An igf2 fusion antibody and its construction method and application

Abstract

This invention discloses an IGF2 fusion antibody, its construction method, and its applications. Using a PD-L1 antibody as a template, the IGF2 domain is fused monovalently or bivalently to the heavy or light chain end of the PD-L1 antibody, self-assembling to form four conformations of fusion antibodies: Type A, Type A-GS, Type B, or Type B-Fab. By constructing IGF2 fusion antibodies with different conformations, the optimal IGF2 fusion antibody conformation is obtained through affinity activity and protein degradation screening. Specifically, the IGF2 of the fusion antibody exhibits nanomolar affinity activity with the IGF2 receptor on the cell surface, achieving efficient degradation of various cell surface membrane proteins (such as PD-L1, HER2, EGFR, and GPC3). This invention provides a new technological platform for membrane protein degradation.

Description

Technical Field

[0001] This invention relates to an IGF2 fusion antibody, its construction method, and its application, particularly to an IGF2 fusion antibody, its construction method, and its application in the degradation of various membrane proteins, belonging to the biomedical field. Background Technology

[0002] In recent years, targeted protein degradation technology has developed rapidly, providing new options for improving the efficacy of tumor treatment. In particular, lysosomal pathway-based targeted protein degradation strategies have further expanded the possibilities for degrading extracellular proteins and proteins associated with cell membrane diseases. Early methods utilized chemical coupling to create lysosomal-targeting chimeras (LYTACs) by combining glycopeptides bound to lysosomal receptors (IGF2R or ASGPR) with target antibodies, and later, bifunctional nucleic acid aptamers, can mediate the entry of target proteins into lysosomes and their degradation. However, the complex synthetic steps or limited in vivo half-life of these LYTAC molecules restrict their further application. 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 recombination and high protein degradation efficiency show potential for clinical applications. However, despite the same target, the structures on the surfaces of different cancer cells differ, and whether these strategies are applicable to the degradation of more tumor target proteins requires further investigation. Therefore, more effective protein degradation strategies need to be explored. Summary of the Invention

[0003] Objectives of the Invention: To address the shortcomings of existing technologies, the first objective of this invention is to provide an IGF2 fusion antibody. The second objective is to provide a method for constructing this IGF2 fusion antibody. The third objective is to provide the application of this IGF2 fusion antibody in membrane protein degradation.

[0004] Technical solution: The present invention discloses an IGF2 fusion antibody, wherein the IGF2 fusion antibody is formed by using a PD-L1 antibody as a template, and fusing the IGF2 domain in a monovalent or bivalent form to the heavy or light chain end of the PD-L1 antibody, thereby self-assembling into four configurations of fusion antibody: Type A, Type A-GS, Type B, or Type B-Fab.

[0005] Furthermore, the heavy chain sequence of Type A is shown in SEQ ID NO.4, the light chain sequence of Type A-GS is shown in SEQ ID NO.5, and the IGF2- light chain sequence of Type A is shown in 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 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.

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

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

[0010] Furthermore, Type A is an IGF2 protein gene sequence fused to the N-terminus of the heavy chain Fc domain of the PD-L1 antibody via monovalent gene recombination.

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

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

[0013] Furthermore, TypeB-Fab retains the single Fab binding domain between the antibody and the PD-L1 protein by fusing the IGF2 protein gene sequence in a bivalent manner to the N-terminus of the heavy and light chains of the PD-L1 antibody.

[0014] The application of the IGF2 fusion antibody described in this invention 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 comprising the IGF2 fusion antibody described herein.

[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] This invention first targets PD-L1 as the protein and designs several monovalent (Type A, Type A-GS) or bivalent (Type B, Type B-Fab) fusion antibodies of IGF2 fused with PD-L1 monoclonal antibodies. Comparison of their affinity for cells and target protein degradation effects demonstrates that the bivalent IGF2 fusion antibody conformation is optimal, efficiently degrading PD-L1 protein in various cell types under low concentration conditions. Furthermore, it verifies that the design strategy of the 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. Attached Figure Description

[0020] Figure 1 SDS-PAGE images of IGF2 fusion antibodies with different configurations;

[0021] Figure 2 Figure 1 shows the expression results of PD-L1 in MDA-MB-231 and SK-Hep-1 cells and the affinity results of IGF2 with SK-Hep-1 cells; where a is the expression of PD-L1 in MDA-MB-231 and SK-Hep-1 cells as determined by flow cytometry, and b is the affinity of IGF2 with IGF2R positive cells (SK-Hep-1) as determined by flow cytometry.

