A bovine serum albumin-binding nanoantibody and its application

Abstract

The present invention discloses a bovine serum albumin-binding nanoantibody and its preparation method and application. The amino acid sequence of the bovine serum albumin-binding nanoantibody Nb1 is shown in SEQ.ID No.1. The nanoantibody Nb not only has a high affinity for bovine serum albumin, but also has good binding activity to human serum albumin and mouse serum albumin. It has a good broad-spectrum application range and can be widely used in enhancing drug half-life, tumor targeted therapy and imaging, immune detection or diagnosis, recombinant albumin purification or enrichment, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody engineering, and in particular to a bovine serum albumin-binding nano antibody and applications thereof. Background Art

[0002] Serum albumins (such as human serum albumin (HSA), bovine serum albumin (BSA), or mouse serum albumin (MSA)) are ideal targets for nanobody (VHH)-mediated drug modification due to their highly conserved gene sequence and structure (the three share 70-80% homology). By designing nanobodies that specifically bind to serum albumin and conjugating them to therapeutic drugs, a "nanobody-serum albumin-drug" ternary complex system can be constructed, significantly optimizing pharmacokinetic and therapeutic efficacy. Modifying drugs with serum albumin-binding nanobodies, especially nanobody-drug conjugates, can extend drug half-life, achieve targeted drug delivery, reduce drug dosage, enhance drug biocompatibility, promote drug absorption, and maximize drug efficacy.

[0003] Bovine serum albumin (BSA), the primary component of bovine serum, boasts excellent water solubility, high stability, and abundant chemical modification sites. It is widely used in the biomedical field, serving as a protein standard and drug carrier. Nanobodies, derived from camelids and lacking the variable region of heavy-chain antibodies, possess unique biological properties such as small molecular weight, high affinity, strong stability, and ease of expression and modification. Their unique structure enables them to recognize epitopes inaccessible to traditional antibodies, demonstrating significant potential for applications in immunodiagnosis and targeted therapy.

[0004] While research on both BSA and nanobodies has progressed, systematic studies on the preparation, characterization, and biological applications of BSA nanobodies remain relatively limited. This is particularly true regarding the efficient preparation of high-affinity, highly stable BSA nanobodies and their application in practical immunoassays and targeted therapies. Further research into this area is expected to provide new strategies and approaches for the diagnosis and treatment of BSA-related diseases. Summary of the Invention

[0005] The core goal of this invention is to prepare high-affinity and stable bovine serum albumin nanobodies, fully characterize them, and explore their potential for biomedical applications. The invention details are as follows:

[0006] In a first aspect, the present invention provides a bovine serum albumin-binding nanobody, the amino acid sequence of the bovine serum albumin nanobody is shown in SEQ ID NO.1.

[0007] Furthermore, the bovine serum albumin-binding nanobody can also bind to human serum albumin (HSA) and mouse serum albumin (MSA).

[0008] In a second aspect, the present invention provides a nucleic acid encoding the bovine serum albumin-binding Nanobody, comprising a currently disclosed nucleotide sequence or a nucleotide sequence that has been codon-optimized as needed. Preferably, the nucleotide sequence is a nucleotide sequence optimized according to the codon preference of Escherichia coli. More preferably, the nucleic acid sequence is as shown in SEQ ID NO. 2 or has 95% homology to the sequence shown in SEQ ID NO. 2.

[0009] The third aspect of the present invention provides a biomaterial, wherein the biomaterial is selected from any one of the following:

[0010] 1. a vector comprising the nucleic acid;

[0011] II. A transformant comprising the vector described in I;

[0012] III. Recombinant cells capable of producing the bovine serum albumin-binding Nanobody and / or containing the nucleic acid.

[0013] Furthermore, the vector includes 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.

[0014] Furthermore, the recombinant cells include at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0015] The fourth aspect of the present invention provides the use of the bovine serum albumin-binding nanobody, the nucleic acid, or the biomaterial in any of the following aspects:

[0016] A1. Application in the preparation of products that extend drug half-life;

[0017] A2. Application in the preparation of tumor targeted therapy and imaging products;

[0018] A3. Application in the preparation of immunoassay or diagnostic products;

[0019] A4. Application in the preparation of products that promote the purification or enrichment of recombinant albumin.

[0020] Furthermore, the drugs used to extend the half-life of the drug include protein drugs, antibody fragments, small molecule chemotherapy drugs, and other biologics. Preferably, the protein drugs include at least one of IL-6R targeted drugs, TNF-α targeted drugs, and IL-17A / IL-17F targeted drugs; and the small molecule chemotherapy drugs include at least one of doxorubicin, sorafenib, and sirolimus. In a specific embodiment of the present invention, the drug is interferon.

