Nano antibody chimera Endobody capable of degrading membrane protein as well as preparation method and application of nano antibody chimera Endobody

Abstract

The invention discloses a nano antibody chimera Endobody capable of degrading membrane protein as well as a preparation method and application of the nano antibody chimera Endobody, and belongs to the technical field of biology. The invention aims to provide the nano antibody chimera Endobody for degrading the membrane protein, and the nano antibody chimera Endobody does not need a transport receptor or endocytosis receptor binding module, is simple in structure and is easy to prepare. The invention provides a nano antibody chimera Endobody for degrading a membrane protein. The nano antibody is obtained by connecting a coding gene of the nano antibody with a coding gene of a cell transmembrane peptide and then expressing the protein. The efficiency of different cell transmembrane peptides for delivering target protein into lysosome and the action mechanism of Endobody for degrading cell membrane protein are shown.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a nano antibody chimera Endobody for degrading membrane protein, and a preparation method and application thereof. Background Art

[0002] Targeted protein degradation (TPD) is a revolutionary technology that shows great potential as a new therapeutic strategy. Compared with traditional small molecule inhibitors, the TPD strategy shows obvious advantages, including targeting non-degradable proteins, higher therapeutic efficacy, catalytic properties and overcoming drug resistance.

[0003] In recent years, research on the development of more effective extracellular targeted protein degradation (eTPD) technologies has received increasing attention. These extracellular protein degradation strategies, such as the lysosome-targeted chimera LYTAC, can be combined with the delivery of cell membrane-associated proteins or secreted proteins to lysosomes for degradation, greatly expanding the scope of application of TPD. LYCTAC consists of an antibody and a chemically synthesized glycopeptide ligand, which is an agonist of CI-M6PR. The antibody can bind to the extracellular domain of the target protein, while the glycopeptide ligand carries multiple units of mannose-6-phosphonate (M6Pn) motifs, recruiting CI-M6PR and internalizing the target protein into the lysosome for subsequent degradation. Similar methods for extracellular target protein degradation delivered by transporters or receptors include: dendritic DNA chimeras DENTACs, integrin-promoted lysosomal degradation IFLD, cytokine receptor-targeted chimeras KineTAC, AbTAC targeting membrane-bound E3 ligases, proteolysis-targeted antibodies PROTAB, and signal-mediated lysosomal targeting chimeras SignalTAC. Although such technologies have great prospects in drug development and therapeutic potential, they still have some disadvantages and challenges. For example, such methods all require the inclusion of an additional transport receptor or endocytic receptor binding module, resulting in complex composition, relatively difficult design and implementation, requiring precise control of the design of binding domains and chemical molecules, and higher costs. In addition, the expression level and distribution of transport receptors or endocytic receptors required for the delivery of target proteins may vary greatly due to differences in cell types and species, thereby affecting the delivery efficiency and degradation of target proteins. Therefore, it is of great significance to design a cell membrane-targeted protein degrader that does not require a transport receptor or endocytic receptor binding module, has a simple structure, and is easy to prepare. Summary of the invention

[0004] The purpose of the present invention is to provide a nano antibody chimera Endobody for degrading membrane proteins, which does not require a transport receptor or endocytosis receptor binding module, has a simple structure, and is an easy-to-prepare cell membrane targeted protein degrader.

[0005] The present invention provides a nano antibody chimera Endobody for degrading membrane proteins. The nano antibody is obtained by connecting a coding gene of the nano antibody with a coding gene of a cell transmembrane peptide to express a protein.

[0006] It is further defined that the nanobody is a nanobody that binds to a protein target outside the cell membrane; a fluorescent protein nanobody or a nanobody that binds to an intracellular protein target; and the cell transmembrane peptide is a cationic cell transmembrane peptide or an amphipathic cell transmembrane peptide.

[0007] It is further defined that the nanobodies are green fluorescent protein nanobodies, epidermal growth factor nanobodies and programmed death ligand-1 nanobodies.

[0008] It is further defined that the cell transmembrane peptide is R9, TAT, pVEC or Bac7.

[0009] It is further defined that the amino acid sequence of the nanobody chimera Endobody is shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.7.

[0010] The present invention provides a method for preparing the above-mentioned nano antibody chimera Endobody, and the preparation method is as follows:

[0011] Step 1: constructing a vector: removing the CBD sequence in the pTXB1 vector, inserting the His6 tag sequence to obtain a recombinant vector 1, and connecting the gene sequence of the above-mentioned nanoantibody chimera Endobody to the recombinant vector to obtain a recombinant vector 2;

[0012] Step 2: Using E. coli to express the recombinant vector 2 obtained in step 1 to obtain a nano antibody chimera Endobody carrying the Intein protein;

[0013] Step 3: Cut the Intein protein in the Nanobody chimera Endobody with Intein protein obtained in Step 2, and purify the Nanobody chimera Endobody obtained.

