CN112898427A

CN112898427A - anti-c-Met single-arm antibody and preparation method and application thereof

Info

Publication number: CN112898427A
Application number: CN201911221523.8A
Authority: CN
Inventors: 蒋明; 房健民; 尹衍新; 郭佳
Original assignee: SUZHOU RESEARCH INSTITUTE OF TONGJI UNIVERSITY
Current assignee: SUZHOU RESEARCH INSTITUTE OF TONGJI UNIVERSITY
Priority date: 2019-12-03
Filing date: 2019-12-03
Publication date: 2021-06-04
Anticipated expiration: 2039-12-03
Also published as: CN112898427B

Abstract

The invention discloses an anti-c-Met single-arm antibody, and a preparation method and application thereof, and belongs to the technical field of immune engineering. The invention discloses an anti-c-Met single-arm antibody, which consists of 3 chains: the light chain is composed of the light chain variable region 1H9D6-V of the parent antibody_LSplicing with a human Kappa light chain constant region Homoc Kappa to obtain the polypeptide; the heavy chain is composed of the heavy chain variable region 1H9D6-V of the parent antibody_HThe gene is obtained by splicing with a human IgG1-hole-Flag sequence, wherein a human IgG1 heavy chain constant region generates 3 amino acid mutations to form a mortar-shaped structure; the constant region of human IgG1 heavy chain in Fc-knob-His chain is mutated by 1 amino acid to form a pestle shapeAnd (5) structure. The anti-c-Met single-arm antibody constructed by the invention solves the problem of c-Met dimerization, and can be prepared into an anti-tumor drug.

Description

anti-c-Met single-arm antibody and preparation method and application thereof

Technical Field

The invention relates to the technical field of immune engineering, in particular to an anti-c-Met single-arm antibody and a preparation method and application thereof.

Background

Hepatocyte Growth Factor (HGF) and its receptor human mesenchymal epithelial transformation factor (c-Met) play an important role in tumorigenesis and development. In solid tumors, hypoxia can cause high expression of HGF and c-Met, which favors tumor angiogenesis and stimulates growth, motility, and invasion of cancer cells. In hepatoma cells, the protein level of c-Met is 25% -100% higher than that of normal liver cells, which indicates that c-Met is a potential target for treating hepatoma. Blocking HGF/c-Met signal transduction has become an important strategy for the research of antitumor drugs. c-Met inhibitors such as tivatinib, golvatinib and cabozantinib are all activated by blocking c-Met signaling. However, the side effects of these drugs and the resistance of hepatoma cells to these drugs remain to be solved. The research and development of antibody medicines for blocking HGF/c-Met signal transduction are hot spots, and the traditional bivalent monoclonal antibody can cause dimerization of c-Met after being combined with the c-Met so as to activate a tumor cell signal transduction pathway.

Therefore, it is an urgent problem for those skilled in the art to provide an anti-c-Met single-arm antibody, and a preparation method and application thereof.

Disclosure of Invention

In view of the above, the present invention provides an anti-c-Met single-arm antibody, which can bind to a c-Met extracellular domain protein without causing c-Met dimerization, and a preparation method and applications thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

an anti-c-Met single-arm antibody consisting of 3 chains: 1 light chain, 1 heavy chain and 1 Fc-knob-His chain; the light chain is composed of the light chain variable region 1H9D6-V of the parent antibody_LSplicing with a human Kappa light chain constant region Homoc Kappa to obtain the polypeptide; the heavy chain is formed by the heavy chain variable region 1H9D6-V of the parent antibody_HThe gene is obtained by splicing with a human IgG1-hole-Flag sequence, wherein a human IgG1 heavy chain constant region generates 3 amino acid mutations to form a mortar-shaped structure; the constant region of human IgG1 heavy chain in the Fc-knob-His chain is subjected to 1 amino groupAcid mutation, forming a pestle structure.

Further, the human IgG1 heavy chain constant region was mutated at 3 amino acids to: T366S: L368A: Y407V; the human IgG1 heavy chain constant region was mutated from 1 amino acid to T366Y.

Further, the preparation method of the anti-c-Met single-arm antibody comprises the following specific steps:

(1) constructing an anti-c-Met single-arm antibody light chain lentivirus expression vector pCMV-2E 6-L: PCR amplification of the light chain variable region V of the parent antibody 1H9D6_LGene 1H9D6-V_LThe human Kappa light chain constant region HomoC Kappa is synthesized by the whole gene, and the HomoC Kappa is mixed with 1H9D6-V_LSplicing the vector into a lentivirus expression vector pRRL-CMV by a homologous recombination method, and transforming to obtain pCMV-2E 6-L;

(2) constructing an anti-c-Met single-arm antibody heavy chain lentivirus expression vector pCMV-2E 6-H: PCR amplification of the heavy chain variable region V of the parent antibody 1H9D6_HGene 1H9D6-V_HThe IgG1-hole-Flag sequence is synthesized by the whole gene, and 1H9D6-V_HSplicing the sequence with an IgG1-hole-Flag sequence into a lentivirus expression vector pRRL-CMV by a homologous recombination method, and transforming to obtain pCMV-2E 6-H;

(3) construction of lentiviral expression vector pCMV-Fc-knob-His: Fc-knob-His is synthesized through a whole gene, and is connected with a lentivirus expression vector pRRL-CMV and is transformed to obtain pCMV-Fc-knob-His;

(4) and co-transfecting the pCMV-2E6-L, pCMV-2E6-H and pCMV-Fc-knob-His into eukaryotic cells, purifying an expression product by using a nickel column, and screening by ELISA to obtain the anti-c-Met single-arm antibody.

