Use of a protein conjugate in membrane protein degradation and method for membrane protein degradation

Abstract

This invention discloses the application of a protein conjugate in membrane protein degradation and a method for membrane protein degradation. The protein conjugate comprises a carrier and an antibody; the antibody can be effectively conjugated to a multivalent antibody via the carrier; membrane protein degradation includes the protein conjugate mediating the cross-linking of the target protein to form aggregates, followed by degradation of the target protein via the proteasome and / or lysosome degradation pathways. This protein conjugate can effectively degrade membrane proteins, and throughout the process, it does not rely on degradation ligands targeting the proteasome or lysosome receptor, thus possessing strong applicability.

Description

Technical Field

[0001] This invention relates to the application of a protein conjugate in membrane protein degradation and a method for membrane protein degradation. Background Technology

[0002] Abnormal protein expression levels are closely related to the development and progression of diseases such as cancer. Most traditional small molecule and antibody drugs require binding to specific sites on target proteins to block or modulate their function. However, the activity of many proteins cannot be altered in this way because 80% of proteins in human cells lack such specific sites. In recent years, protein degraders have offered new opportunities to target these proteins that were traditionally considered undrugable. LYTAC is one such example. [1,2] LYTAC primarily degrades secretory proteins and membrane-associated proteins, which together account for 40% of the human proteome. LYTAC is a bifunctional molecule that simultaneously binds to its target protein and a lysosomal targeting chimera (LTR) receptor on the cell surface, forming a ternary complex. This allows the protein to be internalized via clathrin-mediated endocytosis. After engulfment, a transport vesicle forms, which transports the complex to the lysosome. The low pH conditions within the lysosome cause the complex to dissociate, and the protein substrate is then degraded by protein-degrading enzymes within the lysosome. The LTR itself is then re-transported back to the cell membrane via the Golgi apparatus for recycling. LYTAC degrades proteins before they can function, thus avoiding potential activation of other downstream pathways.

[0003] Currently, in addition to LYTAC utilizing CI-M6PR and ASGPR, various lysosomal targeting technologies based on different lysosomal receptors have been developed, such as IFLD utilizing integrin receptors. [3] DENTAC, utilizing scavenger receptors [4] Using RNF43 AbTAC [5] Using CXCL12R's KineTAC [6] These degradation methods can effectively achieve the degradation of specific membrane proteins.

[0004] However, these membrane protein degradation technologies all rely on lysosomal targeting receptors, and their practical application still has the following limitations: ① Overactivation of lysosomal targeting receptors may affect their normal biological functions; ② The expression of these lysosomal targeting receptors varies greatly in different cells and tissues. For example, ASGPR is mainly expressed in the liver, thus limiting its application to other cancer types or tissues; ③ Some existing lysosomal targeting degraders are in the form of small molecules. For example, LYTAC is a conjugate of antibody and polysaccharide, and the polysaccharide synthesis process is complex and requires a cumbersome optimization process to achieve excellent lysosomal targeting ability.

[0005] Thanksgiving license plate:

[0006] [1] Zhou Y, Teng P, Montgomery NT, Li X, Tang W. Development oftriantennary N-Acetylgalactosamine conjugates as degraders for extracellularproteins. ACS Cent Sci. 2021; 7: 499–506.

[0007] [2] Ahn G, Banik SM, Miller CL, Riley NM, Cochran JR, Bertozzi CR.LYTACs that engage the asialoglycoprotein receptor for targeted protein degradation. Nat Chem Biol. 2021; 17: 937–946.

[0008] [3] Zheng J, He W, Li J, Feng X, Li Y, Cheng B, Zhou Y, Li M, Liu K,Shao X, et al. Bifunctional compounds as molecular degraders for integrin-facilitated targeted protein degradation. J Am Chem Soc. 2022; 144: 21831–2

[0009] [4] Zhu C, Wang W, Wang Y, Zhang Y, Li J. Dendronized DNA ChimerasHarness Scavenger Receptors To Degrade Cell Membrane Proteins. 2023; 62:e202300694.

[0010] [5] Cotton AD, Nguyen DP, Gramespacher JA, Seiple IB, Wells JA. Development of Antibody-based PROTACs for the degradation of the cell-surfaceimmune checkpoint protein PD-L1. J Am Chem Soc. 2021; 143: 593-598.

[0011] [6] Pance K, Gramespacher JA, Byrnes JR, Salangsang F, Serrano J-AC, Cotton AD, Steri V, Wells JA. Modular cytokine receptor-targeting chimeras for targeted degradation of cell surface and extracellular proteins. NatBiotechnol. 2023; 41: 273-281. Summary of the Invention

[0012] To address the aforementioned shortcomings of existing membrane protein degradation technologies, this invention provides an application of protein conjugates in membrane protein degradation and a method for membrane protein degradation. This protein conjugate can effectively degrade membrane proteins and, through conjugation with different antibodies, can target different sites, thus enabling its application in multiple scenarios. Throughout the process, it does not rely on degradation ligands targeting the proteasome or lysosomal receptor, demonstrating strong applicability.

[0013] To achieve the above objectives, the present invention adopts the following technical solution.

[0014] This invention provides an application of a protein conjugate in membrane protein degradation, the protein conjugate comprising a carrier and an antibody;

[0015] The antibody can be effectively displayed as a multivalent antibody via conjugation to the vector.

[0016] The membrane protein degradation includes the formation of aggregates by cross-linking of the target protein mediated by the protein conjugate, followed by degradation of the target protein via the proteasome and / or lysosome degradation pathways.

[0017] In some implementations, the coupling method is either covalent or non-covalent.

[0018] In some embodiments, the protein conjugate does not include degradation ligands that target the proteasome or lysosomal receptor.

[0019] In some embodiments, the membrane protein degradation includes incubating the protein conjugate with cells to mediate the cross-linking of the target protein to form aggregates, followed by the degradation of the target protein in the cells via a proteasome degradation pathway and / or a targeted lysosomal degradation pathway.

