WO2015089881A1 - 全人源抗cd26抗体及其应用 - Google Patents
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
-
- 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
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
Definitions
- the invention belongs to the technical field of genetic engineering antibodies, in particular to a novel high affinity full human antibody which can specifically bind to CD26, a preparation method and application thereof.
- CD26 is a ubiquitous multifunctional type II transmembrane glycoprotein with multiple biological functions and can also be present in plasma in a dissolved form.
- CD26 is often present as a homodimer with a monomer of 766 amino acids and a relative molecular mass of about 110 kDa. The amino acid residues are divided into five parts from the inside to the outside: intracellular regions (1 to 6), transmembrane regions (7 to 28), highly glycosylated regions (29 to 323), and cysteine-rich regions (324). ⁇ 551) and the C-terminal catalytic domain (552 ⁇ 766), the three-dimensional structure and function of CD26 molecules are closely related.
- CD26 The C-terminal catalytic domain of CD26 exerts the activity of Dipeptidyl peptidase IV (DPPIV), which can hydrolyze various substrates in the body to exert biological effects.
- DPPIV Dipeptidyl peptidase IV
- the cysteine-rich region can interact with various molecules in the body. And thus participate in the immune function in the body.
- CD26 is a molecular marker of T cell activation, also acts as a costimulatory molecule in T cell signaling, and involves a variety of T cell functions, including T cell maturation and migration. , cytokine secretion, T cell-dependent antibody production, B cell immunoglobulin transformation, etc.
- CD26 plays an important role in the development of autoimmune diseases and has become a molecular marker of clinical diseases and is considered as a therapeutic or diagnostic target for certain immune diseases. (Ohnuma et al. (2011) Adv Clin Chem, 53, 51-84)
- CD26 can interact with various proteins, such as ADA, CD45, FAP-alpha, etc., and can also bind to ECM, leading to an increase or decrease in the infiltration activity of CD26 cells. It can be seen that CD26 plays an important role in tumor biology.
- the expression level of CD26 is elevated on the surface or serum of various neoplastic cells.
- CD26 is highly expressed in certain offensive T cell malignancies, malignant mesothelioma, renal tumor, and some colon cancer (Havre et al (2008) Front Biosci, 13, 1634-1645
- Certain CD26 + colon cancer cell subsets, CD26 + malignant mesothelioma cells have distinct tumor stem cell characteristics (Ghani et al. (2011) Biochem Biophys Res Commun, 404, 735-742 and Pang et al. (2010) Cell Stem Cell, 6, 603-615), so CD26 can serve as a molecular marker for a variety of tumors.
- anti-CD26 murine monoclonal antibodies E19 and E26 which exhibit inhibition of cell migration of fibroblasts and wound cells to form a monolayer, inhibit angiogenesis, and invade human skin microvascular endothelial cells And the formation of new capillary branches has an inhibitory effect.
- anti-CD26 monoclonal antibodies can play a role in the treatment of various diseases by altering the activity of CD26.
- mouse monoclonal antibody when the mouse monoclonal antibody is directly applied to human body treatment, it produces a human anti-Mouse Antibody (HAMA), and the human body produces an antibody against the mouse antibody, which not only weakens the therapeutic effect, but also causes acute Sensitive reaction.
- HAMA human anti-Mouse Antibody
- a human-mouse chimeric antibody In order to overcome the shortcomings of murine mAb, genetically engineered antibody technology has developed a human-mouse chimeric antibody.
- the chimeric antibody is spliced into a chimeric gene by the V region gene of the murine antibody and the C region gene of the human antibody.
- the source component is reduced by about 70%, and the humanized antibody is further substituted with the framework region (FR) of the variable region of the human antibody in place of the chimeric antibody, and the mouse component of the antibody is greatly reduced.
- FR framework region
- the sequence of a small amount of residual murine antibody may still potentially trigger HAMA.
- the present invention aims to provide a fully human antibody capable of efficiently and specifically binding to human CD26, and a disease thereof characterized by the preparation of a treatment for the expression of CD26, particularly overexpression, diagnosis New uses in drugs that CD26 variably express disease. more specifically:
- a first object of the present invention is to provide an antibody or a fragment thereof derived from a human, the antibody or fragment thereof specifically binding to human CD26, preferably specifically binding to the extracellular region of CD26, the amino acid sequence of said antibody or fragment thereof Including SEQ ID NO: 2, SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 6 SEQ ID NO: 7
- SEQ ID NO: 8 A conjugate of a monoclonal antibody or a fragment thereof or a fragment thereof of any of the six complementarity determining regions of a set of sequences, or an amino acid sequence obtained by amino acid substitution or modification.
- the amino acid sequence of the antibody or fragment thereof of the present invention contains a complementarity determining region of the heavy chain variable region of the sequences set forth in SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.
- the amino acid sequence of the antibody or fragment thereof of the present invention contains a complementarity determining region of the light chain variable region of the sequences set forth in SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.
- the antibody or fragment thereof of the present invention has an amino acid sequence of the heavy chain variable region comprising the following complementarity determining region: CDRH1 as shown in SEQ ID NO: 2, as SEQ ID NO: 3 CDRH2 as shown, such as CDRH3 as shown in SEQ ID NO: 4; And the amino acid sequence of the light chain variable region thereof comprises the following complementarity determining regions: a CDRL1 as shown in SEQ ID NO: 6 of the sequence, such as the CDRL2 of the sequence SEQ ID NO: 7 and as set forth in SEQ ID NO: 8. CDRL3.
- the antibody or fragment thereof of the present invention contains a heavy chain variable region such as the sequence shown in SEQ ID NO: 1 and the light chain variable region as shown in SEQ ID NO: 5.
- a second object of the present invention is to provide a single-chain antibody derived from a human, the amino acid sequence of which is represented by SEQ ID NO: 11.
- a third object of the present invention is to provide a nucleotide sequence encoding the above single-chain antibody, which is represented by SEQ ID NO: 10.
- a fourth object of the present invention is to provide an expression vector comprising the above nucleotide sequence.
- a fifth object of the present invention is to provide a recombinant host strain comprising the above expression vector.
- a sixth object of the present invention is to provide a method for producing the above single chain antibody, comprising:
- a seventh object of the present invention is to provide a novel use of the above antibody or a fragment thereof for the preparation of a medicament for treating a tumor with a high expression of CD26. It is possible to treat diseases characterized by expression of CD26, particularly overexpression, and to provide an antibody-based method for diagnosing CD26 variability, including but not limited to autoimmune diseases and cancer.
- the full human anti-CD26 single-chain antibody gene sequence is 723 nucleotides in length, and 241 amino acids are expected.
- a heavy chain variable region of 115 amino acids (SEQ ID NO: 1) and a light chain variable region of 111 amino acids (SEQ ID NO: 5), between the heavy chain variable region and the light chain variable region
- a flexible peptide linkage of amino acids (SEQ ID NO: 9).
- Expression vectors and host bacteria containing the CD 26 single-chain antibody gene of the present invention are within the scope of the present invention.
- Primer pairs that amplify any fragment of the single chain antibody gene of the invention are also within the scope of the invention.
- the antibodies or fragments thereof of the invention have a variety of properties, including the ability to bind and neutralize CD26.
- the anti-CD26 single-chain antibody obtained by the present invention has high specific binding with CD26, inhibits tumor cell-mediated cell adhesion by CD26 in vitro, and inhibits tumor cell growth in vitro.
- Chain antibodies can significantly inhibit the proliferation and invasion and metastasis of tumor cells.
- FIG. 1 is a schematic view showing the structure of the single-chain antibody ZHB-2cF9.
- VH represents the heavy chain variable region domain (SEQ ID NO: 1)
- VL represents the light chain variable region domain (SEQ ID NO: 5)
- Linker is a flexible peptide linked to VH, VL (SEQ ID NO: 9) .
- Figure 2 is a map showing the identification of the extracellular domain of CD26.
- A is the SDS-PAGE of the purified protein
- Lane 1 is the standard protein
- Lane 2 is the purified CD26 extracellular domain protein (arrow);
- B is the Western Blot identification map of the purified CD26 extracellular domain protein, Lane 1 As a standard protein
- Lane 2 is a purified CD26 extracellular domain protein (arrow)
- primary antibody is anti-CD26 mouse anti-(purchased from MBL)
- secondary antibody is rabbit anti-mouse-HRP antibody (purchased from life technology).
- Figure 3A and Figure 3B show the results of ELISA assay for binding of phage antibody to CD26 extracellular domain protein.
- Figure 3A is a polyclonal phage ELISA, the CD26 extracellular domain protein coating concentration is lg/mL, diluted 4 rounds of screen-amplified phage, incubated with a plate labeled with CD26 extracellular domain protein, anti-M13-HRP antibody again Incubation, 450 nm, absorbance at 650 nm, and OD 45Qnm - OD 65Qnm as the final value. The results showed that phage displaying a CD26-specific single-chain antibody were significantly enriched.
- Figure 3B shows the results of monoclonal phage ELISA assay.
- Monoclonal to 96-well plates were used to express phage antibodies, incubated with microplates coated with CD26 extracellular domain protein, anti-M13-HRP antibody detection results, and OD 45Qnm -OD 65Qnm As a final value, the results showed that more than 90% of the monoclonals produced positive binding to the CD26 extracellular domain protein.
