Complement protein c3c3b antigenic epitope peptide, and antibody thereof and use thereof

Abstract

Provided are a complement protein C3C3b antigenic epitope peptide, and a related antibody thereof and the use thereof. The provided anti-complement protein C3 / C3b single-domain antibody binds to the MG6α-MG7-C345C domain and / or MG6β-MG6α-MG7-TED domain of complement protein C3 / C3b, and can compete with factor B for binding to C3b, thereby inhibiting the formation of a C3 convertase. Moreover, the anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof has a small molecular weight, exhibits a high affinity for complement protein C3 / C3b and a potent complement inhibitory activity, can effectively block the cleavage of C3 and the activation of complement cascade, and can be potentially used in the treatment of diseases associated with complement activation.

Description

Complement protein C3 C3b antigen epitope peptide, antibody thereof and application TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a complement protein C3 C3b antigen epitope peptide, antibody thereof and application. BACKGROUND

[0002] The complement system is a part of the innate immune system, which plays an important role in resisting microbial invasion and maintaining body homeostasis. The complement pathway involves more than 50 known cell membrane receptors and serum proteins, and its activation process is a cascade of proteolytic reactions of a series of complement proteins, which mediates opsonization, promotes inflammatory response, forms a membrane attack complex to lyse target cells, and rapidly neutralizes pathogenic microorganisms or endogenous stress signals.

[0003] The complement cascade is a highly interactive protein network activated by three pathways, including the classical pathway (CP), the lectin pathway (LP) and the alternative pathway (AP). The classical pathway CP starts from the binding of complement protein C1q to the antigen-antibody complex, which induces the conformational change of C1 complex, and then cleaves complement proteins C4 and C2 to form C3 convertase C4bC2a, which starts the complement cascade. The lectin pathway LP is triggered by the binding of mannose-binding lectin (MBL) to polysaccharides or glycoproteins on the surface of foreign cells or damaged host cells. Similar to CP, LP pathway also forms C4bC2a as C3 convertase of LP pathway. Unlike CP and LP, the initiation of the alternative pathway AP is derived from persistent, low-level C3 activation, known as "C3 tick-over". The activation of C3 is mediated by its spontaneous hydrolysis into C3(H2O), which forms active C3 convertase C3(H2O)Bb under the action of FB and FD factors, thereby starting AP. When the complement cascade is activated, C3 convertases C3(H2O)Bb, C3bBb and C4bC2a cleave C3 to generate C3a and C3b, and the newly generated C3b can further generate new C3 convertase C3bBb, thereby amplifying the activation of complement. C3b formed by different pathways can combine with C3 convertase to form C5 convertase C3bBb(C3b) n and C4bC2a(C3b) nC5 is split into C5a and C5b, C5b eventually forms a membrane attack complex (MAC) to lyse target cells under the action of complement components C6, C7, C8, C9. Activation of the complement cascade can produce effects such as inflammatory response, opsonization and cell death through the formation of MAC and other complement effectors (C3a, C5a, etc.).

[0004] C3 has a molecular weight of about 185 kDa, composed of a β chain (75 kDa) and an α chain (110 kDa), and has a total of 13 domains. Under normal physiological conditions, the concentration of C3 in plasma is about 900-1500 mg / mL, and the concentration in the vitreous is about 6-10 mg / mL, making it one of the most abundant complement proteins. C3 is hydrolyzed by C3 convertase to release anaphylatoxin C3a, and then converted to C3b. C3b retains the α chain and β chain structure of C3, and generates a new active site on the α chain. C3b can bind to factor B to form C3 convertase C3bBb under the action of factor D, and then promote the complement cascade. The thioester domain (TED) in the C3b structure can form a covalent bond with the hydroxyl group on the surface of cells or proteins, recruiting immune cells such as macrophages to exert opsonization. C3a is an anaphylatoxin that, upon binding to its receptor C3aR, has the effect of promoting inflammatory response, promoting the release of histamine or other inflammatory mediators, and recruiting immune cells.

[0005] While a functioning complement system provides a powerful defense against infectious microorganisms, inappropriate regulation or activation of complement is involved in the pathogenesis of a variety of diseases, including, for example, rheumatoid arthritis (RA); lupus nephritis (LN); immunoglobulin A nephropathy (IgA nephraopathy); ischemia reperfusion injury (IRI); paroxysmal nocturnal hemoglobinuria (PNH); atypical hemolytic uremic syndrome (aHUS); dense deposit disease (DDD); age-related macular degeneration (AMD); hemolysis, elevated liver enzymes, and low platelets syndrome (HELLP); thrombotic thrombocytopenic purpura (TTP); spontaneous abortion (SAB); Pauci-immune vasculitis; epidermolysis bullosa (EB); recurrent miscarriage (RM); multiple sclerosis (MS); traumatic brain injury (TBI); and injury resulting from myocardial infarction, cardiopulmonary bypass, and hemodialysis. Thus, inhibition of the over- or uncontrolled activation of the complement cascade can provide clinical benefit to patients suffering from such diseases. In addition, complement C3 is also an important target for delaying the progression of geographic atrophy (GA) disease, and developing safe and efficient complement C3 inhibitors has the potential to further better delay the progression of GA disease.

[0006] The first drug molecule targeting complement C3, APL-2 (pegcetacoplan), was approved by the U.S. Food and Drug Administration (FDA) for the treatment of PNH and GA (trade names Empaveli TM and Syfovre TM respectively in May 2021 and March 2023. Pegcetacoplan contains two identical cyclic peptides in its molecule, which are coupled at both ends of a 40 kDa polyethylene glycol polymer (PEG). The cyclic peptides can inhibit the cleavage of C3 by specifically binding to C3 / C3b, inhibiting the binding of C3 to C3 convertase C3bBb / C4bC2a, and thus achieving the purpose of inhibiting complement activation. Empaveli TM is administered by subcutaneous injection, and the recommended dose is 20 mg (1080 mL), administered twice a week. Phase III clinical trials showed that patients receiving Empaveli TM treatment had a change in hemoglobin levels at week 16 that was superior to Soliris (anti-C5 antibody), with an average increase in hemoglobin levels of 3.84 g / dL after administration (p < 0.0001). Syfovre TM is administered by intravitreal injection, and the recommended dose is 15 mg (150 mg / mL; 0.1 mL), administered once every 25 to 60 days. Syfovre TMTwo phase III clinical trials of pegcetacoplan showed that its effect on inhibiting the expansion of retinal atrophy area of the fundus of patients was limited. After intravitreal injection of pegcetacoplan once a month or once every two months, the expansion rate of the area of the fundus lesion of the patients was only delayed by 17-18% and 18-22% at 24 months. The molecular form of pegcetacoplan is a PEGylated cyclic peptide, and PEG is used to improve the pharmacokinetic properties of the cyclic peptide, but it also brings certain safety risks. It is reported that since April 2023, the American Society of Retinal Specialists has received reports of intraocular inflammation after intravitreal injection of pegcetacoplan. As of January 16, 2024, 14 eyes of 13 patients were diagnosed with retinal vasculitis by imaging examination, and 11 eyes were diagnosed with occlusive retinal vasculopathy.

