A nanobody and its application in the preparation of a drug for treating or diagnosing CD176-positive tumor cancer

Abstract

This invention discloses a nanobody and its application in the preparation of drugs for the treatment or diagnosis of CD176-positive tumor cancer. The nanobody sequentially comprises a CDR1 region, a CDR2 region, and a CDR3 region. The amino acid sequence of the CDR1 region is shown in SEQ ID No. 6, the amino acid sequence of the CDR2 region is shown in SEQ ID No. 7, and the amino acid sequence of the CDR3 region is shown in SEQ ID No. 8. This invention reveals that the nanobody possessing the above three complementarity-determining regions can bind to the CD176 protein (EC176) with high affinity. 50 With a molecular weight of 18.65 nM, it has the characteristics of small molecular weight, low immunogenicity, better solubility and stability, and can effectively detect the expression of CD176 or inhibit the activity of CD176, which has potential value in the detection and treatment of tumors.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a nanobody and its application in the preparation of drugs for the treatment or diagnosis of CD176-positive tumor cancer. Background Technology

[0002] The CD176 antigen, also known as the Thomsen-Friedenreich antigen (TF antigen), is a core type 1 O-glycan structure (Galβ1-3GalNAcα1-Ser / Thr). In normal tissues, this glycan core is usually further modified and masked by other glycosyl groups, thus remaining unrecognized by the immune system. However, in various human cancer cells (reportedly affecting over 80% of cancers), due to abnormal glycosylation processes, the CD176 antigen is exposed on the cell surface in an unmasked form, becoming a broad-spectrum tumor-associated antigen. Aberrant expression of CD176 is closely associated with malignant transformation, proliferation, invasion, and metastasis of tumors and is considered a poor prognostic marker for various cancers, such as breast cancer, colorectal cancer, gastric cancer, lung cancer, and bladder cancer.

[0003] Due to its specific high expression on the tumor surface, CD176 has become an ideal target for developing targeted anticancer therapies and diagnostic reagents. Currently, strategies targeting CD176 include developing traditional monoclonal antibodies targeting this antigen, vaccines, and CAR-T cell therapies. However, traditional antibodies suffer from problems such as large molecular weight, poor tissue penetration, high production costs, and poor thermal stability.

[0004] Nanobodies (Nb) are derived from the variable region (VHH) of heavy chain antibodies from camelids and are the smallest known functional units capable of binding antigens. With a molecular weight of only 12-15 kDa, nanobodies offer significant advantages over traditional antibodies, including small molecular weight, high stability, strong affinity, ease of modification and production, and strong tissue infiltration. Phage display technology allows for the efficient screening of high-affinity nanobodies targeting specific targets (such as CD176) from immunized alpacas. Therefore, developing a novel nanobody capable of specifically binding to the CD176 antigen and possessing excellent physicochemical properties has significant clinical and application value for improving the diagnostic rate and therapeutic efficacy of CD176-positive cancers. Summary of the Invention

[0005] The purpose of this invention is to provide a nanobody and its application in the preparation of drugs for the treatment or diagnosis of CD176-positive tumors and cancers, thus providing a new and effective approach for the diagnosis and treatment of CD176-positive cancers.

[0006] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0007] The present invention first provides a nanobody comprising CDR1, CDR2 and CDR3 regions in sequence, wherein the amino acid sequence of the CDR1 region is shown in SEQ ID No. 6, the amino acid sequence of the CDR2 region is shown in SEQ ID No. 7 and the amino acid sequence of the CDR3 region is shown in SEQ ID No. 8.

[0008] Nanobodies consist of three complementarity-determining regions (CDR1, CDR2, and CDR3) and four framework regions (FR1, FR2, FR3, and FR4). The complementarity-determining regions and framework regions are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Among them, the complementarity-determining regions are the key regions in nanobodies responsible for antigen-specific recognition, and their sequence determines the binding performance of nanobodies to antigens.

[0009] This invention has discovered that nanobodies possessing the aforementioned three complementarity-determining regions can bind to the CD176 protein (EC176) with high affinity. 50 With a molecular weight of 18.65 nM, it has the characteristics of small molecular weight, low immunogenicity, better solubility and stability, and can effectively detect the expression of CD176 or inhibit the activity of CD176, which has potential value in the detection and treatment of tumors.

[0010] Based on this, the present invention also provides the application of the above-mentioned nanobody in the preparation of anti-CD176 positive tumor drugs; and the application of the above-mentioned nanobody in the preparation of CD176 positive cancer diagnostic kits.

