Nanobodies targeting hsp70 and methods of making and using the same

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to a nano antibody targeting HSP70 and a preparation method and application thereof. The application discloses a nano antibody targeting HSP70, and the nano antibody comprises a nano antibody Nb D8, wherein the amino acid sequence of the nano antibody Nb D8 is shown as Seq.ID NO.1. Since the provided nano antibody Nb D8 can be highly combined with HSP70, has high affinity and specificity with HSP70, and can be targeted to HSP70 through the nano antibody Nb D8, a solid theoretical basis is provided for subsequent construction of a PROTAC complex, and the new concept provides a new treatment strategy for clinic.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a nanobody targeting HSP70, its preparation method, and its application. Background Technology

[0002] HSP70 is a heat shock protein (HSP), also known as a stress protein (SP). They are a group of structurally highly conserved polypeptides that are widely found in prokaryotic and eukaryotic cells.

[0003] HSP70 expression is low in normal cells, but it plays a crucial role in maintaining cellular homeostasis and normal life activities. However, with the progress of research, it has been found that increased HSP70 expression is often accompanied by tumor development and progression, and high expression of HSP70 has been observed in various tumor cell types. Tumor cells are a group of abnormally proliferating cells that have deviated from normal physiological regulation. To adapt to the various pressures caused by excessive growth, tumor cells need a "chaperone molecule" to help them overcome difficulties. HSP70 is this powerful "chaperone molecule," acting as a strong buffer system to cope with cellular stress and helping them adapt to various pressures in the microenvironment (increased oxidative stress, nutrient deficiency, hypoxia, increased levels of mutant proteins, etc.).

[0004] Numerous in vitro and in vivo studies have reported that high expression of HSP70 significantly stimulates the proliferation and migration of different types of tumor cells. In colorectal cancer, HSP70 stably participates in the receptor tyrosine kinase (RTK) and WNT signaling pathways to regulate mitotic signaling in intestinal epithelial cells, inducing tumorigenesis. In liver cancer, HSP70 knockout significantly downregulates tumor cell proliferation and migration, with marked cell cycle arrest and downregulation of cyclin expression. In gastric cancer, downregulation of HSP70 expression after anti-oligosense nucleotide treatment is accompanied by tumor cell apoptosis and cell cycle arrest. Besides promoting proliferation, HSP70 can also enhance the anti-immune cell resistance and drug resistance of tumor cells, greatly increasing immune escape and drug resistance. Given the close relationship between HSP70 and tumor development, some scientists now consider it a prognostic and diagnostic biomarker for tumors. For example, HSP70 can serve as a reliable potential short-term biomarker in hepatocellular carcinoma, prostate cancer, esophageal adenocarcinoma, lymph node metastatic colorectal cancer, lung cancer, and ovarian cancer. In addition, HSP70 is closely related to clinical tumor grading and survival. In melanoma, oral cancer and bladder cancer, HSP70 can be used as a reliable indicator to provide relatively objective guidance for clinical diagnosis and treatment.

[0005] In summary, the expression level of HSP70 is inextricably linked to tumor development and progression, and inhibiting HSP70 function appears to be an effective cancer treatment. Currently, drugs developed for HSP70 primarily target its two main structural domains (NBD and SBD), thereby inhibiting HSP70's normal physiological function. VER-155008 is a nucleoside HSP70 inhibitor that binds to the nucleotide binding site and NBD domain of HSP70, inhibiting its ATPase activity and overall framework conformation, thus preventing it from performing its normal physiological function. 2-Phenylacetylsulfonamide (PES) is also a highly selective HSP70 inhibitor that competitively inhibits the binding of HSP70 to other chaperone molecules and substrate proteins by binding to the SBD domain, disrupting intracellular protein homeostasis. In addition, various chemotherapy drugs targeting HSP70 are emerging, providing multiple treatment options for patients with tumors exhibiting high HSP70 expression. However, the aforementioned drugs are prone to off-target effects by inhibiting only a certain domain. In addition, the occurrence of drug resistance is also a problem that cannot be ignored. In order to eliminate HSP70 from the root rather than just inhibiting its function in a certain aspect, it is urgent to develop corresponding nanobodies based on the characteristics of HSP70 to provide a new treatment strategy for subsequent clinical treatment. Summary of the Invention

[0006] The purpose of this application is to provide a nanobody targeting HSP70, its preparation method and application, in order to solve the problem that the existing technology lacks nanobodies targeting HSP70, which makes it impossible to better utilize the HSP70 target for the preparation of related drugs.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows:

[0008] In a first aspect, this application provides a nanobody targeting HSP70, the nanobody comprising nanobody Nb D8, wherein the amino acid sequence of the nanobody Nb D8 is shown in Seq.ID NO.1.

