQ ID NO:1的抗DR5的单域抗体包括以下CDRs：CDR1，对应序列SEQ 3；CDR2对应序列SEQ 4；CDR3对应序列SEQ 5；本发明的全长序列为SEQ ID NO:2的抗DR5的单域抗体包括以下CDRs：CDR1，对应序列SEQ 6；CDR2对应序列SEQ 7；CDR3对应序列SEQ 8；在具体应用中，DR5抗体多肽包括一个或更多的结合结构域CDR区，该结合结构域包括SEQ 3，SEQ 4，SEQ 5，SEQ 6，SEQ 7，和SEQ 8的任意一种或者多种。也包括与SEQ 3，SEQ 4，SEQ 5，SEQ 6，SEQ 7，和SEQ 8同源度至少90％的一种或者多种。
实施例3:两种DR5单域抗体在大肠杆菌
Figure PCTCN2018103799-appb-000007
T7Express中的表达和纯化
所需的溶液配置方法为：
1)LB液体培养基：Trytone 10g，酵母提取物(yeast extract)5g，NaCl 10g，用蒸馏水配至1000mL；
2)氨苄青霉素：100mg/mL；
3)IPTG：1M IPTG(异丙基硫代-β-D-半乳糖苷)：取2.38g IPTG溶于10ml ddH 2O中，0.22μm滤膜抽滤，-20℃保存；
4)上样缓冲液：50mM Tris，150mM Nacl，pH8.0；
5)洗涤缓冲液(Washing Buffer)：50mM Tris，150mM Nacl，pH8.0，30mM咪唑 (Imidazole)；
6)洗脱缓冲液(Elution Buffer)：50mM Tris，150mM Nacl，500mM咪唑(Imidazole)，pH8.0。
将筛选的含有上述第一序列和第二序列(SEQ ID NO:1和SEQ ID NO:2)全长序列的单域抗体DNA序列分别连接到表达载体中，转化至T7Shuffle感受态细胞，之后挑取克隆于50mL LB液体培养基中(含100ug/mL氨苄青霉素)，37℃震荡培养5h。之后分别转到1L的LB液体培养基中(含100ug/mL氨苄青霉素)扩大培养，至OD 600达到0.7的时候加入0.4mM的ITPG诱导表达4h。6000rpm离心15min，弃上清，菌体重悬于上样缓冲液中，并加入DNase搅拌30min后，用高压均质机裂解菌体。然后于4℃12000rpm离心30min，将上清吸至一个干净的容器中，弃沉淀。取10ul上清样品用于SDS-PAGE分析。其余上清样品上Ni-NTA柱，收集流出液，取10ul样品用于SDS-PAGE分析。用Washing Buffer以10-15mL/h流速洗柱，约30倍柱体积，取10ul洗脱开始时的样品用于SDS-PAGE分析。最后用Elution Buffer洗柱，收集洗脱组分，取10ul样品用于SDS-PAGE分析。
实验结果:电泳分析结果参见图3：其中左侧第一泳道：C是诱导后菌体直接上样；第二泳道：S是诱导后高压裂菌后上清；第三泳道：Ft是上Ni-NTA柱后的未结合的流穿组分；第四泳道：W是加洗涤缓冲液后非特异结合的组分。第五泳道：E是纯化后的目的蛋白，呈单一条带，大约在15kDa(带His标签及TEV酶切位点)，与预期的一致。M(Marker)为分子量标准。
上述实验结果说明:本发明公开的两种DR5的单域抗体均可以在大肠杆菌中进行表达，可以用Ni柱进行纯化，制备高纯度的单域抗体。
实施例4:DR5单域抗体与DR5结合的亲和力测定数据
实验方法：等温滴定量热技术(ITC)是一种监测任何由结合成分的添加而起始的化学反应的热力学技术，并以此来充分鉴定生物分子间相互作用。首先将纯化的DR5抗原及上述实施例3制得的第一序列和第二序列的单域抗体透析到同样的缓冲液中(50mM Tris，150mM Nacl，pH8.0)。之后将单域抗体浓缩到1mg/ml以上。依据各组分的分子量计算出其摩尔浓度。在恒温下利用注射器将50微摩尔的单域抗体滴定到含有5微摩尔DR5抗原的溶液的池中。当抗体注射到池中，两种物质相互作用，释放或吸收的热量与结合量成正比。当池中的抗原被抗体饱和时，热量信号减弱，直到只观察到稀释的背景热量。
实验结果:滴定结果参照图4，图4显示，第一序列和第二序列的单域抗体与DR5抗原结合，并放出热量，两者结合的摩尔比(Stoichiometry)约为1。
结果说明：用Origin软件对滴定曲线拟合显示第一序列(SEQ ID NO:1)单域抗体与DR5结合亲和力小于10nM，第二序列(SEQ ID NO:2)单域抗体与DR5结合亲和力也小于10nM，均超出了等温滴定为量热仪(ITC)的精确测量范围。
