A fusion protein and its preparation method and application

Abstract

The present invention belongs to the field of biomedicine and discloses a fusion protein, its preparation method, and application. The fusion protein comprises a polypeptide that specifically binds to the KRas protein, a tumor cell-specific membrane-penetrating peptide, and a lysosomal recognition peptide. For the first time, the present invention designs and constructs a fusion protein with tumor targeting, penetrating properties, and protein-specific degradation for an "undruggable" protein, providing new insights into the development of targeted anticancer drugs. Unlike existing technologies, where a single molecule can only target a single target protein, this fusion protein can simultaneously induce the degradation of wild-type and mutant KRas proteins. Furthermore, by inducing KRas degradation, the fusion protein can increase the sensitivity of KRas mutant tumors to tumor-targeted drugs, thereby expanding the scope of application of existing targeted anticancer drugs and having important implications for the clinical treatment of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a fusion protein and a preparation method and application thereof. Background Art

[0002] As a member of the GTPase superfamily, KRas plays an important role in promoting the development of multiple tumors, such as proliferation, metastasis, and angiogenesis, and is a well-known cancer-promoting factor. Studies have shown that KRas mutations are present in many types of cancer, with the proportion of KRas mutations in colorectal cancer cells reaching as high as 37.9%. Common KRas mutations mainly occur at codons 12, 13, 61, and 146 in exon 2, of which mutations at sites 12 and 13 account for 90% of the total number of mutations. The high mutation rate leads to the continuous activation of multiple tumor-promoting signaling pathways downstream of KRas. These activated physiological processes can weaken or even offset the effects of KRas upstream signaling molecules such as epidermal growth factor receptor (EGFR) inhibitors, resulting in colorectal cancer cells' natural resistance to many current first-line anti-cancer targeted drugs.

[0003] In life sciences and basic medicine, reducing the activation or expression level of proteins is an important strategy for protein function research and targeted drug design. Currently, the inhibition of protein activity mainly focuses on the development of small molecule inhibitors targeting the active sites of target proteins. However, studies have shown that 85% of proteins, including KRas proteins, are "undruggable proteins". These proteins are difficult to develop inhibitors for due to their loose and variable structures, strong competitive binding ability with other small molecules inside the cell, and easy mutation of active sites. In addition, using antibodies to block the active sites of target proteins is also a method to inhibit protein activity. However, the large molecular weight of conventional antibodies, the difficulty of entering the cell membrane, and the need to screen antibodies for specific fragments of the target protein limit their use. As a result, only a few membrane proteins can currently be inhibited by this method.

[0004] In addition to activation levels, inhibiting protein expression levels is also an important means of reducing protein function. DNA editing technologies represented by Crispr and RNA interference technologies represented by siRNA can reduce the expression of target genes at the DNA and RNA levels, respectively, thereby reducing the content of encoded proteins in cells and subsequently inhibiting protein function. Currently, DNA editing and RNA interference have been widely used in basic research in life sciences, and some researchers have also explored the application of these two technologies in clinical treatment. However, in actual applications, it has been found that both DNA editing and RNA interference have off-target effects, and the long duration of action makes cells extremely prone to compensatory effects, which has caused problems for basic research. In addition, the strict action conditions have also limited the clinical use of these two technologies, resulting in the fact that no drugs based on DNA editing and RNA interference have been approved for the clinical treatment of diseases.

[0005] In addition to the DNA and RNA levels, reducing the accumulation of proteins in cells at the protein level is also an important strategy to reduce protein expression, among which protein degradation is an important method to reduce its accumulation in cells. Protein degradation in living cells is a rapid and orderly physiological process, which mainly occurs in two organelles, the proteasome and the lysosome. Current research on targeted protein degradation mainly focuses on the proteasome pathway, by inducing ubiquitin ligase (E3) to bind to the target protein, and then transferring the ubiquitin molecule to the protein and being recognized and degraded by the proteasome. The mainstream protein targeted degradation technology is to connect the target protein and the E3 ligase together by constructing a chimeric molecule, thereby promoting the degradation of the target protein. The typical representative technology is PROTAC (protein degradation targeting chimera). PROTAC chimeric molecules are composed of three parts: a target protein ligand, an E3 ligase ligand, and an intermediate fragment that covalently connects the two. The target protein ligand is responsible for recognizing and binding to the target protein, and the E3 ligase is responsible for binding to the E3 ligase. According to public reports, there are currently many proteins that can be rapidly degraded by PROTAC technology. Among them, PROTAC constructed using the KRas G12C ligand MRTX849 can effectively induce the degradation of KRas proteins with G12C mutations. However, G12C mutations only account for a small part of KRas mutations (taking colorectal cancer as an example, only 6.8% are G12C mutations), and there are no effective ligands for other mutation types, so it does not fundamentally solve the problem of inhibiting "undruggable proteins". In addition, the specificity of KRas ligands cannot be guaranteed, and there is a possibility of incorrect binding to proteins; the synthesis of chimeric molecules is also relatively complex and cannot be produced on a large scale, so it has not yet entered commercial production and application. Summary of the Invention

[0006] The first aspect of the present invention aims to provide a fusion protein.

[0007] The purpose of the second aspect of the present invention is to provide a nucleic acid molecule encoding the fusion protein of the first aspect of the present invention.

[0008] The third aspect of the present invention aims to provide a vector comprising the nucleic acid molecule of the second aspect of the present invention.

[0009] The fourth aspect of the present invention aims to provide a host cell comprising the vector of the third aspect of the present invention.

[0010] The purpose of the fifth aspect of the present invention is to provide a method for preparing the fusion protein of the first aspect of the present invention.

[0011] The purpose of the sixth aspect of the present invention is to provide use of the fusion protein of the first aspect of the present invention in preparing a product.

[0012] The purpose of the seventh aspect of the present invention is to provide a product comprising the fusion protein of the first aspect of the present invention.

[0013] The eighth aspect of the present invention aims to provide a medicine.

[0014] The ninth aspect of the present invention aims to provide a combined drug.

[0015] In order to achieve the above object, the technical solution adopted by the present invention is:

[0016] In a first aspect, the present invention provides a fusion protein comprising a polypeptide that specifically binds to a KRas protein, a tumor cell-specific membrane-penetrating peptide, and a lysosome recognition peptide.

[0017] Preferably, the polypeptide that specifically binds to the KRas protein is an anti-KRas protein nanobody.

[0018] Further preferably, the amino acid sequence of the anti-KRas protein Nanobody is:

[0019] a)

[0020] OR

[0021] b) An amino acid sequence shown in SEQ ID NO. 1 that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0022] Preferably, the tumor comprises colorectal cancer, pancreatic cancer, renal cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gastrointestinal cancer, peritoneal cancer, melanoma, endometrial cancer, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, glioblastoma, brain metastasis, salivary gland cancer, thyroid cancer, brain cancer, lymphoma, myeloma and head and neck cancer.

[0023] Preferably, the tumor cell-specific transmembrane peptide is a ganglioside-binding peptide.

[0024] Further preferably, the amino acid sequence of the ganglioside binding peptide is:

[0025] a)RAGLQFPVGRLLRRLLR(SEQ ID NO.2); or

[0026] b) An amino acid sequence shown in SEQ ID NO. 2 that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0027] Preferably, the amino acid sequence of the lysosome recognition peptide is:

[0028] a)KFERQKILDQRFFE(SEQ ID NO.3); or

[0029] b) An amino acid sequence shown in SEQ ID NO. 3 that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0030] Preferably, the amino acid sequence of the fusion protein is:

[0031] or

[0032] b) An amino acid sequence shown in SEQ ID NO. 4 that has been modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0033] The second aspect of the present invention provides a nucleic acid molecule encoding the fusion protein of the first aspect of the present invention.

[0034] The third aspect of the present invention provides a vector comprising the nucleic acid molecule of the second aspect of the present invention.

[0035] The fourth aspect of the present invention provides a host cell comprising the vector of the third aspect of the present invention.

[0036] Preferably, the host cells include prokaryotic cells and eukaryotic cells.

[0037] The fifth aspect of the present invention provides a method for preparing the fusion protein of the first aspect of the present invention, wherein the host cell of the fourth aspect of the present invention is cultured to obtain the fusion protein.

[0038] The sixth aspect of the present invention provides use of the fusion protein of the first aspect of the present invention in preparing a product.

[0039] Preferably, the product is any one of (1) to (3):

[0040] (1) Preparations for degrading KRas protein;

[0041] (2) Anti-tumor drugs;

[0042] (3) Drugs that increase the sensitivity of KRas mutant tumors to tumor-targeted drugs.

[0043] Preferably, the KRas protein in (1) includes wild-type KRas protein and mutant KRas protein.

[0044] Preferably, the mutant KRas protein includes a KRas protein mutated at one or more amino acid positions of G12, G13, S17, P34, A59, Q61 and A146.

[0045] Preferably, the tumor in (2) is a KRas-related tumor; further, it is at least one of colorectal cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gastrointestinal cancer, peritoneal cancer, melanoma, endometrial cancer, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, glioblastoma, brain metastasis, salivary gland cancer, thyroid cancer, brain cancer, lymphoma, myeloma and head and neck cancer.

[0046] Preferably, the KRas mutant tumor in (3) is a tumor associated with mutant KRas protein.

[0047] Preferably, the mutant KRas protein includes a KRas protein mutated at one or more amino acid positions of G12, G13, S17, P34, A59, Q61 and A146.

[0048] Preferably, the tumor-targeted drug is a human epidermal growth factor receptor (EGFR)-targeted drug.

[0049] The seventh aspect of the present invention provides a product comprising the fusion protein of the first aspect of the present invention.

[0050] Preferably, the product is any one of (1) to (3):

[0051] (1) Preparations for degrading KRas protein;

[0052] (2) Anti-tumor drugs;

[0053] (3) Drugs that increase the sensitivity of KRas mutant tumors to tumor-targeted drugs.

[0054] Preferably, the KRas protein in (1) includes wild-type KRas protein and mutant KRas protein.

[0055] Preferably, the mutant KRas protein includes a KRas protein mutated at one or more amino acid positions of G12, G13, S17, P34, A59, Q61 and A146.

[0056] Preferably, the tumor in (2) is a KRas-related tumor; further, it is at least one of colorectal cancer, pancreatic cancer, kidney cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gastrointestinal cancer, peritoneal cancer, melanoma, endometrial cancer, ovarian cancer, cervical cancer, uterine cancer, bladder cancer, glioblastoma, brain metastasis, salivary gland cancer, thyroid cancer, brain cancer, lymphoma, myeloma and head and neck cancer.

[0057] Preferably, the KRas mutant tumor in (3) is a tumor associated with mutant KRas protein.

[0058] Preferably, the mutant KRas protein includes a KRas protein mutated at one or more amino acid positions of G12, G13, S17, P34, A59, Q61 and A146.

[0059] Preferably, the tumor-targeted drug is an epidermal growth factor receptor (EGFR)-targeted drug; further, it is gefitinib.

[0060] The eighth aspect of the present invention provides a medicine comprising the fusion protein of the first aspect of the present invention and pharmaceutically acceptable excipients.

[0061] The ninth aspect of the present invention provides a combination drug comprising the fusion protein of the first aspect of the present invention and at least one of the drugs of the eighth aspect of the present invention, and a tumor-targeting drug.

[0062] Preferably, the tumor-targeted drug is an epidermal growth factor receptor (EGFR)-targeted drug; further, it is gefitinib.

[0063] The beneficial effects of the present invention are:

[0064] The present invention is the first to design and construct a fusion protein with tumor targeting, penetrability and protein-specific degradation for "undruggable" proteins, providing a new idea for the development of targeted anti-cancer drugs; unlike the existing technology where one molecule can only target one target protein, this fusion protein can simultaneously induce the degradation of wild-type and mutant KRas proteins; at the same time, this fusion protein can increase the sensitivity of KRas mutant tumors to tumor-targeted drugs by inducing KRas degradation, thereby expanding the scope of application of existing targeted anti-cancer drugs, which is of great significance in the clinical treatment of tumors.

[0065] Compared with chimeric molecule-induced target protein degradation technology, the fusion protein provided by the present invention is simple to synthesize, and its prokaryotic or eukaryotic expression makes production convenient and has high efficiency, making it easier to produce on a large scale and apply commercially. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1 This is a flow chart of immunizing alpacas and screening target protein nanoantibodies.

[0067] Figure 2 This is the agarose gel electrophoresis result of total RNA from alpaca peripheral blood lymphocytes: where M represents Marker.

[0068] Figure 3 The figure shows the agarose gel electrophoresis results of different amounts of oligo dT Primer and random 6-mer cDNA: where M represents Marker.

[0069] Figure 4 This is the agarose gel electrophoresis result of different cDNA template amounts: where M represents Marker.

[0070] Figure 5 This is the result of colony PCR agarose gel electrophoresis.

[0071] Figure 6 It is a schematic diagram of the construction of the KRas protein degradation system (TKD) molecule and the process of inducing target protein degradation.

[0072] Figure 7 This is a diagram showing the purification effects of TKD and TKDm.

[0073] Figure 8 Figures 2 and 3 are the results of the in vitro binding between TKD molecules, TKDm molecules and KRas proteins: A is the result of the in vitro binding between TKD molecules and KRas proteins; B is the result of the in vitro binding between TKDm and KRas proteins; **** indicates P < 0.0001 compared with NC1; ns indicates no significant difference compared with NC1.

