A targeting cd19 nanobody and preparation method and application thereof

Abstract

The present application relates to the field of biological medicine, and particularly relates to a kind of targeting CD19 nanobody and its preparation method and application.The present application provides a kind of nanobody targeting CD19, nucleic acid substance encoding the above-mentioned single domain antibody, corresponding expression vector and the host cell capable of expressing the nanobody targeting CD19.The nanobody provided by the present application has excellent specific CD19 binding capacity, and the nanobody affinity can reach 3.855x10 ‑9 ‌, show that the nanobody provided by the present application has highly specific binding activity, and has important application value in the treatment of B cell malignant tumor.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a CD19-targeting nanobody, its preparation method, and its application. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] In the development of monoclonal antibody drugs targeting indications such as atopic diseases and tumors, immunogenicity has always been one of the core issues of concern for researchers. Reducing the immunogenicity of antibodies not only helps to reduce drug side effects but may also improve efficacy and safety. Common strategies for this purpose include modifying the molecular size of antibodies, mutating key amino acid sites, and humanizing them through techniques such as affinity maturation. However, most humanization processes are often accompanied by a decrease in antibody affinity activity or structural stability. Balancing immunogenicity and functional integrity has become a significant challenge in antibody engineering.

[0004] A naturally occurring antibody lacking the light chain exists in alpaca peripheral blood. This antibody contains only a heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Unlike artificially modified single-chain antibody fragments (scFv), it does not easily adhere to each other or even aggregate. More importantly, the VHH structure cloned and expressed individually possesses structural stability and antigen-binding activity comparable to the original heavy chain antibody, making it the smallest known unit capable of binding to target antigens. VHH crystals are 2.5 nm in size, 4 nm in length, and have a molecular weight of only 15 kDa; these are also known as nanobodies (Nb). Compared to conventional four-chain antibody scFvs, nanobodies have comparable affinity, but surpass scFvs in solubility, stability, resistance to aggregation, refoldability, expression yield, DNA manipulation, library construction, and 3D structure determination. Furthermore, due to their small size and high stability, nanobodies can be administered via nebulization, improving the convenience of drug delivery and broadening the application scenarios.

[0005] Monoclonal antibodies and antibody-based immunotherapies are highly effective options in cancer treatment and have significantly improved the treatment outcomes of hematologic malignancies. For B-cell lymphomas and leukemia, various monoclonal antibodies (such as rituximab and tafacitinib) and other antibody-based therapies (such as bispecific T-cell enhancers (BiTEs), antibody-drug conjugates), and chimeric antigen receptor (CAR) T-cell therapies have been approved for clinical use. An attractive target antigen in B-cell lymphomas is represented by differentiation cluster (CD)19. CD19 is a B-cell-specific member of the immunoglobulin superfamily, expressed from early pre-B cells with heavy chain rearrangements until plasma cell differentiation. CD19 consists of 556 amino acid residues, with its extracellular region containing two highly conserved C2-type immunoglobulin-like domains (IgD1: 24-118 aa, IgD2: 119-224 aa), anchored to the cell membrane via a transmembrane region (225-243 aa), and an intermediate non-Ig-like domain. It contains a hydrophobic transmembrane domain. The cytoplasmic domain contains 240 amino acids, and the intracellular tail region contains 9 tyrosine phosphorylation sites (such as Y340, Y391, Y421, etc.), which constitute the core components of the B cell receptor (BCR) co-receptor signaling complex and play a key role in CD19-mediated signal transduction.

[0006] CD19 is expressed in all B cell lineages in the human body and plays a crucial role in B cell activation, signal transduction, and growth regulation, serving as a functional receptor molecule. Functionally, CD19 forms a tetrameric complex with CD21 (complement receptor 2), CD81 (TAPA-1), and the transmembrane protein CD225. This complex recruits kinases such as LYN and VAV, activating the PI3K-AKT-mTOR and RAS-MAPK signaling pathways, thereby amplifying the BCR signal by 10 to 1000 times, driving B cell proliferation and inhibiting apoptosis. Furthermore, CD19 also participates in maintaining peripheral B cell tolerance and preventing autoimmune responses by regulating SHP-1 phosphorylation levels.

[0007] During malignant transformation, CD19 is persistently highly expressed in over 95% of B-cell malignancies, and its abnormal activation pathway is closely related to tumor cell drug resistance. Therefore, CD19 is not only a marker of B lymphocyte development but also an important biomarker for lymphoma diagnosis and a potential target for leukemia immunotherapy. Summary of the Invention

[0008] In view of this, the present invention provides a CD19-targeting nanobody, its preparation method and application.

