Anti-human complement C5 single-domain antibody and application thereof

Abstract

The invention belongs to the field of biological medicine, and discloses an anti-human complement C5 single-domain antibody and application thereof. CDR1-CDR3 of the single-domain antibody are respectively shown as SEQ ID NO.3 to SEQ ID NO.5 or SEQ ID NO.6 to SEQ ID NO.5. The single-domain antibody of some examples has high specificity on human complement C5 adsorption, and provides a new direction for treating diseases caused by complement C5 abnormity.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a single-domain antibody against human complement C5 and an application thereof. Background Art

[0002] The complement (C) system is an important component of the innate and adaptive immune systems. It is essential for its role as a host defense and surveillance system and is mainly composed of plasma proteins produced by the liver or membrane proteins expressed on the cell surface. Complement contains about 30 components and is widely present in serum, tissue fluid and cell membrane surfaces. Generally, most complement components in plasma have biological functions only after being activated. Complement activation can quickly protect the body from infectious attacks and is traditionally considered to be a core part of the host's defense against invading pathogens and the removal of potentially destructive cell debris. However, it is worth noting that it can also be a driving factor or exacerbating factor in many inflammatory or autoimmune diseases. In fact, excessive or dysregulated complement activation is associated with the pathogenesis of a variety of diseases, including eye lesions, acute inflammation (sepsis and ischemic stroke, etc.) and chronic diseases (including autoimmune diseases, neurodegenerative diseases, kidney diseases and chronic hemolytic diseases with thrombotic inflammatory characteristics, etc.).

[0003] Complement can produce a series of cascade enzymatic activation of serine proteases through three independent and cross-talking pathways, including the classical pathway, the alternative pathway, and the lectin pathway. Although these activation products are not necessarily the initiating factors of inflammatory disorders, they seem to be the cause of promoting and continuing inflammatory responses, especially complement C5, which is at the end of the cascade reaction. Normal human serum contains about 38~90mg / L. This protein is composed of α and β polypeptide chains connected by disulfide bonds, with a molecular weight of 190kDa, of which the α chain is 115kDa and the β chain is 75kDa. It is found to be an inactive protein in serum, but can be cleaved into two active peptides (C5a and C5b) by one of two different C5 convertases (C4bC2aC3b or C3bBbC3b). Among them, C5a is one of the most effective inflammatory peptides among complement activation products and has powerful functions. C5a mainly acts through two identified C5a receptors, C5aR and C5L2. C5aR belongs to the G protein-coupled receptor of the rhodopsin family and has seven transmembrane segments; while C5L2 is structurally homologous but lacks G protein coupling. The production of C5a is associated with diseases including viral infection, asthma, atherosclerosis, autoimmune arthritis and sepsis. Studies have found that the level of C5a in the serum of patients with inflammatory diseases is elevated, and its excessive complement activation may affect the health of hundreds of millions of people.

[0004] The cleavage of C3 and C5 will lead to the production of anaphylatoxins and allow the formation of membrane attack complexes, which are the main driving factors of the disease. Targeting C3 and C5 is one of the earliest treatment methods considered. Moreover, C5 is at the end of the cascade reaction, and targeting it can regulate the complement signals activated by all three different pathways, so C5 has become a hot target for the development of complement drugs. At present, most of the therapeutic drugs acting on C5 are anti-C5 inhibitors injected, which are mainly treated by blocking the activity of C5. For example, CN105324485A discloses the use of iRNA such as double-stranded ribonucleic acid (dsRNA), a composition targeting the complement component C5 gene, and the use of such iRNA (such as dsRNA) and a composition to inhibit the expression of C5 and treat a disease associated with a complement component C5. CN101970002A discloses administering an effective amount of anti-factor Bb antibodies to a subject to selectively inhibit the formation of complement alternative activation products C3a, C5a and C5b-9, and inhibit the activation of neutrophils, monocytes and platelets.

[0005] Immunoadsorption therapy is a new technology that has emerged in recent years. It combines highly specific antigens or antibodies with adsorption materials (carriers) to make adsorbents (columns). It uses its specific adsorption properties to selectively or relatively specifically remove endogenous pathogenic factors in the patient's blood, thereby achieving the purpose of alleviating the disease.

