Anti-cd22 single domain antibodies and uses thereof

Abstract

The application relates to an anti-CD22 single-domain antibody and application thereof, and belongs to the technical field of biological medicine. The application carries out immunization on a llama by using CD22 protein as an antigen, extracts RNA in lymphocytes by blood drawing, obtains cDNA by reverse transcription, constructs a phage library, carries out screening, picks ELISA positive clones for sequencing analysis, obtains an anti-CD22 nanobody sequence, constructs an expression vector by using the screened sequence, carries out expression and purification through a Pichia pastoris system, and obtains active anti-CD22 nanobody protein. The antibody has higher stability, is easier to produce in large scale, has lower antigenicity, has stronger permeability, has more recognition sites, can form a multimer, and is suitable for industrialized production and application.

Description

Technical Field

[0001] This invention relates to an anti-CD22 single-domain antibody and its application, belonging to the field of biomedical technology. Background Technology

[0002] In 1989, Raymond Hamers and his colleagues discovered a naturally occurring antibody lacking the light chain in camels, known as a heavy chain antibody, and published their findings in *Nature* in 1993. This is the nanobody currently being studied. Nanobodies, also called single-domain antibodies, are the antigen-binding portion of the Fab segment on traditional monoclonal antibodies. They typically consist of about 130 amino acids, are 15 kDa in size, and have a rugby ball-shaped crystal structure with a diameter of about 2.5 nm and a length of 4.2 nm, much smaller than traditional immunoglobulin antibodies (about 150 kDa). Compared to traditional immunoglobulin antibodies, nanobodies offer significant advantages such as structural stability, high solubility, ease of expression and structural modification in microorganisms, short production cycles, and better economic viability. They are considered promising molecules for biomedical development and represent the next generation of protein therapy technology.

[0003] Because single-domain antibodies were developed relatively late, there are currently few single-domain antibodies used for disease treatment, and most are still in the clinical research stage. Only two single-domain antibodies have completed phase III clinical trials. Caplacizumab (ALX-0081), approved in 2019, is used for the treatment of acquired thrombotic thrombocytopenic purpura (dTTP), while envafolimab (KN035), approved in 2021, is used for the treatment of breast cancer.

[0004] CD22 (Siglec-2) is ubiquitous in both normal B cells and B-cell malignancies. Belonging to the sialic acid-binding immunoglobulin lectin family (Siglecs), it is a type I transmembrane protein that specifically binds to sialic acid-containing glycans via an N-terminal immunoglobulin (Ig) domain. It inhibits B-cell receptor (BCR) signaling through its immunoreceptor tyrosine inhibitory motif (ITIM), thus playing a role in maintaining humoral immune homeostasis.

[0005] CD22 is primarily expressed on mature B cells and is a cell surface adhesion molecule that regulates B cell activation. It helps control the sensitivity of B cells to antigen responses, and its main function is to prevent overactivation of the immune system, thus reducing the risk of autoimmune diseases. CD22 is mainly expressed intracellularly in the early stages of B cell development, including on pro-B and pre-B cells. As B cells mature, this expression shifts to the transmembrane cell surface, but at low levels. It is highly expressed in IgM+ and IgD+ mature B cells, as well as in follicular B cells, mantle cells, and marginal zone B cells, but its expression is eventually downregulated in CD27+ memory B cells, especially plasma cells. CD22 expression is highest in mature B cells and is also present on the surface of a small number of immature B cells. It is expressed in most B-cell malignancies, including acute lymphoblastic leukemia (B-ALL), non-Hodgkin's lymphoma (NHL), and hairy cell leukemia (HCL). CD22 is particularly expressed on leukemia cells in >90% of ALL patients. CD22 inhibits the response of B cells to self-antigens by cross-linking with the BCR, preventing the activation of autoreactive B cells and thus suppressing the occurrence of autoimmune diseases. Impaired expression or function of CD22 plays a role in the pathogenesis of autoimmune diseases, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA).

[0006] Given that CD22 is one of the inhibitory co-receptors on the surface of B cells in tumor cells, it is closely related to the development, differentiation, and function of B cells. CD22 is expressed in a restricted manner on the surface of mature B cells and most B lymphoma cells. Therefore, targeting CD22 to block related signaling pathways and thus restricting tumor growth for tumor immunotherapy has become a hot topic in immunological research. Currently, a variety of traditional antibodies targeting CD22 have been developed. However, due to their long amino acid sequences and complex protein structures, traditional antibodies require more sophisticated and complex instruments and can only be produced in eukaryotic systems. Furthermore, existing traditional antibodies have low expression levels. Therefore, there is an urgent need to develop a single-domain anti-CD22 antibody that is highly stable, can be mass-produced, has lower antigenicity, stronger permeability, more recognition sites, and can form multimers. Summary of the Invention

[0007] The purpose of this invention is to provide an anti-CD22 nanobody that is more stable, can be mass-produced, has lower antigenicity, stronger permeability, more recognition sites, and can form multimers compared to traditional antibodies, as well as its preparation and application.

