Method for constructing nanobody-encoding DNA library, and use

Abstract

Provided is a method for constructing a nanobody-encoding DNA library. The method comprises: using an immunogen to screen peripheral blood mononuclear cells (PBMCs) of an animal to obtain positive cells specifically binding to the immunogen; and amplifying nucleic acid samples derived from the positive cells to obtain a nanobody-encoding DNA library, wherein the nanobody is a nanobody specifically binding to the immunogen, and wherein the amplifying comprises using one or more groups of primer pairs, and each group of primer pairs among the one or more groups of primer pairs comprises one or more forward primers and one or more reverse primers. In the present invention, a large number of diverse DNA sequences containing a large number of low-abundance sequences are obtained. A high-throughput sequencing-based technical route for rapid preparation of a VHH antibody allows for efficient and rapid acquisition of the VHH antibody having high affinity and high specificity, thereby satisfying a raw material requirement of a high-quality diagnostic agent.

Description

Methods and applications for constructing nanobody-encoded DNA libraries Technical Field

[0001] This invention relates to the field of antibody technology, and in particular to a method for constructing a nanobody-encoded DNA library and its application. Background Technology

[0002] With the rapid advancements in modern medicine and biotechnology, high-quality antibodies, with their superior targeting specificity and high affinity, have demonstrated unique advantages and broad application prospects in diseases such as viral infections, tumors, autoimmune diseases, and metabolic disorders. Nanobodies (Nb) are single-domain antibody fragments derived from the variable domain (VHH) of heavy-chain antibodies from camel-like animals (such as camels and alpacas) and sharks. Their molecular weight is only one-tenth that of traditional antibodies, yet they possess numerous advantages, including stronger antigen recognition specificity, higher affinity, better stability, and ease of molecular engineering modification and optimization. In recent years, nanobodies, due to their unique biological characteristics and broad application potential, have become one of the research hotspots in the fields of medicine and biotechnology, and are gradually emerging as a new force in next-generation therapeutic biopharmaceuticals and clinical diagnostic reagents.

[0003] One of the main challenges in nanobody production is selecting and determining the optimal preparation process. Obtaining a library containing the required genetic information is crucial for generating nanobodies with high specificity and affinity. Immunotherapy libraries are the most common choice for generating nanobodies. Active immunization of camels induces in vitro nanobodies through antigen-induced immunization. Following immunization, mRNA is extracted from isolated lymphocytes, amplified with a specific sequence, and inserted into a cloning vector. Expression screening is then performed to isolate the most suitable nanobodies. Currently, the screening and preparation of nanobodies mainly relies on traditional phage display technology. This technology fuses VHH antibody fragments with phage surface proteins, making them visible on the phage surface, thus enabling them to interact with antigen molecules and screen for antibodies with affinity and specificity. Compared to the preparation and screening process of traditional monoclonal antibodies, phage display is simpler and more efficient. However, the phage display method for preparing nanobodies also faces several challenges and inherent limitations: the preparation cycle is lengthy, typically taking more than three months; the screening efficiency is low, usually only a few dozen to a dozen antibodies with blocking activity are obtained from hundreds or even thousands of antibodies, resulting in low efficiency and a low success rate; the phage screening process exhibits certain sequence bias, which may reduce antibody sequence diversity and affect the discovery of high-quality antibodies. Furthermore, phages require multiple rounds of screening, leading to antibody nonspecificity and the risk of false positives. These disadvantages make nanobody development expensive and time-consuming, severely restricting its large-scale application.

[0004] High-throughput sequencing technology, also known as next-generation sequencing (NGS sequencing), can sequence and analyze millions of DNA molecules simultaneously. The large amounts of sequencing data obtained through NGS sequencing platforms are ideal for comprehensive analysis of complex collections of diverse gene fragments, such as antibody libraries.

[0005] Existing technologies involve methods for rapidly obtaining nanobodies using high-throughput sequencing, including obtaining PBMC-positive cells via indirect conjugation and obtaining nanobodies target fragments through two rounds of PCR screening. However, current methods involve numerous screening steps in flow cytometry and PCR reactions, increasing the processing difficulty and posing a risk of generating non-specific antibodies. Furthermore, the primer design in existing methods for PCR reactions mostly targets constant regions of the nanobodies sequence, often only yielding antibody sequences with high abundance, potentially missing many low-abundance but high-performance antibody sequences, lacking broad screening scope, and carrying the risk of missed screening. Summary of the Invention

[0006] To address the aforementioned technical problems in the prior art, this invention provides a method for constructing nanobody-encoded DNA libraries and rapidly preparing nanobodies based on high-throughput sequencing.

[0007] In a first aspect, the present invention provides a method for constructing a nanobody-encoded DNA library, the method comprising:

[0008] The peripheral blood mononuclear cells (PBMCs) of animals were screened using an immunogen to obtain positive cells that specifically bind to the immunogen; and

[0009] The nucleic acid sample derived from the positive cells was amplified to obtain the DNA library encoding the nanobody;

[0010] The nanobody mentioned therein is a nanobody that specifically binds to the immunogen.

[0011] In some embodiments, the amplification includes using one or more primer pairs, each of which contains one or more forward primers and one or more reverse primers.

[0012] In some embodiments, the forward primer contains a nucleotide sequence encoding a nanobody initiation region or a signal peptide initiation region. In some embodiments, the reverse primer contains a nucleotide sequence encoding a nanobody endpoint region.

[0013] In some embodiments, the amplification includes using two or more primer pairs, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more primer pairs.

[0014] In some implementations, the amplification includes using fewer than 60 primer pairs, such as 60, 59, 58, 57, 56, 55, 54, 53, 52, 51 or fewer primer pairs.

[0015] In some embodiments, the forward primer consists of 5 degenerate bases NNNNNNN and the target gene primer sequence, wherein each N is independently A, T, C, G, or absent. In some embodiments, the reverse primer consists of 8 degenerate bases NNNNNNNNN and the target gene primer, wherein each N is independently A, T, C, G, or absent.

[0016] In some embodiments, the one or more forward primers have an added nucleotide sequence selected from SEQ ID NOs:1-5 or having a total of no more than 40, 35, 30, 25, 20, or 15 nucleotides (e.g., a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) nucleotides at their 5' and / or 3' ends; and / or the one or more reverse primers have an added nucleotide sequence selected from SEQ ID NOs:1-5. Nucleotide sequences as shown in NOs:20–25 or having a total of no more than 40, 35, 30, 25, 20, or 15 (e.g., a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) nucleotides added at their 5' and / or 3' ends. Specific information for SEQ ID NOs:1–5 and SEQ ID NOs:20–25 is shown in Table 1.

[0017] Table 1

[0018] Where K represents G or T, R represents A or G, W represents A or T, Y represents C or T, D represents G, A or T, H represents T, A or C, V represents G, A or C, B represents C, G or T, N represents A, T, C, G or none, and X represents G, C or none.

[0019] In some implementations, specific examples of the sequence shown in SEQ ID NO:1 are as follows: NNNNNACCGGAGATTTCGCGGCCCAGC (SEQ ID NO:6), NNNNNTCGCGGCCCAGCCGGCCCAGCCGGCC (SEQ ID NO:7), NNNNNACCAGAGAATTCGCGGCCCAGCCG (SEQ ID NO:8) and NNNNNTCGCGGCCCAGCCGGCC (SEQ ID NO:9).

[0020] In some implementations, specific examples of the sequence shown in SEQ ID NO:2 are as follows: NNNNNCAGTTCAACAGTGGTCCTGGCT (SEQ ID NO:10), NNNNNCAGTTCAACAGTGGTCCTGGCTGC (SEQ ID NO:11), and NNNNNCAGTTCAACAGTGGTCCTGGCTGCTC (SEQ ID NO:12).

[0021] In some implementations, specific examples of the sequence shown in SEQ ID NO:3 are as follows: NNNNNAGATTTCGCGGCCCAG (SEQ ID NO:13), NNNNNAGATTTCGCGGCCCAGCG (SEQ ID NO:14), NNNNNAGATTTCGCGGCCCAGCGGC (SEQ ID NO:15) and NNNNNAGATTTCGCGGCCCAGCGGCC (SEQ ID NO:16).

[0022] In some implementations, specific examples of the sequence shown in SEQ ID NO:4 are as follows: NNNNNTCGGCGCGCCGAGGGGTC (SEQ ID NO:17), NNNNNTCGGCGCGCCGAGGGGT (SEQ ID NO:18), and NNNNNTCGGCGCGCCGAGG (SEQ ID NO:19).

[0023] In some implementations, specific examples of the sequence shown in SEQ ID NO:24 are as follows: NNNNNNNCGACGGTGACCTGCAT (SEQ ID NO:26), NNNNNNNNGACGGTGACCTGGGT (SEQ ID NO:27), NNNNNNNNGACGGTCACCTGGGT (SEQ ID NO:28), NNNNNNGAGACGGTGATTTGGGT (SEQ ID NO:29) and NNNNNNNNGACGGTGACCAGGGT (SEQ ID NO:30).

[0024] In some embodiments, the sequence of each of the one or more forward primers is selected from SEQ ID NOs:5-19, or is a nucleotide sequence having an additional nucleotide sequence having a total of no more than 40, 35, 30, 25, 20, or 15 nucleotides (e.g., a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) nucleotides at its 5' end and / or 3' end, and / or

[0025] The sequence of each of the one or more reverse primers is selected from SEQ ID NOs:20-23, 25-30, or a nucleotide sequence having a total of no more than 40, 35, 30, 25, 20 or 15 (e.g., a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) nucleotides added to its 5' end and / or 3' end.

[0026] In some implementations, each of the one or more primer pairs contains two or more forward primers.

[0027] In some embodiments, each of the one or more primer pairs contains two or more reverse primers. When two or more forward primers or two or more reverse primers are used in combination, it is called a forward primer set or a reverse primer set.

[0028] In some implementations, each primer pair contains the same number of forward and reverse primers.

[0029] In some implementations, the number of forward and reverse primers in each primer pair varies.

[0030] In some embodiments, the amplification includes using a first set of forward primers, which comprises forward primers as shown in SEQ ID NO:10 and SEQ ID NO:6.

[0031] In some embodiments, the amplification includes the use of a second set of forward primers, which contains forward primers as shown in SEQ ID NO:17 and SEQ ID NO:12.

[0032] In some embodiments, the amplification includes the use of a third set of forward primers, which comprises forward primers as shown in SEQ ID NO:13, SEQ ID NO:17, and SEQ ID NO:5.

[0033] In some embodiments, the amplification includes the use of a fourth set of forward primers, which comprises forward primers as shown in SEQ ID NO:9 and SEQ ID NO:5.

[0034] In some embodiments, the amplification includes using a first reverse primer set comprising reverse primers as shown in SEQ ID NO:20 and SEQ ID NO:26.

[0035] In some embodiments, the amplification includes the use of a second set of reverse primers, which comprises reverse primers as shown in SEQ ID NO:21 and SEQ ID NO:25.

