A nanobody targeting serum albumin nanobody 3h8 and derivatives and uses thereof

Abstract

The application discloses a nanobody 3H8 targeting serum albumin and a derivative and application thereof, and belongs to the technical field of antibody engineering. Complementarity determining regions CDR of the nanobody include CDR-H1 shown in SEQ ID NO. 2 (RYCMG), CDR-H2 shown in SEQ ID NO. 3 (TIKTLGGSTYYVESVKG), and CDR-H3 shown in SEQ ID NO. 4 (DAGFWSSGGYCLPNLAYMY). The nanobody is coupled with a therapeutic polypeptide, a protein or an antibody drug to construct a long-acting complex. The complex can significantly delay the renal clearance rate of the drug, resist protease degradation, improve the half-life of the drug, avoid frequent administration, reduce the administration frequency while maintaining the curative effect, improve the patient compliance and the treatment safety, and is used for the development of long-acting polypeptides, proteins and antibody drugs.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of antibody engineering, in particular to a nanobody 3H8 targeting serum albumin 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 reduced 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 exceeds 100 kDa, which hinders tissue penetration and poses a risk of activating complement-dependent cytotoxicity; the human serum albumin (HSA) direct fusion strategy 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. Targeting serum albumin nanobody can obtain long-circulating properties by specific 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 modification, 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 successful 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 out the nanobody Nb3, which can bind to multiple serum albumins (human, bovine, and mouse) and adapt to different animal models, improving 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). Targeting 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 application 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 application 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 polypeptide, protein, 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] In order to solve the above problems, the present application provides a nanobody 3H8 targeting serum albumin nanobody and its derivatives and applications. The nanobody targeting serum albumin 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, a nanobody targeting serum albumin 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 (RYCMG), CDR-H2 as shown in SEQ ID NO. 3 (TIKTLGGSTYYVESVKG), and CDR-H3 as shown in SEQ ID NO. 4 (DAGFWSSGGYCLPNLAYMY). 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) containing a nucleic acid molecule encoding the nanobody;

[0015] B2) containing an expression cassette of B1);

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

[0017] B4) containing a recombinant microorganism of B1), or a recombinant microorganism containing the expression cassette of B2), or a recombinant microorganism containing the recombinant vector of B3);

[0018] B5) recombinant cells containing the recombinant cells of B1), or the recombinant cells containing the recombinant vectors of B2), or the recombinant cells containing the recombinant vectors of B3), or the recombinant cells containing the recombinant microorganisms of B4).

[0019] Further, the nucleic acid molecules in B1) include the nucleic acid molecules encoding the nanobodies disclosed and / or optimized according to actual needs, and optionally, the nucleic acid molecules in B1) include at least one of the nucleotide sequences as 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 respectively codon bias optimized sequences for Pichia pastoris and Escherichia coli; a person skilled in the art can optimize the nucleic acid encoding SEQ ID NO: 1 according to different expression systems (such as other engineering bacteria), and such variants are within the scope of the present application.

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

[0022] Further, the recombinant microorganisms in B4) or the recombinant cells 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 microorganisms include at least one of Escherichia coli and Pichia pastoris species.

[0024] It should be noted that the present application does not limit the recombinant microorganisms or recombinant cells, and any cell capable of expressing exogenous genes through recombinant engineering technology is within the scope of the present application.

[0025] In a third aspect of the present application, an anti-serum albumin antibody containing the nanobody amino acid sequence as a VHH chain is provided.

[0026] In a fourth aspect of the present application, a derivative of the nanobody or the anti-serum albumin antibody is provided, and the derivative includes a modifier combined with the nanobody or the antibody through a covalent bond or a non-covalent bond.

[0027] Further, the modifier optionally includes:

[0028] a particle substance combined through a non-covalent bond, such as colloidal gold, colloidal silver, or colloidal carbon;

[0029] Modifications that are bound by covalent bonds, including but not limited to:

[0030] Microspheres (such as colored microspheres, fluorescent microspheres, magnetic microspheres), chromatographic fillers, chemical small molecules (such as biotin, dye molecules, fluorescent molecules).