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

[0023] Figure 4 The image shows the PD-L1 content of MDA-MB-231 cells and HCC827 cells after 24 hours of treatment with 10 nM fusion antibody by Western blotting in Example 3. In this image, a represents MDA-MB-231 cells and b represents HCC827 cells.

[0024] Figure 5 Figure 1 shows the degradation results of different cells treated with different fusion antibodies in Example 3 using Western blotting analysis. Figure 1a shows the expression level of PD-L1 in MDA-MB-231 cells after treatment with different concentrations of Type A and Type B IGF2-LYTACs for 48 hours. Figures 2b and 3f show the PD-L1 degradation levels in MDA-MB-231, HCC827, MCF-7, SK-OV-3, and A549 cells after treatment with PD-L1 Ab, Type A, and Type B at a concentration of 100 nM for 48 hours.

[0025] Figure 6The immunofluorescence analysis in Example 4 shows the expression pathway of PD-L1 on the surface of MDA-MB-231 cell membranes after 24 hours of treatment with PD-L1 Ab, Type A, and Type B. The scale bar is 20 μm.

[0026] Figure 7 This is a diagram showing how Type B achieves targeted protein degradation via the lysosomal protein degradation pathway in Example 5. In this diagram, a is the localization map of PDL1Ab-TypeB in MDA-MB-231 cells; lysosomes are marked with Lysotracker (green), and endocytosed PDL1Ab-TypeB-Cy5 is shown in red. Scale bar: 10 μm. b is a Western blotting analysis of the effects of the lysosomal inhibitors chloroquine and bafilomycin A1 on PD-L1 degradation in Type B-treated MDA-MB-231 cells. c is an analysis of the interaction between the IGF2 and IGF2R complex (PDB: 6UM2), highlighting the key amino acids phenylalanine (F48) and leucine (L53).

[0027] Figure 8 In Example 6, a is a schematic diagram of HER2, EGFR, and GPC3 proteins on the cell surface; bg is a flow cytometry diagram showing the affinity activities of three different fusion antibodies (HER2Ab-TypeB, EGFRAb-TypeB, and GPC3Ab-TypeB) with IGF2R and target proteins on the cell surface; h is a Western blot analysis of the effect of HER2Ab-TypeB on HER2 degradation after 24 hours of treatment of SK-OV-3 cells; i is a Western blot analysis of the effect of EGFRAb-TypeB on EGFR degradation after 24 hours of treatment of SK-Hep-1 cells at different concentrations; and j is a Western blot analysis of the effect of GPC3Ab-TypeB on GPC3 degradation after 24 hours of treatment of HepG2 cells at different concentrations. Detailed Implementation

[0028] The technical solution of the present invention will be further described below with reference to 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 and 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 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) and human peripheral blood CD3 cells were also cultured. + PanT cells and immature dendritic cells (iDCs) were purchased from Shanghai Ruibai Biotechnology Co., Ltd. (Shanghai, China) and cultured in RPMI-1640 medium containing 10% FBS and 1% penicillin and streptomycin at 5% CO2 and 37°C.

