CN110627904B

CN110627904B - Anti-human GPC3 monoclonal antibody

Info

Publication number: CN110627904B
Application number: CN201911053957.1A
Authority: CN
Inventors: 张军锋; 郭志刚
Original assignee: Nanjing Landun Biotechnology Co ltd
Current assignee: Nanjing Landun Biotechnology Co ltd
Priority date: 2019-10-31
Filing date: 2019-10-31
Publication date: 2020-07-10
Anticipated expiration: 2039-10-31
Also published as: CN110627904A

Abstract

The invention discloses an anti-human GPC3 monoclonal antibody, which is a specific and high-affinity nano antibody developed by taking human-derived GPC3 as a target point and based on a phage display technology. Compared with the prior art, the invention successfully prepares the anti-human GPC3 monoclonal antibody which has good specificity and high affinity and can be combined with human GPC3 expressed on the surface of cells, and the anti-human GPC3 monoclonal antibody is a potential drug for tumor immunotherapy.

Description

Anti-human GPC3 monoclonal antibody

Technical Field

The invention relates to an anti-human GPC3 monoclonal antibody, belonging to the technical field of monoclonal antibodies.

Background

Glypican 3(glypican 3, GPC3) is a heparan sulfate proteoglycan on the surface of a cell membrane, and is a sugar-lipid-protein complex formed by covalently linking three parts, a core protein (GPC), Heparan Sulfate (HS) and Glycosylated Phosphatidylinositol (GPI). HS is linked to the serine residue of GPC by oligosaccharide chain, and GPC protein is anchored to the cell membrane surface via Asp554 site through GPI, wherein the HS chain is close to the cell membrane because the HS linking site is the C-terminal two serines of GPC.

The core protein consists of 580 a.a., has a molecular weight of about 70KD, has a furin protease cleavage point in the middle, can be cleaved by furin from between Arg358 and Cys359 to form a 40KD N-terminal subunit and a 30KD C-terminal subunit, has a secretion signal at the N-terminal, and is anchored on a cell membrane at the C-terminal. There are three functional domains in the GPC3 molecule, which are the N-terminus, C-terminus, and two HS sugar chains of the protein.

GPC3 can bind heparin-binding proteins such as growth factors, extracellular matrix components, cell adhesion molecules and molecules related to degradation pathways, regulate cell growth, differentiation and other behaviors, and play an important role in the growth and development of organisms. The function of GPC3 may be related to its binding to growth factors, HS in the molecule being negatively charged and capable of binding positively charged growth factors, including hepatocyte growth factor, fibroblast growth factor. GPC3 can also interact with the Wnts, Hedgehog, and bone morphogenic protein signaling pathways to affect the biological function of these signaling pathways. During embryonic development, GPC3 strongly influences differentiation of tissues and organs with IGF2 signaling pathways and interactions, such as: GPC3 is highly expressed in embryonic tissues and is not expressed in normal adults; in embryonal tumors and mouse embryonic tissues, GPC3 was found to be co-expressed with IGF2, affecting the development of mesodermal tissues and organs through interaction with the IGF2 signaling pathway; in certain cells, GPC3 can cause apoptosis, which can be inhibited by IGF.

GPC3 was discovered when studying the overgrowth syndrome, and its loss of function was responsible for the overgrowth and aberration syndromes. In the process of the occurrence of the giant tongue-navel bulging syndrome, GPC3 function is lost, so that IGF2 signal pathway function is over-expressed, and competition and balance exist between the two. Even in normally developing tissues, GPC3 is thought to function to inhibit tissue growth (differentiation) by competing for Hedgehog receptors. GPC3 is highly expressed in mesoderm-derived tissues, but is less expressed or not expressed in mature tissues. After overexpression of GPC3, hepatocyte proliferation and liver regeneration were inhibited. These phenomena suggest that GPC3 functions primarily to regulate cell growth and differentiation, appears to act as a growth-inhibiting balancer, interacts with multiple signaling pathways, is functionally complex, and its expression is finely regulated.