[0009] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a VHH chain of a CD19-targeting nanobody, wherein the variable region CDR of the VHH chain includes: a CDR1 region with an amino acid sequence as shown in SEQ ID NO.1, a CDR2 region with an amino acid sequence as shown in SEQ ID NO.2, and a CDR3 region with an amino acid sequence as shown in SEQ ID NO.3.

[0010] Furthermore, any of the above-mentioned amino acid sequences may also include a derived sequence that has optionally been added, deleted, modified, and / or substituted at least one amino acid and is capable of retaining CD19 binding affinity.

[0011] Furthermore, the VHH chain also includes a backbone region FR; the backbone region FR includes: the FR1 region with an amino acid sequence as shown in SEQ ID NO.4, the FR2 region with an amino acid sequence as shown in SEQ ID NO.5, the FR3 region with an amino acid sequence as shown in SEQ ID NO.6, and the FR4 region with an amino acid sequence as shown in SEQ ID NO.7.

[0012] The skeleton region FR is used to separate the variable region CDR.

[0013] Further, the amino acid sequence of the VHH chain is as shown in SEQ ID NO.8; or, the amino acid sequence shown in SEQ ID NO.8, after adding, deleting or replacing one or more amino acids, has a sequence similarity of 80% and still exhibits similar physiological activity.

[0014] In a second aspect, the present invention provides a CD19-targeting nanobody, comprising the CD19-targeting nanobody VHH chain described in the first aspect.

[0015] Furthermore, the CD19-targeting nanobody of the present invention includes monomers, bivalents (bivalent antibodies), and / or multivalents (multivalent antibodies).

[0016] Further, the amino acid sequence of the variable region of the nanobody is shown in SEQ ID NO.8, wherein amino acid sequence 1-25 is FR1 (as shown in SEQ ID NO.4), amino acid sequence 26-33 is CDR1 (as shown in SEQ ID NO.1), amino acid sequence 34-50 is FR2 (as shown in SEQ ID NO.5), amino acid sequence 51-58 is CDR2 (as shown in SEQ ID NO.2), amino acid sequence 59-96 is FR3 (as shown in SEQ ID NO.6), amino acid sequence 97-104 is CDR3 (as shown in SEQ ID NO.3), and amino acid sequence 105-116 is FR4 (as shown in SEQ ID NO.7).

[0017] Furthermore, the nanobody also includes a derivative polypeptide modified from the nanobody 1A10, wherein the modification includes functional group modification or the addition of molecular markers such as polyethylene glycol, streptavidin, biotin, radioisotopes, and fluorescent agents.

[0018] Furthermore, the functional group modification includes modifying the FR region with hydrophilic groups or replacing the hydrophobic residues in the FR region.

[0019] Thirdly, the present invention provides a nucleic acid substance that encodes the VHH chain of the CD19-targeting nanobody described in the first aspect of the present invention or the CD19-targeting nanobody described in the second aspect of the present invention.

[0020] The nucleic acid material described in this invention includes a coding nucleic acid that can be translated into the above-mentioned nanobody due to codon degeneracy. The coding nucleic acid is not limited to DNA or RNA. Preferably, the coding nucleic acid is DNA, including cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded, and can be a coding strand or a non-coding strand.

[0021] Furthermore, the nucleotide sequence of the nucleic acid substance is shown in SEQ ID NO.9.

[0022] Fourthly, the present invention provides an expression vector containing the nucleic acid material described in the third aspect of the present invention.

[0023] Furthermore, the expression vector of the present invention includes DNA, RNA, viral vector, bacteriophage, bacterial plasmid, yeast plasmid, plant cell virus, mammalian cell virus or other vector; specifically, the expression vector is a bacterial plasmid or yeast plasmid.

[0024] Fifthly, the present invention provides a host cell comprising the nucleic acid material described in the third aspect of the present invention and / or the expression vector described in the fourth aspect.

[0025] Furthermore, the host cell is a microbial cell; preferably, Escherichia coli is used as the host cell.

[0026] Sixthly, a method for preparing CD19-targeting nanobodies includes the following steps: (a) Culturing the host cells described in the fifth aspect of the present invention to obtain a culture containing the nanobody targeting CD19; (b) Isolating or recovering the CD19-targeting nanobody from the culture; and (c) Optionally, purify the CD19-targeting nanobody obtained in step (b).

