A nanoantibody targeting CD3ε and its application

Abstract

The present invention belongs to the field of immunology technology, and specifically relates to a nanobody targeting CD3ε and its application. The present invention utilizes the high affinity generated by the unique three complementary determining regions (CDR1, CDR2, and CDR3) of antibodies with naturally missing light chains present in alpaca peripheral blood to target and bind to antigens; an expression vector containing the variable region coding sequence of the nanobody, a host cell containing the expression vector, and the application of the nanobody in the preparation of a drug targeting CD3ε are also provided. Therefore, the nanobody provided by the present invention has specific recognition and binding capabilities for CD3ε, and the affinity of the nanobody can reach 5.293E-09. The nanobody can bind to CD3ε on the cell surface and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunology, and particularly relates to a CD3ε-targeting nanoantibody and applications thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Currently, nearly half of the bispecific antibodies used in clinical trials for cancer treatment target the CD3 antigen, making CD3 the second most approved target for development after PD-1. CD3-related bispecific antibodies possess two antigen-binding regions: one that recognizes and binds to CD3, while the other binds to the target antigen on cancer cells, inducing T cells to target cancer cells. Cancer cells stimulate the TCR / CD3 complex to activate downstream signaling pathways, leading to the expression and release of granzymes, which in turn perforate the tumor cell membrane, causing lysis and apoptosis.

[0004] The CD3 molecule is a key biomarker on the T cell membrane. Composed of four protein chains (CD3γ, CD3δ, CD3ε, and CD3ζ), it participates in T cell antigen recognition, signal transduction, and the regulation of T cell development. CD3ε is crucial for T cell activation and development. The CD3ε chain participates in various life processes, such as cerebellar development and synaptic growth. It also serves as a signaling center and a crucial component of numerous signaling pathways, including apoptosis signaling and G protein-coupled receptor signaling. It also regulates gene expression and apoptosis. In terms of immune regulation, the CD3ε chain promotes the proliferation of α-β T cells and increases the production of IFNγ, IL-2, and IL-4. Therefore, it is crucial to develop CD3ε as a key target for tumor identification and immune regulation, and to identify its potential as an emerging anticancer or immunomodulatory drug.

[0005] However, when it comes to monoclonal antibody applications for atopic diseases or tumors, the immunogenicity of these antibodies remains a top concern for researchers. Existing techniques involve recombinant monoclonal antibodies to reduce their molecular size, followed by humanization through methods such as amino acid mutations at key sites or affinity maturation. However, most humanization modifications reduce the antibody's inherent affinity or stability. Therefore, developing an antibody targeting CD3ε that maintains efficacy while minimizing its immunogenicity remains a pressing challenge for researchers in this field. Summary of the Invention

[0006] In response to the needs of the prior art, the present invention aims to provide a nanobody targeting CD3ε and its application, wherein the nanobody has excellent specific antigen binding ability and can reduce its own immunogenicity, and further provides its application in the preparation of drugs targeting CD3ε.

[0007] Specifically, the present invention provides the following technical solutions:

[0008] In a first aspect of the present invention, a nanobody targeting CD3ε is provided, wherein the variable region of the nanobody has a CDR1 region as shown in SEQ ID NO.1, a CDR2 region as shown in SEQ ID NO.2, and a CDR3 region as shown in SEQ ID NO.3.

[0009] Preferably, the Nanobody further comprises four framework regions FR1, FR2, FR3, and FR4, wherein:

[0010] The amino acid sequence of the framework region FR1 of the Nanobody is shown in SEQ ID NO.4;

[0011] The amino acid sequence of the framework region FR2 of the Nanobody is shown in SEQ ID NO.5;

[0012] The amino acid sequence of the framework region FR3 of the Nanobody is shown in SEQ ID NO.6;

[0013] The amino acid sequence of the framework region FR4 of the nanobody is shown in SEQ ID NO.7.

[0014] Preferably, the amino acid sequence of the variable region of the Nanobody is as follows:

[0015] (1) the amino acid sequence shown in SEQ ID NO. 8;

[0016] (2) An amino acid sequence that still exerts similar physiological activity after adding, deleting or replacing one or more amino acids in the amino acid sequence shown in SEQ ID NO.8.