[0011] As a further preferred embodiment, the amino acid sequence of the above-mentioned nanobody is shown in SEQ ID No. 1.

[0012] The present invention also provides a polynucleotide encoding the above-mentioned nanobody, a nucleic acid construct containing the above-mentioned polynucleotide (promoters, terminators, etc. can be added to the polynucleotide as needed), an expression vector containing the above-mentioned nucleic acid construct, and transformed cells containing the above-mentioned polynucleotide, the above-mentioned nucleic acid construct, or the above-mentioned expression vector.

[0013] Compared with the prior art, the technical effects of the present invention are reflected in:

[0014] The nanobody of the present invention can bind to CD176 protein (EC) with high affinity. 50 With a molecular weight of 18.65 nM, it has the characteristics of small molecular weight, low immunogenicity, better solubility and stability, and can effectively detect the expression of CD176 or inhibit the activity of CD176, which has potential value in the detection and treatment of tumors. Attached Figure Description

[0015] Figure 1 Total RNA purity test of PBMCs in anticoagulated venous blood of alpacas immunized with Ac-Ser(TF)-PEG3-Osu-lactoferrin for five rounds;

[0016] Where M is a DNA marker; 1 is the five immune RNA;

[0017] Figure 2 The results are obtained by agarose gel electrophoresis of the PCR products after the first round of nested PCR of cDNA containing 5 immune RNA.

[0018] Where M is a DNA marker, and 1-5 are VHH fragments obtained from the first round of nested PCR amplification.

[0019] Figure 3 The results of agarose gel electrophoresis of the PCR products from the second round of nested PCR of cDNA containing 5 immune RNA.

[0020] Where M is a DNA marker, and 1-2 are VHH fragments obtained from the first round of nested PCR amplification.

[0021] Figure 4 This is a plate colony diagram of cells transformed with the VHH fragment;

[0022] Among them, NB802B10 -3 Dilute the VHH fragment to 10 for cell transformation -3 Colony image after plating, NB802B10 -4 Dilute the VHH fragment to 10 for cell transformation -4 Colony image after plating;

[0023] Figure 5 To verify the results of colony PCR on cells transformed with the VHH fragment and library diversity;

[0024] Figure 6 SDS-PAGE electrophoresis results of supernatant and precipitate after cell lysis following transformation of each VHH fragment;

[0025] Where M represents the DNA marker, 001 and 001non represent the supernatant and precipitate of cells transformed with the VHH fragment of NB802-Anti-75 after cell lysis; 002 and 002non represent the supernatant and precipitate of cells transformed with the VHH fragment of NB802-Anti-24 after cell lysis; 003 and 003non represent the supernatant and precipitate of cells transformed with the VHH fragment of NB802-Anti-59 after cell lysis; 004 and 004non represent the supernatant and precipitate of cells transformed with the VHH fragment of NB802-Anti-65 after cell lysis; 005 and 005non represent the supernatant and precipitate of cells transformed with the VHH fragment of NB802-Anti-139 after cell lysis; and 006 and 006non represent the supernatant and precipitate of cells transformed with the VHH fragment of NB802-Anti-189 after cell lysis.

[0026] Figure 7 The results are from gel filtration chromatography of nanobodies. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but 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.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials disclosed herein and cited by them are incorporated herein by reference.

[0029] For example, a "nanobody," or "heavy-chain single-domain antibody," contains only one heavy-chain variable region (VHH) and naturally lacks the light chain compared to other antibodies. When referring to ligand / receptor, antibody / antigen, or other binding pairs, "specific" binding means determining the presence of the protein, for example, the binding reaction between the nanobody of this invention and the CD176 protein, within a heterogeneous population of proteins and / or other biological reagents. Therefore, under specified conditions, a specific ligand / antigen binds to a specific receptor / antibody and does not bind in significant amounts to other proteins present in the sample.

[0030] Equivalent technologies of the specific embodiments described herein that are apparent to those skilled in the art through routine experimentation are included in this application.

[0031] Unless otherwise specified, the experimental methods in the embodiments of this invention are conventional methods. Unless otherwise specified, the instruments and equipment used in the following embodiments are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following embodiments are all purchased from conventional biochemical reagent stores.

[0032] The reagents used in the following embodiments of this invention, such as enzymes, culture media, antibiotics, and milk, as well as some commonly used biological materials, such as competent cells, vectors, helper phages, and cells to be transformed, are all commercially available products. Some synthetic biological materials, such as primers and sequences, which require artificial synthesis, were all synthesized by Apak Biotechnology Co., Ltd. The CD176 protein of this invention was obtained in the inventors' laboratory.