[0009] Furthermore, the nanobody includes four framework regions FR1, FR2, FR3, and FR4, and three complementarity-determining regions CDR1, CDR2, and CDR3;

[0010] In the nanobody Nb D8, the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, the amino acid sequence of FR4 is shown in SEQ ID NO.5, the amino acid sequence of CDR1 is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.8.

[0011] Furthermore, the base sequence of the nanobody NbD8 is shown in Seq.ID NO.9.

[0012] Secondly, this application provides a method for preparing HSP70-targeting nanobodies, comprising the following steps:

[0013] The HSP70-NBD gene was designed, synthesized, expressed, and purified to obtain the HSP70-NBD protein.

[0014] The HSP70-NBD protein was coated onto an immunotube for enrichment and screening to obtain a phage library.

[0015] The elution buffer of the phage library was amplified by PCR and verified by ELISA, followed by next-generation sequencing, and the gene sequence of the nanobody was synthesized based on the sequencing results.

[0016] The gene sequence of the nanobody was cloned into an expression vector to obtain a recombinant plasmid. The recombinant plasmid was then transformed into a host cell to induce expression and purified to obtain a nanobody targeting HSP70.

[0017] Furthermore, in the enrichment screening step, 2 to 3 rounds of enrichment screening are performed.

[0018] Furthermore, the step of cloning the gene sequence of the nanobody into an expression vector to obtain a recombinant plasmid also includes: fusing and expressing a hemagglutinin tag for subsequent detection.

[0019] Thirdly, this application provides the use of HSP70-targeting nanobodies in the preparation of contrast agents for tumor tissues expressing HSP70.

[0020] Furthermore, the developing agent includes at least one of an imaging agent and a tracer.

[0021] Fourthly, this application provides the application of HSP70-targeting nanobodies in the preparation of drugs that target and treat tumor diseases expressing HSP70.

[0022] Fifthly, this application provides the application of HSP70-targeting nanobodies in the preparation of protein degradation-targeting conjugate drugs.

[0023] The first aspect of this application provides a nanobody targeting HSP70, the nanobody comprising nanobody NbD8, wherein the amino acid sequence of the nanobody NbD8 is shown in Seq. ID NO.1. Since the provided nanobody NbD8 can highly bind to HSP70, exhibiting high affinity and specificity for HSP70, it can target HSP70, providing a solid theoretical basis for the subsequent construction of the PROTAC complex. Furthermore, this novel concept offers a new treatment strategy for clinical practice.

[0024] The second aspect of this application provides a method for preparing HSP70-targeting nanobodies. This method involves screening a high-capacity nanobodies phage library, and the resulting nanobodies have the advantages of strong binding force and good specificity. Furthermore, the preparation method is simple and convenient, which is conducive to its widespread application.

[0025] The application of the HSP70-targeting nanobody provided in the third aspect of this application in the preparation of a contrast agent for HSP70-expressing tumor tissues. Because the nanobody NbD8 has high affinity and high binding specificity to HSP70, providing the HSP70-targeting nanobody NbD8 for the preparation of a contrast agent for HSP70-expressing tumor tissues is beneficial for improving the contrast rate and for wider application.

[0026] The application of the HSP70-targeting nanobody provided in the fourth aspect of this application in the preparation of drugs targeting HSP70-expressing tumors. Because the nanobody NbD8 has high affinity and high binding specificity to HSP70, providing the HSP70-targeting nanobody NbD8 for the preparation of drugs targeting HSP70-expressing tumors is beneficial for improving drug performance.

[0027] The application of the HSP70-targeting nanobody provided in the fifth aspect of this application in the preparation of protein degradation targeting conjugate drugs. Because the NbD8 nanobody has high affinity and high binding specificity to HSP70, the prepared protein degradation targeting conjugate drug uses the HSP70 nanobody as an E3 ligase conjugate. By binding to CHIP and promoting the ubiquitination and degradation of the target protein, it achieves the effect of eliminating pathogenic protein levels, while also providing a novel tool for the construction of PROTAC complexes. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] in:

[0030] Figure 1 The high-purity target protein HSP70-NBD analysis chromatogram provided in Example 1.