[0017] A preferred embodiment of the Nanobody with this variable region sequence screened in the present invention is Nanobody 2G9, the variable region amino acid sequence of 2G9 is shown in SEQ ID NO.8, wherein the amino acid sequence at positions 1-25 is FR1 (as shown in SEQ ID NO.4), the amino acid sequence at positions 26-33 is CDR1 (as shown in SEQ ID NO.1), the amino acid sequence at positions 34-50 is FR2 (as shown in SEQ ID NO.5), the amino acid sequence at positions 51-58 is CDR2 (as shown in SEQ ID NO.2), the amino acid sequence at positions 59-98 is FR3 (as shown in SEQ ID NO.6), the amino acid sequence at positions 99-105 is CDR3 (as shown in SEQ ID NO.3), and the amino acid sequence at positions 106-117 is FR4 (as shown in SEQ ID NO.7).

[0018] Preferably, the nanobody also includes a derivative polypeptide obtained by modifying the amino acid sequence described in (1) or (2) above, wherein the modification method includes but is not limited to functional group modification or the addition of molecular labels such as polyethylene glycol, streptavidin, biotin, radioactive isotopes, fluorescent agents, etc.

[0019] More preferably, the functional group modification includes modifying the FR region with a hydrophilic group or replacing the hydrophobic residues in the FR region.

[0020] The second aspect of the present invention provides a nucleic acid molecule encoding the Nanobody of the first aspect.

[0021] Preferably, the nucleic acid molecule comprises a nucleic acid encoding the above-mentioned Nanobody that can be translated due to codon degeneracy, and the encoding nucleic acid is not limited to DNA or RNA.

[0022] Further preferably, the coding nucleic acid is DNA, including cDNA, genomic DNA or artificially synthesized DNA; the DNA may be single-stranded or double-stranded, and may be a coding strand or a non-coding strand.

[0023] Further preferably, the nucleic acid of the nanobody is as shown in SEQ ID NO.9.

[0024] The third aspect of the present invention provides an expression vector comprising the nucleic acid molecule described in the second aspect.

[0025] Preferably, the expression vector includes but is not limited to bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vectors.

[0026] Further preferably, the expression vector is a bacterial plasmid or a yeast plasmid.

[0027] The fourth aspect of the present invention provides a host cell comprising the nucleic acid molecule of the second aspect and / or the expression vector of the third aspect.

[0028] Preferably, the host cell is a plant cell or a microbial cell.

[0029] More preferably, the host cell is a microbial cell.

[0030] More preferably, the host cell is Escherichia coli.

[0031] In a fifth aspect, the present invention provides the use of the nanobody described in the first aspect in the preparation of a drug targeting CD3ε.

[0032] Preferably, the application method in the preparation of a drug targeting CD3ε includes but is not limited to any one of the following:

[0033] (1) Used for the preparation of drugs targeting CD3ε;

[0034] (2) Used for the development of biochemical detection reagents.

[0035] In the above aspect (2), the biochemical detection reagent is specifically a CD3ε detection antibody reagent, which is used in a detection method and kit based on antibody-specific recognition.

[0036] The sequences involved in the present invention are as follows:

[0037] Amino acid sequence of the CDR1 region of Nanobody 2G9:

[0038] 5' - GFIFSAYT - 3' (SEQ ID NO. 1).

[0039] Amino acid sequence of the CDR2 region of Nanobody 2G9:

[0040] 5' - ISSTISNS - 3' (SEQ ID NO. 2).

[0041] Amino acid sequence of the CDR3 region of Nanobody 2G9:

[0042] 5' - NVKAGRN - 3' (SEQ ID NO. 3).

[0043] Amino acid sequence of the FR1 region of Nanobody 2G9:

[0044] 5' -QVQLQESGGGLVQPGGSLRVSCAAS - 3' (SEQ ID NO. 4).

[0045] The amino acid sequence of the FR2 region of Nanobody 2G9:

[0046] 5' -MSWYRQAPGKEREQVAS- 3' (SEQ ID NO. 5).

[0047] The amino acid sequence of the FR3 region of Nanobody 2G9:

[0048] 5' -GTNYADSVKGRFTISRDDAKSTLYLEMNSLKPEDTAMYYC- 3' (SEQ ID NO. 6).