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

[0032] The coding sequence for mature human IGF2 (CDS, as shown in SEQ ID NO.1) was obtained from the NCBI database (NM_000612.5), and the PD-L1 antibody sequence (Atz heavy chain sequence as shown in SEQ ID NO.2, and light chain sequence as shown in SEQ ID NO.3) was derived from atezolizumab (Atz). First, the PD-L1 antibody sequence was synthesized on the pCDNA3.4 vector (GENEVIZ, Suzhou, China). Then, the CDS sequence was linked to the light chain or the N-terminus of the Fc domain of the PD-L1 antibody via homologous recombination. The mature IGF2 protein gene sequence was fused monovalently to the N-terminus of the Fc domain of the heavy chain of the humanized PD-L1 antibody (Atezolizumab) via gene recombination, named Type A. To reduce steric hindrance and promote the relatively independent folding and function of IGF2 and the antibody, a flexible linker, G4Slinker, was introduced between IGF2 and the Fc domain. The resulting fusion antibody was named Type A-GS. To enhance the binding activity of IGF2 to cells, a bivalent IGF2 antibody fusion protein was constructed. IGF2 was fused bivalently to the N-terminus of the light chain of a PD-L1 antibody via a flexible G4S linker (SGGGGSGGGG), resulting in Type B and Type B-Fab. Type B-Fab retains only the single Fab binding domain of the antibody and the PD-L1 protein. Using the humanized PD-L1 antibody atezolizumab as a template, the mature IGF2 domain was fused monovalently or bivalently to the heavy or light chain of the antibody, self-assembling to form four conformations of fusion antibodies: Type A, Type A-GS, TType B, and Type B-Fab. Figure 1(As shown). The PD-L1 sequence was replaced with a REGN antibody against the spike protein as a control antibody, namely I2SAB. The anti-HER2 antibody (trastuzumab) was constructed according to the structure 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, centrifuged at 6000 rpm for 20 minutes, and the cell supernatant was collected. After filtration through a 0.45 μm filter membrane, the supernatant was purified using a protein G affinity column (NanoMicro, Suzhou, China). The purity of the purified protein was preliminarily identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). 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 the Type A-GS linker is shown in SEQ ID NO.7. The IGF2-heavy chain sequence of Type B-Fab is shown in SEQ ID NO.8, and the IGF2-light chain sequence of Type B-Fab is shown in SEQ ID NO.9. The heavy chain sequence of Type B is shown in SEQ ID NO.10, and the IGF2-light chain sequence of Type B 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] TypeA-GS linker:

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

[0049] AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSESGGGGSGGGGSGGGGSEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNRYTQKSLSLSPGKHHHHHH;

[0050] TypeB-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 determination of affinity

[0061] MDA-MB-231 cells and SK-Hep-1 cells were cultured to the logarithmic growth phase, respectively. The four IGF2 fusion antibodies obtained in Example 2 were added to each of the two cell types at different concentrations (0-400 nM) (1×10⁻⁶). 6Cells 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 determination of PD-L1 expression on the surface of MDA-MB-231 and SK-Hep-1 cells, the cells were directly incubated with anti-human PD-L1 PE fluorescent antibody at 4°C for 20 minutes, followed by fluorescence intensity analysis. The results are as follows: Figure 2-3 As shown, IGF2R protein is widely expressed on the surface of all cells, but its abundance varies among different cell types. In this experiment, the PD-L1-negative cell line SK-Hep-1 was used as the target cell to determine the affinity activity between the fusion antibody and cell surface IGF2R. The reported affinity of IGF2 for cell surface IGF2R is around 100 nM, which is close to the affinity value of the Type B-GS conformation measured in this experiment, approximately 72 nM (see...). Figure 2 Since both Type A and Type A-GS are IGF2 monovalent fusion proteins, their affinity for IGF2R-positive cells is slightly reduced after fusion with PD-L1 antibody, by approximately 80 nM. Figure 3 (a) but there was no significant difference between the two. To improve the binding activity of IGF2 to cells, this experiment constructed a bivalent IGF2 antibody fusion protein, fusing IGF2 to the N-terminus of the light chain of the PD-L1 antibody, namely TypeB and TypeB-Fab. TypeB-Fab retains only a single Fab binding domain of the antibody and PD-L1 protein. The results showed that its affinity activity for PD-L1 on the cell surface was lower than that of the bivalent TypeB fusion antibody (a). Figure 3 (b) The activity of the bivalent IGF2 fusion antibody was lower than that of the monovalent Type A and Type A-GS. However, in terms of affinity activity with cell surface IGF2R, the bivalent IGF2 fusion antibody showed significantly higher affinity activity with cells than the monovalent Type A and Type A-GS, with Type B reaching 5.4 nM. This indicates that the Type B conformation of the bivalent IGF2 fusion antibody maintained affinity activity with the target protein.

