WO2022134276A1 - 一种抗人骨桥蛋白的抗体及其应用 - Google Patents

一种抗人骨桥蛋白的抗体及其应用 Download PDF

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WO2022134276A1
WO2022134276A1 PCT/CN2021/075062 CN2021075062W WO2022134276A1 WO 2022134276 A1 WO2022134276 A1 WO 2022134276A1 CN 2021075062 W CN2021075062 W CN 2021075062W WO 2022134276 A1 WO2022134276 A1 WO 2022134276A1
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antibody
variable region
opn
heavy chain
light chain
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袁运生
周美琪
邓晴
朱建伟
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上海交通大学
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups

Definitions

  • the invention belongs to the technical field of biomedicine, in particular to an anti-human osteopontin antibody and applications thereof, in particular to an anti-human osteopontin antibody and its binding to a specific osteopontin functional epitope, thereby inhibiting the development and metastasis of tumors Applications.
  • Osteopontin is a negatively charged phosphorylated glycoprotein.
  • OPN includes various binding domains, such as arginine-glycine-aspartate (RGD) cell binding sequence, thrombin cleavage site, etc. (Barry, S.T. et al., "Analysis of the alpHa 4 beta 1 integrin-osteopontin interaction" "EXP CELL RES", No. 258, 2000).
  • Patent CN1688604A provides a recombinant anti-osteopontin antibody that can inhibit the recognition of the RGD sequence.
  • the antibody can inhibit the binding of the integrin that recognizes the SVVYGLR sequence to osteopontin or its fragments, which is a good solution for autoimmune diseases, rheumatism and rheumatoid diseases. Arthritis offers a cure.
  • the target antibody can be obtained by constructing hybridoma cell lines, establishing a phage antibody library, and the like.
  • the target antibody can be obtained by constructing hybridoma cell lines, establishing a phage antibody library, and the like.
  • researchers use naturally extracted high-purity osteopontin to prepare a hybridoma cell line that produces anti-osteopontin monoclonal antibodies.
  • the monoclonal antibody targets osteopontin to treat a variety of autoimmune diseases And a variety of cancers including breast cancer and rectal cancer, and can treat tumors at the molecular level by interfering with OPN to inhibit angiogenesis.
  • hu1A12 has the function of inhibiting the migration and invasion of breast cancer cells MDA-MB-435S.
  • hu1A12 It also showed significant inhibition of tumor growth and metastasis (Guo Yajun et al., "A humanized anti-osteopontin antibody inhibits breast cancer growth and metastasis in vivo.” Cancer Immunol Immunother (2010) 59: 355-366).
  • Shi Jinping combined Avastin (anti-VEGF humanized antibody) with a specific anti-OPN humanized antibody hu1A12 to design a humanized dual variable region that can block both VEGF and OPN at the same time.
  • Antibody DVD (Dual-Variable-Domain)-Ig AVOPN, and confirmed that the antibody can effectively inhibit tumor angiogenesis and tumor growth, and has a good anti-tumor effect (Shi Jinping. Preparation of anti-human VEGF and OPN bispecific antibodies and Biological Activity Research [D].2009.). It is suggested that anti-OPN antibody has the potential to become a therapeutic anti-tumor antibody.
  • OPN-N-terminal protein produced by the cleavage of OPN by thrombin can specifically bind to integrin ⁇ v ⁇ 3 (abnormally expressed in malignant tumors), and the RGD sequence of the OPN-N-terminal protein shows a stronger function of promoting cell adhesion (Bayless K.J. et al. , "OPN is a ligand for the alpHa4beta1integrin," Journal of Cell Science, 1998, Vol. 111, No. 9). Therefore, OPN-N-terminal protein has stronger biological functions than OPN full-length protein, and OPN-N-terminal protein will become a target with more therapeutic potential than OPN-C-terminal protein.
  • OPN As a multifunctional phosphorylated glycoprotein, OPN has been implicated in the occurrence and development of various tumors, including lung cancer.
  • the study of M. Flentje showed that compared with the control group, the cell proliferation of A549 cells transfected with siRNA OPN was significantly inhibited.
  • the cell migration rate of A549 cells downregulated by OPN was reduced by about 28% at 72h (Feldbrin Z et al., "Osteopontin levels in plasma, muscles, and bone in patient with non-healing diabetic foot ulcers: A new player in wound healing process" "Journal of Diabetes and its Complications” 2018, S1056872717315088.).
  • OPN is not only involved in the regulation of tumor cell invasion and metastasis, but studies have found that high levels of OPN promote the formation of liver fibrosis (Wang, X et al., "Osteopontin induces ductular reaction contributing to liver fibrosis” Gut, 2014, Vol. 63, No. 11 ).
  • the purpose of the present invention is to provide an antibody against human osteopontin and its application in order to overcome the deficiencies of the prior art.
  • the present invention Based on the high correlation between the change of OPN concentration and the occurrence and development of lung cancer and the important role of the N-terminal fragment generated after thrombin cleavage of OPN, the present invention describes a specific anti-osteopontin antibody that can specifically bind to the OPN-N-terminal fragment , the antibody recognizes the DLPATVFTP sequence immediately adjacent to the RGD domain in OPN and shows good efficacy against non-small cell lung cancer.
  • using the anti-OPN chimeric antibody can not only measure the content of the OPN-N fragment in the detection system, but also because the antigenic epitope specifically recognized by the anti-OPN antibody in the present invention is adjacent to the RGD functional domain, and has good anti-non-small cell The efficacy of lung cancer, it has potential medicinal value.
  • the present invention provides an antibody against human osteopontin, comprising a light chain variable region and a heavy chain variable region;
  • the CRD region sequence of the light chain variable region includes light chain CDR1, light chain CDR2, Light chain CDR3;
  • the amino acid sequence of the light chain CDR1 is QSIVHSNGNTY,
  • the amino acid sequence of the light chain CDR2 is KVS, and
  • the amino acid sequence of the light chain CDR3 is FQGSHVPPT;
  • the CRD region sequence of the heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3; the amino acid sequence of the heavy chain CDR1 is GFTFSSYA, the amino acid sequence of the heavy chain CDR2 is ITSGGSYT, and the amino acid sequence of the heavy chain CDR3 is for AREGPAWFAY.
  • the base sequence of the variable region of the heavy chain of the antibody is shown in SEQ ID NO.1
  • the base sequence of the variable region of the light chain is shown in SEQ ID NO.2;
  • the antibody is a mutant or humanized antibody comprising the aforementioned light chain variable region and heavy chain variable region, and the mutant comprises the same sequences as the aforementioned light chain variable region and heavy chain variable region.
  • An amino acid sequence with a homology of more than 50%; the humanized antibody is obtained by mutating the non-CDR region sequences in the variable region of the light chain and the variable region of the heavy chain.
  • the base sequence of the light chain variable region is shown in SEQ ID NO.6, and the base sequence of the heavy chain variable region is shown in SEQ ID NO.5.
  • the primers for amplifying the base sequence of the variable region of the light chain are shown in SEQ ID NO.7 and SEQ ID NO.8; the primers for amplifying the base sequence of the variable region of the heavy chain are shown in SEQ ID NO. ID NO.9 and SEQ ID NO.10.
  • the antibody further comprises a light chain constant region and a heavy chain constant region;
  • amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.4;
  • the base sequence of the heavy chain constant region is shown in SEQ ID NO.35, and the base sequence of the light chain constant region is shown in SEQ ID NO.36;
  • the antigenic epitope recognized by the antibody is OPN D139-P148, and the amino acid sequence is shown in SEQ ID NO.26.
  • the primers for amplifying the base sequence of the light chain variable region are shown in SEQ ID NO.11 and SEQ ID NO.12; the primers for amplifying the base sequence of the heavy chain variable region are shown in SEQ ID NO.11 and SEQ ID NO.12 ID NO.13 and SEQ ID NO.14.
  • the base sequence of the light chain DNA of the antibody is shown in SEQ ID NO.15; the base sequence of the heavy chain DNA is shown in SEQ ID NO.16.
  • the heavy chain variable region amino acid sequence of the humanized antibody is shown in SEQ ID NO.27
  • the light chain variable region amino acid sequence is shown in SEQ ID NO.28.
  • the present invention provides a recombinant plasmid comprising the aforementioned anti-human osteopontin antibody.
  • the present invention provides a method for preparing the aforementioned anti-human osteopontin antibody, comprising the following steps:
  • variable region DNA of the mouse antibody As a template, amplify the variable region DNA of the light chain variable region of the mouse antibody and the variable region DNA of the heavy chain of the mouse antibody respectively; the human antibody light chain constant region DNA and the mouse antibody light chain The variable region DNA is connected, the human antibody heavy chain constant region DNA and the mouse antibody heavy chain variable region DNA are connected, and then a recombinant plasmid is formed, which is co-transfected into the host cell;
  • the obtained cell culture supernatant is purified, eluted and dialyzed to obtain the purified anti-human osteopontin antibody.
  • step A the amino acid sequence of the murine antibody light chain variable region DNA is shown in SEQ ID NO.2; the amino acid sequence of the murine antibody heavy chain variable region DNA is shown in SEQ ID NO.1 ;
  • the amino acid sequence of the human antibody light chain constant region DNA is shown in SEQ ID NO.4; the amino acid sequence of the human antibody heavy chain constant region DNA is shown in SEQ ID NO.3.
  • the present invention provides an application according to the aforementioned anti-human osteopontin antibody in determining the content of the OPN-N fragment in the detection system; the base sequence of the OPN-N fragment is as SEQ ID NO.17 shown.
  • the present invention provides a detection kit for determining the content of OPN-N fragments in a detection system, comprising the aforementioned anti-human osteopontin antibody.
