Human and mouse serum albumin cross reaction nanometer antibody derived from fully synthetic antibody library

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a human and mouse serum albumin cross reaction nano antibody derived from a fully synthetic antibody library. Nine nano antibodies capable of being specifically combined with human serum albumin are found through nano antibody synthesis library phage screening. A cross-reactive ELISA experiment is carried out on the related nano antibody clones, the four antibody clones are found to have specific binding to MSA, and Nb-HSA-5 shows relatively strong binding force to MSA; the Nb-HSA-4 does not react with the MSA and the BSA at all under the condition that the Nb-HSA-4 is strongly combined with the HSA, so that the combination of the Nb-HSA-4 and the HSA has strong specificity. The anti-HSA nano antibody can be used for preparing an HSA diagnostic kit, a targeted therapeutic drug, a carrier conjugate for prolonging the plasma half-life period of the drug and the like, and a new tool is provided for diagnosis and treatment of various related diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to nanobodies derived from a fully synthetic antibody library that cross-react with human and mouse serum albumin. Background Technology

[0002] Human serum albumin (HSA) is a highly abundant protein in human plasma, accounting for approximately 50%–60% of total plasma protein. The concentration in normal adult plasma is 35–50 g / L, with a molecular weight of approximately 69.4 kDa. HSA plays a variety of important physiological functions, including maintaining plasma colloid osmotic pressure, transporting fatty acids, hormones, drugs, and metabolites, and is crucial in physiological metabolism and disease development. Furthermore, HSA is essential for endocytosis of albumin mediated by nascent Fc receptors, preventing lysosomal degradation and significantly improving the half-life of protein drugs, making it an ideal drug carrier. Based on the physiological functions and high plasma abundance of HSA, anti-HSA antibodies have broad application prospects in the biomedical field, mainly including diagnostics, treatment, and drug delivery. In disease treatment and diagnosis, anti-HSA antibodies can be used for targeted therapy against abnormal HSA, or as core recognition elements in plasma HSA level detection kits (such as immunoturbidimetry and ELISA), assisting in the diagnosis of liver diseases (such as cirrhosis and hepatitis), kidney diseases (such as nephrotic syndrome), and malnutrition. In addition, anti-HSA antibodies can be conjugated to drug molecules, taking advantage of the long circulation characteristics of HSA (half-life of about 19 days) to prolong the duration of drug action in the body, reduce the frequency of administration, and improve drug bioavailability.

[0003] Nanobodies (Nb), also known as single-domain antibodies (sdAb), are variable domains (VHH) derived from heavy-chain antibodies (HCAbs) from camel-dwelling animals or cartilaginous fish. Although lacking light chains, nanobodies can still bind antigens with high specificity and affinity. They also possess excellent properties such as small molecular weight (only 12-15 kDa), high thermal stability, weak immunogenicity, and good tissue permeability. They are easily engineered for protein production and can recognize hidden epitopes. Furthermore, they can serve as important tool proteins for detecting specific conformations in structural biology, showing promising applications in targeted drug delivery, tumor immunology, treatment of infectious and immune diseases, and bioimaging technologies.

[0004] The sequence discovery of antigen-specific nanobodies can be achieved through screening of immune antibody libraries and synthetic antibody libraries. Among these methods, the construction of synthetic nanobodies can avoid the cumbersome process of animal immunization and blood collection, and the mutation sites and overall diversity of the library can be artificially controlled to simulate the evolutionary process of nanobodies. In recent years, this method has been widely used in antibody discovery.

[0005] Phage display technology refers to the insertion of the gene sequence of a foreign protein or peptide into the gene of a phage coat protein, thereby displaying the expressed protein or peptide on the phage surface and constructing a phage-displayed antibody library. Since George P. Smith first introduced phage display technology in 1985, it has become the preferred method for isolating target-specific antibodies and is widely used in the development of nanobodies. Screening strategies for phage-displayed antibody libraries during antibody development include solid-phase screening using solid-phase matrices such as ELISA plates and immunotubes, cell screening, and magnetic bead screening.

[0006] In the current research field of anti-human serum albumin (HSA) antibodies, although there are relevant patents, there are still many technical problems to be solved in antibody screening from fully synthetic nanobody libraries. Moreover, there are significant gaps in the functional studies of existing anti-HSA antibodies, such as human-mouse cross-reactivity, which to some extent hinders the conduct of preclinical animal experiments and the smooth connection with subsequent clinical trials. Summary of the Invention

[0007] To address the aforementioned problems in existing technologies, this invention employs phage magnetic bead screening technology to screen nine anti-HSA nanobodies from a fully synthetic nanobody library. These nanobodies can be used as conjugates to extend drug half-life or for the development of products such as HSA detection kits.

