WO2022257106A1 - Humanized anti-human gpvi monoclonal antibody fab fragment and application thereof - Google Patents

Humanized anti-human gpvi monoclonal antibody fab fragment and application thereof Download PDF

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WO2022257106A1
WO2022257106A1 PCT/CN2021/099670 CN2021099670W WO2022257106A1 WO 2022257106 A1 WO2022257106 A1 WO 2022257106A1 CN 2021099670 W CN2021099670 W CN 2021099670W WO 2022257106 A1 WO2022257106 A1 WO 2022257106A1
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human
gpvi
amino acid
monoclonal antibody
fab fragment
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PCT/CN2021/099670
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French (fr)
Chinese (zh)
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张慧君
杜勇
杜达仁
陈太标
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江苏丰华生物制药有限公司
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Priority to PCT/CN2021/099670 priority Critical patent/WO2022257106A1/en
Priority to CN202180099255.6A priority patent/CN118043349A/en
Publication of WO2022257106A1 publication Critical patent/WO2022257106A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

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  • the invention belongs to the field of genetic engineering recombinant protein medicine, specifically, belongs to the field of genetic engineering recombinant antibody medicine. It relates to a humanized anti-human GPVI monoclonal antibody Fab fragment and application thereof.
  • the humanized anti-human GPVI monoclonal antibody Fab fragment is composed of the complementarity-determining regions (Complementarity-Determining Regions, CDRs) of the rat (rat) anti-human GPVI monoclonal antibody and the framework of the human IgG antibody Fab fragment .
  • the nucleotide sequence encoding the Fab fragment of the humanized anti-human GPVI monoclonal antibody is transfected into CHO cells via a suitable plasmid vector, the transfected CHO secretes and expresses the Fab fragment of the humanized anti-human GPVI monoclonal antibody.
  • the Fab fragment of humanized anti-human GPVI monoclonal antibody can specifically bind to non-reduced human GPVI protein, and can inhibit the binding of GPVI to collagen, and this inhibition has a dose-effect relationship.
  • the present invention also relates to a nucleotide coding sequence, a recombinant expression plasmid, and a recombinant expression cell line for producing the humanized anti-human GPVI monoclonal antibody Fab fragment antibody.
  • the humanized anti-human GPVI monoclonal antibody Fab fragment disclosed by the present invention can be used for clinical treatment of platelet GPVI-related diseases.
  • Platelet glycoprotein 6 (Glycoprotein VI, GPVI) is a transmembrane glycoprotein receptor on the surface of platelets, belonging to the immunoglobulin-like receptor superfamily, with a molecular weight of about 58-64 kDa, encoded by the GPVI gene.
  • the mature GPVI molecule (the amino acid sequence is shown in SEQ ID NO.1) is composed of three functional regions, namely the extracellular region, the transmembrane region and the intracellular region.
  • monomer and dimer Morphology of which dimer is the main functional form and has a high affinity with collagen.
  • GPVI forms a signaling complex with the Fc receptor ⁇ chain through a salt bridge, and this complex can trigger a cascading tyrosine phosphorylation signaling pathway.
  • exposed collagen forms a procoagulant surface on which platelet aggregation is mediated by GPVI and GPIb-IX-V.
  • GPVI is mainly activated by collagen types I and III, triggering the release of soluble secondary activators such as ADP and thromboxane A 2 (Thromboxane A 2 ), thereby activating and recruiting circulating platelets to aggregate , causing thrombosis.
  • thrombosis induced by platelets in response to vascular injury or inflammation is a normal physiological process.
  • circulating platelets quickly adhere to the exposed collagen, and the thrombosis caused by this platelet aggregation can reduce blood loss, but in pathological conditions, such as atherosclerotic plaque rupture leads to collagen
  • Exposure-stimulated thrombosis has been associated with myocardial infarction and stroke.
  • an ideal antiplatelet antithrombotic drug should have highly effective antiplatelet aggregation activity, but at the same time, it will not affect the normal blood coagulation function and will not cause obvious bleeding side effects in patients.
  • GPVI mimetics such as GPVI-Fc fusion protein dimer
  • GPVI depletion depletion
  • GPVI functional blocking molecules such as aptamer , small molecule drugs, antibodies, etc.
  • Invention patent CN103396492A application specification discloses an anti-GPVI single-chain variable fragment (single-chain variable fragment, SCFV) antibody fragment.
  • SCFV single-chain variable fragment
  • the inventor isolated B lymphocytes with affinity for platelets from patients with thrombocytopenic purpura after blood transfusion, and obtained the gene fragment of anti-GPVI antibody through gene amplification, thus constructing a gene fragment containing 8 histidine tags SCFV antibody fragment, this fragment can prevent the aggregation of platelets.
  • Patent application WO2005054294 discloses a rat anti-human GPVI monoclonal antibody hGP5C4, which can specifically bind to human GPVI, inhibit the binding of GPVI to collagen, and inhibit platelet aggregation .
  • Patent application WO2005111083 (Antibodies specific for glycoprotein vi and methods of producing these antibodies)/CN1964990B discloses 4 strains of anti-GPVI monoclonal antibodies-OM1, OM2, OM3 and OM4, these monoclonal antibodies can prevent the binding of GPVI and collagen and inhibit collagen Protein-induced platelet aggregation.
  • Patent application US20060088531 discloses a fragment of SCFV-10B12, 10B12 can inhibit collagen protein-induced platelet aggregation and platelet-collagen-related peptide (Collagen-Related Peptide, CRP) adhesion attached. Segment specifically binds to loop 9 of domain 2 of human GPVI.
  • Patent application WO2008049928 discloses an SCFV antibody fragment engineered using anti-GPVI monoclonal antibody 9O12.2, which has similar biological activity to the parental antibody 9O12.2.
  • Patent application WO2011073954 Novel antagonist antibodies and their fab fragments against gpvi and uses thereof
  • CN102725309B discloses an anti-GPVI monoclonal antibody 390, which can destroy the GPVI protein on the surface of platelets and cause depletion of GPVI.
  • Patent application WO2017021539 (Novel anti-human gpvi antibodies and uses thereof)/CN108289939A discloses an antibody or antibody fragment that can bind to a conformational epitope composed of positions 114-142 and 165-187 of the human GPVI amino acid chain.
  • Patent application WO2019219765 (Antibodies targeting glycoprotein vi)/CN112118869A discloses an anti-GPVI Fab fragment, which is screened from a non-specific MorphoSys-YlanthiaTM phage library and can inhibit collagen-induced platelet aggregation.
  • the invention discloses a humanized anti-human GPVI monoclonal antibody Fab fragment and application thereof.
  • the humanized anti-human GPVI monoclonal antibody Fab fragment (hereinafter referred to as anti-hGPFab) is composed of the CDRs of the heavy chain and light chain of the rat (rat) anti-human GPVI monoclonal antibody and the framework of the human IgG antibody Fab fragment composition.
  • the present invention also relates to a nucleotide coding sequence, a recombinant expression plasmid, and a recombinant expression cell strain for producing the hGPFab protein.
  • the hGPFab disclosed in the present invention can be used for clinical treatment of platelet GPVI-related diseases.
  • Antibody is a type of macromolecular immunoglobulin produced by B cells after the animal's immune system is stimulated by foreign substances (antigens, such as bacteria, viruses and other foreign microorganisms). It is divided into IgG, IgM, IgA, IgE and There are 5 subtypes of IgD, of which IgG is the main subtype. IgG is a "Y" shaped tetramer composed of two light chains with a molecular weight of 25 kDa and two heavy chains with a molecular weight of 50 kDa.
  • Fab is an antigen-binding fragment (Fab).
  • Fab A complete IgG antibody molecule is decomposed into two identical Fab fragments and an Fc fragment after being degraded by papain ( Figure 1).
  • Fab consists of a complete light chain (VL+CL) and 1/2 heavy chain (VH+CH1) (Fd fragment) combined by a disulfide bond. VL and HL together constitute a monovalent antigen-binding region (paratope).
  • Light chain variable region refers to the N-terminal 1/2 part of the antibody light chain, VL includes 3 hypervariable regions (hypervariable region, HV) and 4 framework regions (framework , FR), when the protein is folded, these three hypervariable regions form three loops (loop), these three loops are often called complementarity determining regions of the light chain.
  • Heavy chain variable region refers to the N-terminal 1/4 part of the antibody heavy chain, and VH includes 3 hypervariable regions (hypervariable region, HV) and 4 framework regions (framework , FR), when the protein is folded, these three hypervariable regions form three loops (loop), these three loops are often called complementarity-determining regions of the heavy chain.
  • Humanized antibody refers to replacing the amino acid sequence outside the antigen-binding site (often referred to as the CDR region) of a non-human monoclonal antibody with the amino acid sequence of a human antibody. Humanized antibodies retain the affinity and specificity of parental non-human monoclonal antibodies while reducing their heterogeneity, which can significantly reduce the human anti-mouse antibody (HAMA) reaction that may be produced by the human body.
  • HAMA human anti-mouse antibody
  • Complementarity-determining regions refer to the three loops formed by the variable regions of the heavy and light chains when the protein is folded, because their shapes are complementary to those of their corresponding antigens. named. The six loops of the heavy chain and the light chain together form an antigen-binding region that can bind a specific epitope.
  • the Fd fragment is the heavy chain part of the Fab fragment, consisting of about 220 amino acids, including the variable region and the constant region CH1 of the heavy chain.
  • the hGPFab of the present invention is a monovalent heterodimer formed by combining humanized IgG heavy chain Fd fragment and light chain through a disulfide bond, where the heavy chain Fd fragment refers to the N-terminal part of the antibody heavy chain amino acid sequence 1/2 part, including heavy chain variable region and constant region CH1 (VH+CH1); light chain refers to the whole molecule of light chain, including light chain variable region and constant region (VL+CL).
  • a non-human anti-human GPVI monoclonal antibody is selected to be humanized.
  • a non-human anti-human GPVI monoclonal antibody it can effectively block the binding of human GPVI and collagen; it can prevent the aggregation of platelets; it does not have the activity of activating GPVI to cause platelet activation.
  • the rat anti-human GPVI monoclonal antibody strain hGP5C4 was determined to be the subject of humanization.
  • hGP5C4 is a monoclonal antibody prepared by immunizing Lou/C rats (rat) with human GPVI-Fc dimer protein as the immunogen.
  • human GPVI-Fc dimer protein as the immunogen.
  • patent WO2005054294 Inhibitors of glycoprotein vi p pl based on monoclonal hgan 5 boclonal hgan 4).
  • the Fab fragment obtained by degrading hGP5C4 with papain can effectively prevent the combination of GPVI and collagen; it can also specifically bind to GPVI on the surface of platelets, inhibit the activation of platelets induced by collagen stimulation, and prevent the activation of its active markers PAC-I and CD62 -P expression; effectively inhibit the aggregation of human platelets and the release of ATP; in the ex vivo (ex vivo) human red blood cell bleeding time test, hGP5C4 has no effect on the bleeding time. All these data indicate that hGP5C4 has specific antiplatelet function, but does not have intrinsic stimulating activity, suggesting that hGP5C4 has good application potential.
  • hGP5C4 antibody is derived from rats, its heterogeneity prevents it from being directly applied to humans.
  • heterologous properties can be changed through humanization, which provides the possibility for clinical application.
  • nucleotide coding sequence of the variable region of the heavy chain of the above antibody strain hGP5C4 is CS114614.1 (nucleic acid sequence of 5C4 heavy chain variable) (SEQ ID NO.2); the variable region of the light chain
  • the nucleotide coding sequence is CS114616.1 (nucleotide sequence of 5C4 light chain variable Domain) (SEQ ID NO.4), and its amino acid sequence (SEQ ID NO.3, SEQ ID NO.5) is deduced.
  • the amino acid sequences of the two heavy chain variable regions and the light chain variable region contain 3 CDR regions respectively, and these 6 CDR regions can form a specific antigen-binding region to specifically bind human GPVI protein.
  • CDRs complementarity determining regions
  • Table 1 in the embodiment shows the positions and sequences of the amino acids in the heavy chain CDR region of hGP5C4 predicted by five methods.
  • CDR1 predicted by different methods consists of 5-12 amino acids
  • CDR2 consists of 6-17 amino acids
  • CDR3 consists of 7-9 amino acids.
  • the maximum sequence of the three CDRs of the heavy chain was determined as follows: CDR1 consists of 13 amino acids at positions 23-35, and its sequence is TASGFTFSDYFMS;
  • CDR2 consists of 20 amino acids at positions 47-66 , whose sequence is WVASISSGGASAYWRDSVKG;
  • CDR3 consists of 9 amino acids at positions 97-105, and its sequence is ARGELDFDY.
  • Table 2 in the embodiment shows the amino acid positions and sequences of the hGP5C4 light chain CDR region predicted by five methods.
  • CDR1 predicted by different methods consists of 6-11 amino acids
  • CDR2 consists of 3-10 amino acids
  • CDR3 consists of 7-8 amino acids.
  • the maximized sequences of the three CDRs of the light chain were determined as follows:
  • CDR1 consists of 13 amino acids at positions 21-33, and its sequence is TASQNVYKNLAWY;
  • CDR2 consists of 11 amino acids at positions 43-53 , whose sequence is LLLYSANSLQT;
  • CDR3 consists of 8 amino acids at positions 86-93, and its sequence is QQYYSGNT.
  • amino acid sequence of the human antibody variable region as a framework is determined.
  • Table 3 and Table 4 are five highly homologous germline human antibody heavy chain variable region sequences searched for hGP5C4 heavy chain amino acid variable region sequences, among which Table 3 shows the sequences covering the amino terminal 98 amino acids The sequence has a homology of 75.5%; Table 4 shows the 14 amino acid sequences near the carboxy-terminus, with a homology of 78.6%. The homology comparison between these sequences and hGP5C4 is shown in Table 5. According to the comparison results in Table 5, the heavy chain variable region framework of humanized hGP5C4 was determined.
  • Table 6 and Table 7 are five highly homologous germline human antibody light chain variable region sequences searched for hGP5C4 light chain amino acid variable region sequences, and Table 6 shows the 86 amino acid sequences covering the amino terminal , with a homology of 70.9%-72.3%; Table 7 shows the 11 amino acid sequences near the carboxyl terminal, with a homology of 81.8%.
  • the homology comparison between these sequences and hGP5C4 is shown in Table 8. According to the comparison results in Table 8, the light chain variable region framework of humanized hGP5C4 was determined.
  • amino acid sequences of the heavy chain Fd and light chain of hGP5C4 humanized antibody were constructed.
  • CDR transplantation refers to replacing the CDR sequence in the variable region of a non-human monoclonal antibody with the corresponding CDR sequence of a human antibody to form a recombinant antibody consisting of the CDR region of a non-human antibody and the framework region of a human antibody.
  • the variable regions of antibody heavy and light chains consist of CDR regions and framework regions (FR).
  • FR framework regions
  • the 6 CDRs of the variable region constitute the region that recognizes and binds to the antigen, and they directly contact with the antigen to determine the specificity of the antibody.
  • the framework region is the part other than the variable region, which mainly plays the role of supporting the CDR, and their amino acid composition and arrangement are relatively conservative.
  • humanization can be achieved by grafting the CDR region of a non-human monoclonal antibody to the framework region of a human monoclonal antibody.
  • This humanized antibody maintains the specificity and affinity of non-human monoclonal antibody and antigen binding, and minimizes the heterogeneity of non-human monoclonal antibody.
  • the amino acid sequence of the CDR region of hGP5C4 determined in the above examples was grafted to the corresponding position of the variable region of the germline human antibody as a framework to obtain the amino acid sequence of the variable region of the humanized antibody.
  • the above humanized heavy chain variable region sequence was connected to the conserved human IgG antibody heavy chain constant region 1 (CH1) sequence to obtain the amino acid sequence of the humanized anti-GPVI Fd fragment.
  • the humanized light chain variable region sequence above was linked to the conserved human IgG antibody light chain constant region (CL) sequence to obtain the amino acid sequence of the humanized anti-GPVI light chain.
  • humanized anti-GPVI Fd and humanized anti-GPVI light chain are folded into a complete Fab molecule through the formation of disulfide bonds by their respective cysteine residues when expressed through genetic engineering recombination, which is the present invention
  • nucleotide sequences encoding these amino acid sequences were constructed.
  • the Fab molecule composed of the Fd fragment and the light chain is expressed through genetic engineering recombination, it is necessary to reversely deduce the respective nucleotide coding sequences based on its amino acid sequence. Due to the degeneracy of nucleotide-coded amino acids, the same amino acid sequence can be reversely deduced into multiple combinations of nucleotide-coded sequences. It is common practice to synthesize the corresponding nucleotide sequence according to the bias of the selected host cell towards the encoded amino acid.
  • Chinese hamster cells were selected as the host cells.
  • the corresponding nucleotide sequences were synthesized according to the amino acid coding bias of the mouse cells, and the nucleosides of the humanized Fd fragment and the humanized light chain were determined. acid coding sequence.
  • the recombinant expression vector of humanized Fd fragment and light chain was constructed
  • the commonly used expression vector for genetically engineered recombinant proteins is a modified recombinant bacterial plasmid (plasmid).
  • plasmid modified recombinant bacterial plasmid
  • these plasmids also contain elements that can synthesize proteins in different host cells (such as Escherichia coli, yeast cells, insect cells, mammalian cells, etc.), such as promoters, ribosome binding sites, enhancers factors, transcription termination signals, etc.
  • the hGPFab in the present invention can use the recombinant plasmid as a vector to recombinantly express the Fab fragment with biological activity in these host cells.
  • the optimized solution is to choose mammalian cells as the carrier, such as engineered cell lines of human or mouse origin, so as to facilitate the correct folding of Fab fragments to form molecules close to the natural configuration.
  • CHO cells are used as hosts for recombinant expression.
  • a Kozak sequence and a signal peptide sequence were added to the 5' end of the coding Fd fragment and the light chain nucleotide sequence, respectively, and a stop codon "TGA” was added to the 3' end. ", and corresponding restriction sites were added at both ends to facilitate the construction of recombinant plasmids.
  • Kozak sequence (Kozak consensus sequence) is a common protein translation initiation site on eukaryotic mRNA transcripts.
  • the commonly used Kozak sequence is (gcc)gccRccAUGG, where AUG is the codon for the starting amino acid (methionine) of the target protein .
  • the signal peptide is an amino acid sequence at the amino terminal of the secreted protein, generally between 16-30 amino acid residues in length.
  • the signal peptide can guide the secretion of the recombinant protein to the outside of the cell, and is cleaved by the signal peptidase during the secretion process to secrete the mature target protein.
  • the signal peptide sequences of the heavy chain and light chain of human antibody are selected.
  • the plasmid used in the present invention is pCDdhfr, and the promoter of the foreign protein on the plasmid is the early promoter of mouse CMV. Screening gene is dihydrofolate reductase (dihydrofolatereductase, dhfr) gene. After the pCDdhfr recombinant plasmid was transfected into dhfr-deficient CHO cells, the plasmid sequence could be randomly integrated into the cell genome.
  • the integrated plasmid needs to be continuously amplified to enhance the expression of dhfr, so that the tandem recombinant protein gene will be amplified accordingly to increase expression.
  • methotrexate methotrexate
  • the synthetic genes encoding humanized Fd fragment and light chain were respectively inserted into the multiple cloning site of pCDdhfr, and recombinant plasmids pCDGPFd (containing humanized Fd fragment encoding gene) and pCDGPL (containing humanized light chain gene) were constructed. Chain coding gene), the sequence of the cloned gene was verified to be the same as the designed and synthesized sequence by DNA sequencing.
  • hGPFab is recombinantly expressed using CHO cells.
  • the hGPFab is a heterodimer spontaneously formed when the humanized Fd fragment and the humanized light chain in the present invention are co-expressed in host cells, and the two are connected by a disulfide bond.