[0027] Furthermore, the CD19-targeting nanobody contains an amino acid sequence as shown in SEQ ID NO.8.

[0028] In a seventh aspect, the present invention provides the use of the CD19-targeting nanobody VHH chain described in the first aspect or the CD19-targeting nanobody described in the second aspect in the preparation of CD19-targeting drugs.

[0029] Furthermore, the CD19-related diseases include cancer, autoimmune diseases, infectious diseases, or combinations thereof. The cancers include solid tumors and B-cell malignancies; the cancers are tumors that highly express CD19. The autoimmune diseases include B-cell-mediated diseases such as systemic lupus erythematosus, rheumatoid arthritis, and multiple sclerosis. The infectious diseases include bacterial and viral infections.

[0030] Eighthly, the present invention provides the application of the CD19-targeting nanobody VHH chain described in the first aspect or the CD19-targeting nanobody described in the second aspect in the development of biochemical detection reagents.

[0031] Furthermore, the biochemical detection reagent is a CD19 detection antibody reagent, used in detection methods and kits based on antibody-specific recognition.

[0032] Compared with the prior art, the present invention has achieved the following beneficial effects: The CD19-targeting nanobody provided by this invention has a specific recognition and binding ability to the CD19 antigen, and the affinity of this nanobody can reach 3.855 × 10⁻⁶. -9 This demonstrates that the nanobody provided by the present invention possesses highly specific binding activity. Furthermore, the CD19-targeting nanobody provided by the present invention has promising applications in the treatment of B-cell malignancies. Attached Figure Description

[0033] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0034] Figure 1 This is an electrophoretic identification image of the total RNA extracted in an embodiment of the present invention; Figure 2 This is an electrophoretic identification image of the variable region gene of the antibody amplified in the first round of PCR in this embodiment of the invention; Figure 3 This is an electrophoretic identification diagram of the variable region gene of the antibody amplified in the second round of PCR in this embodiment of the invention; Figure 4 This is an electrophoresis diagram of colony PCR identification of transformants in an embodiment of the present invention; Figure 5 SDS-PAGE image of purified nanobody; Figure 6 The image shows the results of the binding specificity test of nanobodies to CD19 antigen, where (a) is 1A10, (b) is 2A8, and (c) is 2A2. Detailed Implementation

[0035] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, 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.

[0036] Example 1: Screening of CD19-targeting nanobodies 1.1 Alpaca Immunization One healthy adult alpaca was selected, and recombinant antigen CD19 was mixed with Freund's adjuvant at a 1:1 ratio. The alpaca was immunized by subcutaneous injection at multiple sites on the back at a dose of 6-7 μg / kg, for a total of four immunizations, with an interval of 2 weeks between immunizations. Afterward, 10 mL of peripheral blood was collected from the alpaca for the construction of a phage display library.

[0037] 1.2 Isolation of alpaca lymphocytes The peripheral blood collected from alpacas was used to separate lymphocytes using the camel peripheral blood lymphocyte separation kit (Tianjin Haoyang Company, catalog number LTS1076) according to the instructions. Lymphocytes were separated at a rate of 2.5 × 10⁻⁶ cells per kit. 7 Add 1 mL of RNA separation reagent to each live cell, take 1 mL for RNA extraction, and store the remainder at -80 °C.

[0038] 1.3 RNA Extraction 1 mL of Tipure Isolation Reagent containing lymphocytes was repeatedly pipetted and incubated for 5 min; 200 μL of chloroform was added, vortexed for 30 seconds, and incubated for another 5 min; centrifuged at 12000 g for 15 min at 4 °C, and the aqueous phase was transferred to a new EP tube; an equal volume of isopropanol was added, and incubated for 10 min; centrifuged at 12000 g for 10 min at 4 °C, and the supernatant was discarded; the tube was washed with 1 mL of pre-cooled 70% ethanol, centrifuged at 7500 g for 5 min at 4 °C, the supernatant was discarded, and the tube was dried for 5 min; 30 μL of RNase-free water was added to dissolve the precipitate, and the concentration was adjusted to 1 μg / μL for gel electrophoresis. The results are shown in the figure. Figure 1 .