实施例5:DR5单域抗体的三聚体活性形式的构建及表达和纯化
实验方法:DR5的二聚体及更多聚体具有更好的细胞毒活性，所以本发明中将上述第一序列和第二序列，通过连接肽连接成同源的三聚体形式，连接的方式如图5所示，该连接肽(linker)序列为：GGGGSGGGGSGGGGS(SEQ ID NO:9)。连接是按照设计的方式合成能编码目的蛋白的基因序列后，将其连接到表达载体中，并在T7shuffle的大肠杆菌中表达。在表达及纯化中所需的溶液配置方法为：
1)LB液体培养基：Trytone 10g，酵母提取物(yeast extract)5g，NaCl 10g，用蒸馏水配至1000mL；
2)氨苄青霉素：100mg/mL；
3)IPTG：1M IPTG(异丙基硫代-β-D-半乳糖苷)：2.38g IPTG溶于10ml ddH 2O中,0.22μm滤膜抽滤，-20℃保存；
4)上样缓冲液：50mM Tris，150mM Nacl，pH8.0；
5)洗涤缓冲液(Washing Buffer)：50mM Tris，150mM Nacl，pH8.0，30mM咪唑(Imidazole)；
6)洗脱缓冲液(Elution Buffer)：50mM Tris，150mM Nacl，500mM咪唑(Imidazole)，pH8.0。
实验步骤为：将含有第一序列(SEQ ID NO:1)、第二序列(SEQ ID NO:2)及连接肽(SEQ ID NO:9)的三聚体序列分别连接到表达载体中，转化至T7SHuffle感受态细胞，之后挑取克隆于50mL LB液体培养基中(含100ug/mL氨苄青霉素)，37℃震荡培养5h。之后分别转到1L的LB液体培养基中(含100ug/mL氨苄青霉素)扩大培养，至OD 600达到0.7的时候加入0.4mM的ITPG诱导表达4h。6000rpm离心15min，弃上清，菌体重悬于上样缓冲液中，并加入DNase搅拌30min后，用高压均质机裂解菌体。4℃12000rpm离心30min，将上清吸至一个干净的容器中，并弃沉淀。取10ul上清样品用于SDS-PAGE分析。上清样品上Ni-NTA柱，收集流出液，取10ul样品用于SDS-PAGE分析。用Washing Buffer以10-15mL/h流速洗柱，约30倍柱体积，取10ul洗脱开始时的样品用于SDS-PAGE分析。最后用Elution Buffer洗柱，收集洗脱组分，取10ul样品用于SDS-PAGE分析。
实验结果:电泳分析结果参见图5：Marker为分子量标准，左边第二泳道：Super.是诱导后高压裂菌后上清；第三泳道：Ft是上Ni-NTA柱后的未结合的流穿组分；第四泳道：W是加洗涤缓冲液后非特异结合的组分；第五泳道：E是纯化后的目的蛋白，呈单一条带，大约在50kDa(带His标签及TEV酶切位点)，与预期的一致。
结果说明:本发明公开的两种DR5的单域抗体通过连接肽融合成三聚体形式后均可以在大肠杆菌中进行表达，可以用Ni柱进行纯化，制备高纯度的单域抗体。
实施例6:阴离子交换层析的方法去除表达纯化的两种DR5单域抗体三聚体中的内毒素
实验原理:本发明中公开的DR5的两种单域抗体为大肠杆菌表达(上述实施例5)，故在细胞活性测试前，需将内毒素去除。所使用的方法为：利用阴离子交换柱Q柱结合带负电的内毒素，从而去除内毒素。
实验中所需的缓冲液为：
1)A缓冲液：50mM Tris，50mM Nacl，pH8.0；
2)B缓冲液：50mM Tris，2M Nacl，pH8.0。
具体实验步骤为：在低盐离子浓度的情况下，将稀释的单域抗体以三聚体形式上样，并通过不同浓度梯度的洗脱缓冲液进行洗脱，以分离内毒素及单域抗体。实验过程中所用的所有的器材和管路都为无菌无热源器材。使用Q柱纯化前后的三聚体形式的单域抗体分别用鲎三肽试剂盒(市场购得)进行内毒素含量的测定。
实验结果:Q柱层析的结果如图6A，6B所示。其中A为序列为第一序列(SEQ ID NO:1)的单域抗体三聚体形式的内毒素去除实验结果，鲎三肽试剂盒检测结果表明，经过Q柱分离后内毒素含量将为5EU/ml以下(参见图6C)。其中图6B为第二序列(SEQ ID NO:2)的单域抗体三聚体形式的内毒素去除实验结果，鲎三肽试剂盒检测结果表明，经过Q柱分离后内毒素含量将为5EU/ml以下(参见图6D)。
