Single-domain antibody against human complement c5 and use thereof

Abstract

The application belongs to the field of biological medicine, and discloses a single-domain antibody against human complement C5 and application thereof. CDR1-CDR3 of the single-domain antibody are respectively shown as SEQ ID NO. 3-5 or 6-8. The single-domain antibody of some examples of the application has high specificity for adsorption of human complement C5, and provides a new direction for treating diseases caused by complement C5 abnormality.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological medicine, and particularly relates to a single-domain antibody against human complement C5 and application thereof. BACKGROUND

[0002] The complement (C) system is an important component of the innate and adaptive immune systems, which is essential 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. The complement contains more than 30 components, which widely exist in serum, tissue fluid and cell membrane surface. Generally, most of the complement components in plasma only have biological functions after being activated. Complement activation can quickly protect the body from infectious attacks, and is traditionally considered as the core part of host defense against invading pathogens and clearance of potentially damaging cellular debris, but it is worth noting that it can also be a driving factor or aggravating factor in many inflammatory or autoimmune diseases. In fact, excessive or unregulated complement activation is related to the pathogenesis of many diseases, including ocular disorders, acute inflammation (sepsis and ischemic stroke, etc.) and chronic diseases (including autoimmune diseases, neurodegenerative diseases, kidney diseases and chronic hemolytic diseases with thrombo-inflammatory characteristics, etc.).

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

[0004] Both C3 and C5 cleavage lead to the production of anaphylatoxins and allow the formation of the membrane attack complex, both of which are major drivers of disease, and targeting C3 and C5 is one of the earliest therapeutic approaches considered. Moreover, C5 is at the end of the cascade, and targeting it can modulate complement signaling from all three different pathways of activation, so C5 becomes a popular target for complement drug development. And the current therapeutic drugs acting on C5 are mostly anti-C5 inhibitors by injection, mainly through blocking the activity of C5 to treat, such as CN105324485A discloses the use of iRNA such as double-stranded ribonucleic acid (dsRNA), compositions targeting the complement component C5 gene, and the use of such iRNA (e.g., dsRNA), compositions to inhibit the expression of C5 and treat a complement component C5 associated disease. CN101970002A discloses administering to a subject an effective amount of an anti-factor Bb antibody 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] As a new technology emerging in recent years, immunoadsorption therapy is to combine highly specific antigens or antibodies with adsorbent materials (carriers) to make adsorbents (columns), and to selectively or relatively specifically remove endogenous pathogenic factors in the blood of patients by using the specific adsorption performance of the adsorbents, so as to achieve the purpose of relieving the disease.

[0006] Compared with traditional antibodies, single-domain antibodies are a form of antibodies composed of a single variable domain of antibodies, which are heavy chain antibodies (VHH region), and only one domain with disulfide bonds is folded into a relatively stable structure. Studies have shown that single-domain antibodies can maintain antigen binding ability after incubation at 37℃ for one week, so that they can be used in more harsh 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, and have fast blood clearance rate and resistance to aggregation, causing only low immunogenicity. Single-domain antibodies have shown great potential as tools in different biotechnology fields such as diagnosis and treatment. SUMMARY

[0007] The purpose of the present application 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 application is:

[0009] In a first aspect, the present application provides a single-domain antibody against human complement C5 protein, which is composed of framework regions FR1-FR4 and antigen binding regions CDR1-CDR3, and the amino acid sequences of CDR1-CDR3 are selected from the following group:

[0010] Group 1, the amino acid sequences of CDR1-CDR3 are shown in SEQ ID NO. 3-SEQ ID NO. 5, respectively;

[0011] Group 2, the amino acid sequences of CDR1-CDR3 are shown in SEQ ID NO. 6-SEQ ID NO. 8, respectively.

[0012] In some examples of the single domain antibody, the amino acid sequences of FR1-FR4 are selected from a group consisting of:

[0013] Group 1, the amino acid sequences of FR1-FR4 are shown in SEQ ID NO. 9-SEQ ID NO. 12, respectively;

[0014] Group 2, the amino acid sequences of FR1-FR4 are shown in SEQ ID NO. 13, SEQ ID NO. 10, SEQ ID NO. 14 and SEQ ID NO. 12, respectively.

[0015] In some examples of the single domain antibody, it specifically binds to complement C5.

[0016] In some examples of the single domain antibody, it is a chimeric antibody, a humanized antibody, a single domain antibody fused with Fc fragment, a bivalent or multivalent single domain antibody.