  • pCDGPFd and pCDGPL plasmids were co-transfected into dhfr-deficient CHO cells through liposome-mediated co-transfection, they were detected in the cell culture supernatant by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the combined Fab fragments indicated that the two co-transfected plasmids were expressed in the host cells and secreted outside the cells.
  • the transfected CHO cells were screened by MTX pressure and subcultured to obtain a monoclonal cell line that can stably express hGPFab.
  • the expressed hGPFab is purified from the culture supernatant of the cell line.
  • Commonly used recombinant protein purification methods are various chromatographic techniques, including affinity chromatography, ion exchange chromatography, gel chromatography, ultrafiltration, etc.
  • affinity chromatography is to use the specific combination of target protein and ligand or antibody to achieve the purpose of separation and purification.
  • Protein L is a structural protein produced by Peptostreptococcus magnus, consisting of 719 amino acids. Protein L can specifically bind to the kappa light chains of various animal antibodies, so immobilized protein L is often used to purify Fab fragments of antibodies.
  • the hGPFab stably expressing cell line constructed by the present invention has the culture supernatant purified to the target protein through protein L-agarose column affinity chromatography. SDS-PAGE analysis of the purified protein showed it to be a heterodimer.
  • ELISA detection confirms that the purified hGPFab can specifically bind to human GPVI protein.
  • Enzyme-Linked ImmunoSorbent Assay is an analytical technique commonly used to identify the interaction between proteins, by immobilizing one protein and detecting another protein in the liquid phase on the surface of the solid phase , such as analyzing the binding ability between ligand-receptor and antigen-antibody.
  • the purified GPVI protein was coated (solid-phased) on a 96-well microwell plate, and the cell culture supernatant or purified hGPFab expressing recombinant hGPFab was added, and then Horseradish peroxidase (HRP)-labeled anti-human Fab antibody detects Fab fragments bound to immobilized GPVI.
  • HRP Horseradish peroxidase
  • Western Blot shows that the hGPFab of the present invention can specifically bind non-reducing human GPVI protein.
  • WB is also an analytical technique commonly used to identify the interaction between proteins. By immobilizing one protein, another protein in the liquid phase is detected on the surface of the solid phase, such as analyzing ligand-receptor, antigen-antibody interactions. ability to combine.
  • the difference from ELISA is: WB detection is to first separate a protein by electrophoresis (SDS-PAGE), then transfer the separated protein to a solid-phase membrane, and then react with another protein in the liquid phase, and finally Use enzymes, isotopes, biotin, etc. to detect the reaction between the two. Due to the protein separation in the early stage, the detection results of WB are more specific and intuitive, and are often used for identification analysis after ELISA detection.
  • a reducing agent such as dithiothreitol DTT, ⁇ -mercaptoethanol, etc.
  • the reducing agent will open the disulfide bond between amino acid chains, making the protein a linear structure (reducing protein); in the sample without reducing agent, the disulfide bond between amino acid chains will continue to exist and make the protein
  • the protein assumes a three-dimensional configuration (non-reducing protein). Therefore, the interaction of proteins between different configurations can be analyzed through the action of reducing agents.
  • the GPVI protein samples with and without DTT were separated by SDS-PAGE electrophoresis, and then the separated protein was transferred to a nitrocellulose membrane, then the purified hGPFab solution was added, and finally the HRP-labeled anti-human Fab Antibody testing.
  • the results show that the hGPFab of the present invention has a strong reaction with non-reduced GPVI, but has no obvious reaction with reduced GPVI. It shows that the binding site of hGPFab on GPVI protein is conformation-dependent, that is, its epitope belongs to nonlinear structure.
  • the hGPFab of the present invention can inhibit the binding of human GPVI protein and collagen, and has a dose-effect relationship.
  • the parental antibody hGP5C4 of rat origin has the property of inhibiting the binding of GPVI and collagen.
  • a competitive inhibition ELISA assay for the binding of GPVI and hGPFab to collagen was designed.
  • FIG. 1 Schematic representation of Fab fragments of IgG antibodies. The intact antibody is in a "Y" shape, and after papain degradation, two identical Fab fragments and one crystalline fragment (Fc) are formed
  • Figure 2 The coding nucleotide and amino acid sequence of the humanized heavy chain Fd fragment. Containing the predicted three heavy chain CDR regions of hGP5C4 and the framework region of the Fd fragment of the germline human IgG antibody, the encoded nucleotides are 669bp in full length and encode 223 amino acid residues.
  • Figure 3 The coding nucleotide and amino acid sequence of the humanized light chain. It contains the predicted 3 hGP5C4 light chain CDR regions and the framework region of the germline human IgG antibody light chain.
  • the encoded nucleotides are 639bp in full length and encode 213 amino acid residues.
  • Figure 4 Purification of humanized anti-GPVI antibody Fab fragment (hGPFab).
  • hGPFab humanized anti-GPVI antibody Fab fragment
  • Figure A is the SDS-PAGE electrophoresis image.
  • Lane 1 is the band pattern after DTT treatment, and the molecular weight of the main protein is about 25kD;
  • Lane 2 is the sample without DTT treatment, the molecular weight is about 45kD.
  • Panel B is the peak profile of the protein by UV scanning during elution.
  • FIG. 5 Specific binding of hGPFab to human GPVI protein (ELISA experiment).
  • the hGPFab in the present invention can specifically bind to the coated human GPVI protein, and as the hGPFab concentration increases, the binding signal also increases.
  • the Fab fragment of normal human IgG antibody had no obvious reaction with GPVI.
  • FIG. 6 Western blot experiment (WB) of the specific binding of hGPFab to human GPVI protein.
  • Figure A is the SDS-PAGE electrophoresis of human GPVI protein, wherein lane 1 is GPVI treated with DTT (reduced protein), and lane 2 is GPVI without DTT treatment (non-reduced).
  • Panel B is the result of WB experiment, hGPVI showed a strong reaction with non-reduced GPVI (lane 2), but a weak reaction with reduced GPVI (lane 1).
  • Figure 7 Competitive binding experiment of hGPFab with human GPVI protein and collagen (competitive ELISA).
  • hGPFab can inhibit the combination of GPVI and collagen, and with the increase of hGPFab concentration, its inhibitory effect will be enhanced.
  • the Fab fragment of normal human IgG antibody used as a control has no obvious inhibitory effect.
  • the genetic engineering recombination technology, bacteria/cell culture technology and recombinant protein purification technology involved in the present invention are all conventional technologies.
  • the present invention will be further described below by specific examples. The following examples are just to enable those skilled in the art to better understand the present invention but do not limit the present invention in any way.
  • Example 1 Screening and determination of non-human anti-human GPVI monoclonal antibodies to be humanized.
  • hGP5C4 is a monoclonal antibody prepared by immunizing Lou/C rats (rat) with human GPVI-Fc dimer protein as the immunogen. (Massberg S. et al. Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo.
  • Fab fragment obtained by degrading hGP5C4 with papain can effectively prevent the combination of GPVI and collagen; it can also specifically bind to GPVI on the surface of platelets, inhibit the activation of platelets induced by collagen stimulation, and prevent the activation of its active markers PAC-I and CD62 -P expression; effectively inhibit the aggregation of human platelets and the release of ATP; in the ex vivo (ex vivo) human red blood cell bleeding time test, hGP5C4 has no effect on the bleeding time. All these data indicate that hGP5C4 has specific antiplatelet function, but does not have intrinsic stimulating activity, suggesting that hGP5C4 has good application potential.
  • hGP5C4 antibody is derived from rats, its heterogeneity prevents it from being directly applied to humans. Humanized treatment can change its heterologous properties, which provides the possibility for clinical application.
  • nucleotide coding sequence of the heavy chain variable region of the above antibody strain hGP5C4 is CS114614.1 (nucleic acid sequence of 5C4 heavy chain variable); the nucleotide coding sequence of the light chain variable region It is CS114616.1 (nucleotide sequence of 5C4light chain variableDomain).
  • nucleotide sequence of CS114614.1 (nucleic acid sequence of 5C4 heavy chain variable) is shown in SEQ ID NO.2:
  • nucleotide sequence of CS114616.1 (nucleotide sequence of 5C4 light chain variable Domain) is shown in SEQ ID NO.4:
  • CS114614.1 has a total length of 378 bases (base pair, bp), encoding 126 amino acid residues, without start and stop codons. Its deduced coded amino acid sequence is shown in SEQ ID NO.3:
  • CS114616.1 The total length of CS114616.1 is 342 bases, encoding 114 amino acid residues, without start and stop codons. Its deduced coded amino acid sequence is shown in SEQ ID NO.5:
  • amino acid series encoded by CS114614.1 and CS114616.1 have the structural characteristics of antibody heavy chain and light chain variable regions respectively.
  • amino acid sequences of the two heavy chain variable regions and the light chain variable region contain 3 CDR regions respectively, and these 6 CDR regions can form a specific antigen-binding region to specifically bind human GPVI protein.
  • Table 1 shows the position and sequence of the amino acids in the heavy chain CDR region of hGP5C4 predicted by five methods.
  • CDR1 predicted by different methods consists of 5-12 amino acids
  • CDR2 consists of 6-17 amino acids
  • CDR3 consists of 7-9 amino acids.
  • CDR1 consists of 13 amino acids at positions 23-35, and its sequence is TASGFTFSDYFMS (SEQ ID NO.6); CDR2 consists of 13 amino acids at positions 47-35; The 20 amino acids at position 66 are composed of 20 amino acids, and its sequence is WVASISSGGASAYWRDSVKG (SEQ ID NO.7); CDR3 is composed of 9 amino acids at positions 97-105, and its sequence is ARGELDFDY (SEQ ID NO.8).
  • Table 2 shows the amino acid positions and sequences of the hGP5C4 light chain CDR region predicted by five methods.
  • CDR1 predicted by different methods consists of 6-11 amino acids
  • CDR2 consists of 3-10 amino acids
  • CDR3 consists of 7-8 amino acids.
  • the maximized sequences of the three CDRs of the light chain were determined as follows:
  • CDR1 consists of 13 amino acids at positions 21-33, and its sequence is TASQNVYKNLAWY (SEQ ID NO.9);
  • CDR2 consists of 13 amino acids at positions 43-33.
  • the 11 amino acids at position 53 consist of LLLYSANSLQT (SEQ ID NO.10);
  • the CDR3 consists of 8 amino acids at positions 86-93, and its sequence is QQYYSGNT (SEQ ID NO.11).
  • the CDRs of the variable regions of the heavy and light chains of hGP5C4 determined according to the five prediction methods are as follows:
  • Example 3 Screening and determination of the amino acid sequence of the human antibody variable region as a framework.
  • Table 3 and Table 4 are 5 highly homologous germline human antibody heavy chain variable region sequences searched for hGP5C4 heavy chain amino acid variable region sequences, of which Table 3 shows the sequence covering the amino-terminal 98 amino acids, same as The homology is 75.5%; Table 4 is the 14 amino acid sequences near the carboxyl terminal, and the homology is 78.6%. The homology comparison between these sequences and hGP5C4 is shown in Table 5.
  • Table 6 and Table 7 are five highly homologous germline human antibody light chain variable region sequences searched for hGP5C4 light chain amino acid variable region sequences, of which Table 6 shows the 86 amino acid sequences covering the amino terminal, homologous Sex is 70.9% -
  • Table 7 shows the 11 amino acid sequences near the carboxy-terminus, with a homology of 81.8%. The homology comparison between these sequences and hGP5C4 is shown in Table 8.
  • Table 8 Amino acid sequence comparison of light chain variable region of hGP5C4 and germline human antibody light chain variable region
  • Example 4 Construction of amino acid sequences of hGP5C4 humanized antibody heavy chain Fd and light chain
  • CDR transplantation refers to replacing the CDR sequence in the variable region of a non-human monoclonal antibody with the corresponding CDR sequence of a human antibody to form a recombinant antibody consisting of the CDR region of a non-human antibody and the framework region of a human antibody.
  • the variable regions of antibody heavy and light chains consist of CDR regions and framework regions (FR).
  • FR framework regions
  • the 6 CDRs of the variable region constitute the region that recognizes and binds to the antigen, and they directly contact with the antigen to determine the specificity of the antibody.
  • the framework region is other parts than the variable region, which mainly plays the role of supporting the CDR, and their amino acid composition and arrangement are relatively conservative.
  • the purpose of humanization can be achieved by transplanting the CDR region of a non-human monoclonal antibody to the framework region of a human monoclonal antibody.
  • This humanized antibody maintains the specificity and affinity of non-human monoclonal antibody and antigen binding, and minimizes the heterogeneity of non-human monoclonal antibody.
  • the amino acid sequence of the CDR region of hGP5C4 determined in Example 2 above was transplanted to the corresponding position of the variable region of the germline human antibody (Example 3) as a framework to obtain the amino acid sequence of the variable region of the humanized antibody.
  • the sequence of the humanized heavy chain variable region is as follows, and the underlined part is the transplanted CDR region (SEQ ID NO.12).
  • the sequence of the humanized light chain variable region is as follows, and the underlined part is the transplanted CDR region (SEQ ID NO.13).
  • the above humanized heavy chain variable region sequence was connected to the conserved human IgG antibody heavy chain constant region 1 (CH1) sequence to obtain the amino acid sequence of the humanized anti-GPVI Fd fragment: (SEQ ID NO.14)
  • the above humanized light chain variable region sequence was connected to the conserved human IgG antibody light chain constant region (CL) sequence to obtain the amino acid sequence of the humanized anti-GPVI light chain: (SEQ ID NO.15)
  • humanized anti-GPVI Fd and humanized anti-GPVI light chain are folded into a complete Fab molecule through the formation of disulfide bonds by their respective cysteine residues when expressed through genetic engineering recombination, that is, in the present invention Humanized anti-GPVI Fab fragment—hGPFab.
  • the Fab molecule composed of the Fd fragment and the light chain is expressed through genetic engineering recombination, it is necessary to reversely deduce the respective nucleotide coding sequences based on its amino acid sequence. Due to the degeneracy of nucleotide-coded amino acids, the same amino acid sequence can be reversely deduced into multiple combinations of nucleotide-coded sequences. It is common practice to synthesize the corresponding nucleotide sequence according to the bias of the selected host cell towards the encoded amino acid.
  • CHO Chinese hamster cells
  • corresponding nucleotide sequences were synthesized according to the amino acid coding bias of mouse cells.
  • Nucleotide coding sequence (SEQ ID NO.16) of the humanized Fd fragment SEQ ID NO.16
  • the nucleotide coding sequence of the humanized Fd fragment ( Figure 2) is 669 bp, encoding 223 amino acid residues, including the humanized anti-GPVI heavy chain variable region and CH1 region.
  • the nucleotide coding sequence of the humanized light chain ( FIG. 3 ) is 639 bp, encoding 213 amino acid residues, including the humanized anti-GPVI light chain variable region and constant region.
  • Embodiment 6 Construction of the recombinant expression vector of humanized Fd fragment and light chain
  • the carrier is pCDdhfr plasmid.
  • the promoter of the foreign protein on the pCDdhfr plasmid is the early promoter of mouse CMV. Screening gene is dihydrofolate reductase (dihydrofolatereductase, dhfr) gene. After the pCDdhfr recombinant plasmid was transfected into dhfr-deficient CHO cells, the plasmid sequence could be randomly integrated into the cell genome.
  • the integrated plasmid needs to be continuously amplified to strengthen the expression of dhfr, so that the tandem recombinant protein gene will be amplified accordingly to increase expression.
  • methotrexate methotrexate
  • the operation technology is a conventional technology of genetic engineering recombination, and the specific process is as follows:
  • the recombinant vectors pUCGPFd and pUCGPL respectively containing the anti-GPVI Fd fragment and the light chain gene were obtained by gene synthesis.
  • the following constructions are made: (1) In order to facilitate the secretory expression of the target protein, the 5' ends of the coding Fd fragment and the light chain nucleotide sequence are respectively added Signal peptide coding sequence.
  • the amino acid sequence of the signal peptide of the Fd fragment is MELGLSWVFLVAILEGVQC, and its nucleotide coding sequence is ATG GAA CTG GGC CTG TCT TGG GTG TTT CTG GTG GCC ATC CTG GAA GGCGTG CAG TGT; the amino acid sequence of the signal peptide of the light chain is MDMRVPAQLLGLLLLWLRGARC, and its nucleotide
  • the coding sequence is ATG GAT ATG AGA GTG CCT GCC CAG CTG CTG GGC CTG CTGCTGCTG TGGCTG AGA GGC GCC AGA TGT (2) Add the Kozak sequence GCCACC at the 5' end of the above signal peptide start codon ATG.
  • a stop codon TGA was added to the 3' ends of the two genes.
  • Recognition sequences of restriction endonucleases EcoR I (GAATTC) and NotI (GCGGCCGC) were added to the 5' end of the Kozak sequence of the two genes and the 3' end of the stop codon to facilitate subsequent construction of recombinant expression plasmids.
  • T4 ligase was used to connect pCDdhfr plasmid and Fd gene, pCDdhfr plasmid and light chain gene respectively.
  • Example 7 Recombinant expression of hGPFab in CHO cells
  • hGPFab is a heterodimer spontaneously formed when the humanized Fd fragment and the humanized light chain in the present invention are co-expressed in host cells, and the two are connected by a disulfide bond.
  • dhrf-deficient CHO cells are selected as hosts for recombinant expression, and operations are performed according to conventional cell transfection and stable cell line construction techniques.
  • steps 4-5 double the MTX concentration of the cell line with high expression level for pressure screening until the expression level no longer increases significantly. Subclone the high-expressing cell line, freeze it after identification and continue the follow-up experiment.
  • Example 8 Purification of hGPFab recombinantly expressed in CHO cells
  • Protein L is a structural protein produced by Peptostreptococcus magnus, consisting of 719 amino acids. Protein L can specifically bind kappa light chains of various animal antibodies, so immobilized protein L is often used as a medium for purifying Fab fragments of antibodies.
  • the constructed hGPFab stable expression cell line, its culture supernatant was purified by affinity chromatography on the HiTrap Protein L column (HiTrap Protein L column) on the AKTA system, and the operation steps were carried out according to the instructions.
  • the basic steps are as follows:
  • the purified protein was analyzed by SDS-PAGE. In the non-reduced state, the molecular weight was about 45kD. After being treated with the reducing agent DTT, it became a monomer with a molecular weight of about 25kD, suggesting that the secreted and expressed hGPFab protein structure was a dimer. And the molecular weights of the two monomers are close (about 25kD) ( Figure 4), which is in line with the expected results.
  • Example 9 Specific binding test between hGPFab and human GPVI protein.
  • an enzyme-linked immunosorbent assay (ELISA) method has been established to detect the expressed hGPFab protein.
  • the method of operation is a conventional related technology, and the basic steps are as follows:
  • the coating concentration of GPVI protein is 2 ⁇ g/ml
  • the coating solution is carbonate buffer solution with pH 9.6
  • the coating volume is 100 ⁇ l/well
  • HRP horseradish peroxidase
  • Embodiment 10 Western blot experiment (Western Blot, WB) of hGPFab and human GPVI protein
  • reducing agent such as dithiothreitol DTT, ⁇ -mercaptoethanol, etc.
  • the reducing agent will open the disulfide bond between the amino acid chains, making the protein a linear structure (reducing protein);
  • the disulfide bonds between the amino acid chains will exist to make the protein assume a three-dimensional configuration (non-reducing protein). Therefore, the interaction of proteins between different configurations can be analyzed through the action of reducing agents.
  • Example 11 Competitive inhibition test of hGPFab and human GPVI protein with collagen
  • the parental antibody hGP5C4 of rat origin has the property of inhibiting the binding of GPVI and collagen.
  • a competitive inhibition ELISA assay of GPVI and hGPFab binding to human collagen was designed.