  • the present invention provides the use of the aforementioned anti-human osteopontin antibody in the preparation of a composition for inhibiting tumor proliferation and/or migration.
  • the present invention has the following beneficial effects:
  • the present invention uses the recombinant osteopontin N-terminal active domain (osteopontinI17-R167, OPN-R) as an antigen, and uses an immune library to screen to obtain a specific monoclonal antibody against the target antigen, which has the ability to inhibit non-small cells.
  • the antibody of the present invention binds to a specific functional epitope of osteopontin, can inhibit the development and metastasis of tumor, and has potential medicinal value.
  • the present invention screened out a highly specific anti-osteopontin antibody, which has good affinity with the osteopontin-N-terminal domain, and can specifically recognize the linear epitope of the bases D139-P148 of OPN.
  • the antibody can measure the content of OPN-N fragment in the detection system.
  • Fig. 1 is the affinity sorting result in Example 1;
  • Fig. 2 is the PCR detection result of step 2.1 in embodiment 2;
  • Fig. 3 is the Overlap PCR detection result of step 2.3 in embodiment 2;
  • Fig. 4 is the SDS-PAGE electrophoresis detection result in embodiment 3;
  • Fig. 5 is the ELISA detection result of anti-OPN chimeric antibody affinity detection in embodiment 4.
  • Fig. 6 is ForteBio in embodiment 4 detects anti-OPN chimeric antibody affinity detection result
  • Fig. 7 is the SDS-PAGE electrophoresis detection result of OPN truncated body expression in Example 5;
  • Fig. 8 is the anti-OPN chimeric antibody and the truncated body affinity detection result in embodiment 5;
  • Fig. 9 is the affinity detection result of OPN small peptide and anti-OPN chimeric antibody in Example 5.
  • Figure 10 is the SDS-PAGE electrophoresis detection result of OPN truncated body expression in Example 5;
  • Fig. 11 is the anti-OPN chimeric antibody and the truncated body affinity detection result in embodiment 5;
  • Fig. 12 is the affinity detection result of OPN small peptide and anti-OPN chimeric antibody in Example 5;
  • Figure 13 shows the results of the anti-OPN chimeric antibody (antibody 72B) in Example 6 inhibiting the proliferation of non-small cell lung cancer cell A549 for 48h;
  • Figure 14 is the result of the anti-OPN chimeric antibody (antibody 72B) in Example 6 inhibiting the proliferation of non-small cell lung cancer cell A549 for 72h;
  • Figure 15 is the detection result of the cell scratch test in Example 7; scale bar: 100 ⁇ m;
  • Figure 16 is the Transwell experiment detection result in embodiment 7; Scale bar: 100 ⁇ m;
  • Figure 17 is the SDS-PAGE electrophoresis result of the purified hscFV antibody in Example 8.
  • Figure 18 is the SDS-PAGE electrophoresis result of the purified scFV antibody in Example 8.
  • Figure 19 is the affinity detection result of two antibodies and antigen in Example 9;
  • FIG. 20 is a tumor growth curve obtained in Example 10.
  • the male BALB/c mice involved were purchased from Shanghai Slack Laboratory Animal Co., Ltd.; the recombinant human OPN-R was constructed, expressed and purified by the inventor's laboratory, and the specific method was consistent with the published literature (Acta Biochimica et Biophysica Sinica, 2015, 47(9):758-760); VSCM13 helper phage was purchased from Sanyou Biopharmaceutical (Shanghai) Co., Ltd.; biotin-antigen was purchased from Sanyou Biopharmaceutical (Shanghai) Co., Ltd.; HEK293E Cells were purchased from Invitrogen, USA; expression vector pET-28(a) was purchased from Merck (Novagen), Germany; expression cells (BL21(DE3)) were purchased from Shanghai Weidi Biotechnology; expression vector pcDNA3.1/His A was purchased From the American Thermo Fisher (life Technologies); HEK293 and A549 cells were purchased from the Chinese Academy of Sciences
  • 6-week-old male BALB/c mice were used as immunization objects, and recombinant human OPN-R was used as antigen.
  • the first immunization was emulsified with Freund's complete adjuvant and immunized. After 10 days, incomplete Freund's adjuvant and antigen were used. After emulsification, the 2nd, 3rd, and 4th immunizations were carried out respectively, and the interval of each immunization was 9 days. Three days after the fourth immunization, blood was collected to verify the immunization effect. After the anti-OPN-R antibody titer in the serum is greater than 8000-10000, the next experiment can be carried out.
  • the antibody library was inoculated into 2YT medium, and cultivated in a shaker at 220rpm and 37°C until the OD600 value reached 0.5-0.6.
  • VSCM13 helper phage (VSCM13 helper phage) was added, and after standing at 37°C for 30min, cultured in a shaker at 220rpm and 37°C for 1h. Replaced with C+/K+2YT, and cultivated overnight at 225rpm and 30°C. Centrifuge at 13,500 ⁇ g for 5 min, mix the supernatant with PEG6000/NaCl, and incubate on ice for 1-2 hours or overnight.
  • the input amount refers to the titer of the antibody phage library used for screening, and the amount of phage obtained by binding and elution with the antigen is called the screening amount.
  • the ratio of antibody phages to the antigen The larger the ratio, the greater the amount of specific antibody phages that bind to the target antigen in the antibody phage library.
  • 96 clones were selected from the phages screened in the 4th round for ELISA primary screening, and the target antibody sequence was finally screened.
  • the candidate antibody was screened by ELISA, and two high-affinity positive clones (named Y72B-2 and Y72B-3 respectively) were screened out, and the single-chain antibody was extracted and inserted.
  • the expression plasmid of the antibody DNA was verified after expression in E. coli. After the above two positive clones were cultured in 50 ml volume, 0.2 mM isopropyl ⁇ -D-thiogalactoside ( IPTG), and after culturing for 20 hours, the cells were collected.
  • the cells were resuspended in 2 ml of PBS, and then disrupted on ice with a sonicator (200 W, sonicated for 3 minutes). Centrifuge at 12,000g for 30 minutes at 4°C. After the lysed supernatant was measured for antibody concentration, the obtained supernatant was subjected to gradient dilution, with an initial concentration of 2 ⁇ g/ml, which was mixed and diluted with PBS step by step. At the same time, an affinity ELISA was performed with recombinant human OPN-R coated with a concentration of 2 ⁇ g/ml, and a monoclonal antibody (nM) with an affinity level in the nanomolar order was obtained.
  • nM monoclonal antibody
  • Y72B-2 and Y72B-3 encode the same antibody sequence, which is derived from mouse antibody sequence, hereinafter referred to as mouse antibody.
  • the amino acid sequence of the variable region of the antibody heavy chain encoded by Y72B-2 is shown in SEQ ID NO.1, which contains the key domains CDR1, CDR2, CDR3 and framework domains FR1, FR2, FR3, etc. for recognizing antigens.
  • the amino acid sequence of the light chain variable region encoded by Y72B-2 is shown in SEQID NO.2, which includes the recognition antigen domains CDR1, CDR2, CDR3 and framework domains FR1, FR2, FR3, etc., see Table 2 and Table 3 for details .
  • the results show that the screened antibody has good specific recognition and binding ability to the N-terminal domain of OPN.
  • the DNA sequence (SEQ ID NO.31, coding amino acid sequence SEQ ID) of the variable region of the antibody light chain with secretion signal peptide was synthesized by gene synthesis method. NO.32), using this as a template, primer 1 and primer 2 were used as the upstream primer and downstream primer of the variable region DNA of the mouse antibody light chain; in the same way, the antibody with the secretion signal peptide was synthesized by the gene synthesis method.
  • the DNA sequence of the heavy chain variable region (SEQ ID NO.33, encoding amino acid sequence SEQ ID NO.34) is used as a template, and primer 3 and primer 4 are respectively the upstream primers of the murine antibody heavy chain variable region DNA PCR amplification was carried out with downstream primers.
  • the PCR reaction conditions were: melting at 95°C for 30 seconds; annealing at 60°C for 30 seconds; extension at 72°C for 10 seconds, with 34 cycles.
  • the human IgG1 antibody light chain DNA was PCR amplified with primers 5 and 6, and the human IgG1 antibody heavy chain DNA was PCR amplified with primers 7 and 8, and the PCR reaction conditions were the same as before.
  • human antibody heavy chain constant region DNA sequence (amino acid sequence as shown in SEQ ID NO.3, base sequence as shown in SEQ ID NO.35), human antibody light chain constant region DNA sequence (amino acid sequence as shown in SEQ ID NO.35), human antibody light chain constant region DNA sequence (amino acid sequence as shown in SEQ ID NO. .4, the base sequence is shown in SEQ ID NO.36), the DNA sequence of the variable region of the mouse antibody heavy chain (the amino acid sequence is shown in SEQ ID NO.1, and the base sequence is shown in SEQ ID NO.5). shown), mouse antibody light chain variable region DNA sequence (amino acid sequence shown in SEQ ID NO.2, base sequence shown in SEQ ID NO.6).
  • the PCR detection results are shown in Figure 2, and the results show that the target fragments were successfully obtained.
  • Antibody 72B light chain variable region (VL) band size is 336bp
  • antibody 72B heavy chain variable region (VH) band size is 351bp
  • human IgG1 light chain constant region (IgG1-CL) band size is 333bp
  • human The size of the IgG1 heavy chain constant region (IgG1-CH) band was 1005 bp. The results showed that the size of each gene band was similar to the expected band, indicating that the corresponding target band was successfully amplified.
