IGF2 fusion antibody as well as construction method and application thereof

Abstract

The invention discloses an IGF2 fusion antibody and a construction method and application thereof.A PD-L1 antibody is used as a template, an IGF2 structural domain is fused to the heavy chain or light chain end of the PD-L1 antibody in a monovalent or bivalent mode, and the fusion antibody TypeA, TypeA-GS, TypeB or TypeB-Fab of four configurations is formed through self-assembly. IGF2 fusion antibodies with different configurations are constructed, and the optimal IGF2 fusion antibody configuration is obtained through screening of affinity activity and protein degradation, that is, the IGF2 of the fusion antibody and an IGF2 receptor on the cell surface have nanomole-level affinity activity, and efficient degradation of various membrane proteins (such as PD-L1, HER2, EGFR and GPC3) on different cell surfaces is realized. The invention provides a new technical platform for membrane protein degradation.

Description

Technical Field

[0001] The present invention relates to an IGF2 fusion antibody and a construction method and application thereof, in particular to an IGF2 fusion antibody and a construction method thereof and application thereof in the degradation of various membrane proteins, belonging to the field of biomedicine. Background Art

[0002] In recent years, targeted protein degradation technology has developed rapidly, providing a new option for improving the therapeutic effect of tumors. In particular, the targeted protein degradation strategy based on the lysosomal pathway has further expanded the possibility of degrading extracellular proteins and cell membrane-related disease proteins. The earliest lysosomal targeting chimeras (LYTACs) obtained by chemically coupling glycopeptide molecules that bind to lysosomal receptors (IGF2R or ASGPR) with target antibodies, as well as the later developed bifunctional nucleic acid aptamers, can mediate the entry of target proteins into lysosomes and degradation, but the complex synthesis steps or limited in vivo half-life limit the further application of these LYTAC molecules. Protein degradation chimeras designed based on bispecific antibodies or antibody fusion proteins, such as TransTACs or KineTACs, can also effectively degrade membrane proteins. Their molecular recombinantity and high protein degradation efficiency have potential clinical application prospects, but although the same target has different structures on the surface of different cancer cells, whether its strategy is applicable to the degradation of more tumor target proteins needs further study. Therefore, more effective protein degradation strategies need to be explored. Summary of the invention

[0003] Purpose of the invention: In view of the deficiencies of the prior art, the first purpose of the present invention is to provide an IGF2 fusion antibody. The second purpose of the present invention is to provide a method for constructing the IGF2 fusion antibody. The third purpose of the present invention is to provide the application of the IGF2 fusion antibody in membrane protein degradation.

[0004] Technical solution: The IGF2 fusion antibody described in the present invention uses the PD-L1 antibody as a template, and the IGF2 domain is fused to the heavy chain or light chain end of the PD-L1 antibody in a monovalent or bivalent form, and self-assembles to form four configurations of fusion antibodies Type A, Type A-GS, Type B or Type B-Fab.

[0005] Furthermore, the heavy chain sequence of TypeA is shown as SEQ ID NO.4, the light chain sequence of TypeA-GS is shown as SEQ ID NO.5, and the IGF2-light chain sequence of TypeA is shown as SEQ ID NO.6.

[0006] Furthermore, the TypeA-GS linker sequence is shown in SEQ ID NO.7.

[0007] Furthermore, the IGF2-heavy chain sequence of Type B-Fab is shown as SEQ ID NO.8, and the IGF2-light chain sequence of Type B-Fab is shown as SEQ ID NO.9.

[0008] Furthermore, the heavy chain sequence of Type B is shown as SEQ ID NO.10, and the IGF2-light chain sequence of Type B is shown as SEQ ID NO.11.

[0009] The method for constructing the IGF2 fusion antibody of the present invention comprises synthesizing the PD-L1 antibody sequence on the pCDNA3.4 vector, and then connecting the IGF2 coding sequence to the N-terminus of the light chain or the N-terminus of the heavy chain Fc domain of the PD-L1 antibody by homologous recombination.

[0010] Furthermore, Type A is a type A in which the IGF2 protein gene sequence is genetically recombinantly fused to the N-terminus of the heavy chain Fc domain of the PD-L1 antibody in a monovalent form.

