Albumin-binding nanobody 4c11, derivatives and uses thereof

Abstract

The application discloses an albumin-binding nanobody 4C11 and derivatives and applications thereof, and belongs to the technical field of antibody engineering. The nanobody complementarity determining region CDR includes CDR-H1 shown in SEQ ID NO. 2 (GCVG), CDR-H2 shown in SEQ ID NO. 3 (VIDANGRTTYADSVKG), and CDR-H3 shown in SEQ ID NO. 4 (GLRTITYECRPIRDAFRF). The nanobody is covalently connected with a therapeutic polypeptide, a protein or an antibody drug to construct a long-acting complex. The complex can significantly delay the kidney clearance rate of the drug, enhance the protease resistance, prolong the half-life of the drug, and avoid frequent administration. Under the premise of ensuring the same efficacy, the frequency of administration is greatly reduced, and the patient compliance and treatment safety are effectively improved. The application provides a carrier for long-acting biological agent development in the fields of diabetes and tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody engineering, in particular to an albumin-binding nanobody 4C11 and its derivatives and applications. BACKGROUND

[0002] Therapeutic polypeptides, proteins and antibodies have shown significant clinical value in the field of major diseases such as diabetes and cancer due to their high specificity and low toxicity. However, these drugs generally have the defects of short half-life in vivo, rapid clearance by the kidney and degradation by proteases, which requires frequent and large-dose administration for patients (such as daily injection). This not only reduces patient compliance, but also easily causes safety risks due to fluctuations in blood drug concentration. To overcome this bottleneck, traditional half-life extension techniques such as PEGylation, Fc fragment fusion and albumin direct fusion have achieved certain results, but all have fundamental defects. PEGylation can delay kidney clearance by increasing the hydrated radius of the drug, but the PEG chain can shield the active site of the drug, leading to decreased efficacy, and may induce anti-PEG antibodies to accelerate immune clearance; Fc fusion technology can extend the half-life by utilizing the FcRn-mediated recycling mechanism, but the molecular weight of the fusion molecule exceeds 100 kDa, which hinders tissue penetration and poses a risk of activating complement-dependent cytotoxicity; the direct fusion strategy of human serum albumin (HSA) can rely on the natural long-circulating properties of HSA (half-life of about 19 days), but the introduction of a 66.5 kDa carrier protein can cause a sudden increase in drug molecular weight, change the original pharmacokinetic behavior, and hinder the delivery efficiency of the drug to the target tissue.

[0003] In recent years, the rise of nanobody (VHH) technology has provided a new path to break through the above limitations. This variable domain derived from camelid heavy chain antibodies has unique advantages such as small molecular weight (about 15 kDa), strong tissue penetration, high stability, and easy humanization. Anti-serum albumin nanobodies can obtain long-circulating properties by specifically binding to endogenous HSA, while avoiding excessive increase in molecular weight. The core mechanism is that the nanobody-HSA complex can effectively reduce kidney clearance and lysosomal degradation through the FcRn-mediated circulation pathway. In the evolution of technology, Jiangnan University developed a rhamnolipid modification strategy to recruit endogenous antibodies to increase the hydrated radius, but the efficacy is limited by the abundance of antibodies in the body; Kangzhong Bio has screened a broad-spectrum nanobody Nb3 that can bind to human / bovine / mouse serum albumin across species, significantly improving the adaptability of preclinical models (DOI: 10.1016 / j.jconrel.2024.11.080); the "black hat" bispecific antibody (NbCD4-NbHSA-NbCD4) designed by Nanjing University has confirmed that HSA binding can extend the half-life by 5-8 times (CN120173100A).