[0049] The amino acid sequence of the FR4 region of Nanobody 2G9:

[0050] 5' -YWGQGTQVTVSS- 3' (SEQ ID NO. 7).

[0051] Amino acid sequence of Nanobody 2G9:

[0052] 5'–QVQLQESGGGLVQPGGSLRVSCAASGFIFSAYTMSWYRQAPGKEREQVASISSTISNSGTNYADSVKGRFTISRDDAKSTLYLEMNSLKPEDTAMYYCNVKAGRNYWGQGTQVTVSS – 3’ (SEQ ID NO. 8).

[0053] The nucleic acid sequence of Nanobody 2G9:

[0054] 5'–caggtgcagctgcaggagtctgggggaggcttggtgcagcctggggggtctctgagagtctcctgtgcagcctctggattcatcttcagtgcctataccatgagctggtaccgccaggcgccagggaaggagcgcgagcaggtcgcatctattagtagtactattagtaattcc gggacgaactatgcagactccgtgaagggccgattcaccatctccagagacgatgccaagagtacgttgtatctcgaaatgaacagcctgaaacctgaggacacggccatgtattactgtaatgtaaaagcaggccgtaattactggggccaggggacccaggtcaccgtctcctca -3' (SEQ ID NO.9).

[0055] CALL001 primer sequence:

[0056] 5' - GTCCTGGCTGCTCTTCTACAAGG - 3' (SEQ ID NO. 10).

[0057] CALL002 primer sequence:

[0058] 5' - GGTACGTGCTGTTGAACTGTTCC - 3' (SEQ ID NO.11)

[0059] VHH-Back primer sequence:

[0060] 5' - GATGTGCAGCTGCAGGAGTCTGGRGGAGG - 3' (SEQ ID NO. 12).

[0061] VHH-For primer sequence:

[0062] 5' - CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT - 3' (SEQ ID NO. 13).

[0063] pMES-F primer sequence:

[0064] 5' - GCCGCTGGATTGTTATTACTC - 3' (SEQ ID NO. 14).

[0065] pMES-R primer sequences:

[0066] 5' - CTTTCAACAGTGGAACCGTAG - 3' (SEQ ID NO. 15).

[0067] The beneficial effects achieved by one or more technical solutions of the present invention are as follows:

[0068] (1) The nanobody 2G9 provided by the present invention has specific recognition and binding ability to the CD3ε antigen, and the affinity of the nanobody can reach 5.293E-09, indicating that the nanobody provided by the present invention has highly specific binding activity.

[0069] (2) The CD3ε-targeting nanoantibodies prepared by the present invention can be efficiently expressed in Escherichia coli and can be used in the preparation of CD3ε-targeting drugs and the development of CD3ε molecular biochemical detection reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1 This is the SDS-PAGE image of CD3ε antigen protein;

[0071] Figure 2 This is the electrophoresis identification diagram of the extracted total RNA;

[0072] Figure 3 This is the electrophoresis identification diagram of the first round of PCR amplification of the antibody variable region gene;

[0073] Figure 4 This is the electrophoresis identification diagram of the second round of PCR amplification of the antibody variable region gene;

[0074] Figure 5 This is the electrophoresis identification diagram of the pMES4 vector and VHH double enzyme digestion reaction products;

[0075] Figure 6 This is the electrophoresis identification diagram of transformants identified by colony PCR;

[0076] Figure 7 This is the SDS-PAGE diagram of nanobody purification. DETAILED DESCRIPTION

[0077] The present invention is described in detail below with reference to the accompanying drawings and examples. The examples described below are merely preferred embodiments of the present invention. It should be noted that the following description is merely for the purpose of explaining the present invention and does not limit the present invention in any form. Any simple modifications, equivalent changes, and modifications made to the embodiments based on the technical essence of the present invention fall within the scope of the technical solution of the present invention.

[0078] Example 1 :Nanoantibodies targeting CD3ε

[0079] Expression of S1 and CD3ε recombinant proteins

[0080] The CD3ε sequence was linked to the vector pcDNA3.1(+). Endotoxin-free plasmid was extracted and transfected into 293 cells in logarithmic growth. After culturing the transfected cells for 36 hours, the cell culture medium was poured into a 50 mL centrifuge tube and centrifuged at 12,000 g for 5 minutes. The supernatant was collected, filtered through a 0.22 μm filter, and purified using nickel affinity chromatography. Protein expression was then detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Figure 1 : M is Thermo Fisher protein marker, product number 26616).