[0008] This invention isolates and extracts the DNA sequence of anti-CD22 nanobody from the blood of immunized alpacas, and further translates it into a protein sequence. Specifically, this invention recombinantly expresses and purifies CD22 protein as an antigen, immunizes alpacas sequentially, extracts RNA from lymphocytes in blood samples, reverse transcribes it to obtain cDNA to construct a phage library, screens, selects ELISA-positive clones for sequencing analysis to obtain the anti-CD22 nanobody sequence, and then constructs an expression vector from the selected sequence and expresses and purifies it using a Pichia pastoris system to obtain an active anti-CD22 nanobody protein.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] The present invention first provides an anti-CD22 single-domain antibody, the antibody comprising the amino acid sequence shown in SEQ ID NO:1, 2, 3, 4 or 5, or an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the amino acid sequence shown in SEQ ID NO:1, 2, 3, 4 or 5.

[0011] The present invention also provides a nucleic acid that encodes the anti-CD22 single-domain antibody.

[0012] The present invention also provides an expression vector comprising the nucleic acid.

[0013] The present invention also provides a recombinant cell or engineered bacterium, wherein the recombinant cell or engineered bacterium comprises the nucleic acid, or the expression vector, or is capable of expressing the single-domain antibody.

[0014] The present invention also provides applications of the anti-CD22 single-domain antibody, the applications of which include the preparation of detection reagents targeting CD22 protein, the reagents including in vitro detection reagents and in vivo non-invasive diagnostic reagents.

[0015] The present invention provides the application of the anti-CD22 single-domain antibody, the application of which includes the preparation of drugs for treating B-cell tumors or other drugs that target CD22.

[0016] The present invention also provides a pharmaceutical composition comprising the anti-CD22 single-domain antibody described herein, and may further comprise a pharmaceutically acceptable carrier.

[0017] The present invention also provides a reagent for detecting CD22 protein, the reagent comprising the aforementioned anti-CD22 single-domain antibody.

[0018] The present invention also provides a kit for detecting CD22, the kit comprising the anti-CD22 single-domain antibody described in the present invention.

[0019] The present invention also provides the use of the anti-CD22 single-domain antibody or the pharmaceutical composition thereof in the preparation of diagnostic, preventive and / or therapeutic drugs for cancer.

[0020] The beneficial effects of this invention are:

[0021] This invention uses the Pichia pastoris system to express and purify anti-CD22 single-domain antibodies, overcoming the problem of low expression levels in traditional antibodies. This allows for large-scale industrial production and can be used for the treatment of related cancers.

[0022] Single-domain antibodies, also known as nanobodies, are exceptionally stable both biophysically and chemically, possessing strong solubility, heat resistance, and proteolytic activity. They can survive longer at high temperatures and high pH levels, maintaining sufficient binding capacity even after a week at 37°C—a feat unmatched by traditional antibodies. Nanobodies are smaller, resulting in greater tissue penetration, reaching sites inaccessible to traditional antibodies. Their small size and unique CDR3 structure allow them to interact with epitopes inaccessible to traditional antibodies, binding to targets that traditional antibodies struggle to reach. Traditional antibodies, due to their long amino acid sequences and complex protein structures, require sophisticated and complex instruments and are only achievable in eukaryotic systems. However, nanobodies can be expressed in large quantities in microbial systems, followed by rapid screening using antibody libraries, enabling antibody expression at a lower cost. The weak adhesion between nanobody monomers makes it easier to achieve tandem expression of nanobodies through genetic engineering, forming multimers and significantly increasing their half-life.

[0023] This invention isolates and extracts the DNA sequence of an anti-CD22 single-domain antibody from the blood of immunized alpacas, and further translates it to obtain the protein sequence. The selected sequence is then used to construct an expression vector, which is expressed and purified using a Pichia pastoris system to obtain an active anti-CD22 single-domain antibody protein. The anti-CD22 single-domain antibody of this invention exhibits excellent antigen-binding ability, can target the expressed CD22 protein, and further target tumor cells, enabling its application in the treatment of related diseases. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of the structure of a single-domain antibody.

[0025] Figure 2 The results are obtained by SDS-PAGE electrophoresis of CD22 protein. Lane 1 in the figure shows the CD22-Fc reducing result, and lane 2 shows the CD22-Fc un-reducing result.

[0026] Figure 3 The results are from agarose gel electrophoresis of single-domain antibody amplification products.

[0027] Figure 4 This is the result of measuring the positive rate of a single-domain antibody phage library.

[0028] Figure 5 The result is the result of ELISA identification of the phage library obtained in Example 4.

[0029] Figure 6 The results of SDS-PAGE detection and Western blot verification of the anti-CD22 single-domain antibody obtained in this invention are as follows.

[0030] Table 3 shows the SPR detection statistics of the anti-CD22 single-domain antibody expressed in this invention. Detailed Implementation

[0031] To enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention will be described in detail below. However, the following embodiments do not limit the scope of protection of the present invention.

