Nanobodies against cd5 proteins and uses thereof

Abstract

This invention discloses an anti-CD5 protein nanobody and its applications, relating to the field of nanobody biosynthesis technology. Utilizing a camel-derived nanobody library and targeting mouse CD5 protein, this invention employs phage display technology for in vitro enrichment and panning. Camel-derived nanobody sequences targeting CD5 protein are obtained through monoclonal high-throughput ELISA screening. These five nanobody sequences can be used to detect CD5 protein expression, for the diagnosis and screening of related tumors, and also for the preparation of drugs to treat related tumors.

Description

Technical Field

[0001] This invention relates to the field of nanobody biosynthesis technology, and particularly to nanobodies against CD5 protein. Background Technology

[0002] CD5 protein is a type I transmembrane glycoprotein secreted by T cells and some mature B cells. It serves as a receptor for cells such as T cells, B1a cells, and B-cell lymphocytic leukemia (B-CLL). This receptor acts as both a positive and negative regulator of T cell receptor (TCR) signaling and a negative regulator of B cell receptor (BCR) signaling, regulating cellular activity and promoting cell survival and death. In B lymphocytes, CD5 is a negative regulator of BCR and regulates intracellular Ca2+ by activating signaling pathways such as ERK1 / 2 and PI3K. It also regulates the biological activity of B cells. CD5 can also regulate the expression of genes such as IL-10 and IgM in B lymphocytes and promote tumor cell proliferation by activating pathways such as NFAT2 and STAT3 in B lymphocytes. Some studies have also reported that CD5 can enhance the expression of the p53 gene, preventing uncontrolled autoimmune reactions in T and B lymphocytes, thus affecting prognosis.

[0003] Clinically, CD5 protein is an important marker for the diagnosis of T-cell lymphoma, B-cell lymphoma subtyping, and thymoma. Therefore, developing specific monoclonal antibodies targeting CD5 protein has significant clinical diagnostic value.

[0004] Nanobodies (Nb), also known as variable domain of heavy chain antibody (VHH), were first discovered in camels by Belgian experts Hamers et al. in 1993. They are the smallest naturally occurring fragments capable of binding to antigens. Nanobodies are characterized by their small size, large antigen-binding ring, good stability, and good hydrophilicity. They can be used to construct various types of molecular structures, making them highly practical for medical diagnosis and treatment. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a nanobody targeting CD5 protein. The applicant utilizes a phage vector to clone the antibody's coding gene fragment into an appropriate position in the phage capsid protein structural gene, enabling the exogenous antibody to fuse with a specific phage capsid protein and express, displaying it on the phage surface. Then, using a phage display technology platform, a camel-derived nanobody (VHH) library was constructed. Using mouse CD5 protein as the target protein, this library was enriched and panned in vitro, and screened using a high-throughput monoclonal ELISA, ultimately obtaining a specific camel-derived nanobody (VHH) sequence targeting mouse CD5 protein. We expressed and detected recombinant nanobodies based on this sequence, ultimately obtaining purified camel-derived monoclonal nanobodies that specifically recognize mouse CD5 protein.

[0006] This invention utilizes the camel-derived nanobody (VHH) library (details omitted here) already constructed by our company, targeting mouse CD5 protein, and employs phage display technology for in vitro enrichment and panning. Specific camel-derived nanobody (VHH) sequences targeting mouse CD5 protein are obtained through monoclonal high-throughput ELISA screening.

[0007] Based on this sequence, we constructed the VHH recombinant antibody protein, ultimately obtaining a stable camel-derived nanomonoclonal antibody that specifically recognizes mouse CD5 protein. The full names of these antibodies are Mouse-CD5-R4P1-H1, Mouse-CD5-R4P1-C2, Mouse-CD5-R4P1-F3, Mouse-CD5-R4P1-G3, and Mouse-CD5-R4P1-H4, abbreviated as nanobodies H1, C2, F3, G3, or H4.

[0008] A nanobody against CD5 protein, comprising nanobody H1, C2, F3, G3 or H4; wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2, CDR3 of the heavy chain of nanobody H1 are as shown in SEQ ID NO. 1-3, or have at least 95% sequence identity with the sequences shown in SEQ ID NO. 1-3;

[0009] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of the nanobody C2 are as shown in SEQ ID NO. 4-6, or have at least 95% sequence identity with the sequences shown in SEQ ID NO. 4-6.

[0010] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of the nanobody F3 are as shown in SEQ ID NO. 7-9, or have at least 95% sequence identity with the sequences shown in SEQ ID NO. 7-9.