Further, the anti-c-Met single-arm antibody is applied to the preparation of antitumor drugs.

Further, the tumor is liver cancer or glioma.

Further, the anti-c-Met single-arm antibody is applied to inhibiting HGF/c-Met signal channels.

Further, the use is for inhibiting HGF and c-Met binding.

Further, the application is inhibition of c-Met phosphorylation.

Further, the application is inhibition of Gab-1, ERK1/2, Akt and PKB activity.

Further, the anti-c-Met single-arm antibody is applied to inhibiting angiogenesis or inhibiting cell migration.

According to the technical scheme, compared with the prior art, the invention discloses and provides the anti-c-Met single-arm antibody and the preparation method and the application thereof, and the anti-c-Met single-arm antibody constructed by the invention solves the problem of c-Met dimerization; the anti-c-Met single-arm antibody can block HGF from being combined with c-Met, and inhibit signal transduction downstream of HGF/c-Met in hepatoma cell line HepG2 cells, and comprises the following components: inhibit c-Met phosphorylation, Gab-1, ERK1/2, Akt and PKB activity; and inhibit HepG2 cell proliferation and HGF-mediated cell migration. Meanwhile, the c-Met single-arm antibody can inhibit cell proliferation and microtubule formation of human vascular endothelial cells HUVEC. Research on a nude mouse liver cancer cell HepG2 transplanted tumor model shows that the single-arm antibody inhibits tumor growth and has a good anti-tumor effect.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a schematic diagram showing the structure of an anti-c-Met single-arm antibody according to the present invention;

wherein, V_H: a heavy chain variable region; c_H1: heavy chain constant region 1; v_L: a light chain variable region; c_L: a light chain constant region; knobs: a "pestle" structure; holes: a "mortar" structure;

FIG. 2 is a drawing showing the results of SDS-PAGE electrophoresis of the parent antibody 1H9D6 and the anti-c-Met single-arm antibody 2E6, both denatured and non-denatured according to the present invention;

wherein, the left picture is denatured electrophoresis, and the right picture is non-denatured electrophoresis; m: protein Marker, 1: 1H9D6, 2: 2E 6;

FIG. 3 is a graph showing the ELISA results of binding of 1H9D6 (triangle), 2E6 (square) and human IgG control antibody (diamond) to c-Met extracellular domain fusion protein according to the present invention; 3 replicates were performed per condition and the results are presented as mean ± standard deviation;

FIG. 4 is a graph showing the binding results of the present invention 1H9D6 (dotted line), 2E6 (dotted line) and control group (solid line) to HepG2 cell (human hepatoma cell);

FIG. 5 is a drawing showing the results of Western immunoblotting of cell lysates of CHO cells, HeLa cells and HepG2 cells and an anti-c-Met one-armed antibody 2E6 according to the present invention;

FIG. 6 is a graph showing the effect of 1nM human HGF (human hepatocyte growth factor) binding to the c-Met extracellular domain fusion protein after pre-incubation of various concentrations of 1H9D6 (black circles), 2E6 (black squares) and human IgG control antibody (black diamonds) with the c-Met extracellular domain fusion protein in accordance with the present invention; 3 replicates were performed per condition and the results are presented as mean ± standard deviation;

FIG. 7 is a graph showing the effect of 10nM human HGF of the present invention on the binding of 20nM 2E6 to HepG2 cells, as detected by flow cytometry; wherein, the chain line (2E6+ HGF) is the result of the combination of 2E6 and HepG2 cells after adding HGF; the dashed line (2E6) is the result of binding of 2E6 without HGF to HepG2 cells; the solid line (control) is the result of adding only anti-human IgG secondary antibody;

FIG. 8 is a graph showing the results of measuring phosphorylation levels of c-Met protein, Gab-1 protein, Akt protein and Erk protein after 1nM human HGF stimulates HepG2 cells;

FIG. 9 is a graph showing the results of detecting phosphorylation levels of c-Met protein, Gab-1 protein, Akt protein and Erk protein after 1nM human HGF stimulates HepG2 cells after pre-incubation of HepG2 cells of the present invention with 10nM 2E6 or 1 μ M PF-02341066(c-Met target small molecule inhibitor) for 120 minutes; 3 replicates were performed per condition and the results are presented as mean ± standard deviation;

FIG. 10 is a diagram showing the detection of cell proliferation of HepG2 cells after 1nM human HGF and 2E6 at different concentrations for 72 hours by the CCK8 method according to the present invention; 3 replicates were performed per condition and the results are presented as mean ± standard deviation; wherein P <0.05, P <0.01, P <0.001, the differential analysis of each group was compared to the results of 2E6 untreated samples of each group (both with and without HGF); ns means no significant difference, compared to control sample (no 2E6 added, no HGF sample added);