[0062] Example 3: Western blot assay for target protein degradation

[0063] MDA-MB-231 and HCC827 cells were cultured to the logarithmic growth phase and then inoculated at 2×10⁻⁶ cells / year. 5Cells were seeded per well in 12-well plates and cultured for 24 hours. Then, 10 nM and 100 nM of IGF2 fusion antibodies of different conformations obtained in Example 2 were added. After 24 hours, cells were collected, and RIPA lysis buffer containing 1 mM protease inhibitor was added. Cells were incubated on ice for 30 minutes, followed by high-speed centrifugation to collect the cell lysate supernatant. The supernatant was subjected to SDS-PAGE electrophoresis for 1 hour, then transferred to a membrane under constant current for 90 minutes. The PVDF membrane was blocked with 5% skim milk powder for 1 hour, followed by overnight incubation with anti-human PD-L1 primary antibody at 4°C. Finally, the membrane was incubated with anti-rabbit HRP secondary antibody at room temperature for 1 hour before imaging. The same sample processing method was used for different concentrations (0-1000 nM) of Type A and Type B antibodies degrading PD-L1 on the surface of MDA-MB-231 cells.

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

[0065] Example 4: Immunofluorescence assay for target protein degradation

[0066] Logarithmically growing MDA-MB-231 cells were seeded onto cell slides and incubated with 100 nM Type B fusion antibody for 24 hours. Cells were then washed three times with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed with PBS again, blocked with 5% bovine serum albumin solution at room temperature for 45 minutes, and then incubated overnight at 4°C with anti-human PD-L1 primary antibody. After incubation with anti-rabbit green fluorescent secondary antibody at room temperature for 1 hour, cells were stained with 10 μg / mL DAPI solution for 10 minutes. Finally, the cells were mounted with neutral resin and analyzed for protein degradation using confocal fluorescence imaging. The degradation of HER2 protein on the surface of SK-OV-3 cells by HERAB-TypeB was measured in the same manner as with PD-L1, and the results are shown below. Figure 6 As shown.

[0067] This experiment evaluated the target protein degradation capabilities of fusion antibodies with different conformations. In two PD-L1-positive cell lines (MDA-MB-231 cells and HCC827 cells), 10 nM Type B degraded approximately 50% of the PD-L1 protein, while Type A degraded approximately 25% of PD-L1 (see [link to study].) Figure 4Treatment with PD-L1 monoclonal antibodies did not significantly alter PD-L1 protein levels. However, Type A-GS and Type B-Fab treatments degraded 35% and 50% of PD-L1 in MDA-MB-231 cells, respectively, while no effective PD-L1 degradation was observed in HCC827 cells. Further investigation was conducted on the target protein degradation effects of the IGF2 monovalent fusion antibody Type A and the bivalent Type B to determine the optimal conformation.

[0068] In MDA-MB-231 cells, Type A and Type B exhibited concentration-dependent degradation of PD-L1 protein on the cell surface (see [link to relevant documentation]). Figure 5 In section a), when the fusion protein concentration was 100 nM, Type B could degrade over 70% of PD-L1, while Type A could only degrade 50% of PD-L1 at a concentration of 100 nM. Further, the degradation effects of Type A and Type B at 100 nM concentrations on PD-L1 on the surface of several different cancer cells were investigated. The results showed that Type B exhibited better degradation of 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 line (SK-OV-3). Specifically, 80% of PD-L1 in MCF-7 cells was degraded, while the Type A conformation had almost no degradation effect on PD-L1 on the surface of non-small cell lung cancer cells (see section a). Figure 5 (Zhong bf). Immunofluorescence experiments also demonstrated that Type B exhibited superior degradation of PD-L1 compared to the Type A conformation (see...). Figure 6 These results indicate that Type B is the optimal configuration for the IGF2 fusion antibody.

[0069] Example 5 Lysosomal colocalization experiment

[0070] The IGF2 fusion antibody TypeB 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. After ultrafiltration purification to remove free Cy5, TypeB-Cy5 protein molecules were obtained. Logarithmically growing MDA-MB-231 cells were seeded onto cell slides. After 24 hours, 100 nM TypB-Cy5 was added and incubated at 4°C for 30 minutes. The cells were washed three times with PBS, then 50 nM lysosomal green fluorescent dye (Lysotracker) was added and incubated at 37°C for 30 minutes. The cells were washed three times with PBS, and finally stained with 10 μg / mL Hoechst at room temperature for 10 minutes. After washing with PBS, confocal imaging was performed.

