Nano antibody targeting human serum albumin and application thereof

Abstract

The invention relates to the technical field of biological pharmacy, in particular to a human serum albumin targeted nano antibody and application thereof. An alpaca is immunized by using an HSA protein, cDNA is obtained through reverse transcription of total RNA of peripheral blood lymphocytes to serve as a template to amplify a nano antibody sequence, finally, five nano antibodies are obtained through separation and are named as antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8 and antiHSA-H7 respectively, the five nano antibodies have different antigen complementarity determining regions, and an ELISA result shows that the five nano antibodies have different antigen complementarity determining regions. And the five nano antibodies all show high-affinity binding with human HSA (Human Serum Albumin) protein. The nano antibody provided by the invention is expected to be used for preparing vaccines for treating and / or diagnosing diseases related to HSA, or for prolonging the half-life period of macromolecular drugs in vivo, and has important significance.

Description

Technical Field

[0001] The present invention relates to the technical field of biopharmaceuticals, and in particular to a nano antibody targeting human serum albumin and an application thereof. Technical Background

[0002] Human serum albumin (HSA) is one of the most important proteins in human plasma and is also a multifunctional biomolecule. As a single-chain polypeptide, HSA consists of 585 amino acids and has a molecular weight of approximately 66.5 kDa. HSA is a globular protein consisting of three domains, including an N-terminal domain, a central domain, and a C-terminal domain. The N-terminal and C-terminal domains contain 9 α-helices and 3 β-folds, respectively, while the central domain consists of 7 α-helices and 1 β-fold. In addition, the HSA protein also contains 17 disulfide bonds, which are very important for maintaining its stability and biological activity.

[0003] HSA protein has multiple functions in the blood, including transport, regulation, buffering and binding, among which the most important function is transport. HSA protein can bind to a variety of biological molecules, including lipids, drugs, hormones and metal ions, which can be transported in the blood through HSA protein to reach the target cells or tissues. In addition, HSA protein also has regulatory and buffering functions. It can regulate blood pH and ion balance to maintain the stability of the human body's internal environment. At the same time, HSA protein can also bind to and remove some harmful substances in the blood, such as toxins and free radicals.

[0004] HSA protein has broad application prospects in the fields of drug therapy, diagnosis and bioanalysis. HSA protein can be used as a drug carrier to deliver drugs to target cells or tissues, improve the stability and bioavailability of drugs, and thus improve the therapeutic effect of drugs. Based on this effect, anti-HSA nanoantibodies can be coupled with antibodies targeting specific tissues (such as antibodies that specifically bind to tumor cell markers). This dual-binding site fusion antibody is like a directional train that transports HSA to the target location, which can play a role in directional drug delivery and enhance the functionality of HSA protein as a drug carrier. At present, research on using HSA as a drug carrier to direct the tumor to improve the therapeutic effect is a hot topic in HSA-related research.

[0005] In addition to drug treatment, HSA protein can also be used for diagnosis and bioanalysis. For example, HSA protein is a valuable biomarker for many diseases, including cancer, rheumatoid arthritis, postmenopausal obesity, severe acute graft-versus-host disease, and diseases that require monitoring of blood sugar control. The level of HSA reflects the level of disease to a certain extent. Therefore, anti-HSA nanobodies with color-developing groups (such as horseradish oxidase) can be used to detect HSA protein levels, converting protein levels into visible and accurately detectable and calculated data (through light absorption values), which can be used for diagnostic analysis of HSA-related diseases.

[0006] Furthermore, due to the excellent properties of HSA protein itself, such as high stability (stable structure and maintaining its physical and chemical properties within the pH range of 4-6), high content (35-50g / L in blood), and long half-life (19 days), researchers believe that its combination with drugs can increase the retention time of drugs in plasma and expand tissue distribution, preventing drug clearance and degradation, and this reasonable conjecture has been proven to be feasible through various experiments. This function is of great significance to biological drugs with small molecular weight and short half-life, such as nano-antibody drugs.