[0031] In a fifth aspect, the present application provides a product, which comprises at least one of:

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

[0033] C2) a conjugate constructed by linking the nanobody, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody, with a therapeutic agent;

[0034] C3) a pharmaceutical composition of the nanobody, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody;

[0035] C4) a kit comprising the nanobody, and / or the biomaterial, and / or the anti-serum albumin antibody, and / or the derivative of the nanobody or 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] Further, the fusion protein is prepared by linking a polypeptide or a protein to the N-terminus or C-terminus of the nanobody, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody, and optionally, the polypeptide or the protein and the nanobody, or the anti-serum albumin antibody, or the derivative of the nanobody or the anti-serum albumin antibody are linked by a chemical bond.

[0037] In a sixth aspect, the present application provides a method for preparing the nanobody and / or the fusion protein, characterized in that the method comprises the step of culturing the recombinant microorganism of B4) or the recombinant cell of B5) in the biomaterial under suitable culture conditions, and isolating the nanobody or the fusion protein.

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

[0039] D1) use in the preparation of a product for prolonging the half-life of a drug;

[0040] D2) use in the preparation of an immunoassay or diagnostic product;

[0041] D3) use in the preparation of a product for facilitating the purification or enrichment of serum albumin;

[0042] D4) use in the preparation of a product for qualitatively or quantitatively detecting serum albumin.

[0043] Further, the drug for prolonging the half-life of the drug includes a protein drug, an antibody fragment, a small molecule chemotherapeutic drug, and other biological agents; preferably, the protein drug includes at least one of an IL-6R targeted drug, a TNF-α targeted drug, and an IL-17A / IL-17F targeted drug; the small molecule chemotherapeutic drug includes at least one of doxorubicin, sorafenib, and sirolimus.

[0044] Further, the product for prolonging the half-life of the drug in A1 includes at least one of a fusion protein composed of a serum albumin-targeting nanobody and a protein drug, a conjugate composed of a serum albumin-targeting nanobody and a therapeutic agent, and a pharmaceutical composition of the serum albumin-targeting nanobody.

[0045] Further, the protein drug includes at least one of a polypeptide, an antibody, an antibody fragment, a cytokine, and a tumor marker. In a specific embodiment of the present application, the drug is an interleukin-1-receptor antagonist (IL-1Ra).

[0046] Further, the product further includes an additive acceptable in the medical field. Alternatively, the pharmaceutical composition further includes a pharmaceutically acceptable carrier and / or an excipient.

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

[0048] The present application screens a serum albumin-targeting nanobody capable of highly specifically recognizing serum albumin, which has a high affinity for serum albumin and can be widely applied to enhancing the half-life of a drug, tumor-targeted therapy and imaging, immunoassay or diagnosis, and the purification or enrichment of recombinant albumin, and has a wide application prospect in the medical field.

[0049] The present application also provides two expression preparation methods of the nanobody using Pichia pastoris and Escherichia coli as host cells, which are beneficial to the commercial production. BRIEF DESCRIPTION OF DRAWINGS

[0050] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0051] Figure 1 SDS-PAGE electrophoretogram of each stage of purification of 3H8 expressed by Pichia pastoris in the embodiment of the application, M: marker; 1: before loading; 2: flow-through; 3: elution; 4: desalination.

[0052] Figure 2 SDS-PAGE electrophoretogram of each stage of purification of 3H8 expressed by E. coli in the embodiment of the application, M: marker; 1: desalination; 2: flow-through; 3: before loading.

[0053] Figure 3 ELISA binding verification analysis diagram of 3H8 expressed by Pichia pastoris in the embodiment of the application.

[0054] Figure 4 Human serum albumin-nanobody 3H8 affinity detection result diagram in the embodiment of the application. DETAILED DESCRIPTION

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

[0056] E. coli TG1 competent cells were purchased from Shanghai Moikang Biotechnology Co., Ltd., item number MF2384.

[0057] E. coli BL-21 competent cells were purchased from Shanghai Moikang Biotechnology Co., Ltd., item number MF2391.

[0058] X-33 yeast strain was purchased from Thermo Fisher scientific Co., Ltd., item number C18000.

[0059] Adult male rats were purchased from Jiangsu Jizhuangkang Biotechnology Co., Ltd., item number SN0159897.