[0014] The present invention provides a method for targeted protein degradation for non-therapeutic and non-diagnostic purposes, wherein the nano antibody chimera Endobody is added into cells for culture.

[0015] It is further defined that the concentration range of the nano antibody chimera Endobody is 0.5-100 μM and the degradation time is 24 h.

[0016] It is further defined that when the sequence of the nanoantibody chimera Endobody is as shown in SEQ ID NO.2, the concentration for degrading EGFP-EGFR is 10 μM; when the sequence of the nanoantibody chimera Endobody is as shown in SEQ ID NO.4, the concentration for degrading EGFR is 1 μM; when the sequence of the nanoantibody chimera Endobody is as shown in SEQ ID NO.7, the concentration for degrading PDL1 is also 10 μM.

[0017] The present invention provides a use of the above-mentioned nano-antibody in degrading intracellular proteins for non-therapeutic and non-diagnostic purposes.

[0018] Beneficial effects: Using nanobodies and cell transmembrane peptides, a nanoantibody chimera Endobody for degrading membrane proteins was developed, and EGFP fusion protein, endogenous EGFR and PDL1 were successfully degraded. In addition, the efficiency of different cell transmembrane peptides in delivering target proteins into lysosomes and the mechanism of Endobody's degradation of cell membrane proteins were demonstrated. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the result diagram of GBP-R9 protein after separation and purification;

[0020] Figure 2 This is the result diagram of GBP-R9 degrading EGFP-EGFR in Hela cells;

[0021] Figure 3 The expression and purification results of EGFR Nb-R9 are shown in Figure 1: a, expression of EGFR Nb-R9-Intein-His6; b, expression protein connection (EPL) separation of EGFR Nb-R9-Intein-His6; c, purification of EGFR Nb-R9;

[0022] Figure 4 This is the result diagram of EGFR Nb-R9 degrading endogenous EGFR in Hela cells;

[0023] Figure 5 The expression and purification results of EGFR Nb are as follows: a, expression of EGFR Nb-Intein-His6; b, EPL separation of EGFR Nb-Intein-His6; c, purification of EGFR Nb;

[0024] Figure 6 This is the result diagram showing that EGFR Nb does not affect the endogenous EGFR expression in Hela cells;

[0025] Figure 7 This is the time gradient result diagram of EGFR Nb-R9 degrading endogenous EGFR in Hela cells;

[0026] Figure 8 This is the result diagram of EGFR Nb-R9 entering cells by endocytosis;

[0027] Fig. 9 The results of Dan. and CQ inhibiting the degradation of EGFR by EGFR Nb-R9;

[0028] Fig.10 Results of EGFR Nb-R9 localization in early and late endosomes: a, co-localization of EGFR Nb-R9 with early endosomes; b, co-localization of EGFR Nb-R9 with late endosomes;

[0029] Fig.11 The expression and purification results of EGFR Nb-TAT, EGFR Nb-pVEC and EGFR Nb-Bac7 are shown in Figure 1: a, expression and purification of EGFR Nb-TAT; b, expression and purification of EGFR Nb-pVEC; c, expression and purification of EGFR Nb-Bac7;

[0030] Fig.12 This is a comparison of the effects of different CPPs on Endobody's ability to degrade target proteins;

[0031] Fig.13 The expression and purification results of PDL1 Nb-R9 are as follows: a, expression of PDL1 Nb-R9-Intein-His6; b, EPL separation of PDL1Nb-R9-Intein-His6; c, purification of PDL1 Nb-R9;

[0032] Fig.14 The results of PDL1 Nb-R9 degrading endogenous PDL1 in MDA-MB-231 cells;

[0033] Fig.15 The expression and purification results of PDL1 Nb are as follows: a, expression of PDL1 Nb-Intein-His6; b, EPL separation of PDL1Nb-Intein-His6; c, purification of PDL1 Nb;

[0034] Fig.16 The figure shows that PDL1 Nb does not affect the expression of endogenous PDL1 in MDA-MB-231 cells;

[0035] Fig.17 This is a diagram of the mode of action of Endobody. DETAILED DESCRIPTION

