Targeted human serum albumin nano antibody and application thereof

Abstract

The invention discloses a targeted human serum albumin nano antibody and application thereof. The invention discloses a plurality of targeted human serum albumin nano antibodies capable of being used for constructing long-acting fusion protein drugs and an immune screening method thereof. The antibody is only composed of a heavy chain variable region and can be subjected to fusion expression with interleukin, interferon, tumor necrosis factors and other therapeutic proteins or polypeptide drugs in an escherichia coli expression system, the fusion protein has the activity of specific binding with human serum albumin, the affinity is high, the half-life period of the drugs can be effectively prolonged, and the anti-tumor effect is good. Meanwhile, the biological activity of the recombinant protein medicine is not influenced.

Description

Technical Field

[0001] The present invention relates to the technical field of monoclonal antibodies, and in particular to human serum albumin-targeted nano antibodies and applications thereof. Background Art

[0002] Monoclonal antibodies have occupied an important position in the biological products launched in recent years. Traditional antibodies are composed of two heavy chains and two light chains. At the end of the last century, scientists discovered that there was an antibody with a natural lack of light chains in the peripheral blood of alpacas. The antibody only contained one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. The VHH structure cloned and recombinantly expressed separately has the same structural stability and antigen binding activity as the original heavy chain antibody. It is the smallest unit known to bind to the target antigen. Since this type of heavy chain antibody is only about 2.5nm in size, it is also called a nano antibody. Nano antibodies are highly soluble, not easy to aggregate, and can withstand denaturing conditions such as high temperature, strong acid, and strong alkali. They are suitable for prokaryotic expression and various eukaryotic expression systems. They are widely used in the development of therapeutic antibody drugs, diagnostic reagents, affinity purification matrices, and scientific research. Based on the structural characteristics of nano antibodies, the development of nano antibodies targeting HSA to extend the half-life of drugs has become a research hotspot.

[0003] Protein / peptide 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 efficacy, and poor patient compliance. Therefore, it is urgent to carry out secondary transformation. Therefore, conducting long-acting research is of great significance to the development of such drugs. Long-acting strategies include constructing mutants, glycosylation modification, pegylation modification, and fusion with other proteins through genetic means, among which HSA fusion is a commonly used method.

[0004] HSA, human serum albumin, is a highly soluble and stable protein synthesized by liver cells and secreted into the blood. It has a molecular weight of 66.5kDa and a concentration of about 40mg / mL in human blood. The half-life of HSA is very long, about 19-21 days. The key mechanism for HSA's long half-life in the human body is the rescue 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 bring them back into the body's circulation. Due to the recognized serum stability and longevity of human serum albumin, it has become a highly anticipated long-acting drug carrier. At present, there are many strategies for the long-acting transformation of human serum albumin, mainly in vitro gene fusion and in vivo non-covalent binding. In recent years, the long-acting glucagon-like peptide-1 analog developed by Novo Nordisk, which has achieved great market response, uses fatty acid side chains to achieve non-covalent binding with human serum albumin in vivo to extend the half-life.

[0005] At present, the commonly used strategies for the long-acting of recombinant protein drugs include the construction of mutants, glycosylation modification, polyethylene glycol modification, fatty acid modification, Fc fusion protein construction, HSA fusion protein construction, etc., but these methods all have certain limitations. The construction of mutants requires a full analysis 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 currently a widely used technology, but the realization of protein-directed coupling of polyethylene glycol also has high technical requirements, and the long-acting effect of polyethylene glycol is positively correlated with the molecular weight. Long-term use of drugs can easily lead to the accumulation of polyethylene glycol in the body and produce toxicity; fatty acid modification is currently mostly used for the long-acting of small peptide drugs, which are generally prepared by solid phase synthesis. This method is more expensive when used for the preparation of larger cytokine and hormone drugs; Fc / HSA fusion protein construction leads to a large molecular weight of the drug, which has high requirements for the expression system and cannot use the prokaryotic expression system, and the 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 used to construct HSA binding bridges in vitro. However, scFv is prone to aggregation and precipitation due to the lack of a constant region skeleton, while albumin-binding peptides have a greater problem of immunogenicity because they are isolated from bacteria.