[0011] Furthermore, TypeA-GS is obtained by fusing the IGF2 protein gene sequence to the N-terminus of the heavy chain Fc domain of the PD-L1 antibody in a monovalent form via a linker G4S linker.

[0012] Furthermore, Type B is obtained by fusing the IGF2 protein gene sequence to the N-terminus of the light chain of the PD-L1 antibody in a bivalent form via a linker G4S linker.

[0013] Furthermore, Type B-Fab retains the single Fab binding domain of the antibody and the PD-L1 protein, and the IGF2 protein gene sequence is fused to the N-terminus of the heavy chain and light chain of the PD-L1 antibody in a bivalent form.

[0014] The invention discloses an application of the IGF2 fusion antibody in the preparation of membrane protein degradation drugs.

[0015] Furthermore, the membrane protein is PD-L1 protein, HER2, EGFR and / or GPC3.

[0016] The present invention also includes a pharmaceutical composition, which includes the IGF2 fusion antibody of the present invention.

[0017] Furthermore, the pharmaceutical composition also includes one or more pharmaceutically acceptable carriers, diluents or excipients.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0019] The present invention firstly takes PD-L1 as the target protein, designs several monovalent (TypeA, TypeA-GS) or bivalent fusion antibodies (TypeB, TypeB-Fab) of IGF2 fused PD-L1 monoclonal antibodies, and proves that the bivalent IGF2 fusion antibody has the best configuration through the comparison of its affinity activity to cells and target protein degradation effect, and can efficiently degrade PD-L1 protein in various cells under low concentration conditions. Furthermore, it is verified that the design strategy of bivalent IGF2 fusion antibody is also applicable to the efficient degradation of other tumor-related proteins such as HER2, EGFR and GPC3, providing a new platform for protein degradation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the SDS-PAGE picture of IGF2 fusion antibody with different configurations;

[0021] Figure 2 The results of PD-L1 expression in MDA-MB-231 and SK-Hep-1 cells and the affinity of IGF2 to SK-Hep-1 are shown in Figure 1; wherein, a is the expression of PD-L1 in MDA-MB-231 and SK-Hep-1 cells measured by flow cytometry, and b is the affinity of IGF2 to IGF2R-positive cells (SK-Hep-1) measured by flow cytometry;

[0022] Figure 3 The results of flow cytometry determination of the affinity activity of different fusion antibodies with IGF2R and PD-L1 on the cell surface, where a is IGF2R and b is PD-L1;

[0023] Figure 4 This is a graph showing the PD-L1 content of MDA-MB-231 cells and HCC827 cells after 24-hour treatment with 10 nM fusion antibody as measured by WB in Example 3, wherein a is MDA-MB-231 cells and b is HCC827 cells;

[0024] Figure 5 . is a graph showing the degradation results of different cells treated with different fusion antibodies by WB analysis in Example 3; wherein a is a graph showing the expression level of PD-L1 in MDA-MB-231 cells after being treated with different concentrations of TypeA and TypeB IGF2-LYTACs for 48 hours; bf are graphs showing the degradation level of PD-L1 in MDA-MB-231, HCC827, MCF-7, SK-OV-3 and A549 cells after being treated with PD-L1 Ab, TypeA and TypeB at a concentration of 100 nM for 48 hours;

[0025] Figure 6The immunofluorescence analysis of PD-L1 expression on the surface of MDA-MB-231 cell membrane after 24 hours of treatment with PD-L1 Ab, TypeA and TypeB in Example 4, where the scale bar is 20 μm;

[0026] Figure 7 : This is a diagram of targeted protein degradation of TypeB through the lysosomal protein degradation pathway in Example 5; wherein, a is a localization diagram of PDL1Ab-TypeB in MDA-MB-231 cells; lysosomes are labeled by Lysotracker (green), and the internalized PDL1Ab-TypeB-Cy5 is displayed in red, scale bar: 10 μm, b is a WB analysis of the effects of lysosomal inhibitors chloroquine and bafilomycin A1 on PD-L1 degradation in TypeB-treated MDA-MB-231 cells, and c is an analysis of the interaction between IGF2 and IGF2R complexes (PDB: 6UM2), with key amino acids phenylalanine (F48) and leucine (L53) marked;