[0074] Figure 9These are the results of the SPR binding ability experiment of TKD molecules, TKDm molecules and KRas protein: A is the result of the SPR binding ability experiment of TKD molecules and KRas protein; B is the result of the SPR binding ability experiment of TKDm molecules and KRas protein.

[0075] Figure 10 These are the results of the binding levels of TKD molecules, TKDm molecules and KRas proteins in cells: A is the results of the binding levels of TKD molecules, TKDm molecules and KRas proteins in HT29 (KRas wt) cells; B is the results of the binding levels of TKD molecules, TKDm molecules and KRas proteins in HCT116 (KRas G13D) cells.

[0076] Figure 11 This is a graph showing the results of KRas protein degradation by TKD molecules and TKDm molecules at different concentrations.

[0077] Figure 12 This is a graph showing the results of TKD molecules with different concentrations degrading KRas protein at different times.

[0078] Figure 13 This is a diagram showing the degradation effect of TKD molecules at different concentrations on KRas proteins with mutations at different sites.

[0079] Figure 14 Figures 2 and 3 are flow cytometry results after treatment of TKD molecules with different cells: A is the flow cytometry result after treatment of TKD molecules with normal intestinal epithelial cells FHC; B is the flow cytometry result after treatment of TKD molecules with HT29 cells; C is the flow cytometry result after treatment of TKD molecules with HCT116 cells.

[0080] Figure 15 Figure 3 is the accumulation of TKD molecules in tumors derived from KRas wild-type HT29 cells and KRas mutant HCT116 cells.

[0081] Figure 16 The figures show the inhibition results of TMD molecules and TKDm molecules on colorectal cancer: A shows the inhibition results of different concentrations of TMD molecules and TKDm molecules on HT29 (KRas wt) and HCT116 (KRas G13D); B shows the inhibition results of different concentrations of TMD molecules and TKDm molecules on the clone growth of HT29 (KRas wt) and HCT116 (KRas G13D): ** indicates P < 0.01; *** indicates P < 0.001; ns indicates no significant difference.

[0082] Figure 17Figure 3 is a graph showing the effect of TKD molecules on tumor growth: A is a visual graph showing the effect of TKD molecules on the growth of KRas wild-type (HT29) colorectal cancer; B is a visual graph showing the effect of TKD molecules on the growth of KRas mutant (HCT116) colorectal cancer; C is a statistical graph showing the effect of TKD molecules on the growth of KRas wild-type (HT29) colorectal cancer; D is a statistical graph showing the effect of TKD molecules on the growth of KRas mutant (HCT116) colorectal cancer: **** indicates P < 0.0001; ** indicates P < 0.01; * indicates P < 0.05.

[0083] Figure 18 The diagram below shows the effect of TKD molecules on the mouse body: A shows the effect of TKD molecules on the mouse body weight; B shows the effect of TKD molecules on the mouse lungs, liver, spleen, and kidneys.

[0084] Figure 19 This is a graph showing the retention time of TKD molecules in the body.

[0085] Figure 20 This is a graph showing the effect of gefitinib on the cell viability of colorectal cancer cells HCT116 and HT29: **** indicates P<0.0001; *** indicates P<0.001; ** indicates P<0.01; * indicates P<0.05; ns indicates no significant difference.

[0086] Figure 21 It is a visual representation of the effect of gefitinib on the clonal growth of colorectal cancer cells HCT116, HT29 and normal intestinal epithelial cells FHC.

[0087] Figure 22 The figure shows the effect of gefitinib on KRas wild-type (HT29) colorectal cancer and KRas mutant (HCT116) colorectal cancer: A is a visual diagram of the effect of gefitinib on KRas wild-type (HT29) colorectal cancer and KRas mutant (HCT116) colorectal cancer; B is a statistical result diagram of the effect of gefitinib on KRas wild-type (HT29) colorectal cancer and KRas mutant (HCT116) colorectal cancer: ** indicates P < 0.01; ns indicates no significant difference.

[0088] Figure 23 This figure shows the effect of gefitinib on the Raf / ERK / c-myc signaling pathway in KRas wild-type cells HT29 and KRas mutant cells HCT116.

[0089] Figure 24This is a diagram showing the effects of TMD molecules and TKDm molecules on the EGFR downstream signaling pathway KRas / Raf / ERK / c-myc in KRas wild-type cells HT29 and KRas mutant cells HCT116.

[0090] Figure 25 Graphs showing the effects of the combined use of TMD molecules, TKDm molecules and gefitinib on the growth of HT29 (KRas wt) and HCT116 (KRasG13D): A shows the effects of the combined use of TMD molecules and gefitinib on the growth of HT29 (KRas wt); B shows the effects of the combined use of TMD molecules and gefitinib on the growth of HCT116 (KRas G13D); C shows the effects of the combined use of TKDm molecules and gefitinib on the growth of HT29 (KRas wt); D shows the effects of the combined use of TKDm molecules and gefitinib on the growth of HCT116 (KRas G13D): * indicates P < 0.05; ** indicates P < 0.01; ns indicates no significant difference.

[0091] Figure 26 Graph showing the effects of the combined use of TMD molecules, TKDm molecules, and gefitinib on the clonal growth of HT29 (KRas wt) and HCT116 (KRasG13D): A shows the effects of the combined use of TMD molecules, TKDm molecules, and gefitinib on the clonal growth of HT29 (KRas wt); B shows the effects of the combined use of TMD molecules, TKDm molecules, and gefitinib on the clonal growth of HCT116 (KRas G13D).

[0092] Figure 27 The figures show the effects of the combined use of TKD and gefitinib on HT29 (KRas wt)-derived tumors and HCT116 (KRasG13D)-derived tumors: A is a visual diagram of the effects of the combined use of TKD and gefitinib on HT29 (KRas wt)-derived tumors; B is a visual diagram of the effects of the combined use of TKD and gefitinib on HCT116 (KRas G13D)-derived tumors; C is a statistical result diagram of the effects of the combined use of TKD and gefitinib on HT29 (KRas wt)-derived tumors; D is a statistical result diagram of the effects of the combined use of TKD and gefitinib on HCT116 (KRas G13D)-derived tumors: **** indicates P < 0.0001; *** indicates P < 0.001; ** indicates P < 0.01; * indicates P < 0.05; ns indicates no significant difference. DETAILED DESCRIPTION

[0093] The present invention is further described in detail below through specific examples.

[0094] It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0095] The experimental methods in the following examples, where specific conditions are not specified, were generally performed under conventional conditions or the conditions recommended by the manufacturers. The materials and reagents used in these examples were commercially available unless otherwise specified.

[0096] Example 1 Construction of KRas protein degradation system TKD and verification of its effect

[0097] This example uses the designed and constructed nano-antibody-mediated KRas protein targeted degradation system TKD to quickly and efficiently reduce the expression of wild-type or mutant KRas in colorectal cancer cells as an example to demonstrate and confirm the feasibility and advantages of the technical solution of the present invention. KRas protein plays an important role in maintaining the growth of colorectal cancer cells. Therefore, the design of effective inhibitory drugs against KRas is of great significance for the clinical treatment of colorectal cancer. However, as a member of the "undruggable protein", KRas has not yet been used for clinical treatment of KRas targeted drugs.

[0098] 1. Preparation of fusion protein TKD molecules

[0099] (1) Design and construction of TKD molecular tools

[0100] 1. KRas Nanobody VHH Sequence Screening

[0101] In the early stage of this example, high-purity KRas protein (amino acid sequence: MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM, SEQ ID NO.7) After repeated immunization of alpacas four times (using 5 mg of purified target protein (KRas) to immunize alpacas four times, each immunization took 15 days), the immunized alpaca white blood cells were extracted, and mRNA was isolated to construct an antibody gene phage display library. Phage screening and ELISA detection were performed, and the target antibody VHH sequence was determined by sequencing. Finally, a high-affinity KRas protein nanobody VHH sequence was obtained (the KRas nanobody VHH sequence screening process is as follows) Figure 1 The specific process is as follows:

[0102] 1.1 RNA extraction and reverse transcription

[0103] 1.1.1 RNA extraction:

[0104] (1) Transfer the alpaca peripheral blood lymphocytes preserved with Trizol to a 1.5 mL centrifuge tube and add 1 / 5 volume of chloroform to mix;

[0105] (2) After standing at room temperature for 5 minutes, centrifuge at 12,000 g for 15 minutes at 4°C;

[0106] (3) Transfer the supernatant after centrifugation to a new centrifuge tube;

[0107] (4) Add an equal volume of isopropanol to a new centrifuge tube and mix thoroughly by inversion;

[0108] (5) After standing at room temperature for 10 minutes, centrifuge at 12000g for 10 minutes at 4°C and discard the supernatant;

[0109] (6) Add 75% ethanol to wash the precipitate, centrifuge at 7500g for 5 minutes at 4°C, dry the precipitate at room temperature, and dissolve it in DEPC-treated water. After dissolution, combine all samples to obtain the total RNA extracted (gel electrophoresis results are shown in Figure 2). Figure 2 shown).

[0110] 1.1.2 Reverse transcription of cDNA:

[0111] (1) Reverse transcription of the total RNA using the Takara reverse transcription kit: Divide the total RNA sample into two parts, one using the Oligo dT Primer in the kit as a primer, and the other using the Random 6-mers in the kit as a primer. According to the instructions of the reverse transcription kit, the total RNA obtained in the previous step is used as a template for reverse transcription into cDNA, and the two parts are stored in two centrifuge tubes respectively.

[0112] 1.2 PCR amplification

[0113] 1.2.1 First round of PCR amplification:

[0114] (1) The first round of PCR reaction was carried out using cDNA as template, and Taq DNA Polymerase Hot Start enzyme was used for PCR amplification. In order to determine the optimal template usage, 2 μL, 3 μL, and 5 μL of oligo dT Primer and random 6-mer cDNA were used as templates, respectively. The PCR reaction system was as follows: cDNA 2 / 3 / 5 μL (concentration was 800 ng / uL); CALL 001 / CALL 002 (Shenzhen Kangti Life) 2 μL / 2 μL; dNTP Mix 4 μL; 10× ExTaq Buffer 5 μL; HS Ex Taq 0.25 μL; ddH2O to 50 μL; PCR reaction system was as follows: 98°C for 3 min; 94°C for 50 s, 55°C for 30 s, 72°C for 40 s + 2 s / cycle, 23 cycles; 72°C for 5 min; 4°C ∞;

[0115] (2) After the reaction, 20 μL of PCR product was taken for 1% agarose gel electrophoresis (the results of agarose gel electrophoresis with different amounts of cDNA template are shown in the figure). Figure 3 Finally, the template amount with a single target band and a fragment size of 600 bp in the electrophoresis result was selected as the optimal template amount, and all cDNAs were subjected to PCR reaction using the same conditions according to this template amount;

[0116] (3) All PCR products were subjected to 1% agarose gel electrophoresis, and the gel was cut to recover the target fragment with a size of about 600 bp;

[0117] (4) Collect all the purified and recovered products into a centrifuge tube, which is the first round of PCR amplification products and store at -20℃.

[0118] 1.2.2 Second round of PCR amplification

[0119] (1) The first-round PCR amplification product was used as a template for the second-round PCR reaction. In order to determine the optimal template usage, 0.1 μL, 0.25 μL, 0.5 μL, 1 μL, and 2 μL were used as templates, respectively. The PCR reaction system was as follows: cDNA 0.1 / 0.25 / 0.5 / 1 / 2 μL (concentration was 800 ng / μL); VHH-for / VHH-back (Shenzhen Kangti Life) 2 μL / 2 μL; dNTP Mix 4 μL; 10×ExTaq Buffer 5 μL; HS Ex Taq 0.25 μL; ddH2O to 50 μL; PCR reaction system was as follows: 98°C for 3 min; 94°C for 50 s, 55°C for 30 s, 72°C for 40 s, 11 cycles; 72°C for 5 min; 4°C ∞;

[0120] (2) After the reaction, 20 μL of PCR product was taken for 1% agarose gel electrophoresis (the results of agarose gel electrophoresis with different amounts of cDNA template are shown in the figure). Figure 4 As shown), the template amount with a single target band and a fragment size of 600bp in the electrophoresis result was finally selected as the optimal template amount. 1 / 3 of the volume of the obtained first-round PCR amplification product, a total of 192 reactions, was carried out according to this template amount and all the recovered products were collected into a centrifuge tube using 1.2.2.(1), which was the second-round PCR amplification product. At the same time, 2 μL was taken to detect the concentration of the recovered product using a nucleic acid concentration meter and recorded. The remaining products were stored at -20°C under the same conditions for PCR reaction, and then the PCR reaction solution was purified using a universal DNA purification and recovery kit;

[0121] (3) Collect all recovered products into a centrifuge tube, which is the second round of PCR amplification products. At the same time, take 2 μL and use a nucleic acid concentration meter to detect the concentration of the recovered products and record it. The remaining products are stored at -20°C.

[0122] 1.3 Enzyme digestion and ligation

[0123] 1.3.1 Enzyme digestion of vector and PCR product:

[0124] (1) Using pComb3XSS as a phage plasmid vector, 20 μg of pComb3XSS vector and 10 μg of the second-round PCR amplification product were digested with Spe I and Sac I, respectively, and incubated at 37°C for 4 h;

[0125] (2) Use a DNA recovery and purification kit to purify the pComb3XSS vector and the second-round PCR amplification product and store them at 4°C.