[0020] In some implementations, the proteasome degradation pathway includes the ubiquitination-proteasome pathway.

[0021] In some embodiments, the carrier comprises ferritin and / or nanoparticles.

[0022] In some preferred embodiments, the ferritin includes human ferritin.

[0023] In some preferred embodiments, the ferritin comprises human heavy chain ferritin.

[0024] In some specific embodiments, the amino acid sequence of the ferritin is shown in SEQ ID NO: 1.

[0025] In some preferred embodiments, the nanoparticles include nucleic acid nanoparticles, such as DNA nanoparticles.

[0026] In some implementations, the antibody is a monovalent antibody and / or a multivalent antibody.

[0027] The monovalent antibody may refer to different monovalent antibodies that target the same target protein and / or different monovalent antibodies that target different target proteins.

[0028] In this invention, without a specific carrier, it is difficult to induce cross-linking of membrane proteins when using monovalent antibodies to bind to them; correspondingly, without further linking to degradation ligands targeting the proteasome or lysosomal receptor, it is difficult to achieve membrane protein degradation. However, the applicant's research has found that further conjugation with a specific carrier, based on the carrier's multi-spatial and multi-site characteristics, allows the entire structure to exhibit the characteristics of a multivalent antibody, thereby inducing receptor cross-linking, and the subsequent degradation process is not limited by a specific pathway. Furthermore, the applicant's research has found that when ferritin (containing 24 monomers) is chosen as the carrier, effective degradation of the target protein does not require all 24 monomers to display the antibody; only 30% antibody display is needed.

[0029] In some embodiments, the antibody includes one or more antibodies that specifically bind to EGFR (epidermal growth factor receptor), PD-L1 (programmed death ligand 1), HER2 (human epidermal growth factor receptor-2), and PD-1 (programmed death protein 1).

[0030] In some implementations, the antibody is a single-domain antibody or scFv.

[0031] In some specific embodiments, the amino acid sequence of the antibody that specifically binds to EGFR is shown in SEQ ID NO: 2.

[0032] In some specific embodiments, the amino acid sequence of the antibody that specifically binds to PD-L1 is shown in SEQ ID NO: 3.

[0033] In some specific embodiments, the amino acid sequence of the antibody that specifically binds to HER2 is shown in SEQ ID NO: 4.

[0034] In some embodiments, the coupling method includes one of the following: intima-peptide coupling, enzyme catalysis, and bio-coupling, such as Sortase enzyme catalysis, or SpyCatcher and SpyTag bio-coupling.

[0035] In some preferred embodiments, when the coupling method is the SpyCatcher and SpyTag bioconjugation method, the preparation method of the protein conjugate includes:

[0036] The mixture is prepared by reacting a solution of ferritin fused with SpyCatcher and an antibody fused with SpyTag; or, the mixture is prepared by reacting a solution of ferritin fused with SpyTag and an antibody fused with SpyCatcher.

[0037] In some specific embodiments, the amino acid sequence of the SpyCatcher is preferably as shown in SEQ ID NO: 5.

[0038] In some specific embodiments, the amino acid sequence of the SpyTag is preferably as shown in SEQ ID NO: 6.

[0039] In some preferred embodiments, the molar ratio of the ferritin fusion SpyCatcher to the antibody fusion SpyTag is 1:(0.3-4), more preferably 1:2 or 1:3.

[0040] In some preferred embodiments, the molar ratio of the ferritin fusion SpyTag to the antibody fusion SpyCatcher is 1:(0.3-4), more preferably 1:3.

[0041] In some preferred embodiments, the preparation of the ferritin-fused SpyCatcher includes the following steps:

[0042] S1. Connect the target gene 1 encoding ferritin, which is fused with SpyCatcher, to the vector plasmid to construct expression plasmid 1.

[0043] S2. The expression plasmid 1 is transfected, screened, expressed and purified to obtain the ferritin fusion SpyCatcher.

[0044] In some specific embodiments, the ferritin fusion SpyCatcher further includes a linker; the amino acid sequence of the linker preferably includes one or more of the sequences shown in SEQ ID NO: 7, 8 and 9.

[0045] In some specific embodiments, the amino acid sequence of the ferritin fusion SpyCatcher is shown in SEQ ID NO:10; wherein, the transfection is preferably performed using Escherichia coli; and the purification is preferably performed using a Ni column.

[0046] In some preferred embodiments, the preparation of the antibody fusion SpyTag includes the following steps:

[0047] S1. Connect the target gene 2 encoding the antibody fusion SpyTag to the vector plasmid to construct expression plasmid 2;

[0048] S2. The expression plasmid 2 is transfected, screened, expressed and purified to obtain the ferritin fusion SpyCatcher.

[0049] In some specific embodiments, the antibody fusion SpyTag further includes a linker; the amino acid sequence of the linker preferably includes one or more of the sequences shown in SEQ ID NO: 7, 8 and 9.

[0050] In some specific embodiments, the amino acid sequence of the antibody fusion SpyTag is shown in SEQ ID NO: 12; wherein, the transfection is preferably performed using Escherichia coli or human embryonic kidney cells; and the purification is preferably performed using a nickel column, a Capto L affinity column, or a Ni column.

[0051] In some embodiments, the mixed solution also includes a buffer, preferably a Tris or PBS buffer.

[0052] In some preferred embodiments, the buffer has a pH of 7.0-8.0.

[0053] In some implementations, the reaction temperature is 4-37°C.

[0054] In some implementations, the reaction time is 0.5 h to 24 h.

[0055] In some preferred embodiments, the mixed solution also includes arginine and / or glycerol.

[0056] In some implementations, the reaction is followed by a purification step.

[0057] In some preferred embodiments, the purification step is performed using molecular sieves, preferably using a molecular sieve pre-packed column, such as a Superdex molecular sieve pre-packed column.