[0027] Figure 8 In Example 6, a is a schematic diagram of HER2, EGFR and GPC3 proteins on the cell surface, bg are respectively affinity activity diagrams of three different fusion antibodies (HER2Ab-TypeB, EGFRAb-TypeB, GPC3Ab-TypeB) with cell surface IGF2R and target protein determined by flow cytometry, h is a WB analysis of the effect of HER2Ab-TypeB on HER2 degradation after treating SK-OV-3 cells for 24 hours, i is a WB analysis of the effect of EGFRAb-TypeB on EGFR degradation at different concentrations in SK-Hep-1 cells for 24 hours, j is a WB analysis of the effect of GPC3Ab-TypeB on GPC3 degradation at different concentrations in HepG2 cells for 24 hours. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0029] Example 1 Cell culture

[0030] Expi 293F suspension cells (Thermo Scientific, MA, USA) were cultured in serum-free medium (OPMbiosciences, Shanghai, China) at 37°C, 5% CO2 in a shaker at 150 rpm. Human breast cancer cell lines MDA-MB-231 and MCF-7, human non-small cell lung cancer cell line A549, and human liver cancer cell line SK-Hep-1 were cultured in DMEM medium containing 10% FBS and 1% penicillin-streptomycin, respectively. Human non-small cell lung cancer cell line HCC827 and human ovarian cancer cell line SK-OV-3 were cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin, respectively. All of the above cells were obtained from the cell bank of the Chinese Academy of Sciences and cultured in a constant temperature and humidity incubator at 37°C and 5% CO2. Human peripheral blood lymphocytes (PBMCs) cells, human peripheral blood CD3 + PanT cells and immature dendritic cells (iDC) were purchased from Shanghai Rubai Biotechnology (Shanghai, China) and cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin-streptomycin at 5% CO2 and 37°C.

[0031] Example 2 Expression and purification of IGF2 fusion antibody

[0032] The coding sequence of mature human IGF2 (CDS, sequence as shown in SEQ ID NO.1) comes from the NCBI database (NM_000612.5), and the PD-L1 antibody sequence (Atz heavy chain sequence as shown in SEQ ID NO.2, light chain sequence as shown in SEQ ID NO.3) comes from Atezolizumab (Atz). First, the PD-L1 antibody sequence was synthesized on the pCDNA3.4 vector (GENEVIZ, Suzhou, China), and then the CDS sequence was connected to the light chain or Fc domain N-terminus of the PD-L1 antibody by homologous recombination, and the mature IGF2 protein gene sequence was fused to the N-terminus of the heavy chain Fc domain of the humanized PD-L1 antibody (Atezolizumab) in a monovalent form by gene recombination, named Type A. In order to reduce steric hindrance and promote IGF2 and antibody to fold and function relatively independently, a flexible linker, namely G4Slinker, was introduced between IGF2 and Fc, and the resulting fusion antibody was named TypeA-GS. In order to improve the binding activity of IGF2 to cells, a bivalent IGF2 antibody fusion protein was constructed, that is, IGF2 was fused to the N-terminus of the light chain of the PD-L1 antibody in a bivalent form through a flexible linker G4S linker (SGGGGSGGGG), that is, Type B and Type B-Fab were obtained, among which Type B-Fab only retained a single Fab binding domain of the antibody and the PD-L1 protein. That is, the humanized PD-L1 antibody atezolizumab was used as a template, and the mature domain of IGF2 was fused to the heavy chain or light chain end of the antibody in a monovalent or bivalent form, and self-assembled to form four types of fusion antibodies: Type A, TypeA-GS, TTypeB, TypeB-Fab, (such as Figure 1As shown). The PD-L1 sequence was replaced with the REGN antibody against the spike protein as a control antibody, namely I2SAB. The anti-HER2 antibody (trastuzumab) was constructed according to the structural form of Type B, namely HER2Ab-TypeB. The pCDNA3.4 plasmid was transiently transfected into Expi 293F suspension cells using PEI (Life-iLab, Shanghai, China). On the sixth day after transfection, the cell suspension was collected and centrifuged at 6000 rpm for 20 minutes. The cell supernatant was collected, filtered with a 0.45 micron filter membrane, and purified with a protein G affinity column (NanoMicro, Suzhou, China). The purified protein was preliminarily identified for purity by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Among them, the heavy chain sequence of Type A is shown in SEQ ID NO.4, the light chain sequence is shown in SEQ ID NO.5, and the IGF2-light chain sequence is shown in SEQ ID NO.6. The sequence of TypeA-GS linker is shown in SEQ ID NO.7. The IGF2-heavy chain sequence of TypeB-Fab is shown in SEQ ID NO.8, and the IGF2-light chain sequence of TypeB-Fab is shown in SEQ ID NO.9. The heavy chain sequence of TypeB is shown in SEQ ID NO.10, and the IGF2-light chain sequence of TypeB is shown in SEQ ID NO.11.