[0006] CN119032104A discloses an anti-serum albumin nanobody and its derivatives. A series of anti-serum albumin nanobodies are obtained through large-scale screening. The nanobody has high binding activity with serum albumin and has cross-reactivity with human, monkey and / or mouse serum albumin. The binding of the nanobody to serum albumin is pH-independent, and it can effectively participate in the FcRn-mediated serum albumin circulation, thereby prolonging the in vivo half-life of the nanobody. It also discloses coupling / fusion of the nanobody with a drug molecule (polypeptide drug), thereby prolonging the in vivo half-life of the drug molecule. Summary of the invention

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

[0008] The present invention discloses a variety of human serum albumin-targeted nanoantibodies and immune screening methods thereof that can be used to construct long-acting fusion protein drugs. The antibodies are composed only of heavy chain variable regions and can be fused and expressed with therapeutic proteins or polypeptide drugs such as interleukins, interferons, and tumor necrosis factors in an Escherichia coli expression system. The fusion proteins have the activity of specifically binding to human serum albumin and have high affinity, which can effectively prolong the half-life of the drug without affecting the biological activity of the recombinant protein drug.

[0009] The technical route of the present invention is as follows: human serum albumin is used to immunize alpacas at multiple points for multiple times, a small amount of serum is collected during the immunization process for ELISA detection, a large amount of blood is collected after the titer is qualified to separate peripheral blood lymphocytes, total RNA is extracted, and cDNA is obtained by reverse transcription, phagemids are constructed after primer amplification, the phagemids are transferred into phages to construct a phage library, and multiple rounds of panning are performed to screen and obtain 23 monoclonal antibody sequences that specifically bind to HSA; an Escherichia coli expression system is used to induce expression of 23 nano antibodies, the nano antibodies are purified to perform in vitro affinity detection, and fusion proteins are constructed with therapeutic protein drugs, and Escherichia coli is still used to express the fusion proteins, and the activity and half-life of the fusion proteins are detected after purification.

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

[0011] Preferably, the amino acid sequence of the human serum albumin targeting Nanobody has at least 70% sequence identity with any one of SEQ ID NOs: 1-23; more preferably, it has at least 80% sequence identity with any one of SEQ ID NOs: 1-23; further preferably, it has at least 90% sequence identity with any one of SEQ ID NOs: 1-23; further preferably, it has at least 95% sequence identity with any one of SEQ ID NOs: 1-23; most preferably, it is as shown in any one of SEQ ID NOs: 1-23.

[0012] The invention also discloses a fusion protein comprising a therapeutic protein and the above-mentioned human serum albumin-targeted nano antibody.

[0013] The therapeutic proteins include but are not limited to polypeptides, antibodies, antibody fragments, cytokines, tumor marker molecules, etc.

[0014] Preferably, the therapeutic protein is interleukin-1 receptor antagonist (IL-1Ra) or its analogs and derivatives. The sequence of IL-1Ra is shown in SEQ ID NO: 24: MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGVMVTKFYFQEDE.

[0015] 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 portion of such drugs.

[0016] The antibody fragments mentioned in the therapeutic protein of the present invention include but are not limited to single-chain antibodies, Fab domains and heavy-chain antibodies and their multivalent forms.

[0017] As a further preferred embodiment, the fusion protein of the present invention comprises a connecting short peptide between the human serum albumin targeting nanobody and the therapeutic protein, and the therapeutic protein is connected to the N-terminus, C-terminus, or both of the N-terminus and C-terminus of the nanobody through the connecting short peptide.

[0018] The connecting short peptide has any of the following structures: (GGGS)n, wherein n is any value from 2 to 4; or (EAAAK)n-(GGGS)n, wherein n is any value from 1 to 2; or (EAAAK)n, wherein n is any value from 1 to 4.

