A serum albumin-binding nanobody 4b1 and uses thereof

Abstract

The application discloses a serum albumin-binding nanobody 4B1 and application thereof, and belongs to the technical field of antibody engineering. The serum albumin-binding nanobody 4B1 disclosed by the application comprises specific complementarity determining regions, i.e. CDR-H1 shown in SEQ ID NO. 2, CDR-H2 shown in SEQ ID NO. 3 and CDR-H3 shown in SEQ ID NO. 4, so that the serum albumin-binding nanobody 4B1 can be combined with serum albumin in high affinity and specificity. Based on the property, the nanobody 4B1 can effectively prolong the in-vivo half-life of a polypeptide, a protein or an antibody drug fused with the nanobody 4B1, and significantly improve the treatment effect and practicability of a related biological and pharmaceutical product, and is especially suitable for development of long-acting polypeptides, proteins and antibody drugs.

Description

Technical Field

[0001] This invention relates to the field of antibody engineering technology, specifically to a serum albumin-binding nanobody and its application. Background Technology

[0002] Biologics, such as vaccines, blood products, biotechnological drugs, and nucleic acid drugs, have been widely used in the prevention, diagnosis, and treatment of diseases. However, protein and peptide drugs among them generally have short half-lives in serum, affecting their expected efficacy. This is mainly attributed to clearance processes such as protease degradation and glomerular filtration in the body. To achieve therapeutic effects, high doses or frequent administration are often required, but this may lead to increased drug side effects and decreased patient compliance. Traditional strategies to prolong half-life include PEGylation, Fc fragment fusion, and albumin conjugation, but these methods have significant drawbacks: PEGylation may reduce drug activity and induce immunogenicity; while Fc fusion can activate complement-dependent cytotoxicity and antibody-dependent cell-mediated phagocytosis, it has high production costs and poor stability.

[0003] In recent years, nanobodies have become a focus of novel drug carrier development due to their advantages such as small molecular weight, strong permeability, high stability, and simple humanization. In 1993, Hamers et al. first discovered a heavy chain antibody in camel blood that lacked the CH1 region of the light and heavy chain constant regions of traditional antibodies. The variable domain fragment of the heavy chain antibody is called VHH (Variable domain of heavy chain of heavy-chain antibody). Jiangnan University recruited endogenous anti-rhamnolipid antibodies to form immune complexes by modifying haptens such as rhamnolipid, increasing the hydration radius to slow renal clearance. However, monovalent modification resulted in insufficient affinity, and the efficacy depended on the abundance of antibodies in the patient's body (CN116920086A, 2023.10.24). Peking University developed temperature-responsive nanobody conjugates that improved pharmacokinetics and enhanced tissue permeability and bioavailability through smart material modification (CN117860909A, 2024.04.12).

[0004] Human serum albumin (HSA) is a natural transport protein in blood with a molecular weight of 66.5 kDa, and is the most abundant protein in human plasma. One of the main physiological functions of HSA is its participation in the transport, distribution, and metabolism of various endogenous and exogenous substances. HSA has a long half-life (approximately 19 days), is biodegradable, non-immunogenic, and biocompatible, and is widely used in the delivery of various therapeutic drugs, including small molecule drugs, peptides, proteins, and nucleic acids. HSA can be used to load drugs in single-molecule form, such as by linking therapeutic drugs to HSA molecules through non-covalent, covalent, or gene fusion methods. Currently, HSA has been successfully used to produce various fusion proteins. The "official hat type" anti-CD4 nanobody (NbCD4-NbHSA-NbCD4) developed by Nanjing University significantly prolongs the half-life by binding to HSA (CN118290589A, 2024.07.05). Kangzhong Biotechnology has screened out the Nb3 nanobody, which broadly binds to multiple serum albumins (human, bovine, and mouse), and can be adapted to different animal models and improve drug targeting (CN116023487A, 2023.04.28). The fusion of HSA and peptides significantly increases the molecular weight of peptide biopharmaceuticals, bringing many problems to production and transformation. The increase in molecular weight can also change some pharmacokinetic properties of the drug, making it difficult for it to reach the target site and thus affecting efficacy.

[0005] Future development trends focus on multi-mechanism synergistic effects and intelligent design. Based on this background, this study proposes a nanobody that binds to serum albumin. This invention involves periodically immunizing camels to induce the production of specific antibodies against human albumin. Phage display technology is used for selection, and the antibodies are expressed in yeast and *E. coli* to ultimately obtain a high-affinity single-domain antibody against human albumin. This invention aims to overcome the limitations of developing antibodies based on differences in antigenic epitopes, antibody affinity, or antibody structure (sequence), laying the foundation for extending the half-life of biopharmaceuticals and improving the therapeutic efficacy and practicality of related biopharmaceutical products, especially suitable for the development of long-acting peptides, proteins, and antibody drugs. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a serum albumin-bound nanobody and its application. The serum albumin-bound nanobody disclosed in this invention has a high affinity for albumin and can be used to prolong the half-life of biological drugs, thereby improving the therapeutic effects of biological drugs.