[0011] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of the nanobody G3 are as shown in SEQ ID NO. 10-12, or have at least 98% sequence identity with the sequences shown in SEQ ID NO. 10-12;

[0012] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of the nanobody H4 are as shown in SEQ ID NO. 13-15, or have at least 98% sequence identity with the sequences shown in SEQ ID NO. 13-15.

[0013] Furthermore, the amino acid sequence of the heavy chain of the nanobody H1 is shown in SEQ ID NO.16;

[0014] The amino acid sequence of the heavy chain of the nanobody C2 is shown in SEQ ID NO.17;

[0015] The amino acid sequence of the heavy chain of the nanobody F3 is shown in SEQ ID NO.18;

[0016] The amino acid sequence of the heavy chain of the nanobody G3 is shown in SEQ ID NO.19;

[0017] The amino acid sequence of the heavy chain of the nanobody H4 is shown in SEQ ID NO.20.

[0018] In some embodiments of the nanobodies disclosed in this invention, the heavy chain of the nanobodies (H1, C2, F3, G3, or H4) comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with the corresponding sequences (SEQ ID NO. 17-20) described above.

[0019] Furthermore, the amino acid sequences of the constant regions CH2 and CH3 of the nanobody H1 are shown in SEQ ID NO.21-22;

[0020] The amino acid sequences of the constant regions CH2 and CH3 of the nanobody C2 are shown in SEQ ID NO.23-24;

[0021] The amino acid sequences of the constant regions CH2 and CH3 of the nanobody F3 are shown in SEQ ID NO.25-26;

[0022] The amino acid sequences of the constant regions CH2 and CH3 of the nanobody G3 are shown in SEQ ID NO.27-28;

[0023] The amino acid sequences of the constant regions CH2 and CH3 of the nanobody H4 are shown in SEQ ID NO.29-30.

[0024] In the context of this invention, the terms "nanobody," "VHH," etc., are used indiscriminately and refer to the variable domains of a single heavy chain of antibodies of those types found in camelids, which inherently lack a light chain. In the absence of a light chain, each nanobody has three CDRs, denoted as CDR1, CDR2, and CDR3.

[0025] The nanobodies of the present invention can be, in particular, camel, dromedary camel, Bactrian camel, llama or alpaca nanobodies.

[0026] In some embodiments of the present invention, the nanobody is a camel-derived nanobody.

[0027] The present invention also provides a substance comprising any one of the following aspects:

[0028] (1) A nucleic acid or a carrier containing said nucleic acid, said nucleic acid encoding any of the above-mentioned nanoantibodies provided by the present invention;

[0029] (2) A modified cell or recombinant strain, wherein the modified cell or recombinant strain comprises any of the above-mentioned nanobodies provided by the present invention, or the vector thereof;

[0030] (3) An antibody derivative, wherein the antibody derivative comprises any of the above-mentioned nanoantibodies provided by the present invention;

[0031] (4) A pharmaceutical composition comprising a therapeutically effective amount of any of the above-mentioned nanobodies provided by the present invention, or the nucleic acid, or a carrier containing the nucleic acid, or the antibody derivative;

[0032] (5) A detection product comprising any of the above-mentioned nanoantibodies provided by the present invention, or the nucleic acid, or a carrier containing the nucleic acid, or the antibody derivative.

[0033] Furthermore, the nucleic acid is a DNA-coding sequence or an RNA-coding sequence.

[0034] The term "nucleic acid" as used in this invention can include encoded nucleic acids, or it can include nucleic acids that also include additional coding and / or non-coding sequences. The DNA form of the nucleic acids in this invention includes, but is not limited to, cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. Nucleic acids encoding the nanobodies or antigen-binding fragments described in this invention include, but are not limited to, coding sequences that only encode the structure of mature nanobodies or their antigen-binding fragments; coding sequences that encode mature nanobodies or their antigen-binding fragments and various additional coding sequences; coding sequences (and optional additional coding sequences) that encode the structure of mature nanobodies or their antigen-binding fragments, as well as non-coding sequences, etc.

[0035] In some embodiments of the present invention, the full-length nucleic acid sequence or fragment thereof of the nanobody can typically be obtained by PCR amplification, recombinant methods, or artificial synthesis. Once the relevant sequence is obtained, it can be obtained in large quantities using recombinant methods. Generally, it is cloned into a vector, then transformed into cells, and then the relevant sequence is isolated from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, nanobodies and their antigen-binding fragments, etc.) involved in the present invention include biomolecules existing in isolated forms.

[0036] The vectors include viral vectors (such as adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors) and non-viral vectors (plasmids and transposon vectors).

[0037] In some preferred embodiments, the vector is an expression vector.