[0058] The present invention also provides a method for membrane protein degradation for purposes other than disease diagnosis and / or treatment, comprising the following steps:

[0059] The protein conjugate is incubated with cells; the degradation of the target protein in the cells is mediated by the proteasome and / or lysosome degradation pathways.

[0060] The protein conjugate is as defined in the applications described above.

[0061] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0062] The reagents and raw materials used in this invention are all commercially available.

[0063] The positive and progressive effects of this invention are as follows:

[0064] This invention utilizes a specific carrier to conjugate antibodies, enabling the antibodies to be effectively displayed as multivalent antibodies. This allows for effective receptor cross-linking when recognizing and binding to target proteins. Once aggregates are formed, the target protein is degraded via proteasome and / or lysosome degradation pathways. The entire process can be performed without relying on proteasome degradation ligands and / or lysosome-targeting receptors. By conjugating different antibody fragments, it can be successfully applied to the degradation of multiple targets, exhibiting low limitations and greater applicability. Attached Figure Description

[0065] Figure 1The images show agarose gel electrophoresis analysis of 7D12-Spytag-pet32a and Spycatcher-hFerritin-pet30a after XhoI-ApaI double digestion (Lane 1: 7D12-Spytag-pet32a; Lane 2: 7D12-Spytag-pet32a after XhoI-ApaI double digestion; Lane 3: Spycatcher-hFerritin-pet30a; Lane 4: Spycatcher-hFerritin-pet30a after XhoI-ApaI double digestion).

[0066] Figure 2 Ni affinity column purification of Spycatcher-hFerritin (A) and SDS-PAGE analysis (B) (Peak I is the impurity peak, peak II is the target product peak, sample is the lysate product after E. coli induction expression, FT is flowthrough)).

[0067] Figure 3 Ni affinity column purification of TrxA-Anti-EGFR-Spytag (A) and SDS-PAGE analysis (B) (Peak I is the impurity peak, peak II is the target product peak, sample is the lysate product after E. coli induced expression, FT is flowthrough)).

[0068] Figure 4 SDS-PAGE analysis of Spycatcher-hFerritin and TrxA-Anti-EGFR-Spytag after overnight reaction at molar ratios of 1:0, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, and 0:3.

[0069] Figure 5 The purification results (A) and SDS-PAGE analysis (B) of Ferritac-Anti-EGFR using a size exclusion chromatography column (Superdex™ 200 Increase 10 / 300 GL) are shown.

[0070] Figure 6 WB chromatograms of Spycatcher-hFerritin and Ferritac-Anti-EGFR with different loading masses.

[0071] Figure 7 This is a TEM scan result of Ferritac-Anti-EGFR.

[0072] Figure 8The results of Western blot analysis of the in vitro EGFR degradation capacity of PBS, Anti-EGFR, and Ferritac-Anti-EGFR in six cell lines are shown (β-actin is the internal reference protein).

[0073] Figure 9 The experimental flowchart and related results of the in vivo EGFR and CD51 degradation capacity in mice after injection of PBS, Anti-EGFR, and Ferritac-Anti-EGFR are shown below (A is the flowchart of the mouse experiment; B is the in vivo EGFR immunofluorescence analysis result; C is the in vivo CD51 immunofluorescence analysis result; D is the protein expression level of different groups relative to the PBS group; E is the WB analysis result of the in vivo EGFR and CD51 degradation capacity; β-actin is the internal reference protein).

[0074] Figure 10 The results of the study on the degradation mechanism of EGFR by Ferritac-Anti-EGFR and its related results on the degradation of HER-2 and PD-L1 are shown in Figure A (WB analysis results of U-87 MG treated with MG132, Bafilomycin A1 and Ferritac-Anti-EGFR for EGFR, respectively); Figure B (WB analysis results of U-251 MG treated with MG132, Bafilomycin A1 and Ferritac-Anti-EGFR for EGFR, respectively); Figure C (WB analysis results of HeLa treated with MG132, Bafilomycin A1 and Ferritac-Anti-EGFR for EGFR, respectively). A1 shows the results of Western blot analysis of EGFR after treatment with Chloroquine and Ferritac-Anti-EGFR; D shows the cell imaging of SKOV3 cells after treatment with Ferritac-Anti-HER-2; E shows the flow cytometry results of SKOV3 cells after treatment with Ferritac-Anti-HER-2; F shows the flow cytometry results of MDA-MB-231 cells after treatment with Ferritac-Anti-PD-L1. Detailed Implementation

[0075] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0076] The sequence-related information used in the following examples is as follows:

[0077] (1) Human heavy chain ferritin:

[0078] MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES (SEQ ID NO: 1)

[0079] (2) Antibody specifically binding to EGFR:

[0080] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSALE (SEQ ID NO: 2)

[0081] (3) Antibody specifically binding to PD-L1:

[0082] DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS (SEQ ID NO: 3)

[0083] (4) Antibody specifically binding to HER2:

[0084] EVQLVESGGGLVQAGGSLRLSCAASGITFSINTMGWYRQAPGKQRELVALISSIGDTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCKRFRTAAQGTDYWGQGTQVTVSS (SEQ ID NO: 4)

[0085] (5) Spycatcher:

[0086] VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAH (SEQ ID NO: 5)

[0087] (6) Spytag:

[0088] AHIVMVDAYKPTK (SEQ ID NO: 6)

[0089] (7) Linker 1:

[0090] GGSGGSGGSGGS (SEQ ID NO: 7)

[0091] (8) Linker 2:

[0092] GSGSGHM (SEQ ID NO: 8)

[0093] (9) Linker 3:

[0094] GGGS (SEQ ID NO: 9)

[0095] (10) Spycatcher-hFerritin: [[ID=​​​​​​​​VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGSGGSGGSGGSMTTASTSQVRQNYHQDSEAAINRQINLELYA SEQ ID NO: 11)

[0099] In the above sequence, the underlined parts are thrombin cleavage sites.