[0008] The first aspect of the present invention is to provide a nanobody or antigen-binding fragment of an anti-human serum albumin.

[0009] A second aspect of the present invention is to provide a heavy chain antibody against human serum albumin.

[0010] A third aspect of the present invention is to provide a recombinant protein.

[0011] The fourth aspect of this invention aims to provide related biomaterials.

[0012] The fifth aspect of this invention is to provide a coupling material.

[0013] The sixth aspect of this invention aims to provide applications related to the above aspects.

[0014] The seventh aspect of this invention aims to provide a product.

[0015] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows: In a first aspect, the present invention provides a nanobody against human serum albumin or an antigen-binding fragment thereof, comprising a heavy chain variable region, said heavy chain variable region including CDR-1, CDR-2 and CDR-3.

[0016] In some embodiments of the present invention, CDR-1, CDR-2 and CDR-3 are defined based on the heavy chain variable region sequence.

[0017] In some embodiments of the present invention, the heavy chain variable region sequence includes one of a1) to a9): a1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; a2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5; a3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9; a4) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; a5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; a6) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20; a7) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24; a8) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27; a9) The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 31.

[0018] In some embodiments of the present invention, when IMGT is used as the definition scheme, the CDR-1, CDR-2, and CDR-3 of the heavy chain variable region include one of b1) to b9): b1) The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 2, 3, and 4, respectively; b2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, 7, 8; b3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, 11, 12; b4) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14, 15, 16; b5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 18, 19, 16; b6) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 21, 22, 23; b7) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14, 25, 26; b8) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28, 29, 30; b9) The amino acid sequences of the variable region of the heavy chain are shown in SEQ ID NO: 32, 33, 34.

[0019] In some embodiments of the present invention, those skilled in the art may adopt other definition schemes recognized in the art, such as Kabat, Chothia, and Contact, to obtain other CDR sequences based on the heavy chain variable region of the present invention, and the above scope is still within the protection scope of the present invention.

[0020] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is one of a1) to a9).

[0021] Among them, a1)~a9) correspond to b1)~b9) in sequence.

[0022] A second aspect of the invention provides a heavy chain antibody against human serum albumin, comprising a nanobody or antigen-binding fragment thereof from the first aspect of the invention and the Fc region of a human immunoglobulin.

[0023] A third aspect of the invention provides a recombinant protein comprising: the nanobody or antigen-binding fragment thereof described in the first aspect of the invention; and optionally a tag sequence for assisting expression and / or purification.

[0024] In some embodiments of the present invention, the tag sequence is selected from at least one of the following groups: His tag, GGGS sequence, FLAG tag; further, His tag; and even further, 6×His tag.

[0025] A fourth aspect of the present invention provides biomaterials related to the nanobodies or antigen-binding fragments thereof described in the first aspect of the present invention, the heavy chain antibodies of the second aspect, or the recombinant proteins of the third aspect, said biomaterials comprising at least one of c1) to c13): c1) A nucleic acid molecule encoding the nanobody or antigen-binding fragment thereof as described in the first aspect of the present invention, the heavy chain antibody of the second aspect, or the recombinant protein of the third aspect; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A carrier containing the nucleic acid molecule described in c1); c4) A carrier containing the expression box described in c2); c5) Transgenic cell lines containing the nucleic acid molecules described in c1); c6) Transgenic cell lines containing the expression cassette described in c2); c7) A transgenic cell line containing the vector described in c3); c8) A transgenic cell line containing the vector described in c4); c9) Microorganisms containing the nucleic acid molecules described in c1); c10) Microorganisms containing the expression cassette described in c2); c11) Microorganisms containing the carrier described in c3).

[0026] In some embodiments of the present invention, the transgenic cell line does not contain propagation material.

[0027] A fifth aspect of the present invention provides a conjugate comprising the nanobody or antigen-binding fragment thereof described in the first aspect of the present invention, the heavy chain antibody of the second aspect or the recombinant protein of the third aspect, and a conjugation portion comprising at least one of a detectable marker.

[0028] In some embodiments of the present invention, the coupling portion comprises at least one of a detectable marker, a drug, a toxin, a cytokine, a radionuclide, and an enzyme.