[0081] S2. Immunity of Alpacas

[0082] A healthy adult alpaca was selected and the recombinant antigen CD3ε was mixed with Freund's adjuvant in a 1:1 volume ratio. The alpaca was immunized by multiple subcutaneous injections at the back at a dose of 6-7 μg / kg. The immunizations were repeated four times, with a 2-week interval between immunizations. 10 mL of peripheral blood was then collected from the alpaca for the construction of a phage display library.

[0083] S3. Isolation of Camel-derived Lymphocytes

[0084] The alpaca peripheral blood was collected and lymphocytes were separated using the Camel Peripheral Blood Lymphocyte Separation Kit (Tianjin Haoyang Company, Cat. No. LTS1076) according to the instructions. 7 Add 1 mL of RNA isolation reagent to each living cell, take 1 mL for RNA extraction, and store the rest at -80℃.

[0085] S4. Total RNA extraction

[0086] 1 mL of Tipure Isolation Reagent containing lymphocytes was pipetted repeatedly, allowed to stand for 5 minutes, and then 200 μL of chloroform was added. The tube was vortexed for 30 seconds and allowed to stand for another 5 minutes. The tube was then centrifuged at 12000 g for 15 minutes at 4°C. The aqueous phase was transferred to a new EP tube, and an equal amount of isopropanol was added and allowed to stand for 10 minutes. The tube was centrifuged again at 12000 g for 10 minutes at 4°C, and the supernatant was discarded. The tube was washed with 1 mL of pre-cooled 70% ethanol, and then centrifuged again at 7500 g for 5 minutes at 4°C, the supernatant was discarded, and the tube was dried for 5 minutes. Finally, 30 μL of RNase-free water was added to dissolve the precipitate, and the concentration was adjusted to 1 μg / μL for gel electrophoresis detection. The results are shown in FIG. Figure 2 .

[0087] S5. Reverse transcription and cDNA synthesis

[0088] According to the instructions of the reverse transcription kit (Roche's transcripor first stand cDNA synthesis KIT), reverse transcription of cDNA was performed using the RNA obtained in step S3 as a template.

[0089] S6. Antibody variable region gene amplification

[0090] The cDNA obtained by reverse transcription was used as a template for PCR reaction. Two rounds of amplification were performed. The primer sequences for the first round of PCR were as follows:

[0091] CALL001:GTCCTGGCTGCTCTTCTACAAGG

[0092] CALL002:GGTACGTGCTGTTGAACTGTTCC

[0093] PCR reaction conditions and procedures are as follows:

[0094]

[0095] Use an agarose gel recovery kit to recover a band of about 700 bp, and finally adjust the nucleic acid concentration to 5 ng / μL with water ( Figure 3 : M is Trans 2K DNA Marker; 1 is the first-round PCR product).

[0096] The primer sequences for the second round of PCR are as follows:

[0097] VHH-Back: GATGTGCAGCTGCAGGAGTCTGGRGGAGG

[0098] VHH-For:CTAGTGCGGCCGCTGGAGACGGTGACCTGGGT

[0099] PCR reaction conditions and procedures are as follows:

[0100]

[0101] Purify the PCR product using a PCR product recovery kit ( Figure 4 : M is Trans 2K DNA Marker; 1 is the second-round PCR product).

[0102] S7. Vector Construction

[0103] pMES4 (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 second PCR product were added to 15 μL of T4 DNA ligase, supplemented with buffer and water to a total volume of 150 μL. The ligation reaction was incubated at 16°C overnight and the ligated product was recovered. The product was recovered using a PCR product recovery kit and eluted with 20 μL of water. The results of the double-digestion of the pMES4 vector were checked on a 1% agarose gel electrophoresis. Figure 5 shown.

[0104] S8. Electroporation and Storage Capacity Determination

[0105] Take 10 μL of the purified ligation product and add it to a pre-cooled electroporation cuvette containing 50 μL of E. coli TG1 competent cells. Place it in an electroporator (ECM630 electroporator from BTX, USA) for electroporation. Remove the electroporation cuvette, revive and culture the transformants. Randomly select clones and perform colony PCR identification ( Figure 6 M is a 100 bp DNA marker; N is a negative control; the rest are randomly selected monoclonal PCR identification products. The library capacity was calculated based on the PCR positive rate (liquidity capacity = number of clones × dilution factor × PCR positive rate × 10).