[0007] By engineering the nano-antibody drug and the anti-HSA nano-antibody to fuse, the half-life of the antibody drug in vivo can be effectively increased, and the administration requirements (frequency and dosage) can be significantly reduced. The ScFv, Fab or full IgG anti-HSA antibody molecules in the prior art have complex structures and large molecules. Although they can connect active molecules to HSA, they affect the function of the active molecules, the methods are complex, and the loading efficiency is also low; nano-antibody molecules are small and easy to operate, but the degree of humanization is low and the affinity is not high, and the performance of extending the half-life needs to be further improved.

[0008] Therefore, providing a specific high-affinity anti-HSA nanobody is of great significance for diagnosing HSA-related diseases and solving the problem of the short half-life of nanoantibody drugs in the body. Summary of the invention

[0009] In order to solve the problems in the prior art, one of the objects of the present invention is to provide a nanobody targeting human serum albumin, wherein the nanobody is any one of antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8, and antiHSA-H7, wherein antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8, and antiHSA-H7 all comprise three antigen complementary determining regions CDR1, CDR2, and CDR3, wherein:

[0010] The amino acid sequences of the three antigen complementary determining regions of antiHSA-A6 are amino acid sequences with a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 respectively;

[0011] The amino acid sequences of the three antigen complementary determining regions of the antiHSA-A7 are amino acid sequences having a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively;

[0012] The amino acid sequences of the three antigen complementary determining regions of the antiHSA-B1 are amino acid sequences having a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively;

[0013] The amino acid sequences of the three antigen complementary determining regions of antiHSA-E8 are amino acid sequences that have a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively;

[0014] The three antigen complementary determining region amino acid sequences of antiHSA-H7 are amino acid sequences with homologies greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

[0015] The above homologous sequences also include amino acid sequences having one or more (preferably 1, 2 or 3) conservative amino acid mutations (preferably substitutions, insertions or deletions) compared to the sequences shown in the sequence listing.

[0016] Preferably, the amino acid sequences of antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8, and antiHSA-H7 are as follows:

[0017] Amino acid sequence of antiHSA-A6:

[0018]

[0019] Amino acid sequence of antiHSA-A7:

[0020]

[0021] Amino acid sequence of antiHSA-B1:

[0022]

[0023] Amino acid sequence of antiHSA-E8:

[0024]

[0025] Amino acid sequence of antiHSA-H7:

[0026]

[0027] The amino acid sequences of the three antigen complementary determining regions CDR1, CDR2 and CDR3 of the above-mentioned nanobody are shown in bold lines, namely:

[0028] The amino acid sequence of the antigenic complementary determining region of antiHSA-A6:

[0029] CDR1:GFTFDYYGI (SEQ ID NO: 1)

[0030] CDR2: GVSCIRSTGT (SEQ ID NO: 2)

[0031] CDR3:ATVYNLRCPLIAELNYEPGAY(SEQ ID NO:3)

[0032] The amino acid sequences of the three antigenic complementary determining regions of antiHSA-A7 are:

[0033] CDR1: GGVFSSYIM (SEQ ID NO: 4)

[0034] CDR2: FVAAISWSGGR (SEQ ID NO: 5)

[0035] CDR3:AADRRTRPWSAMSQDGNSYDT(SEQ ID NO:6)

[0036] The amino acid sequences of the three antigenic complementary determining regions of antiHSA-B1 are:

[0037] CDR1: GLTFSWYAM (SEQ ID NO: 7)

[0038] CDR2:SVAGINRSGDR (SEQ ID NO: 8)

[0039] CDR3:AADYYGLGSLNPHEYEF(SEQ ID NO:9)

[0040] The amino acid sequences of the three antigenic complementary determining regions of antiHSA-E8 are:

[0041] CDR1: IRTFESYAM (SEQ ID NO: 10)

[0042] CDR2:FVASVSPSGGA(SEQ ID NO:11)

[0043] CDR3:AARNGLWTYTDYTNWNY(SEQ ID NO:12)

[0044] The amino acid sequences of the three antigenic complementary determining regions of antiHSA-H7 are:

[0045] CDR1: GFTLDYYAI (SEQ ID NO: 13)

[0046] CDR2:GVSCIKNSHES(SEQ ID NO:14)

[0047] CDR3:ATDKTDYCSGSAISYAY(SEQ ID NO:15)

[0048] The present invention also provides an antibody targeting human serum albumin, which has any one of the nanobodies antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8, and antiHSA-H7 as described above and an Fc domain.