  • the basic operation process is as follows:
  • hGPFab at a final concentration of 0.5, 1.0, 2.0, 4.0, and 8.0 ⁇ g/ml to the GPVI sample of 50 ng/ml, and incubate at room temperature for 30 minutes. This is the sample to be tested, and a normal human antibody Fab is used as a control.
  • HRP horseradish peroxidase

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Abstract

Disclosed in the present invention are a humanized anti-human GPVI monoclonal antibody Fab fragment and an application thereof. Complementarity-determining regions (CDRs) of heavy and light chains of a rat anti-human GPVI monoclonal antibody are first analyzed and predicted; amino acid sequences of heavy and light chain variable regions of the rat monoclonal antibody are used as templates; amino acid sequences of heavy and light chain variable regions of a corresponding high-homology human IgG antibody are obtained by screening from a human IgG antibody database; a heavy chain Fd fragment and light chain of a humanized anti-human GPVI monoclonal antibody Fab are constructed by means of CDR grafting. Nucleotide sequences encoding the heavy chain Fd fragment and light chain of the humanized anti-human GPVI monoclonal antibody Fab can be expressed recombinantly; secreted and expressed antibody Fab can specifically bind to a non-reducing human GPVI protein, inhibit binding of human GPVI to human collagen, and can be used for clinical treatment of platelet GPVI-related diseases.

Description

一种人源化抗人GPVI单克隆抗体Fab片段及其应用A kind of humanized anti-human GPVI monoclonal antibody Fab fragment and application thereof 技术领域technical field
本发明属于基因工程重组蛋白质药物领域，具体地，属于基因工程重组抗体药物领域。涉及一种人源化抗人GPVI单克隆抗体Fab片段及其应用。所述的人源化抗人GPVI单克隆抗体Fab片段是由大鼠(rat)抗人GPVI单克隆抗体的互补决定区(Complementarity‐Determining Regions，CDRs)和人源IgG抗体Fab片段的框架所组成。将编码人源化抗人GPVI单克隆抗体Fab片段的核苷酸序列经合适的质粒载体转染到CHO细胞后，转染的CHO分泌表达了人源化抗人GPVI单克隆抗体Fab片段，此人源化抗人GPVI单克隆抗体Fab片段可以与非还原性人GPVI蛋白特异结合，并能够抑制GPVI与胶原蛋白的结合，这种抑制存在着剂量‐效应关系。本发明还涉及用于产生所述人源化抗人GPVI单克隆抗体Fab片段抗体的核苷酸编码序列、重组表达质粒、重组表达细胞株。本发明所公开的人源化抗人GPVI单克隆抗体Fab片段可以用于血小板GPVI相关性疾病的临床治疗。The invention belongs to the field of genetic engineering recombinant protein medicine, specifically, belongs to the field of genetic engineering recombinant antibody medicine. It relates to a humanized anti-human GPVI monoclonal antibody Fab fragment and application thereof. The humanized anti-human GPVI monoclonal antibody Fab fragment is composed of the complementarity-determining regions (Complementarity-Determining Regions, CDRs) of the rat (rat) anti-human GPVI monoclonal antibody and the framework of the human IgG antibody Fab fragment . After the nucleotide sequence encoding the Fab fragment of the humanized anti-human GPVI monoclonal antibody is transfected into CHO cells via a suitable plasmid vector, the transfected CHO secretes and expresses the Fab fragment of the humanized anti-human GPVI monoclonal antibody. The Fab fragment of humanized anti-human GPVI monoclonal antibody can specifically bind to non-reduced human GPVI protein, and can inhibit the binding of GPVI to collagen, and this inhibition has a dose-effect relationship. The present invention also relates to a nucleotide coding sequence, a recombinant expression plasmid, and a recombinant expression cell line for producing the humanized anti-human GPVI monoclonal antibody Fab fragment antibody. The humanized anti-human GPVI monoclonal antibody Fab fragment disclosed by the present invention can be used for clinical treatment of platelet GPVI-related diseases.
背景技术Background technique
血小板糖蛋白6(Glycoprotein VI，GPVI)是血小板表面的跨膜糖蛋白受体，属于免疫球蛋白样受体超家族，分子量约58-64kDa，由GPVI基因编码。成熟的GPVI分子(氨基酸序列如SEQ ID NO.1所示)由3个功能区构成，分别是胞外区、跨膜区和胞内区，血小板表面的GPVI有单体和二聚体两种形态，其中二聚体作为主要功能形态，与胶原蛋白有高的亲和力。GPVI与Fc受体γ链通过盐桥形成信号传导复合体，此复合体可引发瀑布式的酪胺酸磷酸化信号传导途径。在血管损伤时，暴露的胶原蛋白形成促凝血的表面，血小板在这些表面藉由GPVI和GPIb-IX-V介导发生聚集。作为血小板的主要受体，GPVI主要由I型和III型胶原蛋白激活后，触发ADP和血栓素A 2(Thromboxane A 2)等可溶性第二激活剂的释放，从而活化和招募循环的血小板发生聚集，引发血栓形成。 Platelet glycoprotein 6 (Glycoprotein VI, GPVI) is a transmembrane glycoprotein receptor on the surface of platelets, belonging to the immunoglobulin-like receptor superfamily, with a molecular weight of about 58-64 kDa, encoded by the GPVI gene. The mature GPVI molecule (the amino acid sequence is shown in SEQ ID NO.1) is composed of three functional regions, namely the extracellular region, the transmembrane region and the intracellular region. There are two types of GPVI on the surface of platelets: monomer and dimer Morphology, of which dimer is the main functional form and has a high affinity with collagen. GPVI forms a signaling complex with the Fc receptor γ chain through a salt bridge, and this complex can trigger a cascading tyrosine phosphorylation signaling pathway. Upon vascular injury, exposed collagen forms a procoagulant surface on which platelet aggregation is mediated by GPVI and GPIb-IX-V. As the main receptor of platelets, GPVI is mainly activated by collagen types I and III, triggering the release of soluble secondary activators such as ADP and thromboxane A 2 (Thromboxane A 2 ), thereby activating and recruiting circulating platelets to aggregate , causing thrombosis.
通常地，血小板因应血管损伤或炎症而引发的血栓形成是正常的生理过程。在血管损伤时，循环的血小板快速地粘附到暴露的胶原蛋白上，这种血小板聚集而引发的血栓形成可以减 少血液流失，但在病理状态下，如动脉粥样硬化斑块断裂导致胶原蛋白暴露所刺激的血栓形成，则和心肌梗塞和中风相关。Generally, thrombosis induced by platelets in response to vascular injury or inflammation is a normal physiological process. When a blood vessel is damaged, circulating platelets quickly adhere to the exposed collagen, and the thrombosis caused by this platelet aggregation can reduce blood loss, but in pathological conditions, such as atherosclerotic plaque rupture leads to collagen Exposure-stimulated thrombosis has been associated with myocardial infarction and stroke.
血小板的过渡活化和聚集可以引发血管内血栓的形成，从而导致心肌梗塞或中风的发生。临床报告显示，对高危病人进行靶向ADP受体P2Y 12和血栓素的抗血小板治疗可以有效降低这种血栓发生的风险，但同时也增加了病人体内出血的风险。 Excessive activation and aggregation of platelets can trigger the formation of intravascular thrombus, leading to myocardial infarction or stroke. Clinical reports have shown that antiplatelet therapy targeting ADP receptor P2Y 12 and thromboxane in high-risk patients can effectively reduce the risk of such thrombosis, but it also increases the risk of internal bleeding in patients.
因此，理想的抗血小板类抗血栓药物应该是具有高效的抗血小板聚集活性，但同时不影响正常的凝血功能、不会引起病人发生明显的出血副作用。Therefore, an ideal antiplatelet antithrombotic drug should have highly effective antiplatelet aggregation activity, but at the same time, it will not affect the normal blood coagulation function and will not cause obvious bleeding side effects in patients.
诸多的研究发现，以GPVI为作用靶点的抗血小板治疗，符合这种理想药物的要求。GPVI的表达具有高度特异性，仅在血小板和骨髓中的血小板前体细胞上表达，在动物试验中，阻断GPVI显示了明显的抗血栓效果，但同时没有发生明显的体内出血倾向。提示靶向GPVI的抗血小板策略在心肌梗塞、中风等血栓性疾病的治疗中具有良好的潜在前景。Many studies have found that antiplatelet therapy targeting GPVI meets the requirements of this ideal drug. The expression of GPVI is highly specific, and it is only expressed on platelets and platelet precursor cells in bone marrow. In animal experiments, blocking GPVI has shown obvious antithrombotic effect, but at the same time, there is no obvious bleeding tendency in vivo. It is suggested that the antiplatelet strategy targeting GPVI has a good potential prospect in the treatment of thrombotic diseases such as myocardial infarction and stroke.
阻断GPVI与胶原蛋白结合的药物候选物或技术有多种，如GPVI模拟体(如GPVI-Fc融合蛋白二聚体)、GPVI耗竭(depletion)及GPVI功能性阻断分子(如适配体、小分子药物、抗体等)。There are many drug candidates or technologies to block the combination of GPVI and collagen, such as GPVI mimetics (such as GPVI-Fc fusion protein dimer), GPVI depletion (depletion) and GPVI functional blocking molecules (such as aptamer , small molecule drugs, antibodies, etc.).
在这些药物候选物中，以抗体的研究最为突出。Among these drug candidates, research on antibodies is the most prominent.
发明专利CN103396492A申请说明书公开了一种抗GPVI的单链可变区(single-chain variable fragment，SCFV)抗体片段。发明者从输血后的血小板减少性紫癜病人体内分离出对血小板有亲和性的B淋巴细胞，通过基因扩增获得了抗GPVI抗体的基因片段，由此构建了含8个组氨酸标签的SCFV抗体片段，这个片段可以阻止血小板的聚集。Invention patent CN103396492A application specification discloses an anti-GPVI single-chain variable fragment (single-chain variable fragment, SCFV) antibody fragment. The inventor isolated B lymphocytes with affinity for platelets from patients with thrombocytopenic purpura after blood transfusion, and obtained the gene fragment of anti-GPVI antibody through gene amplification, thus constructing a gene fragment containing 8 histidine tags SCFV antibody fragment, this fragment can prevent the aggregation of platelets.
专利申请WO2005054294(Inhibitors of glycoprotein vi based on monoclonal antibody hgp 5c4)公开了一种大鼠抗人GPVI单克隆抗体hGP5C4，这株单抗可以特异结合人GPVI、抑制GPVI与胶原蛋白的结合、抑制血小板聚集。Patent application WO2005054294 (Inhibitors of glycoprotein vi based on monoclonal antibody hgp 5c4) discloses a rat anti-human GPVI monoclonal antibody hGP5C4, which can specifically bind to human GPVI, inhibit the binding of GPVI to collagen, and inhibit platelet aggregation .
专利申请WO2005111083(Antibodies specific for glycoprotein vi and methods of producing these antibodies)/CN1964990B公开了4株抗GPVI单克隆抗体-OM1、OM2、OM3和OM4，这些单抗能够阻止GPVI和胶原蛋白的结合、抑制胶原蛋白诱导的血小板聚集。Patent application WO2005111083 (Antibodies specific for glycoprotein vi and methods of producing these antibodies)/CN1964990B discloses 4 strains of anti-GPVI monoclonal antibodies-OM1, OM2, OM3 and OM4, these monoclonal antibodies can prevent the binding of GPVI and collagen and inhibit collagen Protein-induced platelet aggregation.
在专利申请WO2006118350(Anti-platelet membrane glycoprotein vi monoclonal antibody)/CN102875679中，发明人公开了一组抗人GPVI抗体或其抗原结 合性片段，该抗人GPVI抗体或其抗原结合片段可以和GPVI特异结合、活化或减少血小板的活性微弱，和血小板接触至少使其表面的部分GPVI消失。In the patent application WO2006118350 (Anti-platelet membrane glycoprotein vi monoclonal antibody)/CN102875679, the inventor disclosed a group of anti-human GPVI antibodies or antigen-binding fragments thereof, which can specifically bind to GPVI , Activate or reduce the activity of platelets is weak, contact with platelets at least part of the GPVI on the surface will disappear.
专利申请US20060088531(Human antibodies against human glycoprotein VI and their use)公开了一种SCFV片段-10B12，10B12能够抑制胶原蛋白蛋白诱导的血小板聚集和血小板与胶原蛋白相关肽(Collagen-Related Peptide，CRP)的粘附。段与人GPVI结构域2的环9特异性结合。Patent application US20060088531 (Human antibodies against human glycoprotein VI and their use) discloses a fragment of SCFV-10B12, 10B12 can inhibit collagen protein-induced platelet aggregation and platelet-collagen-related peptide (Collagen-Related Peptide, CRP) adhesion attached. Segment specifically binds to loop 9 of domain 2 of human GPVI.
专利申请WO2008049928公开了利用抗GPVI单克隆抗体9O12.2改造而成的SCFV抗体片段，这个抗体片段和亲本抗体9O12.2有类似的生物学活性。Patent application WO2008049928 discloses an SCFV antibody fragment engineered using anti-GPVI monoclonal antibody 9O12.2, which has similar biological activity to the parental antibody 9O12.2.
专利申请WO2011073954(Novel antagonist antibodies and their fab fragments against gpvi and uses thereof)/CN102725309B公开了一株抗GPVI单克隆抗体390，这株单抗能够破坏血小板表面的GPVI蛋白而造成GPVI的耗竭(depletion)。Patent application WO2011073954 (Novel antagonist antibodies and their fab fragments against gpvi and uses thereof)/CN102725309B discloses an anti-GPVI monoclonal antibody 390, which can destroy the GPVI protein on the surface of platelets and cause depletion of GPVI.
专利申请WO2017021539(Novel anti-human gpvi antibodies and uses thereof)/CN108289939A公开了一种能与人GPVI氨基酸链114-142位、165-187位构成的构象表位结合的抗体或抗体片段。Patent application WO2017021539 (Novel anti-human gpvi antibodies and uses thereof)/CN108289939A discloses an antibody or antibody fragment that can bind to a conformational epitope composed of positions 114-142 and 165-187 of the human GPVI amino acid chain.
专利申请WO2019219765(Antibodies targeting glycoprotein vi)/CN112118869A公开了一种抗GPVI Fab片段，这个Fab片段是从非特异MorphoSys-YlanthiaTM噬菌体文库中筛选得到，可以抑制胶原蛋白诱导的血小板聚集。Patent application WO2019219765 (Antibodies targeting glycoprotein vi)/CN112118869A discloses an anti-GPVI Fab fragment, which is screened from a non-specific MorphoSys-YlanthiaTM phage library and can inhibit collagen-induced platelet aggregation.
分析以上信息可以发现，上述的诸多抗GPVI单克隆抗体及片段在实际应用时都存在着不足之处，如单纯的鼠源抗体会引起人体的HAMA反应而不能直接用于人体治疗；SCFV片段存在特异性、稳定性差、体内半衰期短等问题；从非特异噬菌体库所筛选的抗体片段往往特异性差、亲和力低；一些抗体还表现出内源性活性，可能会引发毒副作用；另外，9O12.2等抗体还具有剪切(Shedding)血小板表面GPVI的活性，造成GPVI耗竭(depletion)，而这种耗竭不可控且不可逆转，可能会对人体产生长期的负面影响。Analyzing the above information, it can be found that many of the above-mentioned anti-GPVI monoclonal antibodies and fragments have deficiencies in practical application. For example, pure mouse antibodies can cause HAMA reactions in the human body and cannot be directly used for human treatment; SCFV fragments exist Specificity, poor stability, short half-life in vivo, etc.; antibody fragments screened from non-specific phage libraries often have poor specificity and low affinity; some antibodies also show endogenous activity, which may cause toxic side effects; in addition, 9O12.2 Such antibodies also have the activity of shearing (Shedding) GPVI on the surface of platelets, resulting in GPVI depletion, which is uncontrollable and irreversible, and may have long-term negative effects on the human body.
迄今为止，仅有9O12.2抗体经过人源化后的Fab片段ACT-017成功进入临床试验，用于缺血性中风的治疗。基于此，目前仍有必要研制高效、特异、稳定、无明显毒副作用的抗GPVI抗体，来满足临床日益需要的抗血栓治疗。So far, only the humanized Fab fragment ACT-017 of the 9O12.2 antibody has successfully entered clinical trials for the treatment of ischemic stroke. Based on this, it is still necessary to develop anti-GPVI antibodies with high efficiency, specificity, stability, and no obvious side effects to meet the increasing clinical needs of antithrombotic therapy.
发明内容Contents of the invention
本发明公开了一种人源化抗人GPVI单克隆抗体Fab片段及其应用。The invention discloses a humanized anti-human GPVI monoclonal antibody Fab fragment and application thereof.
所述的人源化抗人GPVI单克隆抗体Fab片段(以下简称抗hGPFab)是由大鼠(rat)抗人GPVI单克隆抗体重链和轻链的CDRs与人源IgG抗体Fab片段的框架所组成。将编码hGPFab的核苷酸序列经合适的质粒载体转染到CHO细胞后，转染的CHO分泌表达了抗hGPFab蛋白，此hGPFab蛋白可以与非还原性人GPVI蛋白特异结合，并能够抑制人GPVI与人胶原蛋白的结合，这种抑制作用存在着剂量-效应关系。The humanized anti-human GPVI monoclonal antibody Fab fragment (hereinafter referred to as anti-hGPFab) is composed of the CDRs of the heavy chain and light chain of the rat (rat) anti-human GPVI monoclonal antibody and the framework of the human IgG antibody Fab fragment composition. After the nucleotide sequence encoding hGPFab is transfected into CHO cells through a suitable plasmid vector, the transfected CHO secretes and expresses anti-hGPFab protein, which can specifically bind to non-reducing human GPVI protein and can inhibit human GPVI There is a dose-effect relationship for this inhibition of binding to human collagen.
本发明还涉及用于产生所述hGPFab蛋白的核苷酸编码序列、重组表达质粒、重组表达细胞株。本发明所公开的hGPFab可以用于血小板GPVI相关性疾病的临床治疗。The present invention also relates to a nucleotide coding sequence, a recombinant expression plasmid, and a recombinant expression cell strain for producing the hGPFab protein. The hGPFab disclosed in the present invention can be used for clinical treatment of platelet GPVI-related diseases.
本发明中和抗体相关的名词有：In the present invention, terms related to antibodies include:
抗体：抗体是动物的免疫***受到外源物(抗原，如细菌、病毒等外源微生物)刺激后，由B细胞产生的一类大分子免疫球蛋白，分为IgG、IgM、IgA、IgE和IgD 5个亚型，其中IgG是主要亚型。IgG是由两条分子量为25kDa的轻链和两条分子量为50kDa的重链组成的“Y”形状的四聚体。 Antibody : Antibody is a type of macromolecular immunoglobulin produced by B cells after the animal's immune system is stimulated by foreign substances (antigens, such as bacteria, viruses and other foreign microorganisms). It is divided into IgG, IgM, IgA, IgE and There are 5 subtypes of IgD, of which IgG is the main subtype. IgG is a "Y" shaped tetramer composed of two light chains with a molecular weight of 25 kDa and two heavy chains with a molecular weight of 50 kDa.
Fab片段：Fab即抗原结合片段(antigen-binding fragment，Fab)，一个完整的IgG抗体分子经木瓜蛋白酶降解后，分解为两个相同的Fab片段和一个Fc片段(图1)。Fab由一个完整的轻链(VL+CL)和1/2重链(VH+CH1)(Fd片段)通过二硫键结合而成。VL和HL共同构成了单价的抗原结合区(paratope)。 Fab fragment : Fab is an antigen-binding fragment (Fab). A complete IgG antibody molecule is decomposed into two identical Fab fragments and an Fc fragment after being degraded by papain (Figure 1). Fab consists of a complete light chain (VL+CL) and 1/2 heavy chain (VH+CH1) (Fd fragment) combined by a disulfide bond. VL and HL together constitute a monovalent antigen-binding region (paratope).