[0033] Example 1: Obtaining nanobodies bound to CD176 protein

[0034] 1. Alpaca Immunization

[0035] (1) Immunization schedule

[0036] Adult male alpacas in good condition were selected. 10 mL of blood was collected before immunization and used as a negative serum control. 0.5 mg of Ac-Ser(TF)-PEG3-Osu-lactoferrin antigen was dissolved in PBS, and the volume was brought to 250 μL with PBS. This solution was then mixed with an equal volume of adjuvant at a 1:1 ratio and injected subcutaneously. Subsequent immunizations were performed every two weeks. 0.25 mg of Ac-Ser(TF)-PEG3-Osu-lactoferrin antigen was dissolved in PBS, and the volume was brought to 250 μL with PBS. This solution was then mixed with an equal volume of adjuvant at a 1:1 ratio and injected subcutaneously. After the second immunization, procoagulant blood was collected from the alpacas, and serum was separated for ELISA to detect the immunization effect. On day 7 after the fifth immunization, peripheral blood was collected from the jugular vein, and blood lymphocytes were separated.

[0037] (2) Immunological identification:

[0038] Dilute 2 µg / mL hemocyanin (KLH)-Osu with coating buffer (0.05 M carbonate, pH 9.6), add 100 µL to each well of a high-binding ELISA plate, and incubate overnight at 4°C. Add only an equal volume of coating buffer to the blank wells. Discard the coating buffer, wash the plate three times with PBST (0.05% Tween-20), add 200 µL of 5% fetal bovine serum / PBS to each well, and block at 37°C for 40 min. Discard the blocking buffer, wash three more times with PBST, and blot dry with filter paper. Collect whole blood after immunization and place it in a coagulation-promoting tube. Incubate at room temperature for 2 h to allow serum to precipitate naturally. Centrifuge at 3000 rpm for 10 min at 4°C and collect the supernatant. Serum was serially diluted twofold (1:2000–1:128000) with sample dilution buffer (1% BSA–0.05% Tween-20 / PBS), 100 µL per well, and incubated at 37°C for 45 min; washed 5 times. HRP-labeled anti-Illama IgG (1:20000), 100 µL / well, was added and incubated at 37°C for 30 min; washed 5 times. 100 µL of LTMB-hydrogen peroxide urea chromogenic solution was added to each well and incubated at 37°C in the dark for 15 min; immediately, 50 µL of 2 M H₂SO₄ was added to stop the reaction. Absorbance was read within 15 min using a microplate reader at 450 nm (reference 630 nm). The results are shown in Table 1.

[0039] Table 1. Neutralizing effect of alpaca immune serum on CD176 antigen

[0040]

[0041] In Table 1, hemocyanin-Osu is hemocyanin conjugated with the CD176 antigen (to increase the immunogenicity of the target antigen). It can be seen that after five immunizations, alpaca A had a lower titer against hemocyanin-Osu; alpaca B had a titer of 1:64K (OD450>0.2), indicating a mid-to-high level of serum titer. This suggests that alpaca B produced a higher serum titer against the target antigen, indicating an effective humoral immune response. Therefore, PBMCs from alpaca B were used to construct a VHH phage library.

[0042] 2. Antibody library construction

[0043] (1) Library construction

[0044] First, the collected anticoagulated venous blood was diluted 1:1 with 1× PBS. 15 ml of lymphocyte separation medium was added to the bottom of a 50 ml centrifuge tube, and 15 ml of diluted anticoagulated blood was slowly added along the tube wall. The tube was centrifuged at 2000 rpm and 20°C for 20 min. The PBMC layer was then aspirated using a pipette. The cells were washed with 1× PBS, centrifuged at 3000 g to collect the precipitate, and peripheral blood mononuclear cells (PBMCs) were obtained. After counting, the cells were aliquoted into small tubes for later use.

[0045] Then, total RNA was extracted from the isolated PBMCs using RNAiso Plus reagent, and 1 μg of RNA was subjected to gel electrophoresis to determine RNA purity. The results showed that the RNA purity was good. Figure 1 The recovery volume was 30 µL; the recovery concentration was 387.5 ng / µL; A260 / A280 = 2.16; A260 / A230 = 2.06; M: DNA marker; 1: 5-immune RNA.