[0036] In some embodiments, the amplification includes the use of a third set of reverse primers, which comprises reverse primers as shown in SEQ ID NO:23 and SEQ ID NO:27.

[0037] In some embodiments, the amplification includes the use of a fourth set of reverse primers, which comprises reverse primers as shown in SEQ ID NO:22 and SEQ ID NO:28.

[0038] In some embodiments, the amplification includes the use of a fifth set of reverse primers, which comprises reverse primers as shown in SEQ ID NO:20, SEQ ID NO:29, and SEQ ID NO:30.

[0039] In some embodiments, a primer pair is formed by mixing any one of the first to the fourth forward primer sets with any one of the first to the fifth reverse primer sets to perform the amplification.

[0040] In some embodiments, the animal is an animal immunized with the immunogen.

[0041] In some embodiments, the animal is selected from camelids and cartilaginous fish. In some embodiments, the camelid is selected from llamas, alpacas, guanacos, vicuñas, dromedaries, or Bactrian camels. In some embodiments, the cartilaginous fish is selected from nurse sharks, great white sharks, wolverines, dogfish, rays, or bamboo sharks. In some embodiments, the animal is a Bactrian camel or an alpaca. In some preferred embodiments, the immunized animal is an alpaca.

[0042] In some embodiments, at least one of the immunogens is used to immunize the same animal simultaneously. In some embodiments, two of the immunogens are used to immunize the same animal simultaneously. In some embodiments, three of the immunogens are used to immunize the same animal simultaneously. In some embodiments, four of the immunogens are used to immunize the same animal simultaneously. In some embodiments, five of the immunogens are used to immunize the same animal simultaneously. In some embodiments, six of the immunogens are used to immunize the same animal simultaneously.

[0043] In some embodiments, the screening of positive cells is performed using flow cytometry. In some embodiments, the screening is performed using a magnetic cell sorter. In some embodiments, the screening is performed using a microfluidic chip. In some preferred embodiments, the screening is performed using flow cytometry.

[0044] In some embodiments, the immunogen is selected from immunogens directly conjugated to a fluorescent dye for screening positive cells.

[0045] In some embodiments, the immunogen is selected from biotin-labeled immunogens and used to screen for positive cells.

[0046] In some embodiments, the fluorescent dye is selected from FITC, TRITC, Alexa488, Alexa560, PE, PE-TR, Cy5, Percp-cy5.5, APC, V450, BV510, or Cy3. In some preferred embodiments, the fluorescent dye is Alexa488 and / or Alexa560.

[0047] In some embodiments, the screening can simultaneously yield at least one positive cell that specifically binds to the immunogen. In some embodiments, the screening can simultaneously yield two positive cells that specifically bind to two of the immunogens. In some embodiments, the screening can simultaneously yield three positive cells that specifically bind to three of the immunogens. In some embodiments, the screening can simultaneously yield four positive cells that specifically bind to four of the immunogens. In some embodiments, the screening can simultaneously yield five positive cells that specifically bind to five of the immunogens. In some embodiments, the screening can simultaneously yield six positive cells that specifically bind to six of the immunogens.

[0048] In some implementations, the method for constructing a DNA-encoding library includes isolating multiple nucleic acids from screened positive cells.

[0049] In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA.

[0050] In some embodiments, the method of constructing a coding DNA library includes generating a cDNA library. In some embodiments, the cDNA library comprises multiple cDNA molecules corresponding to multiple mRNA molecules derived from positive cells. In some embodiments, the multiple cDNA molecules are double-stranded cDNA molecules.

[0051] In some embodiments, the PCR reaction system contains 2×PCR reaction solution, 100–350 ng of cDNA, and a total concentration of 100 nmol / L–400 nmol / L of forward primers or a total concentration of reverse primers.

[0052] In some implementations, the PCR reaction conditions are as follows: pre-denaturation at 94–100°C (e.g., 94°C, 95°C, 96°C, 97°C, 98°C, 100°C) for 1–15 min (e.g., 1 min, 3 min, 5 min, 8 min, 10 min, 12 min, 15 min); denaturation at 94–100°C (e.g., 94°C, 95°C, 96°C, 97°C, 98°C, 100°C) for 10–30 s (e.g., 10 s, 15 s, 20 s). Annealing extension at 40–55℃ (e.g., 40℃, 45℃, 48℃, 50℃, 52℃, 53℃, 55℃) for 20–90s (e.g., 20s, 30s, 35s, 40s, 45s, 50s, 60s, 70s, 80s, 90s), extending at 70–85℃ (e.g., 70℃, 72℃, 75℃, 78℃, 80℃, 85℃) for 10–50s (e.g., 10s, 15s, 20s, 25s, 30s), anne ...25s, 25s, 30s, 25s, 30s, 25s, 30s, 25s, 25s, 25s, 25s, 25s, 25s, 25s, 25s, 25s, 25s, 25s, 25s, 25s, 2 0s, 40s, 50s), amplification 2-8 cycles (e.g., 2, 3, 4, 5, 6, 8); denaturation 94-100℃ (e.g., 94℃, 95℃, 96℃, 97℃, 98℃, 100℃) 10-30s (e.g., 10s, 15s, 20s, 25s, 30s); annealing extension 55-70℃ (e.g., 55℃, 58℃, 60℃, 62℃, 65℃, 68℃, 70℃) 20-90s (e.g., 2 ... annealing extension 55-70℃ (e.g., 55℃, 58℃, 60℃, 62℃, 65℃, 68℃, 70℃) 20-90s (e.g., 30s, 35s, 40s, 45s, 50s, 60s, 70s, 80s, 90s), extended at 70-85℃ (e.g., 70℃, 72℃, 75℃, 78℃, 80℃, 85℃) for 10-50s (e.g., 10s, 15s, 20s, 25s, 30s, 40s, 50s), amplified for 8-15 cycles (e.g., 8, 10, 12, 13, 14, 15); final extension at 70-75℃ for 5-15 minutes.

[0053] In some preferred embodiments, the PCR reaction conditions are as follows: pre-denaturation, 95℃ for 5 min; denaturation at 95℃ for 20 s, annealing and extension at 50℃ for 30 s, extension at 72℃ for 40 s, amplification for 5 cycles; denaturation at 95℃ for 20 s, annealing and extension at 68℃ for 40 s, extension at 72℃ for 40 s, amplification for 10 cycles; final extension at 72℃ for 10 min.

[0054] In some implementations, the total number of PCR cycles does not exceed 25 cycles. In some implementations, the total number of PCR cycles does not exceed 20 cycles. In some implementations, the total number of PCR cycles does not exceed 18 cycles.

[0055] In a second aspect, the present invention provides a kit for amplification.

[0056] In some embodiments, the kit is used to perform the method described in the first aspect.

[0057] In some embodiments, the kit includes:

[0058] One or more forward primers, wherein the sequences of the forward primers are all different and selected from SEQ ID NOs:1-5, or have an additional nucleotide sequence having a total of no more than 40, 35, 30, 25, 20 or 15 nucleotides (e.g., a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) nucleotides at their 5' and / or 3' ends; and

[0059] One or more reverse primers, wherein the sequences of the reverse primers are all different and are selected from SEQ ID NOs:20-25, or have an additional nucleotide sequence having a total of no more than 40, 35, 30, 25, 20 or 15 (e.g., a total of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) nucleotides at their 5' and / or 3' ends.

[0060] In some embodiments, the one or more forward primers are packaged separately and optionally mixed before use, and / or the one or more reverse primers are packaged separately and optionally mixed before use.

[0061] In some implementations, at least two of the plurality of forward primers are premixed, and / or at least two of the plurality of reverse primers are premixed.

[0062] In some embodiments, the kit may also contain other reagents, such as reagents for PCR amplification, reagents for nucleic acid extraction, etc.

[0063] In some embodiments, the reagents for PCR amplification comprise a nucleic acid polymerase, a working buffer for the enzyme (e.g., a nucleic acid polymerase), dNTPs (labeled or unlabeled), water, and a buffer containing ions (e.g., Mg). 2+ ( ) solutions, or any combination thereof.

[0064] In a third aspect, the present invention provides the method for constructing a nanobody-encoding DNA library as described in the first aspect or the kit as described in the second aspect for the preparation of nanobodies.

[0065] In a fourth aspect, the present invention provides a method for preparing nanobodies, the method comprising constructing a DNA library encoding the nanobodies using the method of the first aspect.

[0066] In some embodiments, the preparation method of the nanobody further includes: detecting the integrity and purity of the encoding DNA library and quantifying the amount of DNA in the library, and performing high-throughput sequencing and bioinformatics analysis on the encoding DNA library to obtain the high-reliability encoding DNA of the nanobody.

[0067] In some embodiments, nucleic acid electrophoresis is used to determine the integrity and purity of the DNA library. Clear, untailed DNA bands on electrophoresis indicate good integrity and purity of the obtained coding DNA library. In some embodiments, a spectrophotometer is used to determine the concentration and purity of the purified library DNA. DNA purity is commonly measured using OD. 260 / OD 280 The ratio of OD to OD of pure double-stranded DNA is used to represent the OD of pure double-stranded DNA 260 / OD 280 The ratio should be 1.8. A ratio below 1.6 or above 1.9 indicates the presence of impurities or other contamination.

[0068] In some embodiments, the amount of DNA can be the total amount of nucleic acid, such as ng. In some embodiments, the amount of DNA can be a concentration, such as ng / μL.

[0069] In some embodiments, the high-throughput sequencing equipment includes, but is not limited to: Ilumina HiSeq2000, HiSeq2500k MiSeq, MiSeqDx, NextSeq500, HiSeq X ten, Life SOLiD, Ion Torrent PGM, Proton, Roche 454, and single-molecule sequencing equipment, thereby obtaining the nucleotide sequence of the nanobody-encoded DNA library rapidly and efficiently with high sequencing throughput and low cost.

[0070] In some embodiments, the analysis of DNA sequencing data includes quality assessment and screening of the sequencing data. In some embodiments, the quality assessment includes monitoring the Q30 value of the sequencing data. In some embodiments, the screening includes assembling the sequencing data to obtain a complete DNA sequence and selecting high-confidence coding DNA sequences from it.

[0071] In some implementations, the Q30 of the sequencing data is ≥80%.

[0072] In some embodiments, a high-confidence coding DNA sequence refers to a sequence that encodes an antibody sequence with a starting amino acid sequence of EVQLV (SEQ ID NO:31) or QVQLV (SEQ ID NO:32) and / or an antibody sequence with a terminal amino acid sequence of TVSS (SEQ ID NO:33).

[0073] In some embodiments, the Igblast antibody sequence analysis website is used to perform antibody sequence alignment analysis on the complete DNA sequence to determine whether it encodes a complete antibody. In some embodiments, IMGT / HighV-QUEST is used to perform antibody sequence alignment analysis on the complete DNA sequence to determine whether it encodes a complete antibody.

[0074] In some embodiments, the method for preparing the nanobody further includes: constructing a vector containing the encoding DNA, and using the vector to guide the expression of the encoding DNA to prepare the nanobody.