[0029] In some embodiments of the present invention, the detectable marker is selected from radioactive isotopes, fluorescent substances, chemiluminescent substances, colored substances, or any combination thereof.

[0030] A sixth aspect of the present invention provides the use of the nanobody or antigen-binding fragment thereof described in the first aspect of the present invention, the heavy chain antibody of the second aspect, the recombinant protein of the third aspect, the biomaterial described in the fourth aspect of the present invention, and the conjugate described in the fifth aspect of the present invention in the preparation of products.

[0031] In some embodiments of the present invention, the product comprises at least one of a drug, a reagent, a detection plate, a reagent kit, and a detection chip.

[0032] In some embodiments of the present invention, the product includes one of d1 to d3: d1) Products for detecting the presence or content of HSA protein in samples; d2) Products that extend the half-life of drugs; d3) Targeted therapy drugs.

[0033] In some embodiments of the present invention, the medicament includes pharmaceutically acceptable excipients.

[0034] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of the following: propellants, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, flow aids, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesion agents, penetration enhancers, pH adjusters, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, encapsulating agents, humectants, absorbents, diluents, flocculants and anti-flocculators, filter aids, and release inhibitors.

[0035] The pharmaceutically acceptable excipients mentioned above are generally recognized for use in this purpose and as inactive ingredients in the pharmaceutical preparation. Compilations of pharmaceutically acceptable excipients can be found in reference books such as the *Handbook of Pharmaceutical Excipients* (2nd edition, edited by A. Wade and PJ Weller; published by the American Pharmaceutical Association, Washington and The Pharmaceutical Press, London, 1994) and the *Pharmacopoeia of the People's Republic of China - List of Pharmaceutical Excipients*.

[0036] In some embodiments of the present invention, the dosage form of the product includes one of the following: powder, tablet, granule, capsule, sustained-release agent, solution, dry suspension, effervescent tablet, emulsion, suspension, syrup, drops, and chewable tablet.

[0037] In some embodiments of the present invention, the product is administered via the gastrointestinal tract or non-gastrointestinal route.

[0038] In some embodiments of the present invention, the gastrointestinal administration includes one of oral administration, sublingual administration, and rectal administration.

[0039] In some embodiments of the present invention, the non-gastrointestinal administration includes one of intravenous injection, subcutaneous injection, and mucosal administration.

[0040] A seventh aspect of the present invention provides a product comprising the nanobody or antigen-binding fragment thereof described in the first aspect of the invention, the heavy chain antibody of the second aspect, the recombinant protein of the third aspect, or the conjugate described in the fifth aspect of the invention.

[0041] In some embodiments of the present invention, the product includes at least one of a drug, a reagent, a detection plate, a reagent kit, and a detection chip.

[0042] The beneficial effects of this invention are: 1. This invention uses streptavidin magnetic beads as a medium for liquid-phase screening of fully synthetic nanobody libraries. It eliminates the need to feed and immunize camel animals, shortens the screening cycle, and effectively avoids animal ethics issues and the limitations of conventional solid-phase screening, such as changes in antigen conformation caused by the carrier surface or the shielding of epitopes, resulting in insufficient antigen-accessible surface.

[0043] 2. Through phage screening of the nanobody synthesis library, nine nanobody sequences that specifically bind to human serum albumin were discovered and named Nb-HSA-1, Nb-HSA-2, Nb-HSA-3, Nb-HSA-4, Nb-HSA-5, Nb-HSA-6, Nb-HSA-7, Nb-HSA-8, and Nb-HSA-9, respectively. Their significant specific binding to human serum albumin (HSA) was verified by ELISA.

[0044] 3. This invention conducted cross-reactivity ELISA experiments on the nanobody clones involved, and found that four antibody clones—Nb-HSA-1, Nb-HSA-2, Nb-HSA-3, and Nb-HSA-5—specifically bound to mouse serum albumin (MSA). Nb-HSA-5 showed a strong binding affinity to mouse serum albumin; while Nb-HSA-4, despite exhibiting a strong binding affinity to HSA, did not react with MSA or BSA at all, indicating that its binding to HSA is highly specific. This invention is beneficial for advancing subsequent animal experiments and for facilitating the clinical trial process.