[0038] The terms “vector,” “cloning vector,” and “expression vector” refer to a vector that can introduce DNA or RNA sequences into a host cell by transforming the host and promoting the expression of the introduced sequence (e.g., transcription and translation).

[0039] Other embodiments of plasmids include replicating plasmids containing the origin of replication, or integrating plasmids such as, for example, pUC, pcDNA, pBR, etc.

[0040] The vector can be a recombinant expression vector or a cloning vector. The vectors (e.g., expression vectors) provided by the present invention contain a nucleic acid sequence provided by the present invention encoding an antibody, at least one promoter operatively linked to the nucleic acid sequence, and / or at least one selection marker.

[0041] Examples of vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, λ phages, M13 phages, plasmids, etc.

[0042] The "modified cell" provided by this invention refers to the introduction of a "foreign" (i.e., external or extracellular) gene or DNA or RNA sequence into a host cell so that the host cell expresses the introduced gene or sequence to produce a substance of interest, typically a protein encoded by the gene or the introduced sequence. The host cell that receives and expresses the introduced DNA or RNA has been "modified".

[0043] In specific embodiments of the present invention, the host cell includes, but is not limited to, mammalian cells, insect cells, plant cells, fungal cells, prokaryotic cells, etc. Representative examples include: *Escherichia coli*, *Streptomyces* spp.; bacterial cells of *Salmonella typhimurium*; fungal cells such as yeast; insect cells of *Drosophila S2* or *Sf9*; animal cells of 293 cells, etc. Other embodiments of the host cell include prokaryotic cells (e.g., bacteria) and eukaryotic cells (e.g., yeast cells, mammalian cells, insect cells, plant cells, etc.).

[0044] Specific implementation schemes include Escherichia coli, Kluyveromyces or yeast, mammalian cell lines (e.g., Vero cells, CHO cells, 3T3 cells, COS cells, etc.) and primary or established mammalian cell cultures (e.g., produced by lymphoblasts, fibroblasts, epithelial cells, nerve cells, adipocytes, etc.).

[0045] The present invention provides an antibody derivative, including the nanobody provided by the present invention, which is directly or indirectly linked to a complex formed by a linker.

[0046] The connectable material includes detectable markers, drugs, toxins, cytokines, radionuclides, or enzymes.

[0047] In specific embodiments of the present invention, the connectable material is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (such as DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL), chemotherapeutic agents (such as cisplatin)) or any form of nanoparticles.

[0048] The term "detectable label" refers to any portion that generates a measurable signal through a change in an optical, electrical, or other physical indicator of the molecular state coupled to that portion. Such physical indicators include spectral, photochemical, biochemical, immunochemical, electromagnetic, radiochemical, and chemical methods, such as, but not limited to, fluorescence, chemiluminescence, and chemiluminescence. In the context of the use of labeled assay reagents, a "direct label" is a detectable label that is attached to the assay reagent by any means. In the context of the use of labeled assay reagents, an "indirect label" is a detectable label that specifically binds to the assay reagent. Therefore, an indirect label includes a portion that is a specific binding partner of the assay reagent. Biotin and avidin are examples of such portions employed, for example, by contacting a biotinylated antibody with labeled avidin to generate an indirectly labeled antibody.

[0049] The pharmaceutical compositions provided by this invention include pharmaceutically acceptable carriers or excipients. In some embodiments, the excipients include pharmaceutically acceptable solvents, dispersants, additives, plasticizers, etc. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable carrier medium. The formulated pharmaceutical compositions can be administered via conventional routes, including, but not limited to, intratumoral, intravenous, or local administration. In other embodiments, the pharmaceutical composition is an aqueous pharmaceutical composition (e.g., an aqueous solution), or nanobodies provided with salts in buffer-free water, or contains aqueous buffers or other types of solvents (e.g., organic solvents).

[0050] The term "effective amount" as used in this invention refers to an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic amount is an amount sufficient to achieve the desired therapeutic effect. This amount may be the same as or different from a preventative effective amount, which is the amount required to prevent the onset of disease or disease symptoms. An effective amount may be administered, applied, or dosed once or multiple times. The "therapeutic effective amount" (i.e., effective dose) of a therapeutic compound depends on the selected therapeutic compound. For example, the composition may be administered once or multiple times daily, once or multiple times weekly, once or multiple times monthly, or once or multiple times annually. Certain factors can affect the dosage and timing required for effective treatment of a subject, including but not limited to the severity of the disease or ailment, prior treatment, the subject's general health condition and / or age, and any other pre-existing conditions.