- Figure 4 is a diagram showing the identification of the single-chain antibody ZHB-2cF9.
- A is the SDS-PAGE of the ZHB-2cF9 purified by nickel column
- Lane 2 is the sample protein of ZHB-2cF9
- the protein arrow indicates the band of the target protein
- Lane 1 is the standard protein
- B is the purified ZHB-2cF9 Western Blot identification map
- the primary antibody is anti-myc mouse anti-antibody
- the secondary antibody is rabbit anti-mouse-HRP antibody
- Lane l ZHB-2cF9 sample
- the protein arrow refers to the target protein size band
- Lane 2 is the standard protein.
- Figure 5 shows a Western Blot identification map of the single-chain antibody ZHB-2cF9 binding to the CD26 extracellular domain protein.
- the primary antibody is anti-myc mouse anti-antibody and the secondary antibody is rabbit anti-mouse-HRP antibody.
- Lane 1 is the standard protein
- Lane 2 is the binding of ZHB-2cF9 to the extracellular domain of CD26
- the arrow indicates the band consistent with the size of the extracellular domain of CD26 in Figure 2.
- the results indicate that the anti-CD26 single chain antibody ZHB-2cF9 can specifically bind to the CD26 extracellular domain protein.
- Figure 6 shows an immunofluorescence map of single chain antibody binding to human mesothelioma cell NCI-H2452.
- A is ZHB-2cF9 Immunofluorescence with NCI-H2452 cells
- B is a negative result of immunofluorescence binding assay of negative control antibody and NCI-H2452 cells, and the antibody concentration was diluted from 5% MPBS to 1 ( ⁇ g/mL).
- Figure 7 shows the results of single-chain antibody inhibition of human mesothelioma cells NCI-H2452 binding to ECM.
- the ECM used in the experiment was fibronectin.
- the experiment was divided into Binding group, Blank group and antibody test group.
- the adhesion of cells to fibronectin after 12 hours of single-chain antibody treatment was investigated.
- OD450 (Binding group) - OD450 (Blank group) fully adherent cell value
- OD450 (antibody test group) - OD450 (Blank group) cell adhesion value of the sample group
- IgG control is a negative control antibody
- Cell adhesion rate (%) cell adhesion value of the sample group / fully adhered cell value
- Figure 8 shows the results of detection of proliferation inhibition of human mesothelioma NCI-H2452 cells by single-chain antibodies.
- the cells were incubated in 96-well plates in the presence of lxlO 4 /well. After 48 h of single-chain antibody ZHB-2cF9, scFv-YSllO and negative control antibody IgG (mouse), the cells were reacted with CCK-8 reagent for 30 min to determine the absorbance at 450 nm. The value, the cell proliferation inhibition rate is expressed as % reduction in OD450nm.
- the experimental results showed that ZHB-2cF9 and the positive control scFv-YSllO had significant inhibitory effects on NCI-H2452 in a concentration-dependent manner.
- Figure 9 shows the results of detection of proliferation inhibition of human colon cancer HCT116 cells by single-chain antibodies.
- the cells were incubated in a 96-well plate at a dose of 8 ⁇ 10 4 /well. After 48 h of the single-chain antibody ZHB-2cF9, scFv-YSlOO and the negative control antibody IgG (mouse), the cells were reacted with CCK-8 reagent for 30 min to determine 450 nm. The absorbance value and the cell proliferation inhibition rate are expressed as % reduction in OD450nm.
- the experimental results showed that ZHB-2cF9 and the positive control scFv-YSllO had significant inhibitory effects on NCI-H2452 in a concentration-dependent manner.
- an “antibody” is an immunoglobulin molecule capable of specifically binding to a target antigen by at least one antigen recognition site in a variable region, such as a sugar, a polynucleotide, a lipid, a polypeptide, and the like.
- the term includes not only intact polyclonal and monoclonal antibodies, but also fragments thereof (eg, Fab, Fab', F(ab') 2, Fv), single chain antibodies (scFv), mutants thereof, fusions comprising antibody portions.
- Antibodies include antibodies of either class, such as IgG, IgA or IgM (or subclasses thereof), and the antibodies need not be of any particular class.
- single-chain antibody refers to a single-stranded fusion protein in which an immunoglobulin heavy chain variable region ( VH ) and a light chain variable region (VL) are joined by a 10 to 25 amino acid polypeptide, a linker peptide. (linker), usually rich in glycine and serine, to facilitate the stability and flexibility of single-chain antibodies.
- linker usually rich in glycine and serine, to facilitate the stability and flexibility of single-chain antibodies.
- the connection can connect the N-terminus of VL to the C-terminus of VH , or vice versa. Although the constant region was removed and the linker was introduced, the single-chain antibody retained the specificity of the immunoglobulin for the antigen.
- “Chimeric and humanized antibodies” generally refers to antibodies that combine regions from more than one species. “Chimeric antibodies” conventionally comprise a variable region from a mouse (or in some cases a rat) and a constant region from a human. “Humanized antibody” generally refers to a non-human (eg, murine) antibody form that is a specific chimeric immunoglobulin, immunoglobulin chain or fragment thereof (eg, Fv, Fab) that contains minimal sequence derived from a non-human immunoglobulin. , Fab', F (ab') 2 or other antigen-binding sequence of the antibody).
- the entire antibody, except for the CDRs, is encoded by a human-derived polynucleotide or has an identity other than the CDRs of an antibody encoded by a human-derived polynucleotide.
- Some or all of the CDRs are encoded by nucleic acids derived from non-human organisms, which are grafted into the lamellar framework regions of the variable regions of human antibodies to produce antibodies whose specificity is determined by the CDRs of the implant.
- human antibody means an antibody having an amino acid sequence corresponding to the amino acid sequence of a human-derived antibody, and/or an antibody prepared by any of the techniques known in the art or prepared for the preparation of a human antibody as disclosed herein.
- Human antibodies can be produced using a variety of techniques known in the art. For example, a human antibody is selected from a phage library, wherein the phage library expresses a human antibody.
- Human antibodies can also be prepared by introducing a human immunoglobulin locus (loci) into a transgenic animal, such as a mouse that has partially or completely extinguished the endogenous immunoglobulin gene.
- human antibodies can be prepared by immortalizing human B lymphocytes that produce antibodies against the target antigen, which can be obtained from an individual or have been immunized in vitro.
- the human antibody is "full human,” which means that the antibody comprises human heavy and light chain polypeptides.
- HLF cells human undifferentiated liver cancer cells
- JCRB cell bank Japanese Collection of Research Bioresources Cell Bank
- NCI-H2452 a human mesothelioma cell
- HCT116 a human colon cancer cell
- HLF cells human undifferentiated liver cancer cells
- Reagents such as medium and buffer: RPMI-1640 (GIBCO, Cat# 31800022, added NaHC0 3 1.5g/L, glucose 2.5g/L, Sodium Pyruvate 0.11 g/L), 90%; high quality fetal bovine serum, (GIBCO ) 10%.
- medium and buffer RPMI-1640 (GIBCO, Cat# 31800022, added NaHC0 3 1.5g/L, glucose 2.5g/L, Sodium Pyruvate 0.11 g/L), 90%; high quality fetal bovine serum, (GIBCO ) 10%.
- 2YT medium 1L contains Trptone (OXID) 16g, Yeast Extract (OXID) lOg, NaCl 5g.
- 2YT-AK medium 2YT contains 10 ( ⁇ g/mL ampicillin and 5 ( ⁇ g/mL kanamycin.
- 2YT-AG medium 2YT contains 10 ( ⁇ g/mL ampicillin and 2% glucose.
- IOXPBS (purchased from Beijing Soleil, Cat#P1022).
- PBST PBS containing 0.1% Tween20 (purchased from Beijing Solebao).
- Amp Ampicillin (purchased from Shanghai Shenggong).
- Kan Kanamycin (purchased from Shanghai Biotech).
- Plasmid p3XFLAG-CMV9 (purchased from Sigma-Aldrich); pHEN2 (; obtained from Medical Research Council (UK))
- the purpose of this study was to transfect eukaryotic cells with recombinant plasmids, obtain stable cell lines by G418 resistance screening, secrete and express CD26 extracellular domain protein, and purify CD26 extracellular domain protein by affinity chromatography.
- the CD26 extracellular domain gene is a synthetic gene selected from the "Extracellular" region of amino acids 29-766 in the Uniprot: P27487 sequence, and the CD26 extracellular region gene is ligated into the plasmid p3XFLAG-CMV9, and the restriction sites are Hind III, Xba I, Construction of recombinant plasmids (J. Sambrook. Guide to Molecular Cloning, Third Edition. Science Press. 2002. P68).
- the recombinant plasmid containing the CD26 extracellular domain gene was transferred into HLF cells using Lipofectamine LTX Reagent (purchased from Invitrogen) according to the method described.
- a stable expression cell line of CD26 extracellular domain was obtained, which was recorded as HLF-4D9, and cultured in a large amount without serum, secreting and expressing CD26 extracellular domain protein, the protein With the Flag tag, the serum-free culture supernatant was purified using ANTI-FLAG M2 Affinity Gel (purchased from Sigma-Aldrich) to obtain CD26 extracellular domain protein, SDS-PAGE (Fig.