[0032] In the embodiments of the present invention, unless otherwise described, conventional experimental methods are used. The processes involved in the embodiments, unless otherwise described, are those that can be understood and easily implemented by those skilled in the art based on product instructions or basic knowledge in the field. The reagents or instruments used, unless the manufacturer is specified, are all conventional products that can be obtained commercially, and therefore will not be described in detail.

[0033] Example 1: CD22 protein expression and purification

[0034] The optimized amino acid sequence of the 20-330V-type domain corresponding to the extracellular domain of CD22 was synthesized, as shown in SEQ ID NO:6, and loaded onto pET-32a to obtain a recombinant plasmid. The recombinant plasmid was transformed into BL21 competent cells, and a single colony was added to 5 mL LB and incubated overnight at 37°C and 200 rpm.

[0035] Activate the bacterial culture obtained from overnight culture: Add 100 μL of pET-32a empty vector bacterial culture and positive bacterial culture with recombinant plasmid to 5 mL of LB liquid medium containing 100 μg / mL AMP, and incubate at 37℃ and 200 rpm for 12-14 h.

[0036] Since the concentration of IPTG affects protein expression levels and the activity of the expression product, it is necessary to determine the optimal IPTG concentration:

[0037] Take 10 μL of activated bacterial culture, add 1 mL of liquid LB (containing 100 μg / mL AMP), and incubate at 250 rpm and 37 °C in a shaker until the OD value reaches 0.6. Then, add 0.5 mM, 1 mM, and 1.5 mM IPTG respectively, and incubate at 18 °C and 130 rpm for 20 h. Take the induced samples for SDS-PAGE electrophoresis detection.

[0038] After testing, the optimal IPTG concentration was determined to be 0.5 mM.

[0039] Bacteria containing the target band were cultured overnight at 37°C in a shaker (no more than 16 h). The bacterial culture was then transferred to 50 mL centrifuge tubes and centrifuged twice at 4400 rpm for 30 min at 4°C to enrich the bacterial culture. The precipitate was weighed, and an appropriate amount of non-denaturing lysis buffer (4 mL lysis buffer / g precipitate) was added. After resuspending the precipitate, lysozyme (CAS NO. 9001-63-2) was added to a final concentration of 1 mg / mL, and lysis was performed for 30 min. Note that the entire process must be performed on ice to prevent protein degradation. Afterwards, lysis was performed by sonication on ice at 150 W, 5 s intervals, 9 s intervals, for a total of 30 min. 200 μL of the lysis buffer was collected and stored for later use. The remaining lysate was centrifuged at 4400 rpm for 30 min at 4°C, and the supernatant and precipitate were collected separately. The sonicated lysis buffer, centrifuged supernatant, and precipitate were treated with 1×PBS and then analyzed by SDS-PAGE electrophoresis. Figure 2 If the supernatant contains the target band, it indicates that the recombinant protein is soluble, and the protein can be purified further. If the precipitate contains a band, it is in the form of inclusion bodies and needs further processing to dissolve the inclusion bodies before purification.

[0040] Prepare the necessary purification solutions, including binding buffer (20mM Na₂HPO₄, 0.5M NaCl, 40mM imidazole, pH 7.4) and elution buffer (20mM Na₂HPO₄, 0.5M NaCl, 500mM imidazole, pH 7.4). After sonication to degas all solutions, filter them through a 0.2μm filter membrane for later use. Use a HisTrap™ FF Crude 1mL column (GE, Cat. No. 17-0004-58) on an Akta protein purifier at a flow rate of 1mL / min. Wash the column with binding buffer for 4 column volumes, then with elution buffer for 4 column volumes, and finally clean the column with binding buffer. Centrifuge the purified target protein at 4000rpm and 4℃ for 10min, then collect 5μL of the supernatant. Dilute 5-fold with 1×PBS and use a BCA protein quantification kit to plot a standard curve and calculate the protein concentration of the sample.

[0041] The optimized amino acid sequence corresponding to the CD22 extracellular domain is SEQ ID NO:6:

[0042] MDSKWVFEHPETLYAWEGACVWIPCTYRALDGDLESFILFHNPEYNKNTSKFDGTRLYE

[0043] STKDGKVPSEQKRVQFLGDKNKNCTLSIHPVHLNDSGQLGLRMESKTEKWMERIHLNVSE

[0044] RPFPPHIQLPPEIQESQEVTLTCLLNFSCYGYPIQLQWLLEGVPMRQAAVTSTSLTIKSVFTRS

[0045] ELKFSPQWSHHGKIVTCQLQDADGKFLSNDTVQLNVKHTPKLEIKVTPSDAIVREGDSVTM

[0046] TCEVSSNPEYTTVSWLKDGTSLKKQNTFTLNLREVTKDQSGKYCCQVSNDVGPGRSEEV

[0047] FLQVQYAHHHHHH

[0048] Example 2: Alpaca Immunization

[0049] Before immunization, the purified CD22 protein and Freund's adjuvant were emulsified 1:1 to form a homogeneous mixture and stored at 4°C. After recording the ear tags of blank alpacas, the immunization experiment began. Each time, injections were made near the lymph nodes in the alpaca's neck, on both sides, with two injection points on each side. Approximately 0.4 mL of the emulsified antigen (i.e., CD22 protein) was injected at each point. After immunization, the alpacas were observed for half an hour to confirm they were in good condition and showed no adverse symptoms. Immunization was performed every two weeks for a total of four immunizations.

