Nanobody Targeting Human Serum Albumin and Its Application

Abstract

The present invention discloses a human serum albumin-targeting nanobody and its application. A variety of human serum albumin-targeting nanobodies that can be used to construct long-acting fusion protein drugs and their immune screening methods are disclosed. The antibody is composed only of the heavy chain variable region and can be fused and expressed with therapeutic proteins or polypeptide drugs such as interleukin, interferon, and tumor necrosis factor in an Escherichia coli expression system. The fusion protein has the activity of specifically binding to human serum albumin, has a relatively high affinity, can effectively extend the drug half-life, and does not affect the biological activity of the recombinant protein drug at the same time.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and particularly to nanobodies targeting human serum albumin and their applications. Background Art

[0002] Monoclonal antibodies play an important role in the biological products marketed in recent years. Traditional antibodies are composed of two heavy chains and two light chains. At the end of the last century, scientists found that there is an antibody in the peripheral blood of alpacas that is naturally lacking in light chains. This antibody only contains a single variable heavy chain region (VHH) and two conventional CH2 and CH3 regions. The VHH structure cloned and recombinantly expressed alone has the same structural stability as the original heavy chain antibody and the binding activity to antigens. It is the smallest unit known to be able to bind to the target antigen. Since the size of this type of heavy chain antibody is only about 2.5 nm, it is also called a nanobody. Nanobodies are highly soluble, not easily aggregated, and can withstand denaturing conditions such as high temperature, strong acid, and strong base. They are suitable for prokaryotic expression and various eukaryotic expression systems, and are widely used in the development of therapeutic antibody drugs, diagnostic reagents, affinity purification matrices, and scientific research. Based on the structural characteristics of nanobodies, the development of nanobodies targeting HSA for extending the drug half-life has become a research hotspot.

[0003] Protein / polypeptide drugs have the advantages of low toxicity, high activity, and strong specificity, and have broad development prospects. However, due to the low stability of these drugs, they are easily metabolized or excreted in the blood, liver, kidneys, etc., resulting in limitations such as short half-life, poor drug efficacy, and poor patient compliance. Therefore, there is an urgent need for secondary modification. Therefore, carrying out research on long-acting drugs is of great significance for the development of such drugs. Long-acting strategies include constructing mutants, glycosylation modification, polyethylene glycolylation modification, and fusing with other proteins by genetic means, etc. Among them, HSA fusion is a commonly used method.

[0004] HSA, human serum albumin, is a highly soluble and stable protein synthesized and secreted into the blood by hepatocytes. It has a molecular weight of 66.5 kDa and a concentration of approximately 40 mg / mL in human blood. The half-life of HSA is very long, about 19 - 21 days. The key mechanism for the long half-life of HSA in the human body is the salvage mechanism mediated by the neonatal Fc receptor (FcRn). FcRn is a transmembrane receptor protein widely distributed in various tissues of the human body. It can bind to HSA and IgG in a pH-dependent manner, rescue HSA and IgG from the metabolic process, and reintroduce them into the body circulation. Due to the recognized serum stability and longevity of human serum albumin, it has become a highly regarded long-acting drug carrier. Currently, there are various strategies for the long-acting modification of human serum albumin, mainly including in vitro gene fusion and in vivo non-covalent binding. The long-acting glucagon-like peptide-1 analog developed by Novo Nordisk, which has achieved great market success in recent years, utilizes fatty acid side chains to achieve non-covalent binding to human serum albumin in vivo, thereby extending the half-life.

[0005] Currently, the commonly used long-acting strategies for recombinant protein drugs include constructing mutants, glycosylation modification, polyethylene glycol modification, fatty acid modification, Fc fusion protein construction, HSA fusion protein construction, etc. However, these methods all have certain limitations. Constructing mutants requires a full understanding of the protein structure, and it is technically difficult to obtain ideal mutants; glycosylation modification has high requirements for the expression system, resulting in high production costs; polyethylene glycol modification is a widely used technology at present, but achieving the directional conjugation of polyethylene glycol to proteins also has high technical requirements, and the long-acting effect of polyethylene glycol is positively correlated with the molecular weight. Long-term drug use is likely to cause the accumulation of polyethylene glycol in the body and produce toxicity; fatty acid modification is currently mostly used for the long-acting modification of small peptide drugs, generally prepared by solid-phase synthesis methods. This method has high costs when used for the preparation of larger cytokine and hormone drugs; the construction of Fc / HSA fusion proteins all results in a relatively large molecular weight of the drug, has high requirements for the expression system, cannot use prokaryotic expression systems, and the relatively large molecular weight also leads to poor tissue permeability of the drug. Currently, single-chain antibodies (scFv) and albumin-binding peptides derived from traditional antibodies are also used for constructing HSA-binding bridges in vitro. However, scFv is prone to aggregation and precipitation due to the lack of a constant region framework, while albumin-binding peptides have a relatively large immunogenicity problem because they are isolated from bacteria.