  • Primer 1 (SEQ ID NO.7) 5'-CTAGTCTAGAATGAGGGTCCTTGCTGAG-3'
  • Primer 2 (SEQ ID NO. 8) 5'-CCGGAATTCGGTCCGCTTGATCTCTTTGATTTCCAGCTTCCGGAATTCGGTCCGCTTGATCTCTTTGATTTCCAGCTT-3'
  • Primer 3 (SEQ ID NO.9) 5'-CTAGTCTAGAGAAGTGATGCTGGTGGAG-3'
  • Primer 4 (SEQ ID NO. 10) 5'-CCGGAATTCCGCGCTGCTCACGGTTGCAGAGACAGTGACCCGGAATTCCGCGCTGCTCACGGTTGCAGAGACAGTGAC-3'
  • Primer 5 (SEQ ID NO. 11) 5'-CTAGTCTAGAAAGCTGGAAATCAAAGAGATCAAGCGGACCCTAGTCTAGAAAGCTGGAAATCAAAGAGATCAAGCGGACC-3'
  • Primer 6 (SEQ ID NO. 12) 5'-CCGGAATTCTCAGCACTCGCCCCGGTT-3'
  • Primer 7 (SEQ ID NO. 13) 5'-CTAGTCTAGAGTCACTGTCTCTGCAACCGTGAGCAGCGCGCTAGTCTAGAGTCACTGTCTCTGCAACCGTGAGCAGCGCG-3'
  • Primer 8 (SEQ ID NO. 14) 5'-CCGGAATTCTCACTTCCCGGGGCTCAG-3'
  • the endotoxin-free anti-OPN chimeric antibody light chain DNA (72B-L) and heavy chain DNA (72B-H) amplified in Example 2 were used to construct an OPN gene eukaryotic expression vector through the pcDNA3.1/His A vector,
  • the obtained cell culture supernatant is purified by Protein A affinity chromatography column, and the column is equilibrated with 10 times the column volume of 20mM phosphate buffer (pH 7.2).
  • the sample was loaded at a speed of 0.5mL/min. After loading the sample, elute with 10 times the volume of 0.1M citric acid buffer (pH 3.0), recover the protein product with an Ep tube pre-added with 1M Tris-HCl buffer, and then equilibrate the column with phosphate buffer. The eluted protein product was placed in a dialysis bag with a pore size of 10KD and placed in a PBS buffer (4°C) for overnight dialysis. Finally, the target protein, the anti-OPN chimeric antibody, was obtained. The SDS-PAGE electrophoresis detection chart of the obtained antibody is shown in Figure 4.
  • the molecular weight of the reduced anti-OPN chimeric antibody (antibody 72B, lane 2 in Figure 4) in the figure is 25KD and 50KD, which is consistent with the theoretical molecular weight of the target protein , indicating that the correct expression product was obtained.
  • the purified target antibody showed only two bands, which were heavy chain and light chain, and almost did not contain other proteins, which proved that the prepared antibody 72B was of high purity.
  • Example 4 Affinity of anti-OPN chimeric antibody to antigen
  • OPN-N-terminal protein (its base sequence is shown in SEQ ID NO. 17)
  • OPN full-length protein (OPN-FL) (its base sequence is shown in SEQ ID NO. 18) with ELISA plates were coated at a concentration of 2 ⁇ g/mL and left overnight at 4°C. After discarding the coating solution, the cells were blocked with 3% BSA at room temperature for 1 h, anti-OPN chimeric antibodies of different concentration gradients were added to the treatment group, and human IgG antibody of the same concentration as the treatment group was added to the control group, and incubated at room temperature for 1 h.
  • OPN-R OPN-N-terminal protein
  • OPN-FL OPN full-length protein
  • the equilibrium dissociation constant KD of the anti-OPN chimeric antibody and the antigen OPN-R was detected by ForteBio method. After the antigen OPN-R was biotinylated with the BLK-NH 2 kit (Dojin Chemical), streptavidin was used. The sensor (SA Sensor) immobilized biotinylated OPN-R on a 96-well plate at a concentration of 100 nmol/L. After equilibrating the probe with PBS, it was mixed with anti-OPN chimeric antibodies (50 nmol/L, 100 nmol/L) diluted in a gradient manner. , 200nmol/L, 300nmol/L) combined. PBS was used as blank control.
  • the binding time was 600s and the dissociation time was 900s.
  • a 1:1 binding model was used to fit the binding and dissociation curves to obtain the affinity constant.
  • the results are shown in Figure 6. The results show that the fitted curve has a high degree of agreement with the actual curve, and the distribution between the curves is even.
  • OPN-N2 OPN-D93
  • OPN-N3 OPN I17-T131
  • OPN-N I17-T169, ie OPN-R in Figure 8
  • the expression vector pET-28(a) was used to synthesize and construct OPN-N2 fusion 6 ⁇ His tag recombinant protein and OPN-N3 fusion 6 ⁇ His tag recombinant protein, and use Escherichia coli (BL21(DE3)) for soluble expression.
  • the results are shown in Fig. 7, which show that the truncated bodies OPN-N2 and OPN-N3 were successfully constructed and expressed.
  • OPN-N2, OPN-N3 and OPN-N were diluted with coating buffer to 2 ⁇ g/mL and coated overnight at 4°C. Pour off the coating solution, pat dry, add PBS 300 ⁇ L/well, 5min each time. Blocked with 1% BSA 100 ⁇ L/well for 1 h at room temperature and then discarded. The antibody 72B was diluted from 100 pmol/mL to 0.0002 pmol/mL, and 100 ⁇ L was added to each well. PBS was used as a negative control. After incubation at room temperature for 1 h, the cells were washed with PBS.
  • the anti-OPN chimeric antibody cannot recognize the OPNI17-T131 However, it has a strong affinity with OPNI17-T169, so it is speculated that its epitope recognition region is located between OPN S129-R168, and based on this, three overlapping peptides hOPN-pt4 (its amino acid sequence) encoding OPN S129-R168 were designed and synthesized. As shown in SEQ ID NO.21), hOPN-pt5 (its amino acid sequence is shown in SEQ ID NO.22), hOPN-pt6 (its amino acid sequence is shown in SEQ ID NO.23), to more precisely locate the antigen table bit.
  • OPN D139-P148 is the epitope recognized by the anti-OPN chimeric antibody.
  • the expression vector pET-28(a) to construct a truncated OPN-148P (namely OPNI17-P148) containing the sequence of OPN D139-P148.
  • the sequence is shown in SEQ ID NO.24
  • the truncated body OPN-138T OPN I17-T138, the sequence is shown in SEQ ID NO.25
  • the expression vector pET-28(a) was used to synthesize and construct OPN-148P fusion 6 ⁇ His tag recombinant protein and OPN-138T fusion 6 ⁇ His tag recombinant protein, and use Escherichia coli (BL21(DE3)) for soluble expression.
  • the SDS-PAGE electrophoresis results of the truncations OPN-148P and OPN-138T are shown in Figure 10, and the results show that the target truncations were successfully constructed and expressed.
  • Figure 11 shows the ELISA results of truncated OPN-148P and OPN-138T for determining the affinity of antibody 72B.
  • hOPN-pt7 (OPN D139-P148) (see SEQ ID NO. 26 for the sequence) was used to further confirm that the anti-OPN chimeric antibody not only recognizes the spatial epitope of OPN D139-P148, but also can specifically bind to the space between OPN D139-P148 Linear structures formed by consecutive residues.
  • the ELISA results of hOPN-pt7 and anti-OPN chimeric antibody (antibody 72B) are shown in FIG. 12 . The results showed that at a certain concentration, hOPN-pt7 had specific affinity with the anti-OPN chimeric antibody 72B. It was demonstrated that antibody 72B can specifically bind to the linear structure formed by consecutive amino acid residues between OPN D139-P148.
  • Example 6 Inhibition of cell proliferation by anti-OPN chimeric antibodies
  • the following method was used to investigate whether anti-OPN chimeric antibodies inhibit the proliferation of A549 cells.
  • First take A549 cells with good growth status, adjust the cell concentration to 2.5 ⁇ 10 4 cells/mL, inoculate 100 ⁇ L per well in a 96-well cell culture plate, and culture in a 37°C, 5% CO 2 incubator overnight, after 24 h The serum-free medium was replaced, and the cells were divided into a blank control group and an anti-OPN chimeric antibody group (the anti-OPN chimeric antibody obtained in Example 3, the concentrations were 1.25, 2.5, 5, 10, and 20 ⁇ g/mL, respectively).
  • Set 5 parallel wells with concentration gradient observe and record cell morphological changes at 48h and 72h after treatment respectively.
  • Example 7 Inhibition of cell migration by anti-OPN chimeric antibodies
  • A549 cells were adjusted to 2 ⁇ 10 5 cells/mL with F-12 medium containing 10% FBS, and 1 mL was inoculated into 24 wells In the plate, after culturing overnight to make the cells adhere, use a yellow pipette tip to scratch the monolayer cultured cells in a "1" shape from top to bottom along the upper part of the culture plate. PBS was washed to remove floating cells in the culture medium, and replaced with serum-free medium.
  • the experimental group was added with an anti-OPN chimeric antibody (obtained in Example 3) at a final concentration of 20 ⁇ g/mL, and the PBS group was added with the same volume of the antibody in the experimental group.
  • PBS record the relative distance of the scratch area 0h under the microscope, and mark the recorded area. Incubate at different time points in a 37°C cell incubator, observe and record the recovery at the same position under a microscope.
  • the results are shown in Figure 15. The results show that the wound healing degree of the experimental group was significantly lower than that of the PBS group without antibody, indicating that the antibody 72B can significantly inhibit the migration of A549 cells at a concentration of 20 ⁇ g/mL.
  • the inhibitory effect of anti-OPN chimeric antibody on the migration of A549 cells was detected by Transwell assay.
  • a 24-well Transwell chamber system polycarbonate membrane pore size was 8 ⁇ m
  • the density of mL was resuspended in serum-free F-12 medium.