[0046] (2) Reverse transcription

[0047] The PrimeScript™ Reverse Transcription II 1st Strand cDNA Synthesis Kit was used according to the kit's instructions, transcribing a total of 5 μg of RNA. Specifically: Remove each group of PrimeScript™ II 1st Strand cDNA Synthesis Kit from the -20°C freezer, thaw, mix, briefly centrifuge, and place on ice. Add the components listed in Table 2 to a 0.2 μL centrifuge tube and mix well.

[0048] Table 2 Reverse transcription reaction system 1

[0049]

[0050] Incubate at 65°C for 5 minutes, then place on ice for at least 1 minute. Prepare the cDNA synthesis system by adding the components listed in Table 3 below and mixing well.

[0051] Table 3 Reverse transcription reaction system 2

[0052]

[0053] Mix gently and proceed with the reaction according to the procedure in Table 4 below.

[0054] Table 4 Reverse transcription reaction procedure

[0055]

[0056] After the reaction is complete, the reaction product is briefly centrifuged, and the cDNA is stored at −20℃.

[0057] (3) Nested PCR amplification

[0058] The first round of PCR was performed using the reaction system in Table 5 and the reaction conditions in Table 6.

[0059] Table 5 First-round PCR reaction system

[0060]

[0061] Table 6. First-round PCR reaction conditions

[0062]

[0063] After the first round of PCR amplification, all PCR amplification products were collected and subjected to 2% agarose gel electrophoresis. Figure 2 The VHH fragment of approximately 750 bp was then harvested from the gel. The components included: M - DNA marker; 1 - VHH fragment amplified in the first round of nested PCR; 2 - VHH fragment amplified in the first round of nested PCR; 3 - VHH fragment amplified in the first round of nested PCR; 4 - VHH fragment amplified in the first round of nested PCR; 5 - VHH fragment amplified in the first round of nested PCR.

[0064] The second round of PCR used Apak's internally optimized primers and optimized PCR system and program to amplify the target gene fragment (i.e., the VHH gene fragment), followed by 2% agarose gel electrophoresis. The results are as follows: Figure 3 As shown, M represents the DNA marker, and Lanes 1-2 represent VHH. The approximately 500 bp VHH fragment was purified and recovered using a DNA product purification kit. The purified VHH gene fragment was stored at -20°C for later use.

[0065] The pComb3XSS vector and the VHH gene fragment were digested with SfiI enzyme and incubated overnight at 50°C. The VHH gene fragment was then recovered using a universal DNA purification kit. After the digestion reaction, ligation was performed using T4 ligase at a Vector:VHH molar ratio of 1:3 at 4°C for 16 hours to obtain the pComb3XSS-VHH recombinant vector.

[0066] 3. Determination of library capacity and abundance

[0067] (1) Determination of phage library capacity

[0068] The pComb3XSS-VHH recombinant vector was mixed with competent E. coli cells and subjected to 10 consecutive electroporation transformations. Immediately after each electroporation, 1 mL of 2YT medium (preheated to 37°C) was added to the electroporation cuvette for recovery. The electroporation product was aspirated and the electroporation cuvette was washed with 2YT medium, yielding a total of 100 mL of recovery product. The cells were then incubated at 37°C and 180 rpm for 45 min. 100 μL of the recovery product was serially diluted to 10⁻⁶. -3 and 10 -4 To determine the library volume, spread the culture onto 90mm plates, centrifuge the remaining culture, resuspend it in 8 mL of 2YT, and spread it onto eight 200mm plates. The next day, count the colonies on the 90mm plates used for volume determination and calculate the library volume (e.g., ...). Figure 4 ).

[0069] Figure 4In the middle, NB802B10 -3 Dilute the VHH fragment to 10 for cell transformation -3 Colony image after plating, NB802B10 -4 Dilute the VHH fragment to 10 for cell transformation -4 Colony image after plating; it can be seen that NB802B10 -4 There were 116 clones on the plate, therefore the number of bacterial library transformants was 1.16 × 10⁻⁶. 9 CFU.

[0070] (2) Phage library titer determination

[0071] Follow these steps:

[0072] 1) Inoculate the bacterial library into 300mL of 2YT+A+G (Amp: 100ug / ml, Glu: 1%) medium until its initial OD600 = 0.1-0.2, and culture at 37℃ and 230rpm until OD600 = 0.8 or higher.

[0073] 2) Add helper phage M13KO7 according to the OD600 value (helper phage: bacteria = 20:1).

[0074] 3) After adding M13KO7, mix well and let stand at 37℃ for 30 minutes.

[0075] 4) Shake gently at 37℃ and 180rpm for 30 minutes.