[0021] Furthermore, the drug half-life extension product described in A1 includes at least one of a fusion protein consisting of a bovine serum albumin-binding nanobody and a protein drug, a conjugate formed by connecting a bovine serum albumin-binding nanobody and a therapeutic agent, and a pharmaceutical composition of the bovine serum albumin-binding nanobody.

[0022] Furthermore, the protein drug includes at least one of polypeptides, antibodies, antibody fragments, cytokines, and tumor marker molecules that can play a therapeutic role.

[0023] Furthermore, the bovine serum albumin-bound nanobody pharmaceutical composition also includes additives acceptable in the medical field.

[0024] The fifth aspect of the present invention provides a method for preparing the bovine serum albumin-binding nanobody, which comprises the step of introducing the nucleic acid into a recombinant cell to express the bovine serum albumin-binding nanobody.

[0025] The recombinant cell is selected from at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0026] In a specific embodiment of the present invention, the purpose of preparing bovine serum albumin nanobody is achieved by cloning the bovine serum albumin nanobody gene (nucleotide sequence as shown in SEQ ID NO.2) into the expression vector pET-22b (containing a His tag) and transforming BL21 (DE3) Escherichia coli to express the bovine serum albumin nanobody.

[0027] The beneficial effects of the present invention include but are not limited to:

[0028] The bovine serum albumin nanoantibody provided by the present invention not only has a high affinity for bovine serum albumin, but also has good binding activity to human serum albumin and mouse serum albumin. It has a good broad-spectrum application range and can be widely used in enhancing drug half-life, tumor targeted therapy and imaging, immune detection or diagnosis, recombinant albumin purification or enrichment, etc., and has broad application prospects in the medical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0030] Figure 1 Schematic diagram of SDS-PAGE detection results in an embodiment of the present invention;

[0031] Figure 2 Schematic diagram of the ELISA test results of Nb1 with BSA, HSA and MSA in an embodiment of the present invention;

[0032] Figure 3 This is a blood activity-time curve of IFNα-2b in an embodiment of the present invention;

[0033] Figure 4 1 is a blood drug activity-time curve of the fusion protein Nb1-IFNα-2b in the embodiment of the present invention. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the examples, but the present invention is not limited to these examples. Unless otherwise specified, the raw materials and catalysts in the examples of the present invention are purchased through commercial channels.

[0035] Phage display library: natural alpaca / camel VHH library, purchased from Pujian Biotechnology (Wuhan) Technology Co., Ltd.

[0036] Bovine serum albumin: produced by Shanghai Xinruite Biopharmaceutical Technology Co., Ltd.

[0037] Coating buffer: PBS (pH 7.4);

[0038] Blocking solution: 3% skim milk in PBST (PBS + 0.05% Tween-20);

[0039] Acidic elution: 0.1 M Glycine-HCl (pH 2.2), immediately neutralized with 1 M Tris-HCl (pH 9.0);

[0040] Competitive elution: 1-5 mg / mL soluble BSA (dissolved in PBS);

[0041] Host bacteria: XL1-Blue Escherichia coli and BL21 (DE3) Escherichia coli were purchased from Beijing Biobo Biotechnology Co., Ltd.

[0042] Helper phage: M13KO7, purchased from Thermo Fisher Scientific;

[0043] Transfer buffer (25 mM Tris, 192 mM Glycine, 20% methanol, pH 8.3);

[0044] PVDF membrane (0.45 μm) or nitrocellulose membrane (NC membrane).

[0045] Example 1: Bovine serum albumin-binding nanobody and its preparation method