[0045] 4. The anti-HSA nanobody of the present invention can be used to prepare HSA diagnostic kits, targeted therapeutic drugs, and carrier conjugates for prolonging the plasma half-life of drugs, providing new tools for the diagnosis and treatment of various related diseases. If the anti-serum albumin antibody of the present invention is used to prolong the half-life of drugs, a human-mouse cross-reactive antibody clone can be used to directly verify its effect on prolonging the half-life of drugs in mouse models, thereby predicting its PK behavior after binding with HSA in humans, avoiding the distortion of preclinical data due to species differences. Attached Figure Description

[0046] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1This is a schematic diagram of the magnetic bead screening cycle process for anti-HSA nanobodies of the present invention. In the diagram, the red circular pattern with a notch represents the biotinylated HSA antigen protein, the gray spherical pattern represents the streptavidin magnetic bead, the orange phage pattern represents the nanobodies displayed by the phage that can specifically bind to the target antigen, i.e., the biotinylated HSA protein, the blue phage pattern represents the nanobodies displayed by the phage that can bind to the streptavidin magnetic beads, and the green phage pattern represents the nanobodies displayed by the phage that cannot specifically bind to either the target antigen or the streptavidin magnetic beads.

[0047] Figure 2 The sequence alignment results are for the nine nanobody clones involved in this invention.

[0048] Figure 3 The image shows the experimental results of monoclonal phage ELISA. The dark blue bars represent the absorbance values ​​of samples coated with HSA protein solution at 450 nm as detected by an ELISA reader. The light blue bars represent the results of samples coated with Streptavidin protein solution. The red dashed line represents the cutoff value, which is the mean ± standard deviation (Avg ± 2SD) of the absorbance values ​​of the negative control samples coated with only 3% PBSM. A value more than 3 times the cutoff value is considered to indicate significant binding to the coated antigen, showing a strong binding signal.

[0049] Figure 4 This is a graph showing the validation results of a monoclonal cross-reaction phage ELISA. The blue, orange, and yellow bars represent the absorbance values ​​detected by an ELISA reader at 450 nm for samples coated with human serum albumin (HSA), mouse serum albumin (MSA), and fetal bovine serum albumin (BSA), respectively. The red dashed line represents the cutoff value, which is the mean ± standard deviation (Avg ± 2SD) of the absorbance of the negative control samples coated only with 3% PBSM. A value more than three times the cutoff value is considered to indicate significant binding to the coated antigen, presenting a strong binding signal. Detailed Implementation

[0050] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0051] Example 1: Phage magnetic bead screening based on human serum albumin 1. Phage magnetic bead screening based on human serum albumin Take streptavidin magnetic beads (SA beads), wash and equilibrate them three times with PBS, then add blocking buffer (3% PBSM) and incubate at room temperature for 5 minutes to achieve pre-equilibration. Take 100 μL (i.e., 1 × 10⁻⁶) 13 Add 900 μL of 3% Milk in PBS (3% PBSM) to a 1.5 ml EP tube and incubate at room temperature for 5 min by rotation. Add pre-equilibrated SA beads and incubate at room temperature for 1 h by rotation. Transfer the supernatant to a new EP tube, add 1.2 μg of biotinylated human serum albumin (HSA-Biotin), and incubate at room temperature for 1 h by rotation. Simultaneously, take another portion of the pre-equilibrated SA beads and incubate in 3% PBSM at room temperature by rotation for 1 h. Discard the blocking supernatant from the magnetic beads, retain the SA beads, add the obtained antigen-antibody co-incubation mixture, and incubate at room temperature by rotation for 1-2 h. Discard the supernatant, wash 5 times with PBST, then wash 3 times with PBS, and finally transfer to a new EP tube and discard the supernatant. Add 500 μL of elution buffer (0.1 M Glycine-HCl, pH 2.5) and mix well. Incubate at room temperature by rotation for 5 min, then separate on a magnetic separator for 2 min. After all adsorption is complete, aspirate the supernatant containing the antigen sample and quickly add it to a 1.5 ml EP tube containing 50 μL of neutralization buffer (1 M Tris-HCl, pH 8.5) for neutralization. Immediately mix by vortexing and store at 4 °C.

[0052] 2. Phage amplification and purification Prepare fresh TG1 plates, pick single clones and incubate them in 2×YT antibiotic-free liquid medium for more than 4 hours to activate them. At the same time, take 1 ml of bacterial culture from each plate and add Kanamycin and Ampicillin antibiotics respectively as a test for no contamination. If no growth is observed in either Kanamycin or Ampicillin culture, take the activated bacterial culture and dilute it 1:100 and shake it until OD600 is reached, which is the logarithmic growth phase (0.5~0.6).