[0050] Blood was collected for immune evaluation before each antigen immunization, with 5 mL of blood collected each time. The blood was centrifuged at 400×g for 30 minutes on the same day using a pre-cooled 25°C centrifuge, and the supernatant serum was separated and preserved for subsequent antibody titer testing. 5-7 days after the fourth immunization, 50 mL of blood was collected from the alpaca's jugular vein. 15 mL of cell separation medium was added to a 50 mL centrifuge tube, followed by the slow addition of 15 mL of blood. The blood was added carefully and slowly to prevent mixing with the cell separation medium. The centrifuge was then pre-cooled to 25°C, centrifuged at 400×g for 30 minutes, and the blood separation was observed. The supernatant serum was stored in a new centrifuge tube at -80°C. The cotton-like supernatant immune cells were carefully aspirated using a pipette and transferred to a new 50 mL centrifuge tube. 10 mL of room-temperature PBS buffer was added to each tube, and the tube was centrifuged at 400×g for 20 minutes at 25°C. Remove the supernatant, add 5 mL of room temperature PBS buffer to each tube, mix gently, count the cells using a hemocytometer, and then centrifuge at 25°C, 400 × g for 20 minutes. Remove the supernatant, and lyse the isolated lymphocytes using RNAiso Plus according to the cell count. 7 / mL cell lysate, stored in Trizol at -80℃.

[0051] Example 3: Construction of phage libraries

[0052] (1) RNA extraction: Peripheral blood lymphocytes preserved with Trizol obtained in Example 2 were thawed on ice and transferred to a 1.5 mL centrifuge tube. 1 / 5 volume of chloroform was added and the mixture was shaken and mixed. After standing at room temperature for 5 minutes, the mixture was centrifuged at 12000g for 15 minutes at 4°C. The supernatant after centrifugation was transferred to a new centrifuge tube. An equal volume of isopropanol was added to the new centrifuge tube. After standing at room temperature for 10 minutes, the mixture was centrifuged at 12000g for 10 minutes at 4°C. The precipitate was washed with 75% ethanol and centrifuged at 7500g for 5 minutes at 4°C. The supernatant was discarded and the precipitate was dried at room temperature and then dissolved in an appropriate amount of RNase-free water.

[0053] (2) Reverse transcription of cDNA: According to the instructions of the reverse transcription kit, the RNA obtained in the previous step was divided into two and reverse transcribed into cDNA. The reverse transcription primers used were Oligo T and random primers, respectively.

[0054] The extracted RNA was reverse transcribed into cDNA using a two-step reverse transcription method. First, RNA, Oligo(dT), and random primers were used to synthesize first-strand cDNA under the action of reverse transcriptase. The reaction mixture system is shown in Table 1.

[0055] Table 1. First-strand cDNA reaction system

[0056] RNA template 6μL Oligo dT primer (50μM) 0.5μL Random 6 mers (100μM) 0.5μL <![CDATA[Sterile and enzyme-free ddH2O]]> 5μL Total volume 12μL

[0057] Table 2. RNA Reverse Transcription Components

[0058] 5X Reaction Buffer 4μL RiboLock RNase Inhibitor(20U / μL) 1μL 10mM dNTP Mix 2μL Revert Aid M-MuLV RT(200U / μL) 1μL Total volume 8μL

[0059] Add each reaction component from Table 1 to a sterile, enzyme-free 200 μL EP tube and place it in a PCR instrument. Run the reaction at 65°C for 5 min. To prevent the newly synthesized single-stranded cDNA from forming a double-stranded secondary structure at high temperature, immediately cool the tube on ice after 5 min. After cooling, add each component from Table 2 to the 12 μL system obtained in the above steps, mix thoroughly, and centrifuge briefly. Place the tube in a PCR instrument and run the reaction at the following conditions: 25°C for 5 min, 42°C for 60 min, 70°C for 5 min, and store at 12°C. Stop the reaction and store the obtained cDNA at -20°C for later use.

[0060] (3) Amplification of antibody fragments: Amplify specific antibody fragments from reverse-transcribed cDNA using PCR amplification with Taq DNA Polymerase Hot Start enzyme.

[0061] The PCR reaction system consisted of: 2 μL cDNA template, 2 μL Alpa001F primer, 2 μL Alpa001R primer, 5 μL 10×Taq Buffer, 4 μL dNTP, 0.25 μL Taq(HS), and ddH2O to a final volume of 50 μL.

[0062] The PCR reaction conditions were: 98℃ for 3 minutes; 95℃ for 30 seconds, 57℃ for 30 seconds, 72℃ for 40 seconds, with an increase of 2 seconds for each cycle, repeated for 22 cycles; 72℃ for 5 minutes.

