A feline serum albumin-bound nanobody and its application

Abstract

This invention discloses a feline serum albumin-bound nanobody, its preparation method, and its applications, relating to the field of nanobody engineering technology. The amino acid sequence of the feline serum albumin-bound nanobody Nb1 is shown in SEQ.ID NO.1. The nanobody Nb1 not only exhibits high affinity for feline serum albumin but also good binding activity to mouse serum albumin, and can be widely applied in enhancing drug half-life during pet treatment, tumor targeted therapy and imaging, immune detection or diagnosis, and recombinant albumin purification or enrichment.

Description

Technical Field

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

[0002] Serum albumin, due to its highly conserved gene sequence and structure (70-80% homology among serum albumin, serum albumin, and drug), is an ideal target for drug modification mediated by nanobodies (VHH). By designing nanobodies that specifically bind to serum albumin and conjugating them with therapeutic drugs, a ternary complex system of "nanobody-serum albumin-drug" can be constructed, significantly optimizing pharmacokinetics and therapeutic effects. Modifying drugs with nanobodies that bind to serum albumin, especially nanobody-drug conjugation, can prolong drug half-life, achieve targeted drug delivery, reduce drug dosage, enhance drug biocompatibility, promote drug absorption, and maximize drug efficacy.

[0003] However, current albumin nanobodies are mostly focused on human serum albumin and bovine serum albumin, with relatively little research on nanobodies for pets. But as people's living standards improve, more and more families are paying attention to the health of their companion pets, leading to deeper research in pet medicine and nutrition. 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, systematic research on the preparation, characterization, and biological applications of FSA nanobodies is relatively limited. In particular, there is a significant research gap in how to efficiently prepare high-affinity, high-stability FSA nanobodies and apply them to practical immune detection and targeted therapy. In-depth research in this area holds the potential to provide new strategies and methods for the diagnosis and treatment of FSA-related diseases. Summary of the Invention

[0005] The core objective of this invention is to prepare cat serum albumin nanobodies with high affinity and stability, and to comprehensively characterize them to explore their application potential in the biomedical field. The specific details of this invention are as follows:

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

[0007] Furthermore, the feline serum albumin-bound nanobody can also bind to mouse serum albumin (MSA).

[0008] In a second aspect, the present invention provides a nucleic acid encoding the aforementioned cat serum albumin-binding nanobody, comprising a currently disclosed nucleotide sequence or a nucleotide sequence codon-optimized according to actual needs. Preferably, the nucleotide sequence is a nucleotide sequence optimized according to the codon preference of *E. coli*, and more preferably, the nucleic acid sequence is as shown in SEQ ID NO. 2 or has 95% homology with the sequence shown in SEQ ID NO. 2.

[0009] A third aspect of the present invention provides a biomaterial, said biomaterial being selected from any of the following:

[0010] I. A vector containing the aforementioned nucleic acid;

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

[0012] III. Capable of producing the aforementioned cat serum albumin-bound nanobody and / or recombinant cells containing the aforementioned nucleic acid.

[0013] Furthermore, the vector 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.

[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 this invention provides the use of the said feline serum albumin-bound nanobody, the said nucleic acid, or the said biomaterial in any of the following aspects:

[0016] A1. Applications in the preparation of products with extended drug half-life;

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

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

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

[0020] Further, the drugs used to extend the half-life of the drug include protein drugs, antibody fragments, small molecule chemotherapeutic drugs, and other biological agents; preferably, the protein drugs include at least one of IL-6R-targeting drugs, TNF-α-targeting drugs, and IL-17A / IL-17F-targeting drugs; the small molecule chemotherapeutic drugs include at least one of doxorubicin, solefenib, and sirolimus. In a specific embodiment of the present invention, the drug is interferon.

[0021] Furthermore, the extended drug half-life product described in A1 includes at least one of the following: a fusion protein composed of a feline serum albumin-bound nanobody and a protein drug; a conjugate formed by linking a feline serum albumin-bound nanobody and a therapeutic agent; and a pharmaceutical composition of the feline serum albumin-bound nanobody.

[0022] Furthermore, the protein-based drugs include at least one of the following: peptides, antibodies, antibody fragments, cytokines, and tumor marker molecules that can exert therapeutic effects.