  • 200 ⁇ L of cell suspension was added to the upper chamber and 600 ⁇ L of F-12 medium supplemented with 10% FBS was added to the lower chamber as a chemotactic agent.
  • the treatment group was added with anti-OPN chimeric antibody (obtained in Example 3) at a final concentration of 20 ⁇ g/mL in the lower chamber, and the control group was added with the same volume of PBS as the experimental group antibody 72B in the lower chamber.
  • the chamber system was incubated in a 37°C cell incubator for 24 hours, the cells in the upper layer of the chamber were wiped off with a cotton swab, the chamber was washed with PBS, and then fixed in 4% paraformaldehyde for 30 min at room temperature. Crystal violet was stained for 30 min, the residual dye was washed away with PBS, and four areas were randomly selected and photographed under a microscope (magnification: ⁇ 100). The results are shown in Figure 16.
  • the human IgG sequence with the highest amino acid sequence similarity was selected as the template, and the non-CDR region sequences in the variable regions of the 72B light and heavy chains were mutated according to the human IgG sequence. , to improve the similarity between the variable region sequence of 72B antibody and the corresponding sequence of human IgG.
  • the mutated heavy chain variable region amino acid sequence is shown in SEQ ID No.27, and the light chain variable region amino acid sequence is shown in SEQ ID No.28.
  • hscFV humanized anti-OPN single-chain antibody
  • the synthetic gene sequence is shown in SEQ ID NO.29.
  • the synthetic hscFV expression sequence was constructed into the commercial expression vector pET28a(+) by a general method.
  • the 72B antibody was used as a template to construct a mouse-derived anti-OPN single-chain antibody (scFV) by a general molecular cloning method.
  • VL and The VHs were linked with 3 repeating GGGGS peptide chains, and the DNA sequence (see SED ID NO.30) was also inserted into pET28a(+).
  • Escherichia coli BL21 (DE3) was used as the host strain, and the induction conditions and expression methods were referred to in the literature (Journal of Biotechnology, 2000, 77: 169–178), and two antibodies, hscFV and scFV, were expressed by the method of inclusion body expression.
  • the SDS-PAGE electrophoresis results of the purified hscFV antibody are shown in Figure 17.
  • lane 1 represents the flow-through fraction
  • 2 and 3 are 40mM imidazole elution fractions
  • 4 and 5 are 80 mM imidazole elution fractions.
  • Fractions, 6 and 7 are 100 mM imidazole eluted fractions
  • 8 and 9 are 200 mM imidazole eluted fractions
  • 10 and 11 are 300 mM imidazole eluted fractions
  • 12 and 13 are 500 mM imidazole eluted fractions.
  • the SDS-PAGE electrophoresis results of the purified scFV antibodies are shown in Figure 18.
  • Lane 1 in the figure represents the flow-through fraction
  • 2 and 3 are 40 mM imidazole elution fractions
  • 4 and 5 are 80 mM imidazole elution fractions
  • 6 and 6 7 is a 100 mM imidazole elution fraction
  • 8 and 9 are a 200 mM imidazole elution fraction
  • 10 and 11 are a 300 mM imidazole elution fraction
  • 12 and 13 are a 500 mM imidazole elution fraction.
  • Example 9 Affinity of anti-OPN hscFV to antigen
  • the samples of scFV or hscFV were diluted by 2 times, the concentration was 10 ⁇ g/mL, and the serial dilution was 7 times, and the sample concentrations were 10, 5, 2.5, 1.25, 0.625, 0.313, 0.16 ⁇ g/mL and other samples, respectively.
  • 100 ⁇ l of each sample was added to the wells coated with OPN-R, and three replicate wells were set up for each sample. Then incubate at 37°C for 2 hours, discard the samples in the ELISA plate, and repeat the washing 4 times for 5 minutes each time. Add 1:20,000 freshly diluted HRP-mouse anti-His antibody (Protech Biotech, USA), 100 ⁇ L/well, incubate at 37° C.
  • Example 10 In vivo antitumor activity of anti-OPN antibody 72B
  • the anti-OPN chimeric antibody 72B obtained in Example 3 was used for in vivo anti-tumor pharmacodynamics experiments.
  • Ten 8-week-old male Nude nude mice were inoculated with 5 ⁇ 10 6 non-small cell lung cancer A549 cells in the armpit of each mouse, and 1 hour after inoculation, all mice were randomly divided into 2 groups with 5 mice in each group.
  • mice in the experimental group were injected with 50 ⁇ g of 72B antibody (OPN-Ab(72B) in Figure 20) via tail vein, and mice in the control group were given an equal volume of PBS as a control (Control in Figure 20). After the start of the experiment, it was administered twice a week until the end of the experiment.