[0039] 1.4 Reverse transcription to synthesize cDNA Reverse transcription of cDNA was performed using the RNA obtained in step 1.3 as a template, according to the instructions of the reverse transcription kit (Roche's transcripor first stand cDNAsynthesis KIT).

[0040] 1.5 Amplification of antibody variable region genes The cDNA obtained from reverse transcription was used as a template for PCR. Two rounds of amplification were performed. The primer sequences for the first round of PCR are as follows: CALL001: GTCCTGGCTGCTCTTCTACAAGG, as shown in SEQ ID NO.10; CALL002: GGTACGTGCTGTTGAACTGTTCC, as shown in SEQ ID NO.11.

[0041] The PCR reaction conditions and procedure were as follows: 95 ℃ for 5 min; 95 ℃ for 30 seconds, 57 ℃ for 30 seconds, 72 ℃ for 30 seconds, 30 cycles; 72 ℃ for 7 min. Bands of approximately 700 bp were recovered using an agarose gel extraction kit. The nucleic acid concentration was finally adjusted to 5 ng / μL with water. Figure 2 M stands for Trans 2K DNA Marker; 1 represents the first-round PCR product.

[0042] The primer sequences for the second round of PCR are as follows: VHH-Back: GATGTGCAGCTGCAGGAGTCTGGRGGAGG, as shown in SEQ ID NO.12; VHH-For: CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT, as shown in SEQ ID NO.13.

[0043] The PCR reaction conditions and program were as follows: 95 ℃ for 5 min; 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, 72 ℃ for 30 seconds, 15 cycles; 72 ℃ for 7 min. The PCR products were purified using a PCR product recovery kit. Figure 3 M represents the Trans 2K DNA Marker; 1 represents the second-round PCR product.

[0044] 1.6 Carrier Construction The pMES4 vector (purchased from Biovector) and the second PCR product were double-digested with PstI and BstEII, respectively. 1.5 μg of the digested vector and 450 ng of the digested second PCR product were added, along with 15 μL of T4 DNA ligase, and buffer and water were added to a total volume of 150 μL. The mixture was incubated overnight at 16 °C, and the ligation product was recovered. The product was recovered using a PCR product recovery kit, and eluted with 20 μL of water.

[0045] 1.7 Electroconversion and Storage Capacity Measurement Take 10 μL of the purified ligation product and add it to a pre-chilled electroporation cuvette containing 50 μL of *E. coli* TG1 competent cells. Place the cuvette in an electroporator (BTX ECM630 electroporator, USA) for electroporation. Remove the cuvette, revive and culture the transformants. Randomly select clones for colony PCR identification. Figure 4 M represents the DL5000 DNA Marker; N represents the negative control; 1-20 represent randomly selected monoclonal PCR identification products. The colony PCR positivity rate was 100%. The library size was calculated based on the PCR positivity rate (library size = number of clones × dilution factor × PCR positivity rate × 10). Primer sequences are as follows: pMES-F: GCCGCTGGATTGTTATTACTC, as shown in SEQ ID NO.14; pMES-R: CTTTCAACAGTGGAACCGTAG, as shown in SEQ ID NO.15.

[0046] 1.8 Amplification of M13 phage The revived bacterial culture was inoculated into YT-AG medium and cultured at 37 ℃ and 200 rpm until the culture OD reached its maximum. 600 =0.5. Take 10 mL of bacterial culture and add 4×10 10VCSM13 cells were statically infected at 37 ℃ for 30 min. The cells were then centrifuged at 4000 rpm for 10 min at room temperature, and the supernatant was discarded. The cells were resuspended in 2×YT-AK medium (containing ampicillin and kanamycin) and incubated overnight at 37 ℃ and 200 rpm. After centrifugation, 40 mL of the supernatant was collected in a tube, and 10 mL of PEG / NaCl (20% / 2.5M) solution was added and mixed thoroughly. The supernatant was discarded after centrifugation, and the precipitate was washed with 1 mL of ice-cold PBS and centrifuged. 250 μL of pre-chilled PEG / NaCl was collected, thoroughly mixed, washed, and resuspended.

[0047] Determine phage titer: Incubate TG1 cells until OD500. 600 =0.4, use LB medium to serially dilute the phage, take the serially diluted phage TG1 culture and mix them for culture. The next day, observe the formation of phage plaques in the culture plate. Count the phage plaques in the dilution gradient plates with the number of phage plaques between 30 and 300 and calculate the phage titer (pfu) according to the following formula: Phage titer (pfu / mL) = dilution factor × number of phage plaques × 100.