[0031] Figure 2 The image shows the three-round enrichment analysis of the phage nanobody library provided in Example 1.

[0032] Figure 3 The image shows the analytical results of the purification of the nanobody provided in Example 1.

[0033] Figure 4 The image shows the analytical results of the purification of the nanobody provided in Example 1.

[0034] Figure 5 The image shows the high binding affinity of the nanobody provided in Example 1 to HSP70.

[0035] Figure 6 The nanobody provided in Example 1 shows a good binding ability with HSP70. Detailed Implementation

[0036] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0037] The first aspect of this application provides a nanobody targeting HSP70, the nanobody comprising nanobody Nb D8, wherein the amino acid sequence of the nanobody Nb D8 is shown in Seq. ID NO.1.

[0038] The first aspect of this application provides a nanobody targeting HSP70, the nanobody comprising nanobody Nb D8, wherein the amino acid sequence of the nanobody Nb D8 is shown in Seq. ID NO.1. Since the provided nanobody Nb D8 can highly bind to HSP70, exhibiting high affinity and specificity for HSP70, it can target HSP70, providing a solid theoretical basis for the subsequent construction of the PROTAC complex. Furthermore, this novel concept offers a new treatment strategy for clinical practice.

[0039] In some embodiments, the amino acid sequence of the nanobody Nb D8 is shown in Seq.ID NO.1, wherein Seq.ID NO.1 specifically comprises:

[0040] MAVQLVESGGGLVQAGGSLRLSCAASGRTFSSYDMGWFRQAPGKEREFV AAISRSGGYTYYADSVKGRFTISRDNAKTTVYLQMNSLKPEDTAVYYCNRFPA PARWGQGTQVTVSS.

[0041] In some embodiments, the nanobody includes four framework regions FR1, FR2, FR3, FR4 and three complementarity-determining regions CDR1, CDR2, CDR3.

[0042] In the nanobody Nb D8, the amino acid sequence of FR1 is shown in SEQ ID NO.2, which is specifically: MAVQLVESGGGLVQAGGSLRLSCAASGRTF.

[0043] The amino acid sequence of FR2 is shown in SEQ ID NO.3, which is specifically: WFRQAPGKEREFVAAI.

[0044] The amino acid sequence of FR3 is shown in SEQ ID NO.4, which is: RFTISRDNAKTTVYLQMNSLKPEDTAVYYCN.

[0045] The amino acid sequence of FR4 is shown in SEQ ID NO.5, which is WGQGTQVTVSS.

[0046] The amino acid sequence of CDR1 is shown in SEQ ID NO.6, which is specifically SSYDMG.

[0047] The amino acid sequence of CDR2 is shown in SEQ ID NO.7, which is SRSGGYTYYADSVKG.

[0048] The amino acid sequence of CDR3 is shown in SEQ ID NO.8, which is RFPAPAR.

[0049] In some embodiments, the base sequence of the nanobody NbD8 is shown in Seq. ID NO.9, specifically:

[0050] ATGGCGGTGCAGCTGGTGGAGTCTGGGGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAGTTATGACATGGGCTGGTTCCGCCAGGCTCCAGGGAAGGAGCGTGAGTTTGTAGCAGCTATTAGTCGGAGTGGTGGTTAC ACATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGACTACGGTGTATCTGCAAATGAACAGCCTGAAACCTGAGGACACGGCCGTCTATTACTGTAACAGATTTCCAGCGCCCGCACGCTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA.

[0051] The second aspect of this application provides a method for preparing a nanobody targeting HSP70, comprising the following steps:

[0052] S01. Design and synthesize the HSP70-NBD gene and express and purify it to obtain the HSP70-NBD protein;

[0053] S02. The HSP70-NBD protein is coated onto an immunotube for enrichment and screening to obtain a phage library;

[0054] S03. The elution buffer of the phage library is subjected to PCR amplification and ELISA verification, followed by next-generation sequencing, and the gene sequence of the nanobody is synthesized based on the sequencing results.

[0055] S04. The gene sequence of the nanobody is cloned into an expression vector to obtain a recombinant plasmid. The recombinant plasmid is then transformed into a host cell to induce expression and purified to obtain a nanobody targeting HSP70.