[0004] Current research focuses on three key technical breakthroughs: first, high-affinity humanization, such as the HSA-32 nanobody developed by the Kim team, which retains sub-nanomolar affinity (KD~10 -9 M) after humanization; second, achieving non-pH-dependent binding to ensure stable binding to HSA at physiological pH (7.4) and endosomal acidic environment (pH 5.5-6.0), avoiding dissociation during FcRn recycling; third, establishing an efficient expression system, such as the soluble expression of anti-HSA nanobody-therapeutic peptide fusion protein in E. coli by the Zhou team, significantly reducing production costs (CN119708221B). These advances have pushed anti-HSA nanobodies towards long-acting and intelligent directions, such as fusion with drugs like interferon and GLP-1 analogs, extending the half-life from hours to days; the "nanoadapter" developed by South China University of Technology significantly enhances tumor killing efficiency by FcγR1-HSA fusion loading of multi-specific antibodies (DOI:10.1038 / s41551-025-01425-5); and Kangzhong Bio has screened a nanobody, Nb3, that binds to multiple serum albumins (human, bovine, and mouse), which can adapt to different animal models and improve drug targeting (CN116023487A, 2023.04.28).

[0005] Despite the promising prospects, the field still faces challenges such as complex spatial steric hindrance, cross-species binding consistency, and large-scale production. Future trends will focus on the development of intelligent delivery systems (such as integrating pH / temperature-responsive elements) and multi-mechanism collaborative design (such as tri-specific antibodies targeting therapeutic targets and HSA simultaneously). Anti-serum albumin nanobodies provide an efficient, safe, and programmable technology platform for long-acting polypeptide drugs by precisely utilizing the human body's natural transport system. With the deep integration of gene editing, artificial intelligence-assisted design, and new expression technologies, this field is expected to completely break through traditional pharmacokinetic limitations and lead the biopharmaceutical industry into a new era of "one-time administration, long-term treatment."

[0006] The present invention adopts the programmed immunized camel strategy to induce the production of high specificity anti-human serum albumin (HSA) heavy chain antibodies, and combines with phage display library high-throughput screening technology to screen candidate single domain antibodies (sdAb) targeting HSA natural conformation from the immune library. Further soluble production is realized through yeast and E. coli expression systems, and finally anti-HSA nanobodies with nanomolar to sub-nanomolar binding affinity are obtained. The present invention aims to break through the development limitations caused by antigen epitope masking, insufficient antibody affinity, or structural heterogeneity in traditional methods, and provides a universal carrier basis for constructing long-acting biological drugs. By fusing the nanobody with therapeutic polypeptides, proteins, or antibody drugs, the in vivo half-life can be significantly prolonged, the drug administration convenience can be improved, and the clinical treatment window can be enhanced. SUMMARY

[0007] To solve the above problems, the present application provides an albumin-binding nanobody and its application. The albumin-binding nanobody disclosed in the present application has high affinity with albumin, and can be used to prolong the half-life of biological drugs, and is conducive to improving the beneficial effects of biological drugs in treatment. The albumin includes serum albumin and recombinant albumin, and the serum albumin is optionally human serum albumin.

[0008] In the first aspect of the present application, an albumin-binding nanobody is provided, which can specifically bind to serum albumin, and the nanobody has the following complementarity determining regions (CDRs): CDR-H1 as shown in SEQ ID NO. 2 (GCVG), CDR-H2 as shown in SEQ ID NO. 3 (VIDANGRTTYADSVKG), and CDR-H3 as shown in SEQ ID NO. 4 (GLRTITYECRPIRDAFRF). Optionally, the serum albumin is human serum albumin.

[0009] Further, the amino acid sequence of the nanobody includes at least one of the following:

[0010] A1) the amino acid sequence includes the amino acid sequence as shown in SEQ ID NO. 1;

[0011] A2) an amino acid sequence having 95% or more identity with the amino acid sequence of the nanobody of A1) obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence of the nanobody of A1);

[0012] A3) an amino acid sequence of a fusion protein having the same function obtained by connecting a tag protein to the N-terminus and / or C-terminus of the amino acid sequence of A1) or A2).

