Cat serum albumin combined nano antibody and application thereof

Abstract

The invention discloses a cat serum albumin combined nano antibody as well as a preparation method and application thereof, and relates to the technical field of nano antibody engineering. Wherein the amino acid sequence of the cat serum albumin binding nano antibody Nb1 is shown as SEQ.ID NO.1, and the nano antibody Nb1 not only has higher affinity to cat serum albumin, but also has better binding activity to mouse serum albumin; the polypeptide can be widely applied to the aspects of drug half-life period enhancement, tumor targeted therapy and imaging, immunodetection or diagnosis, recombinant albumin purification or enrichment and the like in the pet treatment process.

Description

Technical Field

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

[0002] Serum albumin, due to its highly conserved gene sequence and structure (the three share 70-80% homology), is an ideal target for nanobody (VHH)-mediated drug modification. 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, particularly 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] However, current albumin nanoantibodies are mostly focused on human serum albumin and bovine serum albumin, while research on nanoantibodies for pets is relatively rare. However, with the improvement of people's living standards, more and more families are paying more attention to the health of their companion pets, and research in the fields of pet medicine and nutrition has also deepened. Since many human medications cannot be directly used to treat pets, extending the half-life of pet medications, especially the half-life of various cytokines, to promote pet treatment is of great significance in the field of pet medicine.

[0004] Currently, there is relatively little systematic research on the preparation, characterization, and biological applications of FSA nanobodies. In particular, there is a significant research gap in the efficient preparation of high-affinity, highly stable FSA nanobodies and their application in practical immunoassays and targeted therapies. Further research in this area is expected to provide new strategies and methods for the diagnosis and treatment of FSA-related diseases. Summary of the Invention

[0005] The core goal of this invention is to prepare high-affinity and stable feline serum albumin nanobodies, fully characterize them, and explore their potential for biomedical applications. The invention details are as follows: In a first aspect, the present invention provides a feline serum albumin-binding Nanobody, the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] Furthermore, the feline serum albumin-binding Nanobody can also bind to mouse serum albumin (MSA).

[0007] In a second aspect, the present invention provides a nucleic acid encoding the feline serum albumin-binding Nanobody, comprising a currently disclosed nucleotide sequence or a nucleotide sequence 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.

[0008] The third aspect of the present invention provides a biomaterial, wherein the biomaterial is selected from any one of the following: 1. A vector comprising the nucleic acid; II. A transformant comprising the vector described in I; III. A recombinant cell capable of producing the feline serum albumin-binding Nanobody and / or containing the nucleic acid.

[0009] 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.

[0010] Furthermore, the recombinant cells include at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0011] The fourth aspect of the present invention provides the use of the cat serum albumin-binding nanobody or the nucleic acid or the biomaterial in any of the following aspects: A1. Application in the preparation of products that extend drug half-life; A2. Application in the preparation of tumor targeted therapy and imaging products; A3. Application in the preparation of immunoassay or diagnostic products; A4. Application in the preparation of products that promote the purification or enrichment of recombinant albumin.

[0012] 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.

[0013] Furthermore, the drug half-life extension product described in A1 includes at least one of a fusion protein consisting of a feline serum albumin-binding nanobody and a protein drug, a conjugate formed by connecting a feline serum albumin-binding nanobody and a therapeutic agent, and a pharmaceutical composition of the feline serum albumin-binding nanobody.

[0014] Furthermore, the protein drug includes at least one of polypeptides, antibodies, antibody fragments, cytokines, and tumor marker molecules that can play a therapeutic role.

[0015] Furthermore, the pharmaceutical composition of cat serum albumin-binding nanoantibodies also includes additives acceptable in the medical field.

[0016] The fifth aspect of the present invention provides a method for preparing the cat serum albumin binding nanobody, which comprises the step of introducing the nucleic acid into a recombinant cell to express the cat serum albumin binding nanobody.

[0017] Furthermore, the method also includes the steps of separating and purifying the expressed cat serum albumin-binding nanobody.

[0018] 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.

[0019] In a specific embodiment of the present invention, the purpose of preparing feline serum albumin nanoantibody is achieved by cloning the feline serum albumin nanoantibody 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 feline serum albumin nanoantibody.

[0020] The beneficial effects of the present invention include but are not limited to: The cat serum albumin nanoantibody provided by the present invention not only has a high affinity for cat serum albumin, but also has good binding activity to mouse serum albumin. It can be widely used in the process of enhancing drug half-life in pet treatment, tumor targeted therapy and imaging, immune detection or diagnosis, recombinant albumin purification or enrichment, etc., and has broad application prospects in the field of medicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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: Figure 1 Schematic diagram of SDS-PAGE detection results in an embodiment of the present invention; Figure 2Schematic diagram of the ELISA test results of Nb1 with FSA, HSA, BSA and MSA in an embodiment of the present invention; Figure 3 This is a blood activity-time curve of IFNα-2b in an embodiment of the present invention; 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

[0022] 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.