[0075] In some embodiments, preparing the nanobody from the carrier includes: directing the expression of encoding DNA via the carrier, preferably, the carrier replicates and expresses the encoding DNA in a host cell. The target gene (i.e., encoding DNA) can be introduced into a host cell using recombinant expression techniques conventional in the art, and the host cell's biological mechanisms can then induce the expression of a specific protein.

[0076] Modern recombinant protein expression technologies include traditional expression systems based on *E. coli*, yeast, insects, and mammalian cells, as well as newer plant and in vitro expression systems. Different expression systems can be selected depending on the nature of the target protein. In some embodiments, expression systems include, but are not limited to: prokaryotic expression systems such as *E. coli* and *Bacillus*, with *E. coli* strains including BL21(DE3), BL21(DE3)Star, B834(DE3), Origami(OE3), Rosetta, and Rosetta(DE3); in other embodiments, expression systems include, but are not limited to: yeast expression systems such as *Saccharomyces cerevisiae* and *P. pastoris*, with INVSC1 being a commonly used strain in *Saccharomyces cerevisiae* expression systems, and *P. pastoris* strains including X-33, GS115, KM71, MCIO0-3, MD1163, SMD1165, and SMD1168H; baculovirus-insect systems, Insect... Insect cell expression systems, such as the Select stable expression system and the Drosophila expression system, use baculoviruses as expression hosts. Commonly used hosts include Sf9 and Sf21 from the fall armyworm and High-Five and Tn-368 from the white-spotted armyworm. The Drosophila expression system uses S2 Drosophila cells as the expression host. Mammalian cell expression systems commonly use non-lymphocytes such as Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, African green monkey kidney (COS) cells, human kidney cells (293, HEK293), young hamster kidney cells (BHK), and MadIin-Darby canine kidney cell line (MDCK). Suitable expression vectors for mammals include simian vacuolar virus (SV40), adenovirus, adeno-associated virus, human vaccinia virus, baculovirus, coronavirus, Epstein-Barr virus, and herpes simplex virus. Viruses, lentiviruses, polioviruses, retroviruses, Semliki Forest virus, etc.; single plasmid expression vectors such as pcDNA3.1; tetracycline-induced expression vectors such as Tet-On and Tet-Off; pTRE-Tight vector and pTRE-Tight-DsRed2 vector; in vitro expression systems such as Escherichia coli extract, rabbit reticulocyte lysate malt extract, recombinant element system, and malt extract.

[0077] In some preferred embodiments, the methods for introducing the vector into the host cell include, but are not limited to, chemical methods, microinjection, electroporation, and virus-mediated methods.

[0078] In some embodiments, the high-confidence coding DNA comprises at least one nucleotide sequence. Therefore, the method of the present invention further includes constructing multiple vectors for multiple high-confidence coding DNAs, each vector capable of expressing a nanobody. After preparing multiple nanobodies, the method of the present invention further includes identifying one or more characteristics of the nanobodies and selecting the optimal nanobody therefrom.

[0079] In some embodiments, the nanobody is synthesized directly based on the antibody sequence obtained by sequencing.

[0080] In some embodiments, the preparation method further includes identifying one or more characteristics of the nanobody, including equilibrium binding constant, kinetic binding constant, protein stability assay, enzyme activity, or nonspecific binding.

[0081] In some embodiments, the equilibrium binding constant is the dissociation constant (KD). In some embodiments, the equilibrium binding constant is the binding constant (Ka). In some embodiments, the kinetic binding constant is the binding rate constant (kon). In some embodiments, the kinetic binding constant is the dissociation rate constant (koff). In some embodiments, the protein stability measurement is the midpoint denaturation concentration (Cm) of the chemical denaturant.

[0082] In some implementations, identifying one or more characteristics includes performing a binding assay on the nanobody.

[0083] In some implementations, identifying one or more features includes associating the sequencing sequence of the coding DNA library with the binding assay.

[0084] In some embodiments, the binding assay includes at least exposing the immunogen to the nanobody and detecting the interaction between the nanobody and the immunogen.

[0085] In some embodiments, the binding assay includes measuring the binding of the nanobody library to at least one subtype of the immunogen.

[0086] In some implementations, the nanobody kinetics-based detection techniques include, but are not limited to, surface plasmon resonance (SPR), KinExA, or BLI (biomembrane interference) techniques.

[0087] Fifthly, the present invention provides a nanobody or an antigen-binding fragment thereof.

[0088] In some embodiments, the nanobody or its antigen-binding fragment is obtained by the preparation method of the fourth aspect.

[0089] In some embodiments, the nanobody or its antigen-binding fragment is directly synthesized based on the antibody sequence obtained by sequencing.

[0090] In a sixth aspect, the present invention provides a nanobody or antigen-binding fragment thereof that specifically binds to RSV.

[0091] In some embodiments, the nanobody that specifically binds to RSV or its antigen-binding fragment is prepared by the nanobody preparation method described in the fourth aspect.

[0092] In some embodiments, the nanobody that specifically binds to RSV or its antigen-binding fragment comprises:

[0093] (a) CDR1, wherein CDR1 comprises a sequence selected from: SEQ ID NO:34, 38, 42, 46, 50 or 54, or having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO:34, 38, 42, 46, 50 or 54;

[0094] (b) CDR2, wherein CDR2 comprises a sequence selected from: SEQ ID NO:35, 39, 43, 47, 51 or 55, or having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO:35, 39, 43, 47, 51 or 55; and

[0095] (c) CDR3, wherein CDR3 comprises a sequence selected from SEQ ID NO:36, 40, 44, 48, 52 or 56, or having one, two or three amino acid substitutions, deletions or additions compared to SEQ ID NO:36, 40, 44, 48, 52 or 56.

[0096] In some embodiments, the nanobody that specifically binds to RSV or its antigen-binding fragment comprises:

[0097] (1) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35 and CDR3 shown in SEQ ID NO:36;

[0098] (2) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:39 and CDR3 shown in SEQ ID NO:40;

[0099] (3) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43 and CDR3 shown in SEQ ID NO:44;

[0100] (4) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47 and CDR3 shown in SEQ ID NO:48;

[0101] (5) CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:52; or

[0102] (6) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55 and CDR3 shown in SEQ ID NO:56.

[0103] In some embodiments, the nanobody that specifically binds to RSV or its antigen-binding fragment comprises:

[0104] Amino acid sequences such as those shown in any one of SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53 or SEQ ID NO:57; or amino acid sequences that are 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more or more, or 99% or more identical to them.

[0105] In a seventh aspect, the present invention provides a nanobody that specifically binds to TCL1 or an antigen-binding fragment thereof.

[0106] In some embodiments, the nanobody that specifically binds to TCL1 or its antigen-binding fragment is prepared by the nanobody preparation method described in the fourth aspect.

[0107] In some embodiments, the nanobody that specifically binds to TCL1 or its antigen-binding fragment comprises:

[0108] (a) CDR1, wherein CDR1 comprises a sequence selected from: SEQ ID NO:58, 62, 66, 70 or 74, or having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO:58, 62, 66, 70 or 74;

[0109] (b) CDR2, wherein CDR2 comprises a sequence selected from: SEQ ID NO:59, 63, 67, 71 or 75, or having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO:59, 63, 67, 71 or 75; and

[0110] (c) CDR3, wherein CDR3 comprises a sequence selected from SEQ ID NO:60, 64, 68, 72 or 76, or having one, two or three amino acid substitutions, deletions or additions compared to SEQ ID NO:60, 64, 68, 72 or 76.

[0111] In some embodiments, the nanobody that specifically binds to TCL1 or its antigen-binding fragment comprises:

[0112] (1) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59 and CDR3 shown in SEQ ID NO:60;

[0113] (2) CDR1 shown in SEQ ID NO:62, CDR2 shown in SEQ ID NO:63 and CDR3 shown in SEQ ID NO:64;

[0114] (3) CDR1 shown in SEQ ID NO:66, CDR2 shown in SEQ ID NO:67 and CDR3 shown in SEQ ID NO:68;

[0115] (4) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:71, and CDR3 shown in SEQ ID NO:72; or

[0116] (5) CDR1 shown in SEQ ID NO:74, CDR2 shown in SEQ ID NO:75 and CDR3 shown in SEQ ID NO:76.

[0117] In some embodiments, the nanobody that specifically binds to TCL1 or its antigen-binding fragment comprises:

[0118] Amino acid sequences such as those shown in any one of SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:69, SEQ ID NO:73 or SEQ ID NO:77; or amino acid sequences that are 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to them.

[0119] In some embodiments, the nanobody or its antigen-binding fragment described in the fifth, sixth, or seventh aspects further comprises: such as EVQLVESGGGX 13 VX 14 X 15 G or QVQLVESGGGX 13 VX 14 X 15 The starting amino acid sequence shown in G, and / or as in GX 16 GX 17 X 18 VTVSS or GX 16 GX 17 X 18 The terminal amino acid sequence shown in VIVSS, where X 13 Selected from S or L, X 14 Selected from Q, H, K, or P, X 15 Selected from S, A, or P, X 16 Selected from Q, K, or R, X 17 Selected from I or T, X 18 Choose from P, Q, or R.

[0120] In some embodiments, the starting amino acid is an amino acid sequence located at positions 1 to 15 of frame region FR1 (IMGT numbering system), and / or the terminal amino acid is an amino acid sequence located at frame region FR4.

[0121] Eighthly, the present invention provides a nucleic acid molecule that encodes the nanobody or antigen-binding fragment thereof described in the fifth, sixth or seventh aspects.

[0122] In some embodiments, the nucleic acid molecule encodes a nanobody or antigen-binding fragment thereof that specifically binds to RSV as described in the sixth aspect.

[0123] In some embodiments, the nucleic acid molecule encoding the nanobody or its antigen-binding fragment that specifically binds to RSV comprises:

[0124] Nucleotide sequences as shown in SEQ ID NOs:78–83, or nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs:78–83.

[0125] In some embodiments, the nucleic acid molecule encodes the TCL1 nanobody or its antigen-binding fragment as described in the seventh aspect.

[0126] In some embodiments, the nucleic acid molecule encoding a TCL1 nanobody or its antigen-binding fragment specifically binding to it comprises:

[0127] Nucleotide sequences as shown in SEQ ID NOs:84-88, or nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs:84-88.

[0128] In a ninth aspect, the present invention provides an expression vector comprising the nucleic acid molecule described in the eighth aspect.

[0129] In a tenth aspect, the present invention provides a host cell comprising the nucleic acid molecule described in the eighth aspect or the vector described in the ninth aspect. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (e.g., Escherichia coli cells), and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.).

[0130] In some embodiments, the expression vector further includes an expression control element operatively linked to the nucleotide sequence and required for expression (transcription and translation) in a host cell.

[0131] In some embodiments, the expression vector is transfected into host cells, or the expression vector is converted into host cells, or the expression vector is directly expressed in vitro without host cells.

[0132] In one aspect, the present invention provides a conjugate comprising the nanobody described in the fifth aspect, or the nanobody specifically binding RSV or its antigen-binding fragment described in the sixth aspect, or the nanobody specifically binding TCL1 or its antigen-binding fragment described in the seventh aspect, and a conjugate.