[0051] The present invention also provides a detection product, including a reagent kit, test strip, nucleic acid membrane strip, chip, system, or device. This detection product is used to detect the expression and expression level of CD5 protein, and can also be used to detect products for diagnosing T-cell lymphoma, thymoma, or B-cell lymphoma.

[0052] Furthermore, the nucleotide sequences of the nucleic acids encoding the nanobodies H1, C2, F3, G3, or H4 are shown in SEQ ID NO. 31-35, respectively.

[0053] This invention also provides applications of the above-mentioned anti-CD5 protein nanobody, characterized in that the application includes any one of the following aspects:

[0054] (1) Used to prepare products for detecting CD5 protein expression; or, used to prepare products for regulating CD5 protein activity or level;

[0055] (2) Products used to prepare diagnostic T-cell lymphoma, thymoma, or B-cell lymphoma;

[0056] (3) Used for the in vitro detection of CD5 protein expression for non-disease diagnosis and treatment purposes, and for the application of T lymphocyte tumors, thymomas and B-cell lymphomas;

[0057] (4) Drugs used to prepare drugs that inhibit CD5 protein expression, or products for treating T-cell lymphoma, thymoma, or B-cell lymphoma.

[0058] Furthermore, the products used to prepare for the detection of CD5 protein expression include kits, test strips, biochips, or detection probes.

[0059] Furthermore, the kit is an ELISA kit; the test strip is an ELISA test strip.

[0060] A nanobody library for recognizing CD5 protein, used to screen for obtaining any of the nanobodies provided by the present invention; is obtained by extracting total RNA from natural camel-derived PBMCs cells, obtaining the gene sequence of the antibody variable region VHH using reverse transcription and PCR amplification, ligating the gene sequence into a phage expression vector, and transforming it into competent cells.

[0061] Compared with existing technologies, the advantages of this invention are as follows: This invention utilizes a camel-derived nanobody (VHH) library, targeting mouse CD5 protein, and employs phage display technology for in vitro enrichment and panning. Through monoclonal high-throughput ELISA screening, specific camel-derived nanobody (VHH) sequences targeting mouse CD5 protein are obtained. Based on this sequence, we construct a recombinant VHH antibody protein, ultimately obtaining a stable, specific camel-derived nanomonoclonal antibody that recognizes mouse CD5 protein. Attached Figure Description

[0062] Figure 1 The purification results are for the recombinant nanobodies H1, C2, F3, G3 and H4 in Example 2;

[0063] Figure 2 The results are ELISA identification of recombinant nanobodies A6, B8, G9, H9, and E12 in Example 3. Detailed Implementation

[0064] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0065] Example 1: Construction of a camel-derived VHH phage nanobody library, and in vitro enrichment and monoclonal screening of camel-derived nanobodies (VHH) specifically targeting mouse CD5 protein.

[0066] 1. Construction of a camel-derived VHH phage nanobody library

[0067] (1) Animal immunization: A 2-year-old male alpaca was purchased, and 50 ml of blood was collected. Alpaca peripheral blood mononuclear cell (PBMC) cells were isolated from the alpaca blood using an alpaca peripheral blood mononuclear cell separation kit (Tianjin Haoyang Company, catalog number LDS1078). Cells were lysed in 1 ml of trizol solution (Invitvogen, catalog number 15596018) using Thermo's PureLink assay. TM The RNA miniprep kit (catalog number 12183020) extracts total RNA from cells and detects it by 1% agarose gel electrophoresis.

[0068] (2) Total RNA from natural alpaca PBMCs cells was selected as a template, and the following methods were used. III. 1st StrandcDNA Synthesis Kit (+gDNA wiper) (Vazyme, catalog number R312-02) for reverse transcription to obtain cDNA;

[0069] (3) Using cDNA as a template, Max Super-Fidelity DNA Polymerase (Vazyme, catalog number: P505-d3) was used to amplify the gene sequence of the antibody variable region VHH by PCR: Take 2 μl of cDNA, 5 μl of 10×PCR Buffer, 2 μl of MgSO4 (50 mM), 1 μl of dNTP (10 mmol / L), 1 μl of F primer (10 μmol / L), 1 μl of amplification primer (10 μmol / L), 0.1 μl of DNA polymerase, and 37.9 μl to 50 μl of sterile pure water. Vortex to mix, briefly centrifuge, and then perform PCR amplification.

[0070] The reaction conditions were: denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 68℃ for 1 min, 30 cycles; extension at 68℃ for 5 min.

[0071] The nucleotide sequences of the amplification primers are as follows:

[0072] Forward primer (as shown in SEQ ID NO.41): 5'-gtcctggctgctcttctacaagg-3';

[0073] Reverse primer (as shown in SEQ ID NO.42): 5'-ggtacgtgctgttgaactgttcc-3'.