[0056] The second aspect of this application provides a method for preparing HSP70-targeting nanobodies. This method involves screening a high-capacity nanobody phage library, and the resulting nanobodies have the advantages of strong binding force and good specificity. Furthermore, the preparation method is simple and convenient, which is conducive to its widespread application.

[0057] In step S01, the HSP70-NBD gene was designed and synthesized, and then expressed and purified to obtain the HSP70-NBD protein.

[0058] In some embodiments, the HSP70-NBD gene was designed and synthesized, and the expressed and purified protein was used for nanobody screening. The expression and purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction conditions were explored using different concentrations of IPTG at 16°C; b) Large-scale induction expression was performed according to the preliminary induction conditions, and the protein was sterilized using a high-pressure sterilizer at 1000W; c) Centrifugation was performed at 17000g, 4°C for 30 min, and the supernatant was incubated with Ni packing material at 4°C for 1 hour; d) The target protein was eluted with imidazole at gradient concentrations; e) After purification using a Ni column, molecular sieve separation was performed to remove impurities, with AKTA parameters set at a flow rate of 0.5 mL / min, and samples were collected every 1 mL; f) The purity of the target protein was determined based on the electrophoresis results, and the protein concentration was determined by the BCA method.

[0059] In step S02, the HSP70-NBD protein is coated onto an immunoassay tube for enrichment and screening to obtain a phage library.

[0060] In some embodiments, an immunotube method was used to screen a natural alpaca-derived phage-displaying nanobody library, with the selected phage display library having a capacity of 2 x 10⁹. The screening steps were as follows: a) the target protein was coated onto an immunotube at a concentration of 25 μg / mL and subjected to three rounds of enrichment screening; b) the phage elution buffer was used for the third round of plating.

[0061] In step S03, the elution buffer of the phage library is subjected to PCR amplification and ELISA verification, followed by next-generation sequencing, and the gene sequence of the nanobody is synthesized based on the sequencing results.

[0062] In some embodiments, 192 single clones are randomly selected for ELISA verification. The 96-well plate used for ELISA is also coated with BSA as a control. The positive standard is that the ELISA reading is more than 3 times the corresponding BSA reading and the reading is greater than 0.5. c) The positive single clones identified by phage ELISA twice are sent to the company for sequencing to determine the sequence information. The sequence is extracted to obtain the nanobody protein sequence. The sequences are compared and analyzed to obtain the distribution frequency of the positive sequence.

[0063] In step S04, the gene sequence of the nanobody is cloned into an expression vector to obtain a recombinant plasmid. The recombinant plasmid is then transformed into a host cell to induce expression and is purified to obtain a nanobody targeting HSP70.

[0064] In some embodiments, the nanobody gene sequence is cloned into the pcold vector, and a hemagglutinin HA tag is fused for subsequent detection. The expression and purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction is performed at 16°C using 0.2 mM IPTG; b) Large-scale induction expression is performed according to the preliminary induction conditions, and autoclave sterilization is performed at 1000W; c) Centrifugation is performed at 17000g, 4°C for 30 min, and the supernatant is incubated with Ni packing material at 4°C for 1 hour; g) After purification by Ni column, molecular sieve separation is performed, with AKATA parameters set at a flow rate of 0.5 mL / min, and 1 mL is collected each time.

[0065] The third aspect of this application provides the use of HSP70-targeting nanobodies in the preparation of contrast agents for tumor tissues expressing HSP70.

[0066] The application of the HSP70-targeting nanobody provided in the third aspect of this application in the preparation of a contrast agent for HSP70-expressing tumor tissues. Because the nanobody NbD8 has high affinity and high binding specificity to HSP70, providing the HSP70-targeting nanobody NbD8 for the preparation of a contrast agent for HSP70-expressing tumor tissues is beneficial for improving the contrast rate and for wider application.

[0067] In some embodiments, the developer includes at least one of an imaging agent and a tracer.

[0068] The fourth aspect of this application provides the use of HSP70-targeting nanobodies in the preparation of medicaments for the targeted treatment of tumor diseases expressing HSP70.

[0069] The application of the HSP70-targeting nanobody provided in the fourth aspect of this application in the preparation of a drug for targeting and treating HSP70-expressing tumor diseases. Since the nanobody NbD8 has high affinity and high binding specificity to HSP70, providing the HSP70-targeting nanobody NbD8 for the preparation of a drug for targeting and treating HSP70-expressing tumor diseases is beneficial for improving drug performance.