[0006] CN119032104A discloses an anti-serum albumin nanobody and its derivatives. A series of nanobodies against serum albumin are obtained through large-scale screening. The nanobodies have high binding activity with serum albumin and possess cross-reactivity with human, monkey, and / or mouse serum albumin. The binding of the nanobodies to serum albumin is non-pH-dependent, and it can effectively participate in the FcRn-mediated serum albumin circulation, thereby prolonging the in vivo half-life of the nanobodies. It also discloses conjugating / fusing the nanobodies with drug molecules (peptide drugs) to prolong the in vivo half-life of the drug molecules. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, the present invention provides an anti-HSA nanobody for prolonging the therapeutic effect of therapeutic proteins, especially prolonging its half-life, and establishes a long-acting technology route for recombinant protein drugs with simple technology, low cost, safety and high efficiency, and relatively low molecular weight.

[0008] The present invention discloses various target human serum albumin nanobodies that can be used to construct long-acting fusion protein drugs and their immune screening methods. The antibodies are composed only of the heavy chain variable region and can be fused and expressed with therapeutic proteins or peptide drugs such as interleukin, interferon, and tumor necrosis factor in an Escherichia coli expression system. The fusion protein has the activity of specifically binding to human serum albumin, with relatively high affinity, can effectively prolong the drug half-life, and does not affect the biological activity of the recombinant protein drug at the same time.

[0009] The technical route of the present invention: Immunize alpacas with human serum albumin multiple times at multiple points. During the immunization process, a small amount of serum is collected for ELISA detection. After the titer is qualified, a large amount of blood is taken to isolate peripheral blood lymphocytes, total RNA is extracted, and cDNA is obtained by reverse transcription. After amplification using primers, a phagemid is constructed, and the phagemid is transferred into phages to construct a phage library. Multiple rounds of panning are carried out to screen out 23 monoclonal antibody sequences that specifically bind to HSA; Use the Escherichia coli expression system to induce the expression of 23 nanobodies, purify the nanobodies for in vitro affinity detection, and construct fusion proteins with therapeutic protein drugs. Still use Escherichia coli to express the fusion proteins, and after purification, detect the activity and half-life of the fusion proteins.

[0010] The present invention discloses a target human serum albumin nanobody, which contains the heavy chain variable region of a camel-derived antibody targeting HSA and is composed of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Preferably, the CDR regions of the target human serum albumin nanobody can be composed of the three CDR regions of any one of the SEQ ID NO: 1-23 sequences shown in Table 1;

[0011]

[0012] Preferably, the amino acid sequence of the nanobody targeting human serum albumin has at least 70% sequence identity with any one of SEQ ID NOs: 1-23; more preferably, at least 80% sequence identity with any one of SEQ ID NOs: 1-23; further preferably, at least 90% sequence identity with any one of SEQ ID NOs: 1-23; further preferably, at least 95% sequence identity with any one of SEQ ID NOs: 1-23; most preferably, as shown in any one of SEQ ID NOs: 1-23.

[0013] The present invention also discloses a fusion protein comprising a therapeutic protein and the above-mentioned nanobody targeting human serum albumin.

[0014] The therapeutic protein includes but is not limited to polypeptides, antibodies, antibody fragments, cytokines, tumor marker molecules, etc.

[0015] Preferably, the therapeutic protein is interleukin-1-receptor antagonist (IL-1Ra) or its analogs, derivatives. The sequence of IL-1Ra is as shown in SEQ ID NO: 24: MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE.