[0060] Interleukin-1-receptor antagonist (IL-1Ra) was purchased from PeproTech, item number: 200-01RA 1MG, agent: Thermo Fisher Scientific Co., Ltd.

[0061] Recombinant human serum albumin was self-prepared by Tonghua Anruit Biological Pharmaceutical Co., Ltd.

[0062] Enzymatic hydrolysis casein was purchased from Solarbio, item number: C8210-100.

[0063] PBST: diluted from 10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) to 1X, plus 0.1% Tween-20.

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

[0065] The present application produces specific antibodies against human serum albumin by regularly immunizing camels, and uses phage display technology to perform panning, and obtains multiple nanobody sequences through three rounds of panning, one of which is named 3H8, and the amino acid sequence is shown as SEQ ID NO. 1. According to the Kabat definition scheme, the CDR is defined, the CDR-H1 amino acid sequence is shown as SEQ ID NO. 2, the CDR-H2 amino acid sequence is shown as SEQ ID NO. 3, and the CDR-H3 amino acid sequence is shown as SEQ ID NO. 4. In addition, the present application provides two expression modes of Pichia pastoris and Escherichia coli as host cells, and obtains the target serum albumin nanobody 3H8 with high affinity through purification, and the nanobody can be used for prolonging the half-life of biological drugs, and is beneficial to improve the beneficial effect of biological drugs in treatment.

[0066] The present application scheme is described below through specific examples.

[0067] It should be noted that the various biological and chemical reagents used in the embodiments of the present application are obtained through conventional commercial channels unless otherwise specified, and the experimental methods not marked with specific conditions are usually performed according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.

[0068] Example 1 Camel immunization and peripheral blood lymphocyte separation

[0069] The camel was immunized by subcutaneous injection of recombinant human albumin mixed with Freund's adjuvant, a total of 5 times of immunization. Blood was collected before immunization, before the 4th immunization, before the 5th immunization, and 2 weeks after the 5th immunization. After standing and centrifugation, serum was obtained. The ELISA (enzyme linked immunosorbent assay) method was used to detect the titer of the immune antibody. After the 5th immunization, blood was collected and centrifuged to obtain peripheral blood mononuclear cells (PBMC).

[0070] Example 2 Phage library construction

[0071] Total RNA in PBMC was extracted by Trizol method, and cDNA was synthesized by reverse transcription. The single domain antibody fragment was amplified by two PCR, and then connected into phage plasmid after restriction enzyme digestion. The library was transformed into E. coli TG1 competent cells by electroporation method. The library capacity was 5.15×10 9 . 40 monoclonal antibodies were randomly selected for PCR identification, and the positive rate was 39 / 40.

[0072] Example 3 Nanobody screening against human albumin

[0073] Add magnetic beads and enzyme hydrolyzed casein (manufacturer: Solarbio, product number: C8210-100) in an EP tube, and seal for 1 h. Add recombinant human albumin and incubate for 1 h for coating. After blocking with enzyme hydrolyzed casein again, add the phage library for binding. PBST (10X PBS (1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4, pH 7.4) diluted to 1X, and 0.1% Tween-20 was added. Wash 9 times with PBS (pH 7.4) once. Add trypsin for elution, and terminate with enzyme hydrolyzed casein to obtain the elution product. Infect TG1 E. coli with the elution product, plate, and incubate at 37°C overnight. Infect E. coli cultured from all colonies in the logarithmic phase with M13K07, and expand the culture. Cycle the screening for 3 rounds to enrich the library. Plate the strains from the last screening on ampicillin-resistant plates, pick single colonies in sterile cell culture plates, and perform ELISA detection. Select qualified positive single colonies for gene sequencing to obtain multiple nanobody sequences, one of which is named 3H8, and its amino acid sequence is shown as SEQ ID NO. 1.