The expression of GPC3 in placenta and fetal tissues is abundant, such as liver, lung and kidney, and is significantly reduced in normal adult organs, in adults, the expression of GPC3 is prone to the occurrence of malignant transformation tissues, such as squamous cell carcinoma and hepatocellular carcinoma, and is particularly closely related to the occurrence and development of the liver cancer, the detection of Notrhern blocking finds that GPC3 is low or not expressed in normal liver tissues, focal nodular hyperplasia of liver and cirrhosis, the detection rate of GPC3 in hepatocellular carcinoma samples is increased by 21.7, 7.2 and 10.8 times compared with the former three samples, the detection rate of GPC 6345 in non-tumor liver samples, pre-nodular liver samples and hepatocellular carcinoma samples is 9.2%, 16% and 63.6%, the detection rates of GPC3 in hepatocellular carcinoma samples are 9.2%, 16% and 63.6%, the diagnosis indexes of GPC3 are sensitive and specific, the administration of the monoclonal antibody of patients with high expression of GPC3 is compared with those with low expression of non-tumor liver samples, the monoclonal antibody 3 can greatly delay the progression of high expression group tumors, the mouse is anchored in liver cancer, the development of Wnt tumor is inhibited by GPC-expressing liver cancer receptor, and the GPT 27 is also involved in the function of GPT 3, GPI receptor of GPT 3, GPI can be used as a liver cancer receptor for inhibiting effect of GPT receptor for inhibiting tumor, GPI-34 can be used as a liver cancer receptor inhibiting effect after the liver cancer transplantation of GPT receptor for inhibiting tumor, GPI receptor inhibiting tumor, GPI-28 can be used for inhibiting the liver transplantation of GPT, GPI-11 inhibiting tumor, GPI inhibiting function of mouse, GPI-HBT 3 can be used for inhibiting the liver cancer receptor inhibiting function of GPT receptor inhibiting tumor, GPI inhibiting the liver transplantation of GPT receptor inhibiting tumor, GPI inhibiting the liver.

GPC3 has important biological functions of regulating cell growth and differentiation, and has many kinds of interacting molecules and various signal transduction pathways involved in or affected by the interaction. In this sense, GPC3 is taken as a target point, antibody medicines with various action mechanisms can be developed, and besides the traditional blocking/competition of single antibody medicine, ADCC/CDC/ADCP mechanism, antibody coupling medicine, vaccine and adoptive immune cell therapy, other target points with synergistic action with the single antibody medicine can be easily found, so that combined medicine or multi-specific antibody can be developed, the treatment effect can be improved, or the medicine can be conveniently selected.

With respect to the antibodies currently under development, although the known GPC3 monoclonal antibody is available, none can exhibit Complement Dependent Cytotoxicity (CDC) against tumor cells. It follows that it is a difficult matter to obtain antibodies targeting GPC3 with strong CDC effects using conventional hybridoma technology. For example, the antibody obtained by the traditional method has poor tissue penetration, insufficient treatment effect, strong immunogenicity, requirement of humanized modification, poor stability, high transportation and storage requirements and high development and use costs. In conclusion, although the existing traditional antibodies play an extremely important role in the treatment/detection of diseases, the defects thereof are also obvious.

Aiming at GPC3 target, 16 clinical medicines exist at present, wherein 10 medicines belong to Chinese domestic research and development institutions, the treated diseases mainly comprise liver cancer treatment, including one lung squamous cell carcinoma, and the using method mainly comprises CART cell immunotherapy, including one antibody medicine. Some of the 16 antibodies were human-mouse chimeric antibodies, and some were human antibodies, all derived from mice, and obtained by hybridoma technology. For the antibody targeting GPC3, no published report of development of nano-antibody exists at present, and no published report of antibody screening scheme targeting GPC3 based on phage display technology exists at present.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the problems in the prior art, the invention discloses an anti-human GPC3 monoclonal antibody.

The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:

an anti-human GPC3 monoclonal antibody, wherein the antibody is of single-chain structure and comprises an antigen binding part with an amino acid sequence shown as SEQ ID NO. 1.