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Abstract

一种抗人骨桥蛋白的抗体及其应用，所述抗体包括轻链可变区、重链可变区；所述轻链可变区的CRD区序列包括轻链CDR1、轻链CDR2、轻链CDR3；所述重链可变区的CRD区序列包括重链CDR1、重链CDR2、重链CDR3。所述重链可变区氨基酸序列如SEQ ID NO.1所示，轻链可变区氨基酸序列如SEQ ID NO.2所示。抗体可特异结合OPN-N端片段，该抗体识别OPN中紧邻RGD域的DLPATEVFTP序列；利用该抗OPN嵌合抗体可测定检测体系中OPN-N片段的含量。同时，由于抗OPN抗体特异识别的抗原表位紧邻RGD功能域，且具有良好的抗非小细胞肺癌的功效，具有潜在的药用价值。

Description

一种抗人骨桥蛋白的抗体及其应用 技术领域
本发明属于生物医药技术领域，具体涉及一种抗人骨桥蛋白的抗体及其应用，尤其涉及一种抗人骨桥蛋白的抗体及其结合特异的骨桥蛋白功能表位从而抑制肿瘤的发展和转移的应用。
背景技术
骨桥蛋白(Osteopontin,OPN)是一种带负电荷的磷酸化糖蛋白。OPN包括多种结合区，如精氨酸-甘氨酸-天冬氨酸(RGD)细胞结合序列，凝血酶切割位点等(Barry,S.T.等，“Analysis of the alpHa 4 beta 1 integrin-osteopontin interaction”《EXP CELL RES》,2000年第258期)。在生物体内，骨桥蛋白中第168位氨基酸被凝血酶剪切后，骨桥蛋白在L和R残基之间裂解，形成OPN-N端片段和OPN-C端片段，并暴露出隐藏的整合素结合位点(SVVYGLR)，该位点可结合整合素β1诱导细胞迁移。专利CN1688604A提供了一种可抑制识别RGD序列的重组抗骨桥蛋白抗体，该抗体能抑制识别SVVYGLR序列的整合素与骨桥蛋白或其片段结合，为自身免疫性疾病、风湿病及类风湿性关节炎提供了治疗方法。
在单克隆抗体制备过程中，可通过构建杂交瘤细胞株，建立噬菌体抗体库等方式获得目的抗体。例如在专利CN101891814A中，研究者利用天然提取的高纯度骨桥蛋白制备出产生抗骨桥蛋白单克隆抗体的杂交瘤细胞株，该单抗以骨桥蛋白为靶点，治疗多种自身免疫疾病及包括乳腺癌、直肠癌在内的多种癌症，同时可通过干扰OPN以抑制血管形成，从分子水平治疗肿瘤。
在抗体治疗方面，研究者已筛选制备出针对OPN不同表位的抗体，并证实了抗体对OPN部分生物学功能的阻断作用。彭玲等报道了一种抗原决定簇位于OPN-C端片段的鼠源抗人OPN单链抗体-Fc融合蛋白1A12(彭玲.抗骨桥蛋白单链抗体可抑制乳腺癌转移和血管生成[D].第二军医大学,2007.)，并证实了该抗体对乳腺癌转移和肿瘤血管生成的抑制作用。该团队利用计算机建模将互补决定区移植使其人源化得到hu1A12抗体，结果证实hu1A12具有抑制乳腺癌细胞MDA-MB-435S迁移与侵袭的功能，在乳 腺癌小鼠肺转移模型中，hu1A12也显示出明显的抑制肿瘤生长与转移功效(郭亚军等，“A humanized anti-osteopontin antibody inhibits breast cancer growth and metastasis in vivo.”《Cancer Immunol Immunother》(2010)59:355–366)。在此基础上，石金平等将Avastin(抗VEGF人源化抗体)与特异性抗OPN的人源化抗体hu1A12联合应用，设计了可同时阻断VEGF和OPN的双可变区的人源化抗体DVD(Dual-Variable-Domain)-Ig AVOPN，并证实该抗体可有效抑制肿瘤血管的生成与肿瘤生长，具有良好的抗肿瘤作用(石金平.抗人VEGF及OPN双特异性抗体的制备及生物学活性研究[D].2009.)。提示抗OPN抗体有潜力成为治疗性抗肿瘤抗体。
OPN经凝血酶切割后产生的OPN-N端蛋白可特异结合整合素αvβ3(在恶性肿瘤中异常表达)，且OPN-N端蛋白的RGD序列显示出更强的促进细胞黏附功能(Bayless K.J.等，“OPN is a ligand for the alpHa4beta1integrin”《Journal of Cell Science》，1998年第111卷第9期)。因此，OPN-N端蛋白具有比OPN全长蛋白具有更强的生物学功能，OPN-N端蛋白将成为比OPN-C端蛋白更具治疗潜力的靶点。
OPN作为多功能磷酸化糖蛋白与多种肿瘤的发生发展有关，包括肺癌。M.Flentje的研究表明，与siRNA OPN转染的A549细胞与对照组相比，细胞增殖受到明显抑制，Transwell实验中，OPN下调的A549细胞分别在72h细胞迁移率降低了约28％(Feldbrin Z.等，“Osteopontin levels in plasma,muscles,and bone in patient with non-healing diabetic foot ulcers:A new player in wound healing process”《Journal of Diabetes and its Complications》2018年S1056872717315088.)。同时，OPN在非小细胞肺癌患者体内的表达水平也被证实与肿瘤进展密切相关(Hu,Z.等，“Overexpression of Osteopontin Is Associated with More Aggressive PHenotypes in Human Non-Small Cell Lung Cancer”《Clinical Cancer Research》，2005年第11卷第13期)。
OPN不仅参与调控肿瘤细胞侵袭转移调控，研究发现高水平的OPN促进肝脏纤维化的形成(Wang，X等，“Osteopontin induces ductular reaction contributing to liver fibrosis”《Gut》,2014年第63卷第11期)。在丙型肝炎诱导肝纤维化肝脏患者肝脏组织中，免疫组化显示OPN表达在肝实质细胞中，并且通过动物模型研究证实肝细胞表达的OPN可以通过上调HMGB1蛋白表达诱导肝星状细胞分泌I型胶原蛋白，引起肝纤维化(Elena Arriazu等，“Signalling via the osteopontin and high mobility group box-1axis drives the fibrogenic response to liver injury”《Gut》,2017年第66卷第6期)。
发明内容
本发明的目的是为了克服现有技术的不足，提供一种抗人骨桥蛋白的抗体及其应用。基于OPN浓度变化与肺癌发生发展的高度相关性以及凝血酶酶切OPN后产生的N端片段的重要作用，本发明记载了一种可特异结合OPN-N端片段的特异性抗骨桥蛋白抗体，该抗体识别OPN中紧邻RGD域的DLPATEVFTP序列，并显示出良好的抗非小细胞肺癌功效。因此，利用该抗OPN嵌合抗体不仅可测定检测体系中OPN-N片段的含量，同时，由于本发明中抗OPN抗体特异识别的抗原表位紧邻RGD功能域，且具有良好的抗非小细胞肺癌的功效，故具有潜在的药用价值。
本发明的目的是通过以下技术方案实现的：
第一方面，本发明提供了一种抗人骨桥蛋白的抗体，包括轻链可变区、重链可变区；所述轻链可变区的CRD区序列包括轻链CDR1、轻链CDR2、轻链CDR3；所述轻链CDR1的氨基酸序列为QSIVHSNGNTY，轻链CDR2的氨基酸序列为KVS，轻链CDR3的氨基酸序列为FQGSHVPPT；
所述重链可变区的CRD区序列包括重链CDR1、重链CDR2、重链CDR3；所述重链CDR1的氨基酸序列为GFTFSSYA，重链CDR2的氨基酸序列为ITSGGSYT，重链CDR3的氨基酸序列为AREGPAWFAY。
优选地，所述抗体的重链可变区的碱基序列如SEQ ID NO.1所示，轻链可变区的碱基序列如SEQ ID NO.2所示；
或所述抗体为包括以前述轻链可变区和重链可变区为基础的突变体或人源化抗体，所述突变体包括与前述轻链可变区和重链可变区序列具有50％以上同源性的氨基酸序列；所述人源化抗体包括对前述轻链可变区和重链可变区中的非CDR区序列进行突变后得到。
优选地，所述轻链可变区的碱基序列如SEQ ID NO.6所示，重链可变区的碱基序列如SEQ ID NO.5所示。
优选地，扩增所述轻链可变区的碱基序列的引物如SEQ ID NO.7和SEQ ID NO.8所示；扩增所述重链可变区的碱基序列的引物如SEQ ID NO.9和SEQ ID NO.10所示。
优选地，所述抗体还包括轻链恒定区、重链恒定区；
所述重链恒定区的氨基酸序列如SEQ ID NO.3所示，轻链恒定区的氨基酸序列如SEQ ID NO.4所示；
所述重链恒定区的碱基序列如SEQ ID NO.35所示，轻链恒定区的碱基序列如SEQ ID NO.36所示；
所述抗体识别的抗原表位为OPN D139-P148，氨基酸序列如SEQ ID NO.26所示。
优选地，扩增所述轻链可变区的碱基序列的引物如SEQ ID NO.11和SEQ ID NO.12所示；扩增所述重链可变区的碱基序列的引物如SEQ ID NO.13和SEQ ID NO.14所示。
优选地，所述抗体的轻链DNA的碱基序列如SEQ ID NO.15所示；重链DNA的碱基序列如SEQ ID NO.16所示。
优选地，所述人源化抗体的的重链可变区氨基酸序列如SEQ ID NO.27所示，轻链可变区氨基酸序列如SEQ ID NO.28所示。
第二方面，本发明提供了一种包含前述的抗人骨桥蛋白的抗体的重组质粒。
第三方面，本发明提供了一种前述的抗人骨桥蛋白的抗体的制备方法，包括以下步骤：
A、筛选获得分泌抗OPN抗体的单克隆杂交瘤细胞，并获得其鼠源抗体可变区DNA；
B、以鼠源抗体可变区DNA为模板，分别扩增得到鼠源抗体轻链可变区DNA和鼠源抗体重链可变区DNA；将人抗体轻链恒定区DNA和鼠抗体轻链可变区DNA连接、人抗体重链恒定区DNA和鼠抗体重链可变区DNA连接，然后形成重组质粒，共转染宿主细胞；