FIG. 11 is a graph showing the experimental analysis of cell migration performed on 4 hepatoma cell lines, Hep G2, Huh-7, Hep 3B2 and SMMC-7721, in accordance with the present invention; wherein, a cell migration picture of Hep G2 cells is selected as a representative result for displaying, the cells are photographed under a 10-fold objective lens, and a negative control is a group without adding 2E6 and HGF; after counting the number of migrating cells of the 4 cells, the number of migrating cells was compared with the number of migrating cells of the respective control sample (without 2E6, without HGF sample), and the results were displayed in the form of a numerical value, with the control sample set to 1.0;

FIG. 12 is a graph showing the analysis of the anti-tumor effect of the one-armed antibody 2E6 of the present invention in glioma cell lines; a cell migration photograph of U87 was selected as a representative result to be displayed, cells were photographed under a 10-fold objective lens, and negative controls were a group without 2E6 and HGF; after counting the number of migrating cells, the number of migrating cells was compared with the number of migrating cells of the respective control sample (without 2E6, without HGF sample), and the results were displayed in the form of a numerical value, with the control sample set to 1.0;

FIG. 13 is a diagram showing the detection of cell proliferation of HUVEC cells (human umbilical vein endothelial cells) after 72 hours of the action of 1nM human HGF and different concentrations of 2E6 by the CCK8 method according to the present invention; 3 replicates were performed per condition and the results are presented as mean ± standard deviation; wherein P <0.05, P <0.01, differential analysis of each group compared to HGF treated sample alone (with HGF added, without 2E6 sample); # P <0.01, compared to control samples (no 2E6 added, no HGF sample added);

FIG. 14 is a drawing showing that the anti-c-Met single-arm antibody 2E6 inhibits HUVEC cell microtubule production in matrigel according to the present invention; after the HUVEC cells are plated on a 96-well cell culture plate, matrigel is coated, then after 1nM human HGF and 2E6 with different concentrations act for 18 hours, 4x objective lens is used for photographing to observe the generation condition of the microtubes, and ImageJ software is used for analyzing the number of the microtubes nodes of each sample; 3 replicates were performed per condition and the results are presented as mean ± standard deviation; wherein, the left column is added with 2E6 antibody with different concentrations separately, and the right column is added with 2E6 antibody with different concentrations and 1nM HGF; p <0.05, differential analysis of each group compared to HGF treated sample alone (with HGF added, without 2E6 sample); # P <0.05, was compared to a control sample (no addition of 2E6, no addition of HGF sample);

FIG. 15 is a graph showing that BALB/c nude mice according to the present invention were inoculated with HepG2 cells and divided into 3 groups according to 6 groups, respectively, a model group, 2E6 treatment group 1(5mg/kg) and 2E6 treatment group 2(10mg/kg), wherein the inoculation frequency was twice per week; results are presented as mean ± standard deviation of tumor volume;

FIG. 16 is a graph showing that after 42 days of the first injection of 2E6 antibody, animals were sacrificed, tissues were removed, photographed, and the volume was measured; tumor volumes are shown as mean ± standard deviation. P <0.01, P <0.001 compared to PBS control;

FIG. 17 is a graph showing that BALB/c nude mice according to the present invention were inoculated with U87 cells and divided into 2 groups of 6 cells each, namely, a model group and 2E6 treatment group 1(5mg/kg), wherein the frequency of inoculation was twice a week; results are presented as mean ± standard deviation of tumor volume.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The primers were synthesized by Suzhou Jinweizhi Biotechnology, Inc.; escherichia coli competent DH5 α, DNA gel recovery kit, and small plasmid extraction kit were purchased from Axygen corporation.

Tumor cells and HEK293 cell lines were purchased from american type culture collection (ATCC, VA, USA). Human Umbilical Vein Endothelial Cells (HUVECs) were purchased from shanghai cells and cultured using HUVEC cell complete medium (# H-004, allcellco., Ltd.).

The statistical software used for the processing of the experimental data was GraphPad Prism 6 (GraphPad, CA, USA). Statistical differences between the antibody group and the control group were calculated using the T-test. The significance threshold for the P value was 0.05.

Example 1

A BALB/c mouse is immunized by using the c-Met extracellular region fusion protein, and a hybridoma cell strain 1H9D6 which stably secretes a bivalent antibody capable of blocking the combination of HGF and c-Met is screened out by ELISA.

The preservation number of the hybridoma cell strain 1H9D6 is CCTCC NO: c2017124, which is preserved in China Center for Type Culture Collection (CCTCC) for short, and the address: the preservation date of Wuhan university in Wuhan, China is 10 months and 17 days in 2017, and the Wuhan university in Wuhan is classified and named as a mouse hybridoma cell strain 1H9D 6.

The antibody secreted by the hybridoma cell strain 1H9D6 is used as the parent antibody 1H9D6 of the invention.

Example 2

Since bivalent antibodies easily cause activation reaction of Receptor Tyrosine Kinase (RTK) which is not ligand-specific, the structure of monovalent antibodies can effectively ensure inhibition of RTK; but the half-life of the monovalent antibody is short, so the application is limited; while the discovery of the "knob and hole" technique enabled the construction of single-arm antibodies with longer half-lives. Based on this, the invention forms a hole structure by introducing the mutations of the Fc end of the full-length heavy chain (T366S, L368A and Y407V), and simultaneously introduces the mutation of the single-chain Fc fragment (T366W) to form a pestle structure, and particularly refers to FIG. 1. Such mutations can aid in the formation of heterodimerization of full-length heavy chains and single chain Fc fragments, aiding in the assembly of monovalent antibodies. The anti-c-Met one-armed antibody 2E6 was expressed from eukaryotic HEK293, which co-expressed a chimeric 1H9D6 light chain, a full-length heavy chain IgG1-hole-Flag comprising a mortar structure, and an Fc-knob-His fragment comprising a knob structure.