[0007] In order to improve the inhibition of complement activation, the common means in the prior art includes developing new synthetic cyclic peptides or antibody drugs targeting C3 target points to block and inhibit complement activation, but none of them has been clinically proven to have beneficial effects or limited effects. It can be seen that there is still an unmet need for drugs targeting complement C3 with ideal effects in the art. SUMMARY

[0008] In order to overcome the defects of the prior art, the present application provides a new antibody or antigen-binding fragment thereof specifically binding to complement protein C3 / C3b. The inventors of the present application immunize alpacas with human C3 protein as immunogen to produce anti-complement protein C3 / C3b single domain antibody, and establish, screen and select a phage display library of anti-human C3 protein nanobody from peripheral blood mononuclear cells, and accordingly obtain a new antibody or antigen-binding fragment thereof specifically binding to human complement protein C3 / C3b. Through binding property test and classical pathway hemolysis inhibition activity test and alternative pathway hemolysis inhibition activity test, it is shown that the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof of the present application has (1) high affinity to C3 / C3b target antigen; (2) strong complement inhibition activity, which can effectively block the cleavage of C3 and the activation of complement cascade reaction; (3) small molecular weight, under the same mass, single domain antibody has higher molar concentration and can bind more antigen molecules; (4) simple structure, easy to fuse with other target antibodies or antigen-binding fragments thereof, receptor domains or other functional components or compounds to form a fusion construct, which can be potentially applied in the treatment of diseases related to complement activation. Meanwhile, based on the variable region sequence of the antibody or antigen-binding fragment thereof specifically binding to complement protein C3 / C3b, the inventors of the present application obtain new, humanized anti-complement protein C3 / C3b antibodies through humanization modification, which provides a selection of low immunogenicity antibodies for the treatment of corresponding diseases.

[0009] In a first aspect, the present application provides an antigenic epitope peptide of complement protein C3 / C3b, which comprises an antigenic epitope peptide derived from a C345C domain, an MG7 domain, an MG6a domain, an MG6b and / or a TED domain of the complement protein C3 / C3b antigen.

[0010] Preferably, the C345C domain is as shown in SEQ ID NO: 49, the MG7 domain is as shown in SEQ ID NO: 110, the MG6a domain is as shown in SEQ ID NO: 111, the MG6b domain is as shown in SEQ ID NO: 112, and the TED domain is as shown in SEQ ID NO: 113.

[0011] Preferably, the antigenic epitope peptide has a length of 5-20 aa (e.g. 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 aa).

[0012] Preferably, the antigenic epitope peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 100-104 and 114-117.

[0013] In a second aspect, the present application provides a mimetic of the complement protein C3 / C3b antigen, which comprises the antigenic epitope peptide described above.

[0014] Preferably, the mimetic of the complement protein C3 / C3b antigen is not a full-length sequence of the complement protein C3 / C3b antigen.

[0015] In a third aspect, the present application provides a fusion protein, which comprises the antigenic epitope peptide described above and / or the mimetic described above.

[0016] In a fourth aspect, the present application provides a biological material, which comprises:

[0017] 1) a gene encoding the antigenic epitope peptide described above, the mimetic described above and / or the fusion protein described above;

[0018] 2) a vector comprising the gene in 1);

[0019] 3) a host cell comprising the gene in 1) and / or the vector in 2).

[0020] In a fifth aspect, the present application provides an antigen-presenting cell, which comprises the antigenic epitope peptide described above, the mimetic described above, the fusion protein described above, the gene described above and / or the vector described above.

[0021] In a sixth aspect of the present application, there is provided the use of the above-mentioned antigenic epitope peptide, the above-mentioned mimetic, the above-mentioned fusion protein, the above-mentioned biomaterial, and the above-mentioned antigen-presenting cell, which comprises:

[0022] 1) use in screening, detecting, preparing and / or purifying an anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof;

[0023] 2) use in preparing an antigen-antibody complex, which comprises 2-1) the above-mentioned complement protein C3 / C3b antigenic epitope peptide, the above-mentioned mimetic, or the above-mentioned fusion protein, and 2-2) an anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof;

[0024] 3) use in preparing a medicament (preferably a vaccine) and / or a diagnostic reagent for the treatment and / or prevention of a complement protein C3 / C3b antigen-related disease.

[0025] In a seventh aspect of the present application, there is provided an anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof, which is capable of binding to the above-mentioned antigenic epitope peptide and / or the above-mentioned mimetic.

[0026] In an eighth aspect of the present application, there is provided the above-mentioned anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof, which is capable of inhibiting or competing with the binding of an endogenous factor to complement protein C3 / C3b.

[0027] For example, the binding of Factor B to complement protein C3 / C3b is competed, thereby inhibiting the formation of C3 convertase, or the binding of Factor H to complement protein C3 / C3b is competed, thereby inhibiting Factor H-induced degradation of C3b by Factor I.

[0028] In a ninth aspect of the present application, there is provided an anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof, which comprises CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region.