[0133] In some embodiments, the conjugate is selected from disease therapeutic molecules, detectable markers, or any combination thereof.

[0134] In some embodiments, the conjugate is selected from detectable markers. Detectable markers can be any substance detectable by fluorescent, phosphorescent, spectroscopic, photochemical, biochemical, immunological, electrical, optical, or chemical means. Such markers are well known in the art and include, but are not limited to, enzymes, fluorescent dyes, radiolabels, luminescent substances, magnetic beads, calorimetric markers, and biotin for binding avidin modified with the marker (e.g., streptavidin).

[0135] In some embodiments, the conjugate is selected from disease therapeutic molecules, enzymes, fluorescent dyes, radiolabeled substances, luminescent substances, magnetic beads, biotin, toxins, cytokines, or any combination thereof.

[0136] In some embodiments, the nanobody or its antigen-binding fragment is optionally conjugated to the conjugate via chemical bonds or linkers.

[0137] In some embodiments, the conjugate is selected from disease therapeutic molecules. In this case, the conjugate of the present invention forms antibody drug conjugates (ADCs) or radionuclide drug conjugates (RDCs) commonly used in the art.

[0138] In some embodiments, the connector is selected from: CC; (G)nC, (G)nCG, (G)nSC, (GGGGS)nC, C(GGGGS)n, (GGGS)nC, C(GGGS)n, (GGS)nC, C(GGS)n, (GGS)nC, C(GGS)n, (G)nCA, (G)nCGA, where n is an integer from 0 to 4; (A)nC, (A)nCA, where n is an integer from 1 to 4; GSCC; CDV; VDC; LPTEG; GGGGCGGGG; GGGGCGGGGA; MPA-AEEA, Val-Cit-PABC, polyethylene glycol PEG, or any combination thereof.

[0139] In a twelfth aspect, the present invention provides a kit comprising the nanobody or antigen-binding fragment thereof as described in the fifth, sixth or seventh aspect, the nucleic acid molecule as described in the eighth aspect, the expression vector as described in the ninth aspect, the host cell as described in the tenth aspect or the conjugate as described in the eleventh aspect.

[0140] In a thirteenth aspect, the present invention provides a pharmaceutical composition comprising the nanobody or antigen-binding fragment thereof as described in the fifth, sixth or seventh aspect, the nucleic acid molecule as described in the eighth aspect, the expression vector as described in the ninth aspect, the host cell as described in the tenth aspect or the conjugate as described in the eleventh aspect, and a pharmaceutically acceptable carrier and / or excipient.

[0141] In a fourteenth aspect, the present invention provides the use of the nanobody or antigen-binding fragment thereof described in the fifth, sixth or seventh aspect, the nucleic acid molecule described in the eighth aspect, the expression vector described in the ninth aspect, the host cell described in the tenth aspect, the conjugate described in the eleventh aspect, the kit described in the twelfth aspect or the pharmaceutical composition described in the thirteenth aspect in the preparation of formulations for the prevention, diagnosis and / or treatment of related diseases.

[0142] Beneficial effects of the present invention

[0143] 1. This invention combines direct coupling screening of positive cells with diverse PCR primer amplification reactions to efficiently obtain high-quality nanobody-encoded DNA libraries with comprehensive and diverse sequences. A single round of PCR reaction can yield a large number of diverse DNA sequences covering a large number of low-abundance sequences.

[0144] 2. Efficiently obtain a diverse and high-quality VHH antibody sequence library: Utilize high-throughput single-cell sequencing technology to rapidly obtain a wide range of VHH antibody sequence information, and through bioinformatics analysis and screening, provide a rich and high-quality sequence resource library for the subsequent efficient preparation of high-affinity antibodies.

[0145] 3. Successfully obtained high-performance VHH antibody products: Based on antibody sequence information, high-affinity and high-specificity high-quality VHH antibodies were accurately screened. These antibodies outperform traditional monoclonal antibodies and have better application prospects.

[0146] 4. Provides key raw materials for the development of high-quality diagnostic kits: The high-performance VHH antibodies obtained through screening can serve as high-quality raw materials, significantly improving the sensitivity and specificity of diagnostic kits.

[0147] 5. Broad application prospects: Rapid antibody preparation technology is not only suitable for the development of diagnostic reagent products, but also has great application potential in the fields of therapeutic antibody drugs and basic biomedical research, which is expected to accelerate scientific research progress and the transformation of results in these fields.

[0148] 6. Shortened antibody preparation cycle: Traditional phage display technology takes several months to prepare nanobodies. By integrating cutting-edge technologies such as cell sorting and high-throughput sequencing, the antibody preparation cycle has been compressed to only one month, improving the efficiency and speed of antibody development.

[0149] 7. Achieve standardized and large-scale production of antibody preparation: This technical route has a simple and standardized operation process, which is easy to achieve large-scale production and can meet the growing needs of antibody development and industrialization in the future.

[0150] 8. This invention provides nanobodies with high affinity and high specificity for RSV and TCL1. The nanobodies of this invention are characterized by low immunogenicity, small molecular weight, strong tissue penetration, and structural stability, and are of great value for the prevention, diagnosis, or treatment of related diseases. Attached Figure Description

[0151] Figure 1 shows a flowchart of nanobody preparation.

[0152] Figure 2 shows the electrophoresis diagram of the PCR product of the VHH antibody gene that specifically binds to the RSV antigen.

[0153] Figure 3 shows the electrophoresis diagram of the purified DNA library.

[0154] Figure 4 shows the DNA library electrophoresis diagrams of six different antigens.

[0155] Figure 5 shows the VHH protein electrophoresis diagram of the purified RSV.

[0156] Figure 6 shows a comparison of the detection results of RSV nanobodies and RSV antigen affinity obtained by the conventional method and the method of the present invention.

[0157] Figure 7 shows a comparison of the detection results of the affinity between TCL1 nanobodies and TCL1 antigen obtained by the conventional method and the method of the present invention.

[0158] Figure 8 shows the detection results of the affinity between RSV nanobodies obtained by the method of the present invention and RSV antigens.

[0159] Figure 9 shows the detection results of the affinity between the TCL1 nanobody and the TCL1 antigen obtained by the method of the present invention. Detailed Implementation

[0160] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. Such structures and techniques have also been described in many publications.

[0161] definition

[0162] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0163] Unless the context clearly indicates otherwise, references to a specific number herein include their plural forms. For example, references to “cell” include one or more such cells and equivalents known to those skilled in the art, etc.

[0164] The term "variable region" as used herein includes "framework region" (FR) and "hypervariable regions." Hypervariable regions are also known as complementarity determining regions or "CDRs." CDRs are primarily responsible for binding to epitopes of the antigen, while the framework region is used to position and align CDRs in three-dimensional space. The precise boundaries of a specific CDR are determined by known numbering methods. The nanobody of this invention comprises three CDRs (CDR1, CDR2, and CDR3). These can be defined, for example, according to the Kabat numbering system, the Chothia numbering system, or the IMGT numbering system. For a given antibody, those skilled in the art will readily identify the CDRs defined by each numbering system. Furthermore, the correspondence between different numbering systems is well known to those skilled in the art.

[0165] The terms "single-domain antibody (VHH)" and "nanobody (Nb)" used in this article are interchangeable and have the meanings commonly understood by those skilled in the art. They refer to antibody fragments composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), which maintain the ability to specifically bind to the same antigen bound by the full-length antibody. Typically, antibodies lacking the light chain and heavy chain constant region 1 (CH1) are first obtained from alpaca immune serum, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one heavy chain variable region. VHHs have a molecular weight of 15-18 kDa, and their crystal structure is 2.5 nm wide and 4.8 nm long, exhibiting a "nanoscale" size, hence the name nanobody. Furthermore, compared to traditional antibody VHs, VHHs have a longer CDR3 region. The average length of the CDR3 region in human and mouse antibody VHs is 9-12 amino acids, while the CDR3 region of VHHs is typically 16-18 amino acids.

[0166] In some embodiments, the CDR3 length of the nanobody of the present invention is 10-35 amino acids, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids. In some embodiments, the CDR3 length of the nanobody of the present invention is 16-25 amino acids. In some preferred embodiments, the CDR3 length of the nanobody of the present invention is 16-18 amino acids.

[0167] In some embodiments, the total length of the nanobodies of the present invention can be 115-135 amino acids, preferably 123-130 amino acids. However, the portions, fragments, or like of the nanobodies are not limited to their length and / or size.

[0168] In this article, unless the context clearly indicates otherwise, the term “nanobody” includes both the complete nanobody and the antigen-binding fragment of the nanobody.

[0169] The “antigen-binding portion” and “antigen-binding fragment” of an antibody, as used in this article, refer to polypeptides containing fragments of nanobodies, including any naturally occurring, enzymatically obtainable, chemically synthesized, or genetically engineered polypeptides or glycoproteins that specifically bind to antigens to form complexes.

[0170] The twenty common amino acids discussed herein are written in accordance with conventional usage. See, for example, Immunology: A Synthesis (2nd Edition, E.S. Golub and D.S. Green, Sinauer Associates, Sunderland, MA. (1991)), which is incorporated herein by reference. In this invention, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented as A or Ala.

[0171] As used in this article, "immunogen" or "antigen" refers to one or more molecules or parts thereof that can be bound by nanobodies, and which can further induce an animal to produce antibodies that bind to the epitopes of the immunogen. "At least one immunogen" in this article means immunizing the same animal with at least one molecule simultaneously to induce antibody production in the same animal.

[0172] The peripheral blood mononuclear cells (PBMCs) used in this article include T lymphocytes, B lymphocytes, and monocytes. Their volume, morphology, and density differ from other cells. Red blood cells and polymorphonuclear leukocytes have a relatively high density, around 1.090, while lymphocytes and monocytes have a density of 1.075–1.090, and platelets have a density of 1.030–1.035. Density gradient centrifugation was performed using a near-isotonic solution (layering solution) with a density between 1.075 and 1.092 to separate cells according to their density gradient.

[0173] As used herein, “polynucleotide” or “nucleic acid” (e.g., in relation to the polypeptides, antibodies, or antibody cDNAs described herein) refers to a polymer composed of multiple nucleotide units (ribonucleotides or deoxyribonucleotides or related structural variants) linked by phosphodiester bonds. Polynucleotides or nucleic acids can be of any length, typically ranging from about 6 nucleotides to about 10. 9 Nucleotides or larger. Polynucleotides or nucleic acids include RNA, DNA, cDNA, and genomic DNA.

[0174] As used herein, "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of the protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site. For example, the pMES4 vector is an E. coli expression vector. The Tac strong promoter can drive the fusion expression of the GST lysing tag and the target gene. The LacI inhibitor protein and the LacO operon prevent the plasmid from being expressed before the addition of IPTG, thus preventing it from affecting bacterial growth.