[0126] 1.3.2 Connection

[0127] (1) The vector and fragment were ligated. The ligation reaction system was as follows: 1.6 μg of PCR product after enzyme digestion; 4 μg of vector (pComb3XSS) after enzyme digestion; 30 μL of T4 ligase; 200 μL of 10×T4 reaction buffer; ddH2O to 2000 μL;

[0128] (2) Incubate the ligation reaction system at 4°C overnight (approximately 16 hours);

[0129] (3) Purify the ligation reaction solution using a universal DNA purification and recovery kit, detect the concentration of the recovered product, and store it at 4°C.

[0130] 1.4 Construction of bacterial and phage libraries

[0131] 1.4.1 Verification of ligation product conversion rate

[0132] (1) Take a 50 μL aliquot of TG1 competent cells and place on ice for 5-10 minutes to thaw;

[0133] (2) Add 100 ng of ligation product and transfer to a pre-cooled electroporation cuvette with a spacing of 1 mm. Set the parameters in the electroporator (1800 V, 1 mm) and perform electroporation.

[0134] (3) Immediately after electroporation, add 1 mL of SOC culture medium preheated at 37°C, mix well, and shake at 200 rpm at 37°C for 1 h to recover the bacteria.

[0135] (4) Take 100 μL of 1 mL of resuscitated bacterial solution and perform 10-fold serial dilutions and plate. Calculate the number of transformed colonies obtained in each reaction based on the dilution factor and the number of single colonies, which is the transformation efficiency of the ligation product.

[0136] (5) At the same time, 48 monoclonal bacteria were randomly selected for colony PCR. The PCR reaction system was as follows: 2 μL of bacterial solution (OD600 = 49.15); 2 μL / 2 μL of VHH-for / VHH-back (Shenzhen Kangti Life); 4 μL of dNTP Mix; 5 μL of 10×ExTaq Buffer; 0.25 μL of HS Ex Taq; ddH2O to 50 μL; the PCR reaction system was as follows: 98°C for 3 min; 94°C for 50 s, 55°C for 30 s, 72°C for 40 s, 11 cycles; 72°C for 5 min; 4°C ∞; a single band of PCR product around 600 bp was considered a positive clone (the results of colony PCR agarose gel electrophoresis are shown in Figure 2). Figure 5 The monoclonal positive rate was estimated based on the results of the PCR.

[0137] 1.4.2 Bacterial library construction

[0138] (1) Perform electroporation reaction using 20 competent cells as described above;

[0139] (2) After 1 h of resuscitation at 37°C, 100 μL of the culture was diluted 10-fold and plated, and cultured at 37°C overnight.

[0140] (3) Collect all the remaining bacterial liquid and evenly spread it on 6 245 mm square culture plates (2×YT medium containing 100 μg / mL Amp (ampicillin)), and culture at 37°C overnight;

[0141] (4) Calculate the number of transformed colonies obtained from all reactions based on the dilution factor and the number of single colonies, which is the library capacity of the bacterial library;

[0142] (5) At the same time, 60 single clones were randomly selected from the gradient dilution plate for colony PCR. The PCR reaction system was as follows: 2 μL bacterial solution (OD600 = 49.15); VHH-for / VHH-back (Shenzhen Kangti Life) 2 μL / 2 μL; dNTP Mix 4 μL; 10× ExTaq Buffer 5 μL; HS Ex Taq 0.25 μL; ddH2O to 50 μL; PCR reaction system was as follows: 98°C for 3 min; 94°C for 50 s, 55°C for 30 s, 72°C for 40 s, 11 cycles; 72°C for 5 min; 4°C ∞; verify the clone positive rate of the bacterial library;

[0143] (6) Scrape the colonies from the 245 mm square culture plate cultured overnight using 2×YT liquid medium, place them in a 50 mL centrifuge tube, and measure their OD 600 Add glycerol to a final concentration of 20% and store at -80°C.

[0144] 1.4.3 Phage library preparation

[0145] (1) According to the OD of the bacterial library 600 Calculate the volume of bacterial library that needs to be added to 100 mL of 2×YT liquid medium. Inoculate the bacterial library into 100 mL of 2×YT liquid medium (containing 100 μg / mL Amp) based on the calculated results and culture at 37°C and 250 rpm until OD 600 0.5-0.55;

[0146] (2) Add helper phage M13K07 at a ratio of 1:20 (number of bacteria: number of phages) according to the titer of the helper phage, and incubate at 37°C, 250 rpm for 30 min;

[0147] (3) Add Kana (kanamycin) to a final concentration of 50 μg / mL and culture overnight at 30°C and 250 rpm;

[0148] (4) Centrifuge the overnight culture at 4°C, 4000 rpm for 10 min, and transfer the supernatant to a new 50 mL centrifuge tube.

[0149] (5) Add 1 / 4 of the pre-cooled 20% PEG / 2.5M NaCl stock solution, mix well, and incubate on ice for 30 min;

[0150] (6) Centrifuge at 4000 rpm for 20 min at 4°C, discard the supernatant, and invert to dry for 2 min;

[0151] (7) Add 1 mL of PBS and resuspend in a new centrifuge tube. Centrifuge at 12,000 rpm for 20 min at 4°C.

[0152] (8) After centrifugation, transfer the supernatant to a new centrifuge tube, add 1 / 4 volume of pre-cooled 20% PEG / 2.5M NaCl storage solution again, mix well, and incubate on ice for 10 min;

[0153] (9) Centrifuge at 4°C, 12,000 rpm for 10 min, discard the supernatant, resuspend with 1 mL of PBS, centrifuge at 12,000 rpm for 2 min, transfer the supernatant to a new centrifuge tube, and store at -80°C. This is the purified phage library.

[0154] (10) Take 10 μL of the prepared phage library and dilute it 10-fold in a 1.5 mL centrifuge tube, for a total of 12 gradients, that is, take 10 μL of the phage library and dilute it to 100 μL, then take 10 μL from it and dilute it to 100 μL, and so on, for a total of 12 gradients to 10 -12 , oscillate to mix.

[0155] (11) Add 90 μL of TG1 bacterial solution to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 min;

[0156] (12) Take 5 μL from each dilution centrifuge tube and add it dropwise to 2×YT solid medium (Amp) and culture at 37°C overnight;

[0157] (13) Count the number of single colonies on the plate at the dilution ratio at which single colonies can be clearly distinguished, and calculate the phage library titer according to the following formula: Phage library titer = number of single colonies at the corresponding dilution ratio × dilution ratio × 400.

[0158] 2. Immunoscreening

[0159] 2.1 First round of screening

[0160] 2.1.1 Take out the screening antigen KRas protein from the -80℃ freezer and place it on ice to thaw;

[0161] 2.1.2 Coat the screening antigen onto the immunotubes (50 μg / tube, coating solution: CBS buffer, pH 9.6, 2 mL / tube) and rotate slowly at 4°C overnight. Simultaneously coat the tubes with BSA as a control.

[0162] 2.1.3 Discard the liquid in the overnight coated immunotubes and wash the immunotubes three times with 2 ml of PBS buffer at room temperature, rotating for 5 minutes each time.

[0163] 2.1.4 Add 2 mL of blocking solution (3% BSA) and rotate at room temperature for 2 h;

[0164] 2.1.5 Discard the liquid in the blocked immunotube and add 2 mL of PBST buffer (1× PBS plus 0.1% Tween 20, the same below) to wash the immunotube three times at room temperature, rotating for 5 minutes each time;

[0165] 2.1.6 Discard the wash solution in the immunotube, add 1 mL of PBS, and add 32 μL of the prepared phage library as the input phage library for the first round of screening according to the following formula. Incubate with rotation at room temperature for 1 hour:

[0166]

[0167] Where, V is the volume of phage added (unit: μL), T library is the phage titer;

[0168] 2.1.7 Discard the liquid in the immunotube and wash the immunotube 20 times with 2 mL of PBST (1× PBS plus 0.1% Tween 20, the same below) at room temperature, rotating for 5 minutes each time.

[0169] 2.1.8 Discard the liquid in the immunotube and remove as much residual liquid as possible. Add 1 mL of 0.25 mg / mL Trypsin solution and elute with rotation at room temperature for 30 minutes.

[0170] 2.1.9 Add 10 μL of 10% AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride) to terminate elution and transfer the solution in the immunotube to a new 1.5 mL centrifuge tube. This is the phage eluate for the first round of screening.

[0171] 2.2 First round of phage eluate titer detection

[0172] 2.2.1 Streak a single colony of the TG1 strain stored at -80°C onto 2×YT solid medium and culture at 37°C overnight (store at 4°C for one week). Pick a single colony from the single colony plate and transfer it to 5 mL of 2×YT medium and culture at 37°C overnight.

[0173] 2.2.2 Transfer 500 μL of overnight culture to 5 mL of 2×YT liquid medium and incubate at 37°C, 250 rpm for about 45-60 min until the OD 600 0.5-0.55;

[0174] 2.2.3 Take 10 μL of the first round of phage eluate and dilute it 10-fold in a 1.5 mL centrifuge tube. Dilute it 12 times in total. That is, take 10 μL of the first round of phage eluate and dilute it to 100 μL. Then take 10 μL and dilute it to 100 μL. And so on. Dilute it 12 times in total. -12 , oscillate to mix;

[0175] 2.2.4 Add 90 μL of TG1 bacterial solution to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 min.

[0176] 2.2.5 Take 5 μL from each dilution centrifuge tube and add dropwise to 2×YT solid medium (Amp) and incubate inverted at 37°C overnight;

[0177] 2.2.6 Count the number of colonies on the plate at the dilution at which single colonies can be clearly distinguished, and calculate the number of phagemids per milliliter of phage solution, i.e., the phage library titer, according to the following formula:

[0178] T (pfu / ml) = N × D × 400

[0179] Wherein, T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0180] 2.3 Amplification of the first round of phage eluate

[0181] 2.3.1 Streak a single colony of the TG1 strain stored at -80°C onto 2×YT solid medium and culture overnight at 37°C (store at 4°C for one week). Transfer a single colony from the single colony plate to 5 mL of 2×YT medium and culture overnight at 37°C.

[0182] 2.3.2 Transfer 500 μl of overnight culture to 5 mL of 2×YT liquid medium and incubate at 37°C, 250 rpm for about 45-60 min until the OD 600 The value is 0.5-0.55;

[0183] 2.3.3 Add 500 μL of phage eluate obtained after the first round of screening to OD 600 The bacterial solution is 0.5-0.55 (the remaining eluate is stored at 4°C);

[0184] 2.3.4 Continue incubation at 37°C and 250 rpm for 30 min.

[0185] 2.3.5 Spread the entire bacterial suspension evenly onto a 245 mm square plate containing 2% agarose containing 100 μg / mL Amp and incubate overnight at 37°C.

[0186] 2.3.6 Take the square plate from which the culture was overnight and add 6 mL of 2×YT liquid medium (containing 100 μg / mL Amp) to the surface of the culture plate. Use a spreading stick to gently scrape off the colonies on the square plate and collect the bacterial liquid into a 15 mL centrifuge tube. This is the amplified bacterial sublibrary. At the same time, use a spectrophotometer to measure the OD value of the bacterial liquid. 600 The value is the OD of the eluate bacterial library 600 value, and add glycerol with a final concentration of 20%, which is the first round of bacterial library.

[0187] 2.3.7 Calculate the amount of bacterial solution from the eluate bacterial library according to the following formula and transfer it to 100 ml of 2×YT liquid medium (containing 100 μg / mL Amp) to make the initial OD 600 is 0.1:

[0188]

[0189] Wherein, V is the volume of the transfer bacterial solution (unit: μL), OD600 is the OD value of the constructed eluate bacterial library. 600 ;

[0190] 2.3.8 Incubate at 37°C, 250 rpm until the OD value of the bacterial solution reaches 600 Reach 0.5-0.55;

[0191] 2.3.9 Calculate and add helper phage M13K07 according to the following formula so that the number of bacteria: number of phage = 1:20:

[0192]

[0193] Where V is the volume of helper phage added (unit: mL, T helper-phage is the titer of the helper phage used, and OD600 is the OD of the bacterial solution. 600 value;

[0194] 2.3.10 Continue incubation at 37°C and 250 rpm for 30 min.

[0195] 2.3.11 Add Kana to a final concentration of 50 μg / mL and culture overnight at 30°C and 250 rpm.

[0196] 2.4 First round of phage purification

[0197] 2.4.1 Transfer the overnight culture to a new 50 mL centrifuge tube and centrifuge at 4000 rpm at 4°C for 10 min.

[0198] 2.4.2 Transfer the supernatant after centrifugation to a new 50 mL centrifuge tube, add 1 / 4 volume of 4°C pre-cooled 20% PEG / 2.5M NaCl, mix thoroughly, and place on ice for 30 minutes;

[0199] 2.4.3 Centrifuge at 4000 rpm and 4°C for 20 min, discard the supernatant, and invert on paper for 2 min;

[0200] 2.4.4 Add 1 mL of PBS to resuspend the pellet, transfer the resuspension to a new 1.5 mL centrifuge tube, and centrifuge at 13,000 rpm and 4°C for 20 min.

[0201] 2.4.5 Transfer the supernatant after centrifugation to a new 1.5 mL centrifuge tube, add 1 / 4 volume of pre-chilled 20% PEG / 2.5 M NaCl solution, mix well, and place on ice for 10 minutes;

[0202] 2.4.6 Centrifuge at 13,000 rpm and 4°C for 10 min, discard the supernatant, and resuspend the pellet in 1 mL of PBS.

[0203] 2.4.7 Centrifuge at 13,000 rpm and 4°C for 2 minutes. Transfer the supernatant to a new 1.5 mL centrifuge tube. This is the first round of phage sub-library screening. Aliquot 100 μL / tube and store at -80°C for long-term storage or at -20°C for short-term storage (1-2 weeks).