As one embodiment of the invention, the anti-human GPC3 monoclonal antibody comprises a signal peptide and an antigen binding portion, and the amino acid sequence of the monoclonal antibody is shown in SEQ ID NO. 2.

Alternatively, the anti-human GPC3 monoclonal antibody comprises a signal peptide, an antigen binding portion and a human Fc region, and the amino acid sequence of the monoclonal antibody is shown in SEQ ID NO. 3.

Or the anti-human GPC3 monoclonal antibody comprises a signal peptide, an antigen binding part, a flexible connecting peptide and a human Fc region, and the amino acid sequence of the monoclonal antibody is shown as SEQ ID NO. 4.

The preparation method of the anti-human GPC3 monoclonal antibody adopts a method of phage display peptide library screening. The specific method comprises the following steps:

the method comprises the steps of coating GPC3 protein on an immune tube, incubating the immune tube with a nano antibody library, washing off phage which are not combined/weakly combined, eluting phage which are combined with GPC3 protein, repeating the steps for several times, changing the GPC3 protein coating amount and washing conditions each time, eliminating phage which are weakly combined and keeping more phage which are strongly combined as much as possible, coating a solid culture medium plate after infecting host bacteria, performing monoclonal cloning on the obtained phage which are strongly combined, performing E L ISA screening by using monoclonal culture supernatant, determining nucleic acid sequence of the monoclonal, establishing coding sequence (according to an embodiment of the invention, connecting signal peptide of secreted luciferase, nano antibody, namely antigen combining part, flexible connecting peptide, IgG coding sequence of human source 2 and His label), reading in frame, constructing mammal expression vector CHO-K1 cell, taking supernatant, performing transfection, performing positive cell transfection, detecting by flow cytometry, performing affinity chromatography, obtaining the binding protein of a through a GPC 38764 column, and performing fusion on the protein, and obtaining the characteristics of the GPC 38764.

A nucleic acid molecule containing a sequence encoding said anti-human GPC3 monoclonal antibody.

A vector comprising said nucleic acid molecule.

A host cell comprising said nucleic acid molecule or said vector.

A kit comprising said anti-human GPC3 monoclonal antibody.

Use of the anti-human GPC3 monoclonal antibody in the preparation of a kit for detecting the presence or level of GPC3 in a sample.

The application of the anti-human GPC3 monoclonal antibody in preparing anti-cancer or cancer detection or medicaments.

The invention takes human GPC3 as a target spot to develop a specific and high-affinity nano antibody. The nanometer antibody is an antibody which is found in camels and adult cartilaginous fishes (such as sharks) and only contains a heavy chain variable region, and a single heavy chain variable region of the antibody has complete antigen binding capacity. Compared with the traditional antibody, the antibody has the characteristics of small volume, strong tissue penetrating power, high water solubility, high tolerance and stability, low immunogenicity, simple structure, easiness in modification (multivalence and multispecific) and the like, so that the antibody has special significance for treating the target treatment of solid tumors which are hindered by blood brain barriers and have poor CART effect and are difficult for effector molecules to enter and the like. Its smaller volume and larger CDR3 region are prone to bind to specific epitopes and thus produce specific biological effects. Meanwhile, the antibody screening technology adopted in the invention is phage surface display, and the constructed universal nano antibody library is used as a starting material, so that the screening process is greatly shortened, and the time is saved by 90% compared with the traditional hybridoma method. Based on the antibody of the present invention, a cancer therapeutic drug such as a monoclonal antibody drug, an antibody conjugate drug, and a CART immunotherapy, or a detection agent for cancer diagnosis can be developed. Compared with the antibody medicine targeted to GPC3, the nano-antibody of the invention has completely different sequences and structures, and is an antibody with a brand new structure different from the existing antibody medicine.

Has the advantages that: compared with the prior art, the invention successfully prepares the anti-human GPC3 monoclonal antibody which has good specificity and high affinity and can be combined with human GPC3 expressed on the surface of cells, and the anti-human GPC3 monoclonal antibody is a potential drug for tumor immunotherapy.