[0017] In a second aspect of the present application, there is provided a nucleic acid molecule encoding the single domain antibody of the first aspect of the present application.

[0018] In a third aspect of the present application, there is provided an expression vector comprising the nucleic acid molecule of the second aspect of the present application.

[0019] In a fourth aspect of the present application, there is provided a host cell expressing the single domain antibody of the first aspect of the present application, or comprising the nucleic acid molecule of the second aspect of the present application, or comprising the expression vector of the third aspect of the present application.

[0020] In a fifth aspect of the present application, there is provided the use of the single domain antibody of the first aspect of the present application, which use comprises:

[0021] preparing an adsorbent for complement C5;

[0022] preparing a detection reagent for complement C5;

[0023] preparing a medicament for treating and / or preventing a disease caused by complement C5 abnormality.

[0024] In some examples of the use, the adsorbent is obtained by coupling the single domain antibody of the first aspect of the present application on a solid phase carrier.

[0025] In some examples of use, diseases caused by complement C5 abnormalities include paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), asthma, rheumatoid arthritis (RA); anti-phospholipid 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 spontaneous abortion; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis, bullous pemphigoid, Shiga toxin E. coli-associated hemolytic uremic syndrome, C3 glomerulopathy, anti-neutrophil cytoplasmic antibody-associated vasculitis, humoral and vascular transplant rejection, graft 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, anti-phospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / enteric vascular disease, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu's disease (arteritis), venous gas emboli (VGE), and restenosis following stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barre syndrome, and percutaneous transluminal coronary angioplasty (PTCA).

[0026] The present application has the following advantages:

[0027] The single-domain antibodies of some examples of the present application have high specificity for adsorption of human complement C5, providing a new direction for treating diseases caused by complement C5 abnormalities. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 is an electrophoresis result map of SDS-PAGE analysis of the single-domain antibody protein C5-1 of Example 3, wherein the single-domain antibody protein has a molecular weight of 13.6 KDa.

[0029] Figure 2Figure 1 is an electrophoresis result chart of SDS-PAGE analysis of the single domain antibody protein C5-2 of Example 3, wherein the molecular weight of the single domain antibody protein is 13.6 KDa. DETAILED DESCRIPTION

[0030] In a first aspect, the present application provides a single domain antibody against human complement C5 protein, which comprises framework regions FR1-FR4 and antigen binding regions CDR1-CDR3, and the amino acid sequences of CDR1-CDR3 are selected from the following groups:

[0031] Group 1, wherein the amino acid sequences of CDR1-CDR3 are shown in SEQ ID NO. 3-5, respectively;

[0032] Group 2, wherein the amino acid sequences of CDR1-CDR3 are shown in SEQ ID NO. 6-8, respectively.

[0033] In some examples of the single domain antibody, the amino acid sequences of FR1-FR4 are selected from the following groups:

[0034] Group 1, wherein the amino acid sequences of FR1-FR4 are shown in SEQ ID NO. 9-12, respectively;

[0035] Group 2, wherein the amino acid sequences of FR1-FR4 are shown in SEQ ID NO. 13, SEQ ID NO. 10, SEQ ID NO. 14 and SEQ ID NO. 12, respectively.

[0036] In some examples of the single domain antibody, it specifically binds to complement C5.

[0037] In some examples of the single domain antibody, the amino acid sequence is shown in SEQ ID NO. 1 or SEQ ID NO. 2.

[0038] In some examples of the single domain antibody, it is a chimeric antibody, a humanized antibody, a single domain antibody fused with Fc fragment, a bivalent or multivalent single domain antibody. The bivalent 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 linker, preferably a flexible linker. The single domain antibody can also be connected to an antibody Fc fragment, so that the Fc fragment can be used to achieve the purpose of prolonging half-life, facilitating purification, forming dimers, etc. These connection methods are commonly used in the art.

[0039] In a second aspect, the present application provides a nucleic acid molecule encoding the single domain antibody of the first aspect of the present application.

[0040] In a third aspect of the application, there is provided an expression vector comprising the nucleic acid molecule of the second aspect of the application.

[0041] In a fourth aspect of the application, there is provided a host cell expressing the single domain antibody of the first aspect of the application, or comprising the nucleic acid molecule of the second aspect of the application, or comprising the expression vector of the third aspect of the application.