[0204] 2.4.8 The titer of the first round of phage sub-library screening was tested using the same method as in 2.2.

[0205] 2.5 Second round of screening

[0206] The screening method is the same as 2.1. The input phage is 0.25 mL of the phage sub-library obtained in the first round of screening, which is used as the input phage library in the second round of screening to obtain the phage eluate of the second round of screening.

[0207] 2.6 Second round of phage eluate titer detection

[0208] The method is the same as 2.2.

[0209] 2.7 Amplification and purification of the second round of eluate

[0210] The method is the same as 2.3-2.4 to obtain the second round of screening phage sub-library.

[0211] 2.8 Second round of screening of phage sub-library titer detection,

[0212] The method is the same as 2.2.

[0213] 2.9 Third round of screening

[0214] The screening method was the same as in 2.1. The input phage was 0.25 mL of the phage sublibrary obtained in the second round of screening, which was used as the input phage library in the third round of screening to obtain the phage eluate of the third round of screening.

[0215] 2.10 The third round of phage eluate titer detection

[0216] The method is the same as 2.2.

[0217] 2.11 Monoclonal ELISA detection

[0218] 2.11.1 Pre-store the TG1 strain at -80°C on 2×YT solid medium for single colony streaking and incubate at 37°C overnight (store at 4°C for one week). Pick a single colony from the single colony plate and transfer it to 5 mL of 2×YT medium and incubate at 37°C overnight.

[0219] 2.11.2 Transfer 500 μL of overnight culture to 5 mL of 2×YT liquid medium and incubate at 37°C, 250 rpm for about 45-60 minutes until the OD 600 The value is 0.5-0.55;

[0220] 2.11.3 Take 10 μL of the phage eluate after the third round of screening and dilute it 10-fold in a 1.5 mL centrifuge tube. Dilute 12 times in a 10-fold gradient. That is, dilute 10 μL of the phage library to 100 μL, then dilute 10 μL to 100 μL, and so on. Dilute 12 times in a 10-fold gradient. Vortex to mix.

[0221] 2.11.4 Add 90 μL of OD to each dilution centrifuge tube 600 The bacterial solution with a value of 0.5-0.55 is mixed evenly;

[0222] 2.11.5 Continue incubation at 37°C, 250 rpm for 30 min.

[0223] 2.11.6 Spread the bacterial suspension evenly onto a 2×YT solid medium plate containing 100 μg / mL Amp and incubate at 37°C overnight.

[0224] 2.11.7 Randomly pick single colonies from the overnight culture plate and place them in a sterile 96-well cell culture plate (P1-P2). Add 200 μL of 2×YT medium (containing 100 μg / mL Amp) to each well and incubate at 37°C overnight.

[0225] 2.11.8 Transfer 2 μL of the overnight culture to 200 μL of 2×YT liquid medium (containing 100 μg / mL Amp) per well of a new 96-well cell culture plate and incubate at 37°C for 3 h. Store the overnight culture at 4°C before transfer.

[0226] 2.11.9 Calculate the number of bacteria and phages in each well using the following formula and add helper phage M13K07 so that the ratio of bacterial count to phage count is 1:20:

[0227]

[0228] Where V is the volume of helper phage added (unit: ml), T helper-phage is the helper phage titer used;

[0229] 2.11.10 Incubate at 37°C for 30 min, add Kana to a final concentration of 50 μg / mL, and incubate at 30°C overnight.

[0230] 2.11.11 Centrifuge the 96-well culture plate after overnight incubation at 4000 rpm for 10 min at 4°C and store at 4°C until use;

[0231] 2.11.12 Coat the screening antigen onto an ELISA plate (1 ng / μL, coating solution: CBS, pH 9.6, 100 μL / well). Simultaneously coat the plate with BSA as a control. Coating should be carried out overnight at 4°C.

[0232] 2.11.13 Discard the overnight coated ELISA plate contents, add 200 μL of PBS buffer to each well, and wash the plate three times at room temperature for 10 minutes each wash.

[0233] 2.11.14 Add 200 μL of blocking solution (3% BSA) to each well to block the ELISA plate at room temperature for 1 hour.

[0234] 2.11.15 Discard the blocking solution and add 200 μL of PBST (1× PBS plus 0.1% Tween 20, the same below) buffer to each well. Wash the ELISA plate three times at room temperature for 10 minutes each time.

[0235] 2.11.16 Add 120 μl of 3% BSA to each well, followed by 80 μl of the supernatant from centrifugation in step 2.11.11, and incubate at room temperature for 2 h.

[0236] 2.11.17 Discard the liquid in the ELISA plate and add 200 μL PBST buffer to each well and wash three times for 10 minutes each time.

[0237] 2.11.18 Add M13 Bacteriophage Antibody (HRP) and Mouse mAb to each well at a dilution of 1:40,000 in blocking buffer (100 μL / well) and incubate at room temperature for 1 hour.

[0238] 2.11.19 Discard the liquid in the ELISA plate and add 200 μl PBST buffer to each well and wash three times for 10 minutes each time;

[0239] 2.11.20 Add 100 μl TMB single-component colorimetric solution to each well and develop for 2-3 minutes in the dark. Add 100 μl 1M HCl to each well to terminate the reaction and read the OD value using a microplate reader. 450nm value, record and save.

[0240] 2.12 Secondary verification of positive clones by ELISA

[0241] To exclude false positive results, clones initially identified as positive were subjected to secondary ELISA verification using the same method as in 2.11.

[0242] 2.13 Sequencing of positive clones

[0243] Positive monoclonal clones were selected based on ELISA test data and secondary verification data

[0244] Take 5 μL of positive clones from the monoclonal ELISA test plate (2.11.7) and inoculate into 1 mL of 2×YT medium (containing 100 μg / mL Amp), and culture at 37°C and 250 rpm until OD 600 When the concentration reaches 0.8-1.0 (about 6-8 hours), 0.5 mL of bacterial solution was taken for sequencing and the rest of the bacterial solution was stored at 4°C.

[0245] 2.14 Sequence Analysis

[0246] The sequenced sequences were aligned and analyzed using GENtle software, and the antibody sequences were translated into amino acids using GENtle software. The amino acid sequence was DVQLQESGGGLVQAGGSLRLSCVASGRTFSTYPTGWFRQAPGKEREFVARINLSGGITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCGGGSTTWAGGIPTNFDYWGQGTQVTVSSGR (SEQ ID NO. 1).

[0247] 3. Construction of TKD Expression Vector

[0248] DNA sequences containing molecular elements such as BR2, nanobody (VHH), and CTM were synthesized and cloned into the prokaryotic expression vector pET28a (plasmid synthesized and constructed by Suzhou Hongxun Biotechnology Co., Ltd.). The synthesized TKD-pET28a plasmid was transformed into E. coli BL (Rosetta) competent cells. Positive recombinants were screened on cannabinoid-resistant plates and verified by colony PCR and DNA sequencing. Sequencing results were analyzed by NCBI Blast. Plasmids were stored at -20°C for long-term storage. Strains with correct sequencing results were stored at -80°C in 15% glycerol. In a control TKD molecule, the arginine residue at the CTM site was artificially mutated to alanine (QA) to prevent lysosomal recognition. This strain, designated TKDm, was generated using the same method (plasmid synthesized and constructed by Suzhou Hongxun Biotechnology Co., Ltd.) and stored at -80°C in 15% glycerol. The pET28a vector carries a 6*His tag, which allows for the expression and purification of His proteins. Schematic diagram of the construction of TKD molecules and the degradation process Figure 6 shown.

[0249] (II) Expression and purification of the TKD system

[0250] 1. Both TKD and TKDm were induced to express using isopropylthiogalactoside (IPTG), and different IPTG concentrations, action temperatures and action times were used to explore the induction expression conditions. Subsequently, SDS-PAGE and Coomassie Brilliant Blue experiments were used to determine the optimal expression conditions. In this example, the correctly sequenced TKD and control group TKDm bacterial liquids were inoculated into TB culture medium containing kanamycin resistance (50ng / μL) and cultured at 37°C with shaking overnight. The next day, the activated strains were inoculated into fresh culture medium at a ratio of 1:100 to expand the culture, and cultured at 37°C with shaking at 220rpm until the OD 600 =0.6, add isopropylthiogalactoside (IPTG) to a final concentration of 0.5 mM. Continue inducing expression under the same conditions for approximately 4 hours, then collect the cells, centrifuge at 5000 rpm for 30 minutes at 4°C, resuspend the cells in 1× PBS buffer, and centrifuge again to collect the cells. The collected cells are resuspended in 1× PBS buffer to a 10% suspension, freeze-thawed three times in liquid nitrogen and then frozen and thawed three times at 37°C, then sonicated in an ice bath for 30 minutes (5 seconds on, 5 seconds off) until the suspension is completely disrupted (or directly use a high-pressure disrupter at 700-900 Pa at 4°C for 8 minutes). Urea is added to the completely disrupted suspension to a final concentration of 8 M, and incubated at room temperature for at least 30 minutes until the suspension becomes clear and translucent. Centrifuge at 10000 rpm for 1 hour at 4°C, discard the precipitate, and filter the supernatant through a 0.22 mm pore filter for subsequent purification.

[0251] 2. Denaturation purification and renaturation of TKD molecules: Resuspend the supernatant obtained above in Ni-NTA agarose gel and incubate at 4°C for 2 hours (incubate according to the ratio of 5g of bacterial cells to 1mL of 50% Ni-NTA agarose gel). During the incubation process, the Ni-NTA agarose gel changes color from blue to brown. Centrifuge at 1000g at 4°C for 1 minute. Take 20uL of the supernatant for subsequent purification verification. Discard most of the supernatant, retaining only a small amount for resuspending the Ni-NTA agarose gel. Load the column and wash three times with 1× PBS buffer containing 50mM imidazole and 8M urea, each time with 2 column volumes. Collect 20uL of the last wash buffer. Then elute with 1x PBS buffer containing 300mM imidazole and 8M urea. If the Ni-NTA agarose gel turns blue again, elution is basically complete. A 20 μL sample of the eluate was retained and the purification efficiency of the fusion protein was determined by 12% SDS-PAGE. The concentration of the eluted protein denaturation solution was determined by adjusting the protein concentration to approximately 1 mg / mL using 1× PBS buffer containing 8 M urea. 5% glycerol was added, and the mixture was then subjected to gradient cryodialysis. The dialysate concentration consisted of 1× PBS buffer containing 4 M, 2 M, and 0 M urea, respectively, with a 1:10 ratio of protein denaturation solution to dialysate. Each gradient was maintained at -10°C for 12 h. The dialyzed protein solution was centrifuged at 10,000 g for 10 min. The supernatant was concentrated using a 3KD ultrafiltration tube (Millipore, Cat. No. UFC900396) and centrifuged at 4500 g. The supernatant was ultrafiltered to 500 μL at 4°C, and the concentrate was then rinsed three times with 1× PBS and centrifuged three times. The concentration of the obtained concentrated protein was measured using a BCA protein concentration assay kit (Thermo Fisher, catalog number: 23227) and stored at -80°C.

[0252] The purification effects of TKD and TKDm are as follows Figure 7 As shown, the purity has reached more than 90%, which can be used for subsequent experimental verification. The amino acid sequence of the TKD molecule is: (SEQ ID NO. 4) (The single underline is the amino acid sequence of the cancer cell-specific membrane-penetrating peptide BR2, SEQ ID NO. 2; the double underline is the amino acid sequence of the lysosomal recognition peptide CTM, SEQ ID NO. 3). The amino acid sequence of the TKDm molecule is: (SEQ ID NO.5) (wherein, the single underline is the amino acid sequence of the cancer cell-specific membrane-penetrating peptide BR2, SEQ ID NO.2; the double underline is the amino acid sequence of CTMmt that cannot be recognized by lysosomes, SEQ ID NO.6).

[0253] 2. Verification of TKD's ability to recognize and bind wild-type and mutant KRas proteins

[0254] This example first verified the binding ability of the TKD molecule to the KRas protein. In vitro, purified TKD molecules and KRas proteins (the KRas protein purification process was consistent with the "TKD system expression and purification" process) were used to conduct enzyme-linked immunosorbent assays (ELISA) and surface plasmon resonance (SPR) binding ability experiments. Intracellular co-immunoprecipitation (Co-IP) experiments were primarily used to verify the binding level of the TKD molecule and KRas protein in cells.

[0255] 1. ELISA verification of the binding level between TKD molecules and KRas protein in vitro

[0256] Enzyme-linked immunosorbent assay (ELASA) is an immunoassay technique developed based on immunoenzyme technology and can also be used to detect interactions between proteins.

[0257] (1) Control groups were set up, including blank control (NC1), TKD+IgG control (NC2), and IgG+KRas control (NC3). The concentrations and volumes of the components in the control groups were consistent with those in the experimental groups. The experimental groups included TKD and TKDm, which were used to verify the interaction with KRas protein.

[0258] (2) Coating: TKD and TKDm were diluted to a concentration of 1 ng / μL using ELISA coating solution (purchased from NeoBioscience, catalog number: NBC01). 100 μL of coating solution was added to each well of a 96-well ELISA plate and coated at 4°C overnight.