轻链可变区：轻链可变区(variable domain，VL)指的是抗体轻链N端1/2部分，VL包含3个高变区(hypervariable region，HV)和4个框架区(framework，FR)，在蛋白折叠时，这3个高变区形成3个环(loop)，这3个环常被称为轻链的互补决定区。 Light chain variable region : The light chain variable region (variable domain, VL) refers to the N-terminal 1/2 part of the antibody light chain, VL includes 3 hypervariable regions (hypervariable region, HV) and 4 framework regions (framework , FR), when the protein is folded, these three hypervariable regions form three loops (loop), these three loops are often called complementarity determining regions of the light chain.
重链可变区：重链可变区(variable domain，VH)指的是抗体重链N端1/4部分，VH包含3个高变区(hypervariable region，HV)和4个框架区(framework，FR)，在蛋白折叠时，这3个高变区形成3个环(loop)，这3个环常被称为重链的互补决定区。 Heavy chain variable region : The heavy chain variable region (variable domain, VH) refers to the N-terminal 1/4 part of the antibody heavy chain, and VH includes 3 hypervariable regions (hypervariable region, HV) and 4 framework regions (framework , FR), when the protein is folded, these three hypervariable regions form three loops (loop), these three loops are often called complementarity-determining regions of the heavy chain.
人源化抗体：人源化抗体指将非人源单克隆抗体抗原结合位点(常指CDR区)外的氨基酸序列替换为人源抗体的氨基酸序列。人源化抗体保留了亲本非人源单克隆抗体的亲和力和特异性，同时又降低了其异源性，能够显著减轻人体可能产生的人抗鼠抗体(Human anti-mouse antibody，HAMA)反应。 Humanized antibody : Humanized antibody refers to replacing the amino acid sequence outside the antigen-binding site (often referred to as the CDR region) of a non-human monoclonal antibody with the amino acid sequence of a human antibody. Humanized antibodies retain the affinity and specificity of parental non-human monoclonal antibodies while reducing their heterogeneity, which can significantly reduce the human anti-mouse antibody (HAMA) reaction that may be produced by the human body.
互补决定区：互补决定区(complementarity-determining regions，CDRs)指的是重链和轻链可变区在蛋白折叠时分别所形成的3个环，因为它们的形状和其相应抗原的形状互补而得名。重链和轻链的6个环共同形成一个抗原结合区，可以结合一个特异的抗原表位(epitope)。 Complementarity -determining regions: Complementarity-determining regions (CDRs) refer to the three loops formed by the variable regions of the heavy and light chains when the protein is folded, because their shapes are complementary to those of their corresponding antigens. named. The six loops of the heavy chain and the light chain together form an antigen-binding region that can bind a specific epitope.
Fd片段：Fd片段即Fab片段内的重链部分，约由220个氨基酸构成，包含重链的可变区和恒定区CH1。 Fd fragment : The Fd fragment is the heavy chain part of the Fab fragment, consisting of about 220 amino acids, including the variable region and the constant region CH1 of the heavy chain.
本发明所述的hGPFab是由人源化的IgG重链Fd片段和轻链通过二硫键结合而成的单价异源二聚体，这里的重链Fd片段指的抗体重链氨基酸序列N端的1/2部分，包括重链可变区和恒定区CH1(VH+CH1)；轻链指的是轻链全分子，包括轻链可变区和恒定区(VL+CL)。The hGPFab of the present invention is a monovalent heterodimer formed by combining humanized IgG heavy chain Fd fragment and light chain through a disulfide bond, where the heavy chain Fd fragment refers to the N-terminal part of the antibody heavy chain amino acid sequence 1/2 part, including heavy chain variable region and constant region CH1 (VH+CH1); light chain refers to the whole molecule of light chain, including light chain variable region and constant region (VL+CL).
本发明的技术方案如下：Technical scheme of the present invention is as follows:
在一个实施例中，选择出待人源化的非人源抗人GPVI单克隆抗体。In one embodiment, a non-human anti-human GPVI monoclonal antibody is selected to be humanized.
首先，确定选择非人源抗人GPVI单克隆抗体的标准：能够有效阻断人GPVI和胶原蛋白的结合；能够阻止血小板的聚集；不具有激活GPVI而导致血小板活化的活性。根据这3条标准，大鼠抗人GPVI单克隆抗体株hGP5C4被确定为人源化的对象。First, determine the criteria for selecting a non-human anti-human GPVI monoclonal antibody: it can effectively block the binding of human GPVI and collagen; it can prevent the aggregation of platelets; it does not have the activity of activating GPVI to cause platelet activation. According to these three criteria, the rat anti-human GPVI monoclonal antibody strain hGP5C4 was determined to be the subject of humanization.
hGP5C4是以人GPVI-Fc二聚体蛋白为免疫原，免疫Lou/C大鼠(rat)而制备的一株单克隆抗体。(Massberg S.et al.Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo.FASEB J 2004；18:397-9)(专利WO2005054294：Inhibitors of glycoprotein vi based on monoclonal antibody hgp 5c4)。用木瓜蛋白酶降解hGP5C4所得到的Fab片段可以有效阻止GPVI和胶原蛋白的结合；也能够特异结合血小板表面的GPVI，抑制由胶原蛋白刺激所诱导的血小板活化，阻止其活性标记物PAC-I和CD62-P的表达；有效抑制人血小板的聚集和ATP的释放；在离体(ex vivo)人红细胞出血时间试验中，hGP5C4对出血时间没有影响。所有这些数据表明hGP5C4具有特异的抗血小板功能，同时不具有内在的激发活性，提示hGP5C4具有良好的应用潜力。hGP5C4 is a monoclonal antibody prepared by immunizing Lou/C rats (rat) with human GPVI-Fc dimer protein as the immunogen. (Massberg S. et al. Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo. FASEB J 2004; 18:397-9) (patent WO2005054294: Inhibitors of glycoprotein vi p pl based on monoclonal hgan 5 boclonal hgan 4). The Fab fragment obtained by degrading hGP5C4 with papain can effectively prevent the combination of GPVI and collagen; it can also specifically bind to GPVI on the surface of platelets, inhibit the activation of platelets induced by collagen stimulation, and prevent the activation of its active markers PAC-I and CD62 -P expression; effectively inhibit the aggregation of human platelets and the release of ATP; in the ex vivo (ex vivo) human red blood cell bleeding time test, hGP5C4 has no effect on the bleeding time. All these data indicate that hGP5C4 has specific antiplatelet function, but does not have intrinsic stimulating activity, suggesting that hGP5C4 has good application potential.
但由于天然的hGP5C4抗体来源于大鼠，其异源性使之不能直接应用于人体。而通过人源化处理能够改变其异源性质，为临床应用提供了可能性。However, since the natural hGP5C4 antibody is derived from rats, its heterogeneity prevents it from being directly applied to humans. However, its heterologous properties can be changed through humanization, which provides the possibility for clinical application.
在基因数据库GenBank中，我们搜索到上述抗体株hGP5C4重链可变区的核苷酸编码序列为CS114614.1(nucleic acid sequence of 5C4heavy chain variable)(SEQ ID NO.2)；轻链可变区的核苷酸编码序列为CS114616.1(nucleotide sequence of 5C4 light chain variableDomain)(SEQ ID NO.4)，并推导出其氨基酸序列(SEQ ID NO.3、SEQ ID NO.5)。In the gene database GenBank, we found that the nucleotide coding sequence of the variable region of the heavy chain of the above antibody strain hGP5C4 is CS114614.1 (nucleic acid sequence of 5C4 heavy chain variable) (SEQ ID NO.2); the variable region of the light chain The nucleotide coding sequence is CS114616.1 (nucleotide sequence of 5C4 light chain variable Domain) (SEQ ID NO.4), and its amino acid sequence (SEQ ID NO.3, SEQ ID NO.5) is deduced.
理论上，这两个重链可变区和轻链可变区氨基酸序列含有各自的3个CDR区，这6个CDR区能够组成一个特异的抗原结合区而特异结合人GPVI蛋白。Theoretically, the amino acid sequences of the two heavy chain variable regions and the light chain variable region contain 3 CDR regions respectively, and these 6 CDR regions can form a specific antigen-binding region to specifically bind human GPVI protein.
在另一实施例中，确定了hGP5C4重链和轻链的互补决定区(CDRs)In another example, the complementarity determining regions (CDRs) of the hGP5C4 heavy and light chains were determined
目前对人和小鼠抗体的CDR研究最为深入，根据抗体可变区的氨基酸序列预测人或小鼠抗体的CDR区也较为准确。由于对其它动物抗体的研究相对较少，对其CDR区的预测则相对不足。基于hGP5C4的大鼠来源，为最大限度地保证所确定的CDR区与GPVI的亲和力与特异性，我们共采用了Kabat(Kabat,E.A.et al.,In:Sequences of Proteins of Immunological Interest,NIH Publication,1991:91-3242)、IMGT(Lefranc,M.P.,The IMGT unique numbering for Immunoglobulins,T-cell receptors,and Ig-like Domains.The Immunologist,1999:7,132-136)、Chothia(Al-Lazikani B et al.,"Standard conformations for the canonical structures of immunoglobulins".Journal of Molecular Biology.1997:273(4):927–48)、North(North B et al.,A new clustering of antibody CDR loop conformations.Journal of Molecular Biology.2011:406(2):228–56)和Contact(R M MacCallum et al.Antibody-antigen interactions:contact analysis and binding site topographyJMol Biol.1996 Oct 11；262(5):732-45)共5种方法来预测hGP5C4重链和轻链的CDR区。At present, the research on CDR of human and mouse antibodies is the most in-depth, and it is more accurate to predict the CDR regions of human or mouse antibodies based on the amino acid sequences of antibody variable regions. Since there are relatively few studies on other animal antibodies, the prediction of their CDR regions is relatively insufficient. Based on the rat source of hGP5C4, in order to ensure the affinity and specificity of the determined CDR region and GPVI to the greatest extent, we used Kabat (Kabat, E.A. et al., In: Sequences of Proteins of Immunological Interest, NIH Publication, 1991:91-3242), IMGT (Lefranc, M.P., The IMGT unique numbering for Immunoglobulins, T-cell receptors, and Ig-like Domains. The Immunologist, 1999:7, 132-136), Chothia (Al-Lazikani B et al. ,"Standard conformations for the canonical structures of immunoglobulins".Journal of Molecular Biology.1997:273(4):927–48), North(North B et al., A new clustering of antibody CDR loop conformations.Journal of Biomolecular .2011:406(2):228–56) and Contact (R M MacCallum et al. Antibody-antigen interactions: contact analysis and binding site topographyJMol Biol.1996 Oct 11; 262(5):732-45) method to predict the CDR regions of hGP5C4 heavy and light chains.
实施例中表1为5种方法预测的hGP5C4重链CDR区氨基酸的位置和序列。不同方法预测的CDR1由5-12个氨基酸组成、CDR2由6-17个氨基酸组成、CDR3由7-9个氨基酸组成。结合各自预测的位置和序列，确定重链3个CDR的最大化序列为：CDR1由第23-35位的13个氨基酸构成，其序列为TASGFTFSDYFMS；CDR2由第47-66位的20个氨基酸构成，其序列为WVASISSGGASAYWRDSVKG；CDR3由第97-105位的9个氨基酸构成，其序列为ARGELDFDY。Table 1 in the embodiment shows the positions and sequences of the amino acids in the heavy chain CDR region of hGP5C4 predicted by five methods. CDR1 predicted by different methods consists of 5-12 amino acids, CDR2 consists of 6-17 amino acids, and CDR3 consists of 7-9 amino acids. Combined with the respective predicted positions and sequences, the maximum sequence of the three CDRs of the heavy chain was determined as follows: CDR1 consists of 13 amino acids at positions 23-35, and its sequence is TASGFTFSDYFMS; CDR2 consists of 20 amino acids at positions 47-66 , whose sequence is WVASISSGGASAYWRDSVKG; CDR3 consists of 9 amino acids at positions 97-105, and its sequence is ARGELDFDY.
实施例中表2为5种方法预测的hGP5C4轻链CDR区氨基酸的位置和序列。不同方法预测的CDR1由6-11个氨基酸组成、CDR2由3-10个氨基酸组成、CDR3由7-8个氨基酸组成。结合各自预测的位置和序列，确定轻链3个CDR的最大化序列为：CDR1由第21-33位的13个氨基酸构成，其序列为TASQNVYKNLAWY；CDR2由第43-53位的11个氨基酸构成，其序列为LLLYSANSLQT；CDR3由第86-93位的8个氨基酸构成，其序列为QQYYSGNT。Table 2 in the embodiment shows the amino acid positions and sequences of the hGP5C4 light chain CDR region predicted by five methods. CDR1 predicted by different methods consists of 6-11 amino acids, CDR2 consists of 3-10 amino acids, and CDR3 consists of 7-8 amino acids. Combined with their predicted positions and sequences, the maximized sequences of the three CDRs of the light chain were determined as follows: CDR1 consists of 13 amino acids at positions 21-33, and its sequence is TASQNVYKNLAWY; CDR2 consists of 11 amino acids at positions 43-53 , whose sequence is LLLYSANSLQT; CDR3 consists of 8 amino acids at positions 86-93, and its sequence is QQYYSGNT.
在另一个实施例中，确定了作为框架的人源抗体可变区氨基酸序列。In another embodiment, the amino acid sequence of the human antibody variable region as a framework is determined.
分别以上述hGP5C4重链和轻链可变区的氨基酸序列作为搜索序列，在IMGT(ImMunoGeneTics，www.imgt.org)数据库中搜索高同源性的胚系(germline)人抗体可变区氨基酸序列。Using the above-mentioned amino acid sequences of hGP5C4 heavy chain and light chain variable regions as the search sequence, the highly homologous germline (germline) human antibody variable region amino acid sequences were searched in the IMGT (ImMunoGeneTics, www.imgt.org) database.
实施例中表3和表4是以hGP5C4重链氨基酸可变区序列搜索到的5个高同源性胚系人抗体重链可变区序列，其中表3所示的是覆盖氨基端98个氨基酸的序列，同源性为75.5％；表4是近羧基端的14个氨基酸序列，同源性为78.6％。这些序列与hGP5C4的同源性比较见表5。根据表5比较结果，确定了人源化hGP5C4的重链可变区框架。In the examples, Table 3 and Table 4 are five highly homologous germline human antibody heavy chain variable region sequences searched for hGP5C4 heavy chain amino acid variable region sequences, among which Table 3 shows the sequences covering the amino terminal 98 amino acids The sequence has a homology of 75.5%; Table 4 shows the 14 amino acid sequences near the carboxy-terminus, with a homology of 78.6%. The homology comparison between these sequences and hGP5C4 is shown in Table 5. According to the comparison results in Table 5, the heavy chain variable region framework of humanized hGP5C4 was determined.
实施例中表6和表7是以hGP5C4轻链氨基酸可变区序列搜索到的5个高同源性胚系人抗体轻链可变区序列，其中表6所示的是覆盖氨基端的86个氨基酸序列，同源性为70.9％-72.3％；表7是近羧基端的11个氨基酸序列，同源性为81.8％。这些序列与hGP5C4的同源性比较见表8。根据表8的比较结果，确定了人源化hGP5C4的轻链可变区框架。In the examples, Table 6 and Table 7 are five highly homologous germline human antibody light chain variable region sequences searched for hGP5C4 light chain amino acid variable region sequences, and Table 6 shows the 86 amino acid sequences covering the amino terminal , with a homology of 70.9%-72.3%; Table 7 shows the 11 amino acid sequences near the carboxyl terminal, with a homology of 81.8%. The homology comparison between these sequences and hGP5C4 is shown in Table 8. According to the comparison results in Table 8, the light chain variable region framework of humanized hGP5C4 was determined.
在另一实施例中，构建了hGP5C4人源化抗体重链Fd和轻链的氨基酸序列。In another example, the amino acid sequences of the heavy chain Fd and light chain of hGP5C4 humanized antibody were constructed.
CDR移植是指将非人源单克隆抗体可变区中的CDR序列取代人源抗体相应的CDR序列，构成了由非人源抗体的CDR区和人源抗体的框架区所组成的重组抗体。抗体重链和轻链的可变区由CDR区和框架区(FR)组成。其中可变区的6个CDR组成识别和结合抗原的区域，它们直接与抗原接触，决定了抗体的特异性。骨架区是可变区以外的其它部分，主要起着支撑CDR的作用，它们的氨基酸组成和排列相对保守。因此，将非人源单克隆抗体的CDR区移植到人单克隆抗体的骨架区就可以达到人源化的目的。这种人源化抗体保持了非人源单克隆抗体和抗原结合的特异性和亲和力，并最大限度地降低了非人源单抗的异源性。CDR transplantation refers to replacing the CDR sequence in the variable region of a non-human monoclonal antibody with the corresponding CDR sequence of a human antibody to form a recombinant antibody consisting of the CDR region of a non-human antibody and the framework region of a human antibody. The variable regions of antibody heavy and light chains consist of CDR regions and framework regions (FR). Among them, the 6 CDRs of the variable region constitute the region that recognizes and binds to the antigen, and they directly contact with the antigen to determine the specificity of the antibody. The framework region is the part other than the variable region, which mainly plays the role of supporting the CDR, and their amino acid composition and arrangement are relatively conservative. Therefore, the purpose of humanization can be achieved by grafting the CDR region of a non-human monoclonal antibody to the framework region of a human monoclonal antibody. This humanized antibody maintains the specificity and affinity of non-human monoclonal antibody and antigen binding, and minimizes the heterogeneity of non-human monoclonal antibody.
将上述实施例所确定的hGP5C4的CDR区氨基酸序列移植到作为框架的胚系人源抗体可变区相应的位置，获得人源化抗体可变区的氨基酸序列。The amino acid sequence of the CDR region of hGP5C4 determined in the above examples was grafted to the corresponding position of the variable region of the germline human antibody as a framework to obtain the amino acid sequence of the variable region of the humanized antibody.
将上述人源化重链可变区序列，连接上保守的人IgG抗体重链恒定区1(CH1)序列，获得人源化的抗GPVI Fd片段的氨基酸序列。The above humanized heavy chain variable region sequence was connected to the conserved human IgG antibody heavy chain constant region 1 (CH1) sequence to obtain the amino acid sequence of the humanized anti-GPVI Fd fragment.
将上述人源化轻链可变区序列，连接上保守的人IgG抗体轻链恒定区(CL)序列，获得人源化的抗GPVI轻链的氨基酸序列。The humanized light chain variable region sequence above was linked to the conserved human IgG antibody light chain constant region (CL) sequence to obtain the amino acid sequence of the humanized anti-GPVI light chain.
上述人源化的抗GPVI Fd和人源化的抗GPVI轻链在基因工程重组表达时，通过各自的半胱氨酸残基形成二硫键而折叠成一个完全的Fab分子，此即本发明中的人源化抗GPVI Fab片段—hGPFab。The above-mentioned humanized anti-GPVI Fd and humanized anti-GPVI light chain are folded into a complete Fab molecule through the formation of disulfide bonds by their respective cysteine residues when expressed through genetic engineering recombination, which is the present invention The humanized anti-GPVI Fab fragment—hGPFab.
在另一实施例中，根据上述人源化Fd片段和轻链的氨基酸序列，构建了编码这些氨基酸序列的核苷酸序列。In another embodiment, based on the amino acid sequences of the above-mentioned humanized Fd fragment and light chain, nucleotide sequences encoding these amino acid sequences were constructed.