[0100] (12) Anti-EGFR-Spytag:

[0101] QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSALEGGGSAHIVMVDAYKPTK. (SEQ ID NO: 12)

[0102] (13)TrxA-Anti-EGFR-Spytag(29.8KD):

[0103] MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTA PKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA GSGSGHMHHHHHHHHSSG LVPRGS QVKLEESGGGSVQTGGSLRLTCAASGRTSRSYGMGWFRQAPGKEREFVSGISWRGDSTGYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAIYYCAAAAGSAWYGTLYEYDYWGQGTQVTVSSALEGGGSAHIVMVDAYKPTK. (SEQ ID NO: 13)

[0104] In the above sequences, the part marked by a single underline is the TrxA sequence, and the part marked by a double underline is the thrombin restriction site.

[0105] Example 1: Preparation and characterization of ferritin-conjugated EGFR antibody (Ferritac-Anti-EGFR):

[0106] 1. Construction of plasmids expressing ferritin fused with SpyCatcher (Spycatcher-hFerritin) nanocages and expressing antibodies fused with SpyTag (TrxA-Anti-EGFR-Spytag) nanobodies.

[0107] Cloning of the target gene and transformation of the plasmid into E. coli:

[0108] The nucleotide sequences encoding Spycatcher-hFerritin nanocages (corresponding amino acid sequences are SEQ ID NO: 11) and TrxA-Anti-EGFR-Spytag nanobodies (corresponding amino acid sequences are SEQ ID NO: 13) were synthesized by General Biotechnology (Anhui) Co., Ltd., and cloned into pET-30a (+) and pET-32a (+) respectively to obtain the target plasmids (Spycatcher-hFerritin-pet30a and 7D12-Spytag-pet32a). The target plasmids were diluted to a concentration of approximately 200 ng / μL and transformed into E. coli BL21 (DE3) competent cells. The specific transformation steps are as follows: Take the competent strain frozen at -80 ℃, thaw it by standing on ice for 2-3 min, add 5 μL of diluted target plasmid (concentration of 200 ng / μL), mix well, and place it in a constant temperature water bath preheated to 42 ℃ for heat shock treatment for 1-1.5 min, then place it on ice for 2 min. Take 500 μL of antibiotic-free LB medium and add it to the heat-shocked competent strain, mix gently with a pipette, adjust the temperature of the constant temperature shaker to 37 ℃ and the rotation speed to 200 rpm, and shake for 40 min to revive the competent strain. Then, the revived strains were collected by centrifugation at 6000 rpm and room temperature. The supernatant was discarded, and the strains were resuspended in 100 μL of antibiotic-free LB medium. The suspensions were then added to solid LB agar plates containing the corresponding antibiotics. pET-30a (+) and pET-32a (+) corresponded to kanamycin and ampicillin resistance, respectively. The bacterial suspension was spread evenly with a spreader and incubated upside down at 37 ℃ for approximately 8 h. Single colonies were then picked. Plasmid extraction was performed using the Novizum plasmid mini-prep kit. Both pET-30a (+) and pET-32a (+) were digested with ApaI-XhoI and then validated using gel electrophoresis. The successfully validated strains were then sent for DNA sequencing to further verify whether the target plasmid had been successfully transformed. The correctly sequenced bacterial suspensions were stored in 20% glycerol and frozen at -80 ℃ for later use.

[0109] 2. Expression and purification of Spycatcher-hFerritin nanocages and TrxA-Anti-EGFR-Spytag nanobodies

[0110] (1) Expression of the target protein:

[0111] The transformed E. coli were cultured overnight in 5 mL LB medium containing kanamycin or ampicillin at 37 °C and 220 rpm. Then, they were transferred to a 500 mL bottle containing antibiotics and cultured for another 5-6 h at 37 °C and 220 rpm until the OD600 value was between 0.6 and 0.8. Then, 0.2 mM IPTG was added, the temperature of the constant temperature shaker was lowered to 25 °C, the shaking speed was adjusted to 180 rpm, and expression was induced for another 16 h.

[0112] (2) Collection and destruction of bacteria:

[0113] After inducing expression, the *E. coli* bacterial culture was centrifuged at 6500 rpm for 20 min at room temperature, resuspended in an appropriate amount of PBS, and then homogenized at 800–900 MPa for 7–8 min to lyse the bacteria. To prevent the large amount of heat generated during homogenization from denaturing the protein and affecting its activity, the homogenizer was pre-cooled with ice water at 4 °C before lysing. After lysing, the bacterial culture was collected and centrifuged at 12000 rpm for 30 min at 4 °C, and the supernatant was collected. For the TrxA-Anti-EGFR-Spytag nanobody, the supernatant after centrifugation can be directly used for Ni column purification. For Spycatcher-hFerritin nanocages, taking advantage of the thermal stability of ferritin, the supernatant was treated in a 60 °C water bath for 20 min to denature the contaminating proteins, and then centrifuged at 12000 g for 30 min. The supernatant was then used for Ni column purification and loading.

[0114] (3) Ni column purification:

[0115] Spycatcher-hFerritin nanocages and TrxA-Anti-EGFR-Spytag nanobodies were purified using Ni columns. First, the S1, A1, and B1 pumps of the AKTA protein purifier were rinsed with ddH2O for 10 min each, with a flow rate of 5 mL / min. Pump S1 was used for sample loading, while pumps A1 and B1 were used to adjust the imidazole concentration. Then, the flow rate was adjusted to 2 mL / min, and 5 mL of the Ni column was connected to the flow path. The column was rinsed with 5 column volumes of ddH2O, followed by equilibration with 10 column volumes of equilibration buffer. After UV and conductivity values ​​stabilized, the column was loaded using pump S1. After loading, the column was equilibrated again with 10 column volumes of equilibration buffer. The column was then eluted with a gradient of imidazole concentrations, starting at 0 mM and ending at 500 mM. Subsequently, the system was washed with ddH2O to remove residual buffer, and the tubing and column were packed with 20% ethanol to prevent bacterial growth. Generally, low concentrations of imidazole are used to remove non-specific proteins bound to the Ni column, while high concentrations are used to elute the target protein. Proteins eluted from each imidazole concentration are collected and analyzed using 10% SDS-PAGE to determine the imidazole concentration and purity corresponding to the target protein. The eluent corresponding to the target protein is dialyzed against imidazole-free PBS buffer and then concentrated using ultrafiltration tubes to a concentration above 1 mg / mL; TrxA-Anti-EGFR-Spytag nanobody and Spycatcher-hFerritin nanocages are concentrated using 3 kDa and 100 kDa ultrafiltration tubes, respectively. The concentration of the target protein is then determined using the BCA method, and 5–10% glycerol is added before freezing at -20 °C or -80 °C.