[0048] 1.9 Phage Display of Nanobodies Take 1 mL of bacterial culture from the nanobody immunoglobulin library and inoculate it into two 10 mL 2×YT-AG culture media, respectively, and incubate at 37℃ and 200 rpm until OD500. 600 =0.5, add 4×10 to each tube 10 PFU helper phages were statically inoculated at 37 ℃ for 30 min; centrifuged at 4000 rpm for 10 min at room temperature, the supernatant was discarded, and the phages were resuspended in 3 mL of 2×YT-AK medium. Finally, the phages were added to 100 mL of 2YT-AK medium and incubated overnight at 37 ℃ and 200 rpm. The next day, the displayed phages were concentrated and precipitated, and the titer was determined. Phage titer (cfu / mL) = dilution factor × colony count × 100 × 2.

[0049] 1.10 Solid-phase panning of phage display libraries In this embodiment, all EP tubes were pre-filled with PBS buffer containing 1% BSA and allowed to stand at room temperature for 10 minutes to seal the tube walls, thereby reducing the impact of the tube walls on the adsorption of trace proteins and the screening results.

[0050] Dilute CD19 recombinant antigen to 10 μg / mL with CBS, coat each well with 100 μL of the solution, incubate overnight at 4°C, and wash 5 times with PBST. Add 250 μL of 1% BSA to each well, block at 37°C for 1 h, and wash 5 times with PBST. Add 100 μL of the solution diluted to 10 μg / mL to each well. 11CFU display phages were incubated at 37 °C for 2 h, followed by 15–25 washes with PBST. After the final wash, 100 μL of glycine solution was added to each well, and the plates were incubated on a horizontal shaker for 15 min. The eluent from each well was then added to an EP tube containing 15 μL of Tris solution, and the titers were measured after pooling. This process was repeated 3–4 times.

[0051] 1.11. Phage ELISA screening for positive clones Positive clones were screened using an ELISA method. ELISA plates were coated with CD19 recombinant antigen, blocked with 5% BSA, and washed with PBST. 100 μL of phage supernatant was added to each well, and the plates were incubated at 37 °C for 1 h. The supernatant was discarded, and HRP-labeled mouse anti-M13 secondary antibody was added, followed by incubation at 37 °C for 1 h. The supernatant was then discarded, and TMB solution was added. After incubation at room temperature for 15 min, 2 M sulfuric acid stop solution was added to each well, and the readings were taken at 450 nm using a microplate reader. Clones with positive phage ELISA results were selected for sequencing.

[0052] Example 2 Expression and purification of CD19-targeting nanobodies The original TG1 nanobody strain was amplified, and the recombinant nanobody plasmid was transformed into *E. coli* BL21(DE3). Positive clones were selected, and the original TG1 glycerol strain containing nanobody nucleic acid was inoculated at a ratio of 1:1000 into 5 mL of fresh LB-A medium and cultured overnight at 37 ℃ and 200 rpm. The next day, plasmids were extracted using the Plasmid mini kit (OMEGA) according to the manufacturer's instructions. After verification, 1 μL of the plasmid was transformed into 100 μL of competent cells, gently mixed, placed on ice for 30 min, heat-shocked at 42 ℃ for 45 seconds, and cooled on ice for 3 min. 600 μL of LB medium was added to a centrifuge tube, and the mixture was incubated at 37 ℃ with shaking for 60 min. 100 μL of the supernatant was collected and spread onto LB-A plates using a triangular spreader, and incubated inverted at 37 ℃ overnight.

[0053] 2.2 Induced Expression of Nanobodies Pick a positive monoclonal colony obtained in step 2.1 and incubate it overnight at 37°C with shaking. The next day, add 100 mL of fresh LB-A medium to the bacterial culture at a ratio of 1:100, and incubate at 37°C with shaking for 2 h until the bacterial culture reaches OD. 600=Approximately 0.8, add IPTG to a final concentration of 1 mM, and induce overnight at 30 ℃. On the third day, centrifuge at 8000 rpm for 10 min to collect the bacterial cells, and resuspend the precipitate in 1.5 mL of pre-chilled TES buffer. After incubating on ice for 2 min, gently vortex for 30 s, repeating this cycle 6 times. Add 3 mL of TES / 4 (TES diluted 4 times with water), gently vortex for 30 s, and incubate on ice for 2 min, repeating the vortexing and incubation steps 6 times. Centrifuge at 9000 rpm at 4 ℃ for 10 min, and collect approximately 4.5 mL of supernatant (periplasmic extract).