[0076] 5) Centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend the precipitate in an equal volume of 2YT+A (Amp: 100 μg / ml) + K (kanamycin: 50 μg / ml) medium, and incubate overnight at 30℃ and 220 rpm.

[0077] 6) Centrifuge the overnight culture at 4°C and 10,000 rpm for 20 min, collect the supernatant and discard the precipitate. Replace the centrifuge tube and centrifuge at 4°C and 10,000 rpm for 20 min, collect the supernatant.

[0078] 7) Add PEG8000 / NaCl to 1 / 5 of the supernatant volume, mix well, and precipitate in an ice bath for at least 2 hours.

[0079] 8) Centrifuge at 4℃ and 10000rpm for 20min, discard the supernatant, and centrifuge again to remove the supernatant completely. Resuspend the precipitate in 1mL of 1×PBS, add 1 / 5 volume of PEG8000 / NaCl for secondary precipitation for 1h.

[0080] 9) Centrifuge at 4℃, 12000rpm for 10min, discard the supernatant, and centrifuge again to remove the supernatant completely. Depending on the amount of precipitate, add 1×PBS to resuspend the precipitate.

[0081] 10) Add 100% glycerol to a final concentration of 50%, mix well, and dispense into 1.5 mL EP tubes. Store at -80°C.

[0082] 11) Take 10 μL of Klebsiella pneumoniae and serially dilute it with 2 YT, starting from 10 μL... -8 and 10 -9 Add 10 μl of the solution to 90 μL of TG1 bacterial culture and mix gently. Incubate at 37°C for 15 min, then spread the mixture onto Amp resistance plates and incubate overnight.

[0083] 12) The next day, 10 -9 There were 246 clones on the titer plate, therefore the phage library titer was 2.46 × 10⁻⁶. 13 cfu / mL (2.46*10) 9 *100).

[0084] Forty-eight clones were randomly selected from the transformant number titer plate. Colony PCR was used to verify the insertion rate, and library diversity analysis showed that the insertion rate was 100%. Figure 5 Forty-eight positive clones were sent for sequencing. The sequencing results showed that clones numbered 6, 9, and 38 were non-monoclonal clones, while the rest were monoclonal clones.

[0085] Based on the results of sequencing analysis of the number of transformants, insertion rate, and diversity, the constructed phage library has a volume of 1.16 × 10⁻⁶. 9 (116*10 4 *10*100). The library has good capacity and diversity, and can proceed to the next stage of screening.

[0086] 4. Screening, identification, and purification of nanobodies

[0087] (1) Indirect ELISA screening

[0088] 1) Preparation of monoclonal phage supernatant

[0089] Twenty-six monoclonal phages were picked from a plate and inoculated into two 96-well deep-well plates, each containing 1 mL of 2×YTAG medium. The plates were labeled with CD176-1 and CD176-2 phage libraries, respectively, and incubated at 30°C with shaking. After 8 hours, 50 μL of phage suspension from each well was inoculated into 500 μL of 2×YTAG medium and incubated at 37°C with shaking (60 μL of 60% glycerol was added to the remaining phage suspension from the original plate to a final concentration of 15%, and the plate was stored at -80°C). After transferring the plates and incubating at 37°C with shaking for 1 hour, 50 μL of KM13 helper phage was added to each well, and the plates were incubated statically at 37°C for 30 minutes, followed by incubation at 37°C with shaking for 40 minutes. Centrifuge the deep-well plate at 1800×g for 10 min, discard the supernatant, and resuspend the precipitate in 400 μL of 2×YTAK medium in each well. Incubate overnight at 30°C with shaking. The next day, centrifuge at the maximum speed of 2020xg for 20 min, aspirate 250 μL of phage supernatant from each well and transfer it to a new deep-well plate. Add 250 μL of blocking buffer (PBS buffer solution containing 3% BSA) to each well and incubate at room temperature for 1 h, ready for indirect ELISA detection.