Description of the drawings:

FIG. 1 shows the results of the combination of the crude culture with human GPC3 and E L ISA assay, and the repeated validation of phage panning was performed using 50ul and 5ul aliquots of 2 × YT medium containing the virus particles of the phase.

FIG. 2: the human GPC3 antibody overexpression CHO-K1 cell line transfection used in the vector structure diagram, MCS is multiple cloning sites, the target gene inserted into the position.

FIG. 3: CHO-K1 overexpression human GPC3 antibody fusion protein SDS-PAGE detection. In the figure, the left lane, the middle lane and the right lane are respectively the 25ul loading amount of untransfected CHO-K1 cells, the 5ul loading amount of transfected CHO-K1 cells and the 25ul loading amount of transfected CHO-K1 cells, and the frames are expressed antibodies.

FIG. 4: human GPC3 binds to monoclonal antibodies for flow detection. In the figure, the left column and the right column represent binding signals with a Huh7 cell line (very weak expression GPC3 ovarian cancer cell line) and a HepG2 cell line (high expression GPC3 ovarian cancer cell line), respectively.

FIG. 5 shows the detection of purified anti-human GPC3 monoclonal antibody fusion protein titer E L ISA, the purified antibody with concentration gradient configuration is combined with human GPC3, and reacted with HRP-labeled anti-human Fc antibody.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples.

Examples

(1) Phage display antibody library panning

1) Activation of host bacteria TG1 preparation of mini argar Medium plate [1 × M9 salt, 2% glucose, 2mM MgSO4, 0.1mM CaCl₂1mM vitamin B1]The TG1 was streaked overnight in a 37 ℃ incubator.

2) Coating: human GPC3 protein (purchased from Acro Biosystem) was diluted at a concentration of 50ug/ml in 0.1M NaHCO₃(pH8.6), 1.5ml of the solution was added to an immunization tube and the tube was coated overnight at 4 ℃. Removing coating liquid, and adding blocking solution [0.01M PBS (pH7.4), 2% skimmed milk powder]After 1 hour of incubation, the cells were washed 4 times with 0.01M PBS (pH7.4).

3) Combining: get 10¹¹pfu phage virions were added to 1.5ml MPBS [0.01M PBS (pH7.4), 2% skim milk powder in an immune tube]And incubated at room temperature for 1 hour. The liquid was washed clean with PBST [0.01M PBS (pH7.4), 0.1% Tween-20]Washed 8 times and then 8 times with 0.01M PBS (pH7.4).

4) And (3) elution: the tube was repeatedly washed with 1ml of 100mM triethylamine for 10 minutes, mixed with 0.5ml of a neutralizing solution [1M Tris-Cl (pH7.6) ] and temporarily stored at 4 ℃.

5) The titer is determined by mixing 2ul, 0.2ul (2 ul after diluting the stock solution 10 times with 2 × YT medium) and 0.02ul (2 ul after diluting the stock solution 100 times with 2 × YT medium) eluates with 0.2ml of TG1 in the middle logarithmic phase (OD600 ═ 0.5), incubating for 30min at room temperature, spreading on 2 × YT-GA100 (containing 2% glucose and 100ug/ml ampicillin) plates, culturing overnight at 37 ℃, counting the number of clones on about 50 cloned plates, and calculating the titer according to the dilution factor.

6) Phage amplification, inoculating overnight cultured TG1 bacterial liquid into 20ml 2 × YT culture solution, shaking culture at 250rpm at 37 deg.C to middle logarithmic phase (OD600 is 0.5), adding eluent, incubating at room temperature or 37 deg.C for 30min, adding helper phage, incubating at room temperature or 37 deg.C for 30min, adding ampicillin to 100ug/ml, kanamycin to 50 ug/ml.37 deg.C, shaking culture at 220rpm overnight, centrifuging at 6000rpm for 10min, removing thallus, adding 2.5M NaCl/20% PEG8000 in 1/5 volume into supernatant, centrifuging at ice bath for 2 hr, 10000rpm for 10min to obtain phage precipitate, removing residual liquid, adding 0.2ml 0.01M PBS (pH7.4), and resuspending the precipitate as above.