[0007] In a first aspect, the present invention provides a nanobody that binds to serum albumin, the nanobody being specifically capable of binding to serum albumin, and the nanobody having the following complementarity-determining regions (CDRs): CDR-H1 shown in SEQ ID NO.2 (NCMG), CDR-H2 shown in SEQ ID NO.3 (TIYNRGTNTYYADSVKG), and CDR-H3 shown in SEQ ID NO.4 (DPRSILTLSSCRSDEYRW).

[0008] Furthermore, the amino acid sequence of the nanobody includes at least one of the following:

[0009] A1) The amino acid sequence includes the amino acid sequence shown in SEQ ID NO.1;

[0010] A2) is an amino acid sequence obtained by substituting and / or deleting and / or adding amino acid residues from the amino acid sequence of the nanobody described in A1), which has more than 95% identity with the amino acid sequence of the nanobody described in A1.

[0011] A3) An amino acid sequence of a fusion protein with the same function obtained by attaching a tag protein to the N-terminus and / or C-terminus of the amino acid sequence described in A1) or A2).

[0012] In a second aspect, the present invention provides a biomaterial comprising at least any one of the following:

[0013] B1) Contains a nucleic acid molecule encoding the nanobody described above;

[0014] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0015] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0016] 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);

[0017] 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.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] Optionally, the recombinant microorganism includes at least one strain of Escherichia coli and Pichia pastoris.

[0023] 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.

[0024] In a third aspect, the present invention comprises an antiserum albumin antibody containing the aforementioned nanobody amino acid sequence as a VHH chain.

[0025] 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.

[0026] Further, the modification may optionally include:

[0027] Particulate matter bonded by non-covalent bonds, such as colloidal gold, colloidal silver, or colloidal carbon;

[0028] Modifiers that are covalently bonded include, but are not limited to:

[0029] 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).

[0030] In a fifth aspect, the present invention provides a product comprising at least one of the following:

[0031] 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;

[0032] 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;

[0033] C3) A pharmaceutical composition comprising the nanobody, or the antiserum albumin antibody, or a derivative thereof;

[0034] 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).

[0035] 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.

[0036] 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.

[0037] 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:

[0038] D1) Applications in the preparation of products with extended drug half-life;

[0039] D2) Applications in the preparation of immunoassay or diagnostic products;

[0040] D3) Applications in the preparation of products that promote the purification or enrichment of serum albumin;

[0041] D4) Applications in the preparation of products for qualitative or quantitative detection of serum albumin.

[0042] 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.

[0043] 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.

[0044] 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 present invention, the protein-based drug is exendin.

[0045] Furthermore, the product also includes pharmaceutically acceptable additives. Optionally, the pharmaceutical composition further includes pharmaceutically acceptable carriers and / or excipients.

[0046] The beneficial effects of the present invention include, but are not limited to:

[0047] This invention provides a nanobody that can specifically recognize human albumin through screening. This nanobody has a high affinity for 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 medical field.

[0048] 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

[0049] 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:

[0050] Figure 1 This is an SDS-PAGE electrophoresis image of Pichia pastoris expressing 4B1 for 48 hours in an embodiment of the present invention. M: marker; 1: uninduced; 2: induced for 48 hours.

[0051] Figure 2 The images shown are SDS-PAGE electrophoresis diagrams of each stage of purification of Pichia pastoris expression 4B1 in this embodiment of the invention. M: marker; 1: before loading; 2: flow-through; 3: elution; 4: desalting.

[0052] Figure 3 This is an SDS-PAGE electrophoresis image of E. coli expressing 4B1 for 48 hours in an embodiment of the present invention. M: marker; 1: supernatant; 2: bacterial cells.

[0053] Figure 4 The images shown are SDS-PAGE electrophoresis images of each stage of the purification of 4B1 expression in E. coli in this embodiment of the invention. M: marker; 1: elution; 2: flow-through; 3: before loading.

[0054] Figure 5 This is an ELISA binding verification analysis diagram of Pichia pastoris expression of 4B1 in an embodiment of the present invention.

[0055] Figure 6 This is a graph showing the affinity detection results of human albumin-nanobody 4B1 in an embodiment of the present invention. Detailed Implementation

[0056] 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.

[0057] Escherichia coli TG1 competent cells were purchased from Shanghai Maokang Biotechnology Co., Ltd., catalog number MF2384.

[0058] Escherichia coli BL-21 competent cells were purchased from Shanghai Maokang Biotechnology Co., Ltd., catalog number MF2391.

[0059] The X-33 yeast strain was purchased from Thermo Fisher Scientific, catalog number C18000.