- Fig. 2A analysis and Western Blot (JS). Boni Fesnon. Guide to Fine Cell Biology Experiments. Science Press. 2007. P177) Identification (Fig. 2B), primary antibody is anti-CD26 mouse anti (purchased from MBL), secondary antibody is rabbit anti-mouse-HRP antibody (purchased from life technology)
- Fig. 2A is an identification diagram of the purified protein by SDS-PAGE, and a single band (La ne 2) of the electrophoresis pure protein size is obtained by one-step affinity chromatography, and Fig. 2B is a purified protein.
- the Western Blot identification map showed that the band indicated by Lan e 2 was consistent with the SDS-PAGE protein, indicating that the CD26 extracellular domain gene was transfected into eukaryotic cells and expressed, and purified to obtain the electrophoresis pure CD26 extracellular domain. protein.
- the single-chain antibody was obtained from a human single-chain antibody phage display library by solid phase affinity screening of the CD26 extracellular domain protein.
- the human single-chain antibody phage display library was constructed by Jiangsu Zhonghong Bioengineering Medicine Research Institute Co., Ltd.
- the phage display library containing the heavy chain and light chain variable regions of the antibody produced by human cells is constructed from human peripheral blood lymphocytes, and the first round of amplification is performed by using primers specific for the antibody variable region gene, and the second round of amplification will be heavy.
- the light chain variable region of the chain was ligated with a flexible linker gene to form a single-chain antibody gene, and the single-chain antibody gene was cloned into plasmid pHEN2 and transformed into E.
- P51 Dilute CD26 extracellular domain protein to 5 ( ⁇ g/mL; coated into ELISA plate (Maxi-sorp96, Nunc) with blank Control wells (without CD26 extracellular domain protein), followed by blocking.
- the phage single-chain antibody library was suspended in 2% MPBS, and ⁇ was added to the blocked blank wells, and placed at room temperature.
- phage antibody The antibody fragment displayed on the surface of the phage particle is called phage antibody.
- the enrichment of CD26-specific phage antibody after 4 rounds of screening was identified by polyclonal phage ELISA, and then the phage affinity was further identified by monoclonal phage ELISA. Phage antibodies.
- Polyclonal phage ELISA was used to identify the antigen, ie lg/mL CD26 extracellular domain protein to the ELISA plate. After blocking, the phage obtained after each round of screening was diluted with 2% MPBS and added to the antigen-coated enzyme strip. . After incubation for 90 min at room temperature, the mouse anti-M13 phage-HRP antibody (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was added and incubated for 1 h at room temperature. After washing, ⁇ ⁇ coloring solution (purchased from AMRESCO) was added. After incubation for 10 min at room temperature, the reaction was quenched with 1 M dilute sulfuric acid.
- OD 45() and B OD 65() were determined , and OD 45 (r OD 65 () was used as the final test result.
- the BSA-coated ELISA plate was used as a negative control, with the number of rounds selected. Increasingly, the binding of the phage antibody to the CD26 extracellular domain protein was enhanced, indicating that the phage bearing the CD26-specific antibody was significantly enriched.
- phage-containing culture supernatant obtained by centrifugation at 1800 g for 10 min was incubated with 1/10 volume of 20% MPBS for 1 h at room temperature. Then added to the ELISA plate coated with the extracellular domain of recombinant CD26, identified by ELISA (method and reagent identification with polyclonal phage ELISA). OD 45Q and OD 65Q were determined , and OD 45Q -OD 65Q was used as the final test result. Clones with high reads were selected for DNA sequencing.
- Figure 3B shows the results of partial monoclonal phage ELSIA, with more than 90% of the monoclonals showing positive binding, further indicating that by 4 rounds of screening, antibodies with CD26 specific antibodies were used.
- pHEN2 is a bifunctional phagemid vector with an amber stop codon (Amber) TAG between the c-my tag and the coat protein gene.
- Amber amber stop codon
- the phage is infected with an amber mutation (SupE) inhibitory strain, such as TG1, the TAG codon is translated into glutamate, the sequence can be read through translation, and the antibody fragment is fused to the coat protein p3 for expression on the surface of the phage; when the phage is infected with a non-amber mutation inhibitory type At the time of the strain, such as HB2151, the translation is terminated at the TAG, and a soluble expressed antibody fragment can be obtained.
- an amber mutation (SupE) inhibitory strain such as TG1
- the TAG codon is translated into glutamate
- the sequence can be read through translation, and the antibody fragment is fused to the coat protein p3 for expression on the surface of the phage; when the phage is inf
- the antibody fragment carries a 6xHis tag and a c-myc tag at the C-terminus, which facilitates purification and detection.
- the single-chain antibody of ZHB-2cF9 was expressed in the periplasmic space of Escherichia coli.
- the periplasmic protein was extracted by high-permeability method, and the target protein with higher purity was obtained by one-step separation and purification by nickel column affinity chromatography.
- a plasmid containing the single-chain antibody ZHB-2cF9 gene (denoted as 2cF9-pHEN2) was extracted from the cell TG1 using a plasmid mini-kit, and used to transform the non-inhibitor E. coli HB2151, which was prepared by calcium chloride method. Transformation competent Escherichia coli HB2151 (J. Sambrook. Molecular Cloning Experimental Guide. Third Edition. Science Press. 2002. P96), the resulting transformed strain is designated HB2151-2cF9.
- hypertonic solution 50 mM Tris-HCl, 20% sucrose, ImM EDTA, pH 8.0
- the supernatant was subjected to nickel column affinity chromatography (purchased from GE), and the purification step was carried out according to GE's standard operating procedure, that is, equilibrating ImL nickel column with an equilibration buffer (Tris 50 mM, NaC1 500 mM, pH 7.5) containing 5 mM imidazole. After 10 column volumes, the N-specifically bound heteroprotein was washed again with an equilibration buffer containing 5 mM imidazole, the non-specific hybrid protein was washed with an equilibration buffer containing 50 mM imidazole, and finally equilibrated with 100 mM imidazole. The buffer is eluted with the protein of interest.
- an equilibration buffer Tris 50 mM, NaC1 500 mM, pH 7.5
- the collected samples were detected by 15% SDS-PAGE, and the single-chain antibody was further identified by Western Blot.
- the primary antibody was anti-c-myc mouse antibody (purchased from Enjing Bio), and the secondary antibody was rabbit anti-mouse-HRP antibody.
- Figure 4A shows that the purified sample contains a band of the desired protein (indicated by the arrow), and the results of Western Blot (Fig. 4B) are determined by SDS-PAGE, indicating that the single-stranded antibody of ZHB-2cF9 was obtained by one-step affinity chromatography on a nickel column. Better purification.
- the purpose of this experiment was to verify the screening of the anti-CD26 single-chain antibody ZHB-2cF9 and CD26 extracellular domain protein. Specific binding.
- Recombinant CD26 extracellular domain protein was subjected to SDS-PAGE electrophoresis, semi-dry transfer method for 1 h, and the protein was transferred to NC membrane (purchased from Millipore); at the end of transfer, the membrane was blocked in 5% MPBS for 1 h at room temperature; with 5% MPBS Dilute ZHB-2cF9 single-chain antibody to lg/mL, incubate for 1 h at room temperature, wash 3 times with TBS; incubate with mouse anti-Myc antibody (purchased from Enjing Bio) for 1 h at room temperature, wash 3 times with TBS; use rabbit anti-mouse-HRP II After incubation for 1 h, the gel imager (ImageQuant LAS4000, GE) was exposed. The results are shown in Figure 5. There is a clear band at the target protein size in the extracellular
- CD26 is highly expressed on the surface of human mesothelioma cells NCI-H2452 (Inamoto et al. (2007) Clin Cancer Res, 13, 4191-200). This experiment identified the immunofluorescence binding of ZHB-2cF9 and NCI-H2452 cells. It was further verified that the single-chain antibody ZHB-2cF9 obtained by screening can specifically bind to NCI-H2452 cells highly expressing CD26, and it is proved that ZHB-2cF9 can specifically bind to CD26 on the cell surface.
- Human mesothelioma cells NCI-H2452 were digested, resuspended after centrifugation, lx lO 5 /well coated in 24-well plates to allow cells to adhere for 12 h, fixed in 4% paraformaldehyde, washed 3 times with PBS, and 5% MPBS was blocked at room temperature for 1 h.
- CD26 mediates adhesion of tumor cells through binding to extracellular matrix proteins (ECMs), one of the common ECMs, and CD26 specifically binds to fibronectin to mediate cell adhesion. This binding can be blocked by anti-CD26 antibodies (Inamoto et al. (2007) Clin Cancer Res, 13, 4191-200), thereby blocking the adhesion of tumor cells.
- ECMs extracellular matrix proteins
- CD26 specifically binds to fibronectin to mediate cell adhesion. This binding can be blocked by anti-CD26 antibodies (Inamoto et al. (2007) Clin Cancer Res, 13, 4191-200), thereby blocking the adhesion of tumor cells.
- YS110 is a humanized antibody against anti-CD26 monoclonal mouse antibody developed by Y's Therapeutics. A clinical trial of anti-CD26 high expression tumors. According to the heavy chain and light chain variable region gene sequence of YS110 antibody (CN 101282994), the single-chain antibody type of YS110, namely scFv-YSlO, was constructed to be the positive control of this experiment, and the expression and purification process was the same as ZHB-2cF9.