The parent antibody 1H9D6 was engineered using the "Knob and Hole" (Knob into Hole) technique to form an anti-c-Met single-armed antibody:

the specific process is as follows:

first, variable region template acquisition

Total cellular RNA was extracted from the mouse hybridoma cell line 1H9D6 by the TRIzol method, and cDNA was obtained by reverse transcription. Then, the heavy chain variable region V of the parent antibody 1H9D6 is amplified by PCR by using cDNA as a template and primers P1/P2 and P3/P4 respectively_H(1H9D6-V_H) And light chain variable region V_LGene (1H9D 6-V)_L). Wherein amplification of V_HThe 5' primer P1 of (a) amplifies V starting from the signal peptide_HThe 3' primer P2 of (a) is complementary to the CH1 sequence of murine IgG 1. For amplifying V_LThe 5 'primer P3 of (1) was derived from the mature peptide of the light chain variable region, the 3' primer P4 was complementary to the murine C kappa (murine kappa light chain constant region) sequence, and the designed primer was used to amplify the V of 1H9D6_HAnd V_LThe gene sizes were 366bp and 336bp, respectively. The amplified product is sent to Suzhou Jinzhi Biotechnology limited company for sequencing verification, the sequencing result accords with the variable region characteristics of the mouse monoclonal antibody, and the homology analysis and comparison determine that the amplified product has uniqueness on the gene sequence or the amino acid sequence.

P1:5’-ATGSARGTRMAGCTGSAGSAGTC-3’；SEQ ID NO.1；

P2:5’-AATTTTCTTGTCCACCTTGGTGCTGCT-3’；SEQ ID NO.2；

P3:5’-GAYATTGTGMTSACCCAGACTCCA-3’；SEQ ID NO.3；

P4:5’-GGATACAGTTGGTGCAGCATCAGCCC-3’；SEQ ID NO.4；

Wherein S represents G/C, R represents A/G, M represents A/C, and Y represents C/T.

The anti-c-Met single-arm antibody 2E6 was designed as shown in FIG. 1, and in FIG. 1, it contained 1 light chain, 1 heavy chain, and 1 Fc-knob-His chain. Wherein the light chain of the anti-c-Met single-arm antibody is the light chain variable region 1H9D6-V of the murine parent antibody_LAnd the human Kappa light chain constant region HomoC Kappa is spliced by a homologous recombination method. The heavy chain of the anti-c-Met single-arm antibody is the heavy chain variable region 1H9D6-V of the murine parent antibody_HSpliced with human IgG1-hole-Flag sequence by a homologous recombination method. Wherein the human Kappa light chain constant region HomoC Kappa, the human IgG1-hole-Flag sequence and the Fc-knob-His are obtained by whole-gene synthesis.

Secondly, constructing an anti-c-Met single-arm antibody light chain lentivirus expression vector pCMV-2E6-L

Synthesizing human Kappa light chain constant region HomoC Kappa through whole gene, wherein the gene sequence is shown as SEQ ID NO.5, and mixing the HomoC Kappa with 1H9D6-V_LSpliced together by homologous recombination. The concrete stepsThe following were used: amplification of 1H9D6-V with P5 and P6, respectively_LThe HomoC kappa is amplified by P7 and P8, a PCR product is purified and recovered after 15g/L agarose electrophoresis, homologous recombination and connection are carried out on the PCR product and a lentiviral expression vector pRRL-CMV which is recovered by BspEI and SalI enzyme digestion through a one-step directional cloning kit, escherichia coli competence DH5 alpha is transformed, and positive clones are screened through sequencing identification. The new one-armed antibody light chain pCMV-2E6-L was purified for use with a plasmid extraction kit for endotoxin removal.

P5：5’-TCACAGGATCTAGTTCCGGAGACATTGTTATGACACAGACTG-3'; SEQ ID No. 6; BspEI restriction sites are underlined;

P6：5’-ACAGATGGTGCAGCCACAGTTCGTTTGATTTCCAGCTTGGTGCCTC-3’；SEQ ID NO.7；

P7：5’-CGAACTGTGGCTGCACCATCTGT-3’；SEQ ID NO.8；

P8：5’-TCCAGAGGTTGATTGTCGACCTAACACTCTCCCCTGTTGAAGC-3'; SEQ ID No. 9; SalI sites are underlined.

Thirdly, constructing a lentiviral expression vector pCMV-Fc-knob-His

The Fc-knob-His chain is a 1-pestle structure formed by replacing 1 small amino acid Threonine (Thr) with 1 large amino acid Tyrosine (Tyrosine, Tyr) in the human IgG1 heavy chain constant region (CH 3) domain, such as T366Y, and adding a 6 × His tag thereafter. The gene sequence is shown in SEQ ID NO. 10. The above sequences were synthesized by whole gene and the above gene fragments were PCR amplified by primers P9 and P10 to form pCMV-Fc-knob-His lentiviral expression vector by ligation to the lentiviral vector pRRLCMV by homologous recombinase.