[0106] The primer sequences are as follows:

[0107] pMES-F: GCC GCT GGA TTG TTA TTA CTC

[0108] pMES-R: CTT TCA ACA GTG GAA CCG TAG.

[0109] S9 and M13 phage amplification

[0110] Inoculate the revived bacterial suspension into YT-AG medium and incubate at 37°C at 200 rpm until the culture reaches an OD600 of 0.5. Remove 10 mL of the bacterial suspension and add 4 × 1010 VCSM13 cells for static infection at 37°C for 30 minutes. Centrifuge at 4000 rpm for 10 minutes at room temperature and discard the supernatant. Resuspend the cells in 2 × YT-AK medium (containing ampicillin and kanamycin) and incubate overnight at 37°C at 200 rpm. Remove the supernatant from the centrifugation and place it in a 40 mL tube. Add 10 mL of PEG / NaCl (20% / 2.5 M) solution and mix thoroughly. Discard the supernatant after centrifugation. Wash the pellet with 1 mL of ice-cold PBS and centrifuge. Remove 250 μL of the supernatant and mix thoroughly, wash, and resuspend in ice-cold PEG / NaCl.

[0111] Determine phage titer: Culture TG1 to OD600 = 0.4, gradient dilute the phage with LB medium, take the serially diluted phage TG1 culture and mix them for culture. Observe the culture plate for plaque formation the next day, count the plaques on the dilution gradient plates with a number of 30-300 and calculate the phage titer (pfu) according to the following formula.

[0112] Phage titer (pfu / mL) = dilution factor × number of plaques × 100

[0113] S10. Phage display of nanobodies

[0114] 1 mL of the nanobody library was inoculated into two 10 mL tubes of 2×YT-AG medium. The cells were cultured at 37°C, 200 rpm / min, to an OD600 of 0.5. 4 × 1010 pfu of helper phage were added to each tube and statically infected at 37°C for 30 min. The cells were centrifuged at 4000 rpm for 10 min at room temperature. The supernatant was discarded and the suspension was resuspended in 3 mL of 2×YT-AK medium. Finally, the suspension was added to 100 mL of 2YT-AK medium and cultured overnight at 37°C, 200 rpm / min. The displayed phage were concentrated and precipitated the next day, and the titer was determined.

[0115] S11. Solid-phase panning of phage display libraries

[0116] Dilute CD3ε antigen protein to 10 μg / mL in CBS, coat the plate with 100 μL per well, incubate overnight at 4°C, and wash the plate five times with PBST. Add 100 μL of 1% BSA to each well, block at 37°C for 1 hour, and wash the plate five times with PBST. Add 100 μL of displayed phage diluted to 1011 c.fu to each well, incubate at 37°C for 2 hours, and wash the plate 15-25 times with PBST. After the final wash, add 100 μL of glycine solution to each well and incubate on a horizontal shaker for 15 minutes. Add the eluate from each well to an EP tube pre-filled with 15 μL of Tris solution, combine, and titer. Perform panning for a total of 3-4 times.

[0117] S12. Phage ELISA screening of positive clones

[0118] Screen positive clones by ELISA. Coat an ELISA plate with recombinant CD3ε antigen, block with 5% BSA, and wash with PBST. Add 100 μL of phage supernatant to each well and incubate at 37°C for 1 hour. Discard the supernatant and add HRP-conjugated mouse anti-M13 secondary antibody. Incubate at 37°C for 1 hour. Discard the supernatant and add TMB solution. Incubate at room temperature for 5 hours. Add 2M sulfuric acid stop buffer to each well and read at 450 nm using a microplate reader. Select clones with positive phage ELISA results and send for sequencing.