[0033] IGF2 (SEQ ID NO.1):

[0034] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCDRSCDDALLETYCATPAKSES;

[0035] Atz:

[0036] Heavy chain (SEQ ID NO.2):

[0037] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK;

[0038] Light chain (SEQ ID NO.3):

[0039] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0040] TypeA:

[0041] Heavy chain (SEQ ID NO.4):

[0042] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK;

[0043] Light chain (SEQ ID NO.5):

[0044] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0045] IGF2 - Light chain (SEQ ID NO.6):

[0046] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK;

[0047] Type A - GS linker:

[0048] IGF2 - light chain (GS linker) (SEQ ID NO.7):

[0049] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGKHHHHHH;

[0050] Type B - Fab:

[0051] IGF2 - heavy chain (SEQ ID NO.8):

[0052] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVHHHHHH;

[0053] IGF2 - light chain (SEQ ID NO.9):

[0054] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECHHHHHH;

[0055] Type B:

[0056] Heavy chain (SEQ ID NO.10):

[0057] EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGT LVTVSSASTKGSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK;

[0058] IGF2-light chain (SEQ ID NO.11):

[0059] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0060] Example 2 Flow cytometry to determine affinity

[0061] MDA-MB-231 cells and SK-Hep-1 cells were cultured to the logarithmic growth phase, and the four IGF2 fusion antibodies obtained in Example 2 were added to the two cells at different concentrations (0-400 nM) (1×10 6The cells were incubated at 4°C for 30 minutes, washed with 2% FBS-PBS, and then incubated with anti-human IgG (H+L) fluorescent antibody at 4°C for 20 minutes. After washing with 2% FBS-PBS, the cells were resuspended and the affinity activity of the fusion antibody to the cells was measured by flow cytometry (CytoFLEX, Beckman, USA). For the expression of PD-L1 on the surface of MDA-MB-231 and SK-Hep-1 cells, the fluorescence intensity was analyzed after direct incubation with anti-human PD-L1 PE fluorescent antibody at 4°C for 20 minutes. The results are shown in Figure 2. Figure 2-3 As shown. IGF2R protein is ubiquitously expressed on the surface of all cells, but the abundance varies from cell to cell. In this experiment, the PD-L1 negative cell line SK-Hep-1 was used as the target cell to determine the affinity activity of the fusion antibody and the cell surface IGF2R. The reported affinity of IGF2 to the cell surface IGF2R is about 100nM, which is close to the affinity value of the TypeB-GS configuration measured in this experiment, about 72nM (see Figure 2 Since TypeA and TypeA-GS are both IGF2 monovalent fusion proteins, their affinity to IGF2R-positive cells is slightly reduced after fusion with PD-L1 antibody, about 80nM ( Figure 3 In a), there was no significant difference between the two. In order to improve the binding activity of IGF2 to cells, this experiment constructed a bivalent IGF2 antibody fusion protein, fused IGF2 to the N-terminus of the light chain of the PD-L1 antibody, namely Type B and Type B-Fab. Among them, Type B-Fab only retains a single Fab binding domain of the antibody and PD-L1 protein. The results showed that its affinity activity with cell surface PD-L1 was lower than that of the bivalent Type B fusion antibody ( Figure 3 b), and also lower than TypeA and TypeA-GS. However, in terms of affinity activity with cell surface IGF2R, the affinity activity of the bivalent IGF2 fusion antibody with cells was significantly higher than that of the monovalent TypeA and TypeA-GS, of which TypeB was 5.4nM, indicating that the bivalent IGF2 fusion antibody TypeB configuration maintained affinity activity with the target protein.