P9：5’-TCACAGGATCTAGTTCCGGAGACAAAACTCACACATGCCCAC-3'; SEQ ID No. 11; BspEI restriction sites are underlined;

P10：5’-TCCAGAGGTTGATTGTCGACCTAGTGATGGTGATGGTGATGTTTAC-3'; SEQ ID No. 12; SalI sites are underlined.

Fourthly, constructing an anti-c-Met single-arm antibody heavy chain lentivirus expression vector pCMV-2E6-H

Human IgG1 heavyThe chain constant region (CH 3) domain has 3 small amino acids substituted for 3 large amino acids, i.e., T366S: L368A: Y407V, 1 mortar-shaped structure, namely, "hole", was formed on the CH3 domain, and a Flag tag, named IgG1-hole-Flag, was added, the gene sequence of which is shown in SEQ ID NO.13, and the IgG1-hole-Flag sequence was synthesized by whole gene. The heavy chain of the anti-c-Met single-arm antibody is 1H9D6-V_HSpliced with human IgG1-hole-Flag sequence by a homologous recombination method. The method comprises the following specific steps: amplification of 1H9D6-V with P11 and P12, respectively_HIgG1-hole-Flag is amplified by P13 and P14, a PCR product is purified and recovered after 15g/L agarose electrophoresis, homologous recombination and connection are carried out on the PCR product and a lentiviral expression vector pRRL-CMV which is recovered after BspEI and SalI enzyme digestion through a one-step directional cloning kit, escherichia coli competence DH5 alpha is transformed, and positive clones are screened through sequencing identification. The new single-arm antibody heavy chain pCMV-2E6-H was purified for use with a plasmid extraction kit for endotoxin removal.

P11：5’-TCACAGGATCTAGTTCCGGACAGGTTCAGCTGGAGCAGTCTG-3'; SEQ ID No. 14; BspEI restriction sites are underlined;

P12：5’-GATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCGTGGTC-3’；SEQ ID NO.15；

P13：5’-GCCTCCACCAAGGGCCCATC-3’；SEQ ID NO.16；

P14：5’-TCCAGAGGTTGATTGTCGACCTACTTATCGTCGTCATCCTT-3'; SEQ ID No. 17; SalI sites are underlined.

Co-transfecting HEK-293 cells with pCMV2E6-L, pCMV-2E6-H and pCMV-Fc-knob-His, collecting an expression supernatant, purifying an expression product by a nickel column, and screening the purified antibody by indirect ELISA (coating c-Met ED protein) to obtain the anti-c-Met single-arm antibody 2E 6.

The electrophoresis result of the denatured SDS-PAGE shows that the electrophoresis result of the natural unmodified 1H9D6 is shown in the first lane of FIG. 2A; the anti-c-Met single-arm antibody 2E6 has a light chain size of about 30kDa, a heavy chain of about 53kDa, and an Fc fragment of about 35kDa, as shown in the second lane of FIG. 2A; namely, the sizes of the light chain and the heavy chain of the anti-c-Met single-arm antibody 2E6 are consistent with the electrophoresis result of the natural 1H9D6 before modification. Electrophoresis results show that the molecular weights of the light chain and the Fc fragment of the anti-c-Met single-arm antibody 2E6 are both larger than the theoretical molecular weight obtained by amino acid series calculation, and show that the eukaryotic expression retains the posttranslational glycosylation modification of the antibody. Non-denaturing electrophoresis shows a large deviation from the expected position due to the fact that protein markers are usually designed for denaturing electrophoresis. Then, the natural antibody 1H9D6 was used as a control protein for calibration, and further subjected to non-denaturing electrophoresis, the results of which are shown in fig. 2B; wherein, lane 1 is the 1H9D6 parent antibody, lane 2 is the 2E6 antibody; the results show that the difference between the molecular weights of 1H9D6 and 2E6 is about 52kDa, consistent with amino acid sequence calculations. In summary, the anti-c-Met one-armed antibody 2E6 (about 102kDa), the overexpressed heavy chain (about 53kDa), and the relatively lower proportion of the light chain (about 24kDa) were all detected in 2E 6.

Example 3

The binding activity of the antibody was determined by enzyme-linked immunosorbent assay (ELISA) using the antibody and the c-Met extracellular domain fusion protein.

c-Met extracellular region fusion protein 1 ug/mL was added to a 96-well plate dedicated to ELISA and incubated overnight at 4 ℃. The plate was then washed with 0.05% Tween-20 in saline and blocked with 1% BSA in saline. Then, 0.1. mu.g/mL of anti-c-Met antibody protein was used as a primary antibody, and incubation was continued with a secondary HRP-conjugated antibody after one hour of incubation. The final color development was achieved by the addition of tetramethylbenzidine (3,3,5, 5' -tetramethylbenzidine, cloudy day). The sample absorption peak value is 450nm, and the detection instrument is a Bio-Rad iMark enzyme-labeling instrument.