- Adhesion experiments were performed using 24-well plates, five parallel experiments, 4 replicate wells per group, one of which was blank control group (Blank), directly blocked with 2% BSA, and the other four groups were treated with l ( ⁇ g/mL fibrosis) protein (purchased from BD Biosciences) were coated at room temperature 20 ⁇ 25 ° C 90min. PBS was added after washing three times with 2% BSA 37 ° C closed lh.
- the five groups were divided into three groups: the antibody test group, and the cells were treated with RPMI-1640 medium containing 5 ( ⁇ g/mL of ZHB-2cF9, positive control scFv-YS110, negative control antibody IgG (mouse) for 2 h, respectively.
- the two groups were the control group (Binding group, Blank group).
- the rats were aliquoted into five groups of closed 24-well plates, and the culture incubator was further cultured for 12 hours.
- Example 7 Inhibition of proliferation of human mesothelioma cell NCI-H2452 by anti-CD26 single chain antibody
- the cultured cells were divided into three groups, and the corresponding active antibodies were: different concentrations of single-chain antibody ZHB-2cF9, positive control scFv-YSlOO and negative control antibody IgG (mouse).
- the final concentration of the active antibody was grouped into 0, 0.1, 1.0, l ( ⁇ g/mL, 5 experimental duplicate wells per group.
- the culture incubator was further cultured for 48 hours, and the cell growth status was observed, and CCK-8 of ⁇ was added to each well.
- IgG has no obvious inhibitory effect on NCI-H2452, indicating that ZHB-2cF9 can inhibit the proliferation of mesothelioma cells with high expression of CD26, and can be used as a potential treatment for CD26 high expression tumors.
- the drug compared to the positive control scFv-YS110, ZHB-pA12 is a fully humanized antibody that eliminates the effects of foreign proteins on the human immune system.
- Example 8 Inhibition of proliferation of human colon cancer cell line HCT116 by anti-CD26 single chain antibody
- the colon cancer cell line HCT116 with high expression of CD26 was also used to investigate the inhibitory effect of single-chain antibody on cell proliferation. .
- HCT116 cells 8 ⁇ 10 4 /well were seeded into 96-well cell culture plates, 100 ⁇ 7 wells, and cultured for 12 hours, and then the original medium was replaced with 1% calf serum medium (containing antibody).
- the cultured cells were divided into three groups, and the corresponding antibody to be added was: different concentrations of single-chain antibody ZHB-2cF9, positive control scFv-YSl lO and negative control antibody IgG (mouse).
- the final concentration of the active antibody was grouped into 0, 0.1, 1.0, l ( ⁇ g/mL, 5 experimental duplicate wells per group.
- the culture incubator was further cultured for 48 hours, and the cell growth status was observed, and CCK-8 of ⁇ was added to each well.
- Negative control IgG to HCT116 There was no significant inhibition, indicating that ZHB-2cF9 can inhibit the proliferation of colon cancer cells with high expression of CD26, and once again proved that ZHB-2cF9 can be used as a potential therapeutic drug for CD26 high expression tumors, and compared with the positive control scFv-YSllO, ZHB-pA12 is a fully humanized antibody that eliminates the effects of foreign proteins on the body's immune system.
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Abstract
提供一种可与CD26特异性结合的全人源抗体、制备方法及其应用。所述的来源于人源的抗体或其片段特异性结合人CD26，优选特异性结合CD26胞外区，所述抗体或其片段的氨基酸序列包括选自含有SEQ ID NO:2、SEQ ID NO:3，SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:7，SEQ ID NO:8一组序列的6个互补决定区的任何区域的单克隆抗体或其片段或其片段的偶联物，或通过氨基酸置换或者修饰得到的氨基酸序列。
Description
全人源抗 CD26抗体及其应用
技术领域
本发明属于基因工程抗体技术领域， 尤其是涉及一种新的可与 CD26特异性结合的 高亲和力全人源抗体、 制备方法及其应用。
背景技术
CD26是一种普遍存在的多功能 II型跨膜糖蛋白， 具有多种生物学功能， 也可以以溶 解形式存在于血浆中。 CD26常以同源二聚体形式存在， 其单体含 766个氨基酸， 相对分 子质量约 110kDa。 氨基酸残基从内向外分为 5个部分： 胞内区 (1〜6)、 跨膜区 (7〜28)、 高 度糖基化区 (29〜323)、 富含半胱氨酸区 (324〜551)和 C端催化结构域 (552〜766)， CD26 分子三维结构与功能密切相关。 CD26的 C端催化结构域发挥二肽基肽酶 IV (Dipeptidyl peptidase 4, DPPIV) 活性， 可以水解体内多种底物发挥生物学作用， 富含半胱氨酸区可 以和体内多种分子相互作用， 从而参与体内免疫功能。 CD26在免疫调节中的作用已被广 泛研究， CD26是 T细胞活化的分子标志， 也在 T细胞信号转导过程中作为共刺激分子， 还涉及多种 T细胞功能， 包括 T细胞发生成熟及迁移， 细胞因子分泌， T细胞依赖的抗 体产生， B细胞免疫球蛋白转型等。 CD26在自身免疫性疾病的发生发展过程中发挥重要 作用， 已成为临床疾病的分子标志， 并被认为是某些免疫性疾病的治疗或诊断靶点。 (Ohnuma et al.(2011) Adv Clin Chem, 53, 51-84)