[0116] (4) SDS-PAGE analysis:

[0117] Take a 10% SDS-PAGE gel, dilute the sample to be analyzed to 1× with 5× bromophenol blue loading buffer, boil the sample in a 95℃ metal bath for 15 min, then centrifuge at 12000 rpm for 3 min. Add the supernatant to the wells of the gel. Electrophoresis is first performed at 90V for 30 min to allow the protein markers to spread, then the voltage is adjusted to 120V, and electrophoresis is continued for approximately 60 min until the bromophenol blue is approximately 1 cm from the bottom edge. After electrophoresis, stain with Coomassie Brilliant Blue solution with shaking for 30 min. To improve staining efficiency, the gel can also be heated on high in a microwave for 10 s. Then, discard the staining solution, add destaining solution, and continue shaking for approximately 30 min, changing the destaining solution once. The target band is then clearly visible, and the gel is ready for gel imaging analysis.

[0118] 3. Preparation of Ferritac-Anti-EGFR

[0119] Spycatcher-hFerritin nanocages and TrxA-Anti-EGFR-Spytag nanobodies were incubated at different molar ratios (1:0, 1:0.5, 1:1, 1:1.5, 1:2, 1:3, or 0:3) at 4°C for 12 hours. A suitable volume of the reaction product was then diluted to 1× with 5× bromophenol blue loading buffer and boiled in a 95°C metal bath for 15 minutes for SDS-PAGE analysis. Specific experimental procedures for SDS-PAGE are described in section 2.2.2.1. To prepare large quantities of TrxA-Ferritin-Anti-EGFR, the optimal molar ratio was selected to ensure complete Spycatcher-hFerritin reaction. To prevent the N-terminal TrxA from interfering with the binding of the target protein, thrombin was added overnight for enzymatic digestion to remove the N-terminal TrxA protein.

[0120] The enzyme digestion products were concentrated by ultrafiltration in a 50 kDa ultrafiltration tube for further purification. During SEC200 purification, the flow rate was first set to 1.5 mL / min, and the column was washed sequentially with ddH2O and 0.2 M sodium hydroxide. The column was then equilibrated with PBS buffer. After UV and conductivity equilibrium was reached, the concentrated enzyme digestion products were loaded onto a 5 mL loop, and the corresponding proteins were collected according to the UV peak. Subsequently, the system was washed with ddH2O to remove residual buffer, and the tubing and column were packed with 20% ethanol to prevent bacterial growth. The collected eluent was analyzed by SDS-PAGE to determine the peak position and purity of the target protein.

[0121] The corresponding Ferritac-Anti-EGFR was obtained after purification.

[0122] 4. Characterization of Ferritac-Anti-EGFR nanocages

[0123] 4.1 The monomer molecular weight of Ferritac-Anri-EGFR was verified by immunoblotting.

[0124] The specific steps are as follows:

[0125] (1) Reduced SDS-PAGE: Keep the loading mass of cell lysates consistent (10~30 μg) and control the loading volume of each sample between 10~20 μL. For EGFR, use 4~12% precast gel for gel electrophoresis analysis. Electrophoresis at 110 V for about 80 min will allow the bromophenol blue to run to about 1 cm from the bottom edge.

[0126] (2) Transfer: After the 0.45 μm PVDF membrane is fully activated by soaking in methanol for 10 s, it is immersed in the anodic transfer solution for 15 min; the SDS-PAGE gel is immersed in ddH2O for 3 min, and then immersed in the cathodic transfer solution for 3 min; two transfer pads are prepared and immersed in the anodic and cathodic transfer solutions respectively. The sandwich structure is built in the order of anodic transfer pad-PVDF membrane-gel-cathodic transfer pad, and placed in a semi-dry transfer apparatus. The current is set to be constant at 1.5 A and the transfer time is 650 s.

[0127] (3) Blocking, antibody incubation and imaging: Place the transferred PVDF membrane in a blocking box, add an appropriate volume of rapid blocking solution to cover the PVDF membrane, and shake on a shaker at room temperature for about half an hour to block. After blocking, wash the membrane 3 times with TBST, add antiferritin heavy chain antibody (ab65080), and incubate at room temperature for 1 h or at 4 ℃ for 12 h; wash 3 times with TBST, add secondary antibody, incubate at room temperature for 1 h, and wash 3 times with TBST again; add equal volumes of ECL chromogenic solution A and B to a 15 mL centrifuge tube, mix well, add to the blocking box, gently shake to spread the chromogenic solution evenly on the PVDF membrane, and then use an imager for chromogenic imaging.

[0128] 4.2 The particle size of Ferritac-Anri-EGFR was determined by TEM.

[0129] The TEM detection process was as follows: the protein sample was dialyzed into 1×PBS buffer and diluted to a concentration of 0.1~0.2 mg / mL. A hydrophilically treated copper mesh was used as a support, and 3 μL of the diluted protein sample was dropped onto the mesh. After adsorption for 1 min, excess protein sample was removed from the edge with filter paper. Then, 3 μL of phosphotungstic acid was added for staining for 45 s. After removing excess staining solution from the edge with filter paper, the sample was observed using a transmission electron microscope with an accelerating voltage of 120 kV.