[0029] the amino acid sequence of CDR-H1 comprises any one of the amino acid sequences set forth in TFSSWHM (SEQ ID NO: 21), SDDAVG (SEQ ID NO: 22), SLPMG (SEQ ID NO: 24), VYTMA (SEQ ID NO: 28), X1YX2X3G (SEQ ID NO: 96) or an amino acid sequence that has at least 80% identity to any one of the amino acid sequences set forth in TFSSWHM (SEQ ID NO: 21), SDDAVG (SEQ ID NO: 22), SLPMG (SEQ ID NO: 24), VYTMA (SEQ ID NO: 28), X1YX2X3G (SEQ ID NO: 96);

[0030] the amino acid sequence of CDR-H2 comprises any one of the amino acid sequences set forth in AIYSX4X5X6TNYX7DSVKG (SEQ ID NO: 97), X8ITSGX9RIX 10 YVDSVKG (SEQ ID NO: 98), GFTSGGRTSYVDSVKG (SEQ ID NO: 29), AIYGSGRTNYVDSVKG (SEQ ID NO: 30), AITIGGSTYYADSVKG (SEQ ID NO: 33), SSGSSTSYADSVKG (SEQ ID NO: 37) or an amino acid sequence that has at least 80% identity to any one of the amino acid sequences set forth in AIYSX4X5X6TNYX7DSVKG (SEQ ID NO: 97), X8ITSGX9RIX 10 YVDSVKG (SEQ ID NO: 98), GFTSGGRTSYVDSVKG (SEQ ID NO: 29), AIYGSGRTNYVDSVKG (SEQ ID NO: 30), AITIGGSTYYADSVKG (SEQ ID NO: 33), SSGSSTSYADSVKG (SEQ ID NO: 37) or an amino acid sequence that has at least 80% identity to any one of the amino acid sequences set forth in AIYSX4X5X6TNYX7DSVKG (SEQ ID NO: 97), X8ITSGX9RIX

[0031] the amino acid sequence of CDR-H3 comprises any one of the amino acid sequences set forth in LGEFX 11 GRPP (SEQ ID NO: 99), LGSKSD (SEQ ID NO: 39), QRPPYNTNSLRAAPRYDY (SEQ ID NO: 42), LGSLVNREVLP (SEQ ID NO: 43), LGLPLSHG (SEQ ID NO: 44), LGPKKS (SEQ ID NO: 45), ALWTGGRMGNY (SEQ ID NO: 46) or an amino acid sequence that has at least 80% identity to any one of the amino acid sequences set forth in LGEFX 11an amino acid sequence having at least 80% identity to any one of the amino acid sequences represented by SEQ ID NO: 21-28, SEQ ID NO: 29-37, SEQ ID NO: 38-46, SEQ ID NO: 93-95, SEQ ID NO: 96-99.

[0032] In SEQ ID NO: 96-99, X can be any natural amino acid residue, for example, alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), threonine (T), proline (P), serine (S), tryptophan (W), tyrosine (Y), valine (V).

[0033] In one specific embodiment of the present application, X1in SEQ ID NO: 96 represents T or M; X2represents H or A; X3represents M or K; X4in SEQ ID NO: 97 represents D or G; X5represents S or G; X6represents S or R; X7represents A or V; X8in SEQ ID NO: 98 represents A or G; X9represents L or G; X 10 represents S or N; X 11 represents P, A or S.

[0034] Preferably, the CDR-H1 comprises an amino acid sequence represented by any one of SEQ ID NO: 21-28 or an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 21-28;

[0035] The CDR-H2 comprises an amino acid sequence represented by any one of SEQ ID NO: 29-37, 93-95 or an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 29-37, 93-95;

[0036] The CDR-H3 comprises an amino acid sequence represented by any one of SEQ ID NO: 38-46 or an amino acid sequence having at least 80% identity to any one of SEQ ID NO: 38-46.

[0037] In one embodiment of the present application, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 comprise any one of the following groups (see Table 1 for details):

[0038] A) SEQ ID NO: 21, 29, 38;

[0039] B) SEQ ID NO: 22, 30, 39;

[0040] C) SEQ ID NO: 23, 31, 40;

[0041] D) SEQ ID NO: 23, 32, 41;

[0042] E) SEQ ID NO: 24, 33, 42;

[0043] F) SEQ ID NO: 23, 34, 43;

[0044] G) SEQ ID NO: 25, 35, 44;

[0045] H) SEQ ID NO: 26, 35, 44;

[0046] I) SEQ ID NO: 27, 36, 45;

[0047] J) SEQ ID NO: 28, 37, 46;

[0048] K) SEQ ID NO: 25, 93, 44;

[0049] L) SEQ ID NO: 25, 94, 44;

[0050] M) SEQ ID NO: 25, 95, 44.

[0051] Table 1 CDR sequences of anti-complement protein C3 / C3b antibodies

[0052] Preferably, the amino acid sequences of CDR-H1, CDR-H2 and CDR-H3 are arranged in the order from N-terminal to C-terminal. In the present application, the division of amino acids of antibody CDR region adopts Kabat numbering system.

[0053] The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof has a structure of single-domain antibody, chimeric antibody, Fab fragment, Fab' fragment, Fd fragment, Fv fragment, dAb fragment, F(ab')2 fragment, single-chain antibody or linear antibody.

[0054] The single-domain antibody includes nanobody.

[0055] The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof can be a humanized antibody.

[0056] Preferably, the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof comprises a sequence after humanization.

[0057] In one embodiment of the present application, the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof is a single-domain antibody, which has a higher molar concentration and can bind more antigen molecules than full-length IgG antibodies, Fabs, and scFvs at the same mass.

[0058] Preferably, the amino acid sequence of the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences in SEQ ID NO: 11-20, 51-71, or has at least 80% identity to any one of the amino acid sequences in SEQ ID NO: 11-20, 51-71.

[0059] In one embodiment of the present application, the amino acid sequence of the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof is as shown in any one of the amino acid sequences in SEQ ID NO: 11-20, 51-71.

[0060] The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof can be obtained by using existing conventional technical methods. For example, expression using eukaryotic or prokaryotic expression systems.

[0061] In a tenth aspect of the present application, the use of the above-mentioned anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof is provided, and the use comprises:

[0062] A use in preparing a fusion construct comprising the above-mentioned anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof and other biologically active effector molecules, wherein the other biologically active effector molecules include antibodies or antigen-binding fragments thereof, receptor domains, or other functional components of other targets other than the above-mentioned anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof;

[0063] B use in detecting complement C3 / C3b.

[0064] Preferably, the other functional components include, but are not limited to, one or more combinations of serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, fatty chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and their markers, PEG, complement factors, or Fc fragments.

[0065] Further preferably, the complement factors include complement factor FH, complement factor FI, complement decay accelerating factor CD55, membrane cofactor protein MCP, complement regulatory protein CD59, C4b binding protein C4BP or Cl inhibitor ClINH.