[0054] 2.3 Purification and Identification of Nanobodies After resuspending the IMAC Sepharose (GE), add 2 mL to a gravity column and let it stand for 30 min to allow the sepharose to settle naturally to the bottom of the column. Then, elute with the preservation buffer. Add 2 column volumes of 0.1 M nickel sulfate solution and elute at a flow rate of approximately 8 s / drop. Add 10 column volumes of equilibration buffer to equilibrate and wash the sepharose, maintaining a constant flow rate. Dilute the sample 2-fold with equilibration buffer and add it to the gravity column, adjusting the flow rate to 6 s / drop, and collect the breakthrough. Add 10 column volumes of wash buffer to wash the sepharose, maintaining a constant flow rate, and collect the wash. Add 3 column volumes of elution buffer, maintaining a flow rate of 6 s / drop, and collect the eluent containing the target protein. Finally, add 10 column volumes of equilibration buffer, 10 column volumes of pure water, and 10 column volumes of 20% ethanol sequentially to wash the sepharose, and finally retain 4 mL of 20% ethanol to preserve the column. The collected samples were analyzed by SDS-PAGE. Figure 5 M stands for Thermo Fisher protein marker, catalog number 26616; lanes 1-3 contain purified nanobodies 2A8, 1A10, and 2A2. Results are as follows: Figure 5 The nanobody was well expressed.

[0055] The nanobody screened in this embodiment was named "1A10". The DNA sequencing results are as follows.

[0056] The nucleic acid sequence of nanobody 1A10 is as follows: 5’-CAGGTGCAGCTGCAGGAGTCTGGAGGAGGATTGGTGCAGGCTGGGGGCTCTCTGAGACTCTCCTGTGCAGCCTCTGGACGCACCTTCAGTAACTATGTCATGGCCTGGTTCCGTCAGGCTCCAGGGAAGGAGCGTGAGTTTGTATCAGCTATTAACTGGAGTCGTGGTAGTACATACTATACAGAGTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACACGATATATCTGCAAATGAACAACCTGAAACCTGAGGACACGGCCGTTTATTACTGTGCAGCAGATCTGGTACATAATCGAGGGGAGTATATGTACTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA-3’, as shown in SEQ ID NO.9.

[0057] The amino acid sequence of nanobody 1A10 is: 5’-QVQLQESGGGLVQAGGSLRLSCAASGRTFSNYVMAWFRQAPGKEREFVSAINWSRGSTYYTESVKGRFTISRDNAKNTIYLQMNNLKPEDTAVYYCAADLVHNRGEYMYWGQGTQVTVSS-3’, as shown in SEQ ID NO.8.

[0058] In the amino acid sequence of the nanobody 1A10, amino acid positions 1-25 are FR1, with the amino acid sequence: 5'-QVQLQESGGGLVQAGGSLRLSCAAS-3', as shown in SEQ ID NO.4; amino acid positions 26-33 are CDR1, with the amino acid sequence: 5'-GRTFSNYV-3', as shown in SEQ ID NO.1; amino acid positions 34-50 are FR2, with the amino acid sequence: 5'-MAWFRQAPGKEREFVSA-3', as shown in SEQ ID NO.5; amino acid positions 51-58 are CDR2, with the amino acid sequence: 5'-INWSRGST-3', as shown in SEQ ID NO.2; amino acid positions 59-96 are FR3, with the amino acid sequence: 5'-YYTESVKGRFTISRDNAKNTIYLQMNNLKPEDTAVYYC-3', as shown in SEQ ID NO. As shown in NO.6; the amino acid sequence from position 97 to 104 is CDR3, with the amino acid sequence: 5'-AADLVHNR-3', as shown in SEQ ID NO.3; the amino acid sequence from position 105 to 116 is FR4, with the amino acid sequence: 5'-GEYMYWGQGTQVTVSS-3', as shown in SEQ ID NO.7.