[0259] (3) Blocking: Wash the ELISA plate once for 5 minutes using phosphate buffered saline (PBS) (PBS formula: 4 g NaCl, 0.1 g KCl, 1.815 g sodium phosphate dibasic dodecahydrate, 0.12 g potassium phosphate dibasic, dissolved in 400 mL of ultrapure water and diluted to 500 mL before use); add 300 μL of 5% (w / v) bovine serum albumin to each well; block at 37°C for 2 hours.

[0260] (4) Adding purified protein KRas: 6.25, 12.5, 25, 50, and 100 μg of purified protein were pre-mixed and diluted in 200 μL PBS, and the diluted KRas protein was added to the wells in sequence. The plate was shaken slowly at room temperature for 5 hours. The ELISA plate was then washed 5 times for 5 minutes each time according to the above-mentioned washing method.

[0261] (5) Add anti-KRas (catalog number: 05-516, purchased from Abcam) antibody: dilute 1 μg of antibody in 2000 μL 5% BSA at a ratio of 1:2000 to obtain antibody diluent, add 100 μL of antibody diluent to each well, and incubate at room temperature for 2 hours or at 4°C overnight.

[0262] (6) Wash off excess antibodies: Wash as above, 5 times, 5 minutes each time.

[0263] (7) Incubation with secondary antibody: dilute the secondary antibody goat anti-mouse IgG HRP with horseradish peroxidase (catalog number: ab6721, purchased from Abcam) at a mass volume ratio of 1:2000, that is, dilute 1 μg of antibody into 2000 μL of 5% BSA to obtain antibody diluent, and add 100 μL of antibody diluent to each well for incubation.

[0264] (8) Color development: 100 μL of ELISA color development solution TMB (purchased from NeoBioscience, catalog number: TMS.12) was added to each well, incubated at 37°C for 1 minute, and immediately stopped with 50 μL of stop solution (purchased from NeoBioscience, catalog number: EST001).

[0265] (9) Read the value: After the color is stable, use a spectrophotometer to perform dual wavelength measurement and press OD 450 -OD 630 The absorbance value of each well is calculated and the drug effect is analyzed. Figure 8 As shown, compared with the control group, both TKD and the control molecule TKDm can interact more strongly with KRas protein.

[0266] 2. In vitro SPR binding assays were performed using purified TKD molecules and KRas proteins. The SPR assay was performed using a Biacore T200. For detailed procedures, please refer to the Biacore T200 Protein-Protein Binding Assay Guide. The key points of this example are as follows:

[0267] (1) Ligand coupling: First, calculate the target coupling amount according to the following formula: Wherein, Rmax is the maximum binding capacity on the chip surface, which is usually substituted with 100RU in protein testing. In this example, a coupled TKD molecule (17kD, analyte MW) was selected and the protein KRas (23kD, ligand MW) was passed through. m is the stoichiometric ratio, select 1 if unknown; R L is the ligand coupling level. The actual coupling amount in the experiment is 1.5 times of R L ; So after calculation, R LThe target coupling amount of TKD molecules is 74RU.

[0268] (2) Ligand preconcentration: TKD molecules were diluted to 10 μg / mL (at least 10-fold dilution ratio) with sodium acetate at pH 5.0, 4.5, and 4.0, respectively. 100 μL of each was prepared. Preconcentration experiments determined that pH 5.0 was the optimal coupling condition. Therefore, 200 μL of 10 μg / mL was prepared using sodium acetate at pH 5.0 for the formal coupling operation.

[0269] (3) Ligand coupling:

[0270] a. Open the Biacore T200 Control Software, click Open / New wizard template under File, and select immobilization. In the dialog box, select CM5 in Chip type and 1 in Flow cell cycle. Check Flow cell 2, select amine coupling as method, enter the ligand name, select aim for immobilized level, and enter the calculated 110 RU as target level. Select Flowpath 2 in Flow path (if chip channel 2 is in use, select channel 4). Then press Next, check prime, and select the experimental temperature, which is generally 25°C by default.

[0271] b. Select Sample and Reagent Rack1 from the left drop-down menu. The system will automatically arrange the sample placement positions (you can rearrange them by dragging the mouse); prepare a sufficient volume of sample according to the sample rack position table (if using EP tubes with caps, all caps must be cut off); place 100μL of EDC in R1D3, 100μL of NHS in R1D4, an empty tube in R1D5, 140μL of ethanolamine in R1D6, and 166μL of 10μg / mL ligand protein in R1D1; close the test tube rack lid and return the sample rack to the sample chamber; click Next. The Prepare Run Protocol dialog box will pop up. Confirm that the running buffer volume is greater than the minimum requirement in the table and click Start.

[0272] (4) Sample testing process:

[0273] a. In the Kinetics / Affinity interface, set the Flowpath to 2-1 or 4-3 and the Chiptype to CM5. In the Setup interface, under Startup, enter HBS-EP in the Solution column and change the Number of cycles to 3. In the Kinetics / Affinity-injection Parameter interface, in the Sample column, set the Contact time to 120 s, the Flow rate to 30 μL / min, the Dissociation time to 120 s, the Regeneration condition to Glycine 2.0, and the Regeneration time to 30 s.

[0274] b. In the Kinetics / Affinity-Sample interface, enter the analyte information: enter the sample name in Sampleid, the molecular weight in MW (Da), the first Concentration is the molar concentration and is adjusted to μM, and the second is the mass concentration. The mass concentration will be automatically calculated after the molar concentration is entered (select a concentration for each sample for repeated injections, and enter the sample concentrations from low to high);

[0275] c. In the Kinetics / Affinity-System Preparations interface, click Next to enter the RackPosition interface. Change ReagentRack to SampleandReagentRack1. Click Menu and select AutomaticPositioning. Change all Pooling fields to Yes. Adjust VialSize as needed. For 1.5mL EP tubes, select medium. Prepare and place samples according to their locations. Dilute the KRas protein serially with HBS-EP running buffer. Click Next, save the method, and then save the data path. The instrument will then begin running automatically.

[0276] The results are as follows Figure 9 As shown, the SPR detection results proved that both TKD and the control molecule TKDm had strong binding abilities to KRas protein in vitro, and the binding of TKD to KRas protein reached the antigen-antibody binding (nM) level.

[0277] 3. Verify the binding level between TKD molecules and KRas protein in cells.

[0278] Co-immunoprecipitation (Co-IP) is an important method for examining protein interactions within cells. To further investigate the mechanism and efficacy of TKD molecules in degrading target proteins, this example performed a co-immunoprecipitation (Co-IP) experiment to further investigate the effects of TKD. The specific steps for Co-IP are as follows:

[0279] (1) One million colon cancer HT29 (KRas wt) and HCT116 (KRas G13D) cells (from the American Type Culture Collection (ATCC) (Cat. No.: HTB-38, CCL-247)) were plated in cell culture dishes in advance, and TKD and its control molecule TKDm were added to a final concentration of 20 μg / mL. The experiment was performed in the continuous presence of the lysosomal inhibitor Lys05 at a final concentration of 10 μM.

[0280] (2) The treated cells were cultured at 37°C for 24 hours, then washed twice with PBS. 1 mL of 0.25% trypsin was added to each culture dish to digest the cells and remove them from the culture dish wall. The digestion was then terminated with 2 mL of complete culture medium per well. The cells were centrifuged at 300 g for 3 minutes at room temperature, and the cells were collected and resuspended in 5 mL of PBS. The centrifugation was repeated once.

[0281] (3) Collect the washed colon cancer cells into a 1.5 mL EP tube. Add the prepared lysis buffer, which is a western and IP lysis buffer containing 1% PMSF, 1% PI and phosphatase inhibitors (Cat. No.: P0013, purchased from Beyotime Biotechnology Co., Ltd.). Lyse on ice, invert upside down several times every 5 minutes, gently, do not vortex. After lysis for 30 minutes, centrifuge at 12000 rpm and 4°C for 30 minutes.

[0282] (4) The supernatant was transferred to a new EP tube and the protein concentration was measured using the BCA method. Then, 1 mg of protein was taken from each group for Co-IP reaction. 30 μL of Protein A / G agarose and 1 μg of mouse IgG (Cat. No. 10284-1-AP; purchased from Proteintech) were added to each group and incubated with rotation at 4°C for 1 hour. The supernatant was then centrifuged at 2500 rpm for 5 minutes at 4°C and the supernatant protein sample was transferred to a new EP tube. This step was to remove proteins in the sample that could nonspecifically bind to the His-tag antibody (Cat. No. 66005-1-Ig; purchased from Proteintech) in the next experiment.

[0283] (5) The supernatant protein samples obtained in step (4) were divided into three groups: IgG group, experimental group and Input group: 2 μg of mouse IgG antibody was added to the IgG group, 2 μg of His-tag antibody was added to the protein samples in the experimental group, and the Input group was left untreated and incubated with rotation at 4°C for 16 to 18 hours.

[0284] (6) Add 30 μL of Protein A / G agarose beads to each sample in the IgG group and the experimental group, and incubate with rotation at 4°C for 4 h.

[0285] (7) The samples of the IgG group and the experimental group were centrifuged at 2500 rpm for 5 minutes, and the supernatant was removed; the beads were gently washed with western and IP lysis buffer, and then centrifuged at 2500 rpm for 5 minutes, repeated three times; the supernatant was removed, and 30 μL of SDS lysis buffer (Cat. No.: P0013G; purchased from: Biyuntian Biotechnology Co., Ltd.) was added to each group. At the same time, 30 μL of protein sample was also taken from the input group; vortexed for one minute, boiled in boiling water for 10 minutes; centrifuged, and the supernatant was collected in a new 1.5 mL EP tube.

[0286] (8) The protein concentration was measured using the BCA method, and 30 μg of protein from each group was subjected to SDS denaturing gel electrophoresis. After transfer and blocking with 5% skim milk for 1 hour, KRas (Cat. No.: 12063-1-AP; purchased from: Proteintech) primary antibody was added and shaken at 4°C overnight. The antibody was removed and the membrane was washed 3 times with TBST, each time for 10 minutes. Subsequently, the secondary antibody labeled with horseradish peroxidase was incubated with goat anti-mouse secondary antibody at room temperature for 1 hour, and the membrane was washed 3 times with TBST, each time for 10 minutes. The membrane was then developed in the dark room using an ELC luminescence kit.

[0287] The results are as follows Figure 10 As shown, co-immunoprecipitation (Co-IP) experiments showed that TKD molecules can pass through the cell membrane and bind to intracellular wild-type and mutant KRas proteins indiscriminately.

[0288] 3. Verify the degradation effect of TKD molecules targeting KRas on wild-type and mutant KRas proteins.

[0289] This example demonstrates that the degradation of wild-type and mutant KRas proteins by TKD molecules is time- and concentration-dependent. Western blotting (WB) was used to assess the KRas degradation efficacy of TKD molecules. The specific steps are as follows:

[0290] 1. The concentration dependence of KRas protein degradation by TKD and TKDm molecules.

[0291] (1) Cell source and culture: Human colon cancer cells HT29 (KRas wt) and HCT116 (KRasG13D) were obtained from the American Type Culture Collection (ATCC) (catalog numbers: HTB-38, CCL-247). Both HT29 and HCT116 cells were cultured in 1640 medium supplemented with 10% fetal bovine serum (Invitrogen) at 5% CO2 and 37°C.

[0292] (2) Colon cancer cells HCT116 and HT29 were plated in six-well plates, with 500,000 cells per well. Each well was divided into two groups. In the experimental group, TKD was added to the culture wells to a final concentration of 0, 0.01, 0.1, 1, 10, and 20 μg / mL (the 0 concentration was replaced by an equal volume of PBS). In the control group, TKD was replaced by TKDm and treated in the same way. 2000 μL of DMEM complete culture medium was added to each well, the culture medium was mixed, and the treated cells were cultured at 37°C for 24 hours.

[0293] (3) Wash the cells twice with PBS, add 0.5 mL of 0.25% trypsin to each well to digest the cells and make them fall off the culture dish wall; then stop the digestion with 1 mL of complete culture medium per well, collect the cell suspension into a 1.5 mL EP tube, centrifuge at 300 g for 3 minutes at room temperature, collect the cells and resuspend them in 1 mL of PBS, and repeat the centrifugation once.

[0294] (4) Remove the supernatant and add 100 μL of lysis buffer (Cat. No.: P0013G; purchased from Beyotime Biotechnology Co., Ltd.) to each group. Lyse on ice, vortex every 5 minutes. After 30 minutes of lysis, centrifuge at 12,000 rpm at 4°C for 30 minutes, and collect the supernatant in a new 1.5 mL EP tube.

[0295] (5) The protein concentration was measured using the BCA method, and 30 μg of protein from each group was subjected to SDS denaturing gel electrophoresis. After transfer and blocking with 5% skim milk for 1 hour, KRas (source: Proteintech; catalog number: 12063-1-AP) and β-tublin (source: Proteintech; catalog number: 10094-1-AP) primary antibodies were added at a dilution ratio of 1:2000, shaken at 4°C overnight, and the antibodies were removed. The membrane was washed 3 times with TBST, each time for 10 minutes; the corresponding HRP-labeled rabbit secondary antibody (source: Quanshijin Company; catalog number: HS101-01) was diluted 1:3000 and incubated with shaking at room temperature for 1 hour, and the membrane was washed 3 times with TBST, each time for 10 minutes; the ELC luminescence kit was used for luminescence development in the dark room; the concentration of all primary antibodies was 1:2000 (1 μg protein diluted in 2000 μL primary antibody diluent).

[0296] The results are as follows Figure 11As shown in the results, TKD can cause the degradation of wild-type and mutant KRas protein expression levels in colorectal cancer cells, and the degree of degradation increases with increasing concentration. The control molecule TKDm, which cannot be recognized by lysosomes, cannot play a similar role, indicating that TKD molecules can induce lysosome-mediated protein degradation of internal cellular proteins.