[0066] Preferably, the receptor domains include complement receptor CR1, complement receptor CR2, complement receptor CR3, complement receptor CR4 or immunoglobulin family complement receptor CRIg.

[0067] Preferably, the other targets are selected from VEGFA, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, TGF, Integrin, Integrin receptor, interleukins (such as IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptors (such as IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL10R, IL12R, IL15Rα, IL17R, IL23R, etc.), PCSK9, TNF-α, TNFR, RANKL, complement protein C5, complement factor FB, complement factor FD, complement factor FP, complement protein C1q, complement protein C1s, complement protein C4b, complement protein C2, complement protein MASP2, complement protein MASP3, complement receptor C3aR, complement receptor C5aR1, complement receptor C5aR2, GPCR, GLP1R, CD3, CD105, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD47, CD80, CD86, CD96, CD99, CD111, CD112, CD123, CD133, CD138, CD155, CD171, Claudin 18.2, OX40, ICOS, CTLA4, 4-1BB, TCR, BTLA, TIM-3, LAG3, Galectin-9, PD-L1, PD-L2, PD-1, TIGIT, EGFR, Her2, PSCA, CEA, FAP, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, CS1, ROR1, recombinant Mycobacterium tuberculosis fusion protein, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL 3 or 5T4.

[0068] In an eleventh aspect, the present application provides a fusion construct comprising the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof described above.

[0069] Preferably, the fusion construct further comprises other bioactive effector molecules, which include antibodies or antigen-binding fragments thereof, receptor domains or other functional components targeting other targets other than the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof described above.

[0070] Preferably, the other functional components include, but are not limited to, one or more of serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, fatty chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and their labels, PEG, complement factors or Fc fragments, or combinations thereof.

[0071] Further preferably, the complement factors include complement factor FH, complement factor FI, complement decay accelerating factor CD55, membrane cofactor protein MCP, complement regulatory protein CD59, C4b-binding protein C4BP or Cl inhibitor Cl INH.

[0072] Preferably, the receptor domains include complement receptor CR1, complement receptor CR2, complement receptor CR3, complement receptor CR4 or immunoglobulin family complement receptor CRIg.

[0073] Preferably, the other target is selected from VEGFA, VEGFB, VEGFC, VEGFD, VEGFR, FGF, FGFR, PlGF, PDGF, ANG2, TGF, Integrin, Integrin receptor, interleukin (such as IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), interleukin receptor (such as IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL10R, IL12R, IL15Rα, IL17R, IL23R, etc.), PCSK9, TNF-α, TNFR, RANKL, complement protein C5, complement factor FB, complement factor FD, complement factor FP, complement protein C1q, complement protein C1s, complement protein C4b, complement protein C2, complement protein MASP2, complement protein MASP3, complement receptor C3aR, complement receptor C5aR1, complement receptor C5aR2, GPCR, GLP1R, CD3, CD105, CD19, CD20, CD22, CD25, CD27, CD28, CD30, CD33, CD38, CD40, CD47, CD80, CD86, CD96, CD99, CD111, CD112, CD123, CD133, CD138, CD155, CD171, Claudin18.2, OX40, ICOS, CTLA4, 4-1BB, TCR, BTLA, TIM-3, LAG3, Galectin-9, PD-L1, PD-L2, PD-1, TIGIT, EGFR, Her2, PSCA, CEA, FAP, EGFRVIII, BCMA, PSMA, CA125, EphA2, C-met, L1CAM, CS1, ROR1, recombinant Mycobacterium tuberculosis fusion protein, NY-ESO-1, MUC1, MUC16, mesothelin, LewisY, GPC3, GD2, EPG, DLL 3, or 5T4.

[0074] The antibody or antigen-binding fragment thereof has a structure of a single-domain antibody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single-chain antibody, or a linear antibody.

[0075] Preferably, the fusion construct comprises one or more anti-complement protein C3 / C3b antibodies or antigen-binding fragments thereof, which can be the same or different.

[0076] Preferably, the fusion construct comprises one or more other bioactive effector molecules, which can be the same or different bioactive effector molecules.

[0077] wherein the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof is directly or indirectly linked to the other bioactive effector molecule.

[0078] Preferably, the indirect linkage is via a linker, a functional domain and / or a linker for conjugation.

[0079] wherein the linker is selected from the group consisting of a connecting peptide, an oligopeptide, an oligopeptide polymer, a polypeptide, a polypeptide polymer, PEG, nucleic acid, polysaccharide, a fatty chain, biotin, streptavidin or avidin.

[0080] The functional domain is a combination of one or more of an Fc fragment, serum albumin, a cytokine, transferrin or a scaffold protein.

[0081] The linker for conjugation comprises a functional group linker.

[0082] The functional group linker comprises a thiol, an amino, a hydroxyl and / or a carboxyl reactive group, which allows covalent conjugation between the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof and the bioactive effector molecule.

[0083] Preferably, the direct or indirect linkage is to the N-terminus, C-terminus and / or internal residues of the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof and / or the antibody to the other target.

[0084] The linkage order of the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof and the antibody to the other target in the fusion construct can be that the N-terminus, C-terminus and / or internal residues of one antibody are linked to the N-terminus, C-terminus and / or internal residues of the other antibody.

[0085] Preferably, the fusion construct further comprises a tag.

[0086] Preferably, the tag is linked to the N-terminus or C-terminus of the fusion construct.

[0087] In a twelfth aspect, the present application provides a nucleic acid encoding the above-mentioned antibody or antigen-binding fragment or the above-mentioned fusion construct. For example, the nucleic acid comprises DNA and / or mRNA.

[0088] In some embodiments, the nucleic acid is DNA encoding the above-mentioned antibody or antigen-binding fragment or the above-mentioned fusion construct.

[0089] More preferably, the nucleotide sequence encoding the anti-complement protein C3 / C3b antibody or antigen binding fragment thereof comprises any one of SEQ ID NOs: 1-10, 72-92, or a degenerate sequence thereof, or a nucleotide sequence having at least 80% identity to any one of SEQ ID NOs: 1-10, 72-92, and having the function of encoding the anti-complement protein C3 / C3b antibody or antigen binding fragment thereof.

[0090] In a thirteenth aspect of the present application, a vector is provided, wherein the vector comprises the nucleic acid described above.

[0091] The vector can be expressed in prokaryotic or eukaryotic cells.