[0062] Example 3 Immunoblotting to determine target protein degradation

[0063] MDA-MB-231 and HCC827 cells were cultured to the logarithmic growth phase and then plated at 2×10 5Each well was inoculated into a 12-well plate. After culturing for 24 hours, 10 nM and 100 nM of IGF2 fusion antibodies of different configurations obtained in Example 2 were added respectively. After 24 hours, the cells were collected, and RIPA lysis buffer containing 1 mM protease inhibitor was added respectively. The cells were placed on ice for 30 minutes, and then the cell lysis supernatant was collected by high-speed centrifugation. The supernatant was subjected to SDS-PAGE electrophoresis for 1 hour, and then the membrane was transferred at a constant flow for 90 minutes. The PVDF membrane was blocked with 5% skim milk powder for 1 hour, and then anti-human PD-L1 primary antibody was added and incubated overnight at 4°C. Finally, the membrane was developed and imaged after incubation at room temperature for 1 hour with anti-rabbit HRP secondary antibody. For the degradation of PD-L1 on the surface of MDA-MB-231 cells at different concentrations (0-1000 nM) of TypeA and TypeB, the samples were processed in the same way.

[0064] For TypeA and TypeB, a working concentration of 100nM was used to degrade PD-L1 on the surface of A549, MCF-7, and SK-OV-3 cells. For TypeB, a working concentration of 10nM was used to degrade PD-L1 on the surface of MDA-MB-231 cells at different time points (0, 2, 6, 12, 24, 48h). The degradation of HER2 protein on the surface of SK-OV-3 cells by HERAB-TypeB was the same as that of PD-L1. The results are shown in Figure 4-5 shown.

[0065] Example 4 Immunofluorescence assay for target protein degradation

[0066] MDA-MB-231 cells in the logarithmic growth phase were inoculated onto cell slides, and 100 nM Type B fusion antibody was added 24 hours later for incubation for 24 hours. The cells were then washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed with PBS, blocked with 5% bovine serum albumin solution at room temperature for 45 minutes, and then incubated with anti-human PD-L1 primary antibody at 4°C overnight. After incubation with anti-rabbit green fluorescent secondary antibody at room temperature for 1 hour, 10 μg / mL DAPI solution was added for staining for 10 minutes. Finally, the slides were sealed with neutral resin for confocal fluorescence imaging to analyze the protein degradation effect. The degradation assay of HERAB-TypeB on the surface of HER2 protein on SK-OV-3 cells is the same as that of PD-L1, and the results are shown in Figure 6 shown.

[0067] This experiment evaluated the target protein degradation ability of fusion antibodies of different configurations. In two PD-L1 positive cell lines (MDA-MB-231 cells and HCC827 cells), 10nM Type B degraded about 50% of PD-L1 protein, and Type A degraded about 25% of PD-L1 (see Figure 4), while PD-L1 monoclonal antibody treatment did not cause significant changes in PD-L1 protein levels. However, TypeA-GS and TypeB-Fab treatment groups were able to degrade 35% and 50% PD-L1 in MDA-MB-231 cells, respectively, while no effective PD-L1 degradation was observed in HCC827 cells. Further studies were conducted on the target protein degradation effects of IGF2 monovalent fusion antibodies TypeA and bivalent TypeB to determine the optimal configuration.

[0068] In MDA-MB-231 cells, Type A and Type B showed concentration-dependent degradation of cell surface PD-L1 protein (see Figure 5 In a), when the fusion protein concentration is 100nM, TypeB can degrade more than 70% of PD-L1, while TypeA can only degrade 50% of PD-L1 at a concentration of 100nM. Furthermore, the degradation effects of TypeA and TypeB at a concentration of 100nM on the surface of PD-L1 in several different cancer cells were investigated. The results showed that TypeB showed a good degradation effect on PD-L1 in breast cancer cell lines (MDA-MB-231, MCF-7), non-small cell lung cancer cell lines (HCC827, A549) and ovarian cancer cell lines (SK-OV-3), among which 80% of PD-L1 was degraded in MCF-7, while the TypeA configuration had almost no degradation effect on PD-L1 on the surface of non-small cell lung cancer cells (see Figure 5 Immunofluorescence experiments also demonstrated that Type B is superior to Type A in degrading PD-L1 (see Figure 6 ). These results indicate that Type B is the optimal configuration for IGF2 fusion antibody.