C、待宿主细胞活率为50％以下时，将获得的细胞培养上清液进行纯化、洗脱、透析，即得纯化后的抗人骨桥蛋白的抗体。
优选地，步骤A中，所述鼠源抗体轻链可变区DNA的氨基酸序列如SEQ ID NO.2所示；鼠源抗体重链可变区DNA的氨基酸序列如SEQ ID NO.1所示；
所述人抗体轻链恒定区DNA的氨基酸序列如SEQ ID NO.4所示；人抗体重链恒定区DNA的氨基酸序列如SEQ ID NO.3所示。
第四方面，本发明提供了一种根据前述的抗人骨桥蛋白的抗体的在测定检测体系中OPN-N片段含量中的应用；所述OPN-N片段的碱基序列如SEQ ID NO.17所示。
第五方面，本发明提供了一种测定检测体系中OPN-N片段含量的检测试剂盒，包括前述的抗人骨桥蛋白的抗体。
第六方面，本发明提供了一种根据前述的抗人骨桥蛋白的抗体的在制备抑制肿瘤增殖和/或迁移的组合物中的应用。
现有技术相比，本发明具有如下的有益效果：
1、本发明以重组骨桥蛋白N端活性结构域(osteopontinI17-R167,OPN-R)作为抗原，使用免疫库进行筛选，得到针对靶点抗原的特异性单克隆抗体，其具有抑制非小细胞肺 癌细胞A549增殖与迁移的功能。
2、本发明的抗体结合特异的骨桥蛋白功能表位，可抑制肿瘤的发展和转移，具有潜在药用价值。
3、本发明筛选出一种高特异性抗骨桥蛋白抗体，它与骨桥蛋白-N端结构域具有良好亲和力，可特异性识别OPN第D139-P148碱基的线性表位，因此，利用该抗体可测定检测体系中OPN-N片段的含量。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为实施例1中的亲和力排序结果；
图2为实施例2中步骤2.1的PCR检测结果；
图3为实施例2中步骤2.3的Overlap PCR检测结果；
图4为实施例3中的SDS-PAGE电泳检测结果；
图5为实施例4中的ELISA检测抗OPN嵌合抗体亲和力检测结果；
图6为实施例4中的ForteBio检测抗OPN嵌合抗体亲和力检测结果；
图7为实施例5中OPN截短体表达的SDS-PAGE电泳检测结果；
图8为实施例5中的抗OPN嵌合抗体与截短体亲和力检测结果；
图9为实施例5中的OPN小肽与抗OPN嵌合抗体亲和力检测结果；
图10为实施例5中OPN截短体表达的SDS-PAGE电泳检测结果；
图11为实施例5中的抗OPN嵌合抗体与截短体亲和力检测结果；
图12为实施例5中的OPN小肽与抗OPN嵌合抗体亲和力检测结果；
图13为实施例6中抗OPN嵌合抗体(抗体72B)抑制非小细胞肺癌细胞A549增殖48h的结果；
图14为实施例6中抗OPN嵌合抗体(抗体72B)抑制非小细胞肺癌细胞A549增殖72h的结果；
图15为实施例7中细胞划痕实验检测结果；比例尺：100μm；
图16为实施例7中Transwell实验检测结果；比例尺：100μm；
图17为实施例8中纯化后hscFV抗体的SDS-PAGE电泳结果；
图18为实施例8中纯化后的scFV抗体的SDS-PAGE电泳结果；
图19为实施例9中两个抗体与抗原的亲和力检测结果；
图20为实施例10中获得的肿瘤生长曲线。
具体实施方式
下面结合具体实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进。这些都属于本发明的保护范围。
本发明的实施例中，涉及的雄性BALB/c小鼠购自上海斯莱克实验动物有限公司；重组人OPN-R由发明人所在实验室构建、表达和纯化获得，具体方法与已发表文献一致(Acta Biochimica et Biophysica Sinica,2015,47(9):758-760)；VSCM13 helper phage购自三优生物医药(上海)有限公司；biotin-抗原购自三优生物医药(上海)有限公司；HEK293E细胞购自美国invitrogen公司；表达载体pET-28(a)购自德国默克公司(Novagen)；表达细胞(BL21(DE3))购自上海唯地生物科技；表达载体pcDNA3.1/His A购自美国赛默飞世尔公司(life Technologies)；HEK293和A549细胞购自中国科学院细胞库；抗体纯化介质(proteinA)购自美国通用生物公司(GE)。
实施例1：小鼠单克隆抗体的筛选
1.1小鼠免疫
6周龄的雄性BALB/c小鼠作为免疫对象，重组人OPN-R作为抗原，第一次免疫与弗氏完全佐剂乳化后进行免疫，10天之后，采用弗氏不完全佐剂和抗原乳化后分别进行第2、3、4次免疫，每次免疫间隔9天。第4次免疫后3天，采血验证免疫效果。血清中抗OPN-R抗体滴度大于8000-10000后即可进行下一步实验。
1.2从抗体库筛选结合OPN的噬菌体
将抗体库接种到2YT培养基中,220rpm,37℃摇床中培养直到OD600值达到0.5-0.6。加入VSCM13辅助噬菌体(VSCM13 helper phage)，37℃静止30min后，220rpm 37℃摇床中培养1h。置换成C+/K+2YT，225rpm 30℃培养过夜。13,500×g离心5min,将上清与PEG6000/NaCl混合，冰上孵育1-2小时或过夜。13,500×g，4℃，离心10min，去上清；适当体积的PBS重悬。将重悬后得到的噬菌体悬液与biotin-抗原包被得Dynabeads孵育，并按照Kingfisher磁珠筛选系统方法进行结合和清洗。用Trypsin洗脱噬菌体。洗脱后噬菌体溶液与对数期SS320细胞充分混合后37℃静置培养。涂布在2YT-Car+-Tet+平板上，同时进行噬菌体滴度检测，37℃培养箱过夜培养。计算噬菌体筛出量，投入量等，刮板并进行2-3轮筛选。由此得到抗体库海选的噬菌体滴 度测定结果如表1所示。
表1
筛选轮次 筛出量 投入量 筛出量/筛出量
1 2.00E+08 7.00E+12 0.0029％
2 1.05E+09 7.50E+12 0.014％
3 5.00E+06 1.00E+11 0.005％
4 1.50E+08 5.00E+11 0.030％
由表1的结果可见，投入量是指用于筛选的抗体噬菌体库滴度，经过和抗原结合洗脱获得的噬菌体量称为筛出量，筛出量和投入量的比值反映了带有结合抗原的抗体噬菌体的比例，这个比值越大说明了抗体噬菌体库中带有结合目的抗原的特异性抗体噬菌体量越多。通过4轮筛选后，从第4轮筛选出的噬菌体中，选择96个克隆进行ELISA初筛，最后筛选出目的抗体序列。
1.3筛选出单链抗体与OPN的N端结构域的反应性
对于1.2步骤中第4轮筛出的单链抗体噬菌体展示库采用ELISA法进行候选抗体筛选，筛选出2个高亲和力的阳性克隆(分别命名为Y72B-2,Y72B-3)，抽取插入单链抗体DNA的表达质粒，并进行大肠杆菌表达后验证，将上述2个阳性克隆分别进行50ml体积培养后，在20℃条件下，加入0.2mM的异丙基β-D-硫代半乳糖苷(IPTG)，继续培养20小时后，收集菌体。用PBS洗涤菌体后，用2ml的PBS重新悬浮菌体，然后在冰上，用超声波破碎仪进行破碎(200W，超声3分钟)。在4℃条件下12000g离心力下离心30分钟，对裂解的上清进行测定抗体浓度后，对得到的上清分别进行梯度稀释，初始浓度为2微克/毫升，按照与PBS进行逐级混合稀释。同时与包被有2微克/毫升浓度重组人OPN-R进行亲和力ELISA测定，得到了亲和力水平在纳摩尔数量级的单克隆抗体(nM)。经过DNA测序分析，Y72B-2和Y72B-3编码的是同一个抗体序列,来源于小鼠的抗体序列，下文称之为鼠源抗体。Y72B-2编码的抗体重链可变区氨基酸序列如SEQ ID NO.1所示，其中包含识别抗原的关键结构域CDR1、CDR2、CDR3和框架结构域FR1、FR2、FR3等。Y72B-2编码的轻链可变区氨基酸序列如SEQID NO.2所示，其中包含识别抗原结构域CDR1、CDR2、CDR3和框架结构域FR1、FR2、FR3等，详见下表2和表3。如图1所示，其结果显示，筛选出的抗体与OPN的N端结构域具有良好的特异性识别和结合能力。
表2
重链CDR区序列  
CDR1 GFTFSSYA
CDR2 ITSGGSYT
CDR3 AREGPAWFAY
重链的框架区序列  
FR1 EVMLVESGGGLLKPGGSLKLSCAAS
FR2 MSWVRQTPEKRLEWVAT
FR3 NY SDSVKGRITISRDNAKNTLYLQMSSLRSEDTAMYYC
表3
Figure PCTCN2021075062-appb-000001
实施例2：抗OPN嵌合抗体表达载体构建
2.1 PCR扩增得到人抗体恒定区和鼠抗体可变区DNA
将实施例1中1.3步骤得到的编码Y72B-2抗体序列信息，通过基因合成方法合成了带有分泌信号肽的抗体轻链可变区的DNA序列(SEQ ID NO.31,编码氨基酸序列SEQ ID NO.32)，以此作为模板，以引物1和引物2分别为鼠源抗体轻链可变区DNA的上游引物和下游引物；同样方法，通过基因合成方法合成了带有分泌信号肽的抗体重链可变区的DNA序列(SEQ ID NO.33,编码氨基酸序列SEQ ID NO.34)，以此作为模板，以引物3和引物4分别为鼠源抗体重链可变区DNA的上游引物和下游引物，进行PCR扩增，PCR反应条件为：解链95℃，30秒；退火60℃，30秒；延伸72℃，10秒，循环34次。将人IgG1抗体轻链DNA用引物5和引物6进行PCR扩增，人IgG1抗体重链DNA用引物7 和引物8进行PCR扩增，PCR反应条件同前。分别得到人抗体重链恒定区DNA序列(氨基酸序列如SEQ ID NO.3所示,碱基序列如SEQ ID NO.35所示)，人抗体轻链恒定区DNA序列(氨基酸序列如SEQ ID NO.4所示,碱基序列如SEQ ID NO.36所示)，鼠源抗体重链可变区DNA序列(氨基酸序列如SEQ ID NO.1所示，碱基序列如SEQ ID NO.5所示)，鼠抗体轻链可变区DNA序列(氨基酸序列如SEQ ID NO.2所示，碱基序列如SEQ ID NO.6所示)。PCR检测结果如图2所示，其结果表明：成功获得了各目的片段。抗体72B轻链可变区(VL)条带大小为336bp，抗体72B重链可变区(VH)条带大小为351bp，人IgG1轻链恒定区(IgG1-CL)条带大小为333bp，人IgG1重链恒定区(IgG1-CH)条带大小为1005bp，结果显示各基因条带与预期条带大小相近，表明成功扩增了相应目的条带。
引物1(SEQ ID NO.7)5’-CTAGTCTAGAATGAGGGTCCTTGCTGAG-3’