[0059] Example 3: Determination of the affinity between nanobodies and antigens 3.1. Chip antigen conjugation CD19 was prepared into a 50 μg / mL working solution using sodium acetate buffers at different pH values ​​(pH=5.5, pH=5.0, pH=4.5, pH=4.0). A 50 mM NaOH regeneration solution was also prepared. The template method in the Biacore T100 protein interaction analysis system was used to analyze the electrostatic binding between the antigen and the chip (GE) surface under different pH conditions. A 5-fold increase in signal strength (RL) was used as the standard to select a suitable, near-neutral pH system, and the antigen concentration was adjusted as needed for coupling. The chip was coupled according to the instrument's built-in template method: channel 1 was selected in blank coupling mode, and channel 2 was selected in target coupling mode, with the target set to the designed theoretical coupling amount. The coupling process took approximately 60 minutes.

[0060] 3.2 Exploration of Analyte Concentration Setting Conditions and Optimization of Regeneration Conditions Manual injection was used, selecting channel 1 and channel 2 in 2-1 mode, with a flow rate of 30 μL / min. Injection conditions were 120 s and 30 μL / min for both. Regeneration conditions were 30 s and 30 μL / min for both. First, the run buffer was continuously run empty until all baselines were stable. Nanobody solutions with a wide concentration range were prepared using the run buffer, with recommended concentrations of 200 μg / mL, 150 μg / mL, 100 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, and 2 μg / mL. Regeneration solutions were prepared using four pH gradients of the glutamate hydrochloride system: 1.5, 2.0, 2.5, and 3.0. A 200 μg / mL analyte sample was manually injected, and channel 2 was observed. Regeneration was performed using the most neutral pH regeneration buffer until the response line in channel 2 returned to the same height as the baseline. Manually inject another 200 μg / mL analyte sample, observe the signal change in channel 2-1 and record the binding amount. Regenerate using the regeneration solution used in the previous step to bring the response line back to baseline. Manually inject another 200 μg / mL analyte sample, observe the signal change in channel 2-1 and record the binding amount. Compare this to the previous binding amount. If the deviation is less than 5%, the regeneration solution at this pH is considered the optimal regeneration solution. If the binding amount after the second injection is lower, continue the experiment using a regeneration buffer with a lower pH. Use the selected optimal regeneration solution as the chip surface regeneration reagent after each injection. Inject samples of the analyte concentrations set above and analyze the binding amount at each concentration to ultimately determine the concentration gradient required for affinity testing.

[0061] 3.3 Affinity Test Following the optimized sample concentration gradient, the regeneration solution was prepared, and the affinity between the nanobody and the antigen was tested using the instrument's built-in template method (with injection conditions set to 60 s, 30 μL / min; dissociation time: 600 s; regeneration conditions: 30 s, 30 μL / min). The signal in channel 2-1 was monitored continuously. The affinity test process took approximately 200 minutes.

[0062] 3.4 Results Analysis Binding and dissociation curves at several suitable concentration gradients were selected and fitted using a 1:1 binding mode to obtain the affinity values, binding constants, dissociation constants, and other important parameters. The results are shown in Table 1. The results show that the 1A10 nanobody can specifically bind to the CD19 protein conjugated on the chip, with an affinity of 3.855 × 10⁻⁶. -9 The affinity data are shown in Table 1.

[0063] Table 1. Values ​​of binding constant, dissociation constant, and affinity.

[0064] Example 4: Binding Ability Analysis of Nanobodies 4.1 Specificity Analysis Individual Nbs were tested to measure their specificity and potential cross-reactivity with structure-related compounds such as casein, porcine submandibular mucin (PSM), skim milk, BSA, HER2, VHH, and Endoline. Several types of antigens were coated in microtiter plates (1 µg / mL) and detected with different concentrations of anti-CD19 Nb.

[0065] The coated microplates were incubated overnight at 4 °C to ensure the antigen was firmly adsorbed onto the well surface. The next day, the microplates were blocked with a PBS solution of 1% BSA and 3% skim milk powder to prevent non-specific binding. After incubation at 37 °C for 2 h, different concentrations of nanobodies (e.g., 2 µg / mL) were added to each well, and the plates were incubated at 37 °C for 1 h to allow the nanobodies to bind to the coated antigen. The microplates were washed three times with 0.05% PBST (PBS containing 0.05% Tween-20) to remove unbound nanobodies. Then, 100 µL of HRP-labeled anti-His-tagged antibody (diluted 1 / 10000) was added, and the plates were incubated at 37 °C for 2 h. The plates were then washed with PBST and PBS. TMB substrate was added, and after incubation for a period of time, the reaction was terminated by adding 1M H2SO4. The absorbance was measured at 450 nm to analyze the specific binding of the nanobodies to the CD19 antigen. The results are as follows: Figure 6 As shown, this demonstrates the specific binding ability of nanobodies.