[0019] The present invention also discloses a method for screening the above-mentioned human serum albumin-targeting nanoantibody and a method for preparing the fusion protein, which specifically comprises the following steps: (1) Immunizing alpacas with HSA; (2) Isolate alpaca peripheral blood mononuclear cells (PBMCs), extract total RNA, and reverse transcribe to synthesize cDNA; (3) Use primers to perform two rounds of PCR amplification of the target fragment, construct a phagemid, transfer it into phage to construct a phage library, and select positive clones in multiple rounds. The primers are as follows: First round primers F:GTCCTGGCGGCTCATCTCCAAGA (SEQ ID NO:26) R:GGTACGTTCTGATGAAGTGTAGC (SEQ ID NO:27) Second round primers F:CCTGCTGCTGGGCCCAAGCGGCCCAGGTGCATCTGCAGGAGTCTGCRCG (SEQ ID NO: 28) R: GAGGAGGAGGGCCGACGGGGCCTGGTCGGTCCTCGCTGAGTTACG (SEQ ID NO: 29) (4) constructing expression vectors for positive clones, inducing expression and purification using a prokaryotic expression system, and measuring affinity with HSA; thus, the screening of nanoantibodies targeting human serum albumin is completed; (5) The positive clone sequence and the therapeutic protein sequence are connected through a short peptide to construct an expression vector, which is then fused and expressed and purified in a prokaryotic expression system.

[0020] The fusion protein of the present invention has both the ability to bind to HSA and the therapeutic activity of the fused therapeutic protein, and its therapeutic activity is significantly improved.

[0021] Preferably, the present invention constructs a fusion protein of a human serum albumin-targeted nanobody and IL-1Ra or its analogs and derivatives, which can exert rapid and lasting anti-inflammatory activity. There are three main subtypes of IL-1Ra in the human body. The three subtypes of IL-1Ra have the biological activity of antagonizing IL-1 when in excess in the body, and can competitively bind to the IL-1 receptor on the membrane 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., so it has attracted much attention in the treatment of inflammatory diseases.

[0022] Beneficial effects of the present invention: The human serum albumin-targeting nanoantibody involved in the present invention has a completely new amino acid sequence and high specificity for HSA. Compared with traditional monoclonal antibodies (fragments), it has the advantages of stable structure, strong solubility, low molecular weight, etc., and its lack of Fc segment reduces immunogenicity. At the same time, the human serum albumin-targeting nanoantibody does not require post-translational modification and can be expressed by a prokaryotic expression system, with low production cost.

[0023] By constructing a fusion protein with human serum albumin-targeted nanoantibodies and therapeutic proteins, the goal of extending the half-life of the drug while retaining its therapeutic activity is achieved, thereby improving the bioavailability of the drug. Compared with existing half-life extension technologies, a lower molecular weight is maintained while eliminating the need for complex technical processes such as structural modification, PEG modification, microsphere release, fatty acid modification, and introduction of non-natural amino acids for therapeutic proteins. The resulting fusion protein can be expressed by a prokaryotic system, which is a simple technology and greatly reduces production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is the technical roadmap of the nano-antibody targeting human serum albumin of the present invention.

[0025] Figure 2 is an electrophoresis diagram of total RNA, which was extracted from PBMCs obtained in Example 1.

[0026] Figure 3 This is the electrophoresis diagram of the first round of PCR amplification of the antibody gene.

[0027] Figure 4 This is the electrophoresis diagram of the second round of PCR amplification of the antibody gene sequence.

[0028] Figure 5 The anti-inflammatory activity of the fusion protein constructed in Example 4 in a rat model is the paw swelling rate.

[0029] Figure 6 This is the drug curve of the fusion protein constructed in Example 4 in the rhesus monkey model.

[0030] Figure 7 The anti-inflammatory activity of the fusion protein constructed in Example 5 in a rat model is the plantar swelling rate.

[0031] Figure 8 The anti-inflammatory activity of the fusion protein constructed in Example 6 in a rat model is the paw swelling rate. DETAILED DESCRIPTION

[0032] The following examples are only for further specific description of the present invention, and the application scope of the present invention is not limited to the examples. The reagents, instruments, experimental methods, etc. involved in the examples are conventional reagents, instruments, methods, etc. unless otherwise specified.