在基因工程重组表达由此Fd片段和轻链所构成的Fab分子时，需要根据其氨基酸序列反向推导出各自的核苷酸编码序列。由于核苷酸编码氨基酸的简并性，相同的氨基酸序列可以反向推导出多种组合的核苷酸编码序列。通常的做法是根据所选择的宿主细胞对编码氨基酸的偏性而合成相应的核苷酸序列。When the Fab molecule composed of the Fd fragment and the light chain is expressed through genetic engineering recombination, it is necessary to reversely deduce the respective nucleotide coding sequences based on its amino acid sequence. Due to the degeneracy of nucleotide-coded amino acids, the same amino acid sequence can be reversely deduced into multiple combinations of nucleotide-coded sequences. It is common practice to synthesize the corresponding nucleotide sequence according to the bias of the selected host cell towards the encoded amino acid.
本实施例中选择中国仓鼠细胞(CHO)作为宿主细胞，相应地，根据鼠细胞的氨基酸编码偏性合成相应的核苷酸序列，确定了人源化Fd片段和人源化轻链的核苷酸编码序列。In this example, Chinese hamster cells (CHO) were selected as the host cells. Correspondingly, the corresponding nucleotide sequences were synthesized according to the amino acid coding bias of the mouse cells, and the nucleosides of the humanized Fd fragment and the humanized light chain were determined. acid coding sequence.
在另一实施例中，构建了人源化Fd片段和轻链的重组表达载体In another embodiment, the recombinant expression vector of humanized Fd fragment and light chain was constructed
基因工程重组蛋白质常用的表达载体是改造的重组细菌质粒(plasmid)。这些质粒除了能够在细菌内复制外，还含有能在不同宿主细胞(如大肠杆菌、酵母细胞、昆虫细胞、哺乳动物细胞等)内合成蛋白质的元件，如启动子、核糖体结合位点、增强因子、转录终止信号等。The commonly used expression vector for genetically engineered recombinant proteins is a modified recombinant bacterial plasmid (plasmid). In addition to being able to replicate in bacteria, these plasmids also contain elements that can synthesize proteins in different host cells (such as Escherichia coli, yeast cells, insect cells, mammalian cells, etc.), such as promoters, ribosome binding sites, enhancers factors, transcription termination signals, etc.
本发明中的hGPFab能够藉由重组质粒作为载体，在这些宿主细胞内重组表达出具有生物学活性的Fab片段。优化的方案是选择哺乳动物细胞作为载体，如人源或鼠源的工程化细胞系，以利于Fab片段的正确折叠而形成接近天然构型的分子。The hGPFab in the present invention can use the recombinant plasmid as a vector to recombinantly express the Fab fragment with biological activity in these host cells. The optimized solution is to choose mammalian cells as the carrier, such as engineered cell lines of human or mouse origin, so as to facilitate the correct folding of Fab fragments to form molecules close to the natural configuration.
本实施方案采用CHO细胞作为重组表达的宿主。为利于目的蛋白的分泌表达，在编码Fd片段和轻链核苷酸序列的5’端分别添加了Kozak序列和信号肽序列(signal peptide,SP)，在3’端添加了终止密码子“TGA”，并分别在两端添加了相应的酶切位点，以利于重组质粒的构建。In this embodiment, CHO cells are used as hosts for recombinant expression. In order to facilitate the secretory expression of the target protein, a Kozak sequence and a signal peptide sequence (signal peptide, SP) were added to the 5' end of the coding Fd fragment and the light chain nucleotide sequence, respectively, and a stop codon "TGA" was added to the 3' end. ", and corresponding restriction sites were added at both ends to facilitate the construction of recombinant plasmids.
Kozak序列(Kozak consensus sequence)是在真核细胞mRNA转录子上常见的蛋白质翻译起始位点，常用的Kozak序列是(gcc)gccRccAUGG，其中AUG是目的蛋白的起始氨基酸(蛋氨酸)的密码子。Kozak sequence (Kozak consensus sequence) is a common protein translation initiation site on eukaryotic mRNA transcripts. The commonly used Kozak sequence is (gcc)gccRccAUGG, where AUG is the codon for the starting amino acid (methionine) of the target protein .
信号肽是分泌型蛋白质氨基端的一段氨基酸序列，长度一般在16-30个氨基酸残基之间。信号肽可以引导重组蛋白分泌至胞外，并且在分泌过程中被信号肽酶切割，分泌出成熟的目的蛋白。本发明中选用了人抗体重链和轻链的信号肽序列。The signal peptide is an amino acid sequence at the amino terminal of the secreted protein, generally between 16-30 amino acid residues in length. The signal peptide can guide the secretion of the recombinant protein to the outside of the cell, and is cleaved by the signal peptidase during the secretion process to secrete the mature target protein. In the present invention, the signal peptide sequences of the heavy chain and light chain of human antibody are selected.
本发明所采用的质粒是pCDdhfr，质粒上外源蛋白的启动子为鼠CMV早期启动子。筛选基因是二氢叶酸还原酶(dihydrofolatereductase，dhfr)基因。pCDdhfr重组质粒转染入dhfr缺陷型CHO细胞后，质粒序列可以随机整合到细胞基因组内。转染的dhfr缺陷型CHO细胞在叶酸合成抑制剂甲氨蝶呤(methotrexate，MTX)的压力下，整合的质粒需要不断扩增来强化dhfr的表达，从而使串联的重组蛋白基因随之扩增而提高表达量。The plasmid used in the present invention is pCDdhfr, and the promoter of the foreign protein on the plasmid is the early promoter of mouse CMV. Screening gene is dihydrofolate reductase (dihydrofolatereductase, dhfr) gene. After the pCDdhfr recombinant plasmid was transfected into dhfr-deficient CHO cells, the plasmid sequence could be randomly integrated into the cell genome. Under the pressure of the folic acid synthesis inhibitor methotrexate (MTX) in the transfected dhfr-deficient CHO cells, the integrated plasmid needs to be continuously amplified to enhance the expression of dhfr, so that the tandem recombinant protein gene will be amplified accordingly to increase expression.
将合成的编码人源化Fd片段和轻链的基因分别***到pCDdhfr的多克隆位点内，构建了重组质粒pCDGPFd(含人源化的Fd片段编码基因)和pCDGPL(含人源化的轻链编码基因)，经DNA测序验证了克隆基因的序列和设计合成的序列相同。The synthetic genes encoding humanized Fd fragment and light chain were respectively inserted into the multiple cloning site of pCDdhfr, and recombinant plasmids pCDGPFd (containing humanized Fd fragment encoding gene) and pCDGPL (containing humanized light chain gene) were constructed. Chain coding gene), the sequence of the cloned gene was verified to be the same as the designed and synthesized sequence by DNA sequencing.
在另一实施例中，采用CHO细胞重组表达了hGPFab。In another embodiment, hGPFab is recombinantly expressed using CHO cells.
所述的hGPFab是由本发明中的人源化Fd片段和人源化轻链在宿主细胞内共表达时所自发形成的异源二聚体，两者间经由二硫键连接。The hGPFab is a heterodimer spontaneously formed when the humanized Fd fragment and the humanized light chain in the present invention are co-expressed in host cells, and the two are connected by a disulfide bond.
将重组质粒转染到哺乳动物细胞的方法有多种，既有电转染的物理方法，也有利用氯化钙、脂质体等作为介导的化学方法。上述pCDGPFd和pCDGPL质粒经脂质体介导共转染(co-transfection)入dhfr缺陷型CHO细胞后，在细胞培养上清中经酶联免疫吸附试验(ELISA)检测到了能和人GPVI蛋白特异结合的Fab片段，提示共转染的两个质粒在宿主细胞内得到了表达，并分泌至细胞外。转染的CHO细胞经MTX压力筛选、传代培养，获得了能稳定表达hGPFab的单克隆细胞株。There are many methods for transfecting recombinant plasmids into mammalian cells, including physical methods such as electroporation, and chemical methods using calcium chloride, liposomes, etc. as mediators. After the above pCDGPFd and pCDGPL plasmids were co-transfected into dhfr-deficient CHO cells through liposome-mediated co-transfection, they were detected in the cell culture supernatant by enzyme-linked immunosorbent assay (ELISA). The combined Fab fragments indicated that the two co-transfected plasmids were expressed in the host cells and secreted outside the cells. The transfected CHO cells were screened by MTX pressure and subcultured to obtain a monoclonal cell line that can stably express hGPFab.
在另一实施例中，在所述细胞株的培养上清中纯化出了表达的hGPFab。In another embodiment, the expressed hGPFab is purified from the culture supernatant of the cell line.
常用的重组蛋白纯化方法是各类层析技术，包括亲和层析、离子交换层析、凝胶层析、超滤等。其中亲和层析是利用目的蛋白与配体或抗体的特异结合而达到分离、纯化的目的。Commonly used recombinant protein purification methods are various chromatographic techniques, including affinity chromatography, ion exchange chromatography, gel chromatography, ultrafiltration, etc. Among them, affinity chromatography is to use the specific combination of target protein and ligand or antibody to achieve the purpose of separation and purification.
蛋白L(Protein L)是由大消化链球菌(Peptostreptococcusmagnus)产生的一种结构蛋白，由719个氨基酸构成。蛋白L可以特异结合多种动物抗体的kappa轻链，因此固相化的蛋白L常被用来纯化抗体的Fab片段。Protein L (Protein L) is a structural protein produced by Peptostreptococcus magnus, consisting of 719 amino acids. Protein L can specifically bind to the kappa light chains of various animal antibodies, so immobilized protein L is often used to purify Fab fragments of antibodies.
本发明所构建的hGPFab稳定表达细胞株，其培养基上清液经蛋白L-琼脂糖柱亲和层析，纯化到了目的蛋白。所纯化的蛋白经SDS-PAGE分析，显示其为异源二聚体。The hGPFab stably expressing cell line constructed by the present invention has the culture supernatant purified to the target protein through protein L-agarose column affinity chromatography. SDS-PAGE analysis of the purified protein showed it to be a heterodimer.
在另一实施例中，ELISA检测证实所纯化的hGPFab和人GPVI蛋白可以特异结合。In another embodiment, ELISA detection confirms that the purified hGPFab can specifically bind to human GPVI protein.
酶联免疫吸附试验(Enzyme-Linked ImmunoSorbent Assay，ELISA)是一种常用来鉴定蛋白之间相互作用的分析技术，通过固相化一种蛋白，在固相表面检测液相中的另一种蛋白，如分析配体-受体、抗原-抗体之间结合能力。Enzyme-Linked ImmunoSorbent Assay (ELISA) is an analytical technique commonly used to identify the interaction between proteins, by immobilizing one protein and detecting another protein in the liquid phase on the surface of the solid phase , such as analyzing the binding ability between ligand-receptor and antigen-antibody.
为分析本发明中的hGPFab与GPVI的结合性质，将纯化的GPVI蛋白包被(固相化)在96孔的微孔板上，加入重组表达hGPFab的细胞培养上清或纯化的hGPFab，再以辣根过氧化物酶(HRP)标记的抗人Fab抗体检测与固相化GPVI结合的Fab片段。In order to analyze the binding properties of hGPFab and GPVI in the present invention, the purified GPVI protein was coated (solid-phased) on a 96-well microwell plate, and the cell culture supernatant or purified hGPFab expressing recombinant hGPFab was added, and then Horseradish peroxidase (HRP)-labeled anti-human Fab antibody detects Fab fragments bound to immobilized GPVI.
结果显示，细胞培养上清和纯化的hGPFab蛋白都能与微孔板上的GPVI特异结合。随液相中hGPFab浓度的增高，结合反应的酶促信号也随之增强，表明hGPFab与GPVI的结合有浓度-效应反应关系。The results showed that both the cell culture supernatant and the purified hGPFab protein could specifically bind to GPVI on the microwell plate. As the concentration of hGPFab in the liquid phase increases, the enzymatic signal of the binding reaction also increases, indicating that the binding of hGPFab to GPVI has a concentration-effect response relationship.
在另一实施例中，免疫印迹实验(Western Blot，WB)显示本发明的hGPFab能够特异结合非还原性的人GPVI蛋白。In another embodiment, Western Blot (WB) shows that the hGPFab of the present invention can specifically bind non-reducing human GPVI protein.
WB也是一种常用来鉴定蛋白之间相互作用的分析技术，通过固相化一种蛋白，在固相表面检测液相中的另一种蛋白，如分析配体-受体、抗原-抗体之间结合能力。和ELISA不同的是：WB检测是先将一种蛋白进行电泳分离(SDS-PAGE)，然后将分离的蛋白转移到固相膜上，再与液相中的另一种蛋白发生结合反应，最后利用酶、同位素、生物素等检测两者之间的反应。由于有了前期的蛋白分离，使WB的检测结果更特异、直观，常用来进行ELISA检测后的鉴定分析。WB is also an analytical technique commonly used to identify the interaction between proteins. By immobilizing one protein, another protein in the liquid phase is detected on the surface of the solid phase, such as analyzing ligand-receptor, antigen-antibody interactions. ability to combine. The difference from ELISA is: WB detection is to first separate a protein by electrophoresis (SDS-PAGE), then transfer the separated protein to a solid-phase membrane, and then react with another protein in the liquid phase, and finally Use enzymes, isotopes, biotin, etc. to detect the reaction between the two. Due to the protein separation in the early stage, the detection results of WB are more specific and intuitive, and are often used for identification analysis after ELISA detection.
电泳分离蛋白时，可以在待分离的蛋白样品中加入或不加还原剂(如二硫苏糖醇DTT、β-巯基乙醇等)。加入还原剂的样品中，还原剂会打开氨基酸链间的二硫键，使蛋白呈线性结构(还原性蛋白)；不加还原剂的样品中，氨基酸链间的二硫键会持续存在而使蛋白呈现立体构型(非还原性蛋白)。所以，可以通过还原剂的作用来分析不同构型之间蛋白的相互作用。When separating proteins by electrophoresis, a reducing agent (such as dithiothreitol DTT, β-mercaptoethanol, etc.) can be added or not added to the protein sample to be separated. In the sample added with reducing agent, the reducing agent will open the disulfide bond between amino acid chains, making the protein a linear structure (reducing protein); in the sample without reducing agent, the disulfide bond between amino acid chains will continue to exist and make the protein The protein assumes a three-dimensional configuration (non-reducing protein). Therefore, the interaction of proteins between different configurations can be analyzed through the action of reducing agents.
本实施例中，将加和不加DTT的GPVI蛋白样品进行SDS-PAGE电泳分离，再将分离的蛋白转移到硝酸纤维素膜上后，加入纯化的hGPFab溶液，最后以HRP标记的抗人Fab抗体检测。结果显示，本发明中的hGPFab和非还原性GPVI有强烈的反应，而和还原性GPVI没有明显的反应。表明此hGPFab在GPVI蛋白上的结合位点是构象依赖的，即其抗原表位(epitope)属于非线性结构。In this example, the GPVI protein samples with and without DTT were separated by SDS-PAGE electrophoresis, and then the separated protein was transferred to a nitrocellulose membrane, then the purified hGPFab solution was added, and finally the HRP-labeled anti-human Fab Antibody testing. The results show that the hGPFab of the present invention has a strong reaction with non-reduced GPVI, but has no obvious reaction with reduced GPVI. It shows that the binding site of hGPFab on GPVI protein is conformation-dependent, that is, its epitope belongs to nonlinear structure.
在另一实施例中，本发明的hGPFab能够抑制人GPVI蛋白和胶原蛋白的结合，并具有剂量-效应关系。In another embodiment, the hGPFab of the present invention can inhibit the binding of human GPVI protein and collagen, and has a dose-effect relationship.
大鼠源的亲本抗体hGP5C4具有抑制GPVI和胶原蛋白结合的性质。为检测本发明中的hGPFab是否仍然具有此抑制功能，设计了GPVI和hGPFab与胶原蛋白结合的竞争抑制ELISA试验。The parental antibody hGP5C4 of rat origin has the property of inhibiting the binding of GPVI and collagen. In order to detect whether the hGPFab in the present invention still has this inhibitory function, a competitive inhibition ELISA assay for the binding of GPVI and hGPFab to collagen was designed.
在微孔板内包被人胶原蛋白，分别加入人GPVI、人GPVI/hGPFab混合物，再以HRP标记的抗GPVI多克隆抗体检测结合到人胶原蛋白上的GPVI，结果显示，加有hGPFab混合物的样品，其HRP酶信号明显减弱，且随着样品中hGPFab浓度的增加，其减弱越多，表明本发明中的hGPFab保持了亲本抗体的性质，能够抑制GPVI和胶原蛋白的结合，这种抑制作用呈现剂量-效应反应关系。Human collagen was coated in the microwell plate, and human GPVI and human GPVI/hGPFab mixture were added respectively, and then HRP-labeled anti-GPVI polyclonal antibody was used to detect GPVI bound to human collagen. The results showed that the samples added with hGPFab mixture , the HRP enzyme signal is obviously weakened, and with the increase of hGPFab concentration in the sample, the more it weakens, it shows that the hGPFab in the present invention maintains the properties of the parental antibody and can inhibit the combination of GPVI and collagen, and this inhibitory effect appears Dose-effect-response relationship.
附图说明Description of drawings
图1：IgG抗体Fab片段示意图。完整的抗体呈“Y”型，经木瓜蛋白酶降解后，形成两个相同的Fab片段和一个结晶片段(Fc)Figure 1: Schematic representation of Fab fragments of IgG antibodies. The intact antibody is in a "Y" shape, and after papain degradation, two identical Fab fragments and one crystalline fragment (Fc) are formed
图2：人源化重链Fd片段的编码核苷酸及其氨基酸序列。含有预测的3个hGP5C4重链CDR区及胚系人IgG抗体Fd片段的框架区，编码的核苷酸全长为669bp，编码223个氨基酸残基。Figure 2: The coding nucleotide and amino acid sequence of the humanized heavy chain Fd fragment. Containing the predicted three heavy chain CDR regions of hGP5C4 and the framework region of the Fd fragment of the germline human IgG antibody, the encoded nucleotides are 669bp in full length and encode 223 amino acid residues.
图3：人源化轻链的编码核苷酸及其氨基酸序列。含有预测的3个hGP5C4轻链CDR区及胚系人IgG抗体轻链的框架区，编码的核苷酸全长为639bp，编码213个氨基酸残基。Figure 3: The coding nucleotide and amino acid sequence of the humanized light chain. It contains the predicted 3 hGP5C4 light chain CDR regions and the framework region of the germline human IgG antibody light chain. The encoded nucleotides are 639bp in full length and encode 213 amino acid residues.
图4：人源化抗GPVI抗体Fab片段(hGPFab)的纯化。表达hGPFab的CHO细胞株培养上清中，采用蛋白L亲和层析纯化得到的hGPFab蛋白。图A为SDS-PAGE电泳图，泳道 1是经DTT处理后的带型，主要蛋白的分子量约为25kD；泳道2是未经DTT处理的样品，分子量约为45kD。图B是洗脱时紫外扫描的蛋白峰图。Figure 4: Purification of humanized anti-GPVI antibody Fab fragment (hGPFab). In the culture supernatant of the CHO cell line expressing hGPFab, the obtained hGPFab protein was purified by protein L affinity chromatography. Figure A is the SDS-PAGE electrophoresis image. Lane 1 is the band pattern after DTT treatment, and the molecular weight of the main protein is about 25kD; Lane 2 is the sample without DTT treatment, the molecular weight is about 45kD. Panel B is the peak profile of the protein by UV scanning during elution.
图5：hGPFab与人GPVI蛋白的特异性结合(ELISA实验)。本发明中的hGPFab可以和包被的人GPVI蛋白特异结合，且随着hGPFab浓度的增加，其结合的信号也随着增加。作为对照的正常人IgG抗体的Fab片段和GPVI无明显的反应。Figure 5: Specific binding of hGPFab to human GPVI protein (ELISA experiment). The hGPFab in the present invention can specifically bind to the coated human GPVI protein, and as the hGPFab concentration increases, the binding signal also increases. As a control, the Fab fragment of normal human IgG antibody had no obvious reaction with GPVI.