[0119] S13. Inducible expression and purification of nanobodies in Escherichia coli

[0120] Positive clones were selected and the original TG1 glycerol stock containing the nanobody nucleic acid was inoculated into 5 mL of fresh LB-A medium at a 1:1000 ratio. The culture was incubated overnight at 37°C at 200 rpm. The next day, plasmids were extracted using a 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. The cells were gently mixed, placed on ice for 30 minutes, heat-shocked in a 42°C water bath for 90 seconds, and cooled in an ice bath for 3 minutes. 600 μL of LB medium was added to the centrifuge tube and incubated at 37°C with shaking for 60 minutes. 100 μL of the supernatant was spread onto an LB-A plate using a triangular spreader and incubated inverted at 37°C overnight.

[0121] Pick a single colony from the above-mentioned cells and culture them in LB-A medium with shaking at 37°C overnight. The next day, add this culture to 100 mL of fresh LB-A medium at a 1:100 ratio and culture at 37°C with shaking for 3 hours until the OD600 reaches approximately 0.8. Then, add IPTG to a final concentration of 1 mM and induce overnight at 30°C. On the third day, harvest the cells by centrifugation at 8000 rpm for 10 minutes and resuspend the pellet in 1.5 mL of pre-chilled TES buffer. Incubate on ice for 2 minutes, then gently shake for 30 seconds. Repeat this cycle six times. Add 3.0 mL of TES / 4 (TES diluted 4-fold with water), gently shake for 30 seconds, then incubate on ice for 2 minutes. Repeat this shaking and incubation cycle six times. Centrifuge at 9000 rpm for 10 minutes at 4°C and collect approximately 4.5 mL of supernatant (periplasmic extract). The Zhouzhi extract was purified using Ni-NTA resin, eluted and collected using different concentrations of imidazole, and the collected samples were subjected to SDS-PAGE detection ( Figure 7 :M is Thermo Fisher protein marker, product number 26616), and finally the nanobody was replaced with PBS.

[0122] Vector NTI software was used to analyze the sequencing results of antibody light and heavy chain genes to determine the framework regions (FR) and complementary determining regions (CDR) of the variable regions.

[0123] A preferred embodiment of the Nanobody screened by the present invention is named "2G9". Through DNA sequencing, the heavy chain nucleic acid sequence of the Nanobody 2G9 is shown in SEQ ID NO.9, and the variable region amino acid sequence is shown in SEQ ID NO.8, wherein the amino acid sequence at positions 1-25 is FR1, the amino acid sequence at positions 26-33 is CDR1 (as shown in SEQ ID NO.1), the amino acid sequence at positions 34-50 is FR2, the amino acid sequence at positions 51-58 is CDR2 (as shown in SEQ ID NO.2), the amino acid sequence at positions 59-98 is FR3, the amino acid sequence at positions 99-105 is CDR3 (as shown in SEQ ID NO.3), and the amino acid sequence at positions 106-117 is FR4.

[0124] Example 2 :Affinity activity determination of nanoantibodies and antigens

[0125] S1. Chip antigen coupling

[0126] The antigen was prepared in sodium acetate buffer at different pH values ​​(pH = 5.5, 5.0, 4.5, and 4.0) to a working solution of 20 μg / mL. A 50 mM NaOH regeneration solution was also prepared. Electrostatic binding between the antigen and the chip surface (GE) at different pH conditions was analyzed using the template method in the Biacore T100 Protein Interaction Analysis System. The most neutral pH system was selected as the coupling condition, with the antigen concentration adjusted as needed, based on a signal increase of 5-fold relative to the real-time (RL). The chip was coupled using the instrument's built-in template method: blank coupling mode was selected for channel 1, and target coupling mode was selected for channel 2, with the target set to the designed theoretical coupling amount. The coupling process took 60 minutes.

[0127] S2. Exploration of analyte concentration setting conditions and optimization of regeneration conditions