[0042] In a fifth aspect of the application, there is provided the use of the single domain antibody of the first aspect of the application, the use comprising:

[0043] an adsorbent for preparing complement C5;

[0044] a detection reagent for preparing complement C5;

[0045] a medicament for treating and / or preventing a disease caused by abnormal complement C5.

[0046] In some examples of use, the adsorbent is obtained by coupling the single domain antibody of the first aspect of the application to a solid support.

[0047] In some examples of use, the detection reagent is coupled to a tracer.

[0048] In some examples of use, the medicament is a single domain antibody coupled to a drug molecule, in particular a cytotoxic drug molecule. This allows targeting of complement C5.

[0049] In some examples of application, diseases caused by complement C5 abnormalities 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 (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 spontaneous abortion; preeclampsia, traumatic brain injury, myasthenia gravis, cold agglutinin disease, dermatomyositis bullous pemphigoid, Shiga toxin E. coli associated hemolytic uremic syndrome, C3 glomerulopathy, anti-neutrophil cytoplasmic antibody-associated vasculitis, humoral and vascular transplant rejection, graft 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, antiphospholipid syndrome (APS), catastrophic APS (CAPS), cardiovascular disorders, myocarditis, cerebrovascular disease, peripheral vascular disease, renal vascular disease, mesenteric / enteric vascular disease, vasculitis, Henoch-Schonlein purpura nephritis, systemic lupus erythematosus-associated vasculitis, rheumatoid arthritis-associated vasculitis, immune complex vasculitis, Takayasu's disease (arteritis), venous gas emboli (VGE), and restenosis after stent placement, rotational atherectomy, membranous nephropathy, Guillain-Barre syndrome, and percutaneous transluminal coronary angioplasty (PTCA). It has been shown that by acting on complement C5, these diseases can be improved or treated to some extent.

[0050] In order to more clearly illustrate the technical solutions, objectives and advantages of the present application, the present application will be further described in detail below with specific examples.

[0051] Example 1: Establishment of a camelid phage antibody library for complement C5 protein

[0052] A camelid phage antibody library was established according to CN114316051A, and the brief steps are as follows:

[0053] S1) Immunize a llama with human complement C5, and collect peripheral blood mononuclear cells PBMC;

[0054] S2) Extract RNA from PBMC and reverse transcribe into cDNA;

[0055] S3) After PCR amplification of the target sequence, connect it to the phagemid vector pHIAT-1 through HindIII and NotI enzyme cutting sites, then transform to E. coli TG1 to form the original phage library;

[0056] S4) Add helper phage M13K07 to the TG1 strain grown to the logarithmic phase, centrifuge to collect the supernatant after overnight culture;

[0057] S5) Use PEG to precipitate the phagemid, then resuspend the phage through PBS and filter bacteria using a 0.45 μm filter, that is, the phage VHH antibody library is obtained, with a library capacity of 4.30 × 10 13 .

[0058] Example 2: Phage library screening

[0059] (1) First round of screening of phage antibodies

[0060] The titer of the helper phage M13K07 determination was 1.17 × 10 10 pfu / mL, and the TG1 was infected using the helper phage M13K07.

[0061] S1) Coat human complement C5 on the ELISA plate, 0.5 μg / well, 4°C overnight, wash the plate 5 times;

[0062] S2) Use PBST to prepare 5% skimmed milk powder (blocking solution) and block, 200 μL / well, 37°C incubate for 2 h, wash the plate 5 times;

[0063] S3) Add 100 μL of phage library solution to each well, 37°C incubate for 2 h, wash the plate 5 times;

[0064] S4) Add 100 μL of Glycine-HCl buffer to each well, gently shake at room temperature for 10 min, then aspirate the eluent and quickly add 80 μL of 1 M Tris-HCl buffer;

[0065] S5) Take out 10 μL for titer determination, and add the remaining liquid to 5 mL of TG1 liquid grown to the logarithmic phase, 37°C for 30 min;

[0066] S6) Add 2 × YT medium to a total volume of 10 mL, 37°C, 220 rpm culture for 30 min-1 h;

[0067] S7) Add ampicillin (Amp) to a final concentration of 100 μg / mL, and incubate the bacterial solution at 37°C, 250 rpm, until OD600=0.4-0.6;

[0068] S8) Add helper phage M13KO7 to the bacterial solution, and incubate at 37°C for 30 min, then incubate at 37°C, 250 rpm, for 45 min-1 h;