7) Repeating the steps 2) -6) twice, adjusting the coating concentration of the GPC3 protein to 30ug/ml for the first time, adjusting the coating concentration to 15ug/ml for the second time, increasing the washing times and keeping the rest unchanged.

(2) Monoclonal E L ISA

1)0.5ug/ml human GPC3 protein 4 ℃ overnight coated ELISA plate, blocking solution treatment for 2h, and PBS washing 4 times.

2) A monoclonal colony picked from a 2 × YT-GA100 plate is subjected to shaking culture to the middle logarithmic phase, added with helper phage, incubated at room temperature or 37 ℃ for 30 minutes, added with ampicillin to a working concentration of 100ug/ml, added with kanamycin to a working concentration of 50 ug/ml.37 ℃, subjected to shaking culture at 220rpm overnight, and centrifuged at 6000rpm for 10 minutes to remove the thallus.

3) Taking 50ul or 5ul of supernatant, taking MPBS as a medium, supplementing 100ul, adding into an enzyme label plate, incubating for 1 hour, removing liquid, and washing with PBS for 4 times.

4) The HRP-labeled anti-M13 phage antibody was diluted with 1% nonfat dry milk in PBS, 100ul of the diluted antibody was added to the microplate, incubated for 1 hour, drained and washed 4 times with PBS.

5) Adding 100ul TMB developing solution, incubating for 10min at 37 ℃ or fully developing yellow, adding 100ul 1M sulfuric acid to stop the reaction, reading OD450 on a microplate reader, wherein the result is shown in figure 1, the reading value of the monoclonal E L ISA is obviously higher than that of a control, the result is positive, and the monoclonal E L ISA has the function of binding human GPC3 protein at the level of E L ISA.

(3) Preparation and flow detection of human GPC3 monoclonal antibody

1) Single colonies picked from 2 × YT-GA100 plate were cultured overnight with liquid shaking, and phagemid (phagemid) was extracted by the plasmid extraction method.

2) The chemical synthesis nanometer antibody fusion gene sequence is characterized in that a signal peptide of the secreted luciferase, a nanometer antibody, a flexible connecting peptide, human IgG2Fc and a coding sequence of a His label are connected and read in frame, the nucleic acid sequence of the fusion gene sequence is shown as SEQ ID NO.5, and the amino acid sequence of the coded antibody is shown as SEQ ID NO. 6.

3) The above nucleic acid fragments were inserted into the MCS region of an eukaryotic expression vector p L OE-purOR in the order (FIG. 2) to synthesize a fusion protein having a signal peptide at the N-terminus, a nanobody at the middle, and Fc and His tags at the C-terminus.

4) Preparing sterile endotoxin-free plasmid, transfecting CHO-K1 cell according to the requirement of transfection reagent, and collecting supernatant.

5) SDS-PAGE detects the expression of the antibody, and as shown in FIG. 3, the fused antibody protein is successfully expressed.

6) Flow assay for cell binding capacity

① liver cancer cell line Huh7 cell of very weak expression GPC3 and liver cancer cell line HepG2 cell of strong expression human GPC3 were digested thoroughly with 0.25% pancreatin, the digestion was stopped with serum, the cells were collected by centrifugation, and PBS was gently blown to prepare a single cell suspension.

② 10ml PBS washed cells 1 times, 1000rpm centrifugation for 5min, then 1ml PBS suspension cells, cell count.

③ is 2.5 × 10⁵The cells were collected by centrifugation in a 1.5ml centrifuge tube.

④ mu.l of the CHO-K1 cell culture supernatant from step 4) was added, mixed well and incubated at room temperature for 30 min.

⑤ cells were harvested by centrifugation and washed 1 time with 1ml PBS.

⑥ mu.l of 20-fold diluted secondary APC-labeled goat anti-human IgG Fc fragment (2.5. mu.l/2.5 × 10) was added⁵Optionally binding with human IgG) and reacting at room temperature in the dark for 20 min.