[0092] For example, the vector can be introduced into the host cell by a method of transient transfection or stable transfection.

[0093] Preferably, the vector comprises pcDNA3.1, PKS001 or PCMV3.

[0094] In a fourteenth aspect of the present application, a host cell is provided, wherein the host cell comprises the nucleic acid described above or the vector described above.

[0095] The host cell can be a eukaryotic cell or a prokaryotic cell.

[0096] The eukaryotic cell includes animal and plant cells, such as T cells, yeast cells, HEK293 cells, CHO cells, and the like.

[0097] The prokaryotic cell is, for example, Escherichia coli, and the like.

[0098] In a fifteenth aspect of the present application, a method for preparing a host cell is provided, wherein the method comprises introducing the nucleic acid or the vector described above into the host cell.

[0099] In a sixteenth aspect of the present application, a method for preparing an anti-complement protein C3 / C3b antibody or antigen binding fragment thereof or fusion construct is provided, wherein the method comprises culturing the host cell described above to express the anti-complement protein C3 / C3b antibody or antigen binding fragment thereof or the fusion construct.

[0100] In a seventeenth aspect of the present application, a product for treating, preventing and / or diagnosing a disease is provided,

[0101] The product for treating, preventing and / or diagnosing a disease comprises any one of the following:

[0102] A) the anti-complement protein C3 / C3b antibody or antigen binding fragment thereof described above;

[0103] B) the fusion construct described above;

[0104] C) the nucleic acid described above;

[0105] D) the vector described above; or,

[0106] E) the host cell described above.

[0107] In an eighteenth aspect, the present application provides a product for treating, preventing and / or diagnosing a disease, wherein the product comprises any one of:

[0108] a) the antigenic epitope peptide according to any one of claims 1-3;

[0109] b) the mimetic according to claim 4;

[0110] c) the fusion protein according to claim 5;

[0111] d) the biomaterial according to claim 6; or,

[0112] e) the antigen presenting cell according to claim 7.

[0113] Preferably, the product can be a diagnostic kit or a drug or a diagnostic chip, etc.

[0114] The disease is a disease associated with complement activation; preferably, the disease is selected from rheumatoid arthritis; lupus nephritis; immunoglobulin A nephropathy, C3 glomerulonephritis; ischemia-reperfusion injury; paroxysmal nocturnal hemoglobinuria; atypical hemolytic uremic syndrome; dense deposit disease; age-related macular degeneration; geographic atrophy; hemolysis, liver enzyme elevation and thrombocytopenia syndrome; thrombotic thrombocytopenic purpura; spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent miscarriage; multiple sclerosis; traumatic brain injury; and injury caused by myocardial infarction, cardiopulmonary bypass and hemodialysis; CD55-deficient protein loss enteropathy, etc.

[0115] The drug can be an antibody-drug conjugate (ADC), which comprises the anti-complement protein C3 / C3b antibody or antigen-binding fragment or fusion construct of the present application, and other drugs covalently bound thereto.

[0116] In a nineteenth aspect, the use of A)-E) described above in the preparation of a product for treating and / or preventing a disease associated with complement activation, or in the preparation of a diagnostic product or tracer for a disease associated with complement activation.

[0117] In the twentieth aspect of the present application, the use of a)-e) above in the preparation of a product for treating and / or preventing a disease associated with complement activation, or in the preparation of a diagnostic product or tracer for a disease associated with complement activation.

[0118] The disease in the nineteenth and twentieth aspects is a disease associated with complement activation; preferably, the disease is selected from rheumatoid arthritis; lupus nephritis; immunoglobulin A nephropathy, C3 glomerulonephritis; ischemia-reperfusion injury; paroxysmal nocturnal hemoglobinuria; atypical hemolytic uremic syndrome; dense deposit disease; age-related macular degeneration; geographic atrophy; hemolysis, liver enzyme elevation, and thrombocytopenia syndrome; thrombotic thrombocytopenia purpura; spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent spontaneous abortion; multiple sclerosis; traumatic brain injury; and injury caused by myocardial infarction, cardiopulmonary bypass, and hemodialysis; CD55-deficient protein loss enteropathy, and the like.

[0119] The product can be a drug or a vaccine, etc.

[0120] The drug can be an antibody drug conjugate comprising the anti-complement protein C3 / C3b antibody or antigen-binding fragment or fusion construct thereof of the present application, and another drug covalently bound thereto.

[0121] The diagnostic product can be a diagnostic kit or a diagnostic chip.

[0122] Preferably, any of the above products can be screening, detection, diagnosis, purification, tracing, treatment, and / or prevention products, etc.

[0123] In the twenty-first aspect of the present application, a detection method for complement protein C3 / C3b is provided, which comprises combining a sample to be detected with the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof described above, and then detecting the content of the complex formed by complement protein C3 / C3b and the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof.

[0124] The detection method is for detecting the presence or content of complement protein C3 / C3b. The presence indicates qualitative analysis of the presence or absence, and the content can be expression level or protein concentration, etc.

[0125] In the twenty-second aspect of the present application, a method for diagnosing a disease is provided, which comprises taking a sample, combining the sample with the diagnostic product for the disease described above, and detecting the content of the complex formed by complement protein C3 / C3b and the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof.

[0126] The disease is a disease associated with complement activation; preferably, the disease is selected from rheumatoid arthritis; lupus nephritis; immunoglobulin A nephropathy, C3 glomerulonephritis; ischemia-reperfusion injury; paroxysmal nocturnal hemoglobinuria; atypical hemolytic uremic syndrome; dense deposit disease; age-related macular degeneration; geographic atrophy; hemolysis, liver enzymes elevation, and low platelet syndrome; thrombotic thrombocytopenic purpura; spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent spontaneous abortion; multiple sclerosis; traumatic brain injury; and injury caused by myocardial infarction, cardiopulmonary bypass, and hemodialysis; CD55-deficient protein loss enteropathy, and the like.

[0127] In a twenty-third aspect of the present application, there is provided a method of treating and / or preventing a disease, the method comprising administering to an individual the above-mentioned product for treating and / or preventing a disease.