[0130] Example 2: Results of Ferritac-Anti-EGFR preparation and characterization:

[0131] (1) Construction of plasmids expressing Spycatcher-hFerritin nanocages and TrxA-Anti-EGFR-Spytag nanobodies

[0132] The target plasmid returned by General Biotechnology (Anhui) Co., Ltd. was transformed into E. coli BL21 (DE3) competent cells, plated on agar plates, and amplified by selecting single colonies. The plasmid was then extracted and verified by double digestion with ApaI and XhoI. The theoretical molecular weights of the 7D12-Spytag and Spycatcher-hFerritin plasmids after digestion at the ApaI and XhoI sites were 1886 bp and 2042 bp, respectively. Figure 1 As shown, the detection results indicated that the enzyme digestion results were as expected. Furthermore, DNA sequencing results also confirmed the successful construction of the expression plasmid. The bacterial culture was stored in 20% glycerol and frozen at -80 °C.

[0133] (2) Expression and purification of Spycatcher-hFerritin nanocages and TrxA-Anti-EGFR-Spytag nanobodies

[0134] The E. coli expression system, as a typical prokaryotic expression system, is widely used in protein production where post-translational modifications are not critical due to its ease of operation, low cost, rapid expression speed, and suitability for large-scale production. Therefore, this invention employs the E. coli expression system for the expression of nanobodies and ferritin nanocages. Ni column affinity chromatography is based on the specific binding between nickel ions and histidine tags (His-tags) in proteins. By adjusting the imidazole concentration in the elution buffer, imidazole competitively binds to the His-tag, promoting the elution of the target protein from the column, thereby achieving its purification. Using E. coli expression and Ni column affinity chromatography, TrxA-Anti-EGFR-Spytag nanobodies and Spycatcher-hFerritin nanocages were successfully prepared. Figure 2 and Figure 3 As shown, this invention used a reducing SDS-PAGE experiment to detect the molecular weight of proteins eluted with different concentrations of imidazole, demonstrating that the TrxA-Anti-EGFR-Spytag nanobody and Spycatcher-hFerritin nanocages can be successfully eluted by high concentrations of imidazole. Since the eluted protein solution contained a small amount of imidazole, this invention removed the imidazole by dialysis and then performed reducing SDS-PAGE on the concentrated protein. The results showed that the molecular weights of the TrxA-Anti-EGFR nanobody and Spycatcher-hFerritin nanocages were approximately 29.8 kDa and 36.1 kDa, respectively, which are consistent with the theoretical molecular weights, proving the successful expression and purification of both.

[0135] (3) Preparation of Ferritac-Anti-EGFR

[0136] The homogeneity of protein drugs is crucial for manufacturing stability and dosage control in clinical use. Therefore, to obtain a more homogeneous TrxA-Ferritac-Anti-EGFR, this invention investigated the reaction of Spycatcher-hFerritin nanocages and TrxA-Anti-EGFR-Spytag nanobodies at different molar ratios. Figure 4 As shown, with the increase of the molar ratio of Spycatcher-hFerritin nanocages to TrxA-Anti-EGFR-Spytag nanobody, the band of the product TrxA-Ferritac-Anti-EGFR nanocage becomes increasingly concentrated. Furthermore, when the molar ratio of Spycatcher-hFerritin nanocages to TrxA-Anti-EGFR-Spytag nanobody reaches 1:3, the band of Spycatcher-hFerritin essentially disappears, indicating that almost all Spycatcher-hFerritin nanocages have been coupled with TrxA-Anti-EGFR-Spytag nanobody. Therefore, this molar ratio was used in subsequent experiments to prepare TrxA-Ferritac-Anti-EGFR nanocages.

[0137] Because the pET32a-expressed protein has TrxA fused to its N-terminus, to prevent TrxA from interfering with the affinity of Ferritac-Anti-EGFR nanocages for EGFR, this invention treats the reaction product (TrxA-Ferritac-Anti-EGFR) of Spycatcher-hFerritin nanocages and TrxA-Anti-EGFR-Spytag nanobody with thrombin overnight at 4°C. SDS-PAGE analysis shows that the monomeric band of Ferritac-Anti-EGFR is clearly visible at 54 kDa. Since the Spycatcher-hFerritin monomer reaction is essentially complete, only Ferritac-Anti-EGFR nanocages, Anti-EGFR-Spytag nanobody, and TrxA remain in the digestion product. Due to the significant molecular weight difference between the Ferritac-Anti-EGFR nanocages and other components, this invention concentrates the digestion product by ultrafiltration and then purifies it using a size exclusion column (Superdex™ 200 Increase 10 / 300 GL). Figure 5 As shown, after verification by SDS-PAGE reduction, peak 1 is the target product (Ferritac-Anti-EGFR), and peak 2 is the unreacted Anti-EGFR-Spytag nanobody and the cleaved TrxA, indicating the successful separation of the target protein, that is, the successful preparation of Ferritac-Anti-EGFR.

[0138] Furthermore, the applicant's research found that in the entire preparation process of Ferritac-Anti-EGFR, if the antibody and ferritin conjugate is constructed by fusion expression, the expression product has a large molecular weight and significantly alters the original sequence structure of ferritin, which leads to a significant reduction in expression yield, and most of the expression occurs within inclusion bodies. In contrast, the method of expressing the antibody and ferritin separately and then conjugating them in this invention can ensure high yield while maintaining stable expression.

[0139] Furthermore, the applicant's research found that the conjugation products of antibodies and ferritin have poor stability and are prone to precipitation during the conjugation process; when arginine (content not higher than 600mM) and glycerol (e.g. 5%) are added to the conjugation system, the stability of the conjugation products can be further guaranteed.