[0074] The target fragment of 300-500 bp was recovered after 1% agarose gel electrophoresis.

[0075] (4) The recovered target fragment and the vector pCANTAB5E (Amersham Biosciences, phage display system) were digested with SfiI and NotI, and then incubated overnight at 16°C with T4 ligase (NEB) to ligate the target fragment onto the vector pCANTAB5E.

[0076] The reaction solution systems for SfiI and NotI enzyme digestion are shown in Table 1 below:

[0077] Table 1 Reaction solution system for SfiI and NotI enzyme digestion

[0078]

[0079] The connection reaction system is shown in Table 2 below:

[0080] Table 2 Connection Reaction System

[0081]

[0082] (5) The ligation product was transferred into TG1 electrocompetent cells (Lucigen) by electroporation, and then plated on 2YT-Amp-Glucose solid medium (formula as below) and cultured overnight at 37°C.

[0083] The formulation of the 2YT solid culture medium is as follows: 1.6% (W / V) Tryptone, 1% (W / V) Yeast Extract, 0.5% (W / V) NaCl, dissolved in 1L of distilled water.

[0084] The formulation of the 2YT-Amp-Glucose medium is as follows: 100 μg / ml of ampicillin and 2% glucose are added to the 2YT solid medium.

[0085] (6) Scrape the colonies from the culture medium, mix them thoroughly, add the bacterial solution to a 15% glycerol aqueous solution, and store at -80℃; inoculate the bacterial solution into 2YT-Amp-Glucose medium and incubate at 37℃ for 1-2 hours until OD. 600 =0.4-0.6.

[0086] Add helper phage (commercially available) and incubate at 37°C for 45 min-1 h; centrifuge the bacterial culture at 3000-5000 rpm, discard the supernatant, resuspend the bacterial cells in an equal volume of 2YT-Amp-Kan medium, and incubate overnight at 30°C; the next day, centrifuge and transfer the supernatant to a new centrifuge tube; add 1 / 4 volume of 5×PEG / NaCl solution, mix thoroughly, and place on ice or at 4°C for 1-2 h; then centrifuge and discard the supernatant, resuspend the precipitate in approximately PBS to obtain the monoclonal nanobody library, which can be stored at 4°C or -20°C.

[0087] The formulation of the 2YT-Amp-Kan medium is as follows: 100 μg / ml ampicillin and 50 μg / ml kanamycin are added to the 2YT medium.

[0088] 2. In vitro enrichment and monoclonal screening of nanobodies (VHH)

[0089] The reagents and materials listed in Table 3 below were used for in vitro enrichment and monoclonal screening of mouse CD5 protein-specific camel-derived nanobodies.

[0090] Table 3 Experimental Reagents and Materials

[0091]

[0092]

[0093] (1) Immunotube solid-phase panning

[0094] ① Wrapped

[0095] Immunotherapy tubes: Antigen protein mouse CD5 (50 μg / ml, CBS), 1 ml / tube, 2 tubes in total, incubated overnight at 4°C; wash the immunotherapy tubes 3 times with 5 ml PBST.

[0096] Add 1 ml of PBS to the control tube.

[0097] ② Closed

[0098] Incubate 5ml of 5% skim milk / PBST at 30°C for 1 hour; wash once with 5ml of PBS.

[0099] ③Incubation

[0100] Add 500 μl to each tube, for a total volume of 10. 12 (Add the same total amount of nano-library phage to each tube.) Incubate at 30°C for 1.5 hours.

[0101] ④ Washing

[0102] Wash 8-10 times with 5ml PBST.

[0103] ⑤ Washing

[0104] Add 1 ml of Gly-HCl (pH = 2.2) to each tube to elute the phage, and incubate with shaking at room temperature for approximately 6-8 minutes. Add Tris-HCl (pH = 9.6) to neutralize the solution to pH 7.0-8.0.

[0105] (2) Determine the titer of bacteriophages after elution.

[0106] ①Culturing E. coli TG1 until OD 600 =0.4-0.6; mix 10 μL of diluted eluted phage with 190 μL of E. coli TG1.

[0107] ② After incubating the mixture at 37°C for 30 min, pour it onto 2×YT-A (Amp 100μg / ml) solid medium and incubate at 37°C overnight.

[0108] (3) Amplification of eluted phages

[0109] ① Directly aspirate 800 μl of the eluted phage solution and add it to 10 ml of TG1 bacterial culture in the logarithmic phase. Incubate at 37°C for 30 min, then incubate at 220 rpm at 37°C for 30 min-1 h.