[0013] In the second aspect of the present application, a biological material is provided, which includes at least any one of the following:

[0014] B1) contains a nucleic acid molecule encoding the nanobody;

[0015] B2) contains an expression cassette of the nucleic acid molecule of B1);

[0016] B3) contains a recombinant vector of the nucleic acid molecule of B1), or a recombinant vector containing the expression cassette of B2);

[0017] B4) contains a recombinant microorganism containing the nucleic acid molecule of B1), or a recombinant microorganism containing the recombinant vector of B2), or a recombinant microorganism containing the recombinant vector of B3);

[0018] B5) Recombinant cells, wherein the recombinant cells contain B1) nucleic acid molecules, or B2) the expression cassette of the recombinant vector, or B3) the recombinant vector, or B4) recombinant microorganisms.

[0019] Further, the nucleic acid molecule described in B1) includes a publicly disclosed nucleic acid molecule encoding the nanobody and / or an optimized nucleic acid molecule as needed. Optionally, the nucleic acid molecule described in B1) includes at least one of the nucleotide sequences shown in SEQ ID NO.5 or SEQ ID NO.6.

[0020] It should be understood that SEQ ID NO. 5 and SEQ ID NO. 6 are sequences optimized for codon preferences of Pichia pastoris and Escherichia coli, respectively; those skilled in the art can optimize the nucleic acid encoding SEQ ID NO: 1 according to different expression systems (such as other engineered bacteria), and such variants are all within the scope of protection of this application.

[0021] Further, the recombinant vector described in B3) includes at least one of the following: pET series vectors, pBAD vectors, pGEX series vectors, pCAl-n / pCAl-pelB vectors, pPOW3.0 vectors, pPIC series vectors, and pYES2 vectors.

[0022] Furthermore, the recombinant microorganisms described in B4) or the recombinant cells described in B5) include at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0023] Optionally, the recombinant microorganism includes at least one strain of Escherichia coli and Pichia pastoris.

[0024] It should be noted that the present invention does not limit the recombinant microorganisms or recombinant cells mentioned above. Any cell that can express exogenous genes through recombinant engineering technology is protected by the present invention.

[0025] In a third aspect, the present invention comprises an antiserum albumin antibody containing the aforementioned nanobody amino acid sequence as a VHH chain.

[0026] A fourth aspect of the present invention includes the nanobody or a derivative of the antiserum albumin antibody, the derivative comprising a modifier that is covalently or non-covalently bound to the nanobody or the antibody.

[0027] Further, the modification may optionally include:

[0028] Particulate matter bonded by non-covalent bonds, such as colloidal gold, colloidal silver, or colloidal carbon;

[0029] Modifiers that are covalently bonded include, but are not limited to:

[0030] Microspheres (such as colored microspheres, fluorescent microspheres, and magnetic microspheres), chromatography packing materials, and small chemical molecules (such as biotin, dye molecules, and fluorescent molecules).

[0031] In a fifth aspect, the present invention provides a product comprising at least one of the following:

[0032] C1) A fusion protein constructed by fusing the nanobody, or the anti-serum albumin antibody, or a derivative of the nanobody or the anti-serum albumin antibody with a polypeptide or protein;

[0033] C2) A conjugate formed by linking the nanobody, or the antiserum albumin antibody, or a derivative of the nanobody or the antiserum albumin antibody, to a therapeutic agent;

[0034] C3) A pharmaceutical composition comprising the nanobody, or the antiserum albumin antibody, or a derivative thereof;

[0035] C4) A kit comprising the nanobody, and / or the biomaterial, and / or the anti-serum albumin antibody, and / or the nanobody or a derivative of the anti-serum albumin antibody, and / or the fusion protein of C1), and / or the conjugate of C2), and / or the pharmaceutical composition of C3).