[0297] 2. The effect of TKD and TKDm molecule administration time on KRas degradation.

[0298] (1) Figure 11 It was shown that when the concentration of TKD was 10ug / mL and 20ug / mL, the degradation effect of KRas was obvious; therefore, we set time points of 1, 2, 6, 12, and 24h to continuously detect the degradation effect of KRas when the concentration of TKD molecules was 0, 10, and 20μg / mL.

[0299] (2) Following the above concentration-dependent assay procedure, cells were collected after treatment at different time points and lysed promptly. Protein concentration was determined by BCA assay. 30 μg of protein was taken and stored in a -20°C refrigerator for later use.

[0300] (3) The collected protein samples were subjected to Western blot experiments.

[0301] The results are as follows Figure 12 As shown, TKD can rapidly degrade KRas, and the degradation process is time-dependent within a certain range.

[0302] 3. TKD molecules can degrade multiple mutant KRas proteins.

[0303] We then used multiple cancer cells containing common KRas site mutations to verify the effects of the TKD molecule. The cell lines used in this example were SW1116 (KRas G12D), SW480 (KRas G12V), and H358 (KRas G12C), all from ATCC, with catalog numbers: CCL-233, CCL-228, and CRL-5807, respectively. The treatment process of each cell was consistent with the effect of the above-mentioned administration time on KRas degradation. The collected protein samples were subjected to Western blot experiments to verify the degradation effect of the TKD molecule on KRas protein.

[0304] The results are as follows Figure 13 As shown, TKD molecules can induce the degradation of KRas proteins in different mutation states, and the degradation effect is more significant; this shows that TKD molecules can indiscriminately degrade target proteins in different mutation states, and have a wider range of applications and better effects than traditional small molecule targeted drugs.

[0305] 4. Verification of TKD Tumor Cell Targeting

[0306] TKD molecules were labeled with the fluorescent dye Cy5 to form a TKD-Cy5 covalent couple, and then the coupler was used to treat normal intestinal epithelial cells and intestinal cancer cells, respectively. Flow cytometry was used to detect the enrichment of TKD molecules in various cell types, and mice were used to perform in vivo tumor targeting evaluation, demonstrating the tumor cell targeting of TKD molecules.

[0307] 1. Labeling TKD molecules with water-soluble Cy5 NHSester

[0308] (1) 1.0 mg of Cy5 NHS was dissolved in 400 μL of DMSO and added to a glass bottle containing 1.0 mg of TKD protein solution in 400 μL.

[0309] (2) Add 15 μL of triethylamine and stir the reaction mixture overnight at room temperature in the dark.

[0310] (3) The protein was purified by HPLC using a protein C18 column (25 cm × 10 mm), with 2 × 400 μL injections each time and a 30-min gradient elution from 0.1% TFA (trifluoroacetic acid) aqueous solution to MeCN (acetonitrile): H2O (0.1% TFA) = 70:30 at a flow rate of 4 mL / min.

[0311] (4) Collect the target color band peak. The retention time of the labeled peptide is longer than that of the unlabeled peptide.

[0312] (5) Dilute the TKD-Cy5 molecule to 60 μM and store the product in PBS solution at -20°C in the dark.

[0313] 2. Flow cytometry verification of tumor cell targeting of TKD molecules

[0314] (1) Intestinal cancer cell lines HCT116 and HT29 cells, and normal intestinal epithelial cells FHC (purchased from ATCC, catalog number: CRL-1831) were plated at 4×10 5 Cells were seeded at a density of 100 μM in 6-well plates. After overnight culture, the cells were treated with corresponding drugs. Experimental and control groups were set up respectively. The experimental group was treated with 60 nM TKD-Cy5 coupled molecules, and the control group was treated with the corresponding concentration of Cy5 single molecules. Lysosomal inhibitor Lys 05 was added to both the control and experimental groups at the same time with a final concentration of 10 μM. The final volume of culture medium in each well was 1 mL. The cells were incubated in the dark at 37°C and 5% CO2 for 1 hour.

[0315] (2) The cells were collected by trypsin digestion and centrifuged at 1500 rpm for 5 min. The supernatant was removed and the cells were resuspended in PBS buffer. The cells were centrifuged again and the supernatant was discarded. This process was repeated three times. Finally, the washed cells were resuspended in 0.2 mL of PBS, wrapped in tin foil, and then analyzed on a flow cytometer (company: BECKMAN COULTER; model: Cytoflex). The fluorescence channel used was APC-A.

[0316] The results are as follows Figure 14 As shown, flow cytometric analysis showed that the TKD molecule could specifically recognize and pass through the cell membrane into colorectal cancer cells, but could not enter normal intestinal epithelial cells, indicating that the TKD molecule has strong tumor targeting.

[0317] 3. In vivo experiments in mice demonstrate that TKD molecules have tumor targeting properties in vivo

[0318] (1) Ten female Balb / c nude mice aged 4-5 weeks were purchased (Guangdong Provincial Laboratory Animal Center). 500,000 HT29 cells (left) and 500,000 HCT116 cells (right) were subcutaneously injected into the left and right flanks of the mice. One week later, the mice were divided into two groups of 5 mice each, the experimental group and the control group. Mouse husbandry and experiments were performed at the Laboratory Animal Center of Sun Yat-sen University. All operations were in accordance with the Regulations on Laboratory Animal Management of Sun Yat-sen University and adhered to the animal ethics rules.

[0319] (2) The TKD-Cy5 molecules and Cy5 single molecules constructed above were injected into mice via the tail vein at a concentration of 60 μM and a volume of 100 μL. The TKD-Cy5 injection group was the experimental group, and the Cy5 single molecule injection group was the control group.

[0320] (3) 2.5 hours after the tail vein injection, the mice were anesthetized with isoflurane gas, and Cy5 imaging was performed using IVISL Lumina (PerkinElmer). The fluorescence was detected in the form of fluorescence.

[0321] (4) Experimental results are as follows Figure 15 As shown, compared to the Cy5 single molecule control group, TKD-Cy5 was able to accumulate in tumors derived from KRas wild-type HT29 cells and KRas mutant HCT116 cells. Apart from bladder retention due to in vivo metabolism, similar to the control group, it did not target other tissues and organs in the mice. These results indicate that TKD has good tumor targeting properties.

[0322] 5. Verification of the effect of TKD on inhibiting the proliferation of colorectal cancer cells

[0323] Through cell proliferation CCK-8 experiments, clone formation experiments and in vivo tumor formation experiments in mice, it was proved that TKD molecules can inhibit the proliferation of intestinal cancer.

[0324] 1.CCK-8 assay

[0325] CCK-8 is a highly sensitive, non-radioactive colorimetric assay used to determine the number of viable cells in cell proliferation or toxicity assays. The main reagent, WST-8, is oxidized and reduced by intracellular dehydrogenases to generate an orange-yellow formazan dye that can be dissolved in the culture medium. The amount of formazan generated is proportional to the number of viable cells.

[0326] (1) HCT116 and HT29 colorectal cancer cells in the logarithmic growth phase were digested with 0.25% trypsin, counted, and inoculated into 96-well culture plates. Each cell type was divided into 6 groups, with 4 wells in each group. 100 μL of cell suspension was added to each well at a concentration of 1×10 4 At the same time, 100 μL of phosphate buffered saline (PBS) was added around the 96-well cell culture plate to prevent the cell culture medium from evaporating. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h to allow them to adhere to the wall.

[0327] (2) After rinsing with PBS three times, the culture medium containing different concentrations of TKD and TKDm (0, 10, 20 μg / mL) was replaced and cultured for another 24 h. The culture medium was discarded and the cells were washed three times with PBS. 100 μL of CCK-8 diluent (CCK-8: culture medium = 1:10) was evenly added and incubated at 37°C, 5% CO2 for 2 h. The corresponding OD value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA).

[0328] (3) Statistical analysis was performed using GraphPad Prism 8 software. One-way anova was used to compare the means of multiple groups. P < 0.05 was considered statistically significant.

[0329] The results are as follows Figure 16 As shown in Figure A, the cell proliferation CCK-8 experiment showed that TKD could significantly inhibit the growth of HT29 (KRas wt) and HCT116 (KRas G13D), while TKDm could not achieve the same effect, indicating that TKD inhibited the proliferation of colorectal cancer cells through lysosome-mediated KRas protein degradation, rather than through the blocking effect of nanoantibodies on KRas protein activity.

[0330] 2. Cell clone formation experiment

[0331] (1) Colon cancer cells HT29 and HCT116 in the logarithmic growth phase were digested with trypsin and prepared into single-cell suspensions. 500 cells per well were evenly seeded into 12-well plates. Each cell type was divided into 6 groups, with 3 replicate wells per group. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h to allow them to adhere to the plate.

[0332] (2) Each group of cells was treated with their own experimental methods. That is, they were cultured in culture medium containing different concentrations of TKD and TKDm (0, 10, and 20 μg / mL). Cell growth was observed daily, and the cell culture medium was changed every 1-2 days until cell clones (more than 50 cells were considered a clone) were observed under a light microscope.

[0333] (3) After discarding the supernatant of each group, wash twice with PBS buffer and air dry; add an appropriate amount of methanol fixative to each well to fix the cells for 15 minutes, wash off the residual fixative in the well with PBS solution, and air dry; add an appropriate amount of crystal violet staining solution to each well to stain the cells for 15 minutes; wash off the residual stain in the well with PBS solution, air dry, and count the cell clones seen in the duplicate wells of each group under an inverted microscope and calculate the number of clones.

[0334] (4) Statistical analysis was performed using GraphPad Prism 8 software. One-way anova was used to compare the means of multiple groups. P < 0.05 was considered statistically significant.

[0335] The results are as follows Figure 16 As shown in B, the clone formation experiment showed at the cellular level that TKD molecules can significantly inhibit the growth of KRas wild-type and mutant (G13D) colorectal cancer cell clones.

[0336] The above CCK-8 and clone formation experiments both proved that compared with the control group TKDm, TKD can significantly inhibit the proliferation of KRas wild-type and mutant colorectal cancer cells, indicating that the nanoantibody-mediated KRas degradation system TKD degrades the target protein through the lysosomal pathway, which is of great value for the treatment of colorectal cancer.

[0337] 3. In vivo tumor formation experiment

[0338] (1) The mice used in this experiment were female Balb / c nude mice, 4-5 weeks old, purchased from the Guangdong Experimental Animal Center and housed at the Experimental Animal Center of Sun Yat-sen University. The experimental mice were divided into three groups, with 5 mice in each group, namely the phosphate buffered saline (PBS) control group, the TKD-25 mg / kg group, and the TKD-50 mg / kg group.

[0339] (2) HT29 and HCT116 cells in the logarithmic phase were digested with trypsin, and the digestion was terminated with complete culture medium and the cells were counted. After counting, 7.5 million HT29 and HCT116 cells were taken, respectively, and washed twice with PBS. After each wash, the cells were centrifuged at 300g for 5 minutes to collect the cells.

[0340] (3) Prepare cell dilution buffer to resuspend the cells. The buffer composition is PBS: Matrigel (Corning) = 1:1. Then use 1.5 ml of buffer to resuspend HT29 and HCT116 cells respectively and place them on ice until used.

[0341] (4) Subcutaneous tumor formation experiment: Each mouse was anesthetized by intraperitoneal injection of 150 μL of tribromoethanol, and then 100 μL (500,000) of HT29 and HCT116 cell suspensions were subcutaneously injected on the left and right sides of the mouse's back, respectively, with HT29 cells on the left and HCT116 cells on the right.

[0342] (5) The mice were fed for one week before the drug administration experiment began: the drug administration method was intraperitoneal injection, in which the PBS control group was injected with PBS, the TKD-25 mg / kg group was injected with TKD at a concentration of 25 mg / kg, and the TKD-50 mg / kg group was injected with TKD at a concentration of 50 mg / kg. The administration volume was 150 μL, and the administration frequency was once every three days at a fixed time, and the administration was continued for 7 times.

[0343] (6) After the administration, each mouse was injected with 150 μL of tribromoethanol and then killed by cervical dislocation. The tumors on both sides of the mouse were collected and weighed to calculate the tumor weight. Figure 17 As shown, compared with the PBS control group, the TKD-treated group can significantly inhibit the growth of KRas wild-type (HT29) and mutant (HCT116) colorectal cancer, and the high-dose TKD-50 mg / kg group has better effects than the low-dose 25 mg / kg group, indicating that TKD can also effectively inhibit tumor growth in vivo.

[0344] VI. Verification of the Toxic and Side Effects of TKD Molecules

[0345] (1) The mice used in this experiment were female Balb / c nude mice, 4-5 weeks old, purchased from the Guangdong Experimental Animal Center and housed at the Experimental Animal Center of Sun Yat-sen University. The experimental mice were divided into three groups, with 5 mice in each group, namely the phosphate buffered saline (PBS) control group, the TKD-25 mg / kg group, and the TKD-50 mg / kg group.

[0346] (2) PBS and TKD solutions were administered to mice via intraperitoneal injection. The volume of each administration was 150 μL, and TKD was administered at 25 mg / kg and 50 mg / kg, respectively. The administration was performed once every 3 days at a fixed time. The physical condition, mental state, and weight changes of the mice were observed and recorded.