[0090] 2) Identification of specific monoclonal phages

[0091] The reactivity of phage supernatant with CD176 protein was detected by indirect ELISA. The specific method was as follows: An experimental group, a negative control group, and a BSA control group were designed. The experimental and negative control groups used CD176 protein to coat 96-well ELISA plates at a concentration of 2 μg / mL. The BSA control group used BSA protein to coat 96-well ELISA plates at a concentration of 2 μg / mL, 100 μL per well, and incubated overnight at 4°C. The next day, the coating liquid was discarded, and 100 μL of blocking buffer was added to each well, incubating at 37°C for 1 h. The blocking buffer was then discarded, and 100 μL of blocking buffer was added to each well for both the experimental and BSA control groups. The phage supernatant obtained from four rounds of screening was used as the primary antibody. An equal volume of PBS was added to the negative control, and the plates were incubated at 37°C for 1 h. The plates were washed 6 times with PBST. 100 μL of secondary antibody (HRP-M13 Antibody, dilution 1:6000) was added to each well, and the plates were incubated at 37°C for 1 h. Wash the plate 8 times with PBST washing buffer. Add 100 μL of chromogenic substrate to each well and incubate in the dark for 5–15 min. Then, add 50 μL of stop solution to each well to terminate the reaction. Place the 96-well ELISA plate on a plate reader to read the OD450 absorbance. Analyze the ELISA results and identify positive phages.

[0092] Glycerol-containing bacteria corresponding to the positive wells were inoculated into 5 mL of 2×YTAG medium and cultured with shaking at 37°C. The bacterial culture was then sent to a sequencing company for sequencing. After the sequencing results were returned, they were analyzed, and the correctly sequenced strains were selected to repeat the above experiment to verify the positive strains. The recombinant plasmid construction strains were determined based on the ELISA identification results of CD176 monoclonal positive strains (see Table 1). The reactivity of the phage supernatant corresponding to 246 monoclonals with CD176 protein was detected by indirect ELISA. Based on the results of the indirect ELISA experiment, 6 monoclonals were selected, all of which showed good reactivity with CD176 protein (Table 7).

[0093] Table 7. ELISA screening results of CD176 monoclonal nanobodies

[0094]

[0095] 3) Identification of nanobodies

[0096] Six monoclonal bacterial cultures were sent to a sequencing company for sequencing, and the amino acid sequences of each monoclonal nanobody were obtained. The nanobodies in this embodiment all include four frame regions (FRs) and three complementarity-determining regions (CDRs), with the four frame regions (FR1, FR2, FR3, FR4) and the three complementarity-determining regions (CDR1, CDR2, CDR3) arranged alternately in sequence.

[0097] ①NB802-Anti-75, its amino acid sequence is shown in SEQ ID No.1: QLQLVESGGGSVQFGGSLRLSCAAS GRVDSIYAM GWYRQAPGMQRERVAM ITKAGRQTYG NNVKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYC AAD RSYAGTYYSVVPGQYDY WGQGTQVTVSS;

[0098] Among them, FR1 region: QLQLVESGGGSVQFGGSLRLSCAAS (SEQ ID No. 2);

[0099] FR2 area: GWYRQAPGMQRERVAM (SEQ ID No. 3);

[0100] FR3 region: NNVKGRFTISRDNAKNTVYLQMNSLKLEDTAVYYC (SEQ ID No. 4);

[0101] FR4 area: WGQGTQVTVSS (SEQ ID No.5);

[0102] The area between FR1 and FR2 is CDR1: GRVDSIYAM (SEQ ID No. 6);

[0103] The area between FR2 and FR3 is CDR2: ITKAGRQTYG (SEQ ID No. 7);

[0104] The area between FR3 and FR4 is CDR3: AADRSYAGTYYSVVPGQYDY (SEQ ID No.8).

[0105] ②NB802-Anti-24, its amino acid sequence is shown in SEQ ID No. 9:

[0106] EVQVVESGGGLVQAGGSLRLSCVAS GRTFSPNTM AWFRQAPGKQRELVAV ITSGGITNYA DSVKGRFTVSRDNDKNFVYLQMDNLKDADTALYSC NIYHRVIRFT WGPGTQVTVSS;

[0107] The underlined parts are CDR1, CDR2, and CDR3 in sequence; the parts without underlined parts are FR1, FR2, FR3, and FR4 in sequence.

[0108] ③NB802-Anti-59, its amino acid sequence is shown in SEQ ID No. 10: QVQLVESGGGLVQAGGSLRLSCAAS GRTISSRAM GWFRQAPGKEREFVAS ISWSGGSTYY ADSVKGRFAISRDNAKNTLYLQMGSLKPEDTAVYYC A ARTSIINRF WGQGTQVTVSS;

[0109] The underlined parts are CDR1, CDR2, and CDR3 in sequence; the parts without underlined parts are FR1, FR2, FR3, and FR4 in sequence.

[0110] ④NB802-Anti-65, its amino acid sequence is shown in SEQ ID No. 11:

[0111] EVQVVESGGGLVQAGGSLRLSCAAS GRTFSSYGL GWFRQAPGKQRELVAR ITARDITNYA DSVKGRFTISRDNAKNTMYLQMNTLKPEDTAVYYC NIVSAWSHG WGRGTQVTVSS;

[0112] The underlined parts are CDR1, CDR2, and CDR3 in sequence; the parts without underlined parts are FR1, FR2, FR3, and FR4 in sequence.