[0175] The term "B cell" or "B lymphocyte" as used in this article refers to pluripotent stem cells derived from bone marrow. B cells possess various membrane surface molecules that recognize antigens and interact with immune cells and molecules, serving as crucial evidence for the isolation and identification of B cells. Based on immunological knowledge, when humans and animals are stimulated by exogenous immunogens such as bacteria, viruses, and non-homologous proteins, acquired immunity is activated. In the synergistic action of immune cells such as macrophages, dendritic cells, and T lymphocytes, B lymphocytes produce immunoglobulins targeting the pathogen (or immunogen). After a series of maturation and differentiation processes, B lymphocytes ultimately express the B cell receptor (BCR) for the recognition of specific antigens (or immunogens). Therefore, through screening for B lymphocyte surface markers and immunogen specificity, single B lymphocytes targeting specific antigens can be obtained.

[0176] The term "cell sorting" or "cell screening" as used in this article refers to the separation of a single cell type from a multicellular sample. Cell sorting includes, but is not limited to: flow cytometry screening (FACS), which selectively separates specific cell subpopulations based on their physical characteristics (such as size and particle size) or chemical characteristics (such as antigen expression); magnetic cell sorting (MACS), where magnetic microbeads bind to specific antigens on the cell surface, and labeled cells are separated from unlabeled cells by a magnetic field gradient; and microfluidic sorting, which achieves cell focusing, sorting, and enrichment by adjusting the fluid flow in microfluidic channels. Microfluidic single-cell sorting methods include inertial sorting, electrophoretic sorting, optical sorting, magnetic sorting, and acoustic sorting.

[0177] The "fluorescent labeling technique" used in this article refers to the use of fluorescent substances covalently bound or physically adsorbed onto a specific group of the molecule under study, utilizing its fluorescence properties to provide information about the object of study. This technique features high sensitivity, high stability, and high selectivity.

[0178] In some embodiments, the fluorescent dyes include, but are not limited to, fluorescein dyes and their derivatives (e.g., including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc., or their analogues), rhodamine dyes and their derivatives (e.g., including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc., or their analogues), C-series dyes and their derivatives (e.g., including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy3, etc., or their analogues), and Alexa-series dyes and their derivatives (e.g., including but not limited to Alexa dyes). Fluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 633, 647, 680, 700, 750, etc. or their analogues) and protein dyes and their derivatives (e.g., including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), polydinoflavin-chlorophyll protein (preCP), etc.).

[0179] The term "flow cytometry cell sorting" as used in this article refers to a single-cell fluorescence sorting technique where cells to be tested are stained with a specific fluorescent dye and placed in a sample tube. The cells are dispersed into individual particles by a flowing sheath fluid. When the single-cell suspension passes through a laser channel, its fluorescence signal is collected and displayed. Combined with fluorescent labeling technology, flow cytometry cell sorting can separate target cells from the whole cell while maintaining high biological activity, making it a powerful tool for biological screening.

[0180] Current technology uses biotin-labeled antigens coupled with streptavidin and fluorescent dyes to indirectly label positive cells for cell sorting. Indirect labeling not only requires more manipulation and washing steps, increasing experimental time and repetitive operations, leading to cell loss; it also suffers from non-specific binding, increasing background signal and causing cell sorting errors, thus increasing the false positive rate; more importantly, streptavidin has a large molecular weight, which may affect the activity of the antigen or its binding to positive cells after coupling with the antigen.

[0181] In some embodiments, flow cytometry is used to sort positive cells. Specifically, in the flow cytometer, PBMC cells are sorted using an immunogen directly conjugated to a fluorescent dye to obtain positive cells. This invention uses a directly conjugated small-molecule dye to label positive cells, which does not create a shielding effect on the structure or binding site of the antigen. It involves fewer steps, saves time, reduces non-specific binding, effectively lowers the false-positive rate of cell sorting, improves the specificity of labeling, and achieves targeted screening of specific cells.

[0182] The term "initiation region" as used in this article refers to a signaling region that promotes translation initiation and serves as a ribosome binding site.

[0183] The term "signal peptide" as used in this article refers to a short amino acid sequence (i.e., signal peptide) that appears at the NH2-terminus of certain proteins and is usually transported by the cell to non-cytoplasmic locations (such as secretion) or becomes a membrane component.

[0184] As used in this article, "polymerase chain reaction (PCR)" refers to a widely accepted and practiced laboratory method used to replicate or amplify the concentration of nucleic acids (NAs) such as DNA in a test tube. Replication / amplification occurs in an aqueous solution containing a specific concentration of DNA molecules. Pre-defined amounts of polymerase, primers, and triphosphates or substrates of four nucleic acids are added to the aqueous solution, followed by three thermal steps called denaturation and annealing / extension; or the annealing and extension steps are combined into two thermal steps: denaturation and annealing.

[0185] As used in this article, a "primer" refers to a natural or synthetic nucleotide sequence that, when combined with a polynucleotide template to form a double helix, serves as the starting point for nucleic acid synthesis and extends from its 3' end along the template to form an extended double helix. The nucleotide sequence added during extension is determined by the sequence of the template polynucleotide. Primers can be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more nucleotides. Primers can be single-stranded or double-stranded.

[0186] The terms "forward" and "reverse" used in this article, as commonly understood by those skilled in the art, are merely for the purpose of describing and distinguishing the two primers in a primer pair; they are relative and do not have any particular meaning.

[0187] The term "DNA" as used in this article refers to deoxyribonucleic acid. DNA can be single-stranded or double-stranded. DNA typically contains four nucleotides: cytosine (C), guanine (G), adenine (A), and thymine (T).

[0188] This document provides DNA molecular sequences comprising one or more degenerate nucleotides. As used herein, a “degenerate nucleotide” refers to a nucleotide or a series of nucleotides capable of performing the same function or producing the same output as a structurally different nucleotide. Non-limiting examples of degenerate nucleotides include G or T nucleotides (K); A or C nucleotides (M); A or G nucleotides (R); G or C nucleotides (S); A or T nucleotides (W); C or T nucleotides (Y); A or C or T nucleotides (H); A or C or G nucleotides (V); G or C or T nucleotides (B); A or G or T nucleotides (D); or A, T, C, or G nucleotides (N). The inventors obtained a richer and more comprehensive DNA library, achieving sequence diversity, by performing overlap extension PCR on various mutant fragments of the antibody variable region.

[0189] The term "identity" as used in this article refers to the sequence matching between two polypeptides or two nucleic acids. The percentage identity between two sequences is determined by the number of identical positions shared by the sequences (i.e., percentage identity = number of identical overlapping positions / total number of positions × 100%). It can be measured using sequence comparison algorithms or by visual inspection.

[0190] In some embodiments, the present invention employs nested PCR to amplify nucleic acid samples derived from the positive cells. Nested PCR generally requires two rounds of PCR reactions. In the first round, a set of outer primers is used to obtain a longer amplification product, and in the second round, one or more sets of inner primers are used, with the PCR product from the first round as a template for the PCR reaction.

[0191] In some embodiments, the reaction procedure for nested PCR is shown in Tables 2 and 3.

[0192] Table 2. First-round procedure for nested PCR:

[0193] Table 3. Second round procedure for nested PCR:

[0194] In some implementations, the amplification is performed using a single round of PCR to amplify a nucleic acid sample derived from the positive cells.

[0195] The term "respiratory syncytial virus (RSV)" as used in this article refers to an RNA virus belonging to the genus Pneumovirus of the family Paramyxoviridae. It is divided into two subtypes, A and B. Although there are minor antigenic differences between subtypes A and B, most studies have found no significant differences between the two subtypes. The nucleotide and amino acid identity of the N gene within the same subtype can reach over 95%. RSV infection is the leading cause of hospitalization for viral respiratory infections in infants and young children, seriously endangering children's health, especially premature infants, infants with congenital heart disease, or those with primary immunodeficiency.

[0196] In this article, "T-Cell Leukemia / Lymphoma Protein 1A," "TCL1A," or "TCL1" refers to a signal transduction protein composed of 99 amino acids with a molecular weight of approximately 14 kDa. It is encoded by the TCL1 gene located on chromosome 14. The TCL1 gene acts as a proto-oncogene in leukemia and plays a crucial role in human hematologic malignancies. The TCL1 protein participates in various cellular biological processes, including cell proliferation, apoptosis, and signal transduction.

[0197] As used herein, "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen or immunogen. The specific binding properties between molecules can be determined using methods known in the art. The strength or affinity of a specific binding interaction can be expressed using the equilibrium dissociation constant (KD). KD values ​​can be measured using any effective method. In this invention, the term "KD" refers to the dissociation constant of a specific antibody-antigen interaction, used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the stronger the antibody-antigen binding and the higher the affinity between the antibody and the antigen. The dissociation constant can be measured using surface plasmon resonance (SPR), or alternatively, biomembrane interferometry or KinExA. In some embodiments, affinity is measured by competitive radioimmunoassay. In some embodiments, affinity is determined by ELISA. In some embodiments, the affinity KD is measured using surface plasmon resonance.

[0198] Phage display, as used in this article, is a widely applied technique across various biological disciplines. Phage display is used in peptide (i.e., polypeptide) library screening protocols, such as screening for peptide ligands and monoclonal antibodies. In this technique, a DNA library is cloned as a genetic fusion with a gene encoding a phage capsid protein. When the fusion protein is expressed and integrated into the viral capsid, the polypeptide library is "displayed" on the surface of the virus. This configuration establishes a physical link between the displayed peptide and its encoding DNA, allowing for library amplification in the bacterial host and convenient peptide sequencing after several rounds of target-directed selection.

[0199] As used in this article, “nucleic acid sequencing” refers to determining the nucleotide sequence in a nucleic acid molecule or a sample of nucleic acid molecules.

[0200] As used in this article, "high-throughput sequencing or next-generation sequencing (NGS)" refers to high-throughput sequencing methods that allow for the parallel sequencing of millions to billions of molecules. Examples of next-generation sequencing methods include sequencing by synthesis, ligation sequencing, hybridization sequencing, polymerase cloning sequencing, ion semiconductor sequencing, and pyrosequencing. By ligating primers to a solid matrix and attaching complementary sequences to nucleic acid molecules, the nucleic acid molecules can hybridize with the solid matrix via primers. Then, polymerases amplify the molecules by generating multiple copies in discrete regions on the solid matrix (these clusters are sometimes called polymerase clones). Therefore, during the sequencing process, nucleotides at a specific location can be sequenced multiple times (e.g., hundreds or thousands of times)—this depth of coverage is called "deep sequencing." Examples of high-throughput nucleic acid sequencing technologies include platforms provided by Illumina, BGI, Qiagen, Thermo-Fisher, and Roche, including formats such as parallel bead arrays, sequencing synthesis, ligation sequencing, capillary electrophoresis, electronic microarrays, “biochips,” microarrays, parallel microarrays, and single-molecule arrays (see, for example, RFService, The Race for the $1000 Genome, Science, 311, 1544–1546).