[0128] The disease is a disease associated with complement activation; preferably, the disease is selected from rheumatoid arthritis; lupus nephritis; immunoglobulin A nephropathy, C3 glomerulonephritis; ischemia-reperfusion injury; paroxysmal nocturnal hemoglobinuria; atypical hemolytic uremic syndrome; dense deposit disease; age-related macular degeneration; geographic atrophy; hemolysis, liver enzymes elevation, and low platelet syndrome; thrombotic thrombocytopenic purpura; spontaneous abortion; pauci-immune vasculitis; epidermolysis bullosa; recurrent spontaneous abortion; multiple sclerosis; traumatic brain injury; and injury caused by myocardial infarction, cardiopulmonary bypass, and hemodialysis; CD55-deficient protein loss enteropathy, and the like.

[0129] The present application provides a new anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof, which has the potential of lower immunogenicity in the human body based on the advantages of single-domain antibodies themselves. The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof provided by the present application can completely inhibit the cleavage of C3 by inhibiting the assembly of C3 convertase C3bBb, thereby inhibiting the complement cascade. The complement inhibitory activity of the series of single-domain antibodies is stronger than the active monomer component APL-1 of the current marketed product Pegcetacoplan, i.e., a single cyclic peptide molecule. Pegcetacoplan is the only drug targeting C3 approved for marketing worldwide for the treatment of complement-related diseases, and its molecular form is a PEGylated cyclic peptide. Due to the limited complement inhibitory activity of the drug, the clinical use has a high concentration of drug administration, a short half-life, and a high frequency of drug administration. Compared with the PEGylated cyclic peptide form, the single-domain antibody molecule of the present application has small molecular weight, good water solubility, is easy to produce, has simple structure, and is easy to construct a fusion construct with other target antibodies or antigen-binding fragments thereof, receptor domains, or other functional components or compounds, and can be potentially applied to the treatment of complement-related diseases.

[0130] In particular, the present application provides a series of anti-complement protein C3 / C3b single domain antibodies with high complement protein C3 / C3b binding affinity (KD= 12.9-77.3 pM by SPR test; EC 50 =70-540 pM), while further humanized single domain antibodies have similar or better performance. In C3 convertase assembly and C3 cleavage inhibition activity experiments, the series of single domain antibodies can completely inhibit C3 cleavage by inhibiting the assembly of C3 convertase C3bBb. In hemolysis inhibition experiments, the series of single domain antibodies can inhibit the complement cascade activated by the classical pathway CP and the alternative pathway AP to inhibit hemolysis, and the activity is better than APL-1. Some of the anti-complement protein C3 / C3b single domain antibodies provided by the present application bind to the MG6a-MG7-C345C or MG6b-MG6a-MG7-TED domain of complement protein C3 / C3b, and can compete with factor B to bind C3b to inhibit the formation of C3 convertase.

[0131] The "C3 / C3b" in the "anti-complement protein C3 / C3b antibody or antigen binding fragment thereof" described in the present application refers to the relationship between C3 and / or C3b, and "C3C3b" in the present application also refers to C3 and / or C3b.

[0132] The "fusion construct" described in the present application defines the fusion of the anti-complement protein C3 / C3b antibody or antigen binding fragment thereof of the present application with another compound. The fusion construct can comprise one or more anti-complement protein C3 / C3b antibodies or antigen binding fragments thereof, and the plurality of anti-complement protein C3 / C3b antibodies or antigen binding fragments thereof can be the same or different. The fusion construct can comprise one or more additional compounds, and the plurality of additional compounds can also be the same or different. The compound can be a protein compound or a non-protein compound. In the case of a protein compound or a fusion construct comprising only a plurality of anti-complement protein C3 / C3b antibodies or antigen binding fragments thereof, the fusion construct can also be referred to as a fusion protein. In the case of a compound being fused to an anti-complement protein C3 / C3b antibody or antigen binding fragment thereof in a conjugated form, the fusion construct can also be referred to as a conjugate.

[0133] The "medicament" of the present application can be used to treat a human or a non-human animal, such as a non-human mammal. The medicament can comprise a pharmaceutically acceptable carrier, adjuvant or salt commonly known in the art. The medicament can be administered by any suitable route, such as a gastrointestinal route (e.g., oral) or a non-gastrointestinal route (e.g., intravenous, intramuscular, subcutaneous, intradermal, intraorgan, intranasal, intraocular, instillation, intracerebral, intrathecal, transdermal, intrarectal, etc.). The medicament can be in any suitable dosage form, such as a gastrointestinal dosage form or a non-gastrointestinal dosage form, preferably including but not limited to tablets, pills, powders, granules, capsules, lozenges, syrups, liquids, emulsions, microemulsions, suspensions, injections, sprays, aerosols, powder sprays, lotions, ointments, plasters, pastes, patches, eye drops, nose drops, sublingual tablets, suppositories, aerosols, effervescent tablets, dripping pills, gels, etc. The various dosage forms of the medicament can be prepared according to conventional methods in the pharmaceutical field. The medicament can contain the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, etc. of the present application in an amount of 0.01-99.5% (e.g., 0.01%, 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%) by weight. The medicament can be prepared as a reagent having a protein concentration of 1-300 mg / mL (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300 mg / mL). The single administration dose of the medicament can be 0.1-1000 mg, such as 0.1, 0.2, 0.5, 0.75, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 3, 5, 10, 20, 50, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 mg.

[0134] The "pharmaceutically acceptable" of the present application means neither significantly stimulating the organism nor inhibiting the biological activity and properties of the active substance of the administered product.

[0135] The "method" or "application" subject matter described in the present application can be replaced by each other, and can be for the purpose of diagnosis, prevention and / or treatment of diseases, or for the purpose of diagnosis, prevention and treatment of non-diseases.

[0136] The "cell" involved in the present application includes cells that can develop into individuals or cannot develop into individuals.

[0137] The "antigen binding fragment" described in the present application is a part of the antibody that retains the specific binding activity of the antibody, that is, any part of the antibody can specifically bind to the epitope on the target molecule of the antibody. It includes, for example, Fab, Fab', F(ab')2, Fv, Fd and variants of these fragments. For example, the heavy chain and / or light chain of the antibody, the heavy chain variable region and / or the light chain variable region of the antibody, or a single or more than 2 CDRs from the heavy chain or light chain of the antibody. Among them,

[0138] Single-domain antibody refers to the variable domain of heavy chain (VHH) of antibody, and the antibody has independent antigen binding activity. Single-domain antibody includes nanobody.