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

[0075] (1) Expression of nanobody 3H8 in Pichia pastoris

[0076] The 3H8 antibody gene was codon-optimized for Pichia pastoris and cloned into the yeast vector pPICZαA. The optimized nucleotide sequence is shown in SEQ ID NO. 5. Sac I After enzyme digestion and linearization, the product was electroporated into the X-33 yeast strain, and single colonies were screened on Zeocin-resistant plates. Secretion expression was induced by inoculating the culture medium and adding methanol at a final concentration of 0.5% every 24 h. The sample was collected after 72 h of induction, and the supernatant was collected by centrifugation. The expression of nanobody 3H8 was determined by SDS-PAGE, and it was confirmed that nanobody 3H8 was expressed correctly.

[0077] (2) Purification of nanobody 3H8 expressed in Pichia pastoris

[0078] The yeast-expressed fermentation supernatant was added to a final concentration of 25 mM imidazole, and after dissolution, it was filtered with a 0.45 μm filter for loading. The nickel column was equilibrated with loading buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0 adjusted with NaOH), and after the baseline was leveled, the sample was loaded and the flow-through was collected. The column was washed with loading buffer until the baseline was leveled again, and then elution buffer was used for elution, and the eluate was collected. The desalting column was equilibrated with desalting buffer for 4 column volumes, and the eluate was loaded onto the desalting column, and the first peak that appeared was collected as the final antibody solution. If the protein concentration was low after desalting, the sample was concentrated using an ultrafiltration tube. The collected samples at each stage of purification were tested for purity, and the SDS-PAGE results are shown in Figure 1 . The results show that the main band of the target protein is clear after purification, and there is no obvious impurity band.

[0079] Example 5 Expression and purification of nanobody in E. coli

[0080] (1) Expression of nanobody 3H8 in E. coli

[0081] The 3H8 antibody gene was codon-optimized for E. coli and synthesized into the pET-28A(+) expression vector. The optimized nucleotide sequence is shown in SEQ ID NO. 6, and the vector was transformed into E. coli BL-21 competent cells. The positive colonies of the transformed BL-21 were inoculated into LB medium and cultured to an OD 600 =0.6, and IPTG was added at a final concentration of 1 mM for induction at 20°C and 250 rpm for 48 h. The bacterial cells were collected by centrifugation, ultrasonicated, and the supernatant was discarded after centrifugation. The inclusion bodies were renatured. The expression of nanobody 3H8 was determined by SDS-PAGE, and the protein expression was good after IPTG induction.

[0082] (2) Purification of Escherichia coli expressing nanobody 3H8

[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 =1.09E-03 μg / mL.

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

[0087] The affinity of human serum albumin-nanobody 3H8 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 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 = 5.24E-010 M indicates that the nanobody 3H8 has a strong binding to human serum albumin.

[0088] Example 8 Half-life extension test

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

[0090] Nanobody 3H8 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 fibroblast growth factor 21 (IL-1Ra) as an example to construct the IL-1Ra-3H8 fusion protein (SEQ ID NO.7). IL-1Ra and nanobody 3H8 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 protein 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 protein 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 IL-1Ra-3H8 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 2mg / kg IL-1Ra-3H8

[0094] Control group: subcutaneous injection of 2mg / kg free IL-1Ra

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

[0096] The concentrations of IL-1Ra-3H8 and IL-1Ra in serum were quantitatively detected by ELISA, and the half-life of each sample in vivo was calculated, and the results are shown in Table 1 below.

[0097] Table 1: Results of half-life extension test

[0098]

[0099] The half-life of the IL-1Ra-3H8 fusion protein (30.0 h) is significantly prolonged to 10 times that of free IL-1Ra (3.0 h), confirming that the targeted serum albumin nanobody 3H8 can effectively prolong the retention time of IL-1Ra in vivo, providing a technical basis for the development of long-acting drugs.

[0100] The above only describes the embodiments of the present application, and the protection scope of the present application is not limited by these specific embodiments, but is determined by the claims of the present application. The present application can have various changes and variations for those skilled in the art. Any modification, equivalent replacement, improvement, etc. within the technical idea and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A nanobody targeting serum albumin, 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 (RYCMG), CDR-H2 shown in SEQ ID NO.3 (TIKTLGGSTYYVESVKG), and CDR-H3 shown in SEQ ID NO.4 (DAGFWSSGGYCLPNLAYMY).

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 at least one of the following: C1) A kit comprising 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, the biomaterial of any one of claims 3-5, 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 IL-1Ra 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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