[0046] The natural alpaca-derived phage-displayed nanoantibody library was screened using the immunotube method. The screening steps are as follows: (1) BSA coating and blocking: BSA was completely dissolved in PBS solution to prepare a BSA solution with a final concentration of 25 μg / mL. The BSA solution was coated on the immunotube and incubated at room temperature for 2 hours. The coating solution was discarded and the tube was washed 3 times with PBST (1 min each time). The blocking solution was added and blocked at room temperature for 1 hour and then washed 3 times with PBST; (2) Phage library pre-clearing: The phage library was incubated with the immunotube not coated with BSA for 30 minutes (room temperature) to remove non-specifically bound phages and collect the unbound phage supernatant for subsequent screening; (3) Three rounds of affinity enrichment screening: Specifically bound phages were collected. The screening process is shown in Table 1; (4) Amplification: The eluted phages were infected with XL1-Blue Escherichia coli, and the helper phage M13KO7 was added. The cells were shaken and cultured at 37°C for 12-16 hours. The phages were purified by NaCl precipitation and used for the next round of screening. (5) Identification of positive clones: The single clones screened in step (4) were picked and inoculated into 96-well plates for culture. Helper phage M13KO7 was added. After culture, the supernatant (containing phage) was collected and coated with BSA (concentration 10 μg / mL) on ELISA plates. The plates were incubated at 4°C overnight. The phage supernatant was added and incubated at room temperature for 1 h. The binding signal was detected to obtain ELISA-positive clones. (6) Amplification and sequencing: After amplifying the positive clones, the obtained nanoantibodies were sequenced by a biological company.

[0047] Table 1

[0048]

[0049] As a result, a bovine serum albumin-binding nanobody with the amino acid sequence Nb1 shown in SEQ ID NO.1 was obtained.

[0050] Example 2: Expression and purification of bovine serum albumin-binding nanobodies

[0051] LB medium formula: tryptone 10 g / L, yeast extract powder 5 g / L, sodium chloride 10 g / L, ampicillin 100 mg / L, pH 7.4.

[0052] Fermentation medium: tryptone 12 g / L, yeast extract 24 g / L, glycerol 4 mL / L, K2HPO4 12.54 g / L, KH2PO4 2.31 g / L, pH 7.0.

[0053] Feed medium formula: trace elements 100 mL / L, glucose 400 g / L, yeast powder 100 g / L, magnesium sulfate 15.5 g / L.

[0054] The nucleotide sequence encoding the nanobody was codon-optimized according to the preference of Escherichia coli to obtain the corresponding nucleotide sequence, wherein the nucleotide sequence encoding the bovine serum albumin nanobody Nb1 is shown in SEQ ID NO.2.

[0055] A biological company was commissioned to clone the bovine serum albumin nanobody gene (nucleotide sequence as SEQ ID NO. 2) into the expression vector pET-22b (containing His tag, His6-TEV site-nanobody), and the expression vector pET-22b was used to transform BL21 (DE3) Escherichia coli, spread on LB agar plates containing ampicillin, and cultured at 37°C for 12 hours.

[0056] Pick a single colony and inoculate it into 5 mL of LB medium (containing Amp), shake and culture at 37℃ for 12 hours. Transfer it to 500 mL of LB medium at a ratio of 1:100 and culture at 37℃ until OD 600 =1.2. Obtain seed solution; transfer the seed solution to a 5L fermentation tank filled with 2.5L fermentation medium at a 5% inoculum volume. Set the temperature to 37°C, ventilation to 25L / min, pH to 7.0, pressure to 0.050MPa, and speed to 200rpm. Set the control mode to automatic. During fermentation, adjust the speed and ventilation to control the dissolved oxygen to above 30%. When OD 600 When growth ceased, feed was initiated to maintain a glucose concentration >5 g / L. When OD600 reached ≥30, feed was discontinued. The temperature of the tank was lowered to 20°C over 1 hour. IPTG was added to a final concentration of 0.5 mM. After 16 hours of cold induction at 25°C, the cells were harvested and centrifuged at 8,000 × g for 10 minutes at 4°C. The supernatant was discarded and the pellet washed with PBS (pH 7.4). The cells were resuspended in lysis buffer (containing 1 mM PMSF and 10 mM imidazole) and sonicated on ice (300 W power, 3 seconds on / 5 seconds off, for 20 minutes). The pellet was centrifuged at 12,000 × g for 30 minutes at 4°C, and the supernatant collected.

[0057] A Ni-NTA column was equilibrated with binding buffer (20 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, pH 8.0). The crude extract was applied to the column at a flow rate of 1 mL / min to bind the His-tagged protein. Nonspecifically bound proteins were eluted with buffers containing 20 mM and then 50 mM imidazole. The nanobody was eluted with a buffer containing 250 mM imidazole, and the eluted peak was collected. The eluate was transferred to a dialysis bag (7 kDa molecular weight cutoff) and dialyzed against PBS for 24 hours (4°C, with three changes of buffer) to remove the imidazole and obtain the purified BSA-bound nanobody. TEV protease (1 U / mg protein) was added and incubated in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 8.0, at room temperature for 4 hours. The column was then passed through a Ni column a second time to remove the His tag and protease, obtaining the purified BSA-bound nanobody.