CD26由于可以和多种蛋白相互作用， 如 ADA、 CD45、 FAP-alpha等， 还可以结合 ECM, 导致表达 CD26细胞浸润活性的增加或降低， 可见 CD26在肿瘤生物学发挥重要 作用。 CD26的表达量在多种的新生瘤细胞表面或血清中会升高， 例如， CD26高表达于 某些进攻性 T细胞恶性肿瘤， 恶性间皮瘤， 肾瘤， 某些结肠癌 （Havre et al. (2008) Front Biosci, 13, 1634-1645 某些 CD26+结肠癌细胞亚群， CD26+恶性间皮瘤细胞都有明显肿 瘤干细胞特征（Ghani et al. (2011) Biochem Biophys Res Commun, 404， 735-742 and Pang et al. (2010) Cell Stem Cell, 6， 603-615 ), 因此 CD26可作为多种肿瘤的分子标志。
已有多种结合 CD26的鼠源抗体报道（Havre et al. (2008) Front Biosci, 13, 1634-1645 ), 在治疗某些 CD26高表达癌症， 以及抑制细胞迁移， 血管形成方面都能发挥作用。 有文 献报道抗 CD26的鼠单克隆抗体 1F7与 CD26的结合导致细胞周期停滞在 G1/S限制点，
并且 CD26的结合通过增强表达细胞周期调节蛋白质 p21来诱导 CD26 Jurkat转染子停滞 在 Gl， 用 1F7抗体治疗可以抑制 CD26+肿瘤的形成， 并在小鼠模型中增强存活率。 其他 抗 CD26的鼠单克隆抗体 E19和 E26， 这些抗体表现出抑制成纤维细胞和创伤细胞的细 胞迁移来形成单层的抑制作用、 对血管形成的抑制效果、 以及对人皮肤微血管内皮细胞 的侵入和毛细管新支的形成具有抑制效果。 由此可见， 抗 CD26单克隆抗体可通过改变 CD26的活性在治疗多种疾病中发挥作用。但是， 鼠单克隆抗体在直接应用于人体治疗时 会产生人抗鼠抗体反应 （ Human Anti-Mouse Antibody, HAMA )， 人体会产生抗小鼠抗体 的抗体， 不仅会减弱疗效， 也会导致急性致敏反应。 为了克服鼠源单抗的缺点， 基因工 程抗体技术发展出了人-鼠嵌合抗体， 嵌合抗体由鼠源性抗体的 V 区基因与人抗体的 C 区基因拼接为嵌合基因， 使鼠源性成分减少 70%左右， 以及人源化抗体， 在嵌合抗体的 基础上进一步用人抗体可变区的骨架区 （FR) 替代鼠 FR， 大大减少了抗体的鼠源成分。 但残留的少量鼠抗体的序列仍有可能潜在引发 HAMA。
发明内容
本发明所要解决的技术问题： 本发明旨在提供一种能有效的、 特异性结合人 CD26 的全人源抗体， 及其在制备治疗以 CD26 的表达， 特别是过量表达为特征的疾病， 诊断 CD26变化性表达疾病的药物中的新用途。 更具体地说：
本发明第一个目的是提供一种来源于人源的抗体或其片段， 所述抗体或其片段特异 性结合人 CD26, 优选特异性结合 CD26胞外区， 所述抗体或其片段的氨基酸序列包括选 自含有 SEQ ID NO: 2、 SEQ ID NO: 3 SEQ ID NO: 4 SEQ ID NO: 6 SEQ ID NO: 7
SEQ ID NO: 8一组序列的 6个互补决定区的任何区域的单克隆抗体或其片段或其片段的 偶联物， 或通过氨基酸置换或者修饰得到的氨基酸序列。
优选的是本发明中的抗体或其片段的氨基酸序列含有如 SEQ ID NO: 2、 SEQ ID NO: 3和 SEQ ID NO: 4所示序列的重链可变区的互补决定区。
优选的是本发明中的抗体或其片段的氨基酸序列含有如 SEQ ID NO:6、SEQ ID NO: 7 和 SEQ ID NO: 8所示序列的轻链可变区的互补决定区。
更为优选的是本发明中的抗体或其片段， 其重链可变区的氨基酸序列含有以下的互 补决定区：如序列 SEQ ID NO: 2所示的 CDRH1 ,如序列 SEQ ID NO: 3所示的 CDRH2, 如序列 SEQ ID NO: 4所示的 CDRH3;
以及其轻链可变区的氨基酸序列含有以下的互补决定区： 如序列 SEQ ID NO: 6所 示的 CDRL1 , 如序列 SEQ ID NO: 7所示的 CDRL2和如序列 SEQ ID NO: 8所示的 CDRL3。 更为优选的是本发明中的抗体或其片段含有重链可变区如 SEQ ID ΝΟ:1所示序列和 轻链可变区如 SEQ ID NO:5所示序列。 本发明第二个目的是提供一种来源于人源的单链抗体， 所述单链抗体的氨基酸序列 如 SEQ ID NO: 11所示。 本发明第三个目的是提供一种编码上述单链抗体的核苷酸序列， 所述核苷酸序列如 SEQ ID NO: 10所示。 本发明第四个目的是提供一种含有上述核苷酸序列的表达载体。
本发明第五个目的是提供一种含有上述表达载体的重组宿主菌。
本发明第六个目的是提供一种生产上述单链抗体的方法， 包括：
1 ) 在合适的条件下培养上述重组宿主菌表达抗体；
2) 然后从宿主菌中纯化、 收集抗体。
本发明第七个目的是提供上述抗体或其片段在制备治疗 CD26高表达肿瘤的药物中 的新用途。 可治疗以 CD26的表达， 特别是过量表达为特征的疾病， 并为诊断 CD26变 化性表达疾病提供了基于抗体的方法， 相关疾病包括但不限于自身免疫病和癌症。
发明进一步说明：
本发明中全人源抗 CD26单链抗体基因序列全长 723个核苷酸， 预期有 241个氨基 酸。具有 115个氨基酸的重链可变区（SEQ ID NO: 1 )和 111个氨基酸的轻链可变区（SEQ ID NO: 5 )，重链可变区和轻链可变区之间由 15个氨基酸的柔性肽连接（SEQ ID NO: 9)。
含有本发明 CD26单链抗体基因的表达载体和宿主菌均属于本发明的保护范围。 扩 增本发明单链抗体基因的任意片段的引物对也在本发明的保护范围之内。
本发明的有益效果有：
本发明中的抗体或其片段具有多种特性， 包括结合并中和 CD26的能力。 具体而言， 本发明得到的抗 CD26单链抗体与 CD26具有高特异性结合， 体外可以抑制肿瘤细胞通 过 CD26介导的细胞粘附作用， 体外抑制肿瘤细胞生长实验结果表明， 本发明得到的单
链抗体可以明显抑制肿瘤细胞的增殖和侵袭转移。
附图说明
图 1是单链抗体 ZHB-2cF9的结构示意图。 VH代表重链可变区结构域（SEQ ID NO: 1 )， VL代表轻链可变区结构域（SEQ ID NO: 5 )， Linker为连接 VH， VL的柔性肽（SEQ ID NO: 9)。
图 2是 CD26胞外区蛋白的鉴定图。 A是纯化后蛋白的 SDS-PAGE图， Lane 1为标 准蛋白， Lane 2为纯化后 CD26胞外区蛋白 （箭头所指）； B是纯化后 CD26胞外区蛋白 的 Western Blot鉴定图， Lane 1为标准蛋白， Lane 2为纯化后 CD26胞外区蛋白 （箭头所 指），一抗为抗 CD26鼠抗（购自 MBL)，二抗为兔抗鼠 -HRP抗体（购自 life technology)。
图 3A、 图 3B是噬菌体抗体与 CD26胞外区蛋白结合的 ELISA测定结果。 图 3A是 多克隆噬菌体 ELISA, CD26胞外区蛋白包被浓度为 l g/mL, 稀释 4轮筛选扩增的噬菌 体， 与包被 CD26胞外区蛋白的酶标板孵育， 抗 M13-HRP抗体再次孵育， 测定 450nm， 650nm吸光值， 并以 OD45Qnm-OD65Qnm作为最终值。 结果表明， 展示有 CD26特异性单链 抗体的噬菌体得到了明显的富集。 图 3B是单克隆噬菌体 ELISA测定结果， 挑选单克隆 至 96孔板中表达噬菌体抗体， 与包被 CD26胞外区蛋白的酶标板孵育， 抗 M13-HRP抗 体检测结果， 测定 OD45Qnm-OD65Qnm作为最终值， 结果显示 90%以上的单克隆与 CD26胞 外区蛋白产生阳性结合作用。
图 4是单链抗体 ZHB-2cF9的鉴定图。 A是镍柱纯化后的 ZHB-2cF9的 SDS-PAGE 图， Lane 2为 ZHB-2cF9样品蛋白， 蛋白箭头所指为目的蛋白大小条带； Lane 1为标准 蛋白； B是纯化后 ZHB-2cF9的 Western Blot鉴定图， 一抗为抗 myc鼠抗， 二抗为兔抗鼠 -HRP抗体， Lane l为 ZHB-2cF9样品， 蛋白箭头所指为目的蛋白大小条带， Lane 2为标 准蛋白。
图 5展示单链抗体 ZHB-2cF9与 CD26胞外区蛋白结合的 Western Blot鉴定图。一抗 为抗 myc鼠抗， 二抗为兔抗鼠 -HRP抗体。 Lane 1为标准蛋白， Lane 2为 ZHB-2cF9与 CD26胞外区蛋白结合作用，箭头所指显示了与图 2中 CD26胞外区蛋白大小一致的条带。 结果表明表明抗 CD26单链抗体 ZHB-2cF9可以特异性与 CD26胞外区蛋白结合。
图 6展示单链抗体与人间皮瘤细胞 NCI-H2452结合的免疫荧光图。 A是 ZHB-2cF9
与 NCI-H2452细胞的免疫荧光， B是阴性对照抗体与 NCI-H2452细胞免疫荧光结合检测 的阴性结果， 抗体浓度均由 5% MPBS稀释至 l(^g/mL。
图 7展示单链抗体抑制人间皮瘤细胞 NCI-H2452结合 ECM的检测结果。 实验所用 ECM为纤连蛋白， 实验分为 Binding组， Blank组， 抗体试验组， 考察单链抗体作用 12h 后的细胞对纤连蛋白的粘附情况。