[0033] Example 1: Alpaca immunization and serum titer determination A total of 4 immunizations were performed on an alpaca over 1 year old. Freund's complete adjuvant (CFA) and Freund's incomplete adjuvant (IFA) were used as immune adjuvants, and the administration route was through multiple subcutaneous injections. 2mL of negative serum was taken before immunization, and ear studs were marked for use. After 3-5 days of observation, immunization was started, and the immunization dose was 1mg / time / head. The immunization and blood collection process is shown in Table 2. The serum titer ELISA test results are shown in Table 3. Peripheral blood mononuclear cells (PBMC) were isolated after the titer was qualified;

[0034] Example 2: Phage display library establishment and single clone screening 1. Acquisition of target genes Total RNA was extracted from the PBMC obtained in Example 1, and the total RNA electrophoresis results were as follows: Figure 2 Total RNA was reverse transcribed into cDNA using a reverse transcription kit.

[0035] In the first round of PCR, cDNA was used as template. The amplification system (20 µL) of each primer pair was shown in Table 4. The amplification procedure was shown in Table 5. The electrophoresis results of the amplification were shown in Table 5. Figure 3 shown.

[0036] First round primers F:GTCCTGGCGGCTCATCTCCAAGA (SEQ ID NO:26) R:GGTACGTTCTGATGAAGTGTAGC (SEQ ID NO:27)

[0037] The second round of PCR amplification of the antibody gene sequence used the 700 bp target fragment of the first round of PCR product as a template. The amplification system (20 µL) of each primer pair was shown in Table 6, the amplification program was shown in Table 7, and the amplification results were electrophoretically analyzed as shown in Table 7. Figure 4shown.

[0038] Second round primers F:CCTGCTGCTGGGCCCAAGCGGCCCAGGTGCATCTGCAGGAGTCTGCRCG (SEQ ID NO: 28) R: GAGGAGGAGGGCCGACGGGGCCTGGTCGGTCCTCGCTGAGTTACG (SEQ ID NO: 29)

[0039] 2 Phage library construction The second round amplified fragments were recovered using a gel recovery kit, and the recovered fragments and pcomb3X vector were digested with sfiI enzyme respectively. After digestion, the fragments were directly purified using a gel recovery purification column, and the pcomb3X vector was recovered as a linear vector using a gel recovery kit; the target fragment was mixed with the pcomb3X vector and connected using T4 ligase at 4°C overnight, and then the ligation product was electroporated into the XL1-Blue competent medium to obtain the antibody bacterial liquid library; VCSM13 helper phage was added to the library bacterial liquid and allowed to infect for half an hour for further expression and amplification, and the phage precipitate was collected to obtain the phage library.

[0040] 48 single clones were randomly selected from the library plate for sequencing, 44 clones were correct, 2 had no signal sequence, and 2 had stop codon sequence, with a sequence accuracy of 91.7%. The concentration of phage after library expansion was 2.64×10 12 PFU / mL.

[0041] 3 Monoclonal screening High affinity ELISA plates were coated with HSA overnight, and then blocked after washing. 5×10 12 PFU phage, after incubation and elution, adjust the pH to 7.4; mix the eluate with E. coli XL1-Blue, rescue the phage and shake and culture overnight at 30°C to obtain the amplified antibody 1st library; repeat the above process to obtain the 2nd library; use ELISA to detect the titer of the obtained library, and select the one with higher titer for monoclonal screening. Select monoclones from the library, and the supernatant after phage expression is used for Elisa detection.

[0042] 192 monoclonal clones were selected for testing, and 23 specific sequences were finally screened and named CN001-CN023.

[0043] Example 3: Verification of recombinant expression of human serum albumin-targeted nanobodies The 23 specific sequences obtained in Example 2 were induced for expression and purification, the expression vector was PET-28a, the expression strain was Escherichia coli BL21, and a small amount of samples were obtained by affinity chromatography purification. The affinity was determined using the instrument Biacore 1K and HSA. The dilution gradient of each sample was 1000nM, 500nM, 250nM, 125nM, 62.5nM, 31.25nM. The affinity results are shown in Table 8;

[0044] Affinity Kd The lower the value, the lower the concentration at which the nanoantibody can form a stable complex with HSA, reflecting a stronger binding ability.