[0049] Preferably, the Fc domain is a human IgG1 Fc domain, the amino acid sequence is shown in SEQ ID NO:21, and the nucleotide sequence encoding the amino acid is shown in SEQ ID NO:22.

[0050] The present invention also provides a polynucleotide, which encodes the nanoantibody antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8 or antiHSA-H7 as described above, or encodes the antibody with an Fc domain as described above; wherein the nucleotide sequence encoding antiHSA-A6 is shown in SEQ ID NO: 23, the nucleotide sequence encoding antiHSA-A7 is shown in SEQ ID NO: 24, the nucleotide sequence encoding antiHSA-B1 is shown in SEQ ID NO: 25, the nucleotide sequence encoding antiHSA-E8 is shown in SEQ ID NO: 26, and the nucleotide sequence encoding antiHSA-H7 is shown in SEQ ID NO: 27.

[0051] The present invention also provides an expression vector comprising the polynucleotide as described above, and a host cell comprising the expression vector as described above. Preferably, the host cell is a host cell for expressing foreign proteins, such as bacteria, yeast, insect cells, and mammalian cells.

[0052] The present invention provides a pharmaceutical composition, which contains any one or more of the nanobodies antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8, and antiHSA-H7 as described above, or contains the antibody having an Fc domain as described above.

[0053] Preferably, the pharmaceutical composition is in a dosage form suitable for administration by subcutaneous injection, intradermal injection, intravenous injection, intramuscular injection or intralesional injection.

[0054] The present invention provides the use of the above-mentioned nanobody / antibody in the preparation of a vaccine for preventing and / or treating diseases associated with human serum albumin.

[0055] The present invention also provides the use of the above-mentioned nanobody / antibody in prolonging the half-life of macromolecular drugs in vivo.

[0056] Preferably, the Nanobody / antibody further comprises a second antibody, which comprises a detectable label, such as a radioisotope, a luminescent substance, a colored substance, an enzyme or polyethylene glycol.

[0057] The beneficial effects of the present invention are:

[0058] 1. Alpacas were immunized three times with HSA protein, and then blood was drawn to separate peripheral blood lymphocytes (PBMCs). Total RNA was extracted from the isolated PBMCs and then immediately reverse transcribed into cDNA. The cDNA was used as a template to amplify the nanoantibody sequence. Finally, five nanoantibodies were isolated and named antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8 and antiHSA-H7.

[0059] The nano antibody (VHH) is derived from natural alpaca heavy chain antibodies, and has the advantages of 1) simple structure and small molecular weight, which is conducive to expression and use; 2) convenient and efficient large-scale expression in Escherichia coli and various eukaryotic systems; 3) since it has only one binding site, it is a single-domain antibody, and has better permeability, specificity and detection linearity as a diagnostic reagent; 4) it is easy to couple with various fusion proteins or be more easily labeled with various markers; 5) it is easier to prepare bifunctional antibodies, which is more conducive to targeted drug development and directional transport of cell targets; 6) as a drug development, it has the advantages of low immunogenicity to humans and is not easy to produce immune rejection.

[0060] 2. The five nanobodies provided by the present invention have different antigen complementary determining regions. The antibody binding to human HSA protein is expressed and secreted by mammalian cells (HEK293F), and the antibody is fused with human IgG1 Fc and cloned into the mammalian expression vector pTT5. The vector is transfected into mammalian cells 293F, and the supernatant is collected after 4 days of culture. The fusion protein in the supernatant is purified using a Protein A column, and the yield of the five nanobodies is greater than 50 mg / L.

[0061] ELISA results showed that all five nanoantibodies showed high affinity binding to human HSA protein, among which the EC 50 At 1.406 nM, the EC of antiHSA-A7-IgG1 Fc 50 At 1.140 nM, the EC of antiHSA-B1-IgG1 Fc 50 At 5.016 nM, the EC of antiHSA-E8-IgG1 Fc 50 At 4.388 nM, the EC of antiHSA-H7-IgG1 Fc 50 At 3.307nM.