[0036] Furthermore, the fusion protein is prepared by linking a polypeptide or protein to the N-terminus or C-terminus of the nanobody, or the anti-serum albumin antibody, or a derivative of the nanobody or the anti-serum albumin antibody. Optionally, the polypeptide or protein and the nanobody, or the anti-serum albumin antibody, or a derivative of the nanobody or the anti-serum albumin antibody are linked by chemical bonds.

[0037] In a sixth aspect, the present invention provides a method for preparing the nanobody and / or the fusion protein, characterized in that the method includes the steps of culturing the recombinant microorganism (B4) or the recombinant cell (B5) in the biological material under suitable culture conditions, and isolating the nanobody or the fusion protein.

[0038] In a seventh aspect, the present invention provides the nanobody, or the biomaterial, or the anti-serum albumin antibody, or a derivative of the nanobody or the anti-serum albumin antibody, or the product, or the method thereof, and its use in any of the following aspects:

[0039] D1) Applications in the preparation of products with extended drug half-life;

[0040] D2) Applications in the preparation of immunoassay or diagnostic products;

[0041] D3) Applications in the preparation of products that promote the purification or enrichment of serum albumin;

[0042] D4) Applications in the preparation of products for qualitative or quantitative detection of serum albumin.

[0043] Furthermore, the drugs used to extend the half-life of the drugs include protein drugs, antibody fragments, small molecule chemotherapeutic drugs, and other biological agents; preferably, the protein drugs include at least one of IL-6R targeted drugs, TNF-α targeted drugs, and IL-17A / IL-17F targeted drugs; the small molecule chemotherapeutic drugs include at least one of doxorubicin, solefenib, and sirolimus.

[0044] Furthermore, the extended drug half-life product described in A1 includes at least one of the following: a fusion protein composed of a serum albumin-bound nanobody and a protein drug; a conjugate formed by linking a serum albumin-bound nanobody and a therapeutic agent; and a pharmaceutical composition of the serum albumin-bound nanobody.

[0045] Furthermore, the protein-based drug includes at least one of the following: polypeptides, antibodies, antibody fragments, cytokines, and tumor marker molecules capable of therapeutic effects. In one specific embodiment of the invention, the drug is nerve growth factor (NGF).

[0046] Furthermore, the product also includes pharmaceutically acceptable additives. Optionally, the pharmaceutical composition further includes pharmaceutically acceptable carriers and / or excipients.

[0047] The beneficial effects of the present invention include, but are not limited to:

[0048] This invention obtains an albumin-binding nanobody that can specifically recognize serum albumin through screening. This nanobody has a high affinity for serum albumin and can be widely used in enhancing drug half-life, tumor targeted therapy and imaging, immune detection or diagnosis, purification or enrichment of recombinant albumin, etc., and has broad application prospects in the pharmaceutical field.

[0049] This invention also provides two methods for preparing the nanobody using Pichia pastoris and Escherichia coli as host cells, which is beneficial for commercial production. Attached Figure Description

[0050] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings:

[0051] Figure 1 The images shown are SDS-PAGE electrophoresis images of each stage of purification of Pichia pastoris expression 4C11 in this embodiment of the invention. M: marker; 1: before loading; 2: flow-through; 3: elution; 4: desalting.

[0052] Figure 2 The images shown are SDS-PAGE electrophoresis images of the purification of Escherichia coli expressing 4C11 at various stages in this embodiment of the invention. M: marker; 1: desalting; 2: flow-through; 3: before loading.

[0053] Figure 3 This is an ELISA binding verification diagram of Pichia pastoris expression of 4C11 in an embodiment of the present invention.

[0054] Figure 4 This is a graph showing the affinity detection results of human serum albumin-nanobody 4C11 in an embodiment of the present invention. Detailed Implementation

[0055] The present invention is described in detail below with reference to the embodiments, but the present invention is not limited to these embodiments. Unless otherwise specified, the raw materials and catalysts in the embodiments of the present invention are all purchased through commercial channels.

[0056] Escherichia coli TG1 competent cells were purchased from Shanghai Maokang Biotechnology Co., Ltd., catalog number MF2384.