[0006] Compared with traditional antibodies, single-domain antibodies are a form of antibody composed of a single variable domain of an antibody, which is a heavy chain antibody (VHH region) with only one domain with a disulfide bond, folded into a relatively stable structure. Studies have shown that single-domain antibodies still maintain antigen binding ability after incubation at 37°C for one week, allowing them to use more stringent chemical and physical conditions during treatment or modification. In addition, single-domain antibodies are small in size, which is conducive to reducing the number of potential immunogenic epitopes. They also have a rapid blood clearance rate and the ability to resist the formation of aggregates, causing only low immunogenicity. Single-domain antibodies have shown great potential as tools in different biotechnology fields such as diagnosis and treatment. Summary of the invention

[0007] The object of the present invention is to overcome at least one deficiency of the prior art and to provide a single domain antibody against human complement C5 and its application.

[0008] The technical solution adopted by the present invention is: The first aspect of the present invention provides: a single domain antibody against human complement C5 protein, which consists of framework regions FR1 to FR4 and antigen binding regions CDR1 to CDR3, wherein the amino acid sequences of CDR1 to CDR3 are selected from the following group: Group 1, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.3 to SEQ ID NO.5, respectively; Group 2, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.6 to SEQ ID NO.8, respectively.

[0009] In some examples of single domain antibodies, the amino acid sequence of FR1 to FR4 is selected from the following group: Group 1, the amino acid sequences of FR1 to FR4 are shown in SEQ ID NO.9 to SEQ ID NO.12, respectively; Group 2, the amino acid sequences of FR1 to FR4 are shown in SEQ ID NO.13, SEQ ID NO.10, SEQ ID NO.14 and SEQ ID NO.12, respectively.

[0010] In some examples of single domain antibodies, they specifically bind complement C5.

[0011] In some examples of single domain antibodies, they are chimeric antibodies, humanized antibodies, single domain antibodies fused to an Fc fragment, bivalent or multivalent single domain antibodies.

[0012] The second aspect of the present invention provides: a nucleic acid molecule encoding the single domain antibody described in the first aspect of the present invention.

[0013] The third aspect of the present invention provides: an expression vector comprising the nucleic acid molecule described in the second aspect of the present invention.

[0014] The fourth aspect of the present invention provides: a host cell, which expresses the single domain antibody described in the first aspect of the present invention, or includes the nucleic acid molecule described in the second aspect of the present invention, or contains the expression vector described in the third aspect of the present invention.

[0015] The fifth aspect of the present invention provides: the use of the single domain antibody described in the first aspect of the present invention, the use comprising: preparing an adsorbent for complement C5; preparing a detection reagent for complement C5; Preparation of drugs for treating and / or preventing diseases caused by abnormal complement C5.

[0016] In some application examples, the adsorbent is obtained by coupling the single domain antibody described in the first aspect of the present invention to a solid phase carrier.

[0017] In some examples of application, the diseases caused by abnormal complement C5 include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration (AMD); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent abortions; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis, bullous pemphigoid, Shiga toxin-producing Escherichia coli-associated hemolytic-uremic syndrome, C3 nephropathy, antineutrophil cytoplasmic antibody-associated vasculitis, humoral and vascular transplant rejection, transplant Vegetative dysfunction, myocardial infarction, xenotransplantation, sepsis, coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos' disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disease , peripheral vascular disease, renovascular disease, mesenteric / intestinal vascular disease, vasculitis, Henoch-Schönlein purpura nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vascular disease, Kawasaki's disease (arteritis), venous gas embolism (VGE), and restenosis after stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barre syndrome, and percutaneous transluminal coronary angioplasty (PTCA).

[0018] The beneficial effects of the present invention are: The single domain antibodies of some examples of the present invention have high specificity for adsorption to human complement C5, providing a new direction for treating diseases caused by abnormal complement C5. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the electrophoresis result diagram of SDS-PAGE analysis of the single-domain antibody protein C5-1 in Example 3, wherein the molecular weight of the single-domain antibody protein is 13.6 KDa.