[0036] GBP, i.e., the amino acid sequence of green fluorescent protein (EGFP) nanobody (SEQ ID NO.1); the amino acid sequence of GBP-R9 (SEQ ID NO.2); EGFR Nb, i.e., the amino acid sequence of epidermal growth factor receptor (EGFR) nanobody (SEQ ID NO.3); the amino acid sequence of EGFR Nb-R9 (SEQ ID NO.4); the amino acid sequence of EGFP-EGFR (SEQ ID NO.5); PDL1 Nb, i.e., the amino acid sequence of programmed death ligand-1 (PDL1) nanobody (SEQ ID NO.6); the amino acid sequence of PDL1 Nb-R9 (SEQ ID NO.7); the amino acid sequence of Intein-His6 (SEQ ID NO.8); the amino acid sequence of mCherry-Rab5 (SEQ ID NO.9); the amino acid sequence of mCherry-Rab7 (SEQ ID NO.10); EGFR Nb-TAT (SEQ ID NO.11); EGFR Nb-pVEC (SEQ ID NO.12); EGFR Nb-Bac7 (SEQ ID NO.13); GBP, i.e., the gene sequence of green fluorescent protein (EGFP) nanobody (SEQ ID NO.14); the gene sequence of GBP-R9 (SEQ ID NO.15); EGFR Nb, i.e., the gene sequence of epidermal growth factor receptor (EGFR) nanobody (SEQ ID NO.16); the gene sequence of EGFR Nb-R9 (SEQ ID NO.17); the gene sequence of EGFP-EGFR (SEQ ID NO.18); PDL1 Nb, i.e., the gene sequence of programmed death ligand-1 (PDL1) nanobody (SEQ ID NO.19); the gene sequence of PDL1 Nb-R9 (SEQ ID NO.20); the gene sequence of Intein-His6 (SEQ ID NO.21); the gene sequence of mCherry-Rab5 (SEQ ID NO.22); the gene sequence of mCherry-Rab7 (SEQ ID NO.23); EGFR Nb-TAT (SEQ ID NO.24); ID NO.24); EGFR Nb-pVEC (SEQ ID NO.25); EGFR Nb-Bac7 (SEQ ID NO.26).

[0037] 1. Experimental cells

[0038] The cells used in this study include Hela cells and MDA-MB-231 cells. The competent cells used for prokaryotic expression of the target protein are Rosetta cells, and the competent cells used for plasmid amplification or new cloning vector construction are XL10-Gold cells.

[0039] 2. Plasmid Construction

[0040] The eukaryotic cell expression vector used in this study was the pEGFP-C1 (Solarbio: #VT000086) vector, and the protein expression prokaryotic vector used was the pTXB1 (NEB: #N6707S) vector. A total of 11 plasmids were used, including EGFP-EGFR, mCherry-Rab5, and mCherry-Rab7 (the basic vector of the three plasmids GFP-EGFR, mCherry-Rab5, and mCherry-Rab7 is the pEGFP-C1 vector. The three plasmids were prepared by inserting EGFP-EGFR, mCherry-Rab5, or mCherry-Rab7 fragments into the pEGFP-C1 vector), pTXB1_GBP-R9-Intein-His6, pTXB1_EGFR Nb-R9-Intein-His6, pTXB1_EGFR Nb-TAT-Intein-His6, pTXB1_EGFR Nb-pVEC-Intein-His6, pTXB1_EGFR Nb-Bac7-Intein-His6, pTXB1_PDL1Nb-R9-Intein-His6, pTXB1_PDL1 Nb-Intein-His6 (the vector used for pTXB1 related plasmids is pTXB1 (NEB: #N6707S). After two-step cloning, the chitin binding domain sequence CBD on the vector was first removed, and the His6 tag was inserted to obtain the pTXB1_Intein-His6 vector. Then, after double enzyme digestion, GBP-R9, EGFR Nb-R9 and other sequences were inserted into the vector to obtain the corresponding vector.

[0041] 3. Protein expression and purification

[0042] (1) The plasmid for protein expression (pTXB1 vector) was transformed into Escherichia coli Rosetta cells and cultured in a 37°C constant temperature incubator for 24 h;

[0043] (2) Pick a single colony on the agar plate and inoculate it into 50 mL of LB liquid medium containing 100 mg / L ampicillin, and shake it at 240 rpm in a shaker at 37°C overnight;

[0044] (3) The preculture was further inoculated into 1.8 L of fresh LB liquid medium containing 100 mg / L ampicillin and 33 mg / L chloramphenicol, and cultured in a shaker at 37°C and 180 rpm for 1.5-2 h until the absorbance at 600 nm reached 0.5-0.6;

[0045] (4) When the absorbance at 600 nm reached 0.5-0.6, 0.5 mL of isopropyl β-D-thiogalactoside (IPTG) stock solution was added and protein expression was induced at 16°C overnight;

[0046] (5) The next day, transfer the culture to a centrifuge bucket for collection. Centrifuge at 8000 rpm for 15 min at 4°C to harvest the cell culture.