[0053] Take TG1 bacterial culture in the logarithmic growth phase and add it to the selection eluent (containing phages displaying the target antibody) at a ratio of 1:10. Infect at 37℃ and 250 rpm for 45 min. Take 4 mL of the bacterial culture from the infection eluent and add it to 40 mL of 2×YT(A) liquid medium. Shake at 37℃ and 250 rpm until the culture reaches the logarithmic growth phase (OD600 = 0.5~0.6). Add 20 times the number of clones (approximately 3.2×10⁻⁶). 11Infect the bacteria with M13KO7 phage (pfu) at 37°C and 250 rpm for 45 min, then add 40 μL of 50 mg / mL Kanamycin and 8 μL of 1 mM IPTG, and shake at 30°C and 250 rpm overnight (16 h ~ 20 h).

[0054] Transfer the overnight cultured bacterial culture to 50ml centrifuge tubes at 40mL / tube, centrifuge at 3200g, 4℃ for 40min; transfer the supernatant to a new 50ml centrifuge tube, add 1 / 4 volume of 4℃ pre-chilled 20% PEG6000 / 2.5M NaCl (i.e., 10mL), mix thoroughly, and incubate on ice for 30min. Perform a second separation and filtration on the cultured bacterial culture, centrifuge at 3200g, 4℃ for 20min, discard the supernatant, and invert the tube on paper for 2min; resuspend the precipitate in 10ml PBS, mix well, and filter through a 0.45μM filter membrane. Transfer the filtrate to a new centrifuge tube, add 1 / 4 volume of 4℃ pre-chilled 20% PEG / 2.5M NaCl (i.e., 2.5mL), mix well, and incubate on ice for 30min. The resulting suspension was centrifuged at 3200g at 4℃ for 20min, the supernatant was discarded, and the tube was inverted on paper for 2min. The precipitate was resuspended in 1ml of 1×PBS per tube, aliquoted into 1.5ml EP tubes, and 1 / 4 volume (250μL) of pre-chilled 20% PEG / 2.5M NaCl at 4℃ was added. The mixture was incubated on ice for 30min, then centrifuged at 13000g at 4℃ for 10min. The supernatant was discarded, and the precipitate was resuspended in 500μl of PBS per tube. This suspension serves as a new phage library, suitable for phage assays and a new round of screening. This antigen underwent a total of 5 rounds of screening.

[0055] 3. Phage titer determination Prepare fresh TG1 plates, pick single clones and incubate them in 2×YT antibiotic-free liquid medium for at least 4 hours to activate them. At the same time, take 1 ml of bacterial culture from each plate and add Kanamycin and Ampicillin antibiotics respectively as a test for no contamination. If no growth is observed in either Kanamycin or Ampicillin culture, take the activated bacterial culture and dilute it 1:100 and shake it until OD600 is reached, which is the logarithmic growth phase (0.5~0.6).

[0056] Take 10 μL of bacteriophage culture and dilute it in 90 μL of 1×PBS to obtain a 10⁻⁶ solution. 1 Similarly, the screening eluent is diluted to 10. 8 The phage amplification solution was diluted to 10. 12Add 90 μL / tube of TG1 bacterial suspension in the logarithmic phase to the diluted bacterial suspension, mix well, and incubate at 37°C for 45 min for infection. Spot 5 μL of the infected bacterial mixture onto a pre-arranged Amp-resistant 2×YT solid plate, designated as the Amp plate. Separately, add 20 μL of the infected bacterial mixture to a semi-solid 2×YT medium that has been thawed and cooled to below 55°C, mix well, and pour onto a 2×YT plate without antibiotics, designated as the TOP plate.

[0057] Incubate overnight at 37°C. The gradient of individual clones can be distinguished on the Amp plate; phage titer = number of clones * dilution gradient * 400. Simultaneously, phage plaques can be observed on the corresponding gradient TOP plate.

[0058] 4. Experimental Results A schematic diagram of the magnetic bead screening cycle of anti-HSA nanobodies is shown below. Figure 1 As shown, phages displaying the target nanoantibody can be separated through this screening and elution process, and enrichment can be achieved through phage amplification.

[0059] The results of five rounds of screening for anti-HSA nanobodies using a self-built fully synthetic antibody library are shown in Table 1. The recovery rate is calculated as follows: Recovery Rate = output / input; the enrichment effect is calculated as follows: Enrichment effect in round n+1 = (Recovery Rate in round n+1) / (Recovery Rate in round n).