引物2(SEQ ID NO.8)5’-CCGGAATTCGGTCCGCTTGATCTCTTTGATTTCCAGCTTCCGGAATTCGGTCCGCTTGATCTCTTTGATTTCCAGCTT-3’
引物3(SEQ ID NO.9)5’-CTAGTCTAGAGAAGTGATGCTGGTGGAG-3’
引物4(SEQ ID NO.10)5’-CCGGAATTCCGCGCTGCTCACGGTTGCAGAGACAGTGACCCGGAATTCCGCGCTGCTCACGGTTGCAGAGACAGTGAC-3’
引物5(SEQ ID NO.11)5’-CTAGTCTAGAAAGCTGGAAATCAAAGAGATCAAGCGGACCCTAGTCTAGAAAGCTGGAAATCAAAGAGATCAAGCGGACC-3’
引物6(SEQ ID NO.12)5’-CCGGAATTCTCAGCACTCGCCCCGGTT-3’
引物7(SEQ ID NO.13)5’-CTAGTCTAGAGTCACTGTCTCTGCAACCGTGAGCAGCGCGCTAGTCTAGAGTCACTGTCTCTGCAACCGTGAGCAGCGCG-3’
引物8(SEQ ID NO.14)5’-CCGGAATTCTCACTTCCCGGGGCTCAG-3’
2.2将人抗体恒定区和鼠抗体可变区连接成抗OPN嵌合抗体(命名为抗体72B)
将上述步骤2.1扩增得到的人抗体轻链恒定区DNA和鼠抗体轻链可变区DNA混合。以该混合DNA为模板，以引物1和引物6分别为轻链DNA扩增的上游引物和下游引物进行PCR扩增，反应条件同步骤2.1，得到包括人抗体轻链恒定区DNA序列及鼠抗体轻链 可变区DNA序列的嵌合抗体轻链DNA(72B-L，碱基序列如SEQ ID NO.15所示)。Overlap PCR检测结果如图3所示，其结果表明：利用overlap PCR，将72B-VL与IgG1-CL连接得到大小为669bp的完整轻链片段，将72B-VH和IgG1-CH连接得到大小为1356bp的完整重链片段，电泳结果与预期一致。
将上述步骤2.1扩增得到的人抗体重链恒定区DNA和鼠抗体重链可变区DNA混合。以该混合DNA为模板，以引物3和引物8为分别为重链DNA扩增的上游引物和下游引物进行PCR扩增，反应条件同步骤2.1，得到包括人抗体重链恒定区DNA序列及鼠抗体重链可变区DNA序列的嵌合抗体重链DNA(72B-H，碱基序列如SEQ ID NO.16所示)。
实施例3：抗OPN嵌合抗体的表达纯化
将实施例2扩增得到的无内毒素抗OPN嵌合抗体轻链DNA(72B-L)及重链DNA(72B-H)通过pcDNA3.1/His A载体来构建OPN基因真核表达载体，获得重组质粒，利用PEI瞬时转染方法以轻链：重链＝2：1的比例共转染到HEK293E细胞中。待细胞活率为50％以下时，将获得的细胞培养上清液利用Protein A亲和层析柱进行纯化，以10倍柱体积的20mM磷酸盐缓冲液(pH 7.2)平衡柱体后，以0.5mL/min速度上样。上样完毕后，以10倍体积的0.1M柠檬酸缓冲液(pH 3.0)洗脱，用预先加入1M Tris-HCl缓冲液的Ep管回收蛋白产物，再磷酸盐缓冲液平衡柱体。将洗脱得到的蛋白产物置于孔径大小为10KD的透析袋中，放置在PBS缓冲液中(4℃)透析过夜。最终获得目的蛋白，即抗OPN嵌合抗体。所获抗体的SDS-PAGE电泳检测图如图4所示，图中还原型抗OPN嵌合抗体(抗体72B，图4中的泳道2)的分子量为25KD和50KD，与理论的目的蛋白分子量一致，说明获得了正确的表达产物。纯化后的目的抗体仅出现两条条带，分别为重链和轻链，几乎不含其他蛋白，证明制备的抗体72B纯度较高。
实施例4：抗OPN嵌合抗体与抗原的亲和力
4.1酶联免疫法(ELISA)测试抗OPN嵌合抗体与抗原的亲和力
利用ELISA实验检测纯化后的抗OPN嵌合抗体与抗原的特异性结合能力。将OPN-N端蛋白(OPN-R)(其碱基序列如SEQ ID NO.17所示)和OPN全长蛋白(OPN-FL)(其碱基序列如SEQ ID NO.18所示)以2μg/mL的浓度包被于ELISA板中，4℃放置过夜。弃去包被液后，用3％BSA室温封闭1h，向处理组加入不同浓度梯度的抗OPN嵌合抗体，向对照组加入与处理组相同浓度的人IgG抗体，室温孵育1h。然后加入1：20000稀释的HRP驴 抗人IgG二抗进行结合反应，37℃孵育1h。最后加入TMB于37℃避光作用5min，并用2M硫酸终止反应，测A450值。结果如图5所示，该结果显示：抗原结合活性与抗体浓度呈正相关，随着抗体72B浓度增高，抗体结合抗原OPN-R的活性变高，证明抗体72B具有较高的抗原结合活性。另外，OPN全长蛋白OPN-FL与抗体72B几乎无结合活性，表明抗体72B仅特异性结合OPN-N端蛋白。
4.2 FoeteBio法测试抗OPN嵌合抗体与抗原的亲和力
采用ForteBio法检测抗OPN嵌合抗体与抗原OPN-R的平衡解离常数KD，将抗原OPN-R利用BLK-NH 2试剂盒(同仁化学)生物素化标记处理之后，采用链霉亲和素传感器(SA Sensor)以100nmol/L浓度将生物素化的OPN-R固定在96孔板上，用PBS平衡探针后，再与梯度稀释的抗OPN嵌合抗体(50nmol/L，100nmol/L，200nmol/L，300nmol/L)结合。以PBS作为空白对照。结合时间600s，解离时间900s，通过对照传感器扣除本底信号后，采用1∶1结合模型去拟合结合和解离曲线，得到亲和力常数。结果如图6所示，该结果显示：拟合曲线与实际曲线吻合度高，曲线之间分布均匀，用ForteBio系统软件分析得到抗原抗体亲和力常数KD＝4.55×10-9M，R 2＝0.989，表明抗原抗体之间有良好的亲和力。
实施例5：抗OPN嵌合抗体表位鉴定
为了鉴定抗OPN嵌合抗体识别的表位，根据编码人OPN-R的DNA序列信息(长度为504bp)，去除48个碱基的信号肽序列后，利用去信号肽后的OPN-R序列设计两个截短体，分别为OPN-N2(OPNI17-D93)(其碱基序列如SEQ ID NO.19所示)、OPN-N3(OPN I17-T131)(其碱基序列如SEQ ID NO.20所示)，以及OPN-N(I17-T169，即图8中的OPN-R)。使用表达载体pET-28(a)，合成和构建OPN-N2融合6×His标签重组蛋白及OPN-N3融合6×His标签重组蛋白，利用大肠杆菌(BL21(DE3))可溶性表达。结果如图7所示，该结果显示：成功构建并表达出截短体OPN-N2及OPN-N3。
将OPN截短体OPN-N2、OPN-N3及OPN-N用包被缓冲液稀释为2μg/mL，在4℃包被过夜。倒去包被液，拍干，加入PBS 300μL/孔，每次5min。用1％BSA 100μL/孔室温封闭1h后倒去。将抗体72B从100pmol/mL倍比稀释至0.0002pmol/mL，每孔加样100μL，用PBS作为阴性对照，室温孵育1h后用PBS洗涤。于反应孔中，加入1：20000比例新鲜稀释的辣根过氧化物酶标记的驴抗人IgG抗体100μL，37℃孵育60min，洗涤。加TMB，每孔100μL，室温避光放置10min。加2M硫酸每孔50μL终止反应，酶标仪读取OD450吸 光值。抗OPN嵌合抗体(抗体72B)与截短体OPN-N2、OPN-N3和OPN-N的ELISA结合反应结果如图8所示，该结果说明抗OPN嵌合抗体无法识别OPNI17-T131之间的序列，但与OPNI17-T169有较强亲和力，故推测其表位识别区域位于OPN S129-R168之间，并据此设计合成编码OPN S129-R168的三个重叠肽hOPN-pt4(其氨基酸序列如SEQ ID NO.21所示)、hOPN-pt5(其氨基酸序列如SEQ ID NO.22所示)、hOPN-pt6(其氨基酸序列如SEQ ID NO.23所示)，以更精确定位抗原表位。
三段多肽hOPN-pt4、hOPN-pt5、hOPN-pt6采用前述的方法与抗OPN嵌合抗体(抗体72B)结合反应的ELISA结果如图9所示，结果显示抗体72B识别的OPN表位区域位于hOPN-pt5和hOPN-pt6的共同区域即OPN 139D-148P。故多肽序列包含OPN D139-P148时，多肽与抗OPN嵌合抗体有较强的亲和力。
为进一步验证OPN D139-P148为抗OPN嵌合抗体识别的抗原表位，我们利用表达载体pET-28(a)，分别构建了包含OPN D139-P148序列的截断体OPN-148P(即OPNI17-P148，序列见SEQ ID NO.24)及不包含OPN D139-P148序列的截断体OPN-138T(OPN I17-T138，序列见SEQ ID NO.25)。使用表达载体pET-28(a)，合成和构建OPN-148P融合6×His标签重组蛋白及OPN-138T融合6×His标签重组蛋白，利用大肠杆菌(BL21(DE3))可溶性表达。截短体OPN-148P及OPN-138T的SDS-PAGE电泳结果如图10所示，结果显示成功构建并表达出目的截短体。截短体OPN-148P及OPN-138T对抗体72B亲和力测定的ELISA结果如图11所示，结果显示OPN-148P及OPN-R(图11中的OPN-N)对抗OPN嵌合抗体72B的亲和力较高，而OPN-138T与抗体72B几乎无亲和力，证实抗体72B可特异性识别OPN D139-P148之间的序列。
hOPN-pt7(OPN D139-P148)(序列见SEQ ID NO.26)被用来进一步确定抗OPN嵌合抗体不仅识别OPN D139-P148的空间表位，同时可特异结合由OPN D139-P148之间连续残基形成的线性结构。hOPN-pt7与抗OPN嵌合抗体(抗体72B)的ELISA结果如图12所示。结果显示，在一定浓度下，hOPN-pt7与抗OPN嵌合抗体72B之间具有特异性亲和力。证明抗体72B可特异结合由OPN D139-P148之间连续氨基酸残基形成的线性结构。
实施例6：抗OPN嵌合抗体对细胞增殖的抑制