As a result, it was found that the binding activity of 2E6 to the coated c-Met extracellular region fusion protein was concentration-dependent as in native 1H9D6, as shown in FIG. 3. The EC50 value for 1H9D6 was 0.2716nM and the EC50 value for 2E6 was 0.2716nM, and the difference between the two values is most likely due to the difference in the CDR/MW ratio.

Example 4

Specific analysis of anti-c-Met single-arm antibody 2E6 with target: the binding activity of 2E6 and 1H9D6 to the c-Met receptor expressed by the natural membrane of the cell was tested by flow cytometry using a cell line HepG2 highly expressing the c-Met protein. About 1X 10⁶The cells were digested, washed and then mixed with an excess of 2E6 or 1H9D6 primary antibody was incubated on ice for 30 minutes in PBS (pH 7.4) buffer containing 2% FBS. After completion of incubation, washes were added with Alexa Fluor 488 goat anti-mouse IgG1 secondary antibody (1:100 dilution; Invitrogen, USA) and incubated for 15 min on ice. The finished cells were incubated and then incubated with the fluorescent marker-conjugated monoclonal antibody for 30 minutes on ice. The cell suspension was subjected to detection on a machine after three washes, and the detection apparatus was a FACS Calibur flow cytometer (BD immunocytometer Systems, NJ, USA). The results show that the two effects are close, see fig. 4. The concentration of 1H9D6 was 13.3nM, the concentration of 2E6 was 20nM, and the control group was a secondary antibody alone at 20 nM.

By detecting the expression of c-Met protein in lysates of three cell lines, CHO (Chinese hamster ovary cancer cell line), Hela (human cervical cancer cell line) and HepG2 (human liver cancer cell line), using 2E6 as a primary antibody, the specificity of 2E6 as an antibody can be determined. The c-Met β subunit band after activation at about 135kDa is evident, whereas the c-Met not glycosylated at about 100kDa also shows an implicit band in high expressing c-Met cell lines. No bands were evident in the CHO cell line 2E6 assay, see FIG. 5. The results show that the expression level of c-Met protein in CHO cell line is low, or the cross reaction of 2E6 and c-Met from Chinese hamster is low. The results show high specificity of 2E6 in the cell line.

Example 5 anti-c-Met Single-arm antibodies are able to reduce HGF/c-Met signaling

The anti-c-Met single-arm antibody is obtained by modifying and transforming a wild-type antibody 1H9D 6. And a part of antigen epitope of the wild-type antibody before modification is overlapped with the antigen binding epitope of HGF, so that the inhibitor effect is achieved by blocking the binding of HGF/c-Met in the process of competitively antagonizing HGF/c-Met signal transmission. 2E6 was first tested for its effect against HGF using a ligand binding assay.

The recombinant protein c-Met extracellular region fusion protein 1 mug/mL is added into a 96-well plate special for ELISA, and the mixture is incubated overnight at 4 ℃. The plate was then washed with 0.05% Tween-20 in saline and blocked with 1% BSA in saline. Then 0.1 μ g/mL of recombinant HGF protein was used for co-incubation with different concentrations of 2E6 or 1H9D6, and after one hour incubation was continued with HRP-conjugated anti-human HGF antibody (American Qulex, CA, USA). The final color development was achieved by the addition of tetramethylbenzidine (3,3,5, 5' -tetramethylbenzidine, cloudy day). The sample absorption peak value is 450nm, and the detection instrument is a Bio-Rad iMark enzyme-labeling instrument.

Consistent with expectations, 2E6 was directly proportional to the inhibitory effect and concentration of the binding of the recombinant HGF protein to the fusion protein coating the c-Met extracellular region, with an EC50 value of 4.326 nM; the unmodified 1H9D6 protein had an EC50 value of 2.159nM under the same conditions, see fig. 6. In order to further confirm that 2E6 can still antagonize HGF in living cells, the invention adds HGF (10nM) in a HepG2 cell line highly expressing c-Met and then continues to add 2E6(20nM), and flow experiments detect results. As a result, it was found that the addition of HGF greatly affected the binding of 2E6 to HepG2 cells, see fig. 7.

To examine the effect of 2E6 on HGF-induced phosphorylation of c-Met protein and downstream signaling, the phosphorylation response of HGF induced intracellularly was examined using HepG2 cell line. Phosphorylation results of c-Met, Gab-1, Akt and Erk1/2 proteins at different time points after co-incubation with HGF (1nM) were detected by immunoblot experiments.

Cells incubated with 1nM HGF were lysed using RIPA lysate (Beyotime Biotechnology; Cat No. P0013B) to obtain whole protein. Protein concentration was determined using the bicinchoninic acid (BCA) protein concentration assay kit (Biyun Tian; Cat. P0012, Lot No. 040717170810). After electrophoresis of the proteins, the proteins were transferred to PVDF Membrane by electrotransfer (Immobilon-P Transfer Membrane, Millipore Corporation, USA; Cat No. IPVH00010, Lot No. K3EA8230FK). Finally, development was performed by using an ultra-high sensitivity enhanced chemiluminescence ECL substrate (Thermo Scientific, USA; Cat No.34096, Lot No. TC263090).