[0060] Table 1

[0061] Example 2: Efficacy test of anti-HSA nanobody 1. Monoclonal Phage ELISA Each monoclonal sample was used as a group in the ELISA plate. For each experiment, one group of M13KO7 phage was set up as a negative control. Each group consisted of 4 wells. The experimental wells were coated with HSA, and the control wells were coated with a mixture of HSA+SA co-incubation solution, SA protein, and 3% PBSM solution, respectively, at 100 μL (4 ng / μL) per well. The plates were incubated overnight at 4 °C. The overnight coating solution was discarded, and the plates were washed three times with PBST. After discarding the washings, 200 μL of 3% PBSM solution was added to each well for blocking, and the plates were incubated horizontally at 37 °C for 1 h. The blocking solution was discarded, and the plates were washed three times with PBST per well. The amplified monoclonal phages were centrifuged overnight, and 50 μL of phage-containing supernatant was added to each well. The plates were incubated horizontally at 37 °C for 2 h. The plate liquid was discarded, and the plates were washed four times with PBST. 100 μL of HRP-conjugated M13Bacteriophage antibody was added to each well, and the plates were incubated horizontally at 37 °C for 1 h. Discard the liquid in the plate and wash four times with PBST. Add 100 μL of TMB chromogenic solution to each well, incubate in the dark for 5-10 minutes, then add 100 μL of ELISA Stop Buffer to terminate the reaction. Measure the absorbance at 450 nm using a microplate reader. Single clones showing a clear binding signal in the sample wells, but not in the corresponding control wells or the M13KO7 negative control wells, are considered positive clones. The sequences of positive clones are obtained through Sanger sequencing, and clones with unique sequences are compared with each other.

[0062] 2. Validation of monoclonal cross-reactive phage ELISA Each monoclonal sample was used as a group in the ELISA plate. For each experiment, one group of M13KO7 phage was set up as a negative control. Each group consisted of 4 wells, coated with HSA, MSA (mouse serum albumin), or BSA (fetal bovine serum albumin) solutions respectively. The control wells were coated with 100 μL of 3% PBSM solution (4 ng / μL per well) and incubated overnight at 4 °C. The overnight coating solution was discarded, and the plate was washed three times with PBST. After discarding the washings, 200 μL of 3% PBSM solution was added to each well for blocking, and the plate was incubated horizontally at 37 °C for 1 h. The blocking solution was discarded, and the plate was washed three times with PBST. The amplified monoclonal phage was centrifuged overnight, and 50 μL of phage-containing supernatant was added to each well. The plate was incubated horizontally at 37 °C for 2 h. The plate liquid was discarded, and the plate was washed four times with PBST. 100 μL of HRP-conjugated M13Bacteriophage antibody was added to each well, and the plate was incubated horizontally at 37 °C for 1 h. Discard the liquid in the plate and wash four times with PBST. Add 100 μL of TMB chromogenic solution to each well, incubate in the dark for 5-10 minutes, then add 100 μL of ELISA Stop Buffer to terminate the reaction. Measure the absorbance at 450 nm using a microplate reader. Single clones showing a clear binding signal in the sample wells, but not in the corresponding control wells or the M13KO7 negative control wells, are considered positive clones. The sequences of positive clones are obtained through Sanger sequencing, and clones with unique sequences are compared with each other.

[0063] 3. Sequence identification results Through phage screening of nanobody synthesis libraries, a total of 9 nanobody sequences that can specifically bind to human serum albumin were discovered and named Nb-HSA-1, Nb-HSA-2, Nb-HSA-3, Nb-HSA-4, Nb-HSA-5, Nb-HSA-6, Nb-HSA-7, Nb-HSA-8, and Nb-HSA-9, respectively.

[0064] The sequence results are as follows: Nb-HSA-1: QVQLQESGGGLVQAGGSLRLSCAASGYISGAIVMGWYRQAPGKEREFVASIAVGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAATREARALQYWGQGTQVTVSS (SEQ ID NO: 1); Its CDR is: CDR1: GYISGAIV (SEQ ID NO: 2); CDR2:IAVGSIT (SEQ ID NO: 3); CDR3: AAATREARALQY (SEQ ID NO: 4).

[0065] Nb-HSA-2: QVQLQESGGGLVQAGGSLRLSCAASGSIFTAPTMGWYRQAPGKERELVATISIGANTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVTTRRIRAHQYWGQGTQVTVSS (SEQ ID NO: 5); Its CDRs are: CDR1: GSIFTAPT (SEQ ID NO: 6); CDR2: ISIGANT (SEQ ID NO: 7); CDR3: AVTTRRIRAHQY (SEQ ID NO: 8).

