HSA nano antibody and application thereof
By constructing a nanobody library targeting HSA and screening for highly efficient binding nanobodies, the problem of HSA-specific nanobodies being difficult to achieve in existing technologies has been solved. This enables the efficient preparation and application of HSA-specific nanobodies, improving drug half-life and disease diagnosis and treatment efficacy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-12-30
- Publication Date
- 2026-04-07
AI Technical Summary
Existing technologies cannot effectively utilize specific nanobodies of human serum albumin (HSA), affecting the effectiveness of drug design and clinical applications.
Develop specific nanobodies against HSA by constructing an immune nanobody library and screening for nanobodies that bind efficiently to HSA, achieve efficient expression and purification in E. coli, and prepare HSA-based drugs or reagents.
This study enabled the efficient preparation of HSA-specific nanobodies, improving drug half-life and disease diagnosis and treatment efficacy, and providing a tool for HSA molecular detection.
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Figure CN121800918A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical or biopharmaceutical technology, and relates to an HSA nanobody and its application. Background Technology
[0002] Human serum albumin (HSA) belongs to the polygenic protein family and is the most abundant protein in human plasma. It has a high affinity for many endogenous and exogenous compounds and is the main soluble protein in the circulatory system.
[0003] HSA is a single-chain, non-glycosylated protein composed of 585 amino acids, with a molecular weight of approximately 66.5 kDa. HSA has a heart-shaped three-dimensional structure containing three homologous domains (I, II, and III). Each domain is further divided into two subdomains, A and B, which are stably folded by 17 disulfide bonds. HSA exhibits a highly flexible conformation, especially the "FA1" and "FA2" regions of domain III, which can dynamically bind various ligands. As a soluble secretory protein, HSA itself lacks a transmembrane structure, but it can achieve transmembrane transport or endocytosis / circulation through receptors (such as FcRn and gp60). The interaction between HSA and the cell membrane depends on the surface charge distribution and specific receptor-binding domains.
[0004] In healthy human plasma, HSA mainly exists in the form of soluble monomers, accounting for 50%–60% of total plasma protein, with a concentration of approximately 35–50 g / L. HSA has a stable structure, maintaining correct folding through disulfide bonds to ensure transport and osmotic regulation functions; however, it may form dimer / multimer forms during long-term storage or at high temperatures, affecting drug binding capacity.
[0005] In addition to its functions of transport, osmotic regulation, and antioxidation, HSA can also mediate inhibitory effects through various mechanisms, affecting drug activity, enzyme function, inflammatory responses, and pathogen infection processes.
[0006] HSA-mediated inhibition can be divided into three aspects: (1) it can bind certain drug molecules with high affinity, reduce their free concentration, thereby weakening the drug effect or delaying the release; (2) it can inhibit the activity of certain enzymes by directly binding or regulating the microenvironment; (3) it can inhibit excessive immune response and inflammation through multiple pathways, such as neutralizing inflammatory mediators, inhibiting complement activation and regulating cytokines.
[0007] HSA has a wide and complex inhibitory effect, which can protect the body (such as anti-inflammatory and detoxification) or interfere with treatment (such as reducing drug activity), and is widely used in drug design and clinical applications.
[0008] Nanobody technology is a revolutionary antibody engineering approach developed by biomedical scientists, combining molecular biology techniques with the concepts of nanoparticle science to create the newest and smallest antibody molecules based on traditional antibodies. In 1993, Hamers et al. first discovered that camels naturally possess a class of heavy chain antibodies lacking both the light chain and the heavy chain constant region 1 (CH1). Cloning their variable region yielded single-domain antibodies consisting solely of a single heavy chain variable region, initially named VHH (heavy-chain antibody variable domain), later collectively referred to as "nanobody" (Nb).