[0016] Analogs or derivatives of IL-1Ra refer to structural analogs that are homologous to IL-1Ra and have the same biological activity, including but not limited to the therapeutic protein moieties of such drugs.

[0017] Antibody fragments mentioned in the therapeutic protein of the present invention include but are not limited to single-chain antibodies, Fab domains, heavy-chain antibodies and their multivalent forms, etc.

[0018] As a further preferred embodiment, the fusion protein of the present invention comprises a linker peptide between the nanobody targeting human serum albumin and the therapeutic protein, and the therapeutic protein is linked to the N-terminus, C-terminus or both the N-terminus and C-terminus of the nanobody through the linker peptide.

[0019] The linker peptide has any one of the following structures:

[0020] (GGGS)n, where n is any value from 2 to 4; or (EAAAK)n-(GGGS)n, where n is any value from 1 to 2; or (EAAAK)n, where n is any value from 1 to 4.

[0021] The present invention also discloses a screening method for the above-mentioned nanobody targeting human serum albumin and a preparation method for the fusion protein, which specifically include the following steps:

[0022] (1) Immunize alpacas with HSA;

[0023] (2) Isolate alpaca peripheral blood mononuclear cells (PBMC), extract total RNA, and reverse transcribe to synthesize cDNA;

[0024] (3) Use primers to perform two rounds of PCR amplification of the target fragment, construct a phagemid, transfer it into a phage to construct a phage library, and perform multiple rounds of panning for positive clones. The primers are as follows:

[0025] Primers for the first round

[0026] F: GTCCTGGCGGCTCATCTCCAAGA (SEQ ID NO:26)

[0027] R: GGTACGTTCTGATGAAGTGTAGC (SEQ ID NO:27)

[0028] Primers for the second round

[0029] F: CCTGCTGCTGGGCCCAAGCGGCCCAGGTGCATCTGCAGGAGTCTGCRCG (SEQ ID NO:28)

[0030] R: GAGGAGGAGGGCCGACGGGGCCTGGTCGGTCCTCGCTGAGTTACG (SEQ ID NO:29)

[0031] (4) Construct an expression vector for the positive clone, induce expression and purification using a prokaryotic expression system, and measure the affinity with HSA; thus, the screening of the nanobody targeting human serum albumin is completed;

[0032] (5) Connect the positive clone sequence and the therapeutic protein sequence through a short peptide to construct an expression vector, and perform fusion expression and purification in a prokaryotic expression system.

[0033] The fusion protein of the present invention has both the binding ability to HSA and the therapeutic activity of the fused therapeutic protein, and its therapeutic activity is significantly improved.

[0034] Preferably, the present invention constructs a fusion protein of a nanobody targeting human serum albumin and IL-1Ra or its analogs and derivatives, which can exert rapid and persistent anti-inflammatory activity. There are mainly three subtypes of IL-1Ra in the human body. When the three subtypes of IL-1Ra are in excess in the body, they all have the biological activity of antagonizing IL-1, can competitively bind to the membrane IL-1 receptor to block the signal transduction of IL-1, block the inflammatory response mediated by IL-1, and reduce tissue damage caused by immune response, infection, inflammatory response, etc. Therefore, it has attracted much attention in the treatment of inflammatory diseases.

[0035] Advantages of the present invention:

[0036] The nanobody targeting human serum albumin involved in the present invention has a brand-new amino acid sequence, has high specificity for HSA, and has advantages such as stable structure, strong solubility, and low molecular weight compared with traditional monoclonal antibodies (fragments). Moreover, due to the lack of the Fc segment, its immunogenicity is reduced. At the same time, the nanobody targeting human serum albumin does not require post-translational modification and can be expressed by a prokaryotic expression system, with low production costs.

[0037] By constructing a fusion protein of a nanobody targeting human serum albumin and a therapeutic protein, the purpose of prolonging the drug half-life while retaining the therapeutic activity is achieved, the bioavailability of the drug is improved. Compared with the existing technologies for prolonging the half-life, while maintaining a lower molecular weight, there is no need for complex technical processes such as structural modification, PEG modification, microsphere release, and fatty acid modification of the therapeutic protein to introduce unnatural amino acids, and the obtained fusion protein can be expressed by a prokaryotic system, with simple technology and greatly reduced production costs. Brief Description of the Drawings

[0038] Figure 1 It is a technical roadmap of the nanobody targeting human serum albumin of the present invention.