[0063] The obtained PCR amplification products were subjected to 1% agarose gel electrophoresis, and one PCR band slightly less than 1000 bp and another slightly less than 750 bp were observed. Figure 3 The approximately 750 bp band was excised from the gel and recovered using a DNA purification and recovery kit according to the manufacturer's instructions. The recovered DNA fragment was then used as a template to amplify the specific antibody fragment again using Taq DNA Polymerase Hot Start Version enzyme. The resulting PCR product was then recovered using a DNA purification and recovery kit according to the manufacturer's instructions.

[0064] The primer sequences involved are as follows:

[0065] Alpa001F primer (SEQ ID NO:7): 5'-gtcctggctgctcttctacaagg-3'

[0066] Alpa001R primer (SEQ ID NO:8): 5'-ggtacgtgctgttgaactgttcc-3'

[0067] (4) Cloning to phage plasmids: The diverse nanobody gene sequences and phage vector pFUW80 obtained in step (3) were digested with enzymes, purified, and ligated. The ligation products were recovered using a DNA purification and recovery kit according to the instructions and dissolved in ultrapure water.

[0068] (5) Transformation of TG1: Pre-cool the electroporation cuvette on ice. After thawing 100 μL of TG1 competent cells, add 100 ng of the recovered ligation product obtained in step (4). Transfer the mixed competent cells and ligation product to the pre-cooled electroporation cuvette and electroporate using the Bacteria transformation program preset on the electroporator. Immediately after electroporation, add 1 mL of SOC medium to the electroporation cuvette. Perform at least 20 electroporations. After thawing the cells at 37°C for 60 minutes, spread them on LB culture plates containing ampicillin for overnight growth. After overnight growth, wash and scrape the cells from the culture plate with 2×YT medium and a spreader, add 20% glycerol, and store at -80°C.

[0069] (6) Amplification and purification of the phage library: After mixing the bacterial cells scraped off in the previous step, the number of phage libraries should be approximately 10. 9 The bacteria were transferred to 100 mL of 2×YT medium pre-added with ampicillin and cultured at 37°C and 220 rpm until the OD600nm reached 0.5. Helper phage was added at a ratio of 20:1 (M13K07:bacterial cells), and the culture was continued at 37°C for 30 minutes. Kanamycin was added to a final concentration of 50 μg / mL, and the culture was incubated overnight at 30°C on a shaker. The overnight cultured bacteria were centrifuged at 13000 rpm for 5 minutes at 4°C. The supernatant was transferred to a new centrifuge tube, and 1 / 4 volume of pre-chilled 5×PEG8000 / NaCl was added. The tube was incubated on ice for 30 minutes. After centrifugation at 13000 rpm for 10 minutes at 4°C, the supernatant was removed, and 1 mL of PBS buffer was added to dissolve the precipitate. 250 μL of 5×PEG8000 / NaCl was added again, and the tube was incubated on ice for 10 minutes. After centrifugation at 16000 g for 15 minutes at 4°C, the supernatant was removed, and the precipitate was dissolved in 1 mL of PBS to obtain the phage library.

[0070] (7) Document quality assessment:

[0071] a) Document size (>5×10) 8The size of the library will be verified by counting individual transformants. Prepare LB plates containing 100 μg / mL ampicillin for plating. A library obtained using 300 ng of the enzyme ligation product is serially diluted 10-fold. Count the resulting colonies to estimate the size of the previous and final libraries.

[0072] b) Insertion rate (>90%): The insertion rate of the library was assessed by PCR amplification of 100 randomly selected colonies.

[0073] c) In-frame percentage (>80%): The in-frame percentage was assessed by sequencing 100 randomly selected positive clones.

[0074] The results of the positive rate determination of the constructed phage library are shown in Figure 4. As can be seen from the figure, the positive rate of the phage library constructed in this invention reaches 90.9%.

[0075] Example 4: Screening and Identification of Nanobodies

[0076] (1) Coating the immunotube: Add 50 μg of CD22 protein to 2 mL of PBS and then to the immunotube. Incubate overnight at 4°C.

[0077] (2) Blocking: Add an appropriate amount of the amplified and purified phage obtained in step (6) of Example 3 to 1 mL of 3% BSA and incubate at room temperature for 2 h by rotation to block. At the same time, add 2-3 mL of 3% BSA to the coated immunotube and incubate at room temperature by rotation for 2 h by rotation to block.

[0078] (3) Antigen and phage incubation: Wash the blocked immunotherapy tubes three times with PBS containing 0.01% Tween, 5 minutes each time. Add the blocked phage library to the blocked immunotherapy tubes, add PBS until 2-3 mL is reached, and incubate at room temperature by rotation for 1 hour.

[0079] (4) Washing: Wash the immunotubes after incubation with antigen and phage 20 times with PBS containing 0.1% Tween, 5 minutes each time.

[0080] (5) Elution: Add 1 mL of 100 mM Trimethymime to the immunotherapy tube, incubate at room temperature for 10 minutes, add 1 M Tris-HCl to neutralize the Trimethymime, and finally transfer 1.5 mL of the eluted phage to a new centrifuge tube.