[0071] To confirm that the IGF2 fusion antibody mediates lysosomal degradation of target proteins through binding to IGF2R, the IGF2 fusion antibody was labeled with Cy5 fluorescence (PDL1Ab-Type B). After treating MDA-MB-231 cells for 2 hours, co-localization analysis with lysosomes in the cells was performed. The results are as follows: Figure 7 As shown, Figure 7 The results showed that the red PDL1Ab-Type B exhibited significant orange-yellow co-localization fluorescence with the green fluorescently labeled lysosomes within the cells (see...). Figure 7 In section a, lysosomes are labeled with Lysotracker (green), and endocytosed PDL1Ab-TypeB-Cy5 is shown in red (scale bar: 10 μm). When MDA-MB-231 cells were treated with the lysosomal inhibitors bafilomycin A1 and chloroquine, subsequent treatment with Type B did not effectively degrade PD-L1 (see [link]). 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 leucine at position 53 (L53) in IGF2 are key amino acids for binding to this receptor (see [link]). Figure 7 In Figure c, c represents the analysis of the interaction between the IGF2 and IGF2R complex (PDB:6UM2), with key amino acids phenylalanine (F48) and leucine (L53) highlighted. Therefore, both F48 and L53 of IGF2 in PDL1Ab-TypeB were mutated to aspartic acid (D), i.e., PDL1Ab-TypeB-FLmut (the IGF2-light chain-FLmut sequence of TypeB-FLmut is shown in SEQ ID NO.12). PDL1Ab-TypeB-FLmut can still bind to PD-L1 molecules on the cell surface, but its binding activity with IGF2R is significantly reduced. In addition, Western blot results showed that the degradation effect of target protein by PDL1Ab-TypeB-FLmut was significantly reduced, with only 15% of PD-L1 being degraded at 500 nM. The above results fully demonstrate that the TypeB conformation of the IGF2 fusion antibody degrades target protein by inducing lysosomal degradation through targeting IGF2R.

[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 antibody

[0076] To demonstrate the universality of the Type B conformation of the bivalent IGF2 fusion antibody, antibodies targeting different proteins were fused with IGF2 for validation. For example... Figure 8 As shown in Figure a, HER2, EGFR, and GPC3 are all membrane proteins highly expressed in various tumors and are closely related to tumor development and progression. Cell affinity analysis results showed that the fusion antibodies formed by fusing the light chains of three humanized target antibodies with IGF2 (HER2Ab-TypeB (heavy chain sequence as SEQ ID NO.13, IGF2-light chain sequence as SEQ ID NO.14), EGFRAb-TypeB (heavy chain sequence as SEQ ID NO.15, IGF2-light chain sequence as SEQ ID NO.16), and GPC3Ab-TypeB (heavy chain sequence as SEQ ID NO.17, IGF2-light chain sequence as SEQ ID NO.18)) all maintained their binding activity to the target antigen, and their affinity for IGF2R-positive cells was within the range of 15-30 nM. Figure 8 (bg). Regarding target protein degradation, HER2Ab-TypeB at 100 nM effectively degraded 73% of HER2 on the surface of SK-OV-3 cells. Figure 8 In the middle (h), EGFRAb-TypeB can degrade 73% of EGFR on the surface of SK-Hep-1 cells at 25 nM. Figure 8 GPC3Ab-TypeB also exhibited good protein degradation activity, degrading 71% of the GPC3 protein on the surface of HepG2 cells at 50 nM. Figure 8(j). In 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 applicable to a wider range of protein degrading agents.

[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 for degrading membrane proteins, characterized in that, The IGF2 fusion antibody is obtained by using a PD-L1 antibody as a template and fusing the IGF2 protein gene sequence to the N-terminus of the light chain of the PD-L1 antibody in a bivalent manner through a G4S linker to obtain Type B; the membrane protein is the PD-L1 protein, the heavy chain sequence of Type B is shown in SEQ ID NO.10, and the IGF2-light chain sequence of Type B is shown in SEQ ID NO.

11.

2. A pharmaceutical composition, characterized in that, Includes the IGF2 fusion antibody as described in claim 1.

3. The pharmaceutical composition according to claim 2, characterized in that, It also includes one or more pharmaceutically acceptable carriers, diluents, or excipients.

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