[0039] Figure 2 It is an electrophoresis diagram of total RNA, and the total RNA is extracted from PBMCs obtained in Example 1.

[0040] Figure 3 It is an electrophoresis diagram of the first-round PCR amplification of the antibody gene.

[0041] Figure 4 It is an electrophoresis diagram of the second-round PCR amplification of the antibody gene sequence.

[0042] Figure 5 It is the anti-inflammatory activity - plantar swelling rate of the fusion protein constructed in Example 4 in a rat model.

[0043] Figure 6 It is the drug curve of the fusion protein constructed in Example 4 in a rhesus monkey model.

[0044] Figure 7 The anti-inflammatory activity of the fusion protein constructed in Example 5 in a rat model - plantar swelling rate.

[0045] Figure 8 The anti-inflammatory activity of the fusion protein constructed in Example 6 in a rat model - plantar swelling rate. Detailed implementation manners

[0046] The following examples are only for further specific illustration of the present invention, and the application scope of the present invention is not limited to the examples. Reagents, instruments, experimental methods, etc. involved in the examples are all conventional reagents, instruments, and methods without specific description.

[0047] Example 1: Alpaca immunization and serum titer determination

[0048] An alpaca over 1 year old was immunized 4 times in total. Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA) were used as immunization adjuvants, and the administration route was through multiple-point subcutaneous injection. 2 mL of negative serum was taken before immunization, and an earring was used for numbering and reserved. After observing for 3 - 5 days, immunization was initiated. The immunization dose for the first immunization was 1 mg / time / head. The immunization and blood collection processes were arranged as shown in Table 2, and the ELISA detection results of the serum titer were shown in Table 3. After the titer was qualified, peripheral blood mononuclear cells (PBMC) were isolated;

[0049]

[0050] Example 2: Establishment of a phage display library and monoclonal screening

[0051] 1. Obtaining the target gene

[0052] Total RNA was extracted from the PBMC obtained in Example 1, and the electrophoresis results of the total RNA are as Figure 2 shown. The total RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0053] For the first-round PCR amplification of the antibody gene, using cDNA as the template, the amplification system (20 µL) for each pair of primers is shown in Table 4, and the amplification program is shown in Table 5. The amplification results were electrophoresed as Figure 3 shown.

[0054] First-round primers

[0055] F: GTCCTGGCGGCTCATCTCCAAGA (SEQ ID NO: 26)

[0056] R: GGTACGTTCTGATGAAGTGTAGC (SEQ ID NO: 27)

[0057]

[0058] For the second-round PCR amplification of the antibody gene sequence, using the 700-bp target fragment of the first-round PCR product as the template, the amplification system (20 µL) for each pair of primers is shown in Table 6, the amplification program is shown in Table 7, and the electrophoresis result of the amplification is as Figure 4 shown.

[0059] Second-round primers

[0060] F: CCTGCTGCTGGGCCCAAGCGGCCCAGGTGCATCTGCAGGAGTCTGCRCG (SEQ ID NO:28)

[0061] R: GAGGAGGAGGGCCGACGGGGCCTGGTCGGTCCTCGCTGAGTTACG (SEQ ID NO:29)

[0062]

[0063] Construction of the phage library

[0064] Use a gel extraction kit to recover the second-round amplified fragment, and use sfiI enzyme to digest the recovered fragment and the pcomb3X vector respectively. After digestion, the fragments are directly purified using a gel extraction and purification column, and the pcomb3X vector is recovered as a linear vector using a gel extraction kit; mix the target fragment with the pcomb3X vector and use T4 ligase to ligate overnight at 4°C, and then electrotransform the ligation product into XL1-Blue competent cells to obtain the antibody bacterial liquid library; add VCSM13 helper phage to the library bacterial liquid and let it stand for half an hour for further expression and amplification, and collect the phage precipitate to obtain the phage library.

[0065] Randomly pick 48 monoclonal colonies from the library-coated petri dishes for sequencing. 44 correct clones, 2 clones without signal sequences, and 2 clones with stop codon sequences, with a sequence accuracy rate of 91.7%. After library amplification, the phage concentration is 2.64×10 12 PFU / mL.