[0057] Escherichia coli BL-21 competent cells were purchased from Shanghai Maokang Biotechnology Co., Ltd., catalog number MF2391.

[0058] The X-33 yeast strain was purchased from Thermo Fisher Scientific, catalog number C18000.

[0059] Adult male rats were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., product number SN0159897.

[0060] Nerve growth factor (NGF) was purchased from the National Institute for Biosafety and Control (NIBSC) in the UK, catalog number: 93 / 556, and distributed by Shenzhen Ailimon Biotechnology Co., Ltd.

[0061] Recombinant human serum albumin: self-supplied by Tonghua Anruit Biopharmaceutical Co., Ltd.

[0062] Enzymatically hydrolyzed casein: purchased from Solarbio, product number: C8210-100.

[0063] PBST: Prepared by diluting 10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) to 1X and then adding 0.1% Tween-20.

[0064] Sample loading buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH adjusted to 8.0 with NaOH.

[0065] This invention involves periodically immunizing camels to induce the production of specific antibodies against human serum albumin. Phage display technology is then used for panning, and multiple nanobody sequences are obtained after three rounds of selection. One of these sequences is named 4C11, and its amino acid sequence is shown in SEQ ID NO. 1. CDRs are defined according to the Kabat protocol: CDR-H1 amino acid sequence is shown in SEQ ID NO. 2, CDR-H2 amino acid sequence is shown in SEQ ID NO. 3, and CDR-H3 amino acid sequence is shown in SEQ ID NO. 4. Furthermore, this invention provides two expression methods using Pichia pastoris and Escherichia coli as host cells. After purification, high-affinity albumin-binding nanobodies are obtained. These nanobodies can be used to extend the half-life of biopharmaceuticals, thereby improving their therapeutic efficacy.

[0066] The present application solution will be described below through specific embodiments.

[0067] It should be noted that, unless otherwise specified, all biological and chemical reagents used in the embodiments of this invention are obtained through conventional commercial channels. Experimental methods without specific conditions are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations.

[0068] Example 1: Camel Immunization and Peripheral Blood Lymphocyte Isolation

[0069] Camels were immunized via subcutaneous injection at multiple sites in the neck after a mixture of recombinant human albumin and Freund's adjuvant. A total of five immunizations were administered. Blood samples were collected before each immunization, before the fourth immunization, before the fifth immunization, and two weeks after the fifth immunization. Serum was obtained after centrifugation, and antibody titers were detected using ELISA (enzyme-linked immunosorbent assay). Peripheral blood mononuclear cells (PBMCs) were obtained after centrifugation following the fifth immunization.

[0070] Example 2: Phage Library Construction

[0071] Total RNA was extracted from PBMCs using the Trizol method, and cDNA was synthesized via reverse transcription. Single-domain antibody fragments were amplified by two PCR assays, digested with restriction endonucleases, and ligated into phage plasmids. The phages were then transformed into *E. coli* TG1 competent cells using electroporation. The library volume was determined to be 5.15 × 10⁻⁶ cells using a serial dilution method. 9 Forty single clones were randomly selected for PCR identification, and the positive rate was 39 / 40.

[0072] Example 3: Screening of anti-human albumin nanobodies

[0073] Magnetic beads and enzymatically hydrolyzed casein (Solarbio, catalog number C8210-100) were added to EP tubes and blocked for 1 h. Recombinant human albumin was added and incubated for 1 h for coating. After blocking again with enzymatically hydrolyzed casein, a phage library was added for binding. The tubes were washed 9 times with PBST (10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) diluted to 1X, then 0.1% Tween-20 was added. The tubes were then washed once with PBS (pH 7.4). Trypsin was added for elution, and the elution was terminated with enzymatically hydrolyzed casein to obtain the elution product. The elution product was used to infect TG1 *E. coli*, plated, and incubated overnight at 37°C. All colonies of *E. coli* in the logarithmic growth phase were infected with M13K07 to expand the culture. The library was enriched by three rounds of cyclic screening. The final screening strains were plated on ampicillin-resistant plates, and single colonies were picked and placed on sterile cell culture plates for ELISA detection. Qualified positive single colonies were selected and their genes were sequenced to obtain multiple nanobody sequences, one of which was named 4C11, and its amino acid sequence is shown in SEQ ID NO. 1.