OD450 (Binding组） -OD450 (Blank组） =完全粘附细胞值；
OD450 (抗体试验组） - OD450 (Blank组） =样品组的细胞粘附值；
IgG control为阴性对照抗体；
细胞粘附率 （％) =样品组的细胞粘附值 /完全粘附细胞值
结果表明， 与阴性对照 IgG control 相比， ZHB-2cF9 及阳性对照 scFv-YSllO 对 NCI-H2452细胞粘附均有明显抑制作用。
图 8展示单链抗体对人间皮瘤 NCI-H2452细胞增殖抑制的检测结果。 细胞以 lxlO4/ 孔的数量孵于 96孔板中， 在单链抗体 ZHB-2cF9， scFv-YSllO及阴性对照抗体 IgG (鼠） 作用 48h后， 采用 CCK-8试剂反应 30min， 测定 450nm的吸光度值， 细胞的增殖抑制率 以 OD450nm 的减少率％表示。 实验结果表明 ZHB-2cF9 及阳性对照 scFv-YSllO 对 NCI-H2452均有明显抑制作用， 且呈浓度依赖性。
图 9展示单链抗体对人结肠癌 HCT116细胞增殖抑制的检测结果。 细胞以 8xl04/孔 的数量孵于 96孔板中， 在单链抗体 ZHB-2cF9， scFv-YSl lO及阴性对照抗体 IgG (鼠） 作用 48h后， 采用 CCK-8试剂反应 30min， 测定 450nm的吸光度值， 细胞的增殖抑制率 以 OD450nm 的减少率％表示。 实验结果表明 ZHB-2cF9 及阳性对照 scFv-YSllO 对 NCI-H2452均有明显抑制作用， 且呈浓度依赖性。
具体实施方式
定义
"抗体"是能够通过可变区内至少一个抗原识别位点特异性结合靶抗原的免疫球蛋 白分子， 所述靶抗原如糖、 多核苷酸、 脂、 多肽等。 该术语不仅包括完整的多克隆和单 克隆抗体， 也包括其片段 （例如 Fab、 Fab'、 F (ab' ) 2、 Fv)、 单链抗体 （scFv)、 其突 变体、 包含抗体部分的融合蛋白和任一包含抗原识别位点的其他改变构型的免疫球蛋白
分子。 抗体包括任一类的抗体， 如 IgG、 IgA或 IgM (或其亚类）， 并且抗体不需要为任 一特定类。
如本文所用， "单链抗体"指的是免疫球蛋白重链可变区 （VH) 和轻链可变区 （VL) 由 10〜25个氨基酸多肽连接形成的单一链融合蛋白， 连接肽 （linker), 通常富含甘氨酸 和丝氨酸， 以利于单链抗体的稳定性与柔韧性。 连接方式可将 VL的 N端连接至 VH的 C 末端， 或者相反。尽管去除了恒定区并引入 linker, 单链抗体依然保留了免疫球蛋白对抗 原的特异性。
"嵌合抗体和人源化抗体"， 一般来说指的是组合了来自不止一个物种的区域的抗 体。 "嵌合抗体"在传统上包含来自小鼠 （或某些情况下为大鼠） 的可变区和来自人的恒 定区。 "人源化抗体"一般是指非人 （例如鼠）抗体形式， 其为含有衍生自非人免疫球蛋 白最小序列的特定嵌合免疫球蛋白，免疫球蛋白链或其片段（例如 Fv、 Fab、 Fab'、 F (ab' ) 2或抗体的其它抗原结合子序列）。 一般而言， 在人源化抗体中， 除 CDR外， 整个抗体是 由人来源的多核苷酸编码，或者与由人来源的多核苷酸编码的抗体在 CDR之外具有同一 性。一些或所有的 CDR是由非人生物体来源的核酸编码的， 将它们移植到人抗体可变区 的片层框架区以产生抗体， 该抗体的特异性由植入的 CDR决定。
如本文所用， "人源抗体"表示具有与人产生的抗体的氨基酸序列对应的氨基酸序列 的抗体、 和 /或用本领域已知或本文公开的制备人源抗体的任一技术制备的抗体。 人源抗 体可以用多种本领域已知技术产生。 例如， 人源抗体选自噬菌体文库， 其中所述的噬菌 体文库表达人源抗体。 人源抗体也可以通过将人免疫球蛋白基因座（loci) 引入转基因动 物来制备，其中所述的转基因动物例如已将内源免疫球蛋白基因部分或完全灭火的小鼠。 备选地， 人抗体可以通过永生化产生抗靶抗原的抗体的人 B淋巴细胞 （该 B淋巴细胞可 以从个人获得或者已在体外进行免疫） 来制备。 在一些实施方案中， 人源抗体为 "全人 源"， 这表示抗体包含人重链和轻链多肽。
现在结合以下实施例说明本发明。 提供这些实施例仅用于说明的目的， 本发明不限 于这些实施例， 而是包含明显由本文提供的教导产生的所有改变。用于构建载体和质粒、 将质粒导入宿主细胞和基因， 以及基因产物的表达鉴定等常规方法的详细描述可以从各 种出版物上获得， 例如《分子克隆实验指南第三版》。 实施例中涉及的百分比， 其中固体 试剂为重量百分比， 液体试剂为体积百分比。
部分材料来源说明于此：
细胞系： HLF 细胞， 是人的未分化的肝癌细胞， 获自 JCRB cell bank (Japanese Collection of Research Bioresources Cell Bank)。 NCI-H2452， 是人间皮瘤细胞， 获自中国 科学院典型培养物保藏委员会细胞库 /中国科学院上海生命科学研究院细胞资源中心。 HCT116,是人结肠癌细胞，获自中国科学院典型培养物保藏委员会细胞库 /中国科学院上 海生命科学研究院细胞资源中心。
培养基和缓冲液等试剂： RPMI-1640(GIBCO， Cat# 31800022, 添加 NaHC03 1.5g/L， glucose 2.5g/L, Sodium Pyruvate 0.11 g/L) , 90%； 优质胎牛血清， （GIBCO) 10%。
2YT培养基： 1L内含 Trptone (OXID) 16g， Yeast Extract (OXID) lOg, NaCl 5g.
2YT-AK培养基： 2YT含 10(^g/mL氨苄青霉素和 5(^g/mL卡那霉素.
2YT-AG培养基： 2YT含 10(^g/mL氨苄青霉素和 2%葡萄糖.
IOXPBS: (购自北京索莱宝， Cat#P1022) .
5%MPBS或 2%MPBS: 含有 5%或 2%脱脂奶粉 （OXID) 的 PBS.
0.1%PBST: 含有 0.1%Tween20 (购自北京索莱宝） 的 PBS.
2%BSA: 含有 2% BSA (购自 MP Biomedicals) 的 PBS
Amp: 氨苄青霉素 （购自上海生工） .
Kan: 卡那霉素 （购自上海生工） .
IPTG: (购自 Amresco) .
其他常见试剂如盐酸， NaCl, Tris, 甘氨酸等购自国药集团化学试剂有限公司。 菌株： TG1 , 大肠杆菌（获自中国微生物菌种网）； HB2151 , 大肠杆菌（获自中国微 生物菌种网）
质粒： p3XFLAG-CMV9 (购自 Sigma-Aldrich )； pHEN2 (；获自 Medical Research Council(UK) )
实施例 1 CD26胞外区蛋白的制备
本研究的目的在于通过重组质粒转染真核细胞， G418抗性筛选获得稳转细胞株， 分 泌表达 CD26胞外区蛋白， 经亲和层析分离纯化出 CD26胞外区蛋白。
CD26胞外区基因是选自 Uniprot: P27487序列中氨基酸 29-766位 "Extracellular"区域 的合成基因， 将 CD26胞外区基因连入质粒 p3XFLAG-CMV9， 酶切位点为 Hind III， Xba I， 构建重组质粒 （J.萨姆布鲁克.分子克隆实验指南.第三版.科学出版社.2002. P68)。
采用 Lipofectamine LTX Reagent (购自 Invitrogen) 并按照说明书方法将含 CD26胞 外区基因的重组质粒转入 HLF细胞。根据稳定转染细胞系构建方法（Current Protocols in Molecular Biology, P9.5.5 ) 获得 CD26胞外区稳定表达细胞株， 记为 HLF-4D9, 无血清 大量培养， 分泌表达 CD26胞外区蛋白， 该蛋白带有 Flag标签， 采用 ANTI-FLAG M2 Affinity Gel (购自 Sigma-Aldrich), 对无血清培养上清进行纯化获得 CD26胞外区蛋白纯 品， SDS-PAGE (图 2A) 分析及 Western Blot(J.S.博尼费斯农.精编细胞生物学实验指南. 科学出版社. 2007. P177)鉴定 （图 2B)， 一抗为抗 CD26鼠抗 （购自 MBL)， 二抗为兔抗 鼠 -HRP抗体 （购自 life technology)
结果如图 2所示， 图 2A是 SDS-PAGE对纯化蛋白的鉴定图， 经一步亲和层析获得 电泳纯级目的蛋白大小的单条带 （Lane2)， 图 2B是对纯化的蛋白进行 Western Blot鉴定 图， Lane2所示条带与 SDS-PAGE鉴定结果蛋白大小一致， 表明 CD26胞外区基因经克 隆重组转染真核细胞并表达， 纯化后获得电泳纯级 CD26胞外区蛋白。
实施例 2全人源抗 CD26单链抗体的分离
该单链抗体从人单链抗体噬菌体展示库， 采用 CD26胞外区蛋白的固相亲和筛选得 到， 该人单链抗体噬菌体展示库由江苏众红生物工程创药研究院有限公司构建。 该含有 人细胞产生的抗体的重链及轻链可变区的噬菌体展示库构建自人外周血淋巴细胞， 通过 使用抗体可变区基因特异的引物进行首轮扩增， 二轮扩增将重链轻链可变区采用柔性连 接肽基因连接形成单链抗体基因，并将单链抗体基因克隆入质粒 pHEN2中转化大肠杆菌 TG1 , 获得 108 p.f.u.噬菌体单链抗体展示库。 （沈倍奋.重组抗体.科学出版社. 2005. P107) 将该单链抗体库原种培养到对数生长阶段，用 M13K07辅助噬菌体感染，在 2YT-AK 培养基中， 30°C摇床培养过夜。 噬菌体用 4% PEG/2.5M NaCl沉淀， 并重悬于 PBS中并 测定抗体库滴度， 获得滴度为 10up.f.u的噬菌体单链抗体库。 （甄永苏.抗体工程药物.化 学工业出版社. 2002. P51 ) 以 PBS稀释 CD26胞外区蛋白至 5(^g/mL; 包被至酶标板 (Maxi-sorp96, Nunc) 中， 同时设置空白对照孔 （不含 CD26胞外区蛋白）， 随后进行封 闭。 将噬菌体单链抗体库悬浮于 2%MPBS， 取 ΙΟΟμΙ^加入到封闭过的空白孔中， 室温放