[0066] Screening of monoclonal clones

[0067] Coat the high-affinity enzyme-linked immunosorbent assay (ELISA) plate with HSA overnight, wash the plate and then block it. Add 5×10 12After incubation and elution of the PFU phage, the pH was adjusted to 7.4. The eluate was mixed with Escherichia coli XL1-Blue for co-culture. After rescuing the phage, it was cultured with shaking overnight at 30 °C to obtain the amplified antibody 1st library. The above process was repeated to obtain the 2nd library in turn. The titer of the obtained library was detected by ELISA, and the one with a relatively high titer was selected for monoclonal screening. Monoclonal colonies were picked from the library, and the supernatant after phage expression was used for Elisa detection.

[0068] 192 monoclonal colonies were picked for detection, and finally 23 specific sequences were screened out and named CN001-CN023.

[0069] Example 3: Verification of recombinant expression of nanobody targeting human serum albumin

[0070] The 23 specific sequences obtained in Example 2 were induced, expressed and purified. The expression vector was PET-28a and the expression strain was Escherichia coli BL21. A small amount of sample was purified by affinity chromatography. The affinity was measured using the instrument Biacore 1K with HSA. The dilution gradient of each sample was 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM. The affinity results are shown in Table 8;

[0071]

[0072] Affinity Kd The lower the value, that is, the nanobody can form a stable complex with HSA at a lower concentration, indicating a stronger binding ability.

[0073] Example 4: Construction of fusion protein of IL-1Ra and nanobody targeting human serum albumin of SEQ ID NO:2

[0074] IL-1Ra was fused with the nanobody targeting human serum albumin of SEQ ID NO:2 in Example 3 of the present invention, and the recombinant expression of the involved amino acid sequence was obtained.

[0075] The amino acid sequence of the fusion protein is shown in SEQ ID NO:25: MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDEGGGSGGGSGGGSQVQLVESGGGLVQAGGSLRLSCSASGLTYSTYIMGWFRQAPGKDREFVAAISWLSGNTYQPDSVKGRFTISRDNAKNTVYLRMNDLKPEDTAVYYCAAALGAGYYKAASQYDYWGQGTQVTVSS。

[0076] IL-1Ra was conjugated with the nanobody targeting human serum albumin of SEQ ID NO:2 through a linker peptide to construct a fusion protein. (The amino acid sequence encoding IL-1Ra is shown in SEQ ID NO:24, and the amino acid sequence of the nanobody targeting human serum albumin is shown in SEQ ID NO:02). The fusion protein sequence was synthesized and inserted into the expression vector pET-28a plasmid. The C-terminus of the synthesized target fragment was inserted with a His tag for subsequent purification. Escherichia coli BL21 was transformed, and induced expression was carried out at 20 °C with 1 mM IPTG. The medium was LB medium. Purification was performed using an AKTA purification system through an IMAC-Ni column. IPTG refers to isopropyl-β-D-thiogalactoside.

[0077] Detection of the activity and half-life of the fusion protein

[0078] 1 In vitro activity detection

[0079] The affinity of the fusion protein with HSA was detected using the instrument Biacore 1K to be 9.92e -10 M, the affinity with IL-1RI was 1.44e -9 M, and the affinity with IL-1RII was 7.9e -8 M, which was at the same level as the affinity of IL-1Ra (IL-1RI: 0.82e -9 M, IL-1RII: 4.8e -8 M). The activity of the IL-1Ra end of the fusion protein was detected using a reporter gene method based on D10G4.1 cells. The results showed that the activity of the fusion protein was approximately 25% of the activity of recombinant IL-1Ra.