[0047] (6) Discard the supernatant and transfer all the precipitate to a 50 mL centrifuge tube. Centrifuge at 4700 rpm for 15 min at 4°C.

[0048] (7) discard the supernatant, resuspend the bacterial pellet in lysis buffer (PBS solution containing 0.5 M NaCl, 3% glycerol, 3 mM β-mercaptoethanol and 1 mM PMSF, pH 8.0), and sonicate on ice at 80 W for 30 min (sonication for 1 s, interval of 3 s) to lyse the bacteria;

[0049] (8) Wash the Ni-NTA column and then elute the His-tag fusion protein using a step gradient of imidazole (50, 100, ..., up to 500 mM) solution;

[0050] (9) The target protein is collected and concentrated in order of purity and concentration using a 50 mL ultrafiltration tube in descending order of concentration. The concentration is generally concentrated to a volume of 500 μL. The resulting protein is quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0051] 4. Protein Expression and Ligation

[0052] Take the preparation of EGFR Nb-R9 as an example.

[0053] (1) EGFR Nb-R9-Intein-His6 protein was expressed and purified in Escherichia coli, and the buffer was replaced three times in buffer A (PBS solution containing 0.5 M NaCl and 3% glycerol) at pH 8.0;

[0054] (2) The concentration of EGFR Nb-R9-Intein-His6 protein is controlled at about 20 mg / mL. For 1 volume of EGFR Nb-R9-Intein-His6 protein, 1 / 2 volume of 2-mercaptoethanesulfonic acid sodium stock solution at pH 8.0 and a concentration of 2M is added as the Intein cleavage reagent and 1 / 2 volume of 4-mercaptophenylacetic acid stock solution at pH 8.0 and a concentration of 1.1M is added as the catalyst for the expression protein connection. Then, a cysteine ​​solution with a final concentration of 0.8 mM is added to the reaction solution. The reactants are centrifuged and filled with argon to prevent the reactants from contacting with air;

[0055] (3) The reaction mixture was incubated at 4°C for 3 days. After 3 days, 1 μL of the reaction mixture was taken and subjected to SDS-PAGE gel electrophoresis to detect whether the Intein protein was cleaved;

[0056] (4) After the Intein protein is cleaved, the ligation product is purified by gradient (0-500 mM imidazole) Ni-IMAC chromatography. During this process, the cleaved Intein-His6 fragment will bind to the resin and the pure EGFR Nb-R9 will be eluted to obtain the EGFR Nb-R9 protein.

[0057] 5. Cell Culture

[0058] HeLa cells or MDA-MB-231 cells were cultured in a proliferation medium according to conventional methods, using DMEM cell culture medium (containing 10% fetal bovine serum, 1% non-essential amino acids and 1% penicillin-streptomycin mixture) at 5% CO2 and 37°C.

[0059] 6. Cell Transfection

[0060] (1) HeLa or MDA-MB-231 cells were seeded at 25,000 cells per well in an 8-well culture dish or 60,000 cells per well in a 24-well culture plate, and transfection was performed when the cells grew to about 60%;

[0061] (2) Transfection was performed using Bio-Tech Lipo 8000 transfection reagent. The transfection system for 8-well culture dishes was 250 ng of plasmid, 0.4 μL of Lipo 8000, and 12.5 μL of Opti medium. The transfection system for 24-well culture plates was 500 ng of plasmid, 0.8 μL of Lipo 8000, and 25 μL of Opti medium. The medium was changed 1.5 h or 2 h after transfection.

[0062] 7. Drug addition

[0063] HeLa or MDA-MB-231 cells were seeded at 25,000 cells per well in an 8-well culture dish or 60,000 cells per well in a 24-well culture plate. When the cells grew to about 80%, different concentrations of GBP-R9 (GR9), EGFR NB-R9 (ER9), EGFR NB (ENB), PDL1 NB-R9 and PDL1 NB (PNB) were added to detect the degradation of EGFP-EGFR, endogenous EGFR and endogenous PDL1. ER9, EGFR NB-TAT (ET), EGFR NB-pVEC (EV) and EGFR NB-Bac7 (EB) were added to detect the effects of different cell transmembrane peptides on EGFR degradation.