[0062] The nanobodies provided by the present invention can be used to detect and analyze HSA protein levels, thereby diagnosing diseases related to HSA; they can also increase the targeting of HSA as a drug carrier and delivery system; the coupled fusion proteins of anti-HSA nanobodies and other nanobodies or peptides and protein drugs with smaller molecular weights can effectively reduce their clearance rate in the human body, solve the problem of too short half-life of nanoantibody drugs in the body, and hopefully provide a way to extend the half-life of protein drugs in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The ELISA results of monoclonal phages in the process of screening for specific binding to human HSA protein nanoantibodies are shown from top to bottom on three different immunoplates. OD 450 The clones with a value greater than 1 were regarded as positive clones and subjected to subsequent sequencing and comparison analysis.

[0064] Figure 2Figure 1 shows the purification results of 5 antibodies. A is the elution graph of nano antibody antiHSA-A6 purified by Protein A column, B is the elution graph of nano antibody antiHSA-A7 purified by Protein A column, C is the elution graph of nano antibody antiHSA-B1 purified by Protein A column, D is the elution graph of nano antibody antiHSA-E8 purified by Protein A column, and E is the elution graph of nano antibody antiHSA-H7 purified by Protein A column.

[0065] Figure 3 The figure shows the results of SDS-PAGE analysis of five antibodies.

[0066] Figure 4 The results of the ELISA analysis of the binding of nanobodies to HSA; the EC values ​​of the five nanobodies 50 The values ​​are antiHSA-A6:EC 50 =1.416nM; antiHSA-A7:EC 50 =1.140nM; antiHSA-B1:EC 50 =5.016nM; antiHSA-E8:EC 50 =4.388nM; antiHSA-H7:EC 50 =3.307nM. DETAILED DESCRIPTION

[0067] For ease of understanding, the technical solution of the present invention is described in more detail below in conjunction with embodiments:

[0068] Example 1

[0069] Preparation of Nanobodies

[0070] 1) Alpacas were immunized with HSA protein (purchased from solarbio, cas 70024-90-7). Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the next two immunizations. Each time, 200 μg of antigen was used, and the antigen and adjuvant were mixed in a volume ratio of 1:1. The first two injections were injected subcutaneously, and the last injection was injected intramuscularly. Alpacas (Vicugna pacos) were immunized 3 times, once every 3 weeks.

[0071] 2) At week 11, venous blood was collected and peripheral blood lymphocytes were separated using Ficoll from Solebol.

[0072] 3) Total RNA was extracted using Omega Biotek's RNA extraction kit and genomic DNA was removed. PrimeScript from Takara was used TMII First Strand cDNA Synthesis Kit, reverse transcribe RNA into cDNA.

[0073] 4) Construction of Nanobody Phage Display Library:

[0074] The coding sequence of the nanobody was obtained by PCR amplification using the above cDNA as a template and specific alpaca VHH primers. The amplified nanobody sequence was inserted into the NcoI and NotI sites of phagemid pR2 by Gibson assembly. The obtained Gibson assembly product was the initial nanobody phage library, and the product was recovered.

[0075] 5) Escherichia coli T1 (MRC Laboratory of Molecular Biology) competent cells were prepared using the 10% glycerol washing method.

[0076] The activated TG1 was cultured in 250 mL 2×YT medium (formula 1 L: 16 g tryptone, 10 g yeast extract, 5 g NaCl) until OD 600 After centrifugation at about 0.6-0.8, 5000g for 15 min, wash three times with 250mL, 250mL, and 100mL of pre-cooled 10% glycerol, and finally resuspend with 1mL of 10% glycerol and dispense into 500μL per tube.

[0077] 6) Use BTX ECM 399 electroporator to transform E. coli TG1 competent cells.