[0066] Nb-HSA-3: QVQLQESGGGLVQAGGSLRLSCAASGTISQFYMGWYRQAPGKERELVASISIGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYERRLRVFGYWGQGTQVTVSS (SEQ ID NO: 9); Its CDRs are: CDR1: GTISQFY (SEQ ID NO: 10); CDR2: ISIGGTT (SEQ ID NO: 11); CDR3: AAYERRLRVFGY (SEQ ID NO: 12).

[0067] Nb-HSA-4 QVQLQESGGGLVQAGGSLRLSCAASGSISKGYGMGWYRQAPGKERELVAGTDHGGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYPGRGYGFKYWGQGTQVTVSS (SEQ ID NO: 13); Its CDRs are: CDR1: GSISKGYG (SEQ ID NO: 14); CDR2: TDHGGST (SEQ ID NO: 15); CDR3: AAYPGRGYGFKY (SEQ ID NO: 16).

[0068] Nb-HSA-5: QVQLQESGGGLVQAGGSLRLSCAASGSISGGYGMGWYRQAPGKEREFVAGIDHGSITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAYPGRGYGFKYWGQGTQVTVSS (SEQ ID NO: 17); Its CDRs are: CDR1: GSISGGYG (SEQ ID NO: 18); CDR2: IDHGSIT (SEQ ID NO: 19); CDR3: AAYPGRGYGFKY (SEQ ID NO: 16). Nb-HSA-6: QVQLQESGGGLVQAGGSLRLSCAASGSISGANVMGWYRQAPGKEREFVASIDDGSITNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAATRQARALHYWGQGTQVTVSS (SEQ ID NO: 20); Its CDRs are: CDR1: GSISGANV (SEQ ID NO: 21); CDR2: IDDGSIT (SEQ ID NO: 22); CDR3: AAATRQARALHY (SEQ ID NO: 23).

[0069] Nb-HSA-7 QVQLQESGGGLVQAGGSLRLSCAASGSISKGYGMGWYRQAPGKERELVASINNGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVYRVVNHFFFYWGQGTQVTVSS (SEQ ID NO: 24); Its CDRs are: CDR1: GSISKGYG (SEQ ID NO: 14); CDR2: INNGGTT (SEQ ID NO: 25); CDR3: AVYRVVNHFFFY (SEQ ID NO: 26).

[0070] Nb-HSA-8 QVQLQESGGGLVQAGGSLRLSCAASGYISGGYVMGWYRQAPGKEREFVAGTDVGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADTREAYGFQYWGQGTQVTVSS (SEQ ID NO: 27); Its CDR is: CDR1: GYISGGYV (SEQ ID NO: 28); CDR2: TDVGGIT (SEQ ID NO: 29); CDR3:AADTREAYGFQY (SEQ ID NO: 30).

[0071] Nb-HSA-9 QVQLQESGGGLVQAGGSLRLSCAASGSIFNGYCMGWYRQAPGKERELVAGIDFGGTTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAELGPDYGFGYWGQGTRVTVSI (SEQ ID NO: 31) Its CDR is: CDR1: GSIFNGYC (SEQ ID NO: 32) CDR2: IDFGGTT (SEQ ID NO: 33) CDR3: AAELGPDYGFGY (SEQ ID NO: 34).

[0072] 4. Experimental Results The sequence alignment results of the nine nanobody clones involved in this invention are as follows: Figure 2 As shown, the median sequence values ​​26-34 represent CDR1, 46-59 represent CDR2, and 96-108 represent CDR3. This indicates that the nine nanobody clones obtained through screening exhibit good sequence diversity in the CDR region (the CDR sequence information involved was obtained using the CDR definition scheme of IMGT).

[0073] The experimental results of monoclonal phage ELISA are as follows: Figure 3As shown in the figure, the nine nanobodies with different sequences—Nb-HSA-1, Nb-HSA-2, Nb-HSA-3, Nb-HSA-4, Nb-HSA-5, Nb-HSA-6, Nb-HSA-7, Nb-HSA-8, and Nb-HSA-9—all exhibited strong specific binding to the HSA antigen protein, with the experimental group data exceeding the cutoff value by more than three times. However, they showed no binding to the Streptavidin protein. NC in the figure represents the negative control, i.e., the M13KO7 phage that did not display the antibody. The negative control group showed no binding to either HSA or Streptavidin proteins.