[0140] (4) Characterization of Ferritac-Anti-EGFR nanocages

[0141] This invention characterized Ferritac-Anti-EGFR using Western blot, TEM, and dynamic light scattering (DLS). Western blot results showed that after incubation with antiferritin heavy chain antibody, Spycatcher-hFerritin and Ferritac-Anti-EGFR exhibited specific bands for antiferritin heavy chain antibody at approximately 36 kDa and 54 kDa, respectively, consistent with theoretical molecular weights, indicating successful expression and purification of both. Figure 6 Furthermore, the band above Ferritac-Anti-EGFR has a molecular weight of approximately 108 kDa, which is presumably due to a dimer formed by the incomplete reduction of the Ferritac-Anti-EGFR nanocage.

[0142] Transmission electron microscopy (TEM) is a high-resolution microscopy technique that uses an electron beam to penetrate a sample for imaging. To more intuitively observe the size, shape, and uniformity of Ferritac-Anti-EGFR nanocages, this invention performed TEM scanning on Ferritac-Anti-EGFR. Figure 7 As shown, transmission electron microscopy results indicate that Ferritac-Anti-EGFR is uniformly distributed in spherical form with a particle size of approximately 10 nm, which is close to the theoretical value of ferritin nanoparticles.

[0143] The above experimental results all demonstrate the successful expression of Ferritac-Anti-EGFR in this invention and its effective self-assembly into spherical nanocages with uniform particle size.

[0144] Example 3: In vivo and in vitro degradation effects of Ferritac-Anti-EGFR:

[0145] 1. In vitro degradation effect

[0146] Ferritac-Anti-EGFR (with a molar ratio of Spycatcher-hFerritin and TrxA-Anti-EGFR-Spytag of 1:3) and Anti-EGFR were incubated with six cell lines (U-87 MG, A549, U-251 MG, HeLa, MDA-MB-231, and SK-OV-3) respectively (the concentration of Ferritac-Anti-EGFR and Anti-EGFR was 100 nM, the incubation temperature was 37 °C, and the incubation time was 12 h). The results after incubation were characterized by Western blotting.

[0147] like Figure 8 As shown in the Western blot results, Ferritac-Anti-EGFR significantly reduced EGFR expression levels in all six cell lines compared to Anti-EGFR. Using the PBS-treated group as a baseline (100%), Image J grayscale analysis revealed that the degradation rates of Ferritac-Anti-EGFR in U-87 MG, A549, U-251 MG, HeLa, MDA-MB-231, and SK-OV-3 cells were 72%, 62%, 49%, 54%, 25%, and 72%, respectively.

[0148] 2. In vivo degradation effect

[0149] HeLa cell-bearing mice (BALB / c mice) were divided into three groups: PBS, Anti-EGFR, and Ferritac-Anti-EGFR (with a molar ratio of Spycatcher-hFerritin and TrxA-Anti-EGFR-Spytag of 1:3), with three mice in each group. The mice were injected with the corresponding drugs via the tail vein every other day at a dose of 5 mg / kg for one week. After that, the mice were euthanized by cervical dislocation, and the tumors were removed for immunoblotting and immunofluorescence analysis.

[0150] like Figure 9 As shown, the immunoblotting results indicated that, compared with PBS, the tumor EGFR content in the Anti-EGFR treatment group mice did not change significantly, while the tumor EGFR expression level in the Ferritac-Anti-EGFR treatment group mice was significantly reduced, approximately 50% of that in the PBS group.

[0151] Simultaneously, this invention also used immunoblotting to detect changes in CD51 (integrin family protein) content as a control. Compared with PBS, although the CD51 content of Anti-EGFR and Ferritac-Anti-EGFR decreased slightly, there was no significant difference. The results of immunofluorescence were consistent with the results of immunoblotting. EGFR expressed in tumor tissue was labeled with rabbit anti-human EGFR antibody and FITC-labeled anti-rabbit secondary antibody. Tumors in mice treated with PBS and Anti-EGFR showed extensive green fluorescence, while tumors in mice treated with Ferritac-Anti-EGFR showed only weak green fluorescence, indicating that Ferritac-Anti-EGFR can effectively degrade EGFR in tumor tissue. Immunohistochemical imaging of CD51 also confirmed that the CD51 content in the Anti-EGFR and Ferritac-Anti-EGFR treatment groups did not change significantly compared with the PBS treatment group. The above experimental results indicate that Ferritac-Anti-EGFR can effectively degrade EGFR levels in HeLa tumor-bearing mice, but has no significant effect on CD51 expression levels. In other words, the ferritin-conjugated antibody method does not affect the specificity of the antibody, and can effectively degrade the target protein without affecting other irrelevant target proteins.

[0152] Example 4: Ferritac-Anti-EGFR used in the protein degradation pathway of EGFR protein.

[0153] Intracellular protein synthesis and degradation are crucial for maintaining protein homeostasis and are essential for normal cell growth and function. Intracellular protein degradation primarily occurs through two pathways: the ubiquitination-26S proteasome pathway and the lysosomal pathway. The ubiquitination-proteasome pathway involves E1, E2, and E3 ubiquitin ligases adding ubiquitin tags to target proteins, which are then recognized and degraded by the proteasome. In contrast, the lysosomal pathway relies on its strongly acidic environment for the hydrolytic degradation of proteins.

[0154] To investigate the degradation pathway of EGFR protein by Ferritac-Anti-EGFR (with a molar ratio of Spycatcher-hFerritin and TrxA-Anti-EGFR-Spytag of 1:3), this invention used lysosomal inhibitors chloroquine and Bafilomycin A1, as well as proteasome inhibitor MG132, and observed the effects of these inhibitors on the degradation of EGFR protein by Ferritac-Anti-EGFR.

[0155] like Figure 10As shown, the results indicate that, compared with the Ferritac-Anti-EGFR treatment alone, the expression level of EGFR protein significantly increased when MG132 was co-incubated with Ferritac-Anti-EGFR, suggesting that MG132 inhibits the degradation of EGFR by Ferritac-Anti-EGFR by inhibiting proteasome function. Furthermore, the EGFR protein band was darkened to some extent when chloroquine or Bafilomycin A1 was co-treated with Ferritac-Anti-EGFR, indicating that the EGFR protein expression level was increased compared to Ferritac-Anti-EGFR treatment alone. This also demonstrates that the lysosomal inhibitor chloroquine can partially inhibit the degradation function of Ferritac-Anti-EGFR.