[0023] Furthermore, the cat serum albumin-bound nanobody pharmaceutical composition also includes pharmaceutically acceptable additives.

[0024] The fifth aspect of the present invention provides a method for preparing the aforementioned cat serum albumin-binding nanobody, the method comprising the step of introducing the aforementioned nucleic acid into recombinant cells to express the aforementioned cat serum albumin-binding nanobody.

[0025] Furthermore, the method also includes the steps of separating and purifying the expressed feline serum albumin by combining it with nanobodies.

[0026] The recombinant cells are selected from at least one of hamster ovary cells, silkworm ovary cells, Pichia pastoris, Saccharomyces cerevisiae, Escherichia coli, and Bacillus subtilis.

[0027] In a specific embodiment of the present invention, the feline serum albumin nanobody gene (nucleotide sequence as shown in SEQ ID NO.2) is cloned into the expression vector pET-22b (containing a His tag), and then transformed into BL21(DE3) Escherichia coli to express the feline serum albumin nanobody, thereby achieving the purpose of preparing the feline serum albumin nanobody.

[0028] The beneficial effects of the present invention include, but are not limited to:

[0029] The feline serum albumin nanobody provided by this invention not only has a high affinity for feline serum albumin, but also has good binding activity to mouse serum albumin. It can be widely used in pet treatment to enhance 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 pharmaceutical field. Attached Figure Description

[0030] 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:

[0031] Figure 1 This is a schematic diagram of the SDS-PAGE detection results in an embodiment of the present invention;

[0032] Figure 2 This is a schematic diagram of the ELISA detection results of Nb1 with FSA, HSA, BSA and MSA in an embodiment of the present invention;

[0033] Figure 3 This is a blood drug activity-time curve of IFNα-2b in an embodiment of the present invention;

[0034] Figure 4 This is a blood drug activity-time curve of the fusion protein Nb1-IFNα-2b in an embodiment of the present invention. Detailed Implementation

[0035] 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.

[0036] Phage display library: Natural alpaca / camel VHH library, purchased from Pujian Biotechnology (Wuhan) Technology Co., Ltd.;

[0037] Feline serum albumin: Produced by Shanghai Xinruit Biomedical Technology Co., Ltd.

[0038] Human serum albumin: Tonghua Anruit Biopharmaceutical Co., Ltd.;

[0039] Mouse serum albumin: Produced by Shanghai Xinruit Biomedical Technology Co., Ltd.

[0040] Bovine serum albumin: Produced by Shanghai Xinruit Biomedical Technology Co., Ltd.

[0041] Coating buffer: PBS (pH 7.4);

[0042] Blocking solution: 3% skim milk in PBST (PBS + 0.05% Tween-20);

[0043] Acidic elution: 0.1 M Glycine-HCl (pH 2.2), immediately neutralized with 1 M Tris-HCl (pH 9.0);

[0044] Competitive elution: 1-5 mg / mL soluble FSA (dissolved in PBS);

[0045] Host bacteria: XL1-Blue Escherichia coli and BL21(DE3) Escherichia coli, purchased from Beijing BioBio Biotechnology Co., Ltd.

[0046] Helper phage: M13KO7, purchased from Thermo Fisher Scientific;

[0047] Transfer buffer (25 mM Tris, 192 mM Glycine, 20% methanol, pH 8.3);

[0048] PVDF membrane (0.45 μm) or nitrocellulose membrane (NC membrane).

[0049] Example 1: Feline serum albumin-bound nanobodies and their preparation method