[0064] Example 1. Construction of a Nanobody Chimera Endobody for Degrading Membrane Proteins

[0065] Endobody composition formula: Nanobody-CPP (in no particular order), such as Fig.17 shown.

[0066] 1. Construction of vector: Insert GBP-R9 sequence into pTXB1_Intein-His6 vector to prepare expression vector pTXB1_GBP-R9-Intein-His6

[0067] 2. GBP-R9-Intein-His6 protein was expressed and purified in E. coli, and the buffer was replaced three times in buffer A (PBS solution containing 0.5 M NaCl and 3% glycerol) at pH 8.0;

[0068] 3. Control the concentration of GBP-R9-Intein-His6 protein at about 20 mg / mL. For 1 volume of GBP-R9-Intein-His6 protein, add 1 / 2 volume of 2-mercaptoethanesulfonic acid sodium stock solution at pH 8.0 with a concentration of 2M as the Intein cleavage reagent and 1 / 2 volume of 4-mercaptophenylacetic acid stock solution at pH 8.0 with a concentration of 1.1M as the catalyst for protein expression connection. Then, add cysteine ​​solution with a final concentration of 0.8 mM to the reaction solution. Centrifuge the reactants and fill them with argon to prevent the reactants from contacting with air;

[0069] 4. Incubate the reaction mixture at 4°C for 3 days. After 3 days, take 1 μL of the reaction mixture and perform SDS-PAGE gel electrophoresis to detect whether the Intein protein is cleaved;

[0070] 5. After the Intein protein is cleaved, the ligation product is purified by gradient (0-500 mM imidazole) Ni-IMAC chromatography. During this process, the cleaved Intein-His6 fragment will bind to the resin and the pure GBP-R9 will be eluted to obtain the GBP-R9 protein.

[0071] Results: Expression and purification of GBP-R9: Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase located on the cell surface. It is an important cell signal transduction molecule that plays a key role in cell growth, division, survival and migration, and is closely related to the occurrence and development of various cancers.

[0072] First, we constructed the first Endobody degrader GBP-R9 (abbreviated as GR9) by using green fluorescent protein (EGFP) nanobody (GBP) and cationic cell transmembrane peptide nona-arginine (R9) to degrade EGFR (EGFP-EGFR) expressed in fusion with EGFP on the cell membrane surface.

[0073] The expression vector pTXB1_GBP-R9-Intein-His6 was constructed, and after being expressed and purified by E. coli, the GR9 degradation agent was prepared by the method of expressing protein ligation, such as Figure 1 shown.

[0074] Example 2. GBP-R9 degrades EGFP-EGFR in Hela cells

[0075] Hela cells with good growth status were inoculated in 24-well plates. When the cells grew to 50%-60% confluence, Lipo 8000 transfection reagent (Biyuntian, Shanghai, China) was used to transfect EGFP-EGFR into Hela cells and cultured for 24 hours. Subsequently, GR9 degrader was added to the cells at final concentrations of 0, 5, 10, 25, 50, and 100 μM. After culturing for 24 hours, WB was used to detect the degradation of EGFP-EGFR by GR9. Figure 2 As shown, when the concentration of GR9 is greater than 10 μM, it can effectively degrade EGFP-EGFR on the surface of Hela cell membrane.

[0076] Example 3. Construction of EGFR Nb-R9 protein

[0077] 1. Construction of vector: Insert the EGFR Nb-R9 sequence into the pTXB1_Intein-His6 vector to prepare the expression vector pTXB1_EGFR Nb-R9-Intein-His6

[0078] 2. The EGFR Nb-R9-Intein-His6 protein was expressed and purified in E. coli, and the buffer was replaced three times in buffer A (PBS solution containing 0.5 M NaCl and 3% glycerol) at pH 8.0;

[0079] 3. Control the concentration of EGFR Nb-R9-Intein-His6 protein at about 20 mg / mL. For 1 volume of EGFR Nb-R9-Intein-His6 protein, add 1 / 2 volume of 2-mercaptoethanesulfonic acid sodium stock solution at pH 8.0 with a concentration of 2M as the Intein cleavage reagent and 1 / 2 volume of 4-mercaptophenylacetic acid stock solution at pH 8.0 with a concentration of 1.1M as the catalyst for the expression protein connection. Then, add cysteine ​​solution with a final concentration of 0.8 mM to the reaction solution. Centrifuge the reactants and fill them with argon to prevent the reactants from contacting with the air;

[0080] 4. Incubate the reaction mixture at 4°C for 3 days. After 3 days, take 1 μL of the reaction mixture and perform SDS-PAGE gel electrophoresis to detect whether the Intein protein is cleaved;

[0081] 5. After the Intein protein is cleaved, the ligation product is purified by gradient (0-500 mM imidazole) Ni-IMAC chromatography. During this process, the cleaved Intein-His6 fragment will bind to the resin and the pure EGFR Nb-R9 will be eluted to obtain the EGFR Nb-R9 protein.