[0128] Use manual injection mode, select 2-1 mode injection for channels 1 and 2, and set the flow rate to 30 μL / min. The injection conditions are all 120 seconds, 30 μL / min. The regeneration conditions are all 30 seconds, 30 μL / min. First, continue to empty the running buffer until all baselines are stable. Prepare a nanoantibody solution with a large concentration span, and configure it with running buffer. It is recommended to set it to 200 μL / min, 150 μL / min, 100 μL / min, 50 μL / min, 20 μL / min, 10 μL / min, and 2 μL / min. Prepare the regeneration solution and select the regeneration solution with four pH gradients of the glutamic acid hydrochloride system: 1.5, 2.0, 2.5, and 3.0. Manually inject 200 μg / mL of analyte sample, observe channel 2, and regenerate from the regeneration buffer with the most neutral pH until the response line of channel 2 after regeneration returns 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. After regeneration with the regeneration solution used to return the response line to baseline in the previous step, manually inject another 200 μg / mL analyte sample, observe the signal change in channel 2-1, and record the binding amount. Compare the binding amount with the previous value. If the deviation is less than 5%, the regeneration solution at this pH is considered optimal. If the binding amount of the second injection is low, continue the experiment with a regeneration buffer at a lower pH. Use the selected optimal regeneration solution as the chip surface regeneration reagent after each injection. Inject samples at the analyte concentrations set above and analyze the binding amount at each concentration to determine the concentration gradient required for affinity testing.

[0129] S3. Affinity test

[0130] Following the optimized sample concentration gradient, the solution was regenerated 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 seconds at 30 μL / min; dissociation time: 600 seconds; regeneration conditions: 30 seconds at 30 μL / min). The signal in channel 2-1 was continuously monitored. The affinity test process took approximately 200 minutes.

[0131] S4. Results Analysis

[0132] The binding and dissociation curves of several appropriate concentration gradients were selected and fitted to all curves using the 1:1 binding mode. The affinity values, binding constants, and dissociation constants were finally obtained as shown in Table 1.

[0133] The data showed that the affinity value of anti-CD3ε nanobody 2G9 was 5.293E-09.

[0134] Table 1

[0135]

[0136] Example 3 :Activity analysis of nanobodies

[0137] S1. Flow cytometry determination of the binding activity of nanobodies to T cells

[0138] To test the ability of CD3ε-specific antibodies to bind to the T cell surface molecule CD3ε, the obtained specific antibodies were subjected to flow cytometry analysis (FACS). Jurkat cells are T lymphocytes that have the CD3ε antigen on their cell surface. Flow cytometry was used to detect the specific binding of the antibody protein 2G9 to the CD3ε site on Jurkat cells.

[0139] This study also selected the nanoantibody Nb that specifically targets β-NGF. NGF (Preliminary laboratory screening) served as a negative control. Flow cytometry (NoveCyte 3130, Aisen Bio (Hangzhou) Co., Ltd.) was used to detect the site-specific binding activity of 2G9 antibody to CD3ε in Jurkat cells.

[0140] Dilute the 2G9 antibody with 0.02 mol / L PBS (pH 7.4, containing 1% BSA) to an initial protein concentration of 300 μg / mL. Take a 96-well U-shaped plate and add 50 μL of 0.02 mol / L PBS (pH 7.4, containing 1% BSA) to wells 1 to 3 in row B to serve as blank control wells.

[0141] Add 50 μL of 0.02 mol / L PBS (pH 7.4, containing 1% BSA) to wells 1–11 in rows C, D, and E. Subsequently, add 75 μL of the antibody solution diluted to 300 μg / mL to wells C12, D12, and E12. Pipette 25 μL of the antibody solution from wells C12, D12, and E12 and dilute it in a 3-fold gradient from right to left to wells C1, D1, and E1 in columns C1, D1, and E1. Mix thoroughly, aspirate 25 μL, and discard, leaving a volume of 50 μL per well. The dilution range is 300 μg / mL to 1.694 ng / mL, with a total of 12 dilutions. Prepare a cell density of 2.0 × 10 6 For a cell suspension of 100 μL of Jurkat cell suspension (100 μL / well) at 1 μg / mL, add 100 μL of the Jurkat cell suspension to each of the sample wells, mix well, incubate at 37°C for 60 min, centrifuge, and carefully aspirate the supernatant. For the blank control well B2, add 50 μL of 0.02 mol / L PBS (pH 7.4, containing 1% BSA) to each well. Add 50 μL of mouse anti-human IgG monoclonal antibody diluted to 2 μg / mL to each well. Mix well, incubate at room temperature for 60 min, and centrifuge to carefully aspirate the supernatant. For the blank control well B1, add 50 μL of 0.02 mol / L PBS (pH 7.4, containing 1% BSA) to each well. Add 50 μL of diluted FITC-conjugated goat anti-mouse IgG (1:100) to the sample wells. Mix well, incubate at 37°C in the dark for 60 min, and centrifuge to carefully aspirate the supernatant. Add 170 μL of 0.02 mol / L PBS (pH 7.4) to each well, resuspend the cells, and mix thoroughly. Set the flow cytometer gate to load 10,000 cells at a high flow rate. Measure the cell fluorescence values ​​and calculate the fluorescence values ​​(subtract the blank control fluorescence value). Calculate the mean fluorescence intensity (MFI) using Flowjox software, and process the data using GraphPad Prism 5.0 software.