[0201] In some implementations, sequencing of the encoding DNA library includes sequencing via Illumina sequencing. Illumina sequencing is based on a technique called “bridge amplification,” in which DNA molecules (approximately 500 bp) with appropriate adapters at each end are used as substrates for repeated amplification synthesis reactions on a solid-phase vector containing oligonucleotide sequences complementary to the adapters. The oligonucleotides on the vector are spaced apart so that the DNA is then amplified repeatedly for several rounds, creating clonal “clusters” consisting of approximately 1000 copies of each oligonucleotide fragment. Each vector can include millions of parallel cluster reactions. During the synthesis reaction, modified nucleotides, each corresponding to one of the four bases and with a different fluorescent label, are incorporated and then detected. The nucleotides also act as synthesis terminators for each reaction, opening the next round of synthesis upon detection. The reaction is repeated 300 rounds or more. Compared to camera-based imaging, the use of fluorescence detection increases detection speed due to direct imaging.

[0202] In this article, "Q30" refers to a quality value of 30 for a single base in the sequencing data, which also indicates that the error rate for that base is 0.1% and the accuracy rate is 99.9%. It is an important indicator for measuring sequencing quality.

[0203] As used in this article, "diagnosis" refers to the determination of the presence or nature of a pathological condition, including but not limited to viral infections (such as RSV infection) or the occurrence or development of a tumor. Diagnostic methods vary in sensitivity and specificity. "Sensitivity" refers to the percentage of diseased individuals who test positive (the percentage of true positives); "specificity" refers to the minimum false positive rate, defined as the proportion of disease-free individuals who test positive. While a particular diagnostic method may not provide a definitive diagnosis, it may provide positive indicators that aid in diagnosis.

[0204] The nanobodies or conjugates thereof of the present invention can be used for diagnostic applications, such as for detecting samples to provide diagnostic information. In some embodiments, the samples used include cells, body fluids (e.g., blood, serum, plasma, extracellular fluid, tissue fluid, lymph, cerebrospinal fluid, aqueous humor), excreta (e.g., urine, feces, saliva, sputum), tissue samples, and biopsy specimens.

[0205] In practical use, those skilled in the art can select appropriate markers based on detection conditions or actual needs. Regardless of the marker used, it falls within the protection scope of this invention.

[0206] In some embodiments, the enzymes include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and glucose-6-phosphate deoxygenase.

[0207] In some embodiments, the radioactive markers include, but are not limited to, those mentioned above. 212 Bi、 131 I, 111 In、 90 Y、 186 Re、211At、 125 I, 188 Re、 153 Sm、 213 Bi、 32 P, 94 mTc, 99 mTc, 203 Pb, 67 Ga、 68 Ga、 43 Sc、 47 Sc、 110 mIn, 97 Ru、 62 Cu、 64 Cu、 67 Cu、 68 Cu、 86 Y、 88 Y、 121 Sn、 161 Tb, 166 Ho、 105 Rh、 177 Lu、 172 Lu and 18 F.

[0208] In some embodiments, the magnetic beads include, but are not limited to, nanoparticles, colloids, organic nanoparticles, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0209] As used herein, "pharmaceutically acceptable carriers and / or excipients" refers to carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient, and are well known in the art. This includes, but is not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents for maintaining osmotic pressure, agents for delaying absorption, or preservatives. Those skilled in the art will select appropriate pharmaceutically acceptable carriers and / or excipients for the formulation of pharmaceutical compositions.

[0210] The pharmaceutical composition of the present invention contains a therapeutically effective amount (e.g., 0.001–99 wt%, preferably 0.01–90 wt%, more preferably 0.1–80 wt%) of the nanobody of the present invention. "Therapeutically effective amount" refers to the dosage required to produce the desired effect. The precise amount depends on the therapeutic purpose and can be determined by a physician, taking into account the patient's (subject's) age, weight, condition, degree of infection or metastasis, and individual differences in the disease.

[0211] As used herein, “treatment” means improving one or more existing symptoms or clinical signs associated with the condition, including reducing or improving the progression, severity, and / or duration of existing symptoms or clinical signs. As used herein, “prevention” means limiting the development and / or occurrence of symptoms or clinical signs of the condition to any extent. In some embodiments, prevention also includes treatment in cases of recurrence.

[0212] In some implementations, the symptoms or clinical signs include the development or onset of upper and / or lower respiratory tract RSV infection or related respiratory symptoms in the subject; progression of upper respiratory tract RSV infection to lower respiratory tract RSV infection or related respiratory symptoms caused by the administration of a therapy (e.g., prophylactic or therapeutic agent); and / or, symptoms of respiratory symptoms related to RSV infection (e.g., asthma, wheezing, or a combination thereof).

[0213] In some embodiments, the symptoms or clinical signs include hematologic malignancies such as leukemia and lymphoma. The leukemia includes acute leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocytic, monocytic, and erythroleukemia), chronic leukemia (e.g., chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (painless and high-grade forms), multiple myeloma, Waldenström's macroglobulinemia, heavy chain disease, myelodysplastic syndromes, hairy cell leukemia, and spinal dysplasia.

[0214] The technical route of this invention is shown in Figure 1. First, alpacas are immunized with purified immunogens (e.g., RSV and TCL1). Blood is collected from the alpaca's neck, and peripheral blood lymphocytes are isolated. Flow cytometry is used to sort out positive B cells that specifically bind to the immunogen. The nucleic acid mRNA of the positive cells is extracted and isolated, and then reverse transcribed to generate the corresponding cDNA. Subsequently, multiple primers of this invention, in different combinations, are used for amplification and library construction. High-throughput sequencing technology and bioinformatics analysis are then used for sequence screening and identification, obtaining the DNA sequence of nanobodies that specifically bind to the corresponding antigens in a short time. Finally, the nanobodies are expressed and purified in a suitable expression system, and their affinity is tested. The obtained antibodies can be used for the treatment or prevention of diseases.

[0215] Through in-depth research and optimization of reaction conditions, the inventors discovered that directly conjugating immunogens with fluorescent dyes to sort PBMC cells yields positive cells with higher purity. Building on this, the inventors utilized different combinations of forward primers with nucleotide sequences shown in SEQ ID NOs:1–5 and reverse primers with nucleotide sequences shown in SEQ ID NOs:20–25 to obtain nanobody-encoding DNA libraries via a single-round PCR reaction. Due to the primer design and simplified PCR reaction conditions, the obtained nanobody-encoding DNA sequences exhibit low repetition, ensuring the acquisition of more low-abundance DNA sequences, thus resulting in a more comprehensive and diverse nanobody-encoding DNA library. Ultimately, during high-throughput sequencing, the sequencing data does not need to be particularly large to obtain the DNA sequences of all nanobody-encoding DNA libraries, laying the foundation for screening high-reliability encoding DNA and obtaining high-quality nanobodies. Furthermore, a series of nanobodies specifically binding to RSV and TCL1 were obtained, exhibiting high binding activity with their corresponding antigens and superior neutralizing activity.

[0216] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments and drawings are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention.

[0217] Example

[0218] Unless otherwise specified, the materials, reagents, or instruments used in the examples are all commercially available conventional products.

[0219] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field (e.g., refer to J. Sambrook's "Molecular Cloning: A Laboratory Manual", 4th edition, Science Press) or the product instructions shall be followed.

[0220] Example 1. Preparation of respiratory syncytial virus (RSV) antigen and T-cell leukemia / lymphoma protein 1 (TCL1)

[0221] DNA sequences encoding RSV and TCL1 antigens were inserted into the pET28a vector using molecular cloning techniques, with six consecutive histidine (his) residues in the vector serving as affinity purification tags. The recombinant plasmid was transformed into the *E. coli* BL21(DE3) expression system, and single colonies were picked and inoculated into 20 mL of LB medium, incubated overnight at 37°C. The following day, the culture was transferred to 1 L of LB medium for expansion. When the bacterial culture reached OD... 600When the concentration reaches 0.8, add IPTG inducer to a final concentration of 1 mM and continue incubation overnight at 16°C. Collect the cell pellet by centrifugation at 8000 rpm for 10 minutes and remove the supernatant. Resuspend the cell pellet in 30 mL of 1×PBS buffer and sonicate for 10 minutes; centrifuge at 8000 rpm for 10 minutes and collect the supernatant; filter the supernatant through a 0.45 μm filter membrane and co-incubate the filtered supernatant with a equilibrated nickel affinity chromatography column to bind the target protein. Optimize different concentrations of imidazole solution to remove non-specific binding proteins and improve the purity of the target protein. For example, 5 column volumes of 20 mM imidazole solution remove weakly binding contaminants; 5 column volumes of 50 mM imidazole solution are used for further washing to remove medium-affinity contaminants. To obtain optimal protein elution, imidazole solutions of increasing concentrations were used, such as 3 column volumes of 100mM, 150mM, 200mM, and 250mM imidazole solutions. The eluent was collected, concentrated, and enriched to obtain high-purity RSV and TCL1 antigens, ensuring the integrity and accuracy of the antigen molecular conformation and antigen purity ≥95%. The concentration was then adjusted to 1 mg / mL for use in alpaca immunization and antibody-antigen affinity detection.

[0222] Example 2. Preparation of antibodies by immunizing alpacas

[0223] Purified RSV and TCL1 antigens were emulsified with Freund's adjuvant at a ratio of 1:1:2 and administered to the underside of the alpaca neck. A multiple immunization strategy was employed, with immunizations every two weeks for a total of four immunizations. One week after the fourth immunization, 150 mL of peripheral blood was collected from the alpaca. Peripheral blood mononuclear cells (PBMCs) were separated by Ficoll density gradient centrifugation, washed with 5 volumes of 1×PBS buffer, and collected by centrifugation at 2000 rpm for 5 minutes. The cell pellet was resuspended in 2 mL of 1×PBS buffer and counted; the total count was 3.5 × 10⁻⁶ cells / mL. 8 Each cell.

[0224] Example 3. Sorting of B cells with antigen-specific labels

[0225] 10 μL of RSV antigen labeled with 488 fluorescein molecules and 10 μL of TCL1 antigen labeled with 560 fluorescein molecules were incubated with 2 mL of PBMC cells on ice in the dark for 30 minutes. The cells were washed twice with 5 mL of 1×PBS buffer to prepare flow cytometry-sorted samples. Unstained cells were used as a negative control. Positive cells were collected at 488 nm and 560 nm using laser excitation. The sorted RSV and TCL1 positive B cells were centrifuged to collect the precipitate for RNA extraction.

[0226] Example 4. Obtaining cDNA template

[0227] Total RNA was extracted using an RNA recovery column. Cell lysis buffer was added to the collected RSV and TCL1-positive B cell pellet, every 10... 5 Add 350 μL of Lysis buffer to each cell (following the manufacturer's instructions for the extraction kit, Omega Bio-tek, catalog number: R6831-01), lyse the cells on ice, and centrifuge at 12,000 rpm for 5 min. Collect the supernatant and add it to an RNA extraction column for RNA adsorption and elution to obtain total RNA, with a concentration of approximately 200-300 ng / μL. Verify the integrity and stability of the extracted RNA using nucleic acid electrophoresis.