[0050] An immunotube method was used to screen a library of naturally occurring alpaca-derived phage-displaying nanobodies. The screening steps are as follows: (1) FSA coating and blocking: FSA is completely dissolved in PBS solution to prepare an FSA solution with a final concentration of 25 μg / mL. The FSA solution is coated on the immunotube and incubated at room temperature for 2 hours. The coating solution is discarded, and the PBST is washed 3 times (1 min each time). Blocking solution is added and the PBST is washed 3 times after blocking at room temperature for 1 h. (2) Phage library pre-cleaning: The phage library is incubated with the immunotube without FSA coating for 30 min (room temperature) to remove non-specifically bound phages. The supernatant of unbound phages is collected for subsequent screening. (3) Three rounds of affinity enrichment screening: Specifically bound phages are collected. The screening process is shown in Table 1. (4) Amplification: The eluted phages are infected with XL1-Blue Escherichia coli. Helper phage M13KO7 is added and cultured at 37℃ with shaking for 12-16 h. The phages are purified by NaCl precipitation for the next round of screening. (5) Identification of positive clones: Select the single clones screened in step (4) and inoculate them into 96-well plates for culture. Add helper phage M13KO7. After culture, collect the supernatant (containing phages), coat the ELISA plate with FSA (concentration 10 μg / mL), incubate overnight at 4℃, add phage supernatant, incubate at room temperature for 1 h, and detect the binding signal to obtain ELISA positive clones; (6) Amplification and sequencing: After amplifying the positive clones, entrust a biotechnology company to sequence the obtained nanobodies.

[0051] Table 1

[0052]

[0053] As a result, cat serum albumin-bound nanobodies with the amino acid sequence Nb1, as shown in SEQ ID NO.1, were obtained.

[0054] Example 2: Expression and purification of feline serum albumin-bound nanobodies

[0055] LB medium formula: 10 g / L tryptone, 5 g / L yeast extract powder, 10 g / L sodium chloride, 100 mg / L ampicillin, pH 7.4.

[0056] 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.

[0057] Supplementary culture medium formula: 100 mL / L trace elements, 400 g / L glucose, 100 g / L yeast extract, and 15.5 g / L magnesium sulfate.

[0058] According to the E. coli preference, the nucleotide sequence encoding the nanobody was codon optimized to obtain the corresponding nucleotide sequence, among which the nucleotide sequence encoding the cat serum albumin nanobody Nb1 is shown in SEQ ID NO.2.

[0059] A biotechnology company was commissioned to clone the feline serum albumin nanobody gene (nucleotide sequence as shown in SEQ ID NO.2) into the expression vector pET-22b (containing a His tag and His6-TEV site-nanobody). The expression vector pET-22b was used to transform BL21(DE3) Escherichia coli, which was then plated on LB agar plates containing ampicillin and incubated at 37°C for 12 hours.

[0060] Pick a single colony and inoculate it into 5 mL of LB medium (containing Amp), and incubate at 37°C with shaking for 12 hours. Transfer to 500 mL of LB medium at a 1:100 ratio and incubate at 37°C until OD (outcome limit) is reached. 600 =1.2. Obtain the seed culture; transfer the seed culture at a 5% inoculum to a 5L fermenter containing 2.5L of fermentation medium. Set the temperature to 37℃, aeration rate to 25L / min, pH to 7.0, pressure to 0.050Mpa, and rotation speed to 200rpm. The control mode is automatic. During fermentation, the dissolved oxygen level is maintained above 30% by adjusting the rotation speed and aeration rate. When OD... 600When growth ceases, begin fed-batch feeding to maintain a glucose concentration >5 g / L. When OD600 ≥30, stop feeding and lower the tank temperature to 20°C within 1 hour. Add IPTG to a final concentration of 0.5 mM, induce at 25°C for 16 hours, collect the cells, centrifuge at 4°C and 8000×g for 10 minutes, discard the supernatant, and wash the cell pellet with PBS buffer (pH 7.4). Resuspend the cells in lysis buffer (containing 1 mM PMSF and 10 mM imidazole), and sonicate on ice (300 W, 3 seconds on, 5 seconds off, for 20 minutes). Centrifuge at 4°C and 12000×g for 30 minutes, and collect the supernatant.

[0061] The 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 loaded onto the column at a flow rate of 1 mL / min to bind the His-tagged protein. Non-specifically bound proteins were washed sequentially with buffers containing 20 mM and 50 mM imidazole. The nanobody was eluted with buffer containing 250 mM imidazole, and the elution peak was collected. The eluent was transferred to a dialysis bag (7 kDa molecular weight cutoff) and dialyzed against PBS for 24 hours (4°C, 3 buffer changes) to remove imidazole and obtain purified feline serum albumin-bound nanobody. TEV protease (1 U / mg protein) was added and incubated for 4 hours at room temperature in a buffer of 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 8.0. The nanobody was then passed through a Ni column twice to remove the His tag and protease, yielding purified feline serum albumin-bound nanobody.