置 60 min后， 加入到含有 CD26胞外区蛋白的孔中， 室温放置 2h; 0.1% PBST及 PBS 分别洗涤 10次， 加入 ΙΟΟμΙ^ Ο.ΙΜ 盐酸（用甘氨酸调至 ρΗ2.2)， 室温振荡 10 min， 15 L 1M Tris (pH9.0) 用于迅速中和洗脱下来的噬菌体； 中和后的噬菌体感染 5mL对数期的 大肠杆菌 TG1， 取 100uL， 做 1〜3次 100倍系列稀释， 然后将系列稀释物铺 TYE固体培 养基 （含有 10(^g/mL Amp禾卩 1%葡萄糖）， 剩余的菌液再加入 20mL含 2x l01Q M13K07 辅助噬菌体的 2YT-AG进行扩增和制备噬菌体库， 用于下一轮筛选过程， 共进行 4轮筛 选。
被展示在噬菌体颗粒表面的抗体片段被称为噬菌体抗体， 本实验先通过多克隆噬菌 体 ELISA鉴定 4轮筛选后 CD26特异性噬菌体抗体的富集情况， 后通过单克隆噬菌体 ELISA进一步鉴定挑选出髙亲和力的噬菌体抗体。
多克隆噬菌体 ELISA鉴定，包被抗原即 l g/mL CD26胞外区蛋白至酶标板，封闭后， 取 ΙΟμΙ^每轮筛选后获得的噬菌体， 2% MPBS稀释后加入到抗原包被酶条中。 室温孵育 90min, 洗涤后加入鼠抗 M13噬菌体 -HRP抗体（购自北京义翘神州生物技术公司）室温 孵育 lh， 洗涤后， 加入 ΙΟΟμΙ^ ΤΜΒ显色液（购自 AMRESCO)。 室温孵育 lOmin后， 1M 稀硫酸终止反应。 测定 OD45()禾 B OD65()， 并以 OD45(rOD65()作为最后检测结果。 如图 3A 所示， 以 BSA包被的酶标板作为阴性对照， 随着筛选轮数增加， 噬菌体抗体与 CD26胞 外区蛋白的结合增强， 表明带有 CD26特异性抗体的噬菌体得到了明显的富集。
单克隆噬菌体 ELISA鉴定， 随机挑选第三轮第四轮筛选过程的滴度测定平板上挑取 单克隆到 96孔细菌培养板（购自 Coming)中，每孔已经添加 2YT培养基（含有 10(^g/mL Amp和 1%葡萄糖）， 37°C培养至对数期， 每孔加 109 p.f.u.M13K07辅助噬菌体 37°C静置 感染 30min， 37°C培养 lh。 1800g离心 lOmin,弃上清。将菌体沉淀重悬于 200μΙ^ 2YT-AK 培养基， 30°C摇床培养过夜。次日 1800g离心 lOmin获得的含噬菌体的培养上清，用 1/10 体积的 20%MPBS 室温孵育 lh， 再加入到包被有重组 CD26胞外区蛋白的酶标板中， ELISA 鉴定 （方法及试剂同多克隆噬菌体 ELISA 鉴定）。 测定 OD45Q和 OD65Q， 并以 OD45Q-OD65Q作为最后检测结果。选出读数高的克隆进行 DNA序列测定。图 3B显示了部 分单克隆噬菌体 ELSIA的检测结果， 90%以上的单克隆显示阳性结合作用， 进一步表明 通过 4轮筛选， 带有 CD26特异性抗体的噬菌体得到了明显的富集。 从中挑选 50个读数 高的单克隆测序， 获得核苷酸编码序列如 SEQ ID NO: 10 所示的单链抗体 （记为 ZHB-2cF9 所对应的单链抗体 ZHB-2cF9的氨基酸序列为 SEQ ID NO: 11。
实施例 3抗 CD26单链抗体的可溶性表达及分离纯化
pHEN2是一个双功能噬菌粒载体， 在表达检测标签 （c-my tag) 与外壳蛋白基因之 间有一个琥珀型终止密码子 （Amber) TAG。 如果噬菌体感染琥珀突变 （SupE) 抑制型 菌株， 如 TG1， TAG密码子被翻译为谷氨酸， 序列可以通读翻译， 抗体片段与外壳蛋白 p3 融合表达于噬菌体表面； 当噬菌体感染非琥珀突变抑制型菌株时， 如 HB2151 , 翻译 在 TAG处终止，可得到可溶型表达的抗体片段。抗体片段 C端带有 6xHis tag及 c-myc tag, 利于纯化和检测鉴定。 ZHB-2cF9单链抗体采用大肠杆菌周质空间的可溶性表达， 纯化采 用高渗透法提取细胞周质蛋白， 再利用镍柱亲和层析一步分离纯化获得纯度较高的目的 蛋白。
采用质粒小提试剂盒从菌体 TG1中提取出含有编码单链抗体 ZHB-2cF9基因的质粒 (记为 2cF9-pHEN2)， 用于转化非抑制子大肠杆菌 HB2151 , 转化采用氯化钙法制备和转 化感受态大肠杆菌 HB2151 (J.萨姆布鲁克.分子克隆实验指南.第三版.科学出版社.2002. P96), 所得转化后菌株记为 HB2151-2cF9。
HB2151-2cF9在 2YT-AG培养基中， 37°C培养至对数期时（OD6(K)=0.8)， 加入终浓度 ImM的 IPTG， 30°C诱导过夜（16〜20h)， 表达可溶性单链抗体 ZHB-2cF9。 6000rpm, 4°C 离心 15min， 收集菌体， 高渗溶液 （50mM Tris-HCl， 20%蔗糖， ImM EDTA， pH8.0) 重 悬菌体，缓慢搅拌 lh， 4°C， 10000g离心 lOmin,倒出上清进行镍柱亲和层析（购自 GE)， 纯化步骤按照 GE的标准操作流程进行，即采用含有 5mM咪唑的平衡缓冲液 ( Tris 50mM， NaC1 500mM， pH7.5 ) 平衡 ImL镍柱， 10个柱体积后上样， 再次采用含有 5mM咪唑的 平衡缓冲液洗涤镍柱上非特异性结合的杂蛋白， 用含有 50mM咪唑的平衡缓冲液洗涤非 特异性杂蛋白， 最后用含有 lOOmM咪唑的平衡缓冲液洗脱目的蛋白。 15%SDS-PAGE检 测收集的样品， Western Blot进一步鉴定单链抗体，一抗为抗 c-myc鼠抗（购自恩晶生物）， 二抗为兔抗鼠 -HRP抗体。 图 4A显示纯化后样品中含有目的蛋白大小条带 （箭头所指）， Western Blot (图 4B)鉴定结果与 SDS-PAGE—致，表明经过镍柱一步亲和层析 ZHB-2cF9 单链抗体得到了较好的纯化。
实施例 4抗 CD26单链抗体与 CD26胞外区蛋白结合的 Immunoblot鉴定
该实验目的是为了验证筛选获得的抗 CD26单链抗体 ZHB-2cF9与 CD26胞外区蛋白
的特异性结合作用。 重组 CD26胞外区蛋白进行 SDS-PAGE电泳， 半干转法 lh， 将蛋白 转印到 NC膜（购自 Millipore);转印结束，将膜在 5% MPBS中室温封闭 lh;用 5%MPBS 稀释 ZHB-2cF9单链抗体至 l g/mL, 室温孵育 lh， TBS洗涤 3遍； 用鼠 anti-Myc抗体 (购自恩晶生物） 室温孵育 lh， TBS洗涤 3遍； 用兔抗鼠 -HRP二抗孵育 lh后凝胶成像 仪 （ImageQuant LAS4000, GE) 曝光， 结果如图 5所示， 在 CD26胞外区目的蛋白大小 处有明显条带， 表明抗 CD26单链抗体 ZHB-2cF9可以特异性与 CD26胞外区蛋白结合。
实施例 5可溶性单链抗体 ZHB-2cF9与人间皮瘤细胞 NCI-H2452的免疫荧光
CD26在人间皮瘤细胞 NCI-H2452细胞表面高表达（Inamoto et al. (2007) Clin Cancer Res, 13, 4191-200),本实验通过鉴定 ZHB-2cF9与 NCI-H2452细胞的免疫荧光结合情况， 进一步验证筛选获得的单链抗体 ZHB-2cF9可以与高表达 CD26的 NCI-H2452细胞特异 性结合， 同时证明 ZHB-2cF9可以与细胞表面的 CD26特异性结合。
将人间皮瘤细胞 NCI-H2452消化，离心后重悬， l x lO5/孔包被 24孔板让细胞贴壁 12h， 4%多聚甲醛固定， PBS洗 3次， 5% MPBS室温封闭 lh。 PBS洗 3次， 不同孔分别加入 抗体 ZHB-2cF9或阴性对照抗体 IgG (鼠）（购自 life technology) ， 37 °C孵育 2h， 以 PBS 洗涤 3次， ZHB-2cF9抗体孔加入鼠抗 myc-FITC抗体 （购自 Sigma- Aldrich), 对照抗体 孔加入羊抗鼠荧光二抗 （购自 life technology), 37°C孵育 lh， PBS洗涤 3次， 荧光显微 镜 （Olympus) 观察并拍照， 结果如图 6所示 ZHB-2cF9单链抗体与 NCI-H2452有明显 的荧光显色， 而对照抗体无明显荧光显色， 表明筛选获得的单链抗体 ZHB-2cF9 可以与 高表达 CD26的 NCI-H2452细胞特异性结合， 进一步证明 ZHB-2cF9可以与细胞表面的 CD26特异性结合。
实施例 6抗 CD26单链抗体对人间皮瘤细胞 NCI-H2452的粘附抑制作用
CD26 通过与细胞外基质蛋白 （ECM) 的结合， 介导肿瘤细胞的粘附作用， 纤连蛋 白是常见的 ECM之一， CD26与纤连蛋白发生特异性结合作用， 从而介导细胞的粘附， 而这种结合作用可被抗 CD26 抗体所阻断 （Inamoto et al. (2007) Clin Cancer Res, 13, 4191-200), 从而阻断肿瘤细胞的粘附作用。
YS110为 Y's Therapeutics公司开发的抗 CD26单克隆鼠抗的人源化抗体， 目前已开