[0020] Figure 2This is the electrophoresis result diagram of SDS-PAGE analysis of the single-domain antibody protein C5-2 in Example 3, wherein the molecular weight of the single-domain antibody protein is 13.6 KDa. DETAILED DESCRIPTION

[0021] The first aspect of the present invention provides: a single domain antibody against human complement C5 protein, which consists of framework regions FR1 to FR4 and antigen binding regions CDR1 to CDR3, wherein the amino acid sequences of CDR1 to CDR3 are selected from the following group: Group 1, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.3 to SEQ ID NO.5, respectively; Group 2, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.6 to SEQ ID NO.8, respectively.

[0022] In some examples of single domain antibodies, the amino acid sequence of FR1 to FR4 is selected from the following group: Group 1, the amino acid sequences of FR1 to FR4 are shown in SEQ ID NO.9 to SEQ ID NO.12, respectively; Group 2, the amino acid sequences of FR1 to FR4 are shown in SEQ ID NO.13, SEQ ID NO.10, SEQ ID NO.14 and SEQ ID NO.12, respectively.

[0023] In some examples of single domain antibodies, they specifically bind complement C5.

[0024] In some examples of single-domain antibodies, the amino acid sequence thereof is shown as SEQ ID NO.1 or SEQ ID NO.2.

[0025] In some examples of single domain antibodies, they are chimeric antibodies, humanized antibodies, single domain antibodies fused to Fc fragments, bivalent or multivalent single domain antibodies. The bivalent antibody or multivalent single domain antibody contains at least one single domain antibody against human complement C5 protein. Two or more single domain antibodies are connected together by a connector, preferably a flexible connector. The single domain antibody can also be connected to the antibody Fc fragment, so that the Fc fragment can be used to achieve the purpose of extending the half-life, facilitating purification, forming dimers, etc. These connection methods are commonly used in the art.

[0026] The second aspect of the present invention provides: a nucleic acid molecule encoding the single domain antibody described in the first aspect of the present invention.

[0027] The third aspect of the present invention provides: an expression vector comprising the nucleic acid molecule described in the second aspect of the present invention.

[0028] The fourth aspect of the present invention provides: a host cell, which expresses the single domain antibody described in the first aspect of the present invention, or includes the nucleic acid molecule described in the second aspect of the present invention, or contains the expression vector described in the third aspect of the present invention.

[0029] The fifth aspect of the present invention provides: the use of the single domain antibody described in the first aspect of the present invention, the use comprising: preparing an adsorbent for complement C5; preparing a detection reagent for complement C5; Preparation of drugs for treating and / or preventing diseases caused by abnormal complement C5.

[0030] In some application examples, the adsorbent is obtained by coupling the single domain antibody described in the first aspect of the present invention to a solid phase carrier.

[0031] In some application examples, a tracer is linked to the detection reagent.

[0032] In some application examples, the drug is a single domain antibody coupled with a drug molecule, especially a cytotoxic drug molecule, so that C5 can be targeted.

[0033] In some examples of application, the diseases caused by abnormal complement C5 include, but are not limited to, paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration ( For example, age-related macular degeneration (AMD); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent abortions; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis, bullous pemphigoid, Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome, C3 nephropathy, antineutrophil cytoplasmic antibody-associated vasculitis, humoral and vascular transplant rejection , graft dysfunction, myocardial infarction, allogeneic transplantation, sepsis, coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos' disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disease Disease, peripheral vascular disease, renal vascular disease, mesenteric / intestinal vascular disease, vasculitis, Henoch-Schönlein purpura nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vascular disease, Kawasaki's disease (arteritis), venous gas embolism (VGE), and restenosis after stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barre syndrome, and percutaneous transluminal coronary angioplasty (PTCA). Studies have shown that by acting on complement C5, these diseases can be improved or treated to a certain extent.

[0034] In order to more concisely and clearly demonstrate the technical solutions, objectives and advantages of the present invention, the present invention is further described in detail below in conjunction with specific embodiments.

[0035] Example 1: Establishment of complement C5 protein alpaca phage antibody library Reference CN114316051A to establish an alpaca phage antibody library. The brief steps are as follows: S1) Immunize alpacas with human complement C5 and collect peripheral blood mononuclear cells (PBMC); S2) Extract RNA from PBMC and reverse transcribe it into cDNA; S3) After PCR amplification of the target sequence, it was ligated to the phagemid vector pHIAT-1 through the HindIII and NotI restriction sites, and then transformed into Escherichia coli TG1 to form the original phage library; S4) adding helper phage M13K07 to the TG1 strain grown to the logarithmic phase, culturing overnight, and collecting the supernatant by centrifugation; S5) Phagemids were precipitated with PEG, and then the phages were resuspended in PBS and sterilized by filtration using a 0.45 μm filter membrane to obtain a phage VHH antibody library with a library capacity of 4.30 × 10 13 .