[0074] Example 4 Expression and purification of nanobodies in Pichia pastoris

[0075] (1) Pichia pastoris expression of nanobody 4C11

[0076] The 4C11 antibody gene was cloned into the yeast vector pPICZαA after codon optimization using Pichia pastoris. The optimized nucleotide sequence is shown in SEQ ID NO. 5. Sac I After linearization by enzyme digestion, the nanobody was electroporated into the X-33 yeast strain, and single colonies were screened on Zeocin-resistant plates. Secretory expression was induced by routine inoculation, with methanol added every 24 h to a final concentration of 0.5%. After 72 h of induction, the sample was collected, and the supernatant was collected by centrifugation. The expression of the nanobody 4C11 was confirmed by SDS-PAGE.

[0077] (2) Purification of Pichia pastoris expression nanobody 4C11

[0078] The yeast fermentation supernatant was added to a final concentration of 25 mM imidazole. After dissolution, it was filtered through a 0.45 μm filter membrane for loading. The nickel column was equilibrated with loading buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH adjusted to 8.0 with NaOH). Once the column reached baseline, the sample was loaded, and flow-through was collected. The column was further washed with loading buffer until the baseline returned to level. Elution buffer was then used, and the eluent was collected. The desalting column was equilibrated to four column volumes with desalting buffer. All the eluent was loaded onto the desalting column, and the first peak observed was collected as the final antibody solution. If the protein concentration was low after desalting, it was concentrated using an ultrafiltration tube. The purity of the collected solutions at each purification stage was tested, and the SDS-PAGE results are shown below. Figure 1 As shown in the figure, the main band of the purified target protein was clear, and no obvious impurities were observed.

[0079] Example 5 Expression and purification of nanobodies in Escherichia coli

[0080] (1) Expression of nanobody 4C11 in Escherichia coli

[0081] The 4C11 antibody gene fragment, synthesized after codon optimization in *E. coli*, was ligated into the pET-28A(+) expression vector. The optimized nucleotide sequence is shown in SEQ ID NO. 6. The vector was then transformed into *E. coli* BL-21 competent cells. The transformed BL-21 positive colonies were inoculated into LB medium and cultured until OD200. 600 The concentration of the nanobody 4C11 was approximately 0.6 mg / L. IPTG was added to a final concentration of 1 mM and the expression was induced at 20°C and 250 rpm for 48 h. The bacterial cells were collected by centrifugation, sonicated, and the supernatant was discarded after centrifugation. The inclusion bodies were then renatured. SDS-PAGE analysis of the 4C11 nanobody showed good protein expression after IPTG induction.

[0082] (2) Purification of Escherichia coli expression nanobody 4C11

[0083] The renatured E. coli expression solution was added to a final concentration of 25 mM imidazole. After dissolution, it was filtered through a 0.45 μm filter membrane for sample loading. The nickel column was equilibrated with loading buffer until the baseline was reached, then the sample was loaded and flow-through was collected. The column was washed again with loading buffer until the baseline was reached, then eluted with elution buffer, and the eluent was collected. The desalting column was equilibrated to four column volumes with desalting buffer, and all the eluent was loaded onto the desalting column. The first peak that appeared was collected as the final antibody solution. If the protein concentration was low after desalting, it was concentrated using an ultrafiltration tube. The collected solutions from each stage of protein renaturation and purification were analyzed by SDS-PAGE. The results are as follows: Figure 2 As shown, the main band of the purified target protein is clear, with no obvious impurities.