[0066] Example 5. In vitro experiments of nanobodies 5.1 Cytotoxicity test The in vitro effects of the prepared anti-CD19 nanobody were investigated using Raji cells, and the effects of proteins unrelated to the CD19 target on cell viability were compared with those of proteins used as negative controls to verify the specific effect of the anti-CD19 nanobody on CD19-overexpressing cells in vitro.

[0067] (1) Cell preparation and counting Raji cells were passaged, and cell status was observed and counted. Cells were diluted with complete culture medium to a concentration of 1 × 10⁶ cells / 100 μL. 4 Each cell.

[0068] (2) Inoculation and pre-incubation Add 100 μL of cell suspension to each well of a 96-well plate. Incubate the cells in a 37 °C incubator containing 5% CO2 for 24 h.

[0069] (3) Gradual dilution of drugs CD19-targeting candidate drugs were serially diluted using complete culture medium.

[0070] (4) Incubation with added medicine The serially diluted drug from step (3) was added to Raji cells after pre-incubation, and incubation continued for 24 h. Five replicates were set up for each drug concentration.

[0071] (5) Add CCK-8 reagent Add CCK-8 solution to the 96-well plate to a final volume concentration of 10%. After adding the reagent, gently tap the 96-well plate to ensure thorough mixing.

[0072] (6) Color development incubation and endpoint determination Continue incubation for 1-4 hours, observing the color change during incubation. When the color inside the well turns orange-yellow, the reaction is considered essentially complete.

[0073] (7) Absorbance detection The absorbance at 450 nm was measured using an ELISA reader.

[0074] (8) Data fitting and IC50 calculation The dose-response curve was fitted using a four-parameter logistic model (4PL), and the half-inhibitory concentration (IC50) was calculated. 50 .

[0075] The results showed that Nb-CD19-1A10 had a significant killing effect on CD19-overexpressing cells, with an IC50 value of [missing information]. 50 The concentration was 1211 ng / mL.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A VHH chain of a CD19-targeting nanoantibody, characterized in that, The variable region CDR of the VHH chain includes: the CDR1 region with an amino acid sequence as shown in SEQ ID NO.1, the CDR2 region with an amino acid sequence as shown in SEQ ID NO.2, and the CDR3 region with an amino acid sequence as shown in SEQ ID NO.

3.

2. The VHH chain as described in claim 1, characterized in that, The VHH chain further includes a backbone region FR; the backbone region FR includes: the FR1 region with an amino acid sequence as shown in SEQ ID NO.4, the FR2 region with an amino acid sequence as shown in SEQ ID NO.5, the FR3 region with an amino acid sequence as shown in SEQ ID NO.6, and the FR4 region with an amino acid sequence as shown in SEQ ID NO.

7.

3. The VHH chain as described in claim 1, characterized in that, The amino acid sequence of the VHH chain is shown in SEQ ID NO.8; or, the amino acid sequence shown in SEQ ID NO.8, after adding, deleting or replacing one or more amino acids, has a sequence similarity of 80% and still exhibits similar physiological activity.

4. A nanobody targeting CD19, characterized in that, Includes the VHH chain of the CD19-targeting nanobody as described in any one of claims 1-3.

5. A nucleic acid substance, characterized in that, The nucleic acid material encodes the VHH chain of the CD19-targeting nanobody according to any one of claims 1-3 or the CD19-targeting nanobody according to claim 4.

6. An expression carrier, characterized in that, The expression vector contains the nucleic acid material as described in claim 5.

7. A host cell, characterized in that, The host cell comprises the nucleic acid material of claim 4 and / or the expression vector of claim 5.

8. A method for preparing CD19-targeting nanobodies, characterized in that, Includes the following steps: (a) Culturing the host cells of claim 7 to obtain a culture containing the CD19-targeting nanobody; (b) Isolating or recovering the CD19-targeting nanobody from the culture; and (c) Optionally, purify the CD19-targeting nanobody obtained in step (b).

9. The use of the CD19-targeting nanobody VHH chain according to any one of claims 1-3 or the CD19-targeting nanobody according to claim 4 in the preparation of a CD19-targeting drug.

10. The use of the CD19-targeting nanobody VHH chain according to any one of claims 1-3 or the CD19-targeting nanobody according to claim 4 in the development of biochemical detection reagents.

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