[0069] S9) Centrifuge at 4°C, 5000 rpm, for 20 min, discard the supernatant, and resuspend the bacterial pellet in the same volume of 2 × YT-Amp-kana (kanamycin) medium (containing 100 μg / mL Amp and 30 μg / mL kana), and incubate at 30°C, 220 rpm, overnight;

[0070] S10) Centrifuge at 4°C, 8000 rpm, for 20 min, take the supernatant and add 1 / 5 volume of 20% PEG / NaCl solution, mix well, and incubate at ice or 4°C for 1-2 h;

[0071] S11) Centrifuge at 4°C, 8000 rpm, for 30 min, discard the supernatant, resuspend the precipitate in 1 mL sterile PBS, then centrifuge at 4°C, 8000 rpm, for 10 min, and the precipitate is the phage antibody particles.

[0072] (2) Second round of screening of phage antibodies

[0073] S1) Coat human complement C5 on the ELISA plate at 1 μg / well, 4°C overnight, and wash the plate 6 times;

[0074] S2) Block with blocking solution, 200 μL / well, 37°C for 2 h, and wash the plate 6 times;

[0075] S3) Add 100 μL of the phage library solution screened in the first round to each well, 37°C for 2 h, and wash the plate 6 times; the rest of the operations are the same as in the first round of screening.

[0076] (3) Third round of screening of phage antibodies

[0077] S1) Coat human complement C5 on the ELISA plate at 0.1 μg / well, 4°C overnight, and wash the plate 5 times; block with blocking solution, 200 μL / well, 37°C for 2 h, and wash the plate 5 times;

[0078] S2) Add 100 μL of the phage library solution screened in the second round to each well, 37°C for 2 h, and wash the plate 10 times; the rest of the operations are the same as in the first round of screening.

[0079] (4) Monoclonal phage detection

[0080] S1) The phage after the third round of screening was used to infect TG1, which was diluted and plated on a plate for culture, and then 96 single colonies were picked;

[0081] S2) ELISA was performed using the original library as a negative control, and the preliminary positive clones with OD450 values greater than 2.1 times of the original library were selected for sequencing;

[0082] S3) According to the sequencing results, 2 sequences were selected as candidate sequences after removing the repeated sequences.

[0083] The final screened single-domain antibodies were designated as C5-1 and C5-2, and their amino acid sequences were represented by C5-1: MEVQLQASGGGLVQPGGSLRLSCVASRSIFSDYAMAWYRQAPGKQRELVAWITSGGATYYADSMKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNKSLAGIWGQGTQVTVSS, (SEQ ID NO. 1) and C5-2: MEVQLQASGGGLVQSGGSLTLSCVLSGSIFSINTLGWYRQAPGKQRELVAITTSGGTTKYADSVKGRFTISRDNAKNTVYLRMNNLKPEDTGVYFCYARVPLDYWGQGTQVTVSS (SEQ ID NO. 2). The amino acid sequences of the CDR regions and FR of C5-1 and C5-2 are shown in Table 1.

[0084] Table 1, Sequence analysis results of single-domain antibodies against human complement C5

[0085]

[0086] Example 3: In vitro recombinant expression and purification of antibodies

[0087] S1) After the pET-28a vector was treated with double digestion of NdeI and XhoI, the gene sequence encoding SEQ ID NO. 1 was connected to the pET-28a vector through NdeI and XhoI restriction sites;

[0088] S2) The ligation product was transformed into BL21 (DE3) competent bacteria;

[0089] S3) After the single colony was picked for PCR and sequencing to identify the correct band sequence, the recombinant protein expression was performed:

[0090] S4) The bacterial liquid was plated and single colonies were picked and inoculated in LB liquid medium containing kanamycin, and then cultured at a ratio of 1:100 to OD600=0.6-0.8, and then 1 mM IPTG was added for induction for 6 h. The bacterial cells were collected by centrifugation, and then broken at low temperature and the protein supernatant was collected by centrifugation;

[0091] S5) Recombinant protein purification:

[0092] S51) The supernatant protein after breaking and centrifugation was filtered through a 0.45 μm filter membrane and then purified by affinity chromatography column (nickel ion chelating filler with His tag);

[0093] S52) The chromatography column was equilibrated with 20 mM Tris-HCL buffer, and then the protein was slowly added to the chromatography column, and then 10 mM imidazole buffer and 40 mM imidazole buffer were added respectively for washing, and then 250 mM imidazole buffer was used for elution of the recombinant protein, and then the elution peak was collected, and then the llama single domain antibody protein was obtained.