[0036] Example 2: Phage library screening (1) The first round of phage antibody screening The titer of helper phage M13K07 was 1.17 × 10 10 pfu / mL, and TG1 was infected with helper phage M13K07.

[0037] S1) Coat human complement C5 on ELISA plates at 0.5 µg / well, incubate at 4°C overnight, and wash the plates five times; S2) Prepare 5% skim milk powder (blocking solution) with PBST and block, 200 µL / well, incubate at 37℃ for 2 h, and wash the plate 5 times; S3) Add 100 µL of phage library solution to each well, incubate at 37°C for 2 h, and wash the plate 5 times; S4) Add 100 μL of Glycine-HCl buffer to each well, shake gently at room temperature for 10 min, then aspirate the eluate and quickly add 80 μL of 1 M Tris-HCl buffer; S5) Take out 10 μL for titer determination, add the remaining liquid to 5 mL of TG1 bacterial solution grown to the logarithmic phase, and incubate at 37°C for 30 min; S6) Add 2 × YT medium to a total volume of 10 mL and culture at 37°C and 220 rpm for 30 min-1 h; S7) Add ampicillin (Amp) to a final concentration of 100 μg / mL and culture the bacterial solution at 37°C and 250 rpm until OD600 = 0.4-0.6; S8) Add helper phage M13KO7 to the bacterial solution, incubate at 37°C for 30 min, and then incubate at 37°C and 250 rpm for 45 min-1 h; S9) Centrifuge at 4°C, 5000 rpm for 20 min, discard the supernatant, resuspend the cells in an equal volume of 2 × YT-Amp-kana (kanamycin) medium (containing 100 μg / mL Amp and 30 μg / mL kana), and culture overnight at 30°C, 220 rpm; S10) Centrifuge at 8000 rpm at 4°C for 20 min, take the supernatant and add 1 / 5 volume of 20% PEG / NaCl solution, mix thoroughly, and place on ice or at 4°C for 1-2 h; S11) Centrifuge at 4°C, 8000 rpm for 30 min, discard the supernatant, resuspend the precipitate in 1 mL of sterile PBS, and then centrifuge at 4°C, 8000 rpm for 10 min. The precipitate is the phage antibody particles.

[0038] (2) Second round of phage antibody screening S1) Human complement C5 was coated on the ELISA plate at 1 µg / well, incubated overnight at 4°C, and the plate was washed 6 times; S2) Block with blocking solution, 200 µL / well, incubate at 37°C for 2 h, and wash the plate 6 times; S3) Add 100 µL of the phage library solution from the first round of screening to each well, incubate at 37°C for 2 h, and wash the plate 6 times; the rest of the operations are the same as the first round of screening.

[0039] (3) The third round of phage antibody screening S1) Coat human complement C5 on the ELISA plate, 0.1 µg / well, incubate at 4°C overnight, wash the plate 5 times; block with blocking solution, 200 µL / well, incubate at 37°C for 2 h, wash the plate 5 times; S2) Add 100 µL of the phage library solution after the second round of screening to each well, incubate at 37°C for 2 h, and wash the plate 10 times; the rest of the operations are the same as the first round of screening.

[0040] (4) Monoclonal phage detection S1) The phages after the third round of screening were used to infect TG1, diluted and spread on plates for culture, and then 96 single clones were picked; S2) Using the original library as a negative control, perform ELISA test, and select the clones with OD450 values ​​greater than 2.1 times that of the original library as preliminary positive clones for sequencing; S3) Based on the sequencing results, after removing the duplicate sequences, two of the sequences were selected as candidate sequences.