[0080] 2 In vivo activity detection in rats

[0081] The anti-inflammatory activity of the fusion protein was evaluated using a rat plantar swelling model. After 60 rats were adaptively fed for one week, an acute inflammation model was established by subcutaneous injection of 0.3 mL of 3% paraformaldehyde solution into the right plantar surface of 50 rats. After modeling, the rats were housed individually in cages. 18 h later, they were randomly divided into 5 groups according to the degree of plantar swelling, namely the model control group, the recombinant IL-1Ra group, the high-dose fusion protein group, the medium-dose fusion protein group, and the low-dose fusion protein group. In addition, 10 rats that did not receive paraformaldehyde injection were used as the blank control group. Drugs were administered subcutaneously. The model group and the blank group were injected with normal saline. The dosing doses of the other four groups were as follows: recombinant IL-1Ra group: 18 mg / kg; high-dose fusion protein group: 30 mg / kg; medium-dose fusion protein group: 15 mg / kg; low-dose fusion protein group: 7.5 mg / kg. The dosing dose of the high-dose fusion protein group was the equimolar dose of the recombinant IL-1Ra group. The right foot volume of the rats was measured before drug administration and at 3, 6, 9, 12, and 24 h after drug administration, and the plantar swelling rate was calculated. The results are as Figure 5 shown, demonstrating that the fusion protein has good anti-inflammatory activity at equimolar doses.

[0082] Preliminary evaluation of the half-life

[0083] Two male rhesus monkeys were selected for the experiment. The dosing doses were as follows: 1 in the high-dose group: 3.2 mg / kg, and 1 in the low-dose group: 0.8 mg / kg. The dosing volume was 0.5 mL / kg. Blood was collected at 1 h, 2 h, 6 h, 10 h, 12 h, 24 h, 48 h, 72 h, 96 h, 120 h, 144 h, and 168 h after drug administration. The blood collection volume was 500 - 1000 μL each time. After standing at room temperature for 2 h, it was centrifuged at 4000 rpm for 10 min at 4 °C to separate the serum. An IL-1Ra detection kit was used to detect the concentration of the fusion protein in the serum. The drug-time curve is as Figure 6 shown, and the T 1 / 2 of the high-dose group was approximately 34 h, prolonging the half-life of IL-1Ra by 7 - 8 times.

[0084] Example 5: Construction of the fusion protein of IL-1Ra and the human serum albumin nanobody targeting SEQ ID NO:2

[0085] Different from Example 4, the amino acid sequence of the linker peptide in this example is as shown in SEQ ID NO:30: EAAAKEAAAKGGGS.

[0086] IL-1Ra was fused with the human serum albumin nanobody targeting SEQ ID NO:2 in Example 3 of the present invention, and the involved amino acid sequence was obtained by recombinant expression.

[0087] The amino acid sequence of the fusion protein is shown in SEQ ID NO:30: MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDEEAAAKEAAAKGGGSQVQLVESGGGLVQAGGSLRLSCSASGLTYSTYIMGWFRQAPGKDREFVAAISWLSGNTYQPDSVKGRFTISRDNAKNTVYLRMNDLKPEDTAVYYCAAALGAGYYKAASQYDYWGQGTQVTVSS。

[0088] IL-1Ra was conjugated with the human serum albumin nanobody targeting SEQ ID NO:2 through a linker peptide to construct a fusion protein. (The amino acid sequence encoding IL-1Ra is shown in SEQ ID NO:24, and the amino acid sequence of the human serum albumin nanobody targeting SEQ ID NO:02), the fusion protein sequence was synthesized and transferred into the expression vector PET-28a plasmid. The C-terminus of the synthesized target fragment was inserted with a His tag for subsequent purification, transformed into Escherichia coli BL21, and induced to express at 20 °C with 1 mM IPTG. The medium was LB medium. It was purified using an AKTA purification system through an IMAC-Ni column. IPTG refers to isopropyl-β-D-thiogalactoside.

[0089] Detection of the activity and half-life of the fusion protein

[0090] 1 In vitro activity detection

[0091] Using the instrument Biacore 1K to detect the affinity of the fusion protein with HSA was 7.94e -10 M, the affinity with IL-1RI was 1.18e -9 M, the affinity with IL-1RII was 5.93e -8 M, which was at the same level as the affinity of IL-1Ra (IL-1RI: 0.82e -9 M, IL-1RII: 4.8e -8 M), but was significantly better than the fusion protein in Example 4. Moreover, using the reporter gene method based on D10G4.1 cells to detect the activity of the IL-1Ra end of the fusion protein, the results showed that: the activity of the fusion protein was about 53% of the activity of recombinant IL-1Ra, and was also significantly better than the fusion protein in Example 4.