[0081] The eluted phages were amplified and purified according to the steps (6) in Example 3. After amplification, the screening process of steps (1) to (5) in this example was repeated twice, and the amount of antigen coated on the immune tube was halved each time to obtain eluted phages after 3 screenings.

[0082] The bacteriophages eluted after three screenings can be subjected to NGS sequencing to obtain a library of candidate nanobody DNA sequences that bind to the antigen.

[0083] ELISA identification ( Figure 5 Simultaneously, the phages obtained in the previous step were serially diluted 10-fold, for a total of 10 dilutions. 100 μL of each dilution was added to TG1 bacterial culture with an OD600nm of 0.5. After incubation at 37°C for 30 minutes, the culture was spread onto 2×YT culture plates containing ampicillin and incubated overnight at 37°C to obtain single colonies the next day. At least 192 single colonies were randomly selected and transferred to 96-well cell culture plates containing 2×YT culture medium containing ampicillin. After overnight incubation at 37°C, these were used as seed plates. 2 μL of the bacterial culture was added to a new 96-well plate (each well containing 200 μL of fresh 2×YT culture medium, 100 μg / mL ampicillin). After 5 hours, helper phages (M13K07 helper phage per well to achieve a bacterial count: phage count = 1:20) were added to each well. After standing at 37°C for 30 minutes, kanamycin was added to a final concentration of 50 μg / mL, and the plate was incubated overnight at 30°C. The next day, the overnight culture was centrifuged to obtain the supernatant containing bacteriophages. Wells containing 100 μL of 5 μg / mL CD22 protein and blank control wells without CD22 protein antigen were blocked with 3% BSA and incubated overnight at 4°C. The obtained bacteriophage supernatant was then added, and the plates were incubated at room temperature for 1 hour. After washing three times with PBS containing 0.1% Tween, 100 μL of M13 Bacteriophage Antibody (HRP) and MouseMab diluted 0.1 ng / L in blocking buffer (3% BSA) was added to each well, and the plates were incubated at room temperature for 1 hour. The liquid in the ELISA plate was discarded, and each well was washed three times with 200 μL of PBST buffer for 10 minutes each time. 100 μL of TMB single-component chromogenic solution was added to each well, and the plates were incubated in the dark for 2-3 minutes. 100 μL of 1M HCl was added to each well to stop the reaction. The results were recorded and stored. Figure 5 This example presents the results of ELISA identification of phage libraries at different concentrations, specifically the statistical results of OD450 binding of different concentrations of positive antibodies to CD22 protein. The results show that after three rounds of screening, the OD450 ratio of antibody binding to CD22 protein was greater than 2.

[0084] Colonies with a high absorbance ratio between CD22 antigen-coated wells and blank control wells were selected for sequencing (two independent phage-ELISA analyses) to obtain the gene sequence of the single-domain antibody.

[0085] Five single-domain antibodies were finally selected, and their amino acid sequences are as follows:

[0086] SEQ ID NO:1:127a.a.

[0087] EVQLVESGGGLVQPGGSLRLSCAANGFRFSVYDMSWVRQAPGKGLEWVSAVDAGGGTTYYADSVKGRFTISHVNATNTLFLQMNNLQPEDTAVYYCAAAEIGPFLPRILSPAEYTYWGRGAQVTVSS

[0088] SEQ ID NO: 2: 129 amino acids

[0089] QVKLEESGGGSVQAGGSLKLSCAVSGFTYSTYCMAWIRQIPGKEREVGATIYAGDGGDGSTNYADSVKGRFALSRDNSKNTLDLQMNFLKPDDTAMYYCAAEPAGWAGYCHGGYRYNYWGQGTQVTVSS

[0090] SEQ ID NO: 3: 125 amino acids

[0091] DVQLVESGGGSVQAGGSLTLSCVVSGYIDRTYRNYCMAWVRQGPGKEREGVARIYIGGKSTYYIDSVKGRFTISLDNAERTLYLRMNSLKPEDSATYYCAADVNCQGWARNPDYWGQGTQVTVSS

[0092] SEQ ID NO: 4: 119 amino acids

[0093] QVQLVESGGDSVQSGASLLLSCISSNNEPSCMSWFRQAPGKEREAVARISVDGTIRYADSVKGRFTISRDTATSTLYLQMNNLEPEDTATYYCAADSRRQCVSWDYLYMGHGTQVTVSS

[0094] SEQ ID NO: 5: 127 amino acids

[0095] AVQLVESGGGSVEAGGSLRLSCAVSGWTYCNDMRWYRQAPGKEREFVSDIDSDGRTSYADSVKGRFTISQDNAKNMVYLQMNGLKPEDTAMYHCNMAIGRWSEREGRCEMIDEYGYWGQGTQVTVSS

[0096] Example 5: Expression and Purification of Single-domain Antibodies

[0097] The amino acid sequence of the single-domain antibody obtained in Example 4 was optimized according to the codon preference of Pichia pastoris, synthesized by GenScript, and loaded onto plasmid pGAPZαA. Sequencing results confirmed successful loading. DH5α competent cells were taken from a -80℃ freezer, aliquoted into 50 μL portions, and stabilized on ice. 2 μL of recombinant plasmid pGAPZαA was added, and the mixture was gently rotated in a centrifuge tube and incubated on ice for 30 min. The suspension was then placed in a dry-cell thermostat at 42℃ for 90 s, followed by an ice bath for 3 min. 500 μL of antibiotic-free LB medium was added, and the mixture was incubated on a shaker at 37℃ and 170 rpm for 1 h. 20 μL of the bacterial culture was plated onto LB agar plates containing 100 mg / mL bleomycin (Zeocin). The plates were incubated overnight at 37℃. Single clones were picked for plasmid purification and sequencing.