[0070] The fifth aspect of this application provides the application of HSP70-targeting nanobodies in the preparation of protein degradation-targeting conjugate drugs.

[0071] The application of the HSP70-targeting nanobody provided in the fifth aspect of this application in the preparation of a protein degradation targeting conjugate drug. Because the NbD8 nanobody has high affinity and high binding specificity to HSP70, the prepared protein degradation targeting conjugate drug uses the HSP70 nanobody as an E3 ligase conjugate. By binding to CHIP and promoting the ubiquitination and degradation of the target protein, it achieves the effect of eliminating pathogenic protein levels, while also providing a novel tool for the construction of PROTAC complexes.

[0072] The following description is based on specific embodiments.

[0073] Example 1

[0074] Nanobodies targeting HSP70 and their preparation methods

[0075] Experimental procedure:

[0076] 1) Expression and purification of HSP70-NBD nanobody

[0077] The HSP70-NBD gene was designed, synthesized, expressed, and purified for nanobody screening. The expression and purification steps are as follows: a) To prevent inclusion body formation and protein degradation, induction conditions were explored using different concentrations of IPTG at 16℃; b) Large-scale induction expression was performed according to the preliminary induction conditions, and the protein was sterilized using a high-pressure autoclave at 1000W; c) Centrifugation was performed at 17000g, 4℃ for 30 min, and the supernatant was incubated with Ni packing material at 4℃ for 1 hour; d) The target protein was eluted with imidazole at gradient concentrations; e) After purification using a Ni column, molecular sieve separation was performed to remove impurities, with AKTA parameters set at a flow rate of 0.5 mL / min, and samples collected every 1 mL; f) The purity of the target protein was determined based on electrophoresis results, and the protein concentration was measured using the BCA method.

[0078] 2) Screening of nanobodies and preliminary validation of positive clones by ELISA

[0079] An immunotube method was used to screen a natural alpaca-derived phage-displayed nanobody library. The selected phage display library had a capacity of 2 x 10⁹. The screening steps were as follows: a) The target protein was coated onto immunotubes at a concentration of 25 μg / mL and subjected to three rounds of enrichment screening; b) Using the third round of phage elution buffer, 192 single clones were randomly selected for ELISA verification. A 96-well plate for ELISA was simultaneously coated with BSA as a control. A positive criterion was an ELISA reading greater than three times the corresponding BSA reading and a reading greater than 0.5; c) Positive single clones identified by two phage ELISA tests were sent to a company for sequencing to determine their sequence information. The sequences were extracted to obtain the nanobody protein sequences, and comparative analysis was performed to obtain the distribution frequency of positive sequences.

[0080] 3) Purification and expression of nanobodies

[0081] The nanobody gene sequence was cloned into the pcold vector, and a hemagglutinin (HA) tag was fused for subsequent detection. The expression and purification steps were as follows: a) To prevent inclusion body formation and protein degradation, induction was performed at 16°C using 0.2 mM IPTG; b) Large-scale induction expression was performed according to the preliminary induction conditions, and autoclaving was performed at 1000W; c) Centrifugation was performed at 17000g, 4°C for 30 min, and the supernatant was incubated with Ni packing material at 4°C for 1 hour; g) After purification by Ni column, molecular sieve separation was performed, with AKATA parameters set at a flow rate of 0.5 mL / min, and 1 mL was collected each time.

[0082] 4) ELISA assay of nanobodies

[0083] This experiment was used to verify whether nanobodies expressed and purified in vitro could directly interact with in vitro purified antigen proteins. The steps were as follows: a) Dilute the antigen protein with PBS to 5 μg / ml, and plate each well with 100 μl of each solution. Coat the wells and incubate overnight at 4°C; b) Block with 3% PBS / BSA at room temperature for 2 h, 200 μg / well; c) Prepare nanobodies of different concentrations with 1% BSA / PBST, 100 μg / well, and incubate at room temperature for 1 h; d) Incubate with secondary antibody anti-HA HRP (1:3000) at room temperature for 1 h; e) Develop color with TMB; f) Stop the reaction with stop solution; g) Measure the absorbance at 450 nm using a microplate reader and plot the absorbance curve based on the absorbance values.