图6：hGPFab与人GPVI蛋白特异结合的免疫印迹实验(WB)。图A为人GPVI蛋白的SDS-PAGE电泳图，其中泳道1是经DTT处理(还原性蛋白)的GPVI，泳道2是未经DTT处理的GPVI(非还原性)。图B是WB实验结果，hGPVI与非还原性的GPVI呈现强烈的反应(泳道2)，而和还原性GPVI的反应微弱(泳道1)。Figure 6: Western blot experiment (WB) of the specific binding of hGPFab to human GPVI protein. Figure A is the SDS-PAGE electrophoresis of human GPVI protein, wherein lane 1 is GPVI treated with DTT (reduced protein), and lane 2 is GPVI without DTT treatment (non-reduced). Panel B is the result of WB experiment, hGPVI showed a strong reaction with non-reduced GPVI (lane 2), but a weak reaction with reduced GPVI (lane 1).
图7：hGPFab与人GPVI蛋白与胶原蛋白的竞争性结合实验(竞争性ELISA)。Figure 7: Competitive binding experiment of hGPFab with human GPVI protein and collagen (competitive ELISA).
hGPFab能够抑制GPVI与胶原蛋白的结合，且随着hGPFab浓度的增加，其抑制作用随之增强。而作为对照的正常人IgG抗体Fab片段则没有明显的抑制作用。hGPFab can inhibit the combination of GPVI and collagen, and with the increase of hGPFab concentration, its inhibitory effect will be enhanced. However, the Fab fragment of normal human IgG antibody used as a control has no obvious inhibitory effect.
具体实施方式Detailed ways
本发明所涉及的基因工程重组技术、细菌/细胞培养技术、重组蛋白纯化技术均为常规技术。下面通过具体实施例对本发明作进一步的说明。下例实施例只是为了使本领域的技术人员能够更好地理解本发明但并不对本发明作任何限制。The genetic engineering recombination technology, bacteria/cell culture technology and recombinant protein purification technology involved in the present invention are all conventional technologies. The present invention will be further described below by specific examples. The following examples are just to enable those skilled in the art to better understand the present invention but do not limit the present invention in any way.
实施例1：待人源化的非人源抗人GPVI单克隆抗体的筛选与确定。Example 1: Screening and determination of non-human anti-human GPVI monoclonal antibodies to be humanized.
首先，确定选择非人源抗人GPVI单克隆抗体的标准：⑴能够与人GPVI蛋白特异结合，有效阻断人GPVI和胶原蛋白的结合；⑵能够阻止血小板的聚集；⑶没有内源活性，不具有激活GPVI而导致血小板活化的活性。First, determine the criteria for selecting a non-human anti-human GPVI monoclonal antibody: (1) can specifically bind to human GPVI protein, effectively blocking the binding of human GPVI and collagen; (2) can prevent the aggregation of platelets; (3) have no endogenous activity, no Has the activity of activating GPVI leading to platelet activation.
根据这3条标准，对背景技术中提到的多株单克隆抗体进行了信息分析，结果显示大鼠抗人GPVI单克隆抗体株hGP5C4符合所设定的标准，其被确定为人源化的对象。According to these three standards, the information analysis of the multiple strains of monoclonal antibodies mentioned in the background technology was carried out, and the results showed that the rat anti-human GPVI monoclonal antibody strain hGP5C4 met the set standards, and it was determined to be the subject of humanization .
hGP5C4是以人GPVI-Fc二聚体蛋白为免疫原，免疫Lou/C大鼠(rat)而制备的一株单克隆抗体。(Massberg S.et al.Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo.FASEB J 2004；18:397-9)(专利WO2005054294：Inhibitors of glycoprotein vi based on  monoclonal antibody hgp 5c4)。用木瓜蛋白酶降解hGP5C4所得到的Fab片段可以有效阻止GPVI和胶原蛋白的结合；也能够特异结合血小板表面的GPVI，抑制由胶原蛋白刺激所诱导的血小板活化，阻止其活性标记物PAC-I和CD62-P的表达；有效抑制人血小板的聚集和ATP的释放；在离体(ex vivo)人红细胞出血时间试验中，hGP5C4对出血时间没有影响。所有这些数据表明hGP5C4具有特异的抗血小板功能，同时不具有内在的激发活性，提示hGP5C4具有良好的应用潜力。hGP5C4 is a monoclonal antibody prepared by immunizing Lou/C rats (rat) with human GPVI-Fc dimer protein as the immunogen. (Massberg S. et al. Soluble glycoprotein VI dimer inhibits platelet adhesion and aggregation to the injured vessel wall in vivo. FASEB J 2004; 18:397-9) (patent WO2005054294: Inhibitors of glycoprotein vi pgdyclonalh4 based on monoclonalh 5) The Fab fragment obtained by degrading hGP5C4 with papain can effectively prevent the combination of GPVI and collagen; it can also specifically bind to GPVI on the surface of platelets, inhibit the activation of platelets induced by collagen stimulation, and prevent the activation of its active markers PAC-I and CD62 -P expression; effectively inhibit the aggregation of human platelets and the release of ATP; in the ex vivo (ex vivo) human red blood cell bleeding time test, hGP5C4 has no effect on the bleeding time. All these data indicate that hGP5C4 has specific antiplatelet function, but does not have intrinsic stimulating activity, suggesting that hGP5C4 has good application potential.
但由于天然的hGP5C4抗体来源于大鼠，其异源性使之不能直接应用于人体。而人源化处理能够改变其异源性质，为临床应用提供了可能性。However, since the natural hGP5C4 antibody is derived from rats, its heterogeneity prevents it from being directly applied to humans. Humanized treatment can change its heterologous properties, which provides the possibility for clinical application.
在基因数据库GenBank中，我们搜索到上述抗体株hGP5C4重链可变区的核苷酸编码序列为CS114614.1(nucleic acid sequence of 5C4 heavy chain variable)；轻链可变区的核苷酸编码序列为CS114616.1(nucleotide sequence of 5C4light chain variableDomain)。In the gene database GenBank, we found that the nucleotide coding sequence of the heavy chain variable region of the above antibody strain hGP5C4 is CS114614.1 (nucleic acid sequence of 5C4 heavy chain variable); the nucleotide coding sequence of the light chain variable region It is CS114616.1 (nucleotide sequence of 5C4light chain variableDomain).
CS114614.1(nucleic acid sequence of 5C4 heavy chain variable)核苷酸序列如SEQ ID NO.2所示：The nucleotide sequence of CS114614.1 (nucleic acid sequence of 5C4 heavy chain variable) is shown in SEQ ID NO.2:
Figure PCTCN2021099670-appb-000001
Figure PCTCN2021099670-appb-000001
CS114616.1(nucleotide sequence of 5C4 light chain variable Domain)核苷酸序列如SEQ ID NO.4所示：The nucleotide sequence of CS114616.1 (nucleotide sequence of 5C4 light chain variable Domain) is shown in SEQ ID NO.4:
Figure PCTCN2021099670-appb-000002
Figure PCTCN2021099670-appb-000002
Figure PCTCN2021099670-appb-000003
Figure PCTCN2021099670-appb-000003
CS114614.1全长共378个碱基(base pair,bp)，编码126个氨基酸残基，无起始和终止密码子。其推导的编码氨基酸序列如SEQ ID NO.3所示：CS114614.1 has a total length of 378 bases (base pair, bp), encoding 126 amino acid residues, without start and stop codons. Its deduced coded amino acid sequence is shown in SEQ ID NO.3:
Figure PCTCN2021099670-appb-000004
Figure PCTCN2021099670-appb-000004
CS114616.1全长共342个碱基，编码114个氨基酸残基，无起始和终止密码子。其推导的编码氨基酸序列如SEQ ID NO.5所示：The total length of CS114616.1 is 342 bases, encoding 114 amino acid residues, without start and stop codons. Its deduced coded amino acid sequence is shown in SEQ ID NO.5:
Figure PCTCN2021099670-appb-000005
Figure PCTCN2021099670-appb-000005
上述CS114614.1和CS114616.1编码的氨基酸系列分别具有抗体重链和轻链可变区的结构特征。理论上，这两个重链可变区和轻链可变区氨基酸序列含有各自的3个CDR区，这6个CDR区能够组成一个特异的抗原结合区而特异结合人GPVI蛋白。The above-mentioned amino acid series encoded by CS114614.1 and CS114616.1 have the structural characteristics of antibody heavy chain and light chain variable regions respectively. Theoretically, the amino acid sequences of the two heavy chain variable regions and the light chain variable region contain 3 CDR regions respectively, and these 6 CDR regions can form a specific antigen-binding region to specifically bind human GPVI protein.
实施例2：hGP5C4重链和轻链互补决定区(CDRs)的预测与确定Example 2: Prediction and determination of hGP5C4 heavy chain and light chain complementarity determining regions (CDRs)
目前对人和小鼠抗体的CDR研究最为深入，根据抗体可变区的氨基酸序列预测人或小鼠抗体的CDR区也较为准确。由于对其它动物抗体的研究相对较少，对其CDR区的预测则相对不足。基于hGP5C4的大鼠来源，为最大限度地保证所确定的CDR区与GPVI的亲和力与特异性，我们共采用了Kabat(Kabat,E.A.et al.,In:Sequences of Proteins of Immunological Interest,NIH Publication,1991:91-3242)、IMGT(Lefranc,M.P.,The IMGT unique numbering for Immunoglobulins,T-cell receptors,and Ig-like Domains.The Immunologist,1999:7,132-136)、Chothia(Al-Lazikani B et al.,"Standard conformations for the canonical structures of immunoglobulins".Journal of Molecular Biology.1997:273(4):927–48)、North(North B et  al.,A new clustering of antibody CDR loop conformations.Journal of Molecular Biology.2011:406(2):228–56)和Contact(R M MacCallum et al.Antibody-antigen interactions:contact analysis and binding site topographyJMol Biol.1996Oct 11；262(5):732-45)共5种方法来预测5C4重链和轻链的CDR区。At present, the research on CDR of human and mouse antibodies is the most in-depth, and it is more accurate to predict the CDR regions of human or mouse antibodies based on the amino acid sequences of antibody variable regions. Since there are relatively few studies on other animal antibodies, the prediction of their CDR regions is relatively insufficient. Based on the rat source of hGP5C4, in order to ensure the affinity and specificity of the determined CDR region and GPVI to the greatest extent, we used Kabat (Kabat, E.A. et al., In: Sequences of Proteins of Immunological Interest, NIH Publication, 1991:91-3242), IMGT (Lefranc, M.P., The IMGT unique numbering for Immunoglobulins, T-cell receptors, and Ig-like Domains. The Immunologist, 1999:7, 132-136), Chothia (Al-Lazikani B et al. ,"Standard conformations for the canonical structures of immunoglobulins".Journal of Molecular Biology.1997:273(4):927–48), North(North B et al., A new clustering of antibody CDR loop conformations.Journal of Biolog Molecules .2011:406(2):228–56) and Contact (R M MacCallum et al. Antibody-antigen interactions: contact analysis and binding site topographyJMol Biol.1996Oct 11; 262(5):732-45) a total of 5 methods To predict the CDR regions of the 5C4 heavy and light chains.
根据实施例1的分析结果，hGP5C4重链可变区的氨基酸序列为：According to the analysis results of Example 1, the amino acid sequence of the variable region of the heavy chain of hGP5C4 is:
Figure PCTCN2021099670-appb-000006
Figure PCTCN2021099670-appb-000006
表1为5种方法预测的hGP5C4重链CDR区氨基酸的位置和序列。不同方法预测的CDR1由5-12个氨基酸组成、CDR2由6-17个氨基酸组成、CDR3由7-9个氨基酸组成。结合各自预测的位置和序列，确定重链3个CDR的最大化序列为：CDR1由第23-35位的13个氨基酸构成，其序列为TASGFTFSDYFMS(SEQ ID NO.6)；CDR2由第47-66位的20个氨基酸构成，其序列为WVASISSGGASAYWRDSVKG(SEQ ID NO.7)；CDR3由第97-105位的9个氨基酸构成，其序列为ARGELDFDY(SEQ ID NO.8)。Table 1 shows the position and sequence of the amino acids in the heavy chain CDR region of hGP5C4 predicted by five methods. CDR1 predicted by different methods consists of 5-12 amino acids, CDR2 consists of 6-17 amino acids, and CDR3 consists of 7-9 amino acids. Combined with their respective predicted positions and sequences, the maximized sequences of the three CDRs of the heavy chain were determined as follows: CDR1 consists of 13 amino acids at positions 23-35, and its sequence is TASGFTFSDYFMS (SEQ ID NO.6); CDR2 consists of 13 amino acids at positions 47-35; The 20 amino acids at position 66 are composed of 20 amino acids, and its sequence is WVASISSGGASAYWRDSVKG (SEQ ID NO.7); CDR3 is composed of 9 amino acids at positions 97-105, and its sequence is ARGELDFDY (SEQ ID NO.8).
表1：hGP5C4重链CDR区预测结果Table 1: Prediction results of hGP5C4 heavy chain CDR region
Figure PCTCN2021099670-appb-000007
Figure PCTCN2021099670-appb-000007
根据实施例1的分析结果，hGP5C4轻链可变区的氨基酸序列为：According to the analysis results of Example 1, the amino acid sequence of the hGP5C4 light chain variable region is:
Figure PCTCN2021099670-appb-000008
Figure PCTCN2021099670-appb-000008
表2为5种方法预测的hGP5C4轻链CDR区氨基酸的位置和序列。不同方法预测的CDR1由6-11个氨基酸组成、CDR2由3-10个氨基酸组成、CDR3由7-8个氨基酸组成。结合各自预测的位置和序列，确定轻链3个CDR的最大化序列为：CDR1由第21-33位的13个氨基酸构成，其序列为TASQNVYKNLAWY(SEQ ID NO.9)；CDR2由第43-53位的11个氨基酸构成，其序列为LLLYSANSLQT(SEQ ID NO.10)；CDR3由第86-93位的8个氨基酸构成，其序列为QQYYSGNT(SEQ ID NO.11)。Table 2 shows the amino acid positions and sequences of the hGP5C4 light chain CDR region predicted by five methods. CDR1 predicted by different methods consists of 6-11 amino acids, CDR2 consists of 3-10 amino acids, and CDR3 consists of 7-8 amino acids. Combined with their predicted positions and sequences, the maximized sequences of the three CDRs of the light chain were determined as follows: CDR1 consists of 13 amino acids at positions 21-33, and its sequence is TASQNVYKNLAWY (SEQ ID NO.9); CDR2 consists of 13 amino acids at positions 43-33. The 11 amino acids at position 53 consist of LLLYSANSLQT (SEQ ID NO.10); the CDR3 consists of 8 amino acids at positions 86-93, and its sequence is QQYYSGNT (SEQ ID NO.11).
表2：hGP5C4轻链CDR区预测结果Table 2: Prediction results of hGP5C4 light chain CDR region
Figure PCTCN2021099670-appb-000009
Figure PCTCN2021099670-appb-000009
综上所述，根据5种预测方法所确定的hGP5C4重链和轻链可变区的CDR如下：In summary, the CDRs of the variable regions of the heavy and light chains of hGP5C4 determined according to the five prediction methods are as follows:
Figure PCTCN2021099670-appb-000010
Figure PCTCN2021099670-appb-000010
Figure PCTCN2021099670-appb-000011
Figure PCTCN2021099670-appb-000011
实施例3：作为框架的人源抗体可变区氨基酸序列的筛选与确定。Example 3: Screening and determination of the amino acid sequence of the human antibody variable region as a framework.
分别以上述hGP5C4重链和轻链可变区的氨基酸序列作为搜索序列，在IMGT(ImMunoGeneTics，www.imgt.org)数据库中搜索同源性高的胚系(germline)人抗体可变区氨基酸序列。Using the above-mentioned amino acid sequences of the hGP5C4 heavy chain and light chain variable regions as the search sequence, search for highly homologous germline (germline) human antibody variable region amino acid sequences in the IMGT (ImMunoGeneTics, www.imgt.org) database .
表3和表4是以hGP5C4重链氨基酸可变区序列搜索到的5个高同源性胚系人抗体重链可变区序列，其中表3所示的是覆盖氨基端98个氨基酸的序列，同源性为75.5％；表4是近羧基端的14个氨基酸序列，同源性为78.6％。这些序列与hGP5C4的同源性比较见表5。Table 3 and Table 4 are 5 highly homologous germline human antibody heavy chain variable region sequences searched for hGP5C4 heavy chain amino acid variable region sequences, of which Table 3 shows the sequence covering the amino-terminal 98 amino acids, same as The homology is 75.5%; Table 4 is the 14 amino acid sequences near the carboxyl terminal, and the homology is 78.6%. The homology comparison between these sequences and hGP5C4 is shown in Table 5.
表3:hGP5C4的高同源性胚系人抗体重链可变区序列(氨基端)Table 3: Highly homologous germline human antibody heavy chain variable region sequence (amino terminal) of hGP5C4
SpeciesSpecies Gene and alleleGene and Allele DomainDomain Domain labelDomain label ％identity%identity OverlapOverlap
Homo sapiensHomo sapiens IGHV3-23*03IGHV3-23*03 11 VHVH 75.575.5 9898
Homo sapiensHomo sapiens IGHV3-48*03IGHV3-48*03 11 VHVH 75.575.5 9898
Homo sapiensHomo sapiens IGHV3-7*01IGHV3-7*01 11 VHVH 75.575.5 9898
Homo sapiensHomo sapiens IGHV3-7*02IGHV3-7*02 11 VHVH 75.575.5 9898
Homo sapiensHomo sapiens IGHV3-7*03IGHV3-7*03 11 VHVH 75.575.5 9898
表4:5C4的高同源性胚系人抗体重链可变区序列(羧基端)Table 4: Highly homologous germline human antibody heavy chain variable region sequence (carboxyl terminus) of 5C4
SpeciesSpecies Gene and alleleGene and Allele DomainDomain ％identity%identity OverlapOverlap
Homo sapiensHomo sapiens IGHJ4*01IGHJ4*01 11 78.678.6 1414
Homo sapiensHomo sapiens IGHJ4*02IGHJ4*02 11 78.678.6 1414
Homo sapiensHomo sapiens IGHJ4*03IGHJ4*03 11 78.678.6 1414
Homo sapiensHomo sapiens IGHJ3*01IGHJ3*01 11 78.678.6 1414
Homo sapiensHomo sapiens IGHJ3*02IGHJ3*02 11 78.678.6 1414
表5:hGP5C4重链可变区与胚系人源抗体重链可变区氨基酸序列比较Table 5: Comparison of the amino acid sequences of the heavy chain variable region of hGP5C4 and the germline human antibody heavy chain variable region
Figure PCTCN2021099670-appb-000012
Figure PCTCN2021099670-appb-000012
根据表5的比较结果，确定人源化hGP5C4的重链可变区框架为：According to the comparison results in Table 5, the heavy chain variable region framework of humanized hGP5C4 was determined to be:
Figure PCTCN2021099670-appb-000013
Figure PCTCN2021099670-appb-000013
其中的“xxxxxx”将是CDR的移植位置。Where "xxxxxx" will be where the CDR is ported.
表6和表7是以hGP5C4轻链氨基酸可变区序列搜索到的5个高同源性胚系人抗体轻链可变区序列，其中表6所示的是覆盖氨基端的86个氨基酸序列，同源性为70.9％-Table 6 and Table 7 are five highly homologous germline human antibody light chain variable region sequences searched for hGP5C4 light chain amino acid variable region sequences, of which Table 6 shows the 86 amino acid sequences covering the amino terminal, homologous Sex is 70.9% -
72.3％；表7是近羧基端的11个氨基酸序列，同源性为81.8％。这些序列与hGP5C4的同源性比较见表8。72.3%; Table 7 shows the 11 amino acid sequences near the carboxy-terminus, with a homology of 81.8%. The homology comparison between these sequences and hGP5C4 is shown in Table 8.