[0023] Phage display library: natural alpaca / camel VHH library, purchased from Pujian Biotechnology (Wuhan) Technology Co., Ltd. Feline serum albumin: produced by Shanghai Xinruite Biopharmaceutical Technology Co., Ltd. Human serum albumin: Tonghua Anrui Biopharmaceutical Co., Ltd. Mouse serum albumin: produced by Shanghai Xinruite Biopharmaceutical Technology Co., Ltd. Bovine serum albumin: produced by Shanghai Xinruite Biopharmaceutical Technology Co., Ltd. Coating buffer: PBS (pH 7.4); Blocking solution: 3% skim milk in PBST (PBS + 0.05% Tween-20); Acidic elution: 0.1 M Glycine-HCl (pH 2.2), immediately neutralized with 1 M Tris-HCl (pH 9.0); Competitive elution: 1-5 mg / mL soluble FSA (dissolved in PBS); Host bacteria: XL1-Blue Escherichia coli and BL21 (DE3) Escherichia coli were purchased from Beijing Biobo Biotechnology Co., Ltd. Helper phage: M13KO7, purchased from Thermo Fisher Scientific; Transfer buffer (25 mM Tris, 192 mM Glycine, 20% methanol, pH 8.3); PVDF membrane (0.45 μm) or nitrocellulose membrane (NC membrane).

[0024] Example 1: Cat serum albumin binding nanobody and its preparation method The natural alpaca-derived phage-displayed nanoantibody library was screened using the immunotube method. The screening steps are as follows: (1) FSA coating and blocking: FSA was completely dissolved in PBS solution to prepare an FSA solution with a final concentration of 25 μg / mL. The FSA 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 FSA 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 FSA (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 signals were detected to obtain ELISA-positive clones. (6) Amplification and sequencing: After amplifying the positive clones, the obtained nanoantibodies were sequenced by a biotechnology company.

[0025] Table 1

[0026] As a result, a cat serum albumin-binding nanobody Nb1 with an amino acid sequence as shown in SEQ ID NO.1 was obtained.

[0027] Example 2: Expression and purification of cat serum albumin-binding nanobodies 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.

[0028] 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.

[0029] Feed medium formula: trace elements 100 mL / L, glucose 400 g / L, yeast powder 100 g / L, magnesium sulfate 15.5 g / L.

[0030] 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 feline serum albumin nanobody Nb1 is shown in SEQ ID NO.2.

[0031] A biological company was commissioned to clone the feline serum albumin nanobody gene (nucleotide sequence as SEQ ID NO. 2) into the expression vector pET-22b (containing a 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.

[0032] 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.

[0033] 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 purified FSA-binding nanobody. TEV protease (1 U / mg protein) was added and incubated in 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 purified FSA-binding nanobody.

[0034] Example 3: Characterization of feline serum albumin-binding Nanobodies (1) SDS-PAGE detection 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 15.5 kDa was obtained.

[0035] (2) ELISA binding validation of cat serum albumin-binding nanoantibodies The His tag was fused to the nanobody gene coding sequence, and the His-tagged nanobody ELISA plates were coated with feline 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 consisted of free nanobody, and the negative control group included only FSA. 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 FSA 50 (FSA concentration that inhibits 50% of Nb binding).

[0036] Replace cat serum albumin with human serum albumin (HSA), mouse serum albumin (MSA), and bovine serum albumin (BSA) and repeat the above steps. The results are as follows Figure 2 shown.

[0037] Depend on Figure 2 It can be seen that the IC of Nb1-FSA 50 3.08×10-3 μM / mL, IC of Nb1-MSA 50 8.57×10 -3 μM / mL but its binding ability to HSA and BSA was poor.

[0038] According to IC 50 The equilibrium dissociation constant of Nb1-FSA was estimated to be 3.08×10 -9 The dissociation constant of Nb1-MSA is 8.57×10 -9 , all less than 10 -8 , that is, Nb1 has a good binding ability with FSA and MSA, but a poor binding ability with HSA and BSA.

[0039] Example 4 Drug Half-Life Verification 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.

[0040] E. coli culture steps: 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 600 When 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).

[0041] The purification steps are as follows: 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 a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 8.0, for 4 hours at room temperature. The His tag and protease were removed by a second pass through a Ni column to obtain the purified target protein.

[0042] When IFNα-2b was purified, it was concentrated using a 7 kDa ultrafiltration tube.

[0043] The biological activity of interferon was determined by the reporter gene method (https: / / shuju.qgyyzs.net / yd2020 / details15932GvoILqidTY53492.html) to determine the biological activity of the fusion interferon. The results of the biological activity test are shown in Table 2.

[0044] The results show that the NB1-IFNα-2b obtained by the present invention has relatively ideal biological activity.

[0045] Table 2

[0046] 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 3).

[0047] Table 3

[0048] 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 9.85h, which is significantly prolonged.

[0049] 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 cat serum albumin binding nanobody, characterized in that The amino acid sequence of the feline serum albumin-binding nanobody is shown in SEQ ID NO.

1.

2. The cat serum albumin binding nanobody according to claim 1, characterized in that The feline serum albumin binding Nanobody can also bind to mouse serum albumin (MSA).

3. A nucleic acid, characterized in that The nucleic acid encodes the feline 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 feline 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 cat serum albumin-binding Nanobody according to any one of claims 1 or 2, or 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 feline serum albumin-binding nanobody and a protein drug, and a conjugate formed by connecting a feline serum albumin-binding nanobody and a therapeutic agent.

8. A method for preparing the cat 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 feline serum albumin-binding nanobody of claim 1 or 2.

9. The method according to claim 8, characterized in that The method further comprises the steps of separating and purifying the expressed cat serum albumin-binding nanobody.

10. The method according to any one of claims 8-9, 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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