[0009] As the smallest antibody fragment with complete antigen-binding function, nanobodies have an elliptical crystal structure with a diameter of about 2.5 nm and a length of about 4 nm, and possess a series of unique advantages: (1) Their chemical composition and structure are simpler than those of traditional antibodies, with no significant chemical hydrophobicity, stronger heat resistance and acid and alkali resistance, and they are easy to bind with other molecules or compounds; (2) Nanobodies have a single encoding gene, which can be efficiently expressed by microorganisms; (3) Nanobodies have excellent environmental tolerance, high conformational stability, and small molecular weight, which not only makes them easy to synthesize and lower in cost, but also improves the targeting and efficacy of clinical treatment. These characteristics of nanobodies enable them to demonstrate application potential far exceeding that of traditional antibodies in the fields of precise disease diagnosis and immunotherapy.
[0010] Given the unique advantages of nanobodies, the development of specific nanobodies targeting HSA is a promising area for advancement. These specific nanobodies will demonstrate broad application prospects in areas such as extending drug half-life, developing molecular diagnostic reagents for HSA, and diagnosing and treating diseases. Summary of the Invention
[0011] The purpose of this invention is to provide a nanobody targeting an HSA epitope (hereinafter referred to as an HSA nanobody), and to provide the encoding sequence of the nanobody and its application in the preparation of HSA-based drugs.
[0012] In a first aspect, the present invention provides an HSA nanobody, the HSA nanobody comprising a framework region (FR) and a complementarity-determining region (CDR), wherein: The frame region FR includes FR1, FR2, FR3 and FR4, and the complementarity determination region CDR includes CRD1, CRD2 and CRD3; The HSA nanobody comprises FR1, CRD1, FR2, CRD2, FR3, CRD3 and FR4 from the N-terminus to the C-terminus. The amino acid sequence of FR1 is shown in SEQ ID NO: 1, the amino acid sequence of FR2 is shown in SEQ ID NO: 3, the amino acid sequence of FR3 is shown in SEQ ID NO: 5, and the amino acid sequence of FR4 is shown in SEQ ID NO: 7. The amino acid sequence of CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 is shown in SEQ ID NO: 6.
[0013] Preferably, the HSA nanobody has an amino acid sequence as shown in SEQ ID NO: 9.
[0014] Preferably, the amino acid sequence of the HSA nanobody is as shown in SEQ ID NO: 9.
[0015] In a second aspect, the present invention provides a nucleic acid molecule that encodes the HSA nanobody described in the first aspect of the present invention.
[0016] Preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 8. More preferably, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO: 8.
[0017] In a third aspect, the present invention provides an expression vector that expresses the nucleic acid molecule described in the second aspect of the present invention.
[0018] In a fourth aspect, the present invention provides a host cell expressing the HSA nanobody described in the first aspect of the present invention.
[0019] In a fifth aspect, the present invention provides an HSA-based drug comprising the HSA nanobody described in the first aspect of the present invention, the expression vector described in the third aspect of the present invention, or the host cell described in the fourth aspect of the present invention.
[0020] The present invention provides, in a sixth aspect, the use of the HSA nanobody described in the first aspect, the expression vector described in the third aspect, or the host cell described in the fourth aspect in the preparation of HSA-based drugs or HSA-based reagents, wherein the HSA-based drugs are drugs for intervening in HSA-mediated effects, and the HSA-based reagents are reagents for detecting the presence or abundance of HSA. Preferably, the HSA-based drug is a drug used to intervene in HSA-mediated inhibition, more preferably a drug used to prolong the drug's half-life or a drug used to diagnose and treat diseases.
[0021] This invention first synthesizes HSA protein and constructs a specific immune library targeting HSA molecules. Then, HSA molecules are conjugated to an ELISA plate. Using this antigen in this form, a gene library of immune nanobodies (camel heavy chain antibody phage display gene library) is screened using phage display technology, thereby obtaining HSA-specific nanobodies. This gene is then transferred into *E. coli*, thus establishing a nanobodies strain that can be efficiently expressed in *E. coli*. The nanobodies and related products of this invention can be used for HSA-related detection or treatment, as well as for extending drug half-life, etc. Attached Figure Description
[0022] Figure 1 These are the ELISA results for screening with 6 HSA antibodies. HSA antigen was coated onto a 96-well plate, and 6 HSA nanobodies were used to bind to it, screening for HSA Nb with high binding rates. The results showed that H1-22 had the highest binding rate.