[0110] ② Add the bacterial culture to 30 ml of 2YT-Amp-Glucose medium and incubate at 37°C and 220 rpm until OD reaches 100%. 600 = Approximately 0.4-0.6.

[0111] ③ Add helper phages to the bacterial culture.

[0112] The amount of helper phage added = 0.6 × liquid volume × 5 × 10 8 ×30pfu; after standing at 37℃ for 30min, incubate at 220rpm for 45min-1h.

[0113] ④ Centrifuge the bacterial culture at 3000-5000 rpm and discard the supernatant; resuspend the bacterial cells in an equal volume of 2YT-Amp-Kan medium; incubate overnight at 30℃ and 220 rpm.

[0114] ⑤ The next day, centrifuge the bacterial culture at 8000 rpm, 4℃, for 20 min, and transfer the supernatant to a new centrifuge tube; add 1 / 4 volume of 5×PEG / NaCl solution; mix thoroughly, and place on ice or at 4℃ for 1-2 h.

[0115] ⑥ Centrifuge at 8000 rpm and 4℃ for 30 min, discard the supernatant, and resuspend the precipitate in about 1 ml of PBS.

[0116] ⑦ Centrifuge at 8000rpm-10000rpm, 4℃, for 10min, and transfer the supernatant to a new centrifuge tube.

[0117] (4) Test the titer of the amplified phage.

[0118] The test procedure is the same as step 2 above, "determining the titer of phages after elution".

[0119] Repeat steps 1 to 4 to perform the second to fourth rounds of enrichment screening. See Table 4 below for details.

[0120] Table 4 shows the process of four rounds of enrichment screening.

[0121]

[0122] The results of in vitro enrichment and panning are shown in Table 5 below.

[0123] Table 5. Phage titer test results

[0124]

[0125] (5) Polyclonal phage ELISA detection

[0126] ① Wrapped

[0127] Immunoassay: 100 μl of mouse CD5 antigen protein (4 μg / ml, CBS) coated on the plate, incubated overnight at 4°C.

[0128] Control wells: coated with 100 μl PBS.

[0129] The immunoassay plate and control wells were washed three times with 300 μl PBST.

[0130] ② Closed

[0131] Incubate 300 μl of 5% skim milk / PBST at 30°C for 1 h. Wash 2-3 times with 300 μl of PBST.

[0132] ③Incubation

[0133] Dilute the phages after each round of amplification with PBS, increasing the dilution factor by threefold. The initial concentration was 10. 12pfu / ml. Add 100 μl of diluted amplified phage to each well; incubate at 30°C for 1 h.

[0134] ④ Washing

[0135] Wash 4-6 times with 300μl PBST.

[0136] ⑤ Secondary Antibody

[0137] Add 100 μl of secondary antibody dilution buffer (anti-M13-HRP, 1:5000) and incubate at 30°C for 1 h;

[0138] ⑥ Washing

[0139] Wash 4-6 times with 300μl PBST.

[0140] ⑦ Color development

[0141] Add 100 μl of TMB chromogenic solution and develop in the dark for 3-8 min. Terminate the reaction by adding 100 μl of 2M HCl. Read the microplate reader readings (450 nm - 620 nm).

[0142] The results of polyclonal phage ELISA detection are shown in Table 6 below.

[0143] Table 6. Results of Polyclonal Phage ELISA Detection

[0144]

[0145] (6) Single clonal phage ELISA screening

[0146] Select an appropriate number of rounds (R4), wash out the phage and dilute it to an appropriate concentration, infect TG1 cells in the logarithmic phase, and plate them.

[0147] The following day, 192 single colonies were picked from the plate and inoculated into 96-well plates (600 μl of YT-Amp-Glucose medium was added to each well); the plates were incubated at 37°C with shaking at 250 rpm for 2 hours until the bacterial culture reached OD500. 600 =0.4-0.6.

[0148] Aspirate 100 μl of bacterial culture into each well of a cell culture plate, add sterile glycerol (final concentration 20%-25%) to each well, mix well, and store at -20°C.

[0149] Add helper phage to the remaining 96-well plate medium. The amount of helper phage added = 0.6 × liquid volume × 5 × 10⁻⁶ 8 ×30pfu; incubate at 37℃ for 30 min, then shake at 250 rpm at 37℃ for 45 min-1 h.

[0150] Centrifuge the 96-well plate at 4000 rpm for 5 min and discard the supernatant; resuspend the bacterial culture in 600 μl of 2YT-Amp-Kan medium in each well and incubate overnight at 30°C with shaking at 250 rpm.