用以下方法研究抗OPN嵌合抗体是否抑制A549细胞在增殖。首先，取生长状态良好的A549细胞，调整细胞浓度为2.5×10 4个/mL，按照每孔100μL接种于96孔细胞培养板，于37℃、5％CO 2培养箱中培养过夜，24h后更换无血清培养基，将细胞分为空白对照组以 及抗OPN嵌合抗体组(实施例3得到的抗OPN嵌合抗体，浓度分别为1.25、2.5、5、10、20μg/mL)，每个浓度梯度设置5个平行孔，分别于处理后的48h和72h观察并记录细胞形态变化，弃去原培养液后，每孔加入100μL加入含有10％CCK8的F-12培养基，37℃下避光孵育，待OD为0.9至1.1范围内，测定其OD480值，每组实验重复3次，计算出细胞存活率。结果如图13和图14所示，该结果显示：抗体72B具有较强的抑制A549细胞增殖效果，与对照组相比，在加入抗体72B 48h或72h后，细胞存活率均有明显下降趋势。
实施例7：抗OPN嵌合抗体对细胞迁移的抑制
利用细胞划痕实验检测抗OPN嵌合抗体对A549细胞迁移的抑制作用，首先将A549细胞用含10％FBS的F-12培养基调整至2×10 5个/mL，取1mL接种于24孔板中，培养过夜使细胞贴壁后用黄枪头沿培养板上部自上而下呈“1”字形进行单层培养细胞划痕。PBS洗涤去除培养液中漂浮的细胞，更换为无血清培养基，实验组加入终浓度为20μg/mL的抗OPN嵌合抗体(实施例3得到)，PBS组加入与实验组的抗体相同体积的PBS，显微镜下记录划痕区0h相对距离，并对记录区域进行标记。在37℃细胞培养箱内孵育不同时间点，显微镜下观察相同位置恢复情况并记录。结果如图15所示，该结果显示：实验组划痕愈合程度显著小于未加抗体的PBS组，说明抗体72B在20μg/mL浓度下能显著抑制A549细胞的迁移。
同时，利用Transwell实验检测抗OPN嵌合抗体对A549细胞迁移的抑制作用，首先利用24孔Transwell小室系统(聚碳酸酯膜孔径为8μm)，将生长状态良好的A549细胞以1×10 5个/mL的密度重悬于无血清F-12培养基中。将200μL细胞悬浮液加入到上腔室中，并将600μL添加了10％FBS的F-12培养基作为趋化剂添加到下腔室中。处理组在下腔室加入终浓度为20μg/mL的抗OPN嵌合抗体(实施例3获得)，对照组在下腔室加入与实验组抗体72B相同体积的PBS。小室系统在37℃细胞培养箱内孵育24h后，用棉签擦去小室上层的细胞，将小室用PBS洗涤后，于室温下在4％多聚甲醛中固定30min，洗涤后在室温下用0.5％结晶紫染色30min，用PBS洗去残余的染料，在显微镜(放大倍数：×100)下随机选择四个区域并拍照记录。结果如图16所示，该结果显示：与PBS对照组相比，当Transwell系统的下室存在20μg/mL抗体72B的条件下，A549细胞难以穿过膜到达小室的下层，细胞向下的迁移受到了显著的抑制。
实施例8：抗OPN人源化单链抗体(hscFV)的表达与纯化
通过分别将72B抗体的重链和轻链与人IgG序列数据库进行对比，选择氨基酸序列相似性最高的人IgG序列作为模版，对72B轻重链可变区中非CDR区序列按照人IgG序列进行突变，提高72B抗体可变区序列与人IgG对应序列的相似性。突变后的重链可变区氨基酸序列见SEQ ID No.27，轻链可变区氨基酸序列见SEQ ID No.28。通过基因合成，利用突变后的序列构建人源化抗OPN单链抗体(hscFV)，VL和VH之间用3个重复的GGGGS肽链连接，合成基因序列见SEQ ID NO.29。将合成的hscFV表达序列用通用的方法构建到商业化表达载体pET28a(+)中，同时以72B抗体为模板，通过通用的分子克隆方式构建了鼠源抗OPN单链抗体(scFV)，VL和VH之间用3个重复的GGGGS肽链连接，DNA序列(见SED ID NO.30)，也插入到pET28a(+)。采用大肠杆菌BL21(DE3)作为宿主菌，参考文献诱导条件和表达方法(Journal of Biotechnology,2000,77:169–178)，利用包涵体表达的方法分别表达hscFV和scFV两个抗体，采用镍亲和层析进行分离，获得纯化后hscFV抗体的SDS-PAGE电泳结果见图17，图中泳道1表示流穿组分，2和3是40mM咪唑洗脱组分，4和5是80mM咪唑洗脱组分，6和7是100mM咪唑洗脱组分，8和9是200mM咪唑洗脱组分，10和11是300mM咪唑洗脱组分，12和13是500mM咪唑洗脱组分。获得纯化后的scFV抗体的SDS-PAGE电泳结果见图18，图中泳道1表示流穿组分，2和3是40mM咪唑洗脱组分，4和5是80mM咪唑洗脱组分，6和7是100mM咪唑洗脱组分，8和9是200mM咪唑洗脱组分，10和11是300mM咪唑洗脱组分，12和13是500mM咪唑洗脱组分。
实施例9：抗OPN的hscFV与抗原的亲和力
针对来源72B的抗人OPN抗体进行人源化后是否改变其与OPN-R的亲和力的问题，我们分别测定72B的scFV和人源化后72B的hscFV分别与OPN-R的结合能力。测定方法和实施例4中一样。将OPN-R蛋白用包被缓冲液稀释为2μg/mL，4℃包被过夜。倒去包被液，加入PBS，300μL/孔，静置5分钟后倒出，重复三次。用3％BSA 100μl/孔室温封闭1小时后弃去。对scFV或hscFV的样品进行2倍进行逐级稀释，浓度为10μg/mL，连续稀释7次，得到样品浓度分别是10、5、2.5、1.25、0.625、0.313、0.16μg/mL等样品，取每个样品100μl加入包被了OPN-R的孔内，每个样品设3个重复孔。然后37℃孵育2个小时，弃去酶标板中的样品，重复洗涤4次，每次5分钟。加入1：20000新鲜稀释的HRP-小鼠抗His抗体(美国protech生物技术公司)，100μL/孔，37℃孵育60分钟，重复洗涤4次，每次5分钟。加入100uL/孔单组份TMB显色液(北京索莱宝生物技术公司)，室温避光放置5分钟。每孔加50ul的2M硫酸终止反应，酶标仪读取450nm处吸光值。结果如图19所示， 两个抗体的与抗原的结合能力没有显著差异。表明72B抗体的人源化改造依然保持识别和结合OPN-R的特性。
实施例10：抗OPN抗体72B的体内抗肿瘤活性
采用实施例3中获得的抗OPN嵌合抗体72B用于体内抗肿瘤药效学实验。10只8周龄的雄性Nude裸鼠，每只小鼠腋下接种5X10 6个非小细胞肺癌A549细胞，接种后1小时，所有小鼠被随机分成2组，每组5只。实验开始后实验组小鼠给与尾静脉注射50μg的72B抗体(图20中的OPN-Ab(72B))，对照组小鼠给予等体积的PBS作为对照(图20中的Control)。实验开始后，每周给药两次，直至实验结束。接种7天后开始测量肿瘤体积，以后每隔两天测量一次，并计算肿瘤的体积。接种肿瘤后35天实验结束，测量肿瘤大小，并绘制肿瘤生长曲线。如图20显示，抗OPN抗体显著抑制皮下移植瘤的生长(p＝0.011)，抑制效果达到35％。结果标明72B抗体具有良好的抗肿瘤活性。
本发明具体应用途径很多，以上所述仅是本发明的优选实施方式。应当指出，以上实施例仅用于说明本发明，而并不用于限制本发明的保护范围。对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进，这些改进也应视为本发明的保护范围。

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  1. 一种抗人骨桥蛋白的抗体，其特征在于，包括轻链可变区、重链可变区；所述轻链可变区的CRD区序列包括轻链CDR1、轻链CDR2、轻链CDR3；所述轻链CDR1的氨基酸序列为QSIVHSNGNTY，轻链CDR2的氨基酸序列为KVS，轻链CDR3的氨基酸序列为FQGSHVPPT；
    所述重链可变区的CRD区序列包括重链CDR1、重链CDR2、重链CDR3；所述重链CDR1的氨基酸序列为GFTFSSYA，重链CDR2的氨基酸序列为ITSGGSYT，重链CDR3的氨基酸序列为AREGPAWFAY。
  2. 根据权利要求1所述的抗人骨桥蛋白的抗体，其特征在于，所述抗体的重链可变区的氨基酸序列如SEQ ID NO.1所示，轻链可变区的氨基酸序列如SEQ ID NO.2所示；
    或所述抗体为包括以前述轻链可变区和重链可变区为基础的突变体或人源化抗体，所述突变体包括与前述轻链可变区和重链可变区序列具有50％以上同源性的氨基酸序列；所述人源化抗体包括对前述轻链可变区和重链可变区中的非CDR区序列进行突变后得到。
  3. 根据权利要求1所述的抗人骨桥蛋白的抗体，其特征在于，所述抗体还包括轻链恒定区、重链恒定区；
    所述重链恒定区的氨基酸序列如SEQ ID NO.3所示，轻链恒定区的氨基酸序列如SEQ ID NO.4所示；
    所述抗体识别的抗原表位为OPN D139-P148，氨基酸序列如SEQ ID NO.26所示。
  4. 根据权利要求3所述的抗人骨桥蛋白的抗体，其特征在于，所述抗体的轻链DNA的碱基序列如SEQ ID NO.15所示；重链DNA的碱基序列如SEQ ID NO.16所示。
  5. 根据权利要求2所述的抗人骨桥蛋白的抗体，其特征在于，所述人源化抗体的的重链可变区氨基酸序列如SEQ ID NO.27所示，轻链可变区氨基酸序列如SEQ ID NO.28所示。
  6. 一种包含权利要求1所述的抗人骨桥蛋白的抗体的重组质粒。
  7. 一种根据权利要求1所述的抗人骨桥蛋白的抗体的制备方法，其特征在于，包括以下步骤：
    A、筛选获得分泌抗OPN抗体的单克隆杂交瘤细胞，并获得其鼠源抗体可变区DNA；
    B、以鼠源抗体可变区DNA为模板，分别扩增得到鼠源抗体轻链可变区DNA和鼠 源抗体重链可变区DNA；将人抗体轻链恒定区DNA和鼠抗体轻链可变区DNA连接、人抗体重链恒定区DNA和鼠抗体重链可变区DNA连接，然后形成重组质粒，共转染宿主细胞；
    C、待宿主细胞活率为50％以下时，将获得的细胞培养上清液进行纯化、洗脱、透析，即得纯化后的抗人骨桥蛋白的抗体；
    步骤A中，所述鼠源抗体轻链可变区氨基酸序列如SEQ ID NO.2所示；鼠源抗体重链可变区氨基酸序列如SEQ ID NO.1所示；
    所述人抗体轻链恒定区氨基酸序列如SEQ ID NO.4所示；人抗体重链恒定区氨基酸序列如SEQ ID NO.3所示。
  8. 一种根据权利要求1所述的抗人骨桥蛋白的抗体的在测定检测体系中OPN-N片段含量中的应用；其特征在于，所述OPN-N片段的碱基序列如SEQ ID NO.17所示。
  9. 一种测定检测体系中OPN-N片段含量的检测试剂盒，其特征在于，包括权利要求1所述的抗人骨桥蛋白的抗体。
  10. 一种根据权利要求1所述的抗人骨桥蛋白的抗体的在制备抑制肿瘤增殖和/或迁移的组合物中的应用。
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