结果说明：经过条件模式后的Q柱层析可以很好的去除大肠杆菌表达的单域抗体中的内毒素。减少了后续细胞毒活性测试的干扰作用及其他副作用。
实施例7:两种DR5单域抗体三聚体的癌细胞抑制检测
实验方法:在含有10％FBS的RPMI 1640培养基或含有10％FBS的MEM培养基中，制备每毫升4.4x10 4个细胞的肺癌(A549)，肝癌(HepG2)，乳腺癌(MDA-MB-231)细胞株，每一种按45μl/孔加入透明底96孔微量培养板(Corning Incorporated制造)中，在37℃和5％C0 2的条件下培养过夜。将去除内毒素的第一序列(SEQ ID NO:1)和第二序列(SEQ ID  NO:2)单域抗体的三聚体形式及相同浓度的HSA蛋白向每孔中加入5μl，抗体的最终浓度为2-1250ng/ml(分成5个浓度梯度，如图7所示)。将细胞在37℃和5％CO 2的条件下培养24小时。之后通过染色后活细胞计数的方法测定各孔的活细胞的数目。将阴性对照即加入等浓度的HSA的小孔所测得的值按100％计算来评价所制备的单域抗体的细胞毒活性。在各曲线中，细胞成活率表示为平均值(n＝3)。
实验结果:参见图7，所制备的含有第一序列(SEQ ID NO:1)和第二序列(SEQ ID NO:2)的单域抗体对肺癌(A549)(图7A)，肝癌(HepG2)(图7B)和乳腺癌(MDA-MB-231)(图7C)细胞株都具有细胞毒杀伤作用。在低浓度约10ng/ml的条件下，对三种肿瘤细胞的抑制活性均超过了50％。在高浓度(250ng/ml)的情况下杀伤肿瘤细胞的活性超过90％，而对正常的血管内皮细胞HUVEC(图7D)几乎没有影响。
结果说明:本发明所公开的第一序列(SEQ ID NO:1)和第二序列(SEQ ID NO:2)的三聚体形式的单域抗体以浓度依赖性方式对三种肿瘤细胞系均显示杀细胞作用，而对正常血管内皮细胞几乎都没有影响，证明了其具有特异的肿瘤细胞杀伤的作用。
综上所述，我们经过免疫羊驼并从中筛选得到了两种能特异与DR5分子结合的单域抗体，对应的序列分别为SEQ ID NO:1和SEQ ID NO:2，经ELISA分析和ITC亲和力测定证明其能特异的与DR5分子相结合，并且结合的解离常数低于10nM。这两种单域抗体经重新设计，通过连接肽连接形成三聚体形式，在纯化并去除内毒素后在培养的多种肿瘤细胞中，可以引起特异的肿瘤细胞毒作用。本发明公开的两种DR5单域抗体单独或者经过基因工程等方法改造后，可以作为抗肿瘤药物或药物载体，如偶联药物、毒素或放射性同位素，进行肿瘤的靶向治疗。同时这两种DR5单域抗体也具有偶联荧光标记等小分子对肿瘤细胞及肿瘤病灶进行显影，起到肿瘤诊断和定位的作用。
以上所述仅为本发明较佳的实施例而已，其结构并不限于上述列举的形状，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
参考文献：
Agostini,M.,Tucci,P.,and Melino,G.(2011).Cell death pathology:Perspective for human diseases.Biochem.Biophys.Res.Commun.414,451–455.
Chang,C.-C.,Kuan,C.-P.,Lin,J.-Y.,Lai,J.-S.,and Ho,T.-F.(2015).Tanshinone IIA Facilitates TRAIL Sensitization by Up-regulating DR5 through the ROS-JNK-CHOP Signaling Axis in Human Ovarian Carcinoma Cell Lines.Chem.Res.Toxicol.28,1574–1583.
Hanahan,D.,and Weinberg,R.A.(2000).The hallmarks of cancer.Cell 100,57–70.
Ichikawa,K.,Liu,W.,Zhao,L.,Wang,Z.,Liu,D.,Ohtsuka,T.,Zhang,H.,Mountz,J.D.,Koopman,W.J.,Kimberly,R.P.,et al.(2001).Tumoricidal activity of a novel anti-human DR5 monoclonal antibody without hepatocyte cytotoxicity.Nat.Med.7,954–960.