[0041] The single domain antibodies finally screened were respectively designated as C5-1 and C5-2, and their amino acid sequences were respectively C5-1: MEVQLQASGGGLVQPGGSLRLSCVASRSIFSDYAMAWYRQAPGKQRELVAWITSGGATYYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNKSLAGIWGQGTQVTVSS, (SEQ ID NO.1) and C5-2: MEVQLQASGGGLVQSGGSLTLSCVLSGSIFSINTLGWYRQAPGKQRELVAITTSGGTTKYADSVKGRFTISRDNAKNTVYLRMNNLKPEDTGVYFCYARVPLDYWGQGTQVTVSS (SEQ ID NO.2). The amino acid sequences of the CDR regions and FRs of C5-1 and C5-2 are shown in Table 1.

[0042] Table 1. Sequence analysis results of single domain antibodies against human complement C5

[0043] Example 3: In vitro recombinant expression and purification of antibodies S1) After the pET-28a vector was double-digested with NdeI and XhoI, the gene sequence encoding SEQ ID NO.1 was connected to the pET-28a vector via the NdeI and XhoI restriction sites; S2) The ligation product was transferred into BL21 (DE3) competent bacteria for transformation; S3) Pick a single clone for PCR and sequencing to confirm that the band sequence is correct, and then express the recombinant protein: S4) Streak the bacterial solution on a plate, pick a single clone and inoculate it into LB liquid medium containing kanamycin, then expand the culture at a ratio of 1:100 to OD600 = 0.6-0.8, add IPTG with a final concentration of 1 mM to induce for 6 hours, collect the bacteria by centrifugation, break them at low temperature and collect the protein supernatant by centrifugation; S5) Purification of recombinant protein: S51) the supernatant protein after crushing and centrifugation is filtered through a 0.45 μm filter membrane and then purified by affinity chromatography column (nickel ion chelating filler with His tag); S52) The chromatography column was equilibrated with 20 mM Tris-HCL buffer, and then the protein was slowly added to the chromatography column, and washed with 10 mM imidazole buffer and 40 mM imidazole buffer, respectively, and then the recombinant protein was eluted with 250 mM imidazole buffer, and the elution peak was collected to obtain the alpaca single domain antibody protein.

[0044] The elution peak was analyzed by 15% SDS-PAGE to identify the purity of the target protein. Figure 1 and Figure 2 As shown. Figure 1 and Figure 2 It can be seen that the expressed single domain antibody has good purity.

[0045] Example 4: Synthesis of immunosorbent ① Add 10 mL of activation reaction solution (sodium borohydride: 0.02 g, NaOH: 0.24 g) and 10 mL of BDGE to 10 mL of agarose (Bestarose 6FF) filler, respectively, and activate the reaction at 37°C and 120 rpm for about 2.5 h, then wash to pH 5.0-7.0 and vacuum dry; ② Add 10 mL of coupling buffer (ammonium sulfate: 3.96 g; NaOH: 0.27 g) and 10 mL of the prepared 3 mg / mL alpaca single domain antibody solution to the filler dried in step ①, and perform coupling reaction at 25°C and 120 rpm for about 10 h, then wash more than 10 times and drain; ③ Add 20 mL of 1 M ethanolamine solution to the filler dried in step ②, and perform the end-capping reaction at 37°C and 120 rpm for about 10 h. Then wash it more than 10 times and drain it. Add 20% ethanol and store it for later use.

[0046] Example 5: Detection of the adsorption performance of immunosorbents on human complement Referring to the method described in Example 5 of patent CN111057153A, the adsorption performance of human complement C5 was tested by static adsorption method. The brief steps are as follows: S1) Prepare the immunosorbent according to the above method, add 10 mL of human plasma containing 100 mg of complement C5 to 1 mL of the immunosorbent, place in a shaker, and shake slowly at room temperature for 1 h; S2) After the reaction is completed, the reaction solution is added to a disposable affinity chromatography column, first rinsed with about 100 mL of equilibration solution (PBS), then eluted with 40 mL of eluent (citric acid 2.1 g / L, NaCl 8.0 g / L), and the elution peak is collected. The OD280 value is detected by ultraviolet light. The adsorption performance (mg / mL) = [(OD280 / 1.38) × 40] / 1; S3) Use a biochemical analyzer to detect the levels of immunoglobulins IgA and IgM in plasma and determine the nonspecific adsorption amount.

[0047] The results are shown in Table 2.