[0069] Example 5 Lysosome co-localization experiment

[0070] The IGF2 fusion antibody Type B purified in Example 2 was reacted with Cyanine5-NHS (Cy5-NHS) fluorescein at a molar ratio of 1:10 at room temperature for 1 hour, and then ultrafiltration was purified to remove free Cy5 to obtain TypeB-Cy5 protein molecules. MDA-MB-231 cells in the logarithmic growth phase were inoculated on a cell slide, and 100nM TypB-Cy5 was added after 24 hours and incubated at 4°C for 30 minutes, washed three times with PBS, and then 50nM lysosomal green fluorescent dye (Lysotracker) was added and incubated at 37°C for 30 minutes, washed three times with PBS, and finally stained with 10μg / mL Hoechst at room temperature for 10 minutes, and confocal imaging was performed after washing with PBS.

[0071] To confirm that IGF2 fusion antibody mediates lysosomal degradation of target protein by binding to IGF2R, IGF2 fusion antibody was labeled with Cy5 fluorescence (PDL1Ab-TypeB) and co-localized with lysosomes in MDA-MB-231 cells after treating them for 2 hours. Figure 7 As shown, Figure 7 The results showed that the red PDL1Ab-TypeB and the green fluorescent labeled lysosomes in the cells had obvious orange-yellow co-localization fluorescence (see Figure 7 In a, lysosomes are labeled with Lysotracker (green), and the internalized PDL1Ab-TypeB-Cy5 is shown in red, scale bar: 10 μm). When MDA-MB-231 cells were treated with lysosomal inhibitors bafilomycin A1 and chloroquine, TypeB treatment failed to effectively degrade PD-L1 (see Figure 7 b). Under neutral conditions, IGF2R requires its three domains (6 / 8 / 11) to form a spatial pocket to accommodate the IGF2 molecule, and the phenylalanine at position 48 (F48) and the leucine at position 53 (L53) in IGF2 are the key amino acids for binding to the receptor (see Figure 7 c, c in the figure are for analyzing the interaction between IGF2 and IGF2R complex (PDB: 6UM2), and the key amino acids phenylalanine (F48) and leucine (L53) are marked. Therefore, F48 and L53 of IGF2 in PDL1Ab-TypeB are mutated to aspartic acid (D), that is, PDL1Ab-TypeB-Flmut (the IGF2-light chain-FLmut sequence of TypeB-Flmut is as shown in SEQ ID NO.12). PDL1Ab-TypeB-FLmut can still bind to the PD-L1 molecule on the cell surface, but the binding activity with IGF2R is significantly reduced. In addition, WB results show that the effect of PDL1Ab-TypeB-FLmut in degrading the target protein is significantly reduced, and only 15% of PD-L1 is degraded at 500nM. The above results fully illustrate that the TypeB conformation of IGF2 fusion antibody degrades the target protein by targeting IGF2R to induce lysosomal degradation.

[0072] TypeB-FLmut:

[0073] IGF2-light chain-Flmut (SEQ ID NO.12):

[0074] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCDRSCDDALLETYCATPAKSESGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0075] Example 6 Study on the targeted degradation of different target proteins by IGF2-fusion antibodies