Jayasooriya,R.G.P.T.,Choi,Y.H.,Hyun,J.W.,and Kim,G.-Y.(2014).Camptothecin sensitizes human hepatoma Hep3B cells to TRAIL-mediated apoptosis via ROS-dependent death receptor 5 upregulation with the involvement of MAPKs.Environ.Toxicol.Pharmacol.38,959–967.
Kaplan-Lefko,P.J.,Graves,J.D.,Zoog,S.J.,Pan,Y.,Wall,J.,Branstetter,D.G.,Moriguchi,J.,Coxon,A.,Huard,J.N.,Xu,R.,et al.(2010).Conatumumab,a fully human agonist antibody to death receptor 5,induces apoptosis via caspase activation in multiple tumor types.Cancer Biol.Ther.9,618–631.
Kerr,J.F.,Wyllie,A.H.,and Currie,A.R.(1972).Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics.Br.J.Cancer 26,239–257.
Lemke,J.,von Karstedt,S.,Zinngrebe,J.,and Walczak,H.(2014).Getting TRAIL back on track for cancer therapy.Cell Death Differ.21,1350–1364.
Ola,M.S.,Nawaz,M.,and Ahsan,H.(2011).Role of Bcl-2 family proteins and caspases in the regulation of apoptosis.Mol.Cell.Biochem.351,41–58.
Rosevear,H.M.,Lightfoot,A.J.,and Griffith,T.S.(2010).Conatumumab,a fully human mAb against death receptor 5 for the treatment of cancer.Curr.Opin.Investig.Drugs 11,688–698.
Yuan,X.,Gajan,A.,Chu,Q.,Xiong,H.,Wu,K.,and Wu,G.S.(2018).Developing TRAIL/TRAIL death receptor-based cancer therapies. Cancer Metastasis Rev.
Zhu,Y.,Bassoff,N.,Reinshagen,C.,Bhere,D.,Nowicki,M.O.,Lawler,S.E.,Roux,J.,and Shah,K.(2017).Bi-specific molecule against EGFR and death receptors simultaneously targets proliferation and death pathways in tumors.Sci.Rep.7,2602.

Claims (7)

  1. 一种抗DR5的单域抗体，所述单域抗体包含序列为SEQ ID NO:1或者SEQ ID NO:2或者含有至少一个CDR区域的氨基酸序列的单体或者二聚体或者多聚体，其中所述CDR区域的序列为：SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6，SEQ ID NO:7，或SEQ ID NO:8。
  2. 一种抗DR5的单域抗体，所述单域抗体包含与SEQ ID NO:1或者SEQ ID NO:2的全长序列或其至少一个CDR区域的氨基酸序列具有至少95％同源性的氨基酸序列单体，二聚体或者多聚体，其中所述CDR区域的序列为：SEQ ID NO:3，SEQ ID NO:4，SEQ ID NO:5，SEQ ID NO:6，SEQ ID NO:7，或SEQ ID NO:8。
  3. 根据权利要求1或2所述的单域抗体，其特征在于，所述二聚体或者多聚体由连接肽连接而成，所述连接肽序列为SEQ ID NO:9。
  4. 根据权利要求1或2所述的单域抗体用于制备肿瘤或癌症靶向药物的用途，所述肿瘤或癌症为肺癌、肝癌或乳腺癌。
  5. 一种肿瘤或癌症的治疗方法，所述治疗方法包括给予有效量或足量的根据权利要求1所述的抗DR5的单域抗体，所述肿瘤或癌症为肺癌、肝癌或乳腺癌。
  6. 根据权利要求5所述的治疗方法，其特征在于，所述有效量或足量为抗DR5的单域抗体的浓度不低于10ng/ml。
  7. 根据权利要求5或者6所述的治疗方法，其特征在于，所述抗DR5的单域抗体为三聚体的形式。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101717775A (zh) * 2009-11-13 2010-06-02 厦门大学 抗人死亡受体5的单链抗体基因
CN102924600A (zh) * 2012-11-14 2013-02-13 河南大学 死亡受体5激动性多价抗体及其在制备抗肿瘤药物中的应用
CN107922491A (zh) * 2015-07-16 2018-04-17 印希比公司 多价和多特异性结合dr5的融合蛋白

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101717775A (zh) * 2009-11-13 2010-06-02 厦门大学 抗人死亡受体5的单链抗体基因
CN102924600A (zh) * 2012-11-14 2013-02-13 河南大学 死亡受体5激动性多价抗体及其在制备抗肿瘤药物中的应用
CN107922491A (zh) * 2015-07-16 2018-04-17 印希比公司 多价和多特异性结合dr5的融合蛋白

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