[0058] Example 3: Characterization of bovine serum albumin-binding nanobodies

[0059] (1) SDS-PAGE detection

[0060] The purified sample was subjected to SDS-PAGE detection: the purified sample was mixed with the loading buffer, boiled for 5 minutes and then loaded. 12% separation gel electrophoresis (constant voltage 120 V, 1 hour), Coomassie brilliant blue staining, and molecular weight verification were performed. The results are as follows Figure 1 As shown, a band of approximately 14 kDa was obtained.

[0061] (2) ELISA binding verification of bovine serum albumin-bound nanoantibodies

[0062] The His tag was fused to the nanobody gene coding sequence, and the ELISA plates expressing the nanobody with the His tag were coated with bovine serum albumin (5 μM / mL, 100 μL / well) at 4°C overnight. 5% skim milk / PBST was blocked at 37°C for 1 hour, and then the serum albumin-Nb complex was serially diluted. The positive control group was free nanobody, and the negative control group included only BSA. The cells were incubated at 37°C for 1 hour and washed three times with PBST. TMB was added for color development, 2M H2SO4 was used for termination, and the OD was measured. 450 , calculate the IC of BSA 50 (BSA concentration that inhibits 50% of Nb binding).

[0063] Repeat the above steps by replacing BSA with human serum albumin (BSA) and mouse serum albumin (MSA).

[0064] The results are as follows Figure 2 shown.

[0065] Depend on Figure 2 It can be seen that the IC of Nb1-BSA 50 9.08×10 -4 μM / mL; IC of Nb1-HSA 50 1.56×10 -3 μM / mL; IC of Nb1-MSA 50 3.42×10 -3 μM / mL.

[0066] According to IC 50 The equilibrium dissociation constants were estimated and the results are shown in Table 2.

[0067] Table 2

[0068]

[0069] The equilibrium dissociation constants of Nb1 with BSA, HSA and MSA were all less than 10 -8 , that is, Nb1 has good binding affinity with BSA, HSA and MSA. The bovine serum albumin nanoantibody Nb1 of the present application has a good broad spectrum of application.

[0070] Example 4 Drug Half-Life Verification

[0071] The fusion proteins Nb1-IFNα-2b and IFNα-2b were prepared in Escherichia coli. Nanobody Nb1 was linked to the N segment of IFN via a flexible linker peptide ((GGGGS)3) to form the fusion protein Nb1-IFNα-2b (nucleotide sequence shown in SEQ ID NO. 3). The nucleotide sequence of IFNα-2b is shown in SEQ ID NO. 4. The protein was purified and used for subsequent experiments.

[0072] E. coli culture steps:

[0073] Pick a single colony and inoculate it into 5 mL of LB medium (containing Amp), shake and culture at 37℃ for 12 hours. Transfer it to 500 mL of LB medium at a ratio of 1:100 and culture at 37℃ until OD 600 =1.2. Obtain seed solution; transfer the seed solution to a 5L fermentation tank filled with 2.5L fermentation medium at a 5% inoculum volume. Set the temperature to 37°C, ventilation to 25L / min, pH to 7.0, pressure to 0.050MPa, and speed to 200rpm. Set the control mode to automatic. During fermentation, adjust the speed and ventilation to control the dissolved oxygen to above 30%. When OD 600 When the growth stops, start feeding to ensure the glucose concentration is >5 g / L; when OD 600When the pH value was ≥30, the feeding was stopped and IPTG was added to a final concentration of 1 mM. After induction at 37°C for 16 hours, the cells were collected and centrifuged at 4°C and 8000×g for 10 minutes. The supernatant was discarded and the cell pellet was washed with PBS buffer (pH 7.4).

[0074] The purification steps are as follows:

[0075] Binding buffer (50 mM Tris-HCl, 300 mM NaCl, 10-20 mM imidazole, pH 8.0) was added to the recombinant E. coli pellet after centrifugation (5000 × g, 15 min, 4°C). The pellet was sonicated on ice (200 W, 3 s on / 5 s off, 10 min) until clear. The pellet was then centrifuged at 12,000 × g for 30 min, and the pellet was collected. The pellet was washed twice with Tris buffer containing 2 M urea and 1% Triton X-100. Inclusion bodies were solubilized with denaturing buffer (50 mM Tris-HCl, pH 8.0, 10 mM DTT) and stirred at room temperature for 3 h. Insoluble matter was removed by centrifugation at 15,000 × g for 25 min, and the supernatant was collected. 7.5 mM DTT was added. The denatured protein was placed in a dialysis bag and dialyzed against 8 M, 4 M, 2 M, 1 M, and 0 M urea concentrations for 5 hours each. The dialysis buffer used during the dialysis was 50 mM Tris-HCl, 0.5 M L-arginine, 1 mM GSH / GSSG, pH 8.0. The dialyzed sample was loaded onto a Ni-NTA affinity chromatography column and washed for 10-15 CV with a buffer containing 20-50 mM imidazole to remove contaminants. The column was then eluted with PBS to remove the imidazole and concentrated to the target concentration (1 mg / mL) using a 10 kDa ultrafiltration tube. TEV protease (1 U / mg protein) was added and incubated in 50 mM Tris-HCl, 150 mM NaCl at room temperature. ， The protein was incubated in 1 mM CaCl2, pH 8.0 buffer for 4 h, and then passed through a Ni column twice to remove the His tag and protease to obtain the purified target protein.

[0076] When IFNα-2b was purified, it was concentrated using a 7 kDa ultrafiltration tube.

[0077] The biological activity of interferon was determined using the reporter gene method. For detailed procedures, please refer to the following website:

[0078] https: / / shuju.qgyyzs.net / yd2020 / details15932GvoILqidTY53492.html, the biological activity of fusion interferon was determined, and the biological activity test results are shown in Table 3.

[0079] The results show that the NB1-IFNα-2b obtained by the present invention has relatively ideal biological activity.

[0080] Table 3

[0081]

[0082] Eight adult male SD rats weighing about 300 g and eight adult female SD rats weighing about 300 g were randomly divided into two groups, with half of the rats in each group being male and half being female. Each group was injected with 8 × 10 6 IU of IFNα-2b and the above-mentioned Nb1-IFNα-2b fusion protein; blood was collected from the tail vein of mice at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, 48, and 52 hours for the fusion protein group, and serum was collected at 0, 0.25, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 hours for the IFNα-2 group, and serum was collected to detect the biological activity of interferon retained in the serum. The "blood drug activity-time curve" was drawn and the data were fitted using software (such as Figure 3 and Figure 4 Pharmacokinetic parameters were analyzed (Table 4).

[0083] Table 4

[0084]

[0085] The results showed that the half-life of IFNα-2b was 1 / 2β The t of fusion protein Nb1-IFNα-2b was 1.79h. 1 / 2β The half-life is 13.48h, which is significantly prolonged.

[0086] The foregoing is merely an embodiment of the present invention. The scope of protection of the present invention is not limited by these specific embodiments but is determined by the claims of the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, improvements, etc. made within the technical concepts and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A bovine serum albumin-binding nanobody, characterized in that The amino acid sequence of the bovine serum albumin nanobody is shown in SEQ ID NO.

1.

2. The bovine serum albumin-binding nanobody according to claim 1, characterized in that The bovine serum albumin-binding nanobody can also bind to human serum albumin (HSA) and mouse serum albumin (MSA).

3. A nucleic acid, characterized in that The nucleic acid encodes the bovine serum albumin-binding nanobody according to any one of claims 1 or 2.

4. The nucleic acid according to claim 3, characterized in that The nucleic acid sequence is shown as SEQ ID NO.

2.

5. A biomaterial, characterized in that The biological material is selected from any one of the following:

1. A vector comprising the nucleic acid according to claim 3 or 4; II. A transformant comprising the vector described in I; III. A recombinant cell capable of producing the bovine serum albumin-binding Nanobody according to claim 1 or 2, and / or containing at least one nucleic acid according to claim 3 or 4.

6. Use of the bovine serum albumin-binding Nanobody according to any one of claims 1 or 2, the nucleic acid according to any one of claims 3 or 4, or the biomaterial according to claim 5 in any of the following aspects: A1. Application in the preparation of products that extend drug half-life; A2. Application in the preparation of immunoassay or diagnostic products.

7. The use according to claim 6, characterized in that The drug half-life extension product described in A1 includes at least one of a fusion protein consisting of a bovine serum albumin-binding nanobody and a protein drug, and a conjugate formed by connecting a bovine serum albumin-binding nanobody and a therapeutic agent.

8. A method for preparing the bovine serum albumin-binding nanobody according to claim 1 or 2, characterized in that: The method comprises the step of introducing the nucleic acid of claim 3 or 4 into a recombinant cell to express the bovine serum albumin-binding nanobody of claim 1 or 2.

9. The method according to claim 8, characterized in that The recombinant cell is selected from at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

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