[0045] Example 4: Construction of IL-1Ra and SEQ ID NO: 2 Targeting Human Serum Albumin Nanobody Fusion Protein IL-1Ra was fused with the human serum albumin-targeting nanobody of SEQ ID NO: 2 in Example 3 of the present invention, and the amino acid sequences involved were obtained by recombinant expression.

[0046] The amino acid sequence of the fusion protein is SEQ ID NO:25 shows: MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEG VMVTKFYFQEDEGGGSGGGSGGGSQVQLVESGGGLVQAGGSLRLSCSASGLTYSTYIMGWFRQAPGKDREFVAAISWLSGNTYQPDSVKGRFTISRDNAKNTVYLRMNDLKPEDTAVYYCAAALGAGYYKAASQYDYWGQGTQVTVSS.

[0047] IL-1Ra and SEQ ID NO:2 human serum albumin-targeting nanobody were coupled by connecting short peptides to construct a fusion protein. (The amino acid sequence encoding IL-1Ra is SEQ ID NO:24, and the amino acid sequence of the human serum albumin-targeting nanobody is SEQ ID NO:02). The fusion protein sequence was synthesized and transferred into the expression vector PET-28a plasmid. The His tag was inserted into the C-terminus of the synthesized target fragment for subsequent purification. Escherichia coli BL21 was transformed and induced to express with 1mMIPTG at 20°C. The culture medium was LB culture medium. Purification was performed using an IMAC-Ni column using an AKTA purification system. IPTG refers to isopropyl-β-D-thiogalactoside.

[0048] Fusion protein activity and half-life detection 1 In vitro activity assay The affinity of the fusion protein to HSA was detected by Biacore 1K and was 9.92e -10 M, with an affinity of 1.44e for IL-1RI -9 M, with an affinity of 7.9e for IL-1RII -8 M, which is consistent with the affinity of IL-1Ra (IL-1RI: 0.82e -9 M, IL-1RII: 4.8e -8 The activity of the fusion protein IL-1Ra was detected using a reporter gene assay based on D10G4.1 cells. The results showed that the activity of the fusion protein was approximately 25% of the activity of the recombinant IL-1Ra.

[0049] 2 In vivo activity assay in rats The anti-inflammatory activity of the fusion protein was evaluated using a rat paw swelling model. After one week of adaptive feeding, 50 rats were subcutaneously injected with 0.3 mL of 3% paraformaldehyde solution on the right paw to construct an acute inflammation model. After modeling, the rats were housed in single cages and randomly divided into five groups according to the degree of paw swelling 18 hours later, namely, a model control group, a recombinant IL-1Ra group, a high-dose fusion protein group, a medium-dose fusion protein group, and a low-dose fusion protein group. In addition, 10 rats that were not injected with paraformaldehyde served as a blank control group. The rats were injected subcutaneously. The model group and the blank group were injected with normal saline. The other four groups were injected with the following doses: recombinant IL-1Ra group: 18 mg / kg; fusion protein high-dose group: 30 mg / kg; fusion protein medium-dose group: 15 mg / kg; fusion protein low-dose group: 7.5 mg / kg. The dose of the fusion protein high-dose group was the same molar dose as the recombinant IL-1Ra group. The right foot volume of the rats was measured before administration and 3, 6, 9, 12, and 24 hours after administration, and the plantar swelling rate was calculated. The results are shown in Figure 5 As shown, it was demonstrated that the fusion protein had good anti-inflammatory activity at an equimolar dose.

[0050] 3 Preliminary evaluation of half-life Two male rhesus monkeys were selected for the experiment. The dosage was 3.2 mg / kg for one monkey in the high-dose group and 0.8 mg / kg for one monkey in the low-dose group. The dosage volume was 0.5 mL / kg. Blood was collected at 1h, 2h, 6h, 10h, 12h, 24h, 48h, 72h, 96h, 120h, 144h, and 168h after administration. The blood collection volume was 500-1000µL / time. After being placed at room temperature for 2h, the serum was separated by centrifugation at 4000rpm for 10min at 4℃. The fusion protein concentration in the serum was detected using the IL-1Ra detection kit. The drug-time curve is shown in the figure. Figure 6 As shown, the high-dose group T 1 / 2 About 34h, extending the half-life of IL-1Ra by 7-8 times.