[0082] The feasibility of the Endobody degradation strategy was demonstrated by the degradation results of EGFP-EGFR by GR9. The degradation effect of this method on endogenous targets was detected. Using EGFR nanoantibodies, a second Endobody degrader, EGFRNb-R9 (ER9), was constructed to degrade endogenous EGFR in Hela cells.

[0083] The expression vector pTXB1_EGFR Nb-R9-Intein-His6 was constructed, and after being expressed and purified in E. coli, an ER9 degrader was prepared by the expression protein ligation method, such as Figure 3 shown.

[0084] Example 4. ER9 degrades endogenous EGFR in Hela cells

[0085] 1. Inoculate Hela cells in good growth state into 24-well plates. When the cells grow to 70%-80% confluence, add ER9 degrader to the cells with final concentrations of 0, 1, 2.5, 5, 10, and 20 μM. After culturing for 24 hours, WB was used to detect the degradation of endogenous EGFR in Hela cells by ER9. Figure 4 As shown, when the concentration of GR9 is 1 μM, it can effectively degrade EGFR on the surface of Hela cell membrane.

[0086] 2. Expression and purification of EGFR Nb

[0087] To exclude the effect of EGFR nanobody itself on EGFR expression, we expressed EGFR nanobody ENB (EGFR Nb) to detect the effect of ENB on EGFR expression.

[0088] The expression vector pTXB1_EGFR Nb-Intein-His6 was constructed, and after being expressed and purified in Escherichia coli, the ENB protein was prepared by the expression protein ligation method, such as Figure 5 shown.

[0089] 3. Effect of EGFR Nb on EGFR expression in Hela cells

[0090] Hela cells with good growth status were inoculated in 24-well plates. When the cells grew to 70%-80% confluence, ENB was added to the cells at final concentrations of 0, 1, 2.5, 5, 10, and 20 μM. After culturing for 24 hours, the effect of ENB on endogenous EGFR in Hela cells was detected by WB. Figure 6 As shown, ENB did not affect the expression of EGFR in Hela cells.

[0091] Example 5. Screening of the optimal degradation time of EGFR Nb-R9 for EGFR degradation

[0092] After confirming that ER9 can efficiently degrade EGFR, we further explored the optimal degradation time of ER9 to degrade EGFR. Hela cells with good growth status were seeded in 24-well plates. When the cells grew to 70%-80% confluence, 1μM ER9 degrader was added to the cells. Cell samples were collected at 1, 3, 6, 12, 24, and 48 hours after the addition of ER9, and WB was used to detect the degradation of endogenous EGFR in Hela cells by ER9. Figure 7 As shown, EGFR was significantly degraded 3 h after the addition of ER9.

[0093] Example 6. Effect of endocytosis inhibitors on EGFR degradation by EGFR Nb-R9

[0094] Previous studies have shown that CPP can carry proteins or small molecule drugs into cells by endocytosis. In order to explore the mechanism of Endobody degradation of target proteins, we prepared FAM-ER9 with green fluorescence by labeling ER9 with FAM-NHS. We then tested the effects of endocytosis inhibitor dansylcadaverine (Dan.) and lysosomal inhibitor chloroquine (CQ) on the entry of FAM-ER9 into cells. Figure 8 As shown in the figure, confocal microscopy observations revealed that FAM-NHS could not enter the cells, while FAM-ER9 could enter the cells under the action of R9 and showed a dotted distribution. However, after adding FAM-ER9 and dansylcadaverine at the same time, FAM-ER9 could not enter the cells. The transmembrane ability of ER9 was affected by the endocytosis inhibitor, indicating that ER9 entered the cells through endocytosis. Fig. 9 As shown, WB detection found that the endocytosis inhibitor dansylcadaverine and the lysosomal inhibitor chloroquine significantly inhibited the degradation of EGFR in Hela cells by ER9, indicating that ER9 degrades EGFR through the lysosomal pathway.

[0095] Example 7. Exploration of the mechanism of ER9 degradation of EGFR

[0096] Since ER9 is distributed in a punctate manner in cells, we next explored whether Endobody degrades the target protein through the endosomal-lysosomal pathway.