[0062] Example 3: Characterization of feline serum albumin-bound nanobodies

[0063] (1) SDS-PAGE detection

[0064] The purified sample was analyzed by SDS-PAGE: the purified sample was mixed with loading buffer, boiled for 5 minutes, and then loaded onto the plate. Electrophoresis was performed on a 12% separating gel (120 V constant voltage, 1 hour), followed by Coomassie brilliant blue staining to verify the molecular weight. Results are as follows... Figure 1 As shown, a stripe of approximately 15.5 kDa was obtained.

[0065] (2) ELISA validation of feline serum albumin-bound nanobody

[0066] The His tag was fused into the coding sequence of the nanobody gene. ELISA plates expressing the His-tagged nanobody were coated with cat serum albumin (5 μM / mL, 100 μL / well) and incubated overnight at 4°C. Blocking was performed with 5% skim milk / PBST at 37°C for 1 h. The serum albumin-Nb complex was then serially diluted, with the positive control containing free nanobody and the negative control containing only FSA. The plates were incubated at 37°C for 1 h and washed three times with PBST. TMB was added for color development, and the reaction was stopped with 2M H2SO4. OD was measured. 450 Calculate the IC of FSA 50 (Inhibits 50% of Nb-bound FSA concentration).

[0067] Replace cat serum albumin with human serum albumin (HSA), mouse serum albumin (MSA), or bovine serum albumin (BSA), and repeat the above steps. The results are as follows: Figure 2 As shown.

[0068] Depend on Figure 2 It can be seen that the IC of Nb1-FSA 50 3.08×10 -3 μM / mL, IC50 of Nb1-MSA 50 It is 8.57×10 -3 μM / mL, but its binding ability with HSA and BSA is poor.

[0069] According to IC 50 The equilibrium dissociation constant is estimated to be 3.08 × 10⁻⁶. -9 The dissociation constant of Nb1-MSA is 8.57 × 10⁻⁶. -9 All less than 10 -8 That is, Nb1 has a good binding affinity with FSA and MSA, but a poor binding affinity with HSA and BSA.

[0070] Example 4: Validation of drug half-life

[0071] Fusion proteins Nb1-IFNα-2b and IFNα-2b were prepared using *E. coli*. The 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 proteins were purified before subsequent experiments.

[0072] E. coli culture steps:

[0073] Pick a single colony and inoculate it into 5 mL of LB medium (containing Amp), and incubate at 37°C with shaking for 12 hours. Transfer to 500 mL of LB medium at a 1:100 ratio and incubate at 37°C until OD (outcome limit) is reached. 600 =1.2. Obtain the seed culture; transfer the seed culture at a 5% inoculum to a 5L fermenter containing 2.5L of fermentation medium. Set the temperature to 37℃, aeration rate to 25L / min, pH to 7.0, pressure to 0.050Mpa, and rotation speed to 200rpm. The control mode is automatic. During fermentation, the dissolved oxygen level is maintained above 30% by adjusting the rotation speed and aeration rate. When OD... 600 When growth ceases, begin fed a feed stream to maintain a glucose concentration >5 g / L; when OD 600 When the concentration reaches ≥30, stop feeding and add IPTG to a final concentration of 1mM. After induction at 37°C for 16 hours, collect the cells and centrifuge at 4°C and 8000×g for 10 minutes. Discard the supernatant and wash the cell pellet with PBS buffer (pH 7.4).

[0074] The purification steps are as follows:

[0075] Add the recombinant E. coli precipitate after centrifugation (5000×g, 15 min, 4℃) to a buffer containing binding buffer (50 mM Tris-HCl, 300 mM NaCl, 10-20 mM imidazole, pH 8.0) and sonicate on ice (200 W, 3 sec on / 5 sec off, 10 min) until the bacterial culture becomes clear. Centrifuge at 12000×g for 30 min and collect the precipitate. Wash the precipitate twice with Tris buffer containing 2 M urea and 1% Triton X-100. Add denaturing buffer containing 6 M guanidine hydrochloride (50 mM Tris-HCl, pH 8.0, 10 mM DTT) and stir at room temperature for 3 h to dissolve the inclusion bodies. Centrifuge at 15000×g for 25 min to remove insoluble matter and collect the supernatant. Add 7.5 mM DTT. Denatured proteins were placed in dialysis bags and dialyzed stepwise with urea concentrations of 8 M, 4 M, 2 M, 1 M, and 0 M, each for 5 hours. The buffer formulation for each dialysis step was 50 mM Tris-HCl, 0.5 mL L-arginine, 1 mM GSH / GSSG, pH 8.0. The dialyzed sample was loaded onto Ni-NTA affinity chromatography and washed 10–15 CV with buffer containing 20–50 mM imidazole to remove contaminating proteins. A stepwise elution was performed (250 / 300 / 500 mM imidazole), and the main peak was collected. Imidazole was removed by elution with PBS, and the sample was concentrated to the target concentration (1 mg / mL) using a 10 kDa ultrafiltration tube. TEV protease (1 U / mg protein) was added and incubated for 4 hours at room temperature in a buffer of 50 mM Tris-HCl, 150 mM NaCl, 1 mM CaCl2, pH 8.0. The sample was then passed through a Ni column twice to remove the His tag and protease, yielding the purified target protein.

[0076] When purifying IFNα-2b, a 7kDa ultrafiltration tube was used for concentration.

[0077] The biological activity of interferon was determined using the reporter gene assay (https: / / shuju.qgyyzs.net / yd2020 / details15932GvoILqidTY53492.html). The biological activity of the fusion interferon was also determined, and the results are shown in Table 2.

[0078] The results show that the NB1-IFNα-2b obtained in this invention has ideal biological activity.

[0079] Table 2

[0080]

[0081] Eight adult male SD rats weighing approximately 300g and eight adult female SD rats weighing approximately 300g were randomly divided into two groups, with half males and half females in each group. Each group was injected with 8×10 6 IU of IFNα-2b was fused with the aforementioned Nb1-IFNα-2b fusion protein. Blood samples were collected from the fusion protein group via rat tail vein 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, and serum was collected. Blood samples were also collected from the IFNα-2 group via rat tail vein 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, and serum was collected to detect the retained interferon biological activity in the serum. A "blood drug activity-time curve" was plotted, and data fitting was performed using software (e.g., ...). Figure 3 and Figure 4 As shown in Table 3), the pharmacokinetic parameters were analyzed.

[0082] Table 3

[0083]

[0084] The results show that the half-life t of IFNα-2b 1 / 2β The t-value was 1.79 h, and the t-value of the fusion protein Nb1-IFNα-2b was... 1 / 2β The half-life is 9.85 hours, which is significantly prolonged.

[0085] 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 feline serum albumin-bound nanobody, characterized in that, The amino acid sequence of the feline serum albumin-bound nanobody is shown in SEQ ID NO.

1.

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

3. A nucleic acid, characterized in that, The nucleic acid encodes the feline serum albumin-bound nanobody as described in claim 1 or 2.

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

2.

5. A biomaterial, characterized in that, The biomaterial is selected from any one of the following: I. A vector comprising the nucleic acid of claim 3 or 4; II. Transformants comprising the vector described in I; III. Recombinant cells capable of producing cat serum albumin-binding nanobodies as described in claim 1 or 2, and / or containing at least one nucleic acid as described in claim 3 or 4.

6. The use of the feline serum albumin-bound nanobody as described in claim 1 or 2, or the nucleic acid as described in claim 3 or 4, or the biomaterial as described in claim 5, in any of the following aspects: A1. Applications in the preparation of products with extended drug half-life; A2. Applications in the preparation of immunoassay or diagnostic products.

7. The application according to claim 6, characterized in that, The extended drug half-life products described in A1 include at least one of the following: a fusion protein consisting of a feline serum albumin-bound nanobody and a protein drug, and a conjugate consisting of a feline serum albumin-bound nanobody and a therapeutic agent.

8. A method for preparing the cat serum albumin-bound nanobody according to claim 1 or 2, characterized in that, The method includes the step of introducing the nucleic acid of claim 3 or 4 into recombinant cells to express the feline serum albumin-bound nanobody of claim 1 or 2.

9. The method according to claim 8, characterized in that, The method also includes the steps of separating and purifying the expressed feline serum albumin by combining it with nanobodies.

10. The method according to any one of claims 8-9, characterized in that, The recombinant cells are 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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