[0023] Figure 2 This is an SDS-PAGE electrophoresis image of the H1-22 HSA nanobody after purification by nickel column resin gel affinity chromatography, analyzed using gel imaging system software. The results show that the purity of the HSA nanobody can reach over 90% after this purification process. Detailed Implementation
[0024] This invention utilizes HSA conjugation on an ELISA plate to display the correct spatial structure of proteins. This antigen is used to screen an immune nanobody gene library (camel heavy chain antibody phage display gene library), thereby obtaining nanobody strains that can be efficiently expressed in Escherichia coli.
[0025] The present invention will be further described below with reference to specific embodiments.
[0026] Example 1: Construction of a nanobody library targeting HSA This embodiment constructs a nanobody library targeting HSA. The construction includes the following steps: (1) Dissolve 1 mg of HSA antigen protein in 1 mL of physiological saline, then mix with 1 mL of Freund's adjuvant and emulsify before immunizing Xinjiang Bactrian camels: once a week, to induce immune cells to produce specific nanobodies against HSA antigen.
[0027] (2) After 7 immunizations, 100 mL of camel peripheral blood lymphocytes were extracted and total RNA was extracted using an RNA extraction kit.
[0028] (3) RNA was reverse transcribed into cDNA, and then the VHH fragment was amplified by two-step PCR. The amplification product obtained by the first PCR was named VHH-h-CH2-CH3 fragment (approximately 700bp). The amplification product obtained by the first PCR was used as a template for the second PCR, and the amplification product obtained was named VHH fragment (approximately 400bp).
[0029] (4) Use restriction endonucleases PstI and Notl The vector pMECS was digested with enzymes, and then the digested vector pMECS and the amplification product fragment obtained from the second PCR in step (3) were ligated to obtain the ligation product.
[0030] (5) The ligation product was transformed into electrocompetent TG1 cells to construct an HSA nanobody library. 10 μL of the HSA nanobody library (i.e., 10 μL of the HSA nanobody library) was taken. -2 Perform a series of 10-fold serial dilutions (ml), and take 10 ml of the solution. -4 10 -5 and 10 -6 Three serial dilutions of the ampicillin-resistant solution were spread onto ampicillin-resistant plates and incubated overnight at 37°C. The number of single colonies grown was then calculated using the serial dilutions, and the library volume was determined to be 3 × 10⁻⁶. 9 CFU. Meanwhile, 24 clones were randomly selected from the serially diluted plates for colony PCR to detect the insertion rate of the nanobody library. The results showed that the insertion rate reached 95.8%.
[0031] Example 2: Screening of nanobodies targeting HSA In this embodiment, nanobodies targeting HSA are screened. The screening includes the following steps: (1) Dissolve 5 μg / mL HSA protein in 100 mM NaHCO3, coat it onto an ELISA plate, and incubate overnight at 4ºC.
[0032] (2) On the second day, add the blocking solution and seal at room temperature for 2 hours.
[0033] (3) After the blocking was completed, the cells were washed 10 times with PBST, and then the phages of the HSA nanobody library constructed in Example 1 were added and incubated at room temperature for 1 hour.
[0034] (4) After incubation, wash with PBST 5 times to remove non-specific phages.
[0035] (5) The phages that specifically bind to the antigen protein were dissociated using TEA elution buffer and then used to infect Escherichia coli TG1 cells in the logarithmic growth phase. The cells were then cultured at 37ºC for 1 hour. The resulting cultured phages were used for the next round of screening. The same screening process was repeated multiple times until phage clones appeared and enriched. After 4 rounds of screening, the antigen-specific phages achieved an enrichment 64 times higher than the negative control.