[0051] Example 5: Construction of IL-1Ra and SEQ ID NO: 2 Targeting Human Serum Albumin Nanobody Fusion Protein Different from Example 4, the amino acid sequence of the connecting short peptide in this example is shown in SEQ ID NO: 30: EAAAKEAAAKGGGS.

[0052] IL-1Ra was fused with the human serum albumin-targeting nanobody of SEQ ID NO: 2 in Example 3 of the present invention, and the amino acid sequence involved was obtained by recombinant expression.

[0053] The amino acid sequence of the fusion protein is SEQ ID NO:30 shows: MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGV MVTKFYFQEDEEAAAKEAAAKGGGSQVQLVESGGGLVQAGGSLRLSCSASGLTYSTYIMGWFRQAPGKDREFVAAISWLSGNTYQPDSVKGRFTISRDNAKNTVYLRMNDLKPEDTAVYYCAAALGAGYYKAASQYDYWGQGTQVTVSS.

[0054] IL-1Ra and SEQ ID NO:2 human serum albumin-targeting nanobody were coupled by connecting short peptides to construct a fusion protein. (The amino acid sequence encoding IL-1Ra is SEQ ID NO:24, and the amino acid sequence of the human serum albumin-targeting nanobody is SEQ ID NO:02). The fusion protein sequence was synthesized and transferred into the expression vector PET-28a plasmid. The His tag was inserted into the C-terminus of the synthesized target fragment for subsequent purification. Escherichia coli BL21 was transformed and induced to express with 1mMIPTG at 20°C. The culture medium was LB culture medium. Purification was performed using an IMAC-Ni column using an AKTA purification system. IPTG refers to isopropyl-β-D-thiogalactoside.

[0055] Fusion protein activity and half-life detection 1 In vitro activity assay The affinity of the fusion protein to HSA was detected by Biacore 1K and was 7.94e -10 M, with an affinity of 1.18e for IL-1RI -9 M, with an affinity of 5.93e for IL-1RII -8 M, which is consistent with the affinity of IL-1Ra (IL-1RI: 0.82e -9 M, IL-1RII: 4.8e -8 M) is at the same level, but significantly better than the fusion protein of Example 4. In addition, 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 about 53% of the activity of the recombinant IL-1Ra, which was also significantly better than the fusion protein of Example 4.

[0056] 2 In vivo activity assay in rats The anti-inflammatory activity of the fusion protein was evaluated using a rat paw swelling model. After one week of adaptive feeding, 50 rats were subcutaneously injected with 0.3 mL of 3% paraformaldehyde solution on the right paw to construct an acute inflammation model. After modeling, the rats were housed in single cages and randomly divided into five groups according to the degree of paw swelling 18 hours later, namely, a model control group, a recombinant IL-1Ra group, a high-dose fusion protein group, a medium-dose fusion protein group, and a low-dose fusion protein group. In addition, 10 rats that were not injected with paraformaldehyde served as a blank control group. The rats were injected subcutaneously. The model group and the blank group were injected with normal saline. The other four groups were injected with the following doses: recombinant IL-1Ra group: 18 mg / kg; fusion protein high-dose group: 30 mg / kg; fusion protein medium-dose group: 15 mg / kg; fusion protein low-dose group: 7.5 mg / kg. The dose of the fusion protein high-dose group was the same molar dose as the recombinant IL-1Ra group. The right foot volume of the rats was measured before administration and 3, 6, 9, 12, and 24 hours after administration, and the plantar swelling rate was calculated. The results are shown in Figure 7 As shown, it was demonstrated that the fusion protein had good anti-inflammatory activity at an equimolar dose.

[0057] The helical structure of EAAAK interacts with the surrounding environment through electrostatic interaction, stabilizes the surface charge distribution of the fusion protein, promotes the synergistic effect between the HSA binding interface and the IL-1Ra receptor binding interface, and thus enhances affinity. GGGS provides moderate flexibility to avoid structural tension caused by excessive rigidity, while allowing the two domains to dynamically adjust their relative positions, thereby improving the binding efficiency with the target (HSA, IL-1RI / II). In short, using EAAAKAAAKGGGS as the amino acid sequence of the connecting short peptide can synergistically enhance the activity of the fusion protein.