[0113] ⑤NB802-Anti-139, its amino acid sequence is shown in SEQ ID No. 12:

[0114] QVQLVESGGGLVQAGGSLRLACVAS GRTISSY™ TWFRQAPGKEREFVAA INWSGGRTNYA DSVSGRFTISRGFAKRTAYLQMNDVKSEDTGVYYC AVKLSTTTDGVATTGFTF WGRGAQVTVSS;

[0115] The underlined parts are CDR1, CDR2, and CDR3 in sequence; the parts without underlined parts are FR1, FR2, FR3, and FR4 in sequence.

[0116] ⑥NB802-Anti-189, its amino acid sequence is shown in SEQ ID No. 13:

[0117] EVQVVESGGGLVQAGGSLRLSCAAS GSTFSIAAM AWYRQAPGKQRELVAY ISSDAHTNYP DSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC VANRRRWTVGVGKHDYDSR GQGTQVTVSS;

[0118] The underlined parts are CDR1, CDR2, and CDR3 in sequence; the parts without underlined parts are FR1, FR2, FR3, and FR4 in sequence.

[0119] (2) Identification of nanobodies

[0120] First, the coding sequence of 6 histidine tags and the translation stop codon TGA were appended to the nucleotide sequence of the alpaca-derived nanobody. Then, the expression vector was constructed into the pET23a plasmid by using the restriction endonuclease sites EcoRI and XhoI.

[0121] Then, 1 μL of the constructed pET23a plasmid containing the target gene was added to 50 μL of *E. coli* BL21(DE3) competent cells. After incubating on ice for 30 minutes, the cells were heat-shocked in a 42°C water bath for 60 seconds, and then incubated on ice for 5 minutes. 450 μL of LB medium was added to the bacterial culture. After mixing, the cells were incubated at 37°C with shaking at 200 rpm for 1 hour for recovery. Then, 200 μL of the bacterial culture was plated onto an LB + ampicillin (100 µg / mL) agar plate, and the plate was inverted and incubated overnight at 37°C. Single colonies were picked from overnight culture plates and inoculated into 5 mL of LB + ampicillin (100 µg / mL) medium. After 8 hours of shaking at 200 rpm at 37°C, the entire bacterial culture was transferred to 4 L of LB + ampicillin (100 µg / mL) medium and cultured until the logarithmic growth phase. Induction with 1 mM IPTG (isopropyl-β-D-thiogalactopyranoside) was then performed, followed by overnight culture at 16°C. The next day, the bacterial pellet was collected by centrifugation, resuspended in 1×PBS, and sonicated at 112.5 w for 3 seconds with a 5-second pause. Afterward, the pellet was centrifuged at 12000 rpm for 20 minutes at 4°C. The supernatant and pellet were collected separately for SDS-PAGE electrophoresis. The results are shown below. Figure 6 As shown in the figure. SDS-PAGE analysis revealed the presence of nanobodies in the supernatant after fragmentation, demonstrating that the nanobodies can be expressed in soluble form in Escherichia coli BL21(DE3).

[0122] (3) Purification of nanobodies

[0123] The obtained nanobody core coding sequence (NB) was constructed into the pcoldI plasmid and transfected into 293F cells for eukaryotic expression. The cell culture supernatant was collected and purified by nickel affinity chromatography and gel filtration chromatography (Superdex™ 75 Increase Hiload column (GE Healthcare)). The purified protein was identified by SDS-PAGE. The specific methods are as follows:

[0124] Inclusion bodies in the precipitate were collected and dissolved in 10 mmol / L Tris-HCl (pH 7.0, containing 8 mol / L urea, 1 mmol / L LTT). Insoluble matter was removed by centrifugation, and then refolding buffer (100 mmol / L Tris-HCl, pH 8.0, 400 mmol / L arginine, 5 mmol / L reduced glutathione, 0.5 mmol / L oxidized glutathione, 0.1 mmol / L LPMSF) was prepared for dilution and refolding. The refolded product was purified by nickel affinity chromatography followed by gel filtration chromatography (Superdex™ 75 Increase 10 / 300 GL, PBS, 0.5 mL min).- ¹, 280 nm monitoring) analysis, the analysis results are as follows Figure 7 As shown, the SEC chromatogram shows that the main peak retention time is 12.282 min, accounting for 81.00% of the total area. SDS-PAGE confirmed that this peak is a monomeric nanobody, that is, the relatively pure target protein (NB802-Anti-75) was obtained.