[0094] The elution peak was analyzed by 15% SDS-PAGE gel electrophoresis, and the purity of the target protein was identified. The results are shown in Figure 1 and Figure 2 It can be seen from Figure 1 and Figure 2 that the expressed single domain antibody has good purity.

[0095] Example 4: Synthesis of immunoadsorbent

[0096] ① 10 mL of agarose (Bestarose 6FF) filler was added with 10 mL of activated reaction solution (sodium borohydride: 0.02 g, NaOH: 0.24 g) and 10 mL of BDGE respectively, and then activated for about 2.5 h at 37°C and 120 rpm, and then washed to pH 5.0-7.0 and vacuum dried;

[0097] ② 10 mL of coupling buffer (ammonium sulfate: 3.96 g; NaOH: 0.27 g) and 10 mL of the prepared llama single domain antibody solution with a concentration of 3 mg / mL were added to the filler dried in step ①, and then coupled at 25°C and 120 rpm for about 10 h, and then washed for more than 10 times and dried;

[0098] ③ 20 mL of 1 M ethanolamine solution was added to the filler dried in step ②, and then end-capped at 37°C and 120 rpm for about 10 h, and then washed for more than 10 times and dried, and then 20% ethanol was added for storage.

[0099] Example 5: Detection of the adsorption performance of the immunoadsorbent to human complement

[0100] According to the method described in Example 5 of the patent CN111057153A, the static adsorption method is used to detect the adsorption performance of human complement C5, and the brief steps are as follows:

[0101] S1) According to the above method, the immunoadsorbent is prepared, 10 mL of human plasma containing 100 mg of complement C5 is added to 1 mL of immunoadsorbent, and placed in a shaking bed, slowly shaken for 1 h at room temperature;

[0102] S2) After the reaction is completed, the above reaction solution is added to a disposable affinity chromatography column, first washed with about 100 mL of equilibrium liquid (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 adsorption performance (mg / mL) = [(OD280 / 1.38) x 40] / 1 is determined by the value of OD280 detected by ultraviolet detection;

[0103] S3) The content of immunoglobulin IgA and IgM in the plasma is detected by using a biochemical analyzer to determine the non-specific adsorption amount.

[0104] The results are shown in Table 2.

[0105] Table 2, adsorption performance detection of immunoadsorbent to each immunoglobulin

[0106]

[0107] As shown in Table 2, the immunoadsorbent formed by coupling two single domain antibodies with a solid carrier has high adsorption performance for human complement C5, and the adsorption performance of the protein numbered SEQ ID NO. 1 is higher, the overall difference is not big, and the non-specific adsorption of the two single domain antibodies to IgA and IgM is low, and the specificity is good.

[0108] The above is a further detailed description of the present application, which cannot be regarded as a specific implementation of the present application. For ordinary skilled persons in the technical field to which the present application belongs, simple deduction or replacement without departing from the concept of the present application is within the protection scope of the present application.

Claims

1. A single-domain antibody against human complement C5 protein, comprising a framework region FR1–FR4 and an antigen-binding region CDR1–CDR3, characterized in that, Its CDR1–CDR3 amino acid sequences are selected from the following group: Group 1, whose CDR1 to CDR3 amino acid sequences are shown in SEQ ID NO.3 to SEQ ID NO.5, respectively; Group 2, whose CDR1 to CDR3 amino acid sequences 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, Its FR1–FR4 amino acid sequences are selected from the following group: Group 1, whose amino acid sequences of FR1 to FR4 are shown in SEQ ID NO.9 to SEQ ID NO.12, respectively; Group 2, whose 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, characterized in that, These are humanized antibodies, bivalent or multivalent single-domain antibodies.

4. A nucleic acid molecule, characterized in that, Encoding the single-domain antibody according to any one of claims 1 to 3.

5. An expression carrier, characterized in that, It includes the nucleic acid molecule as described in claim 4.

6. A host cell, characterized in that, It expresses the single-domain antibody as described in any one of claims 1 to 3, or includes the nucleic acid molecule as described in claim 4, or contains the expression vector as described in claim 5.

7. The application of the single-domain antibody according to any one of claims 1 to 3, characterized in that, The applications include: Preparation of an adsorbent for complement C5; Preparation of reagents for the detection of complement C5.

8. The application according to claim 7, characterized in that, The adsorbent is obtained by conjugating the single-domain antibody according to any one of claims 1 to 3 onto a solid-phase support.

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