[0142] The results are shown in Table 2. Antibody 2G9 binds to Jurkat cells, while the negative control Nb NGF Does not bind to Jurkat cells.

[0143] Table 2

[0144]

[0145] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A nanobody targeting CD3ε, characterized in that The variable region amino acid sequence of the nanobody has a CDR1 region as shown in SEQ ID NO.1, a CDR2 region as shown in SEQ ID NO.2, and a CDR3 region as shown in SEQ ID NO.

3.

2. The Nanobody according to claim 1, characterized in that The nanobody also includes four framework regions FR1, FR2, FR3, and FR4, wherein: The amino acid sequence of the framework region FR1 of the Nanobody is shown in SEQ ID NO.4; The amino acid sequence of the framework region FR2 of the Nanobody is shown in SEQ ID NO.5; The amino acid sequence of the framework region FR3 of the Nanobody is shown in SEQ ID NO.6; The amino acid sequence of the framework region FR4 of the nanobody is shown in SEQ ID NO.

7.

3. The Nanobody according to claim 1, characterized in that The amino acid sequence of the variable region of the Nanobody is as follows: (1) the amino acid sequence shown in SEQ ID NO. 8; Or, (2) an amino acid sequence shown in SEQ ID NO. 8 that has one or more amino acids added, deleted or replaced, and has a similarity of more than 80% and still exhibits similar physiological activity.

4. The Nanobody according to claim 1, characterized in that The amino acid sequence of the variable region of the anti-CD3ε nanobody is shown in SEQ ID NO.8, wherein CDR1 is shown in SEQ ID NO.1, CDR2 is shown in SEQ ID NO.2, and CDR3 is shown in SEQ ID NO.

3.

5. The Nanobody according to claim 3, characterized in that The nanobody also includes a derivative polypeptide obtained by modifying the variable region amino acid sequence in (1) or (2), wherein the modification method includes functional group modification.

6. The Nanobody according to claim 3, characterized in that The nanobody also includes a derivative polypeptide obtained by modifying the variable region amino acid sequence in (1) or (2), wherein the modification method includes adding polyethylene glycol, streptavidin, biotin, radioactive isotope or fluorescent agent.

7. The Nanobody according to claim 5, characterized in that The functional group modification includes modifying the FR region with a hydrophilic group or replacing the hydrophobic residues in the FR region.

8. A nucleic acid molecule encoding a Nanobody according to any one of claims 1 to 4, characterized in that The nucleic acid molecule includes a nucleic acid encoding the above-mentioned nanobody that can be translated due to codon degeneracy, and the nucleic acid molecule includes DNA or RNA; the nucleotide sequence of the nucleic acid molecule of the nanobody is shown in SEQ ID NO.

9.

9. The nucleic acid molecule according to claim 8, characterized in that The nucleic acid molecule is DNA, including genomic DNA or artificially synthesized DNA; the DNA is single-stranded or double-stranded, and the DNA is the coding strand.

10. The nucleic acid molecule according to claim 9, characterized in that The nucleic acid molecule includes cDNA.

11. An expression vector, characterized in that The method comprises the nucleic acid molecule according to any one of claims 8 to 10.

12. The expression vector according to claim 11, characterized in that The expression vector includes bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus or mammalian cell virus.

13. The expression vector according to claim 12, characterized in that The expression vector is a bacterial plasmid or a yeast plasmid.

14. A host cell comprising the nucleic acid molecule according to any one of claims 8 to 10 or the expression vector according to any one of claims 11 to 13; The host cell is a microbial cell.

15. The host cell according to claim 14, characterized in that The host cell is Escherichia coli.

16. Use of the nanobody according to any one of claims 1 to 7 in the preparation of a reagent for detecting CD3ε antibodies.

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