[0078] The product recovered from Gibson assembly was mixed with 500 μL TG1 competent cells and transferred to a 0.1 cm electroporation cuvette, followed by electrotransformation at 2.5 KV. The electroporation product was resuspended in 20 mL 2× culture medium and incubated at 37°C, 220 rpm for 1 h. 10 μL of bacterial solution was diluted to 990 μL 2×YT and mixed, and then 40 μL of bacterial solution was diluted to 160 μL 2×YT and then 100 μL was plated and cultured overnight at 37°C. The library size was calculated on the second day (library size = number of counts × 10 5 ). The bacteria were spread on five 150 mm 2×YT plates supplemented with 100 μg / ml ampicillin and 2% glucose to amplify the phage library and cultured overnight at 37°C. Next, the transformed colonies were scraped from the plates, vortexed thoroughly with a final concentration of 25% glycerol, snap-frozen in liquid nitrogen in 1 mL aliquots and stored at -80°C, and the size of the phage library was calculated.

[0079] 7) Amplification of Nanobody Phage Display Library

[0080] To amplify the phage library of nanobodies, 0.2 ml of the frozen library was thawed on ice, diluted into 200 mL of 2×YT medium supplemented with 100 μg / mL ampicillin and 2% glucose, and cultured at 37°C at 220 rpm. 12 pfu of KM13 helper phage (MRC Laboratory of Molecular Biology) was added to the culture and incubated at 37°C (water bath) for 45 minutes. The cell pellet was separated by high-speed centrifugation and resuspended in 200 mL of 2×YT medium to which 0.1% glucose, 50 μg / mL kanamycin and 100 μg / mL ampicillin were added. The cells were incubated at 25°C, 220 rpm for 20 hours to amplify the phage library. After centrifugation, polyethylene glycol (PEG) was added to the culture supernatant to precipitate phage particles. The precipitated phage particle pellet was dissolved in 1×PBS (formula: 10 mmol / L Na2HPO4; 1.75 mmol / L KH2PO4; 137 mmol / L NaCl; 2.65 mmol / LKCl; pH 7.2-7.6) and stored in 1 mL aliquots at -80°C in the presence of 25% glycerol.

[0081] 8) Screening

[0082] HSA was diluted with PBS to a final concentration of 0.1 mg / ml and coated into one well of a 96-well immunoplate ELISA (Nunc maxsorp plate), and one well of the ELISA plate was set aside for negative control. After washing three times with 1×PBS, 300 μL of MPBS (1×PBS containing 5% skim milk) was added to each well of the ELISA plate and incubated at room temperature for 2 hours to block unbound sites. Next, the plate was washed three times with 1×PBS and 1×10 11 pfu (diluted in 100 μL MPBS) of phage library was added to each well, and after one hour of incubation at room temperature, the wells were washed three times with PBST (1×PBS containing 0.1% Tween 20). Phages displaying nanobodies specific for HSA were eluted by incubation at room temperature for one hour with trypsin at a final concentration of 0.5 mg / ml.

[0083] 10 μL of eluted phages were added to 1 mL of E. coli TG1 competent cells and incubated at 37°C (water bath) for 45 min for infection, and then the bacterial culture was plated on 2×YT supplemented with 100 μg / mL ampicillin and 2% glucose and incubated at 37°C overnight.

[0084] 9) Preparation of monoclonal phage

[0085] After one round of panning, 48 individual colonies were picked into a 96-well round-bottom culture dish containing 100 μL of 2×YT medium supplemented with 100 μg / mL of ampicillin and 2% glucose (w / v). Incubation was continued at 37°C, 180 rpm for 12 hours. Next, 5 μL of this culture was inoculated into a new 96-well round-bottom culture dish containing 200 μL of 2×YT medium supplemented with 100 μg / mL of ampicillin and 2% glucose (w / v). The freshly inoculated plate was incubated at 37°C, 250 rpm for 1.5 hours until the OD 260 It is about 0.5.