[0084] Example 6: Validation of Nanobody ELISA Binding

[0085] Add 100 μL of 0.05 μg / mL recombinant human albumin to a 96-well plate, coat overnight at 4°C, and blot dry. Wash three times with 300 μL PBST, add 100 μL of 5% skim milk powder, and incubate at 37°C for 1 h for blocking. Discard the solution and blot dry. Add 100 μL of serially diluted nanobodies (initial concentration 1 μg / mL, 12 concentration gradients) dissolved in 5% skim milk powder to each well of a 96-well plate, incubate at 37°C for 1 h, and discard the solution and blot dry. Add 1 μL of HRP-labeled secondary antibody (anti-his) to 10 mL of 5% skim milk powder, mix well, add 100 μL to each well of a 96-well plate, and incubate overnight at 4°C. Wash five times with 300 μL PBST, add 100 μL of chromogenic buffer (TMB), and react in the dark for 10 min. Add 100 μL of stop solution and measure the absorbance at 450 nm. Calculate the absorbance OD corresponding to each concentration of nanobody. 450 Mean, in terms of OD 450 The mean was plotted on the ordinate and antibody concentration on the abscissa. Reaction curves of human albumin-nanobodies at different concentrations were plotted. The concentration of nanobodies corresponding to half the absorbance of the flat segment on each curve was defined as EC50. 50 The result is as follows Figure 3 As shown, the detection results are: yeast expresses EC 50 =4.11E-03 μg / mL.

[0086] Example 7: Human serum albumin-nanobody affinity detection

[0087] The affinity of human serum albumin-nanobody 4C11 was detected using biomembrane interferometry (BLI). Using an NTA biosensor, the sensor was first equilibrated in analytical buffer for 10 min, then activated by immersing it in an EDC-NHS mixture for 5 min. The activated sensor was then incubated in nanobody dilution buffer (100 nM) for 10 min and blocked with ethanolamine (1 M, pH 8.5). The blocked sensor was then zeroed by immersing it in buffer solution. Next, the sensor was sequentially immersed in gradient concentrations of human serum albumin solution (50–0.78 nM) for 5 min to bind, and a complete binding curve was generated. Finally, the sensor was transferred to PBS buffer for dissociation for 5 min. Kinetic analysis was performed using a 1:1 binding model, and the results are as follows: Figure 4 As shown, the dissociation equilibrium constant KD = 7.11E-010 M indicates that the nanobody 4C11 has a strong binding to human serum albumin.

[0088] Example 8 Half-life extension test

[0089] 1) Expression and purification of NGF-nanobody fusion protein

[0090] The 4C11 nanobody can be fused with other peptide molecules for expression. Leveraging the antigen-antibody specific binding characteristic, it binds to endogenous albumin, thereby increasing the drug's half-life. This example uses nerve growth factor (NGF) as an example to construct an NGF-4C11 fusion protein (SEQ ID NO.7). NGF and the 4C11 nanobody were linked using a (G4S)3 flexible linker to synthesize the fusion protein gene fragment (SEQ ID NO.8), which was then cloned into the yeast vector pPICZαA. Sac I After linearization by enzyme digestion, the enzyme was electroporated into the X-33 yeast strain, and single colonies were screened on Zeocin-resistant plates. Secretory expression was induced by routine inoculation, with methanol added every 24 h to a final concentration of 0.5%. After 72 h of induction, the supernatant was collected by centrifugation. The fermentation supernatant was added to imidazole to a final concentration of 25 mM, and after dissolution, filtered through a 0.45 μm filter membrane for loading. The nickel column was equilibrated with loading buffer until the baseline was reached, then the sample was loaded, and flow-through was collected. The column was further washed with loading buffer until the baseline was again reached, then eluted with elution buffer, and the eluent was collected. The desalting column was equilibrated to four column volumes with desalting buffer, and all the eluent was loaded onto the desalting column. The first peak observed was collected as the target protein solution. If the protein concentration was low after desalting, it was concentrated using ultrafiltration to obtain high-purity NGF-4C11 fusion protein.