[0228] Subsequently, using a reverse transcription kit (Thermo Fisher Scientific, catalog number: 18064014), 1 μL of reverse transcriptase II, 3 μg of mRNA, 10 μL of 5× reverse transcription buffer, 1 μL of 10 mM dNTP mix, 1 μL of 100 mM DTT, 1 μL of RNase inhibitor, 1 μL of 50 μM Oligo(dT) primer, and DEPC-treated water were added, with a total volume of 50 μL. The reverse transcription reaction program was set as follows: 25℃ for 5 minutes, 42℃ for 60 minutes, 50℃ for 30 minutes, and 70℃ for 10 minutes, ultimately obtaining RSV and TCL1 cDNA, which were used as templates for subsequent PCR amplification of the VHH gene.

[0229] Example 5. Obtaining the VHH antibody gene

[0230] (1) Design multiple specific primers (synthesized by Ruiboxing Biotechnology Co., Ltd.), mix them in equal proportions according to different combinations. The primer sequences and mixing schemes are shown in Table 4. During amplification, different combinations of forward and reverse primers are performed. The combination schemes are shown in Table 5. The forward and reverse primers are randomly combined to fully ensure the diversity of the VHH encoded DNA library.

[0231] (2) A PCR reaction system was established based on the primer set and cDNA template, as shown in Table 6.

[0232] (3) After vortexing and centrifuging the prepared reaction system, place it in a PCR instrument and run the program. The PCR reaction program is shown in Table 7. The VHH antibody gene is amplified through one round of PCR to obtain the VHH DNA fragment (about 400bp).

[0233] (4) The electrophoresis results are shown in Figure 2. Lanes 1-18 represent different primer combinations (specific combinations are shown in Table 5) to amplify the VHH fragment obtained by PCR amplification of cDNA templates from RSV antigen-positive cells, proving that the amplification efficiency and the size of the target fragment are similar for different primer combinations.

[0234] (5) Cut the gel and recover the target band using Beckman Agencourt AMPure XP Beads (follow the manufacturer's instructions for recovery kit):

[0235] a. Add 0.8× Beckman XP magnetic beads to the PCR product, mix well, and let stand at room temperature for 5 min;

[0236] b. Remove the supernatant, add 200 μL of 80% anhydrous ethanol, let stand for 30 seconds, and remove the supernatant;

[0237] (Step b is repeated twice)

[0238] c. Air dry at room temperature for 1-2 minutes, add DEPC water, and let stand for 5 minutes;

[0239] d. Qubit4 was used to measure DNA concentration, and nucleic acid electrophoresis was used to verify purity. The purity and integrity of the VHH antibody gene amplification fragment were detected to perform quality control for subsequent next-generation sequencing.

[0240] The electrophoresis results are shown in Figure 3. It can be seen that the bands of the RSV and TCL1-encoded DNA libraries are neat and the libraries are intact. The concentrations determined by Qubit4 were 38 ng / μL for the RSV-encoded DNA library and 40 ng / μL for the TCL1-encoded DNA library.

[0241] Figure 4 shows the electrophoresis results of DNA libraries obtained from different antigen samples, with lanes 1-6 representing six different antigens. FluA represents Influenza A virus; FluB represents Influenza B virus; HPIV-1 represents Human Parainfluenza Virus type 1; HPIV-3 represents Human Parainfluenza Virus type 3; RSV and TCL1 are as described above.

[0242] Table 4 Primer sequences and mixing schemes

[0243] Table 5 Primer Combination Scheme

[0244] Table 6 PCR amplification reaction system

[0245] Table 7 PCR reaction procedure

[0246] Example 6. High-throughput sequencing and bioinformatics analysis

[0247] High-throughput sequencing of VHH-encoding DNA libraries was performed using the Illumina next-generation sequencing platform. Quality control was performed before sequencing and after data splitting and quality control were conducted to ensure the accuracy of the sequencing data and a Q30 greater than 80%. The sequencing data were assembled into a complete fastq file using FLASH (Fast Length Adjustment of Short Reads) software, and then converted into a FASTA file using seqkit software. VHH antibody sequences were annotated locally using Igblast, and CDR3 sequences were extracted and their frequency calculated. The VHH phylogenetic tree was analyzed using iTOL (Interactive Tree of Life) software to screen for high-confidence VHH sequences. Combining iTOL with phylogenetic tree construction, heatmap generation, and cluster analysis, the conservation and diversity of VHH sequences were comprehensively evaluated, and high-confidence sequences were selected for subsequent experimental validation.

[0248] Example 7. VHH antibody expression and purification

[0249] Candidate VHH antibody sequences were constructed into a pMES4 vector containing the pelB signal peptide. Mature antibodies expressed in *E. coli* migrate to the periplasmic space (the narrow space between the outer membrane and the cell wall), ensuring antibody activity. The vector plasmid containing the VHH coding sequence was transformed into *E. coli* BL21. Single colonies were picked and cultured overnight, then scaled up to 500 mL of LB medium. When the bacterial culture reached OD... 600When the concentration reaches 1.0, add a final concentration of 1 mM IPTG inducer, incubate overnight at 16°C, and collect the bacteria by centrifugation at 8000 rpm for 15 min. Lyse the *E. coli* cell wall with 25% sucrose solution, rotate at 4°C for 1-2 hours, and centrifuge at 8000 rpm for 10 min to obtain the VHH antibody protein solution in the periplasmic space. Purify the VHH antibody using His-tagged affinity chromatography. Wash with 5 volumes of 20 mM and 50 mM imidazole to remove impurities, then elute the target antibody with 3 volumes of 100 mM, 150 mM, and 250 mM imidazole. After centrifugation at 4000 rpm for 10 min, replace the 1×PBS buffer and concentrate the antibody. SDS-PAGE electrophoresis and Coomassie brilliant blue staining were used to detect the purity of VHH antibodies, ensuring a purity ≥95%. The protein electrophoresis results are shown in Figure 5. In Figure 5, 1: RSV-3, 2: RSV-24, 5: RSV-1, 6: RSV-26, and 7: RSV-24 are RSV VHH antibodies with different sequences obtained by the method of this invention; 3: RSV-E10, 4: RSV-P7, 8: RSV-A2, 9: RSV-E3, and 10: RSV-D5 are RSV VHH antibodies with different sequences obtained through traditional phage display technology as a comparison. As can be seen from Figure 5, the purified RSV VHH antibody protein is approximately 15 kDa, consistent with the design, and has a purity ≥95%.

[0250] Example 8. VHH antibody affinity detection and screening

[0251] Using surface plasmon resonance (SPR) technology, RSV antigen or TCL1 antigen was coupled to a CM5 chip for multi-cycle kinetic assays to evaluate the affinity between VHH antibodies and antigens. Antibodies with an affinity of approximately 10 were initially screened. 8 The affinity assay results for antibodies screened using high-throughput sequencing technology are compared with those obtained using traditional phage display technology, as shown in Figures 6 and 7. The affinity assay results for RSV and TCL1 nanobodies screened using high-throughput sequencing technology are shown in Figures 6A and 7A, while the affinity assay results for RSV and TCL1 nanobodies screened using traditional phage display technology are shown in Figures 6B and 7B. Traditional methods typically achieve nanobodies with an affinity of around 10. -7 M(RSV-E10, RSV-P7; TCL1-2D10, TCL1-2G9), while the nanobodies obtained by combining high-throughput sequencing technology have an affinity as high as 10. -8The affinity of nanobodies for RSV (RSV-3, RSV-24; TCL1-37, TCL1-44) using high-throughput sequencing was increased by approximately one order of magnitude. Figure 8 shows the affinity detection results for RSV nanobodies screened using high-throughput sequencing, and Table 8 shows the specific data. Table 8 shows that the affinity detection results for RSV nanobodies screened using high-throughput sequencing were all high. Figure 9 shows the affinity detection results for TCL1 nanobodies screened using high-throughput sequencing, and Table 9 shows the specific data. Table 9 shows that the affinity detection results for RSV nanobodies screened using high-throughput sequencing were all high.

[0252] Table 8. Results of RSV nanobody affinity assay

[0253] Table 9. Affinity test results of TCL1 nanobodies

[0254] The present invention provides a rapid nanobody preparation method to obtain nanobodies with high affinity and specific binding to RSV antigen and TCL1 antigen. The RSV nanobodies are then applied to a colloidal gold rapid diagnostic kit. This method is simple to operate, has a short cycle time, and can significantly improve the early diagnostic efficiency of respiratory syncytial virus infection, showing broad application prospects.

[0255] As can be seen from the examples, the method for constructing nanobody-encoded DNA libraries of the present invention is universal, enabling the simultaneous screening of positive cells and the acquisition of nanobody-encoded DNA libraries by nanobodies generated against more than one immunogen. Furthermore, by combining this method with a nanobody preparation technology based on high-throughput sequencing, the cycle of traditional antibody preparation is significantly shortened. This method is simple to operate, easy to standardize and scale up production, and conducive to the formation of a complete industrial chain. Through the optimized technical route, nanobody-encoded DNA libraries are obtained efficiently, and high-affinity, highly specific, and high-quality nanobodies are screened for acquisition. This provides key raw materials for the development of highly sensitive and specific diagnostic kits, and also has broad application prospects in the fields of therapeutic antibody drug development and early disease diagnosis. The establishment of this technology provides strong technical support for accelerating the application of nanobodies in medical diagnosis and treatment, and is expected to significantly improve the efficiency of disease diagnosis and treatment. Surface plasmon resonance (SPR) assays clearly showed that the binding signals of nanobodies screened by high-throughput sequencing to antigens such as RSV and TCL1 were significantly higher than those screened by phage display (Figures 6 and 7), fully demonstrating the significant advantages of high-throughput sequencing technology in screening high-affinity antibodies. The acquisition of high-affinity nanobodies not only helps to significantly improve the sensitivity and specificity of RSV and TCL1 diagnostic kits, but also provides high-quality candidate molecules for the development of next-generation, highly effective therapeutic antibody drugs against RSV and other pathogens, showing broad application prospects.

[0256] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for constructing a nanobody-encoded DNA library, the method comprising: The peripheral blood mononuclear cells (PBMCs) of animals were screened using an immunogen to obtain positive cells that specifically bind to the immunogen. as well as The nucleic acid sample derived from the positive cells was amplified to obtain the DNA library encoding the nanobody; The nanobody described herein is a nanobody that specifically binds to the immunogen, and The amplification includes the use of one or more primer pairs, each of which contains one or more forward primers and one or more reverse primers.