[0097] Hela cells with good growth status were inoculated into 8-well dishes. When the cells grew to 50%-60% confluence, mCherry-Rab5 and mCherry-Rab7 were transfected into Hela cells using Lipo 8000 transfection reagent and cultured for 24 hours. Then FAM-ER9 was added to the cells. Fig.10 As shown, FAM-ER9 co-localized with early endosomes and late endosomes, indicating that Endobody degrades target proteins through the endosomal-lysosomal pathway.

[0098] Example 8. Effects of other cell transmembrane peptides on Endobody degradation of target protein

[0099] 1. After exploring the lysosomal delivery ability of the cell transmembrane peptide R9 to the target protein, we tested the effects of three other cell transmembrane peptides on the ability of Endobody to degrade the target protein. They are another cationic cell transmembrane peptide TAT, and amphiphilic cell transmembrane peptides pVEC and Bac7. The sequences of the three CPPs are shown in Table 1.

[0100] Table 1 Names and sequences of CPPs

[0101]

[0102] 2. Expression and purification of EGFR Nb-TAT, EGFR Nb-pVEC and EGFR Nb-Bac7

[0103] After determining the three cell transmembrane peptides, we constructed the expression vectors pTXB1_EGFR Nb-TAT-Intein-His6, pTXB1_EGFR Nb-pVEC-Intein-His6, and pTXB1_EGFR Nb-Bac7-Intein-His6. After expression and purification in E. coli, we prepared the Endobody degraders ET (EGFR Nb-TAT), EV (EGFR Nb-pVEC), and EB (EGFR Nb-Bac7) by the expression protein ligation method, as shown in Figure 2 . Fig.11 shown.

[0104] 3. Comparison of the ability of ER9, ET, EV and EB to degrade EGFR in Hela cells

[0105] After preparing the degraders ET, EV and EB, we compared their ability to degrade EGFR in Hela cells with that of ER9.

[0106] Hela cells with good growth status were inoculated into 24-well plates. When the cells grew to 70%-80% confluence, 1 μM ENB, ER9, ET, EV and EB were added to the cells respectively. After culturing for 24 hours, the cell samples were collected and the degradation of EGFR was detected by WB. Fig.12 As shown, all four transmembrane peptides can degrade Hela cell EGFR, among which ER9 and EB have the best effects.

[0107] Example 9. Expression and purification of PDL1 NB-R9

[0108] 1. Build PDL1 NB-R9

[0109] 1. Construction of vector: Insert the PDL1 Nb-R9 sequence into the pTXB1_Intein-His6 vector to prepare the expression vector pTXB1_PDL1Nb-R9-Intein-His6

[0110] 2. PDL1 NB-R9-Intein-His6 protein was expressed and purified in E. coli, and the buffer was replaced three times in buffer A (PBS solution containing 0.5 M NaCl and 3% glycerol) at pH 8.0;

[0111] 3. Control the concentration of PDL1 NB-R9-Intein-His6 protein at about 20 mg / mL. For 1 volume of PDL1NB-R9-Intein-His6 protein, add 1 / 2 volume of 2M sodium 2-mercaptoethanesulfonate stock solution at pH 8.0 as Intein cleavage reagent and 1 / 2 volume of 1.1M 4-mercaptophenylacetic acid stock solution at pH 8.0 as catalyst for protein expression connection. Then, add cysteine ​​solution with a final concentration of 0.8 mM to the reaction solution. Centrifuge the reactants and fill them with argon to prevent the reactants from contacting with air;

[0112] 4. Incubate the reaction mixture at 4°C for 3 days. After 3 days, take 1 μL of the reaction mixture and perform SDS-PAGE gel electrophoresis to detect whether the Intein protein is cleaved;

[0113] 5. After the Intein protein is cleaved, the ligation product is purified by gradient (0-500 mM imidazole) Ni-IMAC chromatography. During this process, the cleaved Intein-His6 fragment will bind to the resin and the pure PDL1 NB-R9 will be eluted to obtain the PDL1 NB-R9 protein.

[0114] Second, since Endobody is completely genetically encoded, we can target different cell membrane surface targets by simply replacing the nanoantibody. Programmed death ligand 1 (PDL1) is a protein that is highly expressed on the surface of many tumor cells. It inhibits the activity of T cells by binding to the PD-1 receptor on the surface of T cells, preventing the immune system from clearing cancer cells. Based on this, we constructed the third Endobody degrader PDL1 NB-R9 (PR9) using PDL1 nanoantibody and R9 to degrade the PDL1 protein on the surface of cancer cell membranes.