[0151] The next day, the 96-well plate was centrifuged at 4000 rpm for 10-15 minutes, and the supernatant was used for ELISA experiments.

[0152] Immunocoating plates: 100 μl of mouse CD5 antigen protein (4 μg / ml in CBS) was incubated overnight at 4°C.

[0153] Control wells: coated with 100 μl PBS.

[0154] The immunoassay plate and control wells were washed three times with 300 μl PBST.

[0155] Blocking: 300 μl 5% skim milk / PBST, block at 30℃ for 1 h; wash 2-3 times with 300 μl PBST.

[0156] Incubation: Add 100 μl of supernatant phage to each well; incubate at 30°C for 1 h.

[0157] Washing: Wash 4-6 times with 300μl PBST.

[0158] Secondary antibody: Add 100 μl of secondary antibody dilution buffer (anti-M13-HRP, 1:5000) and incubate at 30°C for 1 h.

[0159] Washing: Wash 4-6 times with 300μl PBST.

[0160] Color development: Add 100 μl of TMB color development solution and develop color in the dark for 3-8 min, then add 100 μl of 2M HCl to terminate the reaction; record the microplate reader reading (450 nm-620 nm).

[0161] The results of monoclonal phage ELISA detection are shown in Tables 7 and 8 below.

[0162] Table 7. OD of R4P1 (Round 3, Monoclonal Clones 1-96) 600 (Antigen group)-OD 600 (Control group) Results

[0163] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.03 0.03 0.04 0.03 0.03 0.02 0.03 0.02 0.16 3.33 0.03 0.07 B 0.03 0.03 0.02 0.03 0.04 0.03 0.03 0.02 0.02 0.02 0.02 0.02 C 0.04 2.42 0.04 0.03 0.02 0.03 0.02 0.02 0.02 0.03 0.01 0.03 D 0.04 0.06 3.80 0.05 0.02 0.02 3.74 0.01 0.03 0.02 0.02 0.03 E 0.03 0.03 0.04 2.18 0.02 0.02 0.02 0.02 0.02 0.02 0.03 0.02 F 0.02 0.09 3.68 0.02 0.02 0.01 0.02 0.02 0.03 3.45 3.50 0.02 G 0.05 0.05 3.84 0.05 3.05 3.50 0.02 0.02 0.02 0.07 4.00 2.48 H 2.94 3.21 3.51 3.63 2.97 0.02 0.03 3.71 0.02 3.45 0.03 3.70

[0164] Table 8. OD of R4P2 (third-round monoclonal antibodies 97-192) 600 (Antigen group)-OD 600 (Control group) Results

[0165] 1 2 3 4 5 6 7 8 9 10 11 12 A 0.35 3.02 4.16 0.31 0.05 0.03 0.04 0.03 0.02 0.02 0.02 0.18 B 0.12 0.13 0.15 0.07 0.04 0.02 0.03 0.02 0.03 0.07 3.79 0.03 C 0.10 0.09 0.09 0.04 0.03 0.02 0.02 0.02 0.12 4.13 0.05 0.04 D 0.08 0.21 4.20 0.02 0.02 0.02 0.03 3.12 0.02 0.03 0.04 0.03 E 0.17 2.95 0.10 0.03 0.02 0.02 0.03 0.04 0.02 0.02 0.04 0.04 F 0.12 3.05 0.04 0.04 0.03 0.03 0.08 3.39 0.08 0.07 0.08 3.24 G 0.09 2.95 0.23 3.76 0.03 0.03 0.03 0.06 3.15 3.56 0.04 0.03 H 0.03 0.05 0.16 3.82 0.03 0.03 0.02 0.02 0.03 0.09 0.14 2.89

[0166] We will OD 600 (Antigen group)-OD 600 Clones with a value greater than 1 in the (control group) were defined as positive clones and sent for sequencing. After excluding erroneous and duplicate antibody sequences, five high-affinity antibody sequences were finally obtained: Mouse-CD5-R4P1-H1, Mouse-CD5-R4P1-C2, Mouse-CD5-R4P1-F3, Mouse-CD5-R4P1-G3, and Mouse-CD5-R4P1-H4; abbreviated as nanobodies H1, C2, F3, G3, or H4, respectively, and their amino acid sequences are shown in SEQ ID NO. 36-40.

[0167] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of nanobody H1 are shown in SEQ ID NO. 1-3.

[0168] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of nanobody C2 are shown in SEQ ID NO.4-6;

[0169] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of nanobody F3 are shown in SEQ ID NO.7-9;

[0170] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of nanobody G3 are shown in SEQ ID NO.10-12;

[0171] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of nanobody H4 are shown in SEQ ID NO.13-15.