⑦ cells were washed 1 time with 1ml PBS, centrifuged at 1000rpm for 8min and the supernatant removed.

⑧, adding 200 mu l of PBS to be re-suspended into a single-cell suspension, and detecting the suspension on a flow cytometer, as shown in figure 4, compared with Huh7 cell strain which does not express GPC3 (extremely weakly expresses GPC3 liver cancer cell strain), the signal of a high-expression GPC3 liver cancer cell strain HepG2 is obviously enhanced, the histogram is shifted to the right, and the antibody can be combined with GPC3 on the cell surface.

(4) Preparation of human-derived GPC3 monoclonal antibody from CHO-K1 cell

1) Large quantities of positive plasmids were prepared by transfection of three T25 flasks of CHO-K1 cells.

2) 72 hours after transfection, the supernatant (total 12ml) was collected while cells were trypsinized and collected by centrifugation.

3) Cells were suspended in 5ml cold PBS. The ultrasonic power is set to 25% of the maximum power, the work is carried out for 2s, the pause is carried out for 6s, and the total time is 3 min. Cells were disrupted by sonication in an ice bath and combined with cell culture supernatant after completion. A cocktail protease inhibitor was added.

4) Taking 4ml of Ni-NTA agarose gel, adding the gel into a screening plate of a purification column, and filling a gravity purification column.

5) The column was equilibrated with 10ml of 20mM Tris-HCl buffer (pH 8.0)/500mM NaCl.

6) The combined solution was applied to a purification column at low temperature, and the flow rate was controlled at 3 s/drop.

7) The column was washed with 10ml of 20mM Tris-HCl buffer (pH 8.0)/500mM NaCl solution containing 30mM imidazole.

8) The antigen-binding peptide fusion protein was eluted with 4ml of 20mM Tris-HCl buffer (pH 8.0)/500mM NaCl solution containing 300mM imidazole.

9) Adding into deionized water-wetted ultrafiltration tube with cut-off molecular weight of 10KD, cooling PBS to 10ml, and centrifuging at 4 deg.C and 3000rpm for 10min until the liquid surface is at the upper edge of V-shaped ultrafiltration membrane. The filtrate was aspirated off, 10ml of cold PBS was added to the inner column, and the column was centrifuged until the liquid surface was on the upper edge of the V-shaped ultrafiltration membrane.

10) The upper column was aspirated, the ultrafiltration membrane was washed with 0.4ml of cold PBS, and the combined fluids amounted to about 1.5 ml.

(5) Potency assay for purified human GPC3 monoclonal antibody

1) Concentration of fusion protein by Bradford method

The Quick Start of Bio-Rad was used^TMThe measurement was carried out using Bradford 1x Dye Reagent (cat # 5000205) kit, and the test samples were tested at four concentrations of 2 ×, 5 ×, 10 × and 20 ×, and the absorbance was read at 595nm after completion of the procedure according to the kit.

2) The plate was coated with 100ul of 2ug/ml human GPC3 under 4 ℃ overnight.

3) Wash plate 3 times with 0.05% PBST, block with 200ul 2% skim milk for 1 hour at room temperature, wash plate 3 times with 0.05% PBST.

4) The purified protein was diluted to 1ng/ul, 5ng/ul, 25ng/ul, 125ng/ul, 625ng/ul with 1% nonfat dry milk in PBS, 100ul of the diluted purified protein was added to the ELISA plate, and 1% nonfat dry milk in PBS was added to the control wells.

5) Incubate at room temperature for 2 hours, shaking several times during this period. The liquid was removed and washed 3 times with 0.05% PBST.

6) Secondary antibody of HRP-labeled rabbit anti-human IgG Fc fragment was added and incubated at room temperature for 1 hour. The liquid was removed and washed 3 times with 0.05% PBST.

7) 100ul of TMB substrate was added and incubated at 37 ℃ for about 10 minutes until the yellow color was fully developed.

8) The reaction was stopped by adding 100ul 1M sulfuric acid and the OD450 read on a microplate reader. The results are shown in FIG. 5, where the purified antibody bound to the antigen in vitro, showing a typical antigen-antibody binding concentration-dependent curve.