表6:5C4的高同源性胚系人抗体轻链可变区序列(氨基端)Table 6: Highly homologous germline human antibody light chain variable region sequence (amino terminal) of 5C4
SpeciesSpecies Gene and alleleGene and Allele DomainDomain Domain labelDomain label ％identity%identity OverlapOverlap
Homo sapiensHomo sapiens IGKV1D-43*01IGKV1D-43*01 11 V-KAPPAV-KAPPA 72.372.3 8383
Homo sapiensHomo sapiens IGKV1-NL1*01IGKV1-NL1*01 11 V-KAPPAV-KAPPA 71.671.6 8888
Homo sapiensHomo sapiens IGKV1-39*01IGKV1-39*01 11 V-KAPPAV-KAPPA 71.671.6 8888
Homo sapiensHomo sapiens IGKV1D-39*01IGKV1D-39*01 11 V-KAPPAV-KAPPA 71.671.6 8888
Homo sapiensHomo sapiens IGKV1D-13*01IGKV1D-13*01 11 V-KAPPAV-KAPPA 70.970.9 8686
表7:5C4的高同源性胚系人抗体轻链可变区序列(羧基端)Table 7: Highly homologous germline human antibody light chain variable region sequence (carboxyl terminus) of 5C4
SpeciesSpecies Gene and alleleGene and Allele DomainDomain Domain labelDomain label ％identity%identity OverlapOverlap
Homo sapiensHomo sapiens IGKJ2*01IGKJ2*01 11  the 81.881.8 1111
Homo sapiensHomo sapiens IGKJ2*02IGKJ2*02 11  the 81.881.8 1111
表8：hGP5C4轻链可变区与胚系人源抗体轻链可变区氨基酸序列比较Table 8: Amino acid sequence comparison of light chain variable region of hGP5C4 and germline human antibody light chain variable region
Figure PCTCN2021099670-appb-000014
Figure PCTCN2021099670-appb-000014
根据表8的比较结果，确定人源化hGP5C4的轻链可变区框架为：According to the comparison results in Table 8, the light chain variable region framework of humanized hGP5C4 was determined to be:
Figure PCTCN2021099670-appb-000015
Figure PCTCN2021099670-appb-000015
其中的“xxxxxx”将是CDR的移植位置。Where "xxxxxx" will be where the CDR is ported.
实施例4：hGP5C4人源化抗体重链Fd和轻链的氨基酸序列的构建Example 4: Construction of amino acid sequences of hGP5C4 humanized antibody heavy chain Fd and light chain
CDR移植是指将非人源单克隆抗体可变区中的CDR序列取代人源抗体相应的CDR序列，构成了由非人源抗体的CDR区和人源抗体的框架区所组成的重组抗体。抗体重链和轻链的可变区由CDR区和框架区(FR)组成。其中可变区的6个CDR组成识别和结合抗原的区域，它们直接与抗原接触，决定了抗体的特异性。骨架区是可变区以外的其它部分，主要起着 支撑CDR的作用，它们的氨基酸组成和排列相对保守。因此，将非人源单克隆抗体的CDR区移植到人单克隆抗体的骨架区就可以达到人源化的目的。这种人源化抗体保持了非人源单克隆抗体和抗原结合的特异性和亲和力，并最大限度地降低了非人源单抗的异源性。CDR transplantation refers to replacing the CDR sequence in the variable region of a non-human monoclonal antibody with the corresponding CDR sequence of a human antibody to form a recombinant antibody consisting of the CDR region of a non-human antibody and the framework region of a human antibody. The variable regions of antibody heavy and light chains consist of CDR regions and framework regions (FR). Among them, the 6 CDRs of the variable region constitute the region that recognizes and binds to the antigen, and they directly contact with the antigen to determine the specificity of the antibody. The framework region is other parts than the variable region, which mainly plays the role of supporting the CDR, and their amino acid composition and arrangement are relatively conservative. Therefore, the purpose of humanization can be achieved by transplanting the CDR region of a non-human monoclonal antibody to the framework region of a human monoclonal antibody. This humanized antibody maintains the specificity and affinity of non-human monoclonal antibody and antigen binding, and minimizes the heterogeneity of non-human monoclonal antibody.
将上述实施例2所确定的hGP5C4的CDR区氨基酸序列移植到作为框架的胚系人源抗体可变区(实施例3)相应的位置，获得人源化抗体可变区的氨基酸序列。The amino acid sequence of the CDR region of hGP5C4 determined in Example 2 above was transplanted to the corresponding position of the variable region of the germline human antibody (Example 3) as a framework to obtain the amino acid sequence of the variable region of the humanized antibody.
其人源化重链可变区序列如下，下划线部分为移植入的CDR区(SEQ ID NO.12)。The sequence of the humanized heavy chain variable region is as follows, and the underlined part is the transplanted CDR region (SEQ ID NO.12).
Figure PCTCN2021099670-appb-000016
Figure PCTCN2021099670-appb-000016
其人源化轻链可变区序列如下，下划线部分为移植入的CDR区(SEQ ID NO.13)。The sequence of the humanized light chain variable region is as follows, and the underlined part is the transplanted CDR region (SEQ ID NO.13).
Figure PCTCN2021099670-appb-000017
Figure PCTCN2021099670-appb-000017
将上述人源化重链可变区序列，连接上保守的人IgG抗体重链恒定区1(CH1)序列，获得人源化的抗GPVI Fd片段的氨基酸序列：(SEQ ID NO.14)The above humanized heavy chain variable region sequence was connected to the conserved human IgG antibody heavy chain constant region 1 (CH1) sequence to obtain the amino acid sequence of the humanized anti-GPVI Fd fragment: (SEQ ID NO.14)
Figure PCTCN2021099670-appb-000018
Figure PCTCN2021099670-appb-000018
将上述人源化轻链可变区序列，连接上保守的人IgG抗体轻链恒定区(CL)序列，获得人源化的抗GPVI轻链的氨基酸序列：(SEQ ID NO.15)The above humanized light chain variable region sequence was connected to the conserved human IgG antibody light chain constant region (CL) sequence to obtain the amino acid sequence of the humanized anti-GPVI light chain: (SEQ ID NO.15)
Figure PCTCN2021099670-appb-000019
Figure PCTCN2021099670-appb-000019
上述人源化的抗GPVI Fd和人源化的抗GPVI轻链在基因工程重组表达时，通过各自的半胱氨酸残基形成二硫键而折叠成一个完全的Fab分子，即本发明中的人源化抗GPVI Fab片段—hGPFab。The above-mentioned humanized anti-GPVI Fd and humanized anti-GPVI light chain are folded into a complete Fab molecule through the formation of disulfide bonds by their respective cysteine residues when expressed through genetic engineering recombination, that is, in the present invention Humanized anti-GPVI Fab fragment—hGPFab.
实施例5：人源化的抗GPVI Fd和人源化的抗GPVI轻链编码核苷酸的构建Example 5: Construction of humanized anti-GPVI Fd and humanized anti-GPVI light chain encoding nucleotides
在基因工程重组表达由此Fd片段和轻链所构成的Fab分子时，需要根据其氨基酸序列反向推导出各自的核苷酸编码序列。由于核苷酸编码氨基酸的简并性，相同的氨基酸序列可以反向推导出多种组合的核苷酸编码序列。通常的做法是根据所选择的宿主细胞对编码氨基酸的偏性而合成相应的核苷酸序列。When the Fab molecule composed of the Fd fragment and the light chain is expressed through genetic engineering recombination, it is necessary to reversely deduce the respective nucleotide coding sequences based on its amino acid sequence. Due to the degeneracy of nucleotide-coded amino acids, the same amino acid sequence can be reversely deduced into multiple combinations of nucleotide-coded sequences. It is common practice to synthesize the corresponding nucleotide sequence according to the bias of the selected host cell towards the encoded amino acid.
本实施例中选择中国仓鼠细胞(CHO)作为宿主细胞，相应地，根据鼠细胞的氨基酸编码偏性合成相应的核苷酸序列。In this example, Chinese hamster cells (CHO) were selected as host cells, and corresponding nucleotide sequences were synthesized according to the amino acid coding bias of mouse cells.
人源化Fd片段的核苷酸编码序列(SEQ ID NO.16)：Nucleotide coding sequence (SEQ ID NO.16) of the humanized Fd fragment:
Figure PCTCN2021099670-appb-000020
Figure PCTCN2021099670-appb-000020
上述人源化Fd片段(图2)的核苷酸编码序列为669bp，编码223个氨基酸残基，包括人源化的抗GPVI重链可变区和CH1区。The nucleotide coding sequence of the humanized Fd fragment (Figure 2) is 669 bp, encoding 223 amino acid residues, including the humanized anti-GPVI heavy chain variable region and CH1 region.
人源化轻链的核苷酸编码序列(SEQ ID NO.17)：Nucleotide coding sequence (SEQ ID NO.17) of humanized light chain:
Figure PCTCN2021099670-appb-000021
Figure PCTCN2021099670-appb-000021
Figure PCTCN2021099670-appb-000022
Figure PCTCN2021099670-appb-000022
上述人源化轻链(图3)的核苷酸编码序列为639bp，编码213个氨基酸残基，包括人源化的抗GPVI轻链可变区和恒定区。The nucleotide coding sequence of the humanized light chain ( FIG. 3 ) is 639 bp, encoding 213 amino acid residues, including the humanized anti-GPVI light chain variable region and constant region.
实施例6：人源化Fd片段和轻链的重组表达载体的构建Embodiment 6: Construction of the recombinant expression vector of humanized Fd fragment and light chain
本实施方案中载体选用pCDdhfr质粒。pCDdhfr质粒上外源蛋白的启动子为鼠CMV早期启动子。筛选基因是二氢叶酸还原酶(dihydrofolatereductase，dhfr)基因。pCDdhfr重组质粒转染入dhfr缺陷型CHO细胞后，质粒序列可以随机整合到细胞的基因组内。转染的dhfr缺陷型CHO细胞在叶酸合成抑制剂甲氨蝶呤(methotrexate，MTX)的压力下，整合的质粒需要不断扩增而强化dhfr的表达，从而使串联的重组蛋白基因随之扩增而提高表达量。In this embodiment, the carrier is pCDdhfr plasmid. The promoter of the foreign protein on the pCDdhfr plasmid is the early promoter of mouse CMV. Screening gene is dihydrofolate reductase (dihydrofolatereductase, dhfr) gene. After the pCDdhfr recombinant plasmid was transfected into dhfr-deficient CHO cells, the plasmid sequence could be randomly integrated into the cell genome. Under the pressure of the folic acid synthesis inhibitor methotrexate (MTX) in the transfected dhfr-deficient CHO cells, the integrated plasmid needs to be continuously amplified to strengthen the expression of dhfr, so that the tandem recombinant protein gene will be amplified accordingly to increase expression.
操作技术为基因工程重组的常规技术，具体流程如下：The operation technology is a conventional technology of genetic engineering recombination, and the specific process is as follows:
(1)采用基因合成的方式获得分别含有编码抗GPVI Fd片段和轻链基因的重组载体pUCGPFd和pUCGPL。在实施例五中Fd片段和轻链核苷酸编码序列的基础上，作如下构建：⑴为利于目的蛋白的分泌表达，在编码Fd片段和轻链核苷酸序列的5’端分别添加了信号肽编码序列。Fd片段的信号肽氨基酸序列为MELGLSWVFLVAILEGVQC，其核苷酸编码序列为ATG GAA CTG GGC CTG TCT TGG GTG TTT CTG GTG GCC ATC CTG GAA GGCGTG CAG TGT；轻链的信号肽氨基酸序列为MDMRVPAQLLGLLLLWLRGARC，其核苷酸编码序列为ATG GAT ATG AGA GTG CCT GCC CAG CTG CTG GGC CTG CTGCTGCTG TGGCTG AGA GGC GCC AGA TGT⑵在上述信号肽起始密码子ATG的5’端添加Kozak序列GCCACC。⑶在两个基因的3’端添加终止密码子TGA。⑷在两个基因Kozak序列的5’端和终止密码子的3’端分别添加了限制性内切酶EcoR I(GAATTC)和NotI(GCGGCCGC)的识别序列，以利于后续构建重组表达质粒。(1) The recombinant vectors pUCGPFd and pUCGPL respectively containing the anti-GPVI Fd fragment and the light chain gene were obtained by gene synthesis. On the basis of the Fd fragment and the light chain nucleotide coding sequence in Example 5, the following constructions are made: (1) In order to facilitate the secretory expression of the target protein, the 5' ends of the coding Fd fragment and the light chain nucleotide sequence are respectively added Signal peptide coding sequence. The amino acid sequence of the signal peptide of the Fd fragment is MELGLSWVFLVAILEGVQC, and its nucleotide coding sequence is ATG GAA CTG GGC CTG TCT TGG GTG TTT CTG GTG GCC ATC CTG GAA GGCGTG CAG TGT; the amino acid sequence of the signal peptide of the light chain is MDMRVPAQLLGLLLLWLRGARC, and its nucleotide The coding sequence is ATG GAT ATG AGA GTG CCT GCC CAG CTG CTG GGC CTG CTGCTGCTG TGGCTG AGA GGC GCC AGA TGT (2) Add the Kozak sequence GCCACC at the 5' end of the above signal peptide start codon ATG. (3) A stop codon TGA was added to the 3' ends of the two genes. (4) Recognition sequences of restriction endonucleases EcoR I (GAATTC) and NotI (GCGGCCGC) were added to the 5' end of the Kozak sequence of the two genes and the 3' end of the stop codon to facilitate subsequent construction of recombinant expression plasmids.
(2)对pUCGPFd、pUCGPL和pCDdhfr 3个质粒作EcoR I和NotI双酶切后，进行琼脂糖凝胶电泳分离，并从琼脂糖凝胶中回收酶切后的Fd片段基因(约740bp)、轻链基因(约720bp)和pCDdhfr(约5.6kb)。(2) after EcoR I and NotI double enzyme digestion were performed on the 3 plasmids pUCGPFd, pUCGPL and pCDdhfr, agarose gel electrophoresis was carried out, and the Fd fragment gene (about 740bp) after the digestion was recovered from the agarose gel, Light chain gene (about 720bp) and pCDdhfr (about 5.6kb).
(3)用T4连接酶分别连接pCDdhfr质粒与Fd基因、pCDdhfr质粒与轻链基因。(3) T4 ligase was used to connect pCDdhfr plasmid and Fd gene, pCDdhfr plasmid and light chain gene respectively.
(4)常规CaCl 2转化方法将连接产物转化至大肠杆菌DH5α。用T7(TAATACGACTCACTATAGGG)和T3(ATTAACCCTCACTAAAGGGA)引物作PCR扩增筛选阳性转化菌株。将阳性菌株分别命名为pCDGPFd和pCDGPL。 (4) Conventional CaCl 2 transformation method to transform the ligated product into Escherichia coli DH5α. T7 (TAATACGACTCACTATAGGG) and T3 (ATTAACCCTCACTAAAGGGA) primers were used for PCR amplification to screen positive transformed strains. The positive strains were named pCDGPFd and pCDGPL, respectively.
(5)分别提取纯化pCDGPFd和pCDGPL质粒，进行DNA测序确认所***目的基因的序列。(5) Extract and purify pCDGPFd and pCDGPL plasmids respectively, and perform DNA sequencing to confirm the sequence of the inserted target gene.
实施例7：hGPFab在CHO细胞的重组表达Example 7: Recombinant expression of hGPFab in CHO cells
hGPFab是由本发明中的人源化Fd片段和人源化轻链在宿主细胞内共表达时所自发形成的异源二聚体，两者间经由二硫键连接。本实施方案选用dhrf缺陷型CHO细胞作为重组表达的宿主，按常规细胞转染和稳定细胞株构建技术操作。hGPFab is a heterodimer spontaneously formed when the humanized Fd fragment and the humanized light chain in the present invention are co-expressed in host cells, and the two are connected by a disulfide bond. In this embodiment, dhrf-deficient CHO cells are selected as hosts for recombinant expression, and operations are performed according to conventional cell transfection and stable cell line construction techniques.
(一)pCDGPFd和pCDGPL质粒的共转染(1) Co-transfection of pCDGPFd and pCDGPL plasmids
1.在转染的前一天，按5x10 5/孔接种dhfr缺陷型CHO细胞至6孔细胞培养板。 1. On the day before transfection, inoculate dhfr-deficient CHO cells into 6-well cell culture plates at 5x10 5 /well.
2.按脂质体Lipofectamine说明书进行转染操作。转染时的细胞密度达到80％融合度(confluent)，每孔的质粒用量为1μg的pCDGPFd和2μg的pCDGPL(比例1:2)。转染后6小时，换成含血清的完全培养基(含10％胎牛血清的IMDM培养基)，在5％CO 2、37℃的条件下培养转染的细胞。 2. Carry out the transfection operation according to the instructions of liposome Lipofectamine. The cell density at the time of transfection reached 80% confluent, and the amount of plasmid used in each well was 1 μg of pCDGPFd and 2 μg of pCDGPL (ratio 1:2). Six hours after transfection, the complete medium containing serum (IMDM medium containing 10% fetal bovine serum) was replaced, and the transfected cells were cultured under the conditions of 5% CO 2 and 37°C.
3.转染后48小时，按实施例九中的酶联免疫吸附试验(ELISA)方法，检测细胞培养上清液中分泌表达的hGPFab。3. 48 hours after transfection, according to the enzyme-linked immunosorbent assay (ELISA) method in Example 9, the hGPFab secreted and expressed in the cell culture supernatant was detected.
结果显示在转染细胞的上清中检测到了能和人GPVI蛋白特异结合的hGPFab片段，提示共转染的两个质粒在宿主细胞内得到了表达，并分泌至细胞外。The results showed that hGPFab fragments that could specifically bind to human GPVI protein were detected in the supernatant of transfected cells, suggesting that the two co-transfected plasmids were expressed in the host cells and secreted outside the cells.
(二)hGPFab稳定表达细胞株的构建(2) Construction of hGPFab stable expression cell line
1、按有限稀释法将转染的CHO细胞稀释、转移到96孔细胞培养板，数量为1个细胞/每孔。在5％CO 2、37℃的条件下继续培养。 1. Dilute the transfected CHO cells according to the limiting dilution method, and transfer them to a 96-well cell culture plate, the number is 1 cell/well. The culture was continued under the conditions of 5% CO 2 and 37°C.
2、10天后，显微镜下可以看到生长的单个细胞克隆。ELISA检测每孔内只有一个细胞克隆的培养上清液。2. After 10 days, the growth of single cell clones can be seen under the microscope. ELISA detects the culture supernatant of only one cell clone in each well.
3、将OD 450读数高的细胞克隆转移到24孔板，加入终浓度为10 -8的MTX，在5％CO 2、37℃的条件下继续培养。 3. Transfer the cell clones with high OD 450 readings to a 24-well plate, add MTX with a final concentration of 10 -8 , and continue culturing under the conditions of 5% CO 2 and 37°C.
4、待细胞长满孔后，按1:5稀释比例转移细胞至新的培养板，相同条件下继续培养。4. After the cells fill the wells, transfer the cells to a new culture plate at a dilution ratio of 1:5, and continue to culture under the same conditions.
5、待细胞再次长满孔后，ELISA检测上清液中的hGPFab。5. After the cells filled the well again, ELISA was used to detect hGPFab in the supernatant.
6、按步骤4-5，将表达量高的细胞株倍增MTX浓度进行压力筛选，直至表达量不再明显增加。亚克隆高表达细胞株，鉴定后冻存及继续后续试验。6. According to steps 4-5, double the MTX concentration of the cell line with high expression level for pressure screening until the expression level no longer increases significantly. Subclone the high-expressing cell line, freeze it after identification and continue the follow-up experiment.