[0060] Adult male rats were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd., product number SN0159897.

[0061] Exenatide (Exendin-4) was purchased from MedChemExpress (MCE), catalog number HY-13443.

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

[0063] Recombinant human albumin: self-supplied by Tonghua Anruit Biopharmaceutical Co., Ltd.

[0064] 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.

[0065] Sample loading buffer: 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH adjusted to 8.0 with NaOH.

[0066] This invention involves periodically immunizing camels to induce the production of specific antibodies against human albumin. Phage display technology is used for panning, and multiple nanobody sequences are obtained after three rounds of panning. One of these is named 4B1, 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 anti-human albumin nanobodies are obtained. These nanobodies can be used to extend the half-life of biopharmaceuticals, thereby improving their therapeutic effects.

[0067] The present application solution will be described below through specific embodiments.

[0068] 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.

[0069] Example 1: Camel Immunization and Peripheral Blood Lymphocyte Isolation

[0070] 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.

[0071] Example 2: Phage Library Construction

[0072] 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.

[0073] Example 3: Screening of anti-human albumin nanobodies

[0074] 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 4B1, and its amino acid sequence is shown in SEQ ID NO. 1.

[0075] Example 4 Expression and purification of nanobodies in Pichia pastoris

[0076] (1) Pichia pastoris expression of nanobody 4B1

[0077] The 4B1 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 4B1 was determined by SDS-PAGE, and the results are as follows. Figure 1 As shown, the expression of nanobody 4B1 was confirmed to be correct.

[0078] (2) Purification of Pichia pastoris expression nanobody 4B1

[0079] 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 2 As shown in the figure, the main band of the purified target protein was clear, and no obvious impurities were observed.

[0080] Example 5 Expression and purification of nanobodies in Escherichia coli

[0081] (1) Expression of nanobody 4B1 in Escherichia coli

[0082] The 4B1 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 =Approximately 0.6, IPTG was added to a final concentration of 1 mM and the expression was induced at 20℃ and 250 rpm for 48 h. Bacterial cells were collected by centrifugation, sonicated, and the supernatant was discarded after centrifugation. The inclusion bodies were then renatured. The expression of the nanobody 4B1 was determined by SDS-PAGE, and the results are as follows. Figure 3 As shown, protein expression was good after IPTG induction.

[0083] (2) Purification of Escherichia coli expression nanobody 4B1

[0084] 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 4 As shown, the main band of the purified target protein is clear, with no obvious impurities.

[0085] Example 6: Validation of Nanobody ELISA Binding

[0086] 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-nanobody at different concentrations were plotted. The EC50 was defined as the nanobody concentration corresponding to half the absorbance of the flat segment on each curve. The results are as follows: Figure 5 As shown, the detection results are: yeast expresses EC 50 =1.06E-03 μg / mL.

[0087] Example 7: Human albumin-nanobody affinity detection

[0088] The affinity of human albumin-nanobody 4B1 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 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 6 As shown, the dissociation equilibrium constant KD = 4.82E-010 M indicates that the nanobody 4B1 has a strong binding to human albumin.

[0089] Example 8 Half-life extension test

[0090] 1) Expression and purification of peptide-nanobody fusion protein

[0091] The anti-serum albumin nanobody 4B1 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 exenatide (Exendin-4) as an example to construct the Exendin-4-4B1 fusion protein. Exendin-4 and nanobody 4B1 were linked using a (G4S)3 flexible linker to synthesize the fusion protein gene fragment (SEQ ID NO.7), 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.

[0092] 2) Protein half-life assay: Six adult male rats were randomly divided into two groups;

[0093] Experimental group: subcutaneous injection of 1 mg / kg Exendin-4-4B1

[0094] Control group: Subcutaneous injection of 1 mg / kg free Exendin-4

[0095] Blood samples were collected at different time points to determine the levels of free Exendin-4 and Exendin-4-4B1 in serum, and the half-life of each sample in vivo was calculated. The results are shown in Table 1 below.

[0096] Table 1 Results of Half-Life Extension Test

[0097]

[0098] The results showed that the half-life of the Exendin-4-4B1 fusion protein was extended by about 15 times compared with that of free Exendin-4, indicating that anti-serum albumin nanobodies can significantly increase the half-life of drug molecules in vivo.

[0099] 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. A nanobody that binds to 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 (NCMG), CDR-H2 shown in SEQ ID NO.3 (TIYNRGTNTYYADSVKG), and CDR-H3 shown in SEQ ID NO.4 (DPRSILTLSSCRSDEYRW).

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) are selected from 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 pharmaceutical composition comprising the nanobody of claim 1 or 2; C2) A kit comprising the nanobody of claim 1 or 2.

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) Application in the preparation of products with extended exenatide (Exendin-4) 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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