[0048] Table 2. Adsorption performance test of immunosorbents on various immunoglobulins

[0049] It can be seen from Table 2 that the immunosorbents formed by coupling the two single domain antibodies with the solid phase carrier have high adsorption performance for human complement C5, among which the adsorption performance of the protein numbered SEQ ID NO.1 is higher, and the overall difference is not large. In addition, both single domain antibodies have low nonspecific adsorption for IgA and IgM and good specificity.

[0050] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. A single domain antibody against human complement C5 protein, consisting of framework regions FR1 to FR4 and antigen binding regions CDR1 to CDR3, characterized in that: The amino acid sequences of its CDR1 to CDR3 are selected from the following group: Group 1, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.3 to SEQ ID NO.5, respectively; Group 2, the amino acid sequences of CDR1 to CDR3 are shown in SEQ ID NO.6 to SEQ ID NO.8, respectively.

2. The single domain antibody according to claim 1, characterized in that The amino acid sequence of FR1 to FR4 is selected from the following group: Group 1, the amino acid sequences of FR1 to FR4 are shown in SEQ ID NO.9 to SEQ ID NO.12, respectively; Group 2, the amino acid sequences of FR1 to FR4 are shown in SEQ ID NO.13, SEQ ID NO.10, SEQ ID NO.14 and SEQ ID NO.12, respectively.

3. The single domain antibody according to claim 1 or 2, characterized in that It specifically binds complement C5.

4. The single domain antibody according to claim 1 or 2, characterized in that It is a chimeric antibody, a humanized antibody, a single domain antibody fused to an Fc fragment, or a bivalent or multivalent single domain antibody.

5. A nucleic acid molecule, characterized in that Encoding the single domain antibody according to any one of claims 1 to 4.

6. An expression vector, characterized in that: Comprising the nucleic acid molecule of claim 5.

7. A host cell, characterized in that It expresses the single domain antibody according to any one of claims 1 to 4, or comprises the nucleic acid molecule according to claim 5, or contains the expression vector according to claim 6.

8. The use of the single domain antibody according to any one of claims 1 to 4, characterized in that: The applications include: preparing an adsorbent for complement C5; preparing a detection reagent for complement C5; Preparation of drugs for treating and / or preventing diseases caused by abnormal complement C5.

9. The use according to claim 8, characterized in that: The adsorbent is obtained by coupling the single domain antibody according to any one of claims 1 to 4 to a solid phase carrier.

10. The use according to claim 8, characterized in that: Diseases caused by abnormalities in complement C5 include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); antiphospholipid antibody syndrome; lupus nephritis; ischemia-reperfusion injury; typical or infectious hemolytic uremic syndrome (tHUS); dense deposit disease (DDD); neuromyelitis optica (NMO); multifocal motor neuropathy (MMN); multiple sclerosis (MS); macular degeneration (e.g., age-related macular degeneration); degeneration (AMD); hemolysis, elevated liver enzymes, and low platelets (HELLP) syndrome; thrombotic thrombocytopenic purpura (TTP); spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent abortions; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis, bullous pemphigoid, Shiga toxin-producing Escherichia coli-associated hemolytic uremic syndrome, C3 nephropathy, antineutrophil cytoplasmic antibody-associated vasculitis, humoral and vascular transplant rejection, allograft dysfunction Common, myocardial infarction, allogeneic transplantation, sepsis, coronary artery disease, dermatomyositis, Graves' disease, atherosclerosis, Alzheimer's disease, systemic inflammatory response sepsis, septic shock, spinal cord injury, glomerulonephritis, Hashimoto's thyroiditis, type I diabetes, psoriasis, pemphigus, autoimmune hemolytic anemia (AIHA), ITP, Goodpasture's syndrome, Degos' disease, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disease, peripheral Vascular disease, renovascular disease, mesenteric / enteric vascular disease, vasculitis, Henoch-Schönlein purpura nephritis, vasculitis associated with systemic lupus erythematosus, vasculitis associated with rheumatoid arthritis, immune complex vasculitis, Takayasu's disease, dilated cardiomyopathy, diabetic vascular disease, Kawasaki's disease (arteritis), venous gas embolism (VGE) and restenosis after stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barre syndrome, and percutaneous transluminal coronary angioplasty (PTCA).

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