[0036] Example 3: Screening of HSA-specific single positive clones In this embodiment, an enzyme-linked immunosorbent assay (ELISA) using bacteriophages is used to screen for HSA-specific single positive clones. The screening includes the following steps: (1) 384 single colonies were randomly selected from the bacteriophages obtained in the second round of Example 2 and inoculated into TB medium containing p100 μg / mL Am and cultured in 96-well plates. After the growth reached the logarithmic phase, IPTG was added for induction and cultured overnight at 28ºC.
[0037] (2) Crude antibodies were then obtained by osmotic shock method, and the antibody supernatant was transferred to an enzyme-labeled plate coated overnight (5 μg / mL, HSA antigen) and incubated at room temperature for 1 h.
[0038] (3) Wash away unbound antibodies with PBST, add mouse anti-HA antibody and incubate at room temperature for 1 hour.
[0039] (4) Wash away unbound antibodies with PBST, add Anti-Mouse IgG, and incubate at room temperature for 1 hour.
[0040] (5) Wash away unbound antibodies with PBST, add TMB colorimetric solution, and read the absorbance at 450 nm wavelength on an ELISA instrument.
[0041] (6) When the OD450 of HAS is more than three times that of NaHCO3, it is judged as a positive clone well, and a total of 88 positive clones are obtained.
[0042] (7) The 88 positive clones obtained in step (6) were cultured and sequenced, and sequence analysis was performed using Geneious Prime. Clones with the same CDR1, CDR2, and CDR3 sequences were considered as the same clone, while clones with different CDR1, CDR2, and CDR3 sequences were considered as different clones.
[0043] (8) Six HSA nanobody genes with different CDR3 values were selected from the obtained sequences and transformed into E. coli expression strain WK6 for amplification culture. Then, the expression of nanobodies was induced by IPTG. After the culture was completed, the crude antibody supernatant was obtained by osmotic shock method, and the crude HCMV-gB nanobody extract was purified by nickel column affinity chromatography. The expression of the target protein was observed by SDS-PAGE, and the binding was screened again by ELISA as described in Example 3. The optimal antibody was selected by combining the results of SDS-PAGE and ELISA. The amino acid sequence of the VHH chain of this antibody is shown in SEQ ID NO: 9.
[0044] Example 4: Expression and purification of HSA nanobodies in host bacterium Escherichia coli In this example, the expression and purification of HSA nanobodies in the host bacterium *Escherichia coli* were performed, and the specific process is as follows: (1) Six HSA nanobody genes with different CDR3 were selected from the obtained sequences and transformed into Escherichia coli expression strains. The HSA nanobody expression strains were plated on LB agar plates (containing 100 μg / mL ampicillin and 1% glucose) and cultured overnight at 37°C.
[0045] (2) Select a single colony and inoculate it into 5 mL of LB medium containing 100 μg / mL ampicillin. Incubate overnight at 37°C in a shaker to obtain overnight bacteria.
[0046] (3) Inoculate 1 mL of overnight bacterial culture into 330 mL of LB culture medium and culture in a shaker at 37°C until the OD value drops to 0.6~1. Then add IPTG (final concentration of 1 mM) and culture in a shaker at 28°C overnight to obtain Escherichia coli culture.
[0047] (4) Collect the E. coli culture obtained in step (3) by centrifugation, and then obtain the crude extract of HSA nanobody by permeation method.
[0048] (5) The crude extract of HSA nanobody was purified by nickel column ion affinity chromatography to obtain purified HSA nanobody.