[0092] 2 In Vivo Activity Detection in Rats

[0093] The anti-inflammatory activity of the fusion protein was evaluated using a rat paw swelling model. After 60 rats were adaptively fed for one week, an acute inflammation model was established by subcutaneous injection of 0.3 mL of 3% paraformaldehyde solution into the right hind paw of 50 rats. After modeling, the rats were housed individually in cages. Eighteen hours later, they were randomly divided into 5 groups according to the degree of paw swelling, namely the model control group, the recombinant IL-1Ra group, the high-dose fusion protein group, the medium-dose fusion protein group, and the low-dose fusion protein group. In addition, 10 rats that were not injected with paraformaldehyde were used as the blank control group. Drugs were administered subcutaneously. The model group and the blank group were injected with normal saline. The drug doses for the other four groups were as follows: recombinant IL-1Ra group: 18 mg / kg; high-dose fusion protein group: 30 mg / kg; medium-dose fusion protein group: 15 mg / kg; low-dose fusion protein group: 7.5 mg / kg. The drug dose of the high-dose fusion protein group was the equimolar dose of the recombinant IL-1Ra group. The volume of the right hind paw of the rats was measured before drug administration and at 3, 6, 9, 12, and 24 hours after drug administration, and the paw swelling rate was calculated. The results are as Figure 7 shown, demonstrating that the fusion protein has good anti-inflammatory activity at equimolar doses.

[0094] The helical structure of EAAAK interacts with the surrounding environment through electrostatic interactions, stabilizes the surface charge distribution of the fusion protein, and promotes the synergistic effect between the HSA binding interface and the IL-1Ra receptor binding interface, thereby enhancing the affinity. GGGS provides moderate flexibility, avoids structural tension caused by excessive rigidity, and at the same time allows the two domains to adjust their relative positions dynamically, improving the binding efficiency with the targets (HSA, IL-1RI / II). In short, using EAAAKEAAAKGGGS as the amino acid sequence of the linker peptide can synergistically enhance the activity of the fusion protein.

[0095] Example 6: Construction of the Fusion Protein of IL-1Ra and the Nanobody Targeting Human Serum Albumin with SEQ ID NO:2

[0096] Different from Example 4, the amino acid sequence of the linker peptide in this example is as shown in SEQ ID NO:30: EAAAKEAAAKEAAAK.

[0097] IL-1Ra was fused with the nanobody targeting human serum albumin with SEQ ID NO:2 in Example 3 of the present invention, and the amino acid sequences involved were obtained by recombinant expression.

[0098] The amino acid sequence of the fusion protein is shown in SEQ ID NO: 31: MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDEEAAAKEAAAKEAAAKQVQLVESGGGLVQAGGSLRLSCSASGLTYSTYIMGWFRQAPGKDREFVAAISWLSGNTYQPDSVKGRFTISRDNAKNTVYLRMNDLKPEDTAVYYCAAALGAGYYKAASQYDYWGQGTQVTVSS。

[0099] IL-1Ra was conjugated with the human serum albumin nanobody targeting SEQ ID NO: 2 through a linker peptide to construct a fusion protein. (The amino acid sequence encoding IL-1Ra is shown in SEQ ID NO: 24, and the amino acid sequence of the human serum albumin nanobody targeting SEQ ID NO: 02), the fusion protein sequence was synthesized and transferred into the expression vector pET-28a plasmid. The C-terminus of the synthesized target fragment was inserted with a His tag for subsequent purification. Escherichia coli BL21 was transformed and induced to express at 20 °C with 1 mM IPTG. The medium was LB medium. Purification was carried out using an AKTA purification system through an IMAC-Ni column. IPTG refers to isopropyl-β-D-thiogalactoside.

[0100] Detection of the activity and half-life of the fusion protein

[0101] 1 In vitro activity detection

[0102] The affinity of the fusion protein for HSA was detected using the instrument Biacore 1K to be 1.13e -9 M, the affinity for IL-1RI was 1.76e- 9 M, and the affinity for IL-1RII was 8.50e -8 M, which was at the same level as the affinity of IL-1Ra (IL-1RI: 0.82e -9 M, IL-1RII: 4.8e -8 M). The activity of the IL-1Ra end of the fusion protein was detected using a reporter gene method based on D10G4.1 cells. The results showed that the activity of the fusion protein was approximately 21% of the activity of recombinant IL-1Ra.