2. The method according to claim 1, characterized in that, The sequence of each of the one or more forward primers is selected from NNNNNWCYRGAGAWKTCGCGGCCCAGCXXXXX (SEQ ID NO:1), NNNNNNCAGTTCAACAGTGGTCCTGGCTXXNX (SEQ ID NO:2), NNNNNNAGATTTCGCGGCCCAGXXXXXX (SEQ ID NO:3), NNNNNNTCGGCGCGCCGAGGXXNX (SEQ ID NO:4), NNNNNNGGTGGTCCTGGCTGC (SEQ ID NO:5), or an added nucleotide sequence having a total of no more than 40, 35, 30, 25, 20, or 15 nucleotides at its 5' end and / or 3' end; and / or The sequence of each of the one or more reverse primers is selected from NNNNNNNNGCGCACCTGCGGCCGC (SEQ ID NO:20), NNNNNNNNGGAGACAGTGACCAGG (SEQ ID NO:21), NNNNNNNNCTTGGGTTCTGAGGA (SEQ ID NO:22), NNNNNNNNCTGCGCCGGTGAGGA (SEQ ID NO:23), NNNNNNNNDACRRTGAYYHGVVB (SEQ ID NO:24), NNNNNNNNGCTGTGGTGCGCTGA (SEQ ID NO:25), or an added nucleotide sequence having a total of no more than 40, 35, 30, 25, 20, or 15 nucleotides at its 5' end and / or 3' end; Where K represents G or T, R represents A or G, W represents A or T, Y represents C or T, D represents G, A or T, H represents T, A or C, V represents G, A or C, B represents C, G or T, N represents A, T, C, G or none, and X represents G, C or none.

3. The method according to claim 2, characterized in that, The sequence of each of the one or more forward primers is selected from SEQ ID NOs:5-19, or has an added nucleotide sequence having a total of no more than 40, 35, 30, 25, 20, or 15 nucleotides at its 5' end and / or 3' end; and / or The sequence of each of the one or more reverse primers is selected from SEQ ID NOs:20-23, SEQ ID NOs:25-30, or an added nucleotide sequence having a total of no more than 40, 35, 30, 25, 20 or 15 nucleotides at its 5' end and / or 3' end.

4. The method according to any one of claims 1 to 3, characterized in that, The amplification includes the use of at least two primer pairs, and / or Each of the one or more primer pairs contains two or more forward primers, and / or Each of the one or more primer pairs contains two or more reverse primers, and / or The number of forward primers and the number of reverse primers are the same, or the number of forward primers and the number of reverse primers are different.

5. The method according to any one of claims 1-4, characterized in that, The immunogen is selected from immunogens directly conjugated with fluorescent dyes or biotin-labeled immunogens; and / or The animal is an animal immunized with the immunogen. Preferably, the animal is selected from camels or cartilaginous fish. More preferably, the animal is selected from llamas, alpacas, guanacos, llamas, dromedaries, Bactrian camels, nurse sharks, great white sharks, shad sharks, horned sharks, rays, or striped bamboo sharks.

6. A kit for amplification, said kit comprising: One or more forward primers, wherein the sequence of each forward primer is selected from SEQ ID NOs:1–5, or has an added nucleotide sequence having a total of no more than 40, 35, 30, 25, 20, or 15 nucleotides at its 5' end and / or 3' end; and One or more reverse primers, wherein the sequence of each reverse primer is selected from SEQ ID NOs:20-25, or has an added nucleotide sequence having a total of no more than 40, 35, 30, 25, 20 or 15 nucleotides at its 5' end and / or 3' end.

7. The kit according to claim 6, wherein the one or more forward primers are individually packaged and optionally mixed before use, and / or the one or more reverse primers are individually packaged and optionally mixed before use; and / or At least two of the plurality of forward primers are premixed, and / or at least two of the plurality of reverse primers are premixed.

8. The use of the method of any one of claims 1-5 or the kit of claim 6 or 7 in the preparation of nanobodies.

9. A method for preparing nanobodies, the method comprising: The DNA library encoding the nanobody was obtained using the method described in any one of claims 1-5; The encoded DNA library was subjected to high-throughput sequencing and bioinformatics analysis to obtain the encoding DNA of the nanobody; Construct a vector containing the encoded DNA; and The nanobody is prepared by directing the expression of the encoded DNA via the vector.

10. A nanobody or an antigen-binding fragment thereof, obtained by the method of claim 9.

11. A nanobody or antigen-binding fragment thereof that specifically binds to respiratory syncytial virus (RSV), characterized in that, The nanobody or its antigen-binding fragment that specifically binds to RSV comprises: (a) CDR1, wherein CDR1 comprises a sequence selected from: SEQ ID NO:34, 38, 42, 46, 50 or 54, or having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO:34, 38, 42, 46, 50 or 54; (b) CDR2, wherein CDR2 comprises a sequence selected from: SEQ ID NO:35, 39, 43, 47, 51 or 55, or having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO:35, 39, 43, 47, 51 or 55; and (c) CDR3, wherein CDR3 comprises a sequence selected from SEQ ID NO:36, 40, 44, 48, 52 or 56, or having one, two or three amino acid substitutions, deletions or additions compared to SEQ ID NO:36, 40, 44, 48, 52 or 56.

12. The nanobody or antigen-binding fragment thereof that specifically binds to RSV according to claim 11, characterized in that, The nanobody or its antigen-binding fragment that specifically binds to RSV comprises: (1) CDR1 shown in SEQ ID NO:34, CDR2 shown in SEQ ID NO:35 and CDR3 shown in SEQ ID NO:36; (2) CDR1 shown in SEQ ID NO:38, CDR2 shown in SEQ ID NO:39 and CDR3 shown in SEQ ID NO:40; (3) CDR1 shown in SEQ ID NO:42, CDR2 shown in SEQ ID NO:43 and CDR3 shown in SEQ ID NO:44; (4) CDR1 shown in SEQ ID NO:46, CDR2 shown in SEQ ID NO:47 and CDR3 shown in SEQ ID NO:48; (5) CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:51, and CDR3 shown in SEQ ID NO:52; or (6) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:55 and CDR3 shown in SEQ ID NO:

56.

13. The nanobody or antigen-binding fragment thereof that specifically binds to RSV according to claim 11 or 12, characterized in that, The nanobody or its antigen-binding fragment that specifically binds to RSV comprises: Amino acid sequences such as those shown in any one of SEQ ID NO:37, SEQ ID NO:41, SEQ ID NO:45, SEQ ID NO:49, SEQ ID NO:53 or SEQ ID NO:57; or amino acid sequences that have at least 85% sequence identity with them.

14. A nanobody or antigen-binding fragment thereof that specifically binds to TCL1, characterized in that, The nanobody or its antigen-binding fragment that specifically binds to TCL1 comprises: (a) CDR1, wherein CDR1 comprises a sequence selected from: SEQ ID NO:58, 62, 66, 70 or 74, or having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO:58, 62, 66, 70 or 74; (b) CDR2, wherein CDR2 comprises a sequence selected from: SEQ ID NO:59, 63, 67, 71 or 75, or having one or two amino acid substitutions, deletions or additions compared to SEQ ID NO:59, 63, 67, 71 or 75; and (c) CDR3, wherein CDR3 comprises a sequence selected from SEQ ID NO:60, 64, 68, 72 or 76, or having one, two or three amino acid substitutions, deletions or additions compared to SEQ ID NO:60, 64, 68, 72 or 76.

15. The nanobody or antigen-binding fragment thereof that specifically binds to TCL1 according to claim 14, characterized in that, The nanobody or its antigen-binding fragment that specifically binds to TCL1 comprises: (1) CDR1 shown in SEQ ID NO:58, CDR2 shown in SEQ ID NO:59 and CDR3 shown in SEQ ID NO:60; (2) CDR1 shown in SEQ ID NO:62, CDR2 shown in SEQ ID NO:63 and CDR3 shown in SEQ ID NO:64; (3) CDR1 shown in SEQ ID NO:66, CDR2 shown in SEQ ID NO:67 and CDR3 shown in SEQ ID NO:68; (4) CDR1 shown in SEQ ID NO:70, CDR2 shown in SEQ ID NO:71, and CDR3 shown in SEQ ID NO:72; or (5) CDR1 shown in SEQ ID NO:74, CDR2 shown in SEQ ID NO:75 and CDR3 shown in SEQ ID NO:

76.

16. The nanobody or antigen-binding fragment thereof that specifically binds to TCL1 according to claim 14 or 15, characterized in that, The nanobody or its antigen-binding fragment that specifically binds to TCL1 comprises: Amino acid sequences such as those shown in any one of SEQ ID NO:61, SEQ ID NO:65, SEQ ID NO:69, SEQ ID NO:73 or SEQ ID NO:77; or amino acid sequences that have at least 85% sequence identity with them.

17. The nanobody or antigen-binding fragment of any one of claims 10-16, further comprising: such as EVQLVESGGGX 13 VX 14 X 15 G or QVQLVESGGGX 13 VX 14 X 15 The starting amino acid sequence shown in G, and / or as in GX 16 GX 17 X 18 VTVSS or GX 16 GX 17 X 18 The terminal amino acid sequence shown in VIVSS Where X 13 Selected from S or L, X 14 Selected from Q, H, K, or P, X 15 Selected from S, A, or P, X 16 Selected from Q, K, or R, X 17 Selected from I or T, X 18 Selected from P, Q, or R; and / or The starting amino acid is an amino acid sequence located at positions 1 to 15 of frame region FR1 (IMGT numbering system), and / or the terminal amino acid is an amino acid sequence located at position FR4 of frame region; and / or It is obtained by the preparation method described in claim 9.

18. An isolated nucleic acid molecule encoding a nanobody or an antigen-binding fragment thereof as described in any one of claims 10-17.

19. The nucleic acid molecule according to claim 18, characterized in that, The nucleic acid molecule encodes a nanobody or antigen-binding fragment thereof that specifically binds to RSV as described in any one of claims 11-13, and the nucleic acid molecule comprises: Nucleotide sequences as shown in SEQ ID NOs:78–83, or nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs:78–83; or The nucleic acid molecule encodes a nanobody or antigen-binding fragment thereof that specifically binds to TCL1 as described in any one of claims 14-16, and the nucleic acid molecule comprises: Nucleotide sequences as shown in SEQ ID NOs:84-88, or nucleotide sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleotide sequences shown in SEQ ID NOs:84-88.

20. A vector comprising the nucleic acid molecule of claim 18 or 19.

21. A host cell comprising the nucleic acid molecule of claim 18 or 19 or the vector of claim 20.

22. A conjugate comprising any one of the nanobodies or antigen-binding fragments thereof as claimed in claims 10-17, and a conjugate thereof.

23. The conjugate according to claim 22, characterized in that, The conjugate is selected from disease therapeutic molecules, enzymes, fluorescent dyes, radiolabeled substances, luminescent substances, magnetic beads, biotin, toxins, cytokines, or any combination thereof.

24. A pharmaceutical composition comprising a nanobody or an antigen-binding fragment thereof as described in any one of claims 10-17, a nucleic acid molecule as described in claim 18 or 19, a carrier as described in claim 20, a host cell as described in claim 21, or a conjugate as described in claim 22 or 23, and a pharmaceutically acceptable carrier and / or excipient.

25. Use of the nanobody or antigen-binding fragment thereof of any one of claims 10-17, the nucleic acid molecule of claim 18 or 19, the carrier of claim 20, the host cell of claim 21, the conjugate of claim 22 or 23, or the pharmaceutical composition of claim 24 in the preparation of formulations for the prevention, diagnosis, and / or treatment of related diseases.

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