[0098] Plasmid linearization and yeast competent cell preparation: A large quantity of recombinant plasmid was extracted and linearized by SacI digestion (the 200 μL linearization reaction system consisted of: 145 μL ddH2O, 20 μL 10×Buffer, 30 μL recombinant plasmid (approximately 15 μg), and 5 μL SacI). After thorough mixing, the mixture was digested at 37°C for approximately 5 hours. Then, 2 μL of the digestion product was subjected to agarose gel electrophoresis to check for complete linearization. The linearized plasmid was then extracted to achieve the purity required for electroporation, following the specific steps below:

[0099] a) Add sterile deionized water to the EP tubes undergoing the linearization reaction to a total volume of 500 μL.

[0100] b) Add an equal volume of phenol, a 1:1 mixture of phenol and chloroform, and extract once with chloroform. After each extraction, mix thoroughly and centrifuge at 12,000 rpm for 5 min.

[0101] c) Transfer the supernatant to a new centrifuge tube, add 1 / 10 volume of 3M pH 5.2 NaAC and 2.5 volumes of pre-cooled anhydrous ethanol, mix thoroughly and let stand for 1 hour, centrifuge at 12000 rpm for 5 minutes, wash twice with pre-cooled 70% ethanol, dry and dissolve in 10 μL of sterile double-distilled water, and store at -20℃ for later use.

[0102] Autoclave 200 mL of YPD medium. Resuscitate one tube of Pichia pastoris X33 empty culture from a -80°C freezer. On a clean bench, add 40 μL of the revival solution to the medium and incubate overnight at 28.5°C and 225 rpm. Prepare the treatment solution (10 mM Tris-HCl, used to dissolve 100 mM LiAc and 0.6 M Sorbitol, pH 7.5). When the OD600 value reaches approximately 1.2, place six centrifuge tubes on ice. Add 9 mL of the bacterial solution to each tube and centrifuge at 4°C and 5000 rpm for 5 min, discarding the supernatant. Add 5 mL of ultrapure water to each tube, mix well, and centrifuge at 4°C and 5000 rpm for 5 min, discarding the supernatant. Add 5 mL of the treatment solution to each of the two remaining tubes (combining three precipitates into one tube, resulting in two tubes in total). Add 50 μL of DTT to each tube, mix well, and incubate at room temperature for 20 min on an ice box. Centrifuge at 6000 rpm for 5 min at 4℃, discard the supernatant, add 5 mL of sorbitol, mix well, and centrifuge at 6000 rpm for 5 min at 4℃, then discard the supernatant. Add 500 μL of sorbitol to each tube and mix well to obtain Pichia pastoris X33 competent cells.

[0103] Plasmid and bacterial electroporation: Add 80 μL of X33 competent cells to each of two new electroporation cuvettes, followed by 10 μL of SacI single-enzyme-cutting plasmid. Incubate on ice for 5 min, then perform rapid electroporation using an electroporator with the following parameters: 1.5 kV, 25 μF, 250 ohms. After electroporation, transfer to a sterile work surface and gently add 1 mL of sorbitol to each cuvette. Incubate at 28.5 °C for at least 1 h. After incubation, mix thoroughly by pipetting and spread onto 200 μL of each YPDS plate containing 100 μg / mL Zeocin (until no significant liquid flow is observed on the surface). Incubate at 28.5 °C for approximately 48 h until single-clone strains emerge. Transfer each single clone to 10 mL of YPD medium and incubate overnight at 28.5 °C and 225 rpm. After approximately 24 h of culture, perform SDS-PAGE to confirm the correct single clone. Select and validate high-expression single clones, pick them into 10 mL of YPD medium, and incubate overnight at 28.5°C and 225 rpm. Measure the OD value the next day.

[0104] Expanding the culture: When the OD value of the cultured bacterial suspension reaches 6, 500 μL is transferred to a 1L bottle containing 200 mL of YPD medium and incubated overnight at 225 rpm at 28.5°C. After approximately 48 hours of incubation, the cultured suspension can be stored at 4°C until purification. The remaining bacterial suspension is centrifuged at 8000 rpm for 5 minutes, and the supernatant is stored at -20°C. The Ni column purification procedure is the same as in Example 1 above. The purified nanobody samples are subjected to SDS-PAGE and Western blot analysis to verify whether it is the desired target protein. Figure 6 ), Figure 6 The antibody of the present invention was obtained.