[0086] 50 μL containing 4 × 10 8 2×YT medium with pfu KM13 helper phage was added to each well of the plate and incubated at 37°C for 45 minutes without shaking for infection. After infection, 150 μL of supernatant was discarded and the remaining volume was centrifuged at 3500g for 15 minutes, the supernatant was discarded, and the bacterial pellet was resuspended in 200 mL of 2×YT medium supplemented with 100 μg / mL ampicillin, 50 μg / mL kanamycin and 0.1% glucose (w / v), and incubated overnight at 25°C, 250rpm for about 14-16 hours. The next day, the culture was centrifuged at 3500g for 30 minutes, and then 150 μL of supernatant was transferred to a new 96-well plate and stored at 4°C for screening of nanobodies.

[0087] 10) Phage ELISA test.

[0088] 100 μL HSA at a final concentration of 0.2 μg / mL was used to coat a 96-well round-bottom immunoELISA plate by diluting with 1×PBS and incubating at 4°C for 16 hours. Next, the ELISA plate was washed 3 times with 1×PBS. The plate was blocked with MPBS (1×PBS containing 5% skim milk) at room temperature for 2 hours. After that, the wells were washed 4 times with PBS-0.1% Tween 20. Then 100 μL of 1×10 11 pfu of phage was added to each well and incubated at room temperature for 1 hour. The ELSA plate was then washed 5 times with PBS-0.1% Tween 20. HRP-KM13 diluted in MPBS 1:8000 was added to the ELISA plate at 100 μL per well and incubated at room temperature for 1 hour. The wells were then washed 4 times with PBS-0.1% Tween 20, and 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) was added to each well, incubated in the dark for 7 minutes to develop color, and then 50 μL of 1M H2SO4 was immediately added to terminate the reaction, and the OD was measured by an enzyme reader.450nm Value, the result is Figure 1 shown.

[0089] 11) OD 450nm Positive clones with the ability to bind to HSA and a value greater than 1 were picked out for sequencing, and the obtained antibody sequence results were analyzed to exclude repeated, highly similar sequences and useless sequences that could not be purified and expressed to obtain high-purity antibodies. Finally, 5 nanobodies that specifically bind to HSA protein were determined and named as antiHSA-A6 (amino acid sequence as shown in SEQ ID NO: 16), antiHSA-A7 (amino acid sequence as shown in SEQ ID NO: 17), antiHSA-B1 (amino acid sequence as shown in SEQ ID NO: 18), antiHSA-E8 (amino acid sequence as shown in SEQ ID NO: 19), and antiHSA-H7 (amino acid sequence as shown in SEQ ID NO: 20). These 5 nanobodies have specific CDR regions that bind to HSA protein.

[0090] Example 2

[0091] Expression and purification of nanobody-FC fusion protein

[0092] 1) Design a peptide gene sequence that guides secretion and fuse it to the N-terminus of the nanobody gene to ensure secretion after expression, fuse human IgG1 Fc to the C-terminus of the nanobody gene, and recombine the nanobody gene and human IgG1 Fc and then clone them into the mammalian expression vector pTT5.

[0093] The constructed vector was transfected into HEK293F cells (density about 2.5x10 6 cells / ml), in Freestyle TM The transfected mammalian cells were cultured with 293 expression medium (purchased from Yonglian Biotechnology) at 5% CO2, 150 rpm, and 37°C for 4 days, and then the supernatant of the mammalian cell culture was collected by centrifugation at 2000 rpm for 10 minutes.

[0094] Purify the nanobody-FC fusion protein using a protein A column ( Figure 2 ), and SDS-PAGE electrophoresis analysis was performed, such as Figure 3 As shown, highly pure Nanobody-Fc fusion protein was obtained from the supernatant.

[0095] Example 3

[0096] Analysis of the binding affinity of nanoantibodies to HSA protein

[0097] Immuno MaxiSorb plates (Nunc) were coated with 2 μg / mL HSA protein and allowed to stand at room temperature for 2 h. The ELISA plate was washed three times with 1×PBS and 240 μL of MPBS was added to each well and allowed to stand at room temperature for 2 h to block unbound sites. Next, nanoantibody-Fc was gradient diluted with a diluent containing 5% milk in PBST (starting at 50 nM, three-fold continuous gradient dilution, 12 gradient dilutions). 100 μL was added to each well and incubated at room temperature for 1 h. After 1 h, the ELISA plate was washed three times with 1×PBST and HRP-anti-human IgG Fc (Beijing Yiqiao Shenzhou) was diluted with a diluent containing 5% milk in PBST at a ratio of 1:10000 and incubated at room temperature for 1 h. The ELISA plate was washed three times with 1×PBST and 100 μL of TMB was added to each well and reacted at room temperature in the dark for 7 min. Finally, 50 μL of 1M H2SO4 was added to terminate the reaction and the absorbance at 450 nm was detected.