[0172] Specifically, the amino acid sequences of the heavy chains of nanobodies H1, C2, F3, G3 and H4 are shown in SEQ ID NO.16-20;

[0173] The amino acid sequences of the constant regions CH2 and CH3 of nanobody H1 are shown in SEQ ID NO.21-22;

[0174] The amino acid sequences of the constant regions CH2 and CH3 of nanobody C2 are shown in SEQ ID NO.23-24;

[0175] The amino acid sequences of the constant regions CH2 and CH3 of nanobody F3 are shown in SEQ ID NO.25-26;

[0176] The amino acid sequences of the constant regions CH2 and CH3 of nanobody G3 are shown in SEQ ID NO.27-28;

[0177] The amino acid sequences of the constant regions CH2 and CH3 of nanobody H4 are shown in SEQ ID NO.29-30.

[0178] Example 2: Recombinant expression of nanobodies H1, C2, F3, G3 and H4

[0179] The encoding gene sequences of the five selected nanobody amino acid sequences were subcloned into the pcDNA3.1(+) vector, with the Nhe1 / Not1 restriction enzyme site and human IgG1 Fc added to the C-terminus. Xten CHO cells were transfected, and after 72 h, the cells were removed by centrifugation. The culture supernatant was purified using Protein A resin. The purification results (12% Non-reduced SDS-PAGE) are shown below. Figure 1 As shown.

[0180] Example 3: ELISA identification of recombinant nanobodies A6, B8, G9, H9, and E12

[0181] 1. Immunoplate coating: Antigen protein mouse CD5 (5 μg / ml in CBS), 100 μl, incubated overnight at 4°C; control wells coated with 100 μl PBS. Wash 3 times with 300 μl PBST;

[0182] 2. Blocking: 300 μl 5% skim milk / PBST, block at 30℃ for 1 h, wash 2-3 times with 300 μl PBST;

[0183] 3. Incubation: The recombinant VHH was diluted 5-fold starting from 10 μg / ml, for a total of 7 gradients;

[0184] 4. Washing: Wash 4-6 times with 300μl PBST;

[0185] 5. Secondary antibody: Add 100 μl of secondary antibody dilution buffer (anti-human Fc-HRP, 1:5000) and incubate at 30℃ for 1 h;

[0186] 6. Washing: Wash 4-6 times with 300μl PBST;

[0187] 7. Color development: Add 100 μl of TMB color development solution and develop color in the dark for 3-8 min. Add 100 μl of 2M HCl to stop the reaction; read the microplate reader (450 nm-620 nm).

[0188] The results are as follows Figure 2As shown, the five recombinant VHH molecules bind well to mouse CD5, with R4P1-H1 being the best, having an EC50 of 28.85 ng / ml.

[0189] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A nanobody against CD5 protein, characterized in that, The nanobody H1 is described in SEQ ID NO. 1-3, and the amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain of the nanobody H1 are shown in SEQ ID NO. 1-3.

2. The anti-CD5 protein nanobody according to claim 1, characterized in that, The amino acid sequence of the heavy chain of the nanobody H1 is shown in SEQ ID NO.

16.

3. The anti-CD5 protein nanobody according to claim 2, characterized in that, The amino acid sequences of the constant regions CH2 and CH3 of the nanobody H1 are shown in SEQ ID NO.21-22.

4. A nucleic acid molecule or a carrier containing a nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the nanobody according to any one of claims 1-3.

5. The nucleic acid molecule or carrier containing the nucleic acid molecule according to claim 4, characterized in that, The nucleotide sequence of the nucleic acid molecule encoding the nanobody H1 is shown in SEQ ID NO.

31.

6. A modified cell or recombinant bacterial strain, characterized in that, The modified cells or recombinant strains include the nanobodies according to any one of claims 1-3, or the carrier containing nucleic acid molecules according to claim 4; the modified cells are non-plant cells.

7. A testing product, characterized in that, The detection product includes the nanobody as described in any one of claims 1-3, or includes the nucleic acid molecule as described in claim 4, or includes the carrier containing the nucleic acid molecule as described in claim 4.

8. The application of the anti-CD5 protein nanobody according to any one of claims 1-3, characterized in that, The application includes any one of the following aspects: (1) Used to prepare products for detecting CD5 protein expression; (2) Used for the in vitro detection of CD5 protein expression for purposes other than disease diagnosis and treatment.

9. The application according to claim 8, characterized in that, Products used to prepare for the detection of CD5 protein expression include kits, test strips, biochips, or detection probes.

10. The application according to claim 9, characterized in that, The kit is an ELISA kit; the test strip is an ELISA test strip.

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