[0091] 2) Protein half-life detection

[0092] Six adult male rats were randomly divided into two groups.

[0093] Experimental group: subcutaneous injection of 35 μg / kg NGF-4C11

[0094] Control group: Subcutaneous injection of 35 μg / kg free NGF

[0095] Blood was collected via tail vein at 1, 2, 4, 8, 12, 24, 48, 72, and 96 hours after drug administration, and plasma was separated.

[0096] The concentrations of NGF-4Cl1 and NGF in serum were quantitatively detected by ELISA, and the half-life of each sample in vivo was calculated. The results are shown in Table 1 below.

[0097] Table 1 Results of Half-Life Extension Test

[0098]

[0099] The half-life (20 h) of the NGF-4C11 fusion protein was significantly extended by 8 times compared to the NGF standard (2.47 h), confirming that the anti-serum albumin nanobody 4C11 can effectively increase the retention time of NGF in vivo, providing a technical basis for the development of long-acting NGF drugs.

[0100] The above description is merely an embodiment of the present invention, and the scope of protection of the present invention is not limited to these specific embodiments, but is determined by the claims of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any modifications, equivalent substitutions, improvements, etc., made within the technical concept and principle of the present invention should be included within the scope of protection of the present invention.

Claims

1. An albumin-bound nanobody, characterized in that, The nanobody can specifically bind to serum albumin, and the nanobody has the following complementarity-determining regions (CDRs): CDR-H1 shown in SEQ ID NO.2 (GCVG), CDR-H2 shown in SEQ ID NO.3 (VIDANGRTTYADSVKG), and CDR-H3 shown in SEQ ID NO.4 (GLRTITYECRPIRDAFRF).

2. The nanobody according to claim 1, characterized in that, The amino acid sequence of the nanobody includes at least one of the following: A1) The amino acid sequence includes the amino acid sequence shown in SEQ ID NO.1; A2) An amino acid sequence of a fusion protein with the same function obtained by linking a tag protein to the N-terminus and / or C-terminus of the amino acid sequence described in A1).

3. A biomaterial, characterized in that, The biomaterial includes at least one of the following: B1) A nucleic acid molecule encoding the nanobody of claim 1 or 2; B2) An expression cassette containing the nucleic acid molecule described in B1); B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing nucleic acid molecules of B1), or recombinant microorganisms containing recombinant vectors of expression cassettes of B2), or recombinant microorganisms containing recombinant vectors of B3); B5) Recombinant cells, wherein the recombinant cells are recombinant cells containing the nucleic acid molecules of B1), or recombinant cells containing the recombinant vector of the expression cassette of B2), or recombinant cells containing the recombinant vector of B3).

4. The biomaterial according to claim 3, characterized in that, The nucleic acid molecule described in B1) includes at least one of the nucleotide sequences shown in SEQ ID NO. 5 or SEQ ID NO.

6.

5. The biomaterial according to claim 3, characterized in that, The recombinant microorganisms described in B4) or the recombinant cells described in B5) include at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

6. A product characterized in that, The product includes a kit containing the nanobody of claim 1 or 2, and / or the biomaterial of any one of claims 3-5.

7. A method for preparing the nanobody according to claim 1 or 2, characterized in that, The method includes the steps of culturing the recombinant microorganism (B4) or the recombinant cell (B5) in the biomaterial of claim 3 under suitable culture conditions, and isolating the nanobody.

8. The use of the nanobody of claim 1 or 2, or the biomaterial of any one of claims 3-5, or the product of claim 6, or the method of claim 7, in any of the following aspects: D1) Applications in the preparation of products with extended NGF half-life; D2) Applications in the preparation of products that promote the purification or enrichment of serum albumin; D3) Applications in the preparation of products for qualitative or quantitative detection of serum albumin.

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