实施例8：CHO细胞重组表达的hGPFab的纯化Example 8: Purification of hGPFab recombinantly expressed in CHO cells
蛋白L(Protein L)是由大消化链球菌(Peptostreptococcusmagnus)产生的一种结构蛋白，由719个氨基酸构成。蛋白L可以特异结合多种动物抗体的kappa轻链，因此固相化的蛋白L常被用作纯化抗体Fab片段的介质。Protein L (Protein L) is a structural protein produced by Peptostreptococcus magnus, consisting of 719 amino acids. Protein L can specifically bind kappa light chains of various animal antibodies, so immobilized protein L is often used as a medium for purifying Fab fragments of antibodies.
所构建的hGPFab稳定表达细胞株，其培养上清液在AKTA***上经HiTrap蛋白L纯化柱(HiTrap Protein L column)亲和层析纯化，按说明书操作步骤进行，基本步骤如下：The constructed hGPFab stable expression cell line, its culture supernatant was purified by affinity chromatography on the HiTrap Protein L column (HiTrap Protein L column) on the AKTA system, and the operation steps were carried out according to the instructions. The basic steps are as follows:
1、收集2升hGPFab表达细胞的培养上清，10000rpm离心30分钟以去除细胞碎片。再用0.22μm滤膜真空抽滤，备用。1. Collect 2 liters of culture supernatant of cells expressing hGPFab, and centrifuge at 10,000 rpm for 30 minutes to remove cell debris. Then use a 0.22 μm filter membrane to vacuum filter and set aside.
2、用结合缓冲液(20mM sodium phosphate,150mM NaCl,pH 7.2)平衡HiTrap蛋白L纯化柱，直至紫外扫描和导电率平稳。2. Equilibrate the HiTrap protein L purification column with binding buffer (20mM sodium phosphate, 150mM NaCl, pH 7.2) until the UV scan and conductivity are stable.
3、将步骤1的样品上样。3. Load the sample from step 1.
4、用结合缓冲液洗涤HiTrap蛋白L纯化柱，直至紫外扫描和导电率平稳且回到基线。4. Wash the HiTrap protein L purification column with binding buffer until the UV scan and conductivity stabilize and return to baseline.
5、用洗脱缓冲液洗脱目的蛋白(100mM glycine-HCl,pH 2.5)。5. Elute the target protein with elution buffer (100mM glycine-HCl, pH 2.5).
所纯化的蛋白经SDS-PAGE分析，非还原状态下，分子量约为45kD，经还原剂DTT处理后，变成分子量约为25kD的单体，提示所分泌表达的hGPFab蛋白结构为二聚体，且两个单体的分子量接近(约25kD)(图4)，符合预期结果。The purified protein was analyzed by SDS-PAGE. In the non-reduced state, the molecular weight was about 45kD. After being treated with the reducing agent DTT, it became a monomer with a molecular weight of about 25kD, suggesting that the secreted and expressed hGPFab protein structure was a dimer. And the molecular weights of the two monomers are close (about 25kD) (Figure 4), which is in line with the expected results.
实施例9：hGPFab和人GPVI蛋白的特异结合试验。Example 9: Specific binding test between hGPFab and human GPVI protein.
为分析本发明中hGPFab与GPVI的结合特性，建立了酶联免疫吸附试验(ELISA)方法检测所表达的hGPFab蛋白，操作方法为常规的相关技术，基本步骤如下：In order to analyze the binding characteristics of hGPFab and GPVI in the present invention, an enzyme-linked immunosorbent assay (ELISA) method has been established to detect the expressed hGPFab protein. The method of operation is a conventional related technology, and the basic steps are as follows:
1、将纯化的GPVI蛋白包被在96孔的微孔板上，GPVI蛋白的包被浓度为2μg/ml，包被液为pH 9.6的碳酸盐缓冲液，包被量为100μl/孔，4℃过夜后，用含5％脱脂奶粉的PBS缓冲液室温下封闭2小时，再用含吐温-20(Tween-20)的洗液洗涤微孔3次备用。1. Coat the purified GPVI protein on a 96-well microwell plate, the coating concentration of GPVI protein is 2 μg/ml, the coating solution is carbonate buffer solution with pH 9.6, and the coating volume is 100 μl/well, After staying overnight at 4°C, block with PBS buffer containing 5% skimmed milk powder for 2 hours at room temperature, and then wash the microwells 3 times with washing solution containing Tween-20 for future use.
2、加入100μl的待检样品(如不同稀释度的hGPFab蛋白或细胞培养上清、对照等)，室温下反应2小时，再以洗液洗涤微孔5次。2. Add 100 μl of the sample to be tested (such as different dilutions of hGPFab protein or cell culture supernatant, control, etc.), react at room temperature for 2 hours, and then wash the microwell 5 times with washing solution.
3、加入100μl的辣根过氧化物酶(HRP)标记的抗人Fab抗体(1:2000稀释)，室温下反应2小时，再以洗液洗涤微孔5次。3. Add 100 μl of horseradish peroxidase (HRP)-labeled anti-human Fab antibody (1:2000 dilution), react at room temperature for 2 hours, and then wash the microwells 5 times with washing solution.
4、加入100μl的TMB底物，室温下反应15分钟，加入100μl的2M硫酸终止酶促反应。4. Add 100 μl of TMB substrate, react at room temperature for 15 minutes, add 100 μl of 2M sulfuric acid to terminate the enzymatic reaction.
5、在酶联仪上测定OD 450读数。 5. Determine the OD 450 reading on the enzyme-linked analyzer.
结果显示(图5)，不管是细胞培养上清，还是纯化的hGPFab片段，都能与微孔板上的GPVI特异结合。随液相中hGPFab浓度的增高，结合反应的酶促信号也随之增强，表明hGPFab与GPVI的结合有浓度-效应反应关系。作为对照的正常人IgG抗体Fab片段和包被的GPVI无明显反应。The results showed (FIG. 5) that both the cell culture supernatant and the purified hGPFab fragment could specifically bind to GPVI on the microwell plate. As the concentration of hGPFab in the liquid phase increases, the enzymatic signal of the binding reaction also increases, indicating that the binding of hGPFab to GPVI has a concentration-effect response relationship. As a control, the Fab fragment of normal human IgG antibody had no obvious reaction with the coated GPVI.
实施例10：hGPFab和人GPVI蛋白的免疫印迹实验(Western Blot，WB)Embodiment 10: Western blot experiment (Western Blot, WB) of hGPFab and human GPVI protein
为进一步证实hGPFab与人GPVI蛋白的特异结合性质，设计了此WB实验。In order to further confirm the specific binding properties of hGPFab and human GPVI protein, this WB experiment was designed.
在待分离的蛋白样品中加入还原剂(如二硫苏糖醇DTT、β-巯基乙醇等)时，还原剂会打开氨基酸链间的二硫键，使蛋白呈线性结构(还原性蛋白)；不加还原剂的样品中，氨基酸链间的二硫键会存在而使蛋白呈现立体构型(非还原性蛋白)。所以，可以通过还原剂的作用来分析不同构型之间蛋白的相互作用。When a reducing agent (such as dithiothreitol DTT, β-mercaptoethanol, etc.) is added to the protein sample to be separated, the reducing agent will open the disulfide bond between the amino acid chains, making the protein a linear structure (reducing protein); In the sample without reducing agent, the disulfide bonds between the amino acid chains will exist to make the protein assume a three-dimensional configuration (non-reducing protein). Therefore, the interaction of proteins between different configurations can be analyzed through the action of reducing agents.
本实施例中WB实验的操作为常规技术，主要步骤如下：The operation of the WB experiment in this embodiment is a conventional technique, and the main steps are as follows:
1、取2份各2μg的人GPVI蛋白样品，加入等量的2倍样品缓冲液，其中一份加入终浓度为50mM的DTT，样品煮沸5分钟后，以15％的聚丙烯酰胺凝胶电泳分离样品的蛋白。1. Take 2 samples of human GPVI protein of 2 μg each, add an equal amount of 2-fold sample buffer, and add DTT with a final concentration of 50 mM to one part, boil the sample for 5 minutes, and run it on a 15% polyacrylamide gel electrophoresis Separate the protein from the sample.
2、电泳结束后，在甘氨酸缓冲液中将凝胶中的蛋白电泳转移到0.45μm的硝酸纤维素膜上，稳电流350mA转移60分钟，然后将硝酸纤维素膜在含5％脱脂奶粉的TBS缓冲液中封闭1小时。2. After electrophoresis, transfer the protein in the gel electrophoresis to a 0.45 μm nitrocellulose membrane in a glycine buffer, transfer it at a steady current of 350 mA for 60 minutes, and then place the nitrocellulose membrane in TBS containing 5% skimmed milk powder Buffer for 1 hour.
3、加入纯化的hGPFab至终浓度为0.5μg/ml，室温下轻摇2小时。TBST洗膜3次。3. Add purified hGPFab to a final concentration of 0.5 μg/ml, and shake gently at room temperature for 2 hours. Wash the membrane 3 times with TBST.
4、加入1:500稀释的HRP标记抗人Fab抗体，室温下轻摇2小时。TBST洗膜3次。4. Add 1:500 diluted HRP-labeled anti-human Fab antibody and shake gently at room temperature for 2 hours. Wash the membrane 3 times with TBST.
5、加入DAB显色液，室温显色10分钟。5. Add DAB color developing solution, develop color at room temperature for 10 minutes.
结果显示(图6)，hGPFab和非还原性GPVI有强烈的反应，而和还原性GPVI反应微弱。提示hGPFab片段在GPVI蛋白上的结合位点是构象依赖的，即其抗原表位(epitope)属于非线性结构。The results showed ( FIG. 6 ), that hGPFab strongly reacted with non-reduced GPVI, but weakly reacted with reduced GPVI. It is suggested that the binding site of the hGPFab fragment on the GPVI protein is conformation-dependent, that is, its epitope (epitope) belongs to a non-linear structure.
实施例11：hGPFab和人GPVI蛋白与胶原蛋白的竞争抑制试验Example 11: Competitive inhibition test of hGPFab and human GPVI protein with collagen
大鼠源的亲本抗体hGP5C4具有抑制GPVI和胶原蛋白结合的性质。为检测本发明中的hGPFab是否仍然具有此抑制功能，设计了GPVI和hGPFab结合人胶原蛋白的竞争抑制ELISA试验。The parental antibody hGP5C4 of rat origin has the property of inhibiting the binding of GPVI and collagen. In order to detect whether the hGPFab in the present invention still has this inhibitory function, a competitive inhibition ELISA assay of GPVI and hGPFab binding to human collagen was designed.
基本操作流程如下：The basic operation process is as follows:
1、将人胶原蛋白用pH 9.6的碳酸盐包被缓冲液稀释至2μg/ml，加入100μl/孔到96孔的微孔板内，4℃包被过夜。用含5％脱脂奶粉的PBS缓冲液室温下封闭2小时，再用含吐温-20(Tween-20)的洗液洗涤微孔3次备用。1. Dilute human collagen to 2 μg/ml with pH 9.6 carbonate coating buffer, add 100 μl/well to a 96-well microplate, and coat overnight at 4°C. Block with PBS buffer containing 5% skimmed milk powder at room temperature for 2 hours, and then wash the microwells with washing solution containing Tween-20 (Tween-20) 3 times for use.
2、在50ng/ml的GPVI样品中，加入终浓度为0.5、1.0、2.0、4.0和8.0μg/ml的hGPFab，室温孵育30分钟，此为待测样品，同时用正常人抗体Fab作为对照。2. Add hGPFab at a final concentration of 0.5, 1.0, 2.0, 4.0, and 8.0 μg/ml to the GPVI sample of 50 ng/ml, and incubate at room temperature for 30 minutes. This is the sample to be tested, and a normal human antibody Fab is used as a control.
3、加入100μl的待检样品及对照，室温下反应2小时，再以洗液洗涤微孔5次。3. Add 100 μl of the sample to be tested and the control, react at room temperature for 2 hours, and then wash the microwells 5 times with washing solution.
4、加入100μl的辣根过氧化物酶(HRP)标记的抗人GPVI抗体(1:1000稀释)，室温下反应2小时，再以洗液洗涤微孔5次。4. Add 100 μl of horseradish peroxidase (HRP)-labeled anti-human GPVI antibody (1:1000 dilution), react at room temperature for 2 hours, and then wash the microwells 5 times with washing solution.
5、加入100μl的TMB底物，室温下反应15分钟，加入100μl的2M硫酸终止酶促反应。5. Add 100 μl of TMB substrate, react at room temperature for 15 minutes, add 100 μl of 2M sulfuric acid to terminate the enzymatic reaction.
6、在酶联仪上测定OD 450读数。 6. Determine the OD 450 reading on the enzyme-linked analyzer.
结果显示(图7)，hGPFab能够抑制人GPVI蛋白与胶原蛋白的结合，随着hGPFab加入浓度的增加，其对GPVI的抑制作用随之增强，提示hGPFab的这种抑制作用存在着剂量-效应关系。而作为对照的人IgG抗体Fab片段对GPVI和胶原蛋白的结合无明显作用。The results show (Figure 7), hGPFab can inhibit the binding of human GPVI protein to collagen, and as the concentration of hGPFab increases, its inhibitory effect on GPVI increases, suggesting that the inhibitory effect of hGPFab has a dose-effect relationship . As a control, the Fab fragment of human IgG antibody had no obvious effect on the combination of GPVI and collagen.

Claims (14)

  1. 一种人源化抗人GPVI单克隆抗体Fab片段，其特征在于，所述人源化抗人GPVI单克隆抗体Fab片段的氨基酸序列为SEQ IDNO.14和SEQ ID NO.15所示的氨基酸序列。A humanized anti-human GPVI monoclonal antibody Fab fragment, characterized in that the amino acid sequence of the humanized anti-human GPVI monoclonal antibody Fab fragment is the amino acid sequence shown in SEQ ID NO.14 and SEQ ID NO.15 .
  2. 根据权利要求1所述的人源化抗人GPVI单克隆抗体Fab片段，其特征在于，是由SEQ IDNO.14和SEQ IDNO.15两条氨基酸链所组成的异源二聚体。The humanized anti-human GPVI monoclonal antibody Fab fragment according to claim 1 is characterized in that it is a heterodimer composed of two amino acid chains of SEQ ID NO.14 and SEQ ID NO.15.
  3. 根据权利要求1所述的人源化抗人GPVI单克隆抗体Fab片段，其特征在于，Fab片段的氨基酸序列，其重链的CDR氨基酸序列为SEQ ID NO.6、SEQ ID NO.7和SEQ ID NO.8所示的氨基酸序列。The humanized anti-human GPVI monoclonal antibody Fab fragment according to claim 1 is characterized in that, the amino acid sequence of the Fab fragment, the CDR amino acid sequence of its heavy chain is SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.7 and SEQ ID NO.7 The amino acid sequence shown in ID NO.8.
  4. 根据权利要求1所述的人源化抗人GPVI单克隆抗体Fab片段，其特征在于，Fab片段的氨基酸序列，其轻链的CDR氨基酸序列为SEQ ID NO.9、SEQ ID NO.10和SEQ ID NO.11所示的氨基酸序列。The humanized anti-human GPVI monoclonal antibody Fab fragment according to claim 1 is characterized in that, the amino acid sequence of the Fab fragment, the CDR amino acid sequence of its light chain is SEQ ID NO.9, SEQ ID NO.10 and SEQ ID NO.10 and SEQ ID NO.10 The amino acid sequence shown in ID NO.11.
  5. 根据权利要求1所述的人源化抗人GPVI单克隆抗体Fab片段，其特征在于，Fab片段的氨基酸序列，其重链Fd片段的氨基酸序列和SEQ ID NO.14的同源性大于等于95％。The humanized anti-human GPVI monoclonal antibody Fab fragment according to claim 1, characterized in that, the amino acid sequence of the Fab fragment, the amino acid sequence of its heavy chain Fd fragment and the homology of SEQ ID NO.14 are greater than or equal to 95 %.
  6. 根据权利要求1所述的人源化抗人GPVI单克隆抗体Fab片段，其特征在于，Fab片段的氨基酸序列，其轻链的氨基酸序列和SEQ ID NO.15的同源性大于等于95％。The humanized anti-human GPVI monoclonal antibody Fab fragment according to claim 1, characterized in that the amino acid sequence of the Fab fragment, the amino acid sequence of its light chain and the homology of SEQ ID NO.15 are greater than or equal to 95%.
  7. 根据权利要求1所述的人源化抗人GPVI单克隆抗体Fab片段，其特征在于所述人源化抗人GPVI单克隆抗体Fab片段特异结合非还原性人GPVI蛋白。The humanized anti-human GPVI monoclonal antibody Fab fragment according to claim 1, characterized in that the humanized anti-human GPVI monoclonal antibody Fab fragment specifically binds to non-reducing human GPVI protein.
  8. 一种编码权利要求1所述的人源化抗人GPVI单克隆抗体Fab片段重链Fd片段的核苷酸序列。A nucleotide sequence encoding the heavy chain Fd fragment of the humanized anti-human GPVI monoclonal antibody Fab fragment according to claim 1.
  9. 一种编码权利要求1所述的人源化抗人GPVI单克隆抗体Fab片段轻链的核苷酸序列。A nucleotide sequence encoding the light chain of the Fab fragment of the humanized anti-human GPVI monoclonal antibody according to claim 1.
  10. 一种重组表达质粒载体，其特征在于，包含如权利要求8所述的核苷酸编码序列。A recombinant expression plasmid vector, characterized in that it comprises the nucleotide coding sequence as claimed in claim 8.
  11. 一种重组表达质粒载体，其特征在于，包含如权利要求9所述的核苷酸编码序列。A recombinant expression plasmid vector, characterized in that it comprises the nucleotide coding sequence as claimed in claim 9.
  12. 一种宿主细胞，其特征在于，包含如权利要求10所述的重组表达质粒载体；所述宿主细胞是CHO衍生细胞株。A host cell, characterized in that it comprises the recombinant expression plasmid vector according to claim 10; the host cell is a CHO-derived cell strain.
  13. 一种宿主细胞，其特征在于，包含如权利要求11所述的重组表达质粒载体；所述宿主细胞是CHO衍生细胞株。A host cell, characterized in that it comprises the recombinant expression plasmid vector according to claim 11; the host cell is a CHO-derived cell strain.
  14. 权利要求1所述的人源化抗人GPVI单克隆抗体Fab片段在用于制备人血小板GPVI相关疾病药物中的应用。The application of the humanized anti-human GPVI monoclonal antibody Fab fragment according to claim 1 in the preparation of medicines for human platelet GPVI-related diseases.
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WO2005054294A2 (en) * 2003-12-03 2005-06-16 Trigen Gmbh Inhibitors of glycoprotein vi based on monoclonal antibody hgp 5c4
CN1964990A (en) * 2004-04-29 2007-05-16 大冢制药株式会社 Antibodies specific for glycoprotein VI and methods of producing these antibodies
CN102725309A (en) * 2009-12-18 2012-10-10 赛诺菲 Novel antagonist antibodies and their Fab fragments against GPVI and uses thereof
CN102875679A (en) * 2005-04-28 2013-01-16 持田制药株式会社 Anti-platelet membrane glycoprotein vi monoclonal antibody
CN108289939A (en) * 2015-08-05 2018-07-17 艾科迪科尔生物技术公司 Novel anti-human GPVI antibody and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005054294A2 (en) * 2003-12-03 2005-06-16 Trigen Gmbh Inhibitors of glycoprotein vi based on monoclonal antibody hgp 5c4
CN1964990A (en) * 2004-04-29 2007-05-16 大冢制药株式会社 Antibodies specific for glycoprotein VI and methods of producing these antibodies
CN102875679A (en) * 2005-04-28 2013-01-16 持田制药株式会社 Anti-platelet membrane glycoprotein vi monoclonal antibody
CN102725309A (en) * 2009-12-18 2012-10-10 赛诺菲 Novel antagonist antibodies and their Fab fragments against GPVI and uses thereof
CN108289939A (en) * 2015-08-05 2018-07-17 艾科迪科尔生物技术公司 Novel anti-human GPVI antibody and application thereof

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