[0347] (3) After 7 consecutive doses (3 weeks), the drug was stopped and the mice were euthanized (cervical dislocation). The main organs of the mice, such as lungs, liver, spleen, and kidneys, were collected for hematoxylin-eosin (HE) staining and pathological histological analysis to evaluate the toxic and side effects of TKD on the body. All animal experiments in this study were conducted in accordance with the relevant animal ethics regulations of Sun Yat-sen University.

[0348] The experimental results are as follows Figure 18 As shown, compared to the PBS control group, mice in both TKD-treated groups showed no significant weight changes. Pathological tissue analysis revealed that the TKD molecule did not cause damage to vital organs such as the lungs, liver, spleen, and kidneys. These data demonstrate that the TKD molecule has minimal toxic side effects and holds important clinical value.

[0349] VII. Verification of TKD molecule retention time in vivo

[0350] (1) The mice used in this experiment were female Balb / c nude mice, 4-5 weeks old, purchased from the Guangdong Experimental Animal Center and housed at the Experimental Animal Center of Sun Yat-sen University. The experimental mice were divided into three groups, with 5 mice in each group, namely the phosphate buffered saline (PBS) control group, the TKD-25 mg / kg group, and the TKD-50 mg / kg group.

[0351] (2) PBS and TKD solutions were administered to mice via intraperitoneal injection. The volume of each administration was 150 μL. TKD was administered once at 25 mg / kg and 50 mg / kg, respectively.

[0352] (3) Three days after administration, the mice were euthanized (cervical dislocation), fresh blood was drawn from the mouse heart and centrifuged to obtain serum, and the serum protein concentration was measured using the BCA method (the steps were as described above). Subsequently, 20 μg of serum protein was taken for WB experiment (the experimental process was as described above), and serum whole protein was run on gel and stained with Coomassie Brilliant Blue.

[0353] (4) Experimental results are as follows Figure 19 As shown, three days after administration, the high concentration TKD group could still be detected (50 mg / kg), indicating that the half-life of TKD molecules in the body is long and has important clinical application value.

[0354] Example 2 Verification of the effect of TKD in reversing Gefitinib resistance in colorectal cancer cells

[0355] "Example 1" describes the design of TKD, verifies its role in inducing the degradation of wild-type and mutant KRas proteins, and further verifies its tumor targeting and inhibitory effects on colorectal cancer proliferation. Because KRas protein is an important effector molecule in the EGFR downstream signaling pathway, KRas mutations often lead to clinical resistance of colorectal cancer cells to EGFR-targeted drugs such as gefitinib. Therefore, this example uses TKD to induce KRas protein degradation to reduce KRas expression levels in colorectal cancer cells and evaluates the effect of this mode of action on restoring gefitinib sensitivity in colorectal cancer cells.

[0356] 1. Verification of the efficacy of KRas mutation-induced gefitinib resistance in colorectal cancer cells

[0357] EGFR primarily promotes tumor development and progression by activating two signaling pathways: EGFR / KRas / Raf / ERK and EGFR / PI3K / AKT. KRas mutations typically lead to sustained activation of the KRas / Raf / ERK signaling pathway, thereby counteracting or even weakening the effects of EGFR-targeted drugs. This example uses the EGFR-targeted drug gefitinib as an example, and first explores the effect of KRas mutations on drug resistance in colorectal cancer cells using KRas wild-type cells HT29 (KRas wt) and KRas mutant cells HCT116 (KRas G13D).

[0358] 1. CCK-8 experiments demonstrated that KRas mutant cells HCT116 (KRas G13D) are resistant to gefitinib

[0359] (1) Logarithmic growth phase colorectal cancer cells HCT116 and HT29 and normal intestinal epithelial cells FHC as controls were digested with 0.25% trypsin, counted, and seeded into 96-well culture plates. Each cell type was divided into 6 groups, with 4 wells in each group. 100 μL of cell suspension was added to each well at a concentration of 1×10 4 At the same time, 100 μL of phosphate buffered saline (PBS) was added around the 96-well cell culture plate to prevent the cell culture medium from volatilizing. The cells were cultured in a 37° C., 5% CO 2 incubator for 24 hours to allow them to adhere to the wall.

[0360] (2) After rinsing with PBS three times, the culture medium containing different concentrations of gefitinib (0 μM, 1 μM, 2 μM, 4 μM, 6 μM, and 8 μM) was replaced and cultured for another 24 h (0 μM concentration was replaced with an equal volume of dimethyl sulfoxide (DMSO)). The culture medium was discarded and the cells were washed three times with PBS. 100 μL of CCK-8 diluent (CCK-8: culture medium = 1:10) was evenly added and incubated at 37°C and 5% CO2 for 2 h. The corresponding OD value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay (ELISA).

[0361] (3) Statistical analysis was performed using GraphPad Prism 8 software, and one-way analysis of variance was used to compare the means of multiple groups. P < 0.05 was considered statistically significant.

[0362] The results are as follows Figure 20 As shown, cell proliferation CCK-8 assay confirmed that gefitinib could significantly inhibit the growth of HT29 (KRas wt) but not HCT116 (KRas G13D).

[0363] 2. Cloning experiments demonstrated that KRas mutant cells HCT116 (KRas G13D) are resistant to gefitinib

[0364] (1) Colon cancer cells HT29 (KRas wt), HCT116 (KRas G13D) and normal intestinal epithelial cells FHC in the logarithmic growth phase were digested with trypsin and prepared into single-cell suspensions. 500 cells per well were evenly seeded into 12-well plates. Each cell type was divided into 6 groups, with 3 replicate wells per group. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h to allow them to adhere to the plate.

[0365] (2) Each group of cells was treated with their own experimental methods, i.e., they were cultured in culture medium containing different concentrations of gefitinib (0 μM, 1 μM, 2 μM, 4 μM, 6 μM, 8 μM). Cell growth was observed daily, and the cell culture medium was changed every 1-2 days until cell clones (more than 50 cells were considered a clone) were observed under a light microscope.

[0366] (3) After discarding the supernatant of each group, wash twice with PBS buffer and air dry; add an appropriate amount of methanol fixative to each well to fix the cells for 15 minutes, wash off the residual fixative in the well with PBS solution, and air dry; add an appropriate amount of crystal violet staining solution to each well to stain the cells for 15 minutes; wash off the residual stain in the well with PBS solution, air dry, and count the cell clones seen in the duplicate wells of each group under an inverted microscope and calculate the number of clones.

[0367] (4) Statistical analysis was performed using GraphPad Prism 8 software. One-way anova was used to compare the means of multiple groups. P < 0.05 was considered statistically significant.

[0368] The results are as follows Figure 21 As shown, the clone formation experiment verified at the cellular level that gefitinib could significantly inhibit the growth of KRas wild-type cells HT29 (KRas wt), but had no significant effect on the growth of KRas mutant cells HCT116 (KRas G13D).

[0369] 3. In vivo tumorigenesis experiments demonstrated that KRas mutant HCT116 cells (KRas G13D) are resistant to gefitinib

[0370] (1) The mice used in this experiment were female Balb / c nude mice, 4-5 weeks old, purchased from the Guangdong Experimental Animal Center and housed at the Experimental Animal Center of Sun Yat-sen University. The experimental mice were divided into two groups, 5 in each group, namely the phosphate buffered saline (PBS) control group and the gefitinib group.

[0371] (2) HT29 and HCT116 cells in the logarithmic phase were digested with trypsin, and the digestion was terminated with complete culture medium and the cells were counted. After counting, 5 million HT29 and HCT116 cells were taken respectively and washed twice with PBS. After each wash, the cells were centrifuged at 300g for 5 minutes to collect the cells.

[0372] (3) Prepare cell dilution buffer to resuspend the cells. The buffer composition is PBS: Matrigel (Corning) = 1:1. Then use 1 ml of buffer to resuspend HT29 and HCT116 cells respectively and place them on ice until used.

[0373] (4) Subcutaneous tumor formation experiment: Each mouse was anesthetized by intraperitoneal injection of 150 μL of tribromoethanol, and then 100 μL (500,000) of HT29 and HCT116 cell suspensions were subcutaneously injected on the left and right sides of the mouse's back, respectively, with HT29 cells on the left and HCT116 cells on the right.

[0374] (5) The mice were fed for one week before the drug administration experiment began: the drug administration method was oral administration, in which the PBS control group was injected with PBS, and the gefitinib group was injected with gefitinib at a concentration of 50 mg / kg. The administration volume was 150 μL, and the administration frequency was once every three days at a fixed time, and the administration was continued for 7 times.

[0375] (6) After the administration, each mouse was injected with 150 μL of tribromoethanol and then killed by cervical dislocation. The tumors on both sides of the mouse were collected and weighed to calculate the tumor weight. The experimental results are shown in the figure. Figure 22 As shown, compared with the PBS control group, the gefitinib-treated group could significantly inhibit the growth of KRas wild-type (HT29) colorectal cancer, but had no effect on the growth of KRas mutant (HCT116) colorectal cancer.

[0376] 4. Gefitinib cannot inhibit the activation of the EGFR downstream signaling pathway KRas / Raf / ERK / c-myc in KRas mutant cells.

[0377] (1) Colon cancer cells HCT116 (KRas G13D) and HT29 (KRas wt) were plated in six-well plates, with 500,000 cells per well. The cells were divided into two groups. Gefitinib was added to the culture wells in the experimental groups to a final concentration of 0 μM and 4 μM (the 0 concentration was replaced by an equal volume of dimethyl sulfoxide (DMSO)). 2000 μL of DMEM complete culture medium was added to each well, the culture medium was mixed, and the cells were cultured at 37°C for 24 hours. The concentration of 4 μM was selected because gefitinib does not produce toxic side effects on normal cells at this concentration.

[0378] (2) Wash the cells twice with PBS, add 0.5 mL of 0.25% trypsin to each well to digest the cells and remove them from the culture dish wall; then stop the digestion with 1 mL of complete culture medium per well, collect the cell suspension into a 1.5 mL EP tube, centrifuge at 300 g for 3 minutes at room temperature, collect the cells and resuspend them in 1 mL of PBS, and repeat the centrifugation once.

[0379] (3) Remove the supernatant and add 100 μL of lysis buffer (catalog number: P0013G; purchased from Biyuntian Biotechnology Co., Ltd.) to each group. Lyse on ice and vortex every 5 minutes. After lysis for 30 minutes, centrifuge at 12000 rpm at 4°C for 30 minutes and collect the supernatant in a new 1.5 mL EP tube.

[0380] (4) The protein concentration was measured using the BCA method, and 20 μg of protein from each group was subjected to SDS denaturing gel electrophoresis. After transfer and blocking with 5% skim milk for 1 hour, the primary antibodies β-tublin (source: Proteintech; catalog number: 10094-1-AP), ERK (source: GST; catalog number: 4695s), pEKR (source: GST; catalog number: 8544S), Raf (source: GST; catalog number: 53745s), pRaf (source: GST; catalog number: 9427s), and c-myc (source: GST; catalog number: 9402s) were added at a dilution ratio of 1:2000, respectively. The cells were shaken at 4°C overnight, the antibodies were removed, and the membrane was washed three times with TBST for 10 minutes each time. The corresponding HRP-labeled mouse or rabbit secondary antibody (source: Quanshijin Company; catalog number: HS101-01 and HS201-01) was diluted 1:3000 and incubated with shaking at room temperature for 1 hour. The membrane was washed 3 times with TBST, each time for 10 minutes, and developed by luminescence in the dark room using the ELC luminescence kit. The concentration of all primary antibodies was 1:2000 (1 μg protein was diluted in 2000 μL primary antibody diluent).

[0381] (5) The results are as follows Figure 23 As shown in the results, gefitinib can significantly inhibit the activation of the Raf / ERK / c-myc signaling pathway in KRas wild-type cells HT29, but cannot inhibit the activation of this signaling pathway in KRas mutant cells HCT116.

[0382] The above CCK-8 experiments, clone formation experiments, in vivo mouse experiments and WB experiments all proved that KRas-mutated colorectal cancer cells are resistant to gefitinib.

[0383] 2. TKD-induced KRas degradation can inhibit the activation of the KRas / Raf / ERK / c-myc signaling pathway downstream of EGFR

[0384] (1) Colon cancer cells HCT116 and HT29 were plated in six-well plates, with 500,000 cells per well. The cells were divided into two groups. In the experimental groups, TKD was added to the culture wells to make the final concentrations of 0 μg / mL, 10 μg / mL, and 20 μg / mL (the 0 concentration was replaced by an equal volume of PBS). In the control group, TKD was replaced by TKDm and treated in the same way. 2000 μL of DMEM complete culture medium was added to each well, the culture medium was mixed, and the treated cells were cultured at 37°C for 24 hours.

[0385] (2) Wash the cells twice with PBS, add 0.5 mL of 0.25% trypsin to each well to digest the cells and make them fall off the culture dish wall; then stop the digestion with 1 mL of complete culture medium per well, collect the cell suspension into a 1.5 mL EP tube, centrifuge at 300 g for 3 minutes at room temperature, collect the cells and resuspend them in 1 mL of PBS, and repeat the centrifugation once.

[0386] (3) Remove the supernatant and add 100 μL of lysis buffer (Cat. No.: P0013G; purchased from Beyotime Biotechnology Co., Ltd.) to each group. Lyse on ice, vortexing every 5 minutes. After 30 minutes of lysis, centrifuge at 12,000 rpm at 4°C for 30 minutes. Collect the supernatant in a new 1.5 mL EP tube.