[0074] The results of monoclonal cross-reactive phage ELISA validation are as follows: Figure 4 As shown in the figure, the results of the four experimental groups, Nb-HSA-1, Nb-HSA-2, Nb-HSA-3, and Nb-HSA-5, were higher than the 3-fold cutoff value, indicating that they showed specific binding signals to mouse serum albumin (MSA). Among them, Nb-HSA-5 showed a significantly strong binding signal, far exceeding the 3-fold cutoff value. NC in the figure represents the negative control, i.e., the M13KO7 phage that did not show antibodies. The negative control group showed no binding to HSA, MSA, and BSA proteins.

[0075] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A nanobody against human serum albumin or its antigen-binding fragment, comprising a heavy chain variable region, characterized in that: The heavy chain variable region includes CDR-1, CDR-2 and CDR-3; The CDR-1, CDR-2, and CDR-3 are defined based on the heavy chain variable region sequence; The heavy chain variable region sequence includes one of a1) to a9); a1) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 1; a2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 5; a3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 9; a4) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 13; a5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 17; a6) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 20; a7) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 24; a8) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 27; a9) The amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO:

31.

2. The nanobody or its antigen-binding fragment according to claim 1, characterized in that: When IMGT is used as the definition scheme, the CDR-1, CDR-2 and CDR-3 of the heavy chain variable region include one of b1) to b9); b1) The amino acid sequences of CDR1, CDR2, and CDR3 are shown in SEQ ID NO: 2, 3, and 4, respectively; b2) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, 7, 8; b3) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 10, 11, 12; b4) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14, 15, 16; b5) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 18, 19, 16; b6) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 21, 22, 23; b7) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 14, 25, 26; b8) The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 28, 29, 30; b9) The amino acid sequences of the variable region of the heavy chain are shown in SEQ ID NO: 32, 33, 34.

3. The nanobody or its antigen-binding fragment according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is one of a1) to a9).

4. A heavy chain antibody against human serum albumin, characterized in that: The heavy chain antibody comprises the nanobody or its antigen-binding fragment as described in any one of claims 1 to 3 and the Fc region of human immunoglobulin.

5. A recombinant protein comprising: a nanobody or an antigen-binding fragment thereof as described in any one of claims 1 to 3; and optionally a tag sequence for assisting expression and / or purification.

6. A biomaterial relating to the nanobody or antigen-binding fragment thereof as described in any one of claims 1 to 3, the heavy chain antibody as described in claim 4, or the recombinant protein as described in claim 5, wherein the biomaterial comprises at least one of c1) to c11): c1) A nucleic acid molecule encoding the nanobody or antigen-binding fragment of any one of claims 1 to 3, the heavy chain antibody of claim 4, or the recombinant protein of claim 5; c2) An expression cassette containing the nucleic acid molecule described in c1); c3) A carrier containing the nucleic acid molecule described in c1); c4) A carrier containing the expression box described in c2); c5) Transgenic cell lines containing the nucleic acid molecules described in c1); c6) Transgenic cell lines containing the expression cassette described in c2); c7) A transgenic cell line containing the vector described in c3); c8) A transgenic cell line containing the vector described in c4); c9) Microorganisms containing the nucleic acid molecules described in c1); c10) Microorganisms containing the expression cassette described in c2); c11) Microorganisms containing the carrier described in c3).

7. A conjugate comprising: a nanobody or an antigen-binding fragment thereof as described in any one of claims 1 to 3, a heavy chain antibody as described in claim 4, and a recombinant protein as described in claim 5; And a coupling portion, the coupling portion comprising at least one of detectable markers.

8. The use of the nanobody or antigen-binding fragment of any one of claims 1 to 3, the heavy chain antibody of claim 4, the recombinant protein of claim 5, the biomaterial of claim 6, and the conjugate of claim 7 in the preparation of products; The product includes at least one of the following: drug, reagent, test plate, reagent kit, and test chip.

9. The application according to claim 8, characterized in that: The product includes one of d1 to d3: d1) Products for detecting the presence or content of HSA protein in samples; d2) Products that extend the half-life of drugs; d3) Targeted therapy drugs.

10. A product comprising any one of claims 1 to 3 a nanobody or an antigen-binding fragment thereof, the heavy chain antibody of claim 4, the recombinant protein of claim 5, or the conjugate of claim 7; The product includes at least one of the following: drug, reagent, test plate, reagent kit, and test chip.

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