[0058] Example 6: Construction of IL-1Ra and SEQ ID NO: 2 Targeting Human Serum Albumin Nanobody Fusion Protein Different from Example 4, the amino acid sequence of the connecting short peptide in this example is shown in SEQ ID NO: 30: EAAAKEAAAKEAAAK.

[0059] IL-1Ra was fused with the human serum albumin-targeting nanobody of SEQ ID NO: 2 in Example 3 of the present invention, and the amino acid sequence involved was obtained by recombinant expression.

[0060] The amino acid sequence of the fusion protein is SEQ ID NO:31 shows: MRPSGRKSSKMQAFRIWDVNQKTFYLRNNQLVAGYLQGPNVNLEEKIDVVPIEPHALFLGIHGGKMCLSCVKSGDETRLQLEAVNITDLSENRKQDKRFAFIRSDSGPTTSFESAACPGWFLCTAMEADQPVSLTNMPDEGV MVTKFYFQEDEEAAAKEAAAKEAAAKQVQLVESGGGLVQAGGSLRLSCSASGLTYSTYIMGWFRQAPGKDREFVAAISWLSGNTYQPDSVKGRFTISRDNAKNTVYLRMNDLKPEDTAVYYCAAALGAGYYKAASQYDYWGQGTQVTVSS.

[0061] IL-1Ra and SEQ ID NO:2 human serum albumin-targeting nanobody were coupled by connecting short peptides to construct a fusion protein. (The amino acid sequence encoding IL-1Ra is SEQ ID NO:24, and the amino acid sequence of the human serum albumin-targeting nanobody is SEQ ID NO:02). The fusion protein sequence was synthesized and transferred into the expression vector PET-28a plasmid. The His tag was inserted into the C-terminus of the synthesized target fragment for subsequent purification. Escherichia coli BL21 was transformed and induced to express with 1mMIPTG at 20°C. The culture medium was LB culture medium. Purification was performed using an IMAC-Ni column using an AKTA purification system. IPTG refers to isopropyl-β-D-thiogalactoside.

[0062] Fusion protein activity and half-life detection 1 In vitro activity assay The affinity of the fusion protein to HSA was detected by Biacore 1K and was 1.13e -9 M, with an affinity of 1.76e- 9 M, with an affinity of 8.50e for IL-1RII -8 M, which is consistent with the affinity of IL-1Ra (IL-1RI: 0.82e -9 M, IL-1RII: 4.8e -8 The activity of the fusion protein IL-1Ra was detected using a reporter gene assay based on D10G4.1 cells. The results showed that the activity of the fusion protein was approximately 21% of the activity of the recombinant IL-1Ra.

[0063] 2 In vivo activity assay in rats The anti-inflammatory activity of the fusion protein was evaluated using a rat paw swelling model. After one week of adaptive feeding, 50 rats were subcutaneously injected with 0.3 mL of 3% paraformaldehyde solution on the right paw to construct an acute inflammation model. After modeling, the rats were housed in single cages and randomly divided into five groups according to the degree of paw swelling 18 hours later, namely, a model control group, a recombinant IL-1Ra group, a high-dose fusion protein group, a medium-dose fusion protein group, and a low-dose fusion protein group. In addition, 10 rats that were not injected with paraformaldehyde served as a blank control group. The rats were injected subcutaneously. The model group and the blank group were injected with normal saline. The other four groups were injected with the following doses: recombinant IL-1Ra group: 18 mg / kg; fusion protein high-dose group: 30 mg / kg; fusion protein medium-dose group: 15 mg / kg; fusion protein low-dose group: 7.5 mg / kg. The dose of the fusion protein high-dose group was the same molar dose as the recombinant IL-1Ra group. The right foot volume of the rats was measured before administration and 3, 6, 9, 12, and 24 hours after administration, and the plantar swelling rate was calculated. The results are shown in Figure 8As shown, it was demonstrated that the fusion protein had good anti-inflammatory activity at an equimolar dose.