After 1nM human HGF stimulates HepG2 cells, the phosphorylation level detection results of c-Met protein, Gab-1 protein (growth factor binding receptor protein 2 binding protein 1), Akt protein (v-Akt gene expression protein, also called PKB (protein kinase B)) and Erk protein (extracellular regulatory protein kinase) show the rapidity and the transient response of HGF/c-Met signaling pathway. The highest phosphorylation level occurred after 5 minutes of HGF addition, see fig. 8. Whereas pre-incubation with 2E6 (10nM) before HGF addition greatly reduced the phosphorylation levels of c-Met, Gab-1, Akt and Erk1/2 proteins after 5 min stimulation with HGF (0.5nM), see FIG. 9. The small molecule inhibitor adopted by the experimental positive control is c-Met/Anaplastic Lymphoma Kinase (ALK) Tyrosine Kinase Inhibitor (TKI) PF-02341066(1 mu M) (PF-02341066, the trade name of which is Crizotinib, which is a c-Met small molecule inhibitor). The results show that intracellular kinase activity can be accomplished by antagonizing the HGF/c-Met effect, or by inhibiting kinase activity directly using TKI.

Example 6 anti-tumor Activity of anti-c-Met Single-arm antibodies in HCC cell lines

Cell culture: the cells were cultured using DMEM (Dulbecco's modified Eagle's medium, Gibco Co., Ltd., USA.) medium with the addition of 10% fetal bovine serum (FBS, qualified, Australia # 10099-. Incubator humidity and 5% CO₂Supply, incubation at 37 ℃.

Cell proliferation: after plating the cells in 96-well plates, HGF was added or co-incubated with different concentrations of 2E6 and culture was continued for 72 hours.

The cell proliferation of HepG2 cells after 72 hours of action of 1nM human HGF and various concentrations of 2E6 was examined using the CCK8(WST-8 reagent, collectively referred to as 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazole monosodium salt) method, and 2E6 was found to have an effect on the proliferation of the HCC cell line HepG 2. In the groups with and without HGF, 2E6(1-25nM) has great inhibition effect on cell proliferation, and the result is related to the 2E6 concentration; the addition of exogenous HGF had only a minimal positive effect on cell proliferation and had a minor effect on the inhibition caused by 2E6, see fig. 10. These results indicate that the anti-c-Met one-armed antibody most likely inhibited cell proliferation by inhibiting the HGF autocrine cycle of HepG2 cells.

The present invention uses a series of HCC cells: HepG2, Huh-7, Hep 3B2 and SMMC-7721 were subjected to cell migration experiments: the well-penetrating chamber was purchased from BD corporation and had a density of 2.5X 10 tumor cell plates per upper chamber⁴And (4) cells. The whole experiment was incubated at 37 ℃. The HGF is added into the lower culture medium of the chamber, and the cells in the upper chamber gradually migrate to the lower chamber during 24 hours of cultureAnd (6) moving. After the culture was completed, the mobility of the cells under different conditions was evaluated by counting the number of crystal violet stained cells in a random six fields, wherein a cell migration photograph of Hep G2 cells was selected as a representative result to be displayed, and the cells were photographed under a 10-fold objective lens, and the result is shown in FIG. 11.

Unlike the results of the proliferation experiments in the Transwell experiment, the exogenous HGF greatly promotes the migration and invasion of cells, while the presence of 2E6(1-25nM) can greatly inhibit the effect. When the 2E6 concentration was higher than 5nM, the number of migrating Hep G2 cells was even lower than baseline. The above results show that both endogenous and exogenous HGF-induced cell migration can be inhibited by 2E 6.

Example 7 anti-tumor Activity of anti-c-Met Single-armed antibodies in brain glioma cell lines

The cell migration test was performed on the U87 cell line, the 2E6 antibody was placed in the upper chamber of the transwell, the HGF/SF was placed in the lower chamber of the transwell, the number of cells in the lower surface of the transwell was observed after 48h of culture, and the effect of the 2E6 antibody on the HGF/SF-induced cell migration was studied, and the results are shown in fig. 12; the result shows that 2E6 can effectively inhibit HGF/SF-induced brain glioma U87 cell migration.

Example 8 anti-c-Met one-armed antibodies inhibit HGF-induced angiogenesis

Activation of c-Met is critical in the angiogenesis process of tumors. Since HGF-induced c-Met activation ultimately triggers an endothelial cell response, 2E6 may also have an inhibitory effect on HGF-induced HUVEC cell responses.

HUVEC cells (human umbilical vein endothelial cells) were tested for cell proliferation 72 hours after exposure to 1nM human HGF and varying concentrations of 2E6 using the CCK8(WST-8 reagent, collectively: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazole monosodium salt) protocol. Endothelial cell proliferation experiments show that HGF (0.5nM) can greatly improve the activity of HUVEC cells, and 2E6(2.5-20nM) can inhibit HGF-induced HUVEC proliferation, and the inhibition effect is related to the concentration, as shown in FIG. 13.

After solubilization of Matrigel (Corning inc., MA, USA) at 4 ℃, 100 μ L was added to each well of a 96-well plate, followed by incubation overnight at 37 ℃. HUVEC cells were digested and diluted to3×10⁴100 μ L, adding HGF, antibody, or other drugs into the culture medium. The suspension was added to pre-incubated wells containing Matrigel. After incubation for 18-20 hours at 37 ℃, the capillary-like structures formed by the HUVEC cells were observed microscopically and the microtubular generation effect per well was photographed in the microscope in the bright field. In endothelial cell angiogenesis experiments, the strong pro-angiogenic effect of HGF (0.5nM) was confirmed in vitro Matrigel-mediated angiogenesis experiments, while 2E6(2.5-10nM) inhibited HGF-induced HUVEC microtubule production, see fig. 14.