展抗 CD26高表达肿瘤的临床实验。根据 YS110抗体的重链及轻链可变区基因序列（CN 101282994)，全合成构建 YS110的单链抗体型式，即 scFv-YSl lO作为本实验的阳性对照， 表达及纯化过程同 ZHB-2cF9。
粘附实验采用 24 孔板， 五组平行实验， 每组 4 个复孔， 其中一组为空白对照组 (Blank), 直接用 2%BSA封闭， 其余四组用 l(^g/mL纤连蛋白 （购自 BD Biosciences) 室温 20〜25 °C包被 90min。 PBS洗 3次后加入 2%BSA 37°C封闭 lh。 同时将 lOx lO5个 NCI-H2452细胞用 PBS洗 3 次后分成等量的五组， 三组为抗体试验组， 细胞分别用含 5(^g/mL的 ZHB-2cF9， 阳性对照 scFv-YS110， 阴性对照抗体 IgG (鼠） 的 RPMI-1640 培养基处理 2h， 另外两组为对照组 （Binding组， Blank组） 用与试验组含等量 PBS的 RPMI-1640培养基处理 2h后， 等分加入五组封闭后的 24孔板中， 培养箱继续培养 12h。 用 PBS洗孔 3次，洗掉未粘附的细胞，每孔加入 270μ RPMI-1640及 30μ 的 CCK-8 (购 自同仁化学研究所）， 37°C继续培养 30min， 在酶标仪 450nm波长处测定光吸收值 （OD 值）， OD值的大小与活细胞数量成正比，据此计算细胞的粘附率。细胞粘附率（％) =[OD (抗体试验组） -OD ( Blank组） ]/[ OD (Binding组） -OD (Blank组） ]。 图 7显示， 用 scFv-YSl lO及 ZHB-2cF9处理后的细胞对纤连蛋白的粘附下降，而阴性对照抗体 IgG(鼠） 则不影响细胞粘附， 表明 ZHB-2cF9可以抑制 CD26对 ECM的结合， 抑制 CD26介导的 肿瘤细胞粘附效应， 抑制了肿瘤细胞的向其他器官的侵袭转移。
实施例 7抗 CD26单链抗体对人间皮瘤细胞 NCI-H2452的增殖抑制作用
将 NCI-H2452细胞 l x lO4/孔接种 96孔细胞培养板， 100μΙ7孔， 培养 12h后用 1%小 牛血清培养基 （含作用抗体） 替换原培养基。 将培养的细胞分为三组， 分别对应加入的 作用抗体为：不同浓度的单链抗体 ZHB-2cF9，阳性对照 scFv-YSl lO及阴性对照抗体 IgG (鼠）。 作用抗体终浓度分组为 0, 0.1， 1.0， l(^g/mL， 每组 5个实验复孔。 培养箱继续培 养 48h， 观察细胞生长状况后每孔加入 ΙΟμΙ^的 CCK-8 (购自同仁化学研究所）， 37°C继 续培养 30min， 在酶标仪 450nm波长处测定光吸收值 （OD值）， OD值的大小与活细胞 数量成正比，据此计算单链抗体 ZHB-2cF9对细胞增殖的抑制率。如图 8所示， ZHB-2cF9 及阳性对照 scFv-YS 110对 NCI-H2452均有明显抑制作用， 且呈浓度依赖性， ZHB-2cF9 与阳性对照的活性相当，阴性对照 IgG对 NCI-H2452没有明显抑制作用，表明 ZHB-2cF9 可以对高表达 CD26的间皮瘤细胞增殖有抑制作用， 可用作 CD26高表达肿瘤的潜在治
疗药物， 且相比于阳性对照 scFv-YS110， ZHB-pA12为全人源化抗体， 消除了外源蛋白 对人体免疫系统的影响。
实施例 8抗 CD26单链抗体对人结肠癌细胞 HCT116的增殖抑制作用
除人间皮瘤细胞 NCI-H2452外， 高表达 CD26的结肠癌细胞系 HCT116 (Abe et al. (2011) BMC Cancer, 2011, 11 :51 ) 也被用于考察单链抗体对细胞的增殖抑制效果。
将 HCT116细胞 8 X 104/孔接种 96孔细胞培养板， 100μΙ7孔， 培养 12h后用 1%小牛 血清培养基 （含作用抗体） 替换原培养基。 将培养的细胞分为三组， 分别对应加入的作 用抗体为： 不同浓度的单链抗体 ZHB-2cF9， 阳性对照 scFv-YSl lO及阴性对照抗体 IgG (鼠）。 作用抗体终浓度分组为 0, 0.1， 1.0， l(^g/mL， 每组 5个实验复孔。 培养箱继续培 养 48h， 观察细胞生长状况后每孔加入 ΙΟμΙ^的 CCK-8 (购自同仁化学研究所）， 37°C继 续培养 30min， 在酶标仪 450nm波长处测定光吸收值 （OD值）， OD值的大小与活细胞 数量成正比，据此计算单链抗体 ZHB-2cF9对细胞增殖的抑制率。如图 9所示， ZHB-2cF9 及阳性对照 scFv-YSllO对 HCT116均有明显抑制作用， 且呈浓度依赖性， ZHB-2cF9与 阳性对照的活性相当， 阴性对照 IgG对 HCT116没有明显抑制作用， 表明 ZHB-2cF9可 以对高表达 CD26的结肠癌细胞增殖有抑制作用，再次证明 ZHB-2cF9可用作 CD26高表 达肿瘤的潜在治疗药物， 且相比于阳性对照 scFv-YSllO, ZHB-pA12为全人源化抗体， 消除了外源蛋白对人体免疫系统的影响。
Claims
1、 一种抗 CD26 抗体或其片段， 其特征在于， 所述抗体或其片段特异性结合人 CD26, 所述抗体或其片段的氨基酸序列包括选自含有 SEQ ID NO: 2、 SEQ ID NO: 3、 SEQ ID NO: 4、 SEQ ID NO: 6、 SEQ ID NO: 7、 SEQ ID NO: 8—组序列的 6个互补决定区 的任何区域的单克隆抗体或其片段或其片段的偶联物， 或通过对其氨基酸置换或者修饰 得到的氨基酸序列。
2、 如权利要求 1所述的抗 CD26抗体或其片段， 其特征在于， 所述抗体或其片段的 重链可变区的氨基酸序列含有如 SEQ ID NO: 2、 SEQ ID NO: 3和 SEQ ID NO: 4所示的互 补决定区， 和 /或所述抗体或其片段的轻链可变区的氨基酸序列含有如 SEQ ID NO: 6、 SEQ ID NO: 7和 SEQ ID NO: 8所示的互补决定区。
3、 如权利要求 1所述的抗 CD26抗体或其片段， 其特征在于， 所述抗体或其片段的 重链可变区的氨基酸序列如 SEQ ID ΝΟ:1所示， 所述抗体或其片段的轻链可变区的氨基 酸序列如 SEQ ID NO:5所示。
4、 一种抗 CD26 的单链抗体， 其特征在于， 所述单链抗体的氨基酸序列如 SEQ ID NO: 11所示。
5、 一种编码如权利要求 4所述的单链抗体的核苷酸序列， 其特征在于， 所述核苷酸 序列如 SEQ ID NO: 10所示。
6、 一种含有如权利要求 5所述的核苷酸序列的表达载体。
7、 一种含有如权利要求 6所述表达载体的重组宿主菌。
8、 一种生产如权利要求 4所述单链抗体的方法， 包括：
1 ) 在合适的条件下培养如权利要求 7所述的重组宿主菌并表达抗体；
2) 然后从宿主菌中纯化、 收集抗体。
9、 如权利要求 1至 3中任一项所述的抗体或其片段在制备抑制 CD26高表达的肿瘤 细胞增殖或侵袭转移的药物中的新用途。
10如权利要求 4所述的单链抗体在制备抑制 CD26高表达的肿瘤细胞增殖及侵袭转 移的药物中的新用途。
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| WO2021222587A1 (en) * | 2020-04-29 | 2021-11-04 | HCW Biologics, Inc. | Anti-cd26 proteins and uses thereof |
| US11401324B2 (en) | 2018-08-30 | 2022-08-02 | HCW Biologics, Inc. | Single-chain chimeric polypeptides and uses thereof |
| US11518792B2 (en) | 2018-08-30 | 2022-12-06 | HCW Biologics, Inc. | Multi-chain chimeric polypeptides and uses thereof |
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| US11672826B2 (en) | 2018-08-30 | 2023-06-13 | HCW Biologics, Inc. | Methods of treating aging-related disorders |
| US11738052B2 (en) | 2019-06-21 | 2023-08-29 | HCW Biologics, Inc. | Multi-chain chimeric polypeptides and uses thereof |
| US12024545B2 (en) | 2020-06-01 | 2024-07-02 | HCW Biologics, Inc. | Methods of treating aging-related disorders |
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| CN109709337B (zh) * | 2018-12-28 | 2021-09-14 | 江苏众红生物工程创药研究院有限公司 | 人cd26的免疫组化检测试剂盒及其临床应用 |
| CN110386983B (zh) * | 2019-08-12 | 2021-02-05 | 西南医科大学 | 全人源抗cd20重组抗体 |
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