[0125] Example 2: CD176 antigen-antibody specific binding assay

[0126] KLH-Osu and KLH-his (irrelevant) were coated at a concentration of 2 μg / mL as antigen proteins (50 mM NaHCO3, pH=9.6) at 100 μL / well and incubated overnight at 4°C. After washing three times with PBST, the nanobodies were blocked with 300 μL / well of 5% milk. The plates were then incubated at 37°C for 1 h. After washing once with PBST, each nanobody was serially diluted 1:5 from 100 nM (using 5% milk), and 100 μL of each serially diluted nanobody was added to the wells of an ELISA plate. The plates were incubated at 37°C for 1 h. After washing five times with PBST, the corresponding secondary antibody (HRP-labeled human IgG, 1:10K) diluted with blocking buffer was added, and the plates were incubated at 37°C for 0.5 h. Wash the ELISA plate incubated with secondary antibody five times with PBST, add 100 μL of TMB single-component chromogenic solution to each well, incubate at 37°C for 7 min, and stop the reaction by adding 50 μL of 1M HCl to each well. OD 450 reading.

[0127] The OD450 values ​​of the six candidate nanobodies and isotype controls bound to the antigen (hemocyanin-Osu) at seven concentration gradients are shown in Table 8.

[0128] Table 8 ELISA Results

[0129]

[0130] As can be seen from Table 8, the binding of the isotype control to KLH-Osu was basically the same at all concentrations, indicating that the isotype control did not specifically bind to KLH-Osu; while the binding of the six candidate nanobodies to KLH-Osu increased with increasing concentration, indicating that all six candidate nanobodies specifically bound to the CD176 antigen.

[0131] In this embodiment, human breast cancer cells MCF-7 (Pronosa, CL-0149) were used to detect the binding ability of each candidate nanobody to the cell surface antigen CD176.

[0132] The experimental method was as follows: cells were washed twice with 1×PBS buffer, and then resuspended in 1×PBS buffer to a cell concentration of 3×10⁻⁶. 5Cells / μL, aliquoted into PCR plates, 50 μL per well; candidate nanobodies were diluted to 200 nM with 1×PBS buffer and added to the detection wells, followed by seven 5-fold serial dilutions, with 50 μL / well added to each well, and incubated at 4°C for 1 h; after incubation, cells were washed three times with 1×PBS buffer, and 100 μL of fluorescent secondary antibody (Alexa Fluor 647-labeled anti-human IgG Fc antibody) was added to each well, and incubated at 4°C for 1 h; after incubation, cells were washed once with PBS, transferred to 96-well cell culture plates, and analyzed by flow cytometry. The results are shown in Table 9.

[0133] Table 9. Binding eclampsia of each candidate nanobody to cell surface antigen CD176 50

[0134]

[0135] As shown in Table 9, the candidate nanobodies exhibited different binding energies to the CD176 antigen on human breast cancer cells MCF-7. Among them, NB802-Anti-75 showed the strongest binding energies, with an EC50 of [missing value] for binding to the CD176 antigen. 50 18.65 as low as nM.

[0136] This invention utilizes CD176 protein to immunize alpacas, constructing a library with a size of 1.16 × 10⁻⁶. 9 A phage display antibody library of 7 cfu / mL was used for two rounds of immunopanning to identify six nanobodies with superior affinity for CD176 protein. In particular, NB802-Anti-75 can effectively and specifically bind to CD176 protein. Multivalent nanobodies can be constructed based on this to enhance their binding effect with CD176.

Claims

1. A nanobody comprising in sequence a CDR1 region, a CDR2 region and a CDR3 region, characterized in that, The amino acid sequence of the CDR1 region is shown in SEQ ID No. 6, the amino acid sequence of the CDR2 region is shown in SEQ ID No. 7, and the amino acid sequence of the CDR3 region is shown in SEQ ID No.

8.

2. The Nanobody of claim 1, wherein The amino acid sequence is shown in SEQ ID No.

1.

3. A polynucleotide encoding the Nanobody of claim 1 or 2.

4. A nucleic acid construct comprising the polynucleotide of claim 3.

5. An expression vector comprising the nucleic acid construct of claim 4.

6. A transformed cell comprising the expression vector of claim 5.

7. Use of the Nanobody of claim 1 or 2 for the manufacture of a diagnostic kit for CD176-positive cancer.

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