[0076] To prove the universality of the Type B configuration of the bivalent IGF2 fusion antibody, antibodies of different target proteins were fused with IGF2 for verification. Figure 8 As shown in a, HER2, EGFR and GPC3 are membrane proteins that are highly expressed in a variety of tumors and are closely related to the occurrence and development of tumors. The results of cell affinity activity analysis showed that the fusion antibodies after the three humanized target antibody light chains were fused with IGF2 (HER2Ab-TypeB (heavy chain sequence such as SEQ ID NO.13, IGF2-light chain sequence such as SEQ ID NO.14), EGFRAb-TypeB (heavy chain sequence such as SEQ ID NO.15, IGF2-light chain sequence such as SEQ ID NO.16), GPC3Ab-TypeB (heavy chain sequence such as SEQ ID NO.17, IGF2-light chain sequence such as SEQ ID NO.18)) can all maintain the binding activity to the target antigen, and the affinity with IGF2R positive cells is in the range of 15-30nM ( Figure 8 In terms of target protein degradation, HER2Ab-TypeB can effectively degrade 73% of HER2 on the surface of SK-OV-3 cells at 100nM ( Figure 8 h), while EGFRAb-TypeB could degrade 73% of EGFR on the surface of SK-Hep-1 cells at 25 nM ( Figure 8 GPC3Ab-TypeB also showed good protein degradation, and could degrade 71% of GPC3 protein on the surface of HepG2 cells at 50 nM ( Figure 8In summary, this bivalent IGF2 protein-linked target antibody can maintain the original target antigen binding activity and good protein degradation effect, and its structure is potentially suitable for more protein degraders.

[0077] HER2Ab-TypeB:

[0078] Heavy chain (SEQ ID NO.13):

[0079] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKCLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQ GTLVTVSSASTKGSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK THTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKT ISKAKGQPREPQVCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK;

[0080] IGF2-light chain (SEQ ID NO.14):

[0081] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSDIQMTQSPSSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSR SGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGCGTKVEIKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0082] EGFRAb-Type B:

[0083] Heavy chain (SEQ ID NO.15):

[0084] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK;

[0085] IGF2 - Light chain (SEQ ID NO.16):

[0086] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPCDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC;

[0087] GPC3Ab-Type B:

[0088] Heavy chain (SEQ ID NO.17):

[0089] EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYSMNWVRQAPGKCLEWVSLISSNSSYIYYADSVKGRFTISRDNAKNSLDLQMNSLRAEDTAVYYCLTGGFDYWGQGTLVTVSSASTKGPSVCPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGK;

[0090] IGF2 - light chain (SEQ ID NO.18):

[0091] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSDIVMTQSPLSLPVTLGQPASISCRSSQSLVYNNGNTYLHWFQQRPGQSPRRLIYKVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQHTHWPTFGCGTKVEIKR。

Claims

1. An IGF2 fusion antibody, characterized in that: The IGF2 fusion antibody uses the PD-L1 antibody as a template, and fuses the IGF2 domain to the heavy chain or light chain end of the PD-L1 antibody in a monovalent or bivalent form, and self-assembles to form four configurations of fusion antibodies Type A, Type A-GS, Type B or Type B-Fab.

2. The method for constructing the IGF2 fusion antibody according to claim 1, characterized in that: The method comprises synthesizing the PD-L1 antibody sequence on the pCDNA3.4 vector, and then connecting the IGF2 coding sequence to the N-terminus of the light chain or the N-terminus of the heavy chain Fc domain of the PD-L1 antibody by homologous recombination.

3. The construction method according to claim 2, characterized in that: Type A is a monovalent fusion of the IGF2 protein gene sequence to the N-terminus of the heavy chain Fc domain of the PD-L1 antibody through genetic recombination.

4. The construction method according to claim 2, characterized in that: TypeA-GS is obtained by fusing the IGF2 protein gene sequence to the N-terminus of the heavy chain Fc domain of the PD-L1 antibody in a monovalent form via a linker G4S linker.

5. The construction method according to claim 2, characterized in that: Type B is obtained by fusing the IGF2 protein gene sequence to the N-terminus of the light chain of the PD-L1 antibody in a bivalent form via a linker G4S linker.

6. The construction method according to claim 2, characterized in that: Type B-Fab retains the single Fab binding domain of the antibody and PD-L1 protein, and is obtained by fusing the IGF2 protein gene sequence in a bivalent form to the N-terminus of the heavy chain and light chain of the PD-L1 antibody.

7. Use of the IGF2 fusion antibody according to claim 1 in the preparation of membrane protein degradation drugs.

8. The use according to claim 7, characterized in that: The membrane protein is PD-L1 protein, HER2 protein, EGFR protein and / or GPC3 protein.

9. A pharmaceutical composition, characterized in that Comprising the IGF2 fusion antibody according to claim 1.

10. The pharmaceutical composition according to claim 9, characterized in that One or more pharmaceutically acceptable carriers, diluents or excipients are also included.

Images