[0049] Example 5: Identification of the binding ability of HSA nanobodies to HSA proteins In this embodiment, the binding ability of HSA nanobodies to HSA proteins was identified, and the specific process is as follows: The HSA protein was diluted to 2 μg / mL with 100 mM NaHCO3 (pH 9.6), and 100 μL / well was added to a 96-well microplate. The plate was then coated overnight at 4°C. The coating solution was discarded, and 200 μL / well of PBST buffer containing 1% BSA was added. The plate was blocked at room temperature for 2 h. The blocking solution was discarded, and the plate was washed three times with PBST. The purified HSA nanobodies obtained in Example 4 were added to the wells according to the grouping, 100 μL / well, and incubated at room temperature for 1 h. Unbound HSA nanobodies were washed away with PBST. HRP-labeled anti-VHH (Chengdu Critical Point Biotechnology Co., Ltd.) was added, 100 μL / well, and incubated at room temperature for 1 h. Unbound antibodies were washed away with PBST. TMB chromogenic solution was added, 100 μL / well, and the plate was incubated at room temperature in the dark for 20 min. The reaction was stopped by adding 50 μL / well of TMB stop solution. The absorbance value at 450 nm (OD450) was read using a microplate reader, and the results are as follows: Figure 1 As shown.
[0050] Example 6: Purification and Identification of HSA Nanobodies Based on the HSA nanobody expression and binding to the HSA antigen observed in Examples 4 and 5, the optimal HSA nanobody expression strain (numbered H1-22) was selected. The amino acid sequence of the VHH chain of this expression strain's HSA nanobody is shown in SEQ ID NO: 9. The purified product of the H1-22 HSA nanobody was identified using SDS-PAGE and analyzed using gel imaging software. The results showed that the purity of the H1-22 HSA nanobody after purification reached over 90% (e.g., ...). Figure 2 (As shown).
[0051] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. An HSA nanobody, wherein the HSA nanobody comprises a framework region (FR) and a complementarity-determining region (CDR), characterized in that: The frame region FR includes FR1, FR2, FR3 and FR4, and the complementarity determination region CDR includes CRD1, CRD2 and CRD3; The HSA nanobody comprises FR1, CRD1, FR2, CRD2, FR3, CRD3 and FR4 from the N-terminus to the C-terminus. The amino acid sequence of FR1 is shown in SEQ ID NO: 1, the amino acid sequence of FR2 is shown in SEQ ID NO: 3, the amino acid sequence of FR3 is shown in SEQ ID NO: 5, and the amino acid sequence of FR4 is shown in SEQ ID NO:
7. The amino acid sequence of CDR1 is shown in SEQ ID NO: 2, the amino acid sequence of CDR2 is shown in SEQ ID NO: 4, and the amino acid sequence of CDR3 is shown in SEQ ID NO:
6.
2. The HSA nanobody according to claim 1, characterized in that: The HSA nanobody has the amino acid sequence shown in SEQ ID NO:
9.
3. The HSA nanobody according to claim 2, characterized in that: The amino acid sequence of the HSA nanobody is shown in SEQ ID NO:
9.
4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the HSA nanobody as described in any one of claims 1 to 3.
5. The nucleic acid molecule according to claim 4, characterized in that: The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO:
8.
6. The nucleic acid molecule according to claim 5, characterized in that: The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:
8.
7. An expression carrier, characterized in that, The expression vector contains a nucleotide sequence as shown in SEQ ID NO:
8.
8. A host cell, characterized in that, The host cell expresses the HSA nanobody as described in any one of claims 1 to 3.
9. A drug based on HSA, characterized in that: The drug comprises the HSA nanobody as described in any one of claims 1 to 3, the expression vector as described in claim 7, or the host cell as described in claim 8.
10. The use of the HSA nanobody according to any one of claims 1 to 3, the expression vector according to claim 7, or the host cell according to claim 8 in the preparation of HSA-based drugs or HSA-based reagents, wherein, HSA-based drugs are drugs used to intervene in HSA-mediated effects, and HSA-based reagents are reagents used to detect the presence or abundance of HSA. Preferably, the HSA-mediated effect is an HSA-mediated inhibitory effect; More preferably, HSA-based drugs are drugs used to prolong the half-life of a drug or to diagnose or treat a disease.