[0105] Example 6: Validation of single-domain antibodies

[0106] The SPR experiment used the Biacore T200 instrument. For detailed operating procedures, please refer to the "Biacore T200 Protein and Protein Binding Detection Operation Guide." The specific implementation steps are as follows:

[0107] (1) The target coupling ligand amount is calculated according to the following formula:

[0108] R L =R max / S m *(Ligand MW / Analyte MW)

[0109] Among them, R max To determine the maximum response value of the analyte on the surface, 100 RU was selected in the kinetic analysis of this implementation step; S m For stoichiometry, 1 is selected in this implementation step; Ligand MW is the molecular weight of the conjugated ligand, and Analyte MW is the molecular weight of the analyte. In this experiment, the conjugated ligand is CD22, with a molecular weight of 36 kDa; the analyte is a nanobody with a molecular weight of 13 kDa. Substituting these values ​​into the above formula, the theoretical target conjugation amount R of the ligand can be obtained. L The actual target coupling quantity is set to 1.5 times R, with a set value of 277 RU. L That is, 415RU.

[0110] (2) For the pre-enrichment of ligands, the nanobody was diluted to 10 μg / mL with sodium acetate at pH 5.0, 4.5, and 4.0 (at least a 10-fold dilution ratio), and 100 μL of each was prepared. Through the pre-enrichment experiment, pH 5.0 was determined to be the optimal coupling condition. Therefore, 10 μg / mL of sodium acetate at pH 5.0 was used for the formal coupling operation in 200 μL.

[0111] (3) Ligand Coupling: Open the BiacoreT200 Control Software, set the Chiptype to CM5, select amine coupling, and enter the calculated 415RU as the target level in the aim for immobilized level. Set the coupling temperature to 25℃. Place 100μL of EDC in R1D3, 100μL of NHS in R1D4, an empty tube in R1D5, 140μL of ethanolamine in R1D6, and 166μL of 10μg / mL ligand protein in R1D1. Cover the test tube rack, return the sample rack to the sample chamber, and begin the coupling experiment.

[0112] (4) Sample testing process:

[0113] a. In the Kinetics / Affinity interface, set the Flowpath to 2-1 or 4-3, and select CM5 for Chiptype. In the Setup interface, enter HBS-EP in the Solution field under Startup, and change Numberofcycles to 3. In the Kinetics / Affinity-injectionParameter interface: in the Sample field, set Contacttime to 120s, Flowrate to 30μL / min, Dissociationtime to 120s, regeneration conditions to Glycine 2.0, and regeneration time to 30s.

[0114] b. In the Kinetics / Affinity-Sample interface, fill in the analyte information: Sampleid is the sample name, MW(Da) is the molecular weight, the first Concentration is the molar concentration and should be set to μM, the second is the mass concentration. After the molar concentration is filled in, the mass concentration will be automatically calculated (please select one concentration for each sample for repeated injection, and fill in the sample concentrations from low to high).

[0115] c. In the Kinetics / Affinity-System Preparations interface, click Next to enter the RackPosition interface. Change ReagentRack to SampleandReagentRack1, and select AutomaticPositioning from the Menu. Change all entries in the Pooling section to Yes. Adjust the VialSize as needed; select medium for 1.5mL EP tubes. Prepare and place the sample according to its location. Serially dilute the B protein using the running buffer HBS-EP. After clicking Next, save the method and the data path; the instrument will then begin automatic operation.

[0116] The affinity assay for the antibody against the CD22 protein was performed on all clones detected by the above ELISA. The results are shown in Table 3 below. As can be seen from the results in Table 3, the antibody obtained by this invention has high affinity and can specifically recognize and bind to the CD22 protein.

[0117] Table 3. Statistical results of antibody SPR detection in this invention

[0118] 1 1.46E+05 4.72E-05 3.23E-10 2 1.36E+05 7.39E-04 5.43E-09 3 4.86E+05 1.45E-04 2.98E-10 4 9.82E+05 7.52E-04 7.66E-10 5 2.84E+05 5.52E-04 1.94E-09

Claims

1. An anti-CD22 single-domain antibody, characterized in that, The amino acid sequence of the antibody is the amino acid sequence shown in SEQ ID NO:1, 2, 3, 4 or 5.

2. A nucleic acid, characterized in that, The nucleic acid encodes the anti-CD22 single-domain antibody of claim 1.

3. An expression carrier, characterized in that, The expression vector comprises the nucleic acid of claim 2.

4. A recombinant cell, characterized in that, The recombinant cells comprise the nucleic acid of claim 2 or the expression vector of claim 3.

5. The application of the anti-CD22 single-domain antibody according to claim 1, characterized in that, The application is for preparing detection reagents targeting CD22 protein.

6. The application according to claim 5, characterized in that, The testing reagents include in vitro testing reagents and in vivo non-invasive diagnostic reagents.

7. A reagent for detecting CD22 protein, characterized in that, The reagent includes the anti-CD22 single-domain antibody as described in claim 1.

8. A kit for detecting CD22, characterized in that, The kit includes the anti-CD22 single-domain antibody as described in claim 1.

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