[0084] 5) Surface plasmon resonance (SPR) experiment

[0085] This experiment was used to further verify the binding of antigen and nanobodies and to calculate their equilibrium constant. Purified antigen proteins were immobilized on a chip, and nanobodies of different concentrations were sequentially added to analyze their affinity for the antigen proteins. The reaction signals were recorded over 360 seconds, kinetic curves were generated, and relevant parameters were calculated.

[0086] Results analysis:

[0087] 1. Expression and purification of HSP70-NBD protein

[0088] The HSP70 protein has a molecular weight of approximately 70 kDa. Its signature sequence fragment NBD was extracted and fused with GST and His to form a fusion protein, which was then purified. This fusion protein, approximately 43 kDa, served as a screening target for nanobodies. After codon optimization, the HSP70-NBD fusion gene was synthesized and induced for expression and purification in *E. coli*. To avoid protein degradation and inclusion body formation, induction was performed overnight at 16°C with 0.4 mM IPTG. Finally, the protein was purified using a Ni column and molecular sieve. Figure 1 As shown, high-purity target protein was obtained for subsequent nanobody screening.

[0089] 2. Screening, identification, and purification of HSP70-NBD nanobodies

[0090] We first screened the HSP70-NBD phage nanobody library. After two and three rounds of screening, the library was enriched by nearly 200 and 60 times, respectively. Figure 2 From the library obtained in the third round, approximately 192 clones were selected for preliminary validation using two phage ELISA assays. Eleven positive clones were initially identified and sequenced; nine of these were normal. Based on the pre- and post-sequence sequences of the nanobodies, the nanobody sequences could be obtained from the sequencing results. These nine sequences were translated into amino acids, sorted, and subjected to multiple sequence alignment, yielding six distinct nanobody sequences. These six nanobodies were subsequently purified and validated using Coomassie brilliant blue staining and Western blotting. Figure 3 and Figure 4 In addition, in the preliminary experiments, A9 and F10 were found to be false positive nanobodies, and we excluded them in the subsequent binding affinity verification experiments.

[0091] 3. Affinity Detection of HSP70-NBD Nanobodies

[0092] The binding of HSP70-NBD nanobodies to HSP70 was further detected by ELISA. The results showed that, compared with the control group BSA, four of the purified HSP70-NBD nanobodies had a higher binding affinity to HSP70 in the experimental group, indicating that the nanobodies have a high binding capacity to HSP70. Figure 5 Next, using Biacore, the affinity constants of the above four nanobodies with HSP70 were further detected, yielding one nanobodies, NbD8, with an affinity at the nanomolar level. Figure 6 This further demonstrates that the nanobody we obtained has a good binding ability with HSP70.

[0093] In summary, this application provides a nanobody targeting HSP70, comprising nanobody NbD8, wherein the amino acid sequence of nanobody NbD8 is shown in Seq. ID NO.1. Since the provided nanobody NbD8 can highly bind to HSP70, exhibiting high affinity and specificity for HSP70, it can target HSP70, providing a solid theoretical basis for the subsequent construction of the PROTAC complex. Furthermore, this novel concept offers a new treatment strategy for clinical practice.

[0094] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A nanobody targeting HSP70, characterized in that, The nanobody includes nanobody Nb D8, wherein the amino acid sequence of the nanobody Nb D8 is shown in Seq.ID NO.

1.

2. The HSP70-targeting nanobody according to claim 1, characterized in that, The nanobody includes four framework regions FR1, FR2, FR3, and FR4 and three complementarity-determining regions CDR1, CDR2, and CDR3. In the nanobody Nb D8, the amino acid sequence of FR1 is shown in SEQ ID NO.2, the amino acid sequence of FR2 is shown in SEQ ID NO.3, the amino acid sequence of FR3 is shown in SEQ ID NO.4, the amino acid sequence of FR4 is shown in SEQ ID NO.5, the amino acid sequence of CDR1 is shown in SEQ ID NO.6, the amino acid sequence of CDR2 is shown in SEQ ID NO.7, and the amino acid sequence of CDR3 is shown in SEQ ID NO.

8.

3. The HSP70-targeting nanobody according to claim 1, characterized in that, The base sequence of the nanobody Nb D8 is shown in Seq.ID NO.

9.

4. The use of the HSP70-targeting nanobody as described in any one of claims 1 to 3 in the preparation of a contrast agent for expressing HSP70 tumor tissue.

5. The application according to claim 4, characterized in that, The developing agent includes at least one of an imaging agent and a tracer.

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