[0156] Based on these observations, this invention hypothesizes that Ferritac-Anti-EGFR degrades EGFR protein through a dual pathway of ubiquitination-proteasome and lysosome, with the ubiquitination-proteasome pathway being the primary pathway.

[0157] In addition, by Figure 10 The results show that the strategy of using ferritin-coupled antibodies to promote membrane protein degradation can also be successfully applied to the degradation of HER2 and PD-L1.

Claims

1. The application of a protein conjugate in the degradation of membrane proteins, characterized in that, The protein conjugate includes a carrier and an antibody; The antibody can be effectively displayed as a multivalent antibody via conjugation to the vector. The membrane protein degradation includes the formation of aggregates by cross-linking of the target protein mediated by the protein conjugate, followed by degradation of the target protein via the proteasome and / or lysosome degradation pathways.

2. The application as described in claim 1, characterized in that, The coupling method is either covalent or non-covalent; And / or, the membrane protein degradation includes incubating the protein conjugate with cells to mediate the cross-linking of the target protein to form aggregates, followed by the degradation of the target protein in the cells via the proteasome degradation pathway and / or targeted lysosomal degradation pathway; And / or, the proteasome degradation pathway includes the ubiquitination-proteasome pathway.

3. The application as described in claim 1 or 2, characterized in that, The carrier includes ferritin and / or nanoparticles; The ferritin preferably includes human ferritin; More preferably, the ferritin comprises human heavy chain ferritin; More preferably, the amino acid sequence of the ferritin is shown in SEQ ID NO: 1; Preferably, the nanoparticles include nucleic acid nanoparticles, such as DNA nanoparticles.

4. The application as described in any one of claims 1-3, characterized in that, The antibody is a monovalent antibody and / or a multivalent antibody; And / or, the antibody includes one or more antibodies that specifically bind to EGFR, PD-L1, HER2, and PD-1; And / or, the antibody is a single-domain antibody or scFv; The amino acid sequence of the antibody that specifically binds to EGFR is preferably shown in SEQ ID NO: 2; The amino acid sequence of the antibody that specifically binds to PD-L1 is preferably shown in SEQ ID NO: 3; The amino acid sequence of the antibody that specifically binds to HER2 is preferably shown in SEQ ID NO:

4.

5. The application as described in any one of claims 1-4, characterized in that, The coupling method includes one of the following: peptide coupling, enzyme catalysis, and bio-coupling, such as Sortase enzyme catalysis, or SpyCatcher and SpyTag bio-coupling. When the coupling method is the SpyCatcher and SpyTag bioconjugation method, the preparation method of the protein conjugate preferably includes: The mixture of ferritin fused with SpyCatcher and antibody fused with SpyTag was prepared by reaction; or, the mixture of ferritin fused with SpyTag and antibody fused with SpyCatcher was prepared by reaction. The amino acid sequence of the SpyCatcher is preferably shown in SEQ ID NO: 5; The amino acid sequence of the SpyTag is preferably shown in SEQ ID NO:

6.

6. The application as described in any one of claims 1-5, characterized in that, The molar ratio of the ferritin fusion SpyCatcher and the antibody fusion SpyTag is 1:(0.3-4); Alternatively, the molar ratio of the ferritin fusion SpyTag to the antibody fusion SpyCatcher is 1:(0.3-4).

7. The application as described in any one of claims 1-6, characterized in that, The preparation of the ferritin-fused SpyCatcher includes the following steps: S1. Connect the target gene 1 encoding ferritin, which is fused with SpyCatcher, to the vector plasmid to construct expression plasmid 1. S2. The expression plasmid 1 is transfected, screened, expressed and purified to obtain the ferritin fusion SpyCatcher; Preferably, the ferritin fusion SpyCatcher further includes a linker; the amino acid sequence of the linker preferably includes one or more of the sequences shown in SEQ ID NO: 7, 8 and 9; More preferably, the amino acid sequence of the ferritin fusion SpyCatcher is shown in SEQ ID NO: 10; wherein the transfection is preferably performed using Escherichia coli; and the purification is preferably performed using a Ni column.

8. The application as described in any one of claims 1-7, characterized in that, The preparation of the antibody fusion SpyTag includes the following steps: S1. Connect the target gene 2 encoding the antibody fusion SpyTag to the vector plasmid to construct expression plasmid 2; S2. The expression plasmid 2 is transfected, screened, expressed and purified to obtain the ferritin fusion SpyCatcher; Preferably, the antibody fusion SpyTag further includes a linker; the amino acid sequence of the linker preferably includes one or more of the sequences shown in SEQ ID NO: 7, 8 and 9; More preferably, the amino acid sequence of the antibody fusion SpyTag is shown in SEQ ID NO: 12; wherein, the transfection is preferably performed using Escherichia coli or human embryonic kidney cells; and the purification is preferably performed using a nickel column, a Capto L affinity column, or a Ni column.

9. The application as described in any one of claims 1-8, characterized in that, The mixed solution also includes a buffer, preferably a Tris or PBS buffer. And / or, the pH of the buffer solution is 7.0-8.0; And / or, the temperature of the reaction is 4-37°C; And / or, the reaction time is 0.5h-24h; Preferably, the mixed solution also includes arginine and / or glycerol; And / or, the reaction may further include a purification step; More preferably, the purification step is performed using a molecular sieve, and more preferably using a molecular sieve pre-packed column, such as a Superdex molecular sieve pre-packed column.

10. A method for degrading membrane proteins for purposes other than disease diagnosis and / or treatment, characterized in that, It includes the following steps: The protein conjugate is incubated with cells to mediate the degradation of the target protein in the cells via the proteasome and / or lysosome degradation pathways. The protein conjugate is as defined in any one of claims 1-9.

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