[0387] (4) The protein concentration was measured using the BCA method, and 20 μg of protein from each group was subjected to SDS denaturing gel electrophoresis, transferred and blocked with 5% skim milk for 1 hour, and then KRas (source: Proteintech; catalog number: 12063-1-AP), β-tublin (source: Proteintech; catalog number: 10094-1-AP), ERK (source: GST; catalog number: 4695s), pEKR (source: GST; catalog number: 8544S), Raf (source: GST; catalog number: 53745s), and Primary antibodies were used for pRaf (source: GST; catalog number: 9427s) and c-myc (source: GST; catalog number: 9402s), shaken at 4°C overnight, and the antibodies were removed. The membrane was washed with TBST three times, 10 min each time, and incubated with the corresponding HRP-labeled mouse or rabbit secondary antibody (source: Quanshijin Company; catalog number: HS101-01 and HS201-01) at a dilution of 1:3000 and shaken at room temperature for 1 hour. The membrane was washed with TBST three times, 10 min each time, and developed in the dark room using the ELC luminescence kit. The concentration of all primary antibodies was 1:2000 (1 μg protein diluted in 2000 μL primary antibody diluent).

[0388] (6) The results are as follows Figure 24 As shown in the results, compared with the control group TKDm, TKD could significantly inhibit the activation of the EGFR downstream signaling pathway KRas / Raf / ERK / c-myc in KRas wild-type cells HT29 and KRas mutant cells HCT116.

[0389] 3. Combination of TKD and gefitinib enhances the effect of gefitinib

[0390] 1. CCK-8 experiments have shown that TKD can enhance the effect of gefitinib

[0391] (1) HCT116 and HT29 colorectal cancer cells in the logarithmic growth phase were digested with 0.25% trypsin, counted, and inoculated into 96-well culture plates. Each cell type was divided into 6 groups, with 4 wells in each group. 100 μL of cell suspension was added to each well at a concentration of 1×10 4 At the same time, 100 μL of phosphate buffered saline (PBS) was added around the 96-well cell culture plate to prevent the cell culture medium from volatilizing. The cells were cultured in a 37° C., 5% CO 2 incubator for 24 hours to allow them to adhere to the wall.

[0392] (2) After rinsing with PBS three times, culture medium containing TKD and TKDm (final concentrations of 0 μg / mL, 10 μg / mL, and 20 μg / mL, respectively) and gefitinib was added and cultured for 24 hours (0 μM concentration was replaced with an equal volume of dimethyl sulfoxide (DMSO)). The culture medium was discarded and the cells were washed with PBS three times. 100 μL of CCK-8 diluent (CCK-8: culture medium = 1:10) was evenly added. After incubation at 37°C and 5% CO2 for 2 hours, the corresponding OD value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay. In order to better verify the effect of TKD on restoring the sensitivity of colorectal cancer cells to gefitinib, a lower concentration of gefitinib was used for treatment. The treatment concentration of KRas wild-type cells HT29 was 2 μM, and the treatment concentration of KRas mutant cells HCT116 was 4 μM.

[0393] (3) Statistical analysis was performed using GraphPad Prism 8 software. One-way anova was used to compare the means of multiple groups. P < 0.05 was considered statistically significant.

[0394] The results are as follows Figure 25 As shown, cell proliferation CCK-8 assay confirmed that the combination of TKD and gefitinib could significantly inhibit the growth of HT29 (KRas wt) and HCT116 (KRas G13D), and the effect was better than that of either drug alone.

[0395] 2. Clone formation experiments demonstrated that TKD can enhance the effect of gefitinib

[0396] (1) Colon cancer cells HT29 (KRas wt) and HCT116 (KRas G13D) in the logarithmic growth phase were digested with trypsin and prepared into single-cell suspensions. 500 cells per well were evenly seeded into 12-well plates. Each cell type was divided into 6 groups, with 3 replicate wells per group. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h to allow them to adhere to the plate.

[0397] (2) After rinsing with PBS three times, culture medium containing TKD and TKDm (final concentrations of 0 μg / mL, 10 μg / mL, and 20 μg / mL, respectively) and gefitinib was added and cultured for 24 hours (0 μM concentration was replaced with an equal volume of dimethyl sulfoxide (DMSO)). Cell growth was observed daily, and the cell medium was changed every 1-2 days. The culture was terminated when cell clones (more than 50 cells were considered a clone) were observed under a light microscope. In order to better verify the effect of TKD on restoring the sensitivity of colorectal cancer cells to gefitinib, a lower concentration of gefitinib was used in this experiment. The treatment concentration of KRas wild-type cells HT29 was 2 μM, and the treatment concentration of KRas mutant cells HCT116 was 4 μM.

[0398] (3) After discarding the supernatant of each group, wash twice with PBS buffer and air dry; add an appropriate amount of methanol fixative to each well to fix the cells for 15 minutes, wash off the residual fixative in the well with PBS solution, and air dry; add an appropriate amount of crystal violet staining solution to each well to stain the cells for 15 minutes; wash off the residual stain in the well with PBS solution, air dry, and count the cell clones seen in the duplicate wells of each group under an inverted microscope and calculate the number of clones.

[0399] (4) Statistical analysis was performed using GraphPad Prism 8 software. One-way anova was used to compare the means of multiple groups. P < 0.05 was considered statistically significant.

[0400] The results are as follows Figure 26 As shown, colony formation experiments confirmed that the combination of TKD and gefitinib can significantly inhibit the growth of HT29 (KRas wt) and HCT116 (KRas G13D), and the effect is better than the effect of either drug alone.

[0401] 3. In vivo tumorigenesis experiments demonstrated that TKD can enhance the effect of gefitinib

[0402] (1) The mice used in this experiment were female Balb / c nude mice, 4-5 weeks old, purchased from the Guangdong Provincial Experimental Animal Center and raised in the Experimental Animal Center of Sun Yat-sen University. The experimental mice were divided into 4 groups, with 5 mice in each group, namely phosphate buffered saline (PBS) control group, gefitinib group (25 mg / kg), TKD group (25 mg / kg), and gefitinib (25 mg / kg) and TKD (25 mg / kg) combined treatment group. In order to better demonstrate the effect of the combined use of TKD and gefitinib, this experiment used lower concentrations of TKD and gefitinib to treat mice.

[0403] (2) HT29 and HCT116 cells in the logarithmic phase were digested with trypsin and digested with complete culture medium before cell counting. After counting, 10 million HT29 and HCT116 cells were collected and washed twice with PBS. After each wash, the cells were collected by centrifugation at 300 g for 5 minutes.

[0404] (3) Resuspend the cells in cell dilution buffer (PBS: Matrigel (Corning)) at a ratio of 1:1. Resuspend the HT29 and HCT116 cells in 1 ml of buffer respectively and place on ice until ready to use.

[0405] (4) Subcutaneous tumor formation experiment: Each mouse was anesthetized by intraperitoneal injection of 150 μL of tribromoethanol, and then 100 μL (500,000) of HT29 and HCT116 cell suspensions were subcutaneously injected on the left and right sides of the mouse's back, respectively, with HT29 cells on the left and HCT116 cells on the right.

[0406] (5) After one week of feeding, the mice were administered with PBS and gefitinib via gavage, and TKD via intraperitoneal injection. The dosing volume was 150 μL. The dosing frequency was once every three days at a fixed time for 7 consecutive doses.

[0407] (6) After the administration, each mouse was injected with 150 μL of tribromoethanol and then killed by cervical dislocation. The tumors on both sides of the mouse were collected and weighed to calculate the tumor weight. The experimental results are shown in the figure. Figure 27 As shown, the combined administration of TKD and gefitinib can significantly inhibit the growth of KRas wild-type (HT29) and KRas mutant (HCT116) colorectal cancer, and the inhibitory effect is better than that of the PBS control group and the group using either drug alone.

[0408] These data demonstrate that the combined use of TKD and gefitinib significantly inhibits the proliferation of KRas wild-type and mutant colorectal cancer cells at both the cellular and animal levels, with greater efficacy than either agent alone. In particular, in gefitinib-resistant KRas mutant cells, the combined use of the two agents significantly enhanced the response of colorectal cancer cells to gefitinib. This demonstrates that TKD can restore sensitivity to EGFR-targeted drug-resistant cancer cells by targeting KRas for degradation, which will have significant implications for expanding the use of existing targeted anti-cancer drugs.

[0409] The above embodiments are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered equivalent replacement methods and are included in the scope of protection of the present invention. SEQUENCE LISTING <110> Sun Yat-sen University Cancer Center (Sun Yat-sen University Cancer Hospital, Sun Yat-sen University Cancer Research Institute) <120> A fusion protein and its preparation method and application <130> <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 126 <212> PRT <213> Artificial sequence <400> 1 Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Thr Tyr 20 25 30 Pro Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val 35 40 45 Ala Arg Ile Asn Leu Ser Gly Gly Ile Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Gly Gly Gly Ser Thr Thr Trp Ala Gly Gly Ile Pro Thr Asn Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Arg 115 120 125 <210> 2 <211> 17 <212> PRT <213> Synthetic sequence <400> 2 Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15 Arg <210> 3 <211> 14 <212> PRT <213> Synthetic sequence <400> 3 Lys Phe Glu Arg Gln Lys Ile Leu Asp Gln Arg Phe Phe Glu 1 5 10 <210> 4 <211> 157 <212> PRT <213> Synthetic sequence <400> 4 Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15 Arg Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly 20 25 30 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Thr 35 40 45 Tyr Pro Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 50 55 60 Val Ala Arg Ile Asn Leu Ser Gly Gly Ile Thr Asn Tyr Ala Asp Ser 65 70 75 80 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 85 90 95 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 100 105 110 Cys Gly Gly Gly Ser Thr Thr Trp Ala Gly Gly Ile Pro Thr Asn Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Arg Lys 130 135 140 Phe Glu Arg Gln Lys Ile Leu Asp Gln Arg Phe Phe Glu 145 150 155 <210> 5 <211> 157 <212> PRT <213> Artificial Sequence <400> 5 Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Leu Leu Arg Arg Leu Leu 1 5 10 15 Arg Asp Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly 20 25 30 Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Thr 35 40 45 Tyr Pro Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe 50 55 60 Val Ala Arg Ile Asn Leu Ser Gly Gly Ile Thr Asn Tyr Ala Asp Ser 65 70 75 80 Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val 85 90 95 Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr 100 105 110 Cys Gly Gly Gly Ser Thr Thr Trp Ala Gly Gly Ile Pro Thr Asn Phe 115 120 125 Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Arg Lys 130 135 140 Phe Glu Arg Ala Lys Ile Leu Asp Ala Arg Phe Phe Glu 145 150 155 <210> 6 <211> 14 <212> PRT <213> Artificial Sequence <400> 6 Lys Phe Glu Arg Ala Lys Ile Leu Asp Ala Arg Phe Phe Glu 1 5 10 <210> 7 <211> 189 <212> PRT <213> Artificial sequence <400> 7 Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys 1 5 10 15 Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr 20 25 30 Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly 35 40 45 Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr 50 55 60 Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys 65 70 75 80 Val Phe Ala Ile Asn Asn Thr Lys Ser Phe Glu Asp Ile His His Tyr 85 90 95 Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Glu Asp Val Pro Met Val *100 105 110 Leu Val Gly Asn Lys Cys Asp Leu Pro Ser Arg Thr Val Asp Thr Lys 115 120 125 Gln Ala Gln Asp Leu Ala Arg Ser Tyr Gly Ile Pro Phe Ile Glu Thr 130 135 140 Ser On Lys Thr Arg Gln Arg Val Glu Asp On Phe Tyr Thr Leu Val 145 150 155 160 Arg Glu Ile Arg Gln Tyr Arg Leu Lys Ile Ser Glu Glu Lys 165 170 175 Thr Pro Gly Cys Val Lys Ile Lys Cys Ile Ile Met 180 185

Claims

1. A fusion protein comprising, from N-terminus to C-terminus, a tumor cell-specific membrane-penetrating peptide, a polypeptide that specifically binds to the KRas protein, and a lysosome recognition peptide; The tumor cell-specific membrane-penetrating peptide is a ganglioside-binding peptide; The amino acid sequence of the ganglioside binding peptide is: RAGLQFPVGRLLRRRLLR (SEQ ID NO: 2); The polypeptide that specifically binds to the KRas protein is an anti-KRas protein nanobody; The amino acid sequence of the anti-KRas protein nanobody is: DVQLQESGGGLVQAGGSLRLSCVASGRTFSTYPTGWFRQAPGKEREFVARINLSGGITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCGGGSTTWAGGIPTNFDYWGQGTQVTVSSGR (SEQ ID NO: 1); The amino acid sequence of the lysosome recognition peptide is: KFERQKILDQRFFE (SEQ ID NO: 3).

2. The fusion protein according to claim 1, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID NO:

4.

3. A nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 2. A vector comprising the nucleic acid molecule according to claim 3 . A host cell comprising the vector according to claim 4 .

6. The method for preparing the fusion protein according to any one of claims 1 to 2, characterized in that: The host cell according to claim 5 is cultured to obtain.

7. Use of the fusion protein according to any one of claims 1 to 2 in preparing a product; The product is any one of (1) to (3): (1) Preparations for degrading KRas protein; (2) Anti-tumor drugs; (3) drugs that increase the sensitivity of KRas mutant tumors to tumor-targeted drugs; The tumor is intestinal cancer; The targeted drug is an EGFR targeted drug.

8. A medicine comprising the fusion protein according to any one of claims 1 to 2 and pharmaceutically acceptable excipients.

9. A combined drug comprising the fusion protein according to any one of claims 1 to 2 or the drug according to claim 8, and a tumor-targeting drug.

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