[0115] The expression vector pTXB1_PDL1 NB-R9-Intein-His6 was constructed, and after being expressed and purified by E. coli, the PR9 degradation agent was prepared by the expression protein connection method, such as Fig.13 shown.

[0116] Example 10. PDL1 NB-R9 degrades endogenous PDL1 in MDA-MB-231 cells

[0117] 1. MDA-MB-231 cells with good growth status were seeded in 24-well plates. When the cells grew to 70%-80% confluence, PR9 degraders were added to the cells with final concentrations of 0, 1, 2.5, 5, 10, and 20 μM. After culturing for 24 hours, WB was used to detect the degradation of endogenous EGFR in MDA-MB-231 cells by PR9. Fig.14 As shown, when the concentration of PR9 is 10 μM, it can effectively degrade PDL1 protein on the membrane surface of MDA-MB-231 cells.

[0118] 2. Expression and purification of PDL1 Nb

[0119] In order to exclude the effect of PDL1 nanobody itself on PDL1 expression, PDL1 nanobody PDL1Nb (PNB) was also expressed to detect the effect of PNB on PDL1.

[0120] The expression vector pTXB1_PDL1 Nb-Intein-His6 was constructed, and after being expressed and purified in E. coli, the PNB protein was prepared by the expression protein ligation method, such as Fig.15 shown.

[0121] 3. Effect of PDL1 Nb on PDL expression in MDA-MB-231 cells

[0122] MDA-MB-231 cells with good growth status were inoculated in 24-well plates. When the cells grew to 70%-80% confluence, PNB was added to the cells at final concentrations of 0, 1, 2.5, 5, 10, and 20 μM. After culturing for 24 hours, the degradation of endogenous PDL1 in MDA-MB-231 cells by PNB was detected by WB. Fig.16 As shown, PNB did not affect the expression of PDL1 in MDA-MB-231 cells.

Claims

1. A nanoantibody chimera Endobody for degrading membrane proteins, characterized in that: The nanobody is obtained by connecting the coding gene of the nanobody with the coding gene of the cell transmembrane peptide and then expressing the protein.

2. The nanoantibody chimera Endobody according to claim 1, characterized in that The nanobody is a nanobody that binds to a protein target outside the cell membrane; the cell transmembrane peptide is a cationic cell transmembrane peptide or an amphipathic cell transmembrane peptide.

3. The nanoantibody chimera Endobody according to claim 2, characterized in that The nano antibodies are green fluorescent protein nano antibodies, epidermal growth factor nano antibodies and programmed death ligand-1 nano antibodies.

4. The nanoantibody chimera Endobody according to claim 1, characterized in that The cell transmembrane peptide is R9, TAT, pVEC or Bac7.

5. The nanoantibody chimera Endobody according to claim 1, characterized in that: The amino acid sequence of the nano antibody chimera Endobody is shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13 and SEQ ID NO.

7.

6. The method for preparing the nanoantibody chimera Endobody according to any one of claims 1 to 5, characterized in that: The preparation method is as follows: Step 1: constructing a vector: removing the CBD sequence in the pTXB1 vector, inserting the His6 tag sequence to obtain a recombinant vector 1, and connecting the gene sequence of the nanoantibody chimera Endobody described in claim 1 to the recombinant vector to obtain a recombinant vector 2; Step 2: Using E. coli to express the recombinant vector 2 obtained in step 1 to obtain a nano antibody chimera Endobody carrying the Intein protein; Step 3: The Intein protein tag in the Nanobody chimera Endobody with Intein protein obtained in Step 2 is removed, and the Nanobody chimera Endobody is obtained after purification.

7. A method for targeted protein degradation for non-therapeutic and diagnostic purposes, characterized in that: The nanoantibody chimera Endobody according to any one of claims 1 to 5 is added to cells for culture.

8. The method according to claim 7, characterized in that The concentration range of the nanoantibody chimera Endobody is 0.5-100 μM, and the degradation time is 6-48 h.

9. The method according to claim 8, characterized in that When the sequence of the nanoantibody chimera Endobody is as shown in SEQ ID NO.2, the concentration for degrading EGFP-EGFR is 10 μM; when the sequence of the nanoantibody chimera Endobody is as shown in SEQ ID NO.4, the concentration for degrading EGFR is 1 μM; when the sequence of the nanoantibody chimera Endobody is as shown in SEQ ID NO.7, the concentration for degrading PDL1 is also 10 μM.

10. Use of the Nanobody according to any one of claims 1 to 5 in degrading intracellular proteins for non-therapeutic and diagnostic purposes.

Images