[0064] Thermal stability test Tm (melting temperature), Tm represents the temperature at which the folded protein is equal to the unfolded protein. It reflects the thermal stability of the fusion protein or its mutant to a certain extent, that is, the higher the Tm value, the better the thermal stability. For specific methods and parameters, refer to the methods and parameters disclosed in CN118185902B. The parameters of the fluorescence quantitative PCR instrument are set as follows: the heating rate is set to: 1℃ / 10s, the temperature change range is: 25℃ to 99℃, the fluorescence signal is collected every 1-3℃ during the heating process, and Tm is calculated based on the collected signal analysis;

[0065] As can be seen from the above table, the thermal stability of the fusion protein of Example 5 is significantly better than that of the fusion proteins of Examples 4 and 6. The possible reason is that the α-helical structure of EAAAK enhances the local rigidity of the fusion protein through the formation of a hydrogen bond network and a hydrophobic core, resisting the unfolding caused by thermal perturbations. Furthermore, glutamic acid (E) and lysine (K) in the EAAAK sequence are arranged alternately to form an amphipathic helix, which may form a stable interaction with the solvent (such as a buffer) through charge complementarity, reducing the tendency of protein aggregation at high temperatures. Compared with the fusion protein of the embodiment, (EAAAK)3 also has an α-helical structure, but the excessively long rigid connection may introduce internal stress, resulting in local conformational instability (such as helical stacking defects), making its thermal stability slightly lower than that of (EAAAK)2-GGGS used in the fusion protein of Example 5.

[0066] The preferred specific embodiments of the present invention are described in detail above. It should be understood that a person skilled in the art can make many modifications and changes based on the concept of the present invention without creative work. Therefore, any technical solution that can be obtained by a person skilled in the art through logical analysis, reasoning or limited experiments based on the concept of the present invention on the basis of the prior art should be within the scope of protection 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 has at least 70% sequence identity with any one of SEQ ID NOs: 1-23.

2. The human serum albumin-targeting nanobody according to claim 1, characterized in that: The amino acid sequence of the human serum albumin targeting nanobody has at least 80% sequence identity with any one of SEQ ID NOs: 1-23.

3. The human serum albumin-targeting nanobody according to claim 1, characterized in that: The amino acid sequence of the human serum albumin targeting nanobody has at least 90% sequence identity with any one of SEQ ID NOs: 1-23.

4. The human serum albumin-targeting nanobody according to claim 1, characterized in that: The amino acid sequence of the human serum albumin-targeting nanobody is any one of SEQ ID NOs: 1-23.

5. Use of the human serum albumin targeting nanobody as described in any one of claims 1 to 4 in a fusion protein.

6. The use according to claim 5, characterized in that: The fusion protein comprises a therapeutic protein and the human serum albumin-targeting nanobody according to any one of claims 1 to 4.

7. The use according to claim 6, characterized in that: The therapeutic protein includes any one of a polypeptide, an antibody, an antibody fragment, a cytokine, and a tumor marker molecule.

8. The use according to claim 6, characterized in that: The therapeutic protein is an interleukin-1-receptor antagonist.

9. The use according to claim 8, characterized in that: The sequence of the interleukin-1 receptor antagonist is shown in SEQ ID NO:

24.

10. The use according to claim 6, characterized in that The method for preparing the fusion protein specifically comprises the following steps: (1) Immunizing alpacas with HSA; (2) Isolate alpaca peripheral blood mononuclear cells, extract total RNA, and reverse transcribe to synthesize cDNA; (3) using primers to perform two rounds of PCR amplification of the target fragment, constructing phagemids, transferring them into phages to construct a phage library, and performing multiple rounds of panning for positive clones; The sequences of the primers are: First round primers The forward primer sequence is shown in SEQ ID NO: 26 The reverse primer sequence is shown in SEQ ID NO: 27 Second round primers The forward primer sequence is shown in SEQ ID NO: 28 The reverse primer sequence is shown in SEQ ID NO: 29 (4) constructing expression vectors for positive clones, inducing expression and purification using a prokaryotic expression system, and determining affinity with HSA; thus, the screening of nanoantibodies targeting human serum albumin is completed; (5) The positive clone sequence and the therapeutic protein sequence are connected by short peptides to construct an expression vector, which is then fused and expressed and purified in a prokaryotic expression system.

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