[0139] Chimeric antibody refers to an antibody in which part of the heavy chain and / or light chain is derived from a specific source or species, and the rest of the heavy chain and / or light chain is derived from a different source or species.

[0140] Single-chain antibody is an antibody formed by connecting the variable domain of heavy chain and the variable domain of light chain through a linker.

[0141] Fab, that is, a monovalent fragment composed of VL, VH, CL and CH1 domains.

[0142] Fab', that is, a Fab fragment with one or more cysteine residues at the C-terminal of the CH1 domain.

[0143] F(ab')2, that is, a bivalent fragment containing two Fab fragments connected by a disulfide bond in the hinge region.

[0144] Fd, Fd fragment composed of VH and CH1 domains.

[0145] Fv, Fv fragment composed of VL and VH domains of a single arm of antibody.

[0146] dAb fragment, antibody fragment composed of VH domain.

[0147] The linear antibody of the present application comprises one or more pairs of tandem antibody fragments, which can be Fd segments (VH-CH1), single-chain antibodies (scFv), antibody fragments (Fab), or single-domain antibodies (VHH), which are linked together by a linker peptide to form a continuous antibody structure.

[0148] wherein VH represents the variable region of the heavy chain, VL represents the variable region of the light chain, CH represents the constant region of the heavy chain, and CL represents the constant region of the light chain.

[0149] The “comprising” or “including” of the present application is an open-ended writing, when used to describe the sequence of a protein or nucleic acid, the protein or nucleic acid can be composed of the sequence, or can have additional amino acids or nucleotides at one end or both ends of the protein or nucleic acid, but still has the same or similar activity as the original sequence.

[0150] The “homology” or “identity” of the present application refers to the use of protein sequences or nucleotide sequences, and the skilled person in the art can adjust the sequence according to the actual work needs, so that the use sequence has (including but not limited to) 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% homology / identity compared with the sequence obtained by prior art.

[0151] The “humanized antibody” of the present application refers to the framework region and / or constant region part (such as CH region) of the antibody or all parts of the antibody encoded by human antibody genes.

[0152] The "individual" of the present application can be a human or a non-human mammal, which can be a wild animal, a zoo animal, an economic animal, a pet, a laboratory animal, and the like. Preferably, the non-human mammal includes, but is not limited to, a pig, a cow, a sheep, a horse, an ass, a fox, a raccoon dog, a mink, a camel, a dog, a cat, a rabbit, a mouse (for example, a rat, a mouse, a guinea pig, a hamster, a gerbil, a chinchilla, a squirrel), or a monkey, and the like.

[0153] The "treatment" of the present application means to slow down, interrupt, stop, control, reduce, or reverse the progression or severity of a sign, a symptom, a disorder, a disease, or a disease-related sign, symptom, disorder, or the like, after the disease has started to develop, but does not necessarily involve the complete elimination of all disease-related signs, symptoms, disorders, or the like.

[0154] The "prevention" of the present application means a mode implemented to prevent or delay the occurrence of a disease or a disorder or a symptom in the body.

[0155] The "diagnosis" of the present application means to ascertain whether a patient has had, has, or will have a disease or a disorder, or to ascertain the progression or possible future progression of a disease. BRIEF DESCRIPTION OF DRAWINGS

[0156] Hereinafter, embodiments of the present application will be described in detail with reference to the accompanying drawings.

[0157] Figure 1: shows ELISA results of anti-complement protein C3 / C3b Nanobodies binding to human C3 (Figure 1A) and human C3b (Figure IB).

[0158] Figure 2: shows binding and dissociation curves of anti-complement protein C3 / C3b Nanobodies to complement protein C3 (Figure 2A) or C3b (Figure 2B) in SPR affinity testing.

[0159] Figure 3: shows results of anti-complement protein C3 / C3b Nanobodies on alternative pathway (Figure 3A) and classical pathway (Figure 3B) complement inhibitory activity.

[0160] Figure 4: shows results of anti-complement protein C3 / C3b Nanobodies on inhibition of C3 convertase C3bBb assembly and C3a release.

[0161] Figure 5: shows results of NB472 humanized antibody on classical pathway (Figure 5A) and alternative pathway (Figure 5B) complement inhibitory activity.

[0162] Figure 6: shows results of NB090 humanized antibody on classical pathway (Figure 6A) and alternative pathway (Figure 6B) complement inhibitory activity.

[0163] Figure 7: shows results of NB071 humanized antibody on classical pathway (Figure 7A) and alternative pathway (Figure 7B) complement inhibitory activity.

[0164] Figure 8: shows the binding and dissociation curves of humanized anti-complement protein C3 / C3b Nanobodies to C3 (Figure 8A) and C3b (Figure 8B) in SPR affinity test.

[0165] Figure 9: shows the molecular docking results of NB090-C3 (Figure 9A), NB090-C3b (Figure 9B), NB071-Hz6a-C3 (Figure 9C), NB071-Hz6a-C3b (Figure 9D); Figure 9E-9H are the molecular dynamics simulation results of the above complexes, respectively.

[0166] Figure 10: shows the ELISA binding results of anti-complement protein C3 / C3b Nanobody NB090 to C345C domain of C3.

[0167] Figure 11: shows the functional verification results of anti-complement protein C3 / C3b Nanobody NB090 binding to MG6a-MG7-C345C domain, including competing with Factor B for binding to C3b (Figure 11A), inhibiting Factor B cleavage (Figure 11B), and inhibiting H factor-induced C3b degradation (Figure 11C). DETAILED DESCRIPTION

[0168] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0169] Example 1: Immunization of Llama and Construction of Anti-complement Protein C3 / C3b Nanobody Phage Library

[0170] 1.1 Llama Immunization

[0171] In this embodiment, human C3 protein (purchased from Comptech, item number: A113) was used as an immunogen to immunize a llama to produce anti-complement protein C3 / C3b Nanobodies. Specifically, two adult llamas over 8 months old were selected for immunization at 1 mg of human C3 protein per animal per time. During the immunization process, 1 mg of human C3 protein was mixed and emulsified with Freund's complete adjuvant, and multiple point subcutaneous immunization injections (more than 8 points) were performed on the back of the llama. After the first immunization, the second, third, and fourth immunizations were performed at intervals of 14 days, respectively. Blood was collected 7 days after each immunization, and the serum was separated by centrifugation for ELISA titer detection. After the fourth immunization, ELISA detection was performed to determine that the titers of the two animals both reached the requirements for library construction, and 80 mL of peripheral blood was collected from each animal for mononuclear cell separation and phage library construction.