Sequence listing

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<120> anti-human GPC3 monoclonal antibody

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tgcggggttg ggcacagaat cacagagtat aactactctg gtcaggggac ccaggtcacc 420

gtctcctcag gtggaggcgg ttcaggcgga ggtggctctg gcggtggcgg atcgccagca 480

ccacctgtgg caggaccgtc agtcttcctc ttccccccaa aacccaagga caccctcatg 540

atctcccgga cccctgaggt cacgtgcgtg gtggtggacg tgagccacga agaccccgag 600

gtccagttca actggtacgt ggacggcgtg gaggtgcata atgccaagac aaagccacgg 660

gaggagcagt tcaacagcac gttccgtgtg gtcagcgtcc tcaccgttgt gcaccaggac 720

tggctgaacg gcaaggagta caagtgcaag gtctccaaca aaggcctccc agcccccatc 780

gagaaaacca tctccaaaac caaagggcag ccccgagaac cacaggtgta caccctgccc 840

ccatcccggg aggagatgac caagaaccag gtcagcctga cctgcctggt caaaggcttc 900

taccccagcg acatcgccgt ggagtgggag agcaatgggc agccggagaa caactacaag 960

accacacctc ccatgctgga ctccgacggc tccttcttcc tctacagcaa gctcaccgtg 1020

gacaagagca ggtggcagca ggggaacgtc ttctcatgct ccgtgatgca tgaggctctg 1080

cacaaccact acacgcagaa gagcctctcc ctgtctccgg gtaaatgaca ccaccatcac 1140

caccattga 1149

<210>6

<211>381

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>6

Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu

1 5 10 15

Ser Met Ala Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Ser Val Gln

20 25 30

Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Met Tyr

35 40 45

Ser Thr Asn Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg

50 55 60

Glu Gly Val Ala Ala Ile Asn Phe Gly Ser Gly Arg Thr Asp Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Asn

85 90 95

Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met

100 105 110

Tyr Tyr Cys Ala Ala Asp His Trp Cys Gly Val Gly His Arg Ile Thr

115 120 125

Glu Tyr Asn Tyr Ser Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly

130 135 140

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Ala

145 150 155 160

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

165 170 175

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

180 185 190

Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp

195 200 205

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe

210 215 220

Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp

225 230 235 240

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu

245 250 255

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg

260 265 270

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys

275 280 285

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

290 295 300

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

305 310 315 320

Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

325 330 335

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

340 345 350

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

355 360 365

Leu Ser Leu Ser Pro Gly Lys His His His His His His

370 375 380

Claims

1. An anti-human GPC3 monoclonal antibody, characterized in that the antibody is of single-chain structure and comprises an antigen binding portion with an amino acid sequence shown as SEQ ID NO. 1.

2. The monoclonal antibody against human GPC3 according to claim 1, wherein the monoclonal antibody against human GPC3 comprises a signal peptide and an antigen-binding portion, and the amino acid sequence thereof is represented by SEQ ID No. 2.

3. The monoclonal antibody against human GPC3 according to claim 1, wherein the monoclonal antibody against human GPC3 comprises a signal peptide, an antigen-binding portion and a human Fc region, and the amino acid sequence thereof is represented by SEQ ID No. 3.

4. The monoclonal antibody against human GPC3 according to claim 1, wherein the monoclonal antibody against human GPC3 comprises a signal peptide, an antigen binding portion, a flexible linker peptide and a human Fc region, and the amino acid sequence thereof is represented by SEQ ID No. 4.

5. A nucleic acid molecule comprising a gene encoding the anti-human GPC3 monoclonal antibody of claim 1.

6. A vector comprising the nucleic acid molecule of claim 5.

7. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A kit comprising an anti-human GPC3 monoclonal antibody of any one of claims 1 to 4.

9. The use of the monoclonal antibody against human GPC3 as claimed in any one of claims 1 to 4 in the preparation of an anti-liver cancer drug or in the preparation of a liver cancer detection kit.