[0103] 2 In vivo activity detection in rats

[0104] The anti-inflammatory activity of the fusion protein was evaluated using a rat plantar swelling model. After 60 rats were adaptively fed for one week, an acute inflammation model was established by subcutaneous injection of 0.3 mL of 3% paraformaldehyde solution into the right plantar of 50 rats. After modeling, the rats were housed individually in cages. After 18 h, they were randomly divided into 5 groups according to the degree of plantar swelling, namely the model control group, the recombinant IL-1Ra group, the high-dose fusion protein group, the medium-dose fusion protein group, and the low-dose fusion protein group. In addition, 10 rats that were not injected with paraformaldehyde were used as the blank control group. Drugs were administered subcutaneously. The model group and the blank group were injected with normal saline. The drug doses of the other four groups were as follows: recombinant IL-1Ra group: 18 mg / kg; high-dose fusion protein group: 30 mg / kg; medium-dose fusion protein group: 15 mg / kg; low-dose fusion protein group: 7.5 mg / kg. The drug dose of the high-dose fusion protein group was the equimolar dose of the recombinant IL-1Ra group. The right foot volume of the rats was measured before drug administration and at 3, 6, 9, 12, and 24 h after drug administration, and the plantar swelling rate was calculated. The results are as Figure 8 shown, demonstrating that the fusion protein has good anti-inflammatory activity at equimolar doses.

[0105] Thermal stability test

[0106] Tm (melting temperature), Tm represents the temperature at which the folded protein and the unfolded protein are equal, and it reflects to a certain extent the thermal stability of the fusion protein or its mutant, that is, the higher the Tm value, the better the thermal stability. The specific methods and parameters refer to the methods and parameters disclosed in CN118185902B. The parameters of the fluorescence quantitative PCR instrument were set as follows: the heating rate was set at 1 °C / 10 s, the temperature change range was from 25 °C to 99 °C, and the fluorescence signal was collected every 1 - 3 °C during the heating process. Tm was analyzed and calculated based on the collected signals;

[0107]

[0108] As can be seen from the above table, the thermal stability of the fusion protein in Example 5 is significantly better than that of the fusion proteins in Examples 4 and 6. The possible reasons are as follows: The α-helix structure of EAAAK enhances the local rigidity of the fusion protein through the formation of hydrogen bond networks and hydrophobic cores, resisting the unfolding caused by thermal perturbations. Moreover, glutamate (E) and lysine (K) in the EAAAK sequence are alternately arranged to form an amphiphilic helix, which may form stable interactions with the solvent (such as buffer) through charge complementarity, reducing the tendency of protein aggregation at high temperatures. Compared with the fusion protein in the example, (EAAAK)3 also has an α-helix structure, but the too-long rigid connection may introduce internal stress, leading to local conformational instability (such as helix packing defects), making its thermal stability slightly lower than that of (EAAAK)2-GGGS used in the fusion protein of Example 5.

[0109] The preferred specific embodiments of the present invention have been described in detail above. It should be understood that those of ordinary skill in the art can make many modifications and variations based on the concept of the present invention without creative efforts. Therefore, all technical solutions that can be obtained by those skilled in the art in the technical field according to the concept of the present invention through logical analysis, reasoning or limited experiments on the basis of the prior art should fall within the protection scope determined by the claims.

Claims

1. A nanobody targeting human serum albumin, characterized in that: The invention comprises a heavy chain variable region of a camel-derived antibody targeting HSA, which is composed of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4; The amino acid sequence of the human serum albumin-targeting nanobody is SEQ ID NO:

2.

2. Use of the human serum albumin-targeting nanobody as claimed in claim 1 in the preparation of a fusion protein.

3. The use according to claim 2, characterized in that: The fusion protein comprises a therapeutic protein and the human serum albumin-targeting nanobody according to claim 1; the therapeutic protein is an interleukin-1 receptor antagonist.

4. The use according to claim 3, characterized in that: The sequence of the interleukin-1 receptor antagonist is shown in SEQ ID NO:

24.

5. The use according to claim 3, characterized in that The method for preparing the fusion protein specifically comprises the following steps: constructing an expression vector by connecting the human serum albumin-targeting nanobody described in claim 1 with a therapeutic protein sequence through a short peptide, and performing fusion expression and purification in a prokaryotic expression system.

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