[0098] The results of ELISA analysis are as follows Figure 4 As shown, the EC of HSA protein and nanobody antiHSA-A6-IgG1 Fc 50 At 1.416 nM, the EC of the nanobody antiHSA-A7-IgG1 Fc 50 At 1.140 nM, the EC of the nanobody antiHSA-B1-IgG1Fc 50 At 5.016 nM, the EC of the nanobody antiHSA-E8-IgG1Fc 50 At 4.388 nM, the EC of the nanobody antiHSA-H7-IgG1 Fc 50 At 3.307 nM, the prepared nanobody has a higher affinity to HSA protein.

[0099] The above implementation modes are only used to illustrate the technical solutions of the present invention, but not to limit the present invention. Although the present invention has been described in detail with reference to the above implementation modes, those skilled in the art should understand that any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A nanobody targeting human serum albumin, characterized in that: The nanobody is any one of antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8, and antiHSA-H7, wherein antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8, and antiHSA-H7 all contain three antigen complementary determining regions CDR1, CDR2, and CDR3, wherein: The amino acid sequences of the three antigen complementary determining regions of antiHSA-A6 are amino acid sequences that have a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; The amino acid sequences of the three antigen complementary determining regions of the antiHSA-A7 are amino acid sequences that have a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively; The amino acid sequences of the three antigen complementary determining regions of the antiHSA-B1 are amino acid sequences having a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively; The amino acid sequences of the three antigen complementary determining regions of antiHSA-E8 are amino acid sequences that have a homology of greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively; The three antigen complementary determining region amino acid sequences of antiHSA-H7 are amino acid sequences with a homology greater than or equal to 80% with the amino acid sequences shown in SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO:

15.

2. A nanobody targeting human serum albumin according to claim 1, characterized in that: The three antigen complementary determining region amino acid sequences of antiHSA-A6 are shown as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively; the three antigen complementary determining region amino acid sequences of antiHSA-A7 are shown as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, respectively; the three antigen complementary determining region amino acid sequences of antiHSA-B1 are shown as SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, respectively; the three antigen complementary determining region amino acid sequences of antiHSA-E8 are shown as SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, respectively; the three antigen complementary determining region amino acid sequences of antiHSA-H7 are shown as SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15, respectively.

3. A nanobody targeting human serum albumin according to claim 2, characterized in that: The amino acid sequence of antiHSA-A6 is shown in SEQ ID NO: 16, the amino acid sequence of antiHSA-A7 is shown in SEQ ID NO: 17, the amino acid sequence of antiHSA-B1 is shown in SEQ ID NO: 18, the amino acid sequence of antiHSA-E8 is shown in SEQ ID NO: 19, and the amino acid sequence of antiHSA-H7 is shown in SEQ ID NO:

20.

4. An antibody targeting human serum albumin, comprising any one of the nanobodies antiHSA-A6, antiHSA-A7, antiHSA-B1, antiHSA-E8, and antiHSA-H7 as claimed in claim 1 and an Fc domain.

5. The antibody according to claim 4, characterized in that The Fc domain is a human IgG1 Fc domain.

6. A polynucleotide encoding a Nanobody as claimed in any one of claims 1 to 3, or encoding an antibody as claimed in any one of claims 4 to 5. An expression vector comprising the polynucleotide according to claim 6 . A host cell comprising the expression vector according to claim 7 .

9. Use of the Nanobody according to any one of claims 1 to 3 in the preparation of a vaccine for preventing and / or treating a disease associated with human serum albumin.

10. Use of the nanobody according to any one of claims 1 to 3 in prolonging the half-life of macromolecular drugs in vivo.

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