Example 9 anti-c-Met single-armed antibodies inhibit growth following human tumor HepG2 xenograft

18 BALB/c nude mice between 6 and 8 weeks old were purchased from SLRC Laboratory Animal Co., Ltd and cultured in a pathogen-free Animal house of university of Hospital for one week before the experiment. The mice were closely observed during the culture process and were free to drink filtered tap water and consume standardized commercial feeds. Squirrel cage cleaning and padding replacement are all responsible for by specially-assigned people. Both animal care and experiments were approved by the institutional animal care and use committee of the university of congratulation and were conducted strictly in accordance with the committee's guidelines. All procedures were performed after sodium pentobarbital anesthesia and the operative pain was minimized as much as possible. Injection of 5X 10 per mouse⁶HepG2 cells supplemented with Matrigel (Corning, NY, USA) as adjuvant and injected subcutaneously into the right side of the body. Each group had 6 mice, for a total of 18. The final volume of the exogenous grafted tumor is 50mm³(length × width × height/2). Mice in each group were randomly assigned to control and administration groups and received intraperitoneal injections of either control vehicle or 2E6(5 or 10mg/kg) twice weekly for 4 months. All mice received intraperitoneal injections of sodium pentobarbital (500mg/kg, Sigma-Aldrich, MO, USA) for euthanasia at the time of experimental focus.

The antitumor effect of 2E6 was evaluated by tumor size following HepG2 xenograft injection in mice with 2E6 injected continuously for six weeks. Figure 15 shows that the tumor growth rate was significantly slowed by 2E6 administration. Mice receiving 2E6 injection clearly showed a reduction in xenograft tumor volume, see figure 16. 2E6 at 5mg/kg inhibited tumor volume by 44.5% + -13.3%, while 2E6 at 10mg/kg inhibited tumor volume by 65.8% + -12.5%. There was little difference in body weight among the groups of nude mice, indicating that the administration of 2E6 was relatively safe for the mice (results not shown).

Example 10 Single arm anti-c-Met antibodies inhibit growth of human tumor after U87 xenograft

The U87 nude mouse tumor model has good stability and the tumor formation rate reaches 100%, so the animal model is selected to preliminarily evaluate the anti-tumor effect of the single-arm antibody 2E 6. Administration was started 14 days after U87 cell inoculation, and divided into 2E6 injection group and normal saline group, each of 6 nude mice in each group, 2E6 was intraperitoneally injected at a concentration of 30mg/kg, formulated with normal saline, and injected 1 time at 1 day intervals, and the growth curve of the transplanted tumor (fig. 17) showed that the transplanted tumor growth in the 2E6 injection group was significantly lower than that in the control group, and 2E6 showed good antitumor effect.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

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Claims

1. An anti-c-Met single-arm antibody, consisting of 3 chains: 1 light chain, 1 heavy chain and 1 Fc-knob-His chain; the light chain is composed of the light chain variable region 1H9D6-V of the parent antibody_LSplicing with a human Kappa light chain constant region Homoc Kappa to obtain the polypeptide; the heavy chain is formed by the heavy chain variable region 1H9D6-V of the parent antibody_HThe gene is obtained by splicing with a human IgG1-hole-Flag sequence, wherein a human IgG1 heavy chain constant region generates 3 amino acid mutations to form a mortar-shaped structure; the constant region of the heavy chain of human IgG1 in the Fc-knob-His chain was mutated by 1 amino acid to form a knob-like structure.

2. The anti-c-Met single-arm antibody according to claim 1, wherein the human IgG1 heavy chain constant region has 3 amino acid mutations: T366S: L368A: Y407V; the human IgG1 heavy chain constant region was mutated from 1 amino acid to T366Y.

3. The method for preparing an anti-c-Met single-arm antibody according to claim 1, which comprises the following steps:

4. Use of the anti-c-Met single-arm antibody according to any one of claims 1 to 3 for the preparation of an anti-tumor medicament.

5. The use of the anti-c-Met single-arm antibody in the preparation of an anti-tumor drug according to claim 4, wherein the tumor is liver cancer or glioma.

6. Use of an anti-c-Met single-arm antibody according to any one of claims 1-3 for inhibiting the HGF/c-Met signaling pathway.

7. The use of an anti-c-Met single-arm antibody according to claim 6 for inhibiting the HGF/c-Met signaling pathway, wherein said use is inhibiting HGF and c-Met binding.

8. The use of an anti-c-Met single-arm antibody according to claim 6 for inhibiting HGF/c-Met signaling pathway, wherein said use is inhibition of c-Met phosphorylation.

9. The use of an anti-c-Met single-arm antibody according to claim 6 for inhibiting HGF/c-Met signaling pathway, wherein said use is inhibition of Gab-1, ERK1/2, Akt and PKB activity.

10. Use of an anti-c-Met one-armed antibody according to any one of claims 1 to 3 for inhibiting angiogenesis or inhibiting cell migration.