[0172] 1.2 Construction of anti-complement protein C3 / C3b Nanobody phage library

[0173] The peripheral blood of alpaca was extracted, and total RNA was extracted from the separated alpaca PBMC using Trizol, and cDNA was reversely transcribed. The variable region VH fragment (about 700 bp in length) was amplified using nanobody specific primers, and amplified twice. The above obtained fragment and pcomb3X vector were digested with Sfil enzyme (purchased from: NEB, item number: R0123L), and mixed in appropriate proportions after digestion, and connected with T4 ligase (purchased from: Vazyme, item number: C301). After connection, it was used for electric transformation of XL1-Blue competent cells. According to the colony growth of the competent cell dilution on the resistance plate, the transformation library capacity of the alpaca was calculated: the first alpaca was 3.58*10 8 , and the second alpaca was 7.9*10 8 .

[0174] Example 2 Phage display panning and screening of anti-complement protein C3 / C3b nanobodies

[0175] 2.1 Phage library panning

[0176] In this example, solid phase method was used to wash the binding activity of phage library (binding human complement protein C3, human complement protein C3b, cynomolgus monkey complement protein C3). Specifically, the binding activity panning process was as follows:

[0177] 1) High affinity enzyme-labeled plate was used to coat human complement protein C3 (purchased from: Comptech, item number: A113), human complement protein C3b (purchased from: Comptech, item number: A114) and cynomolgus monkey complement protein C3 (purchased from: Yiqioshenzhou, item number: 90944-C08H) overnight, and after washing the plate with 0.1% PBST, 3% skimmed milk powder was used for blocking at room temperature for 1 h, and the blocking solution was discarded and the plate was washed for use.

[0178] 2) About 5*10 11 number of libraries were added to the above blocked antigen plate, and incubated with the antigen at 37°C for 2 h.

[0179] 3) The plate was washed with 0.1% PBST for 6 times.

[0180] 4) Glycine-HCl was used to elute the bound liquid, and then Tris-HCl was used to adjust the pH to 7.4.

[0181] 5) The above eluate was mixed with E. coli XL1-Blue, and incubated at 37°C for 30 min, and then shaken and cultured.

[0182] 6) Rescue phage and incubate overnight at 30°C with shaking to obtain amplified 1st library.

[0183] 7) Repeat the process of 1) to 6) to obtain subsequent round libraries.

[0184] The amplified libraries obtained by enrichment under the condition of coating human complement protein C3, human complement protein C3b and cynomolgus monkey complement protein C3 were diluted and subjected to antigen binding ELISA test, all of which were positive, as shown in Table 2, indicating that phages binding to human complement protein C3 and human complement protein C3b were enriched, and phage monoclonal binding screening can be carried out.

[0185] Table 2 Detection results of phage libraries obtained by enrichment under different selection conditions for binding to human C3, human C3b and monkey C3

[0186] 2.2 Phage monoclonal screening and sequencing

[0187] After the panning library infects XL1-blue, single clone colonies are selected from the colony plate, and positive clones are screened by detecting the ELISA binding activity of phage expression supernatant to human complement protein C3 and / or human complement protein C3b. Specifically, the experimental procedure is as follows:

[0188] 1) Coat human complement protein C3 and human complement protein C3b at a protein concentration of 0.5 μg / mL at 4°C overnight for ELISA binding activity detection.

[0189] 2) Blocking: 3% milk powder, 200 μL / well, incubate at room temperature for 1 hour.

[0190] 3) Add 10-fold diluted phage expression supernatant, 100 μL / well, incubate at room temperature for 1 hour.

[0191] 4) Add detection antibody: anti-M13 antibody (purchased from Yiqiao God, item number: 11973-MM05T-H), concentration is 0.2 μg / mL, 100 μL / well, incubate at room temperature for 1 hour.

[0192] 5) Add TMB developing solution 200 μL / well, develop for 20 minutes, add stop solution 50 μL / well. Detect OD450 by microplate reader.

[0193] After DNA sequencing and codon translation, the amino acid sequences of the series of nanobodies are obtained, a total of 115 unique sequences, Table 3 and Table 4 provide the full-length nucleic acid sequences and amino acid sequences of representative nanobodies, and the CDR region (CDR-H1, CDR-H2, CDR-H3) amino acid sequences defined according to the kabat principle are shown in Table 5.

[0194] Table 3 Nucleotide sequences of anti-complement protein C3 / C3b Nanobodies

[0195] Table 4 Amino acid sequences of anti-complement protein C3 / C3b Nanobodies

[0196] Table 5 Amino acid sequences of CDR regions of anti-complement protein C3 / C3b Nanobodies

[0197] Example 3 Expression of anti-complement protein C3 / C3b Nanobodies

[0198] The DNA sequences encoding each Nanobody were obtained by gene synthesis with a secretion signal peptide MDAMKRGLCCVLLLCGAVFVSPS (SEQ ID NO: 47) added at the N-terminus of the Nanobody, a linker GGGGS (SEQ ID NO: 48) and 6xHis added at the C-terminus of the Nanobody, and then ligated into the expression vector pCDNA3.1(+) through the restriction sites NheI and XhoI. The plasmids were sequenced correctly and endotoxin-free extracted in large quantities. The plasmids were transfected into suspension 293F cells (purchased from Gibco, Cat# 11-625-019) for transient expression using PEI (purchased from Polysciences, Cat# 02371). The cell culture supernatant was used to purify the target protein using a Ni-NTA affinity chromatography column. The protein purity was detected by reducing and non-reducing SDS-PAGE after purification.

[0199] Example 4 Detection of binding activity of anti-complement protein C3 / C3b Nanobodies

[0200] ELISA method was used to detect the activity of the series of representative nanobodies binding to human complement protein C3 and human complement protein C3b. Specifically, human C3 (purchased from: Comptech, item number: A113c) or C3b (purchased from: Comptech, item number: A114) was diluted to 2 μg / ml with coating buffer (purchased from: Yuan Ye Bio, item number: R20934) and added to an enzyme-labeled plate, 100 μL / well, 4°C coated overnight. After blocking with 5% skim milk and PBST (0.1% Tween 20 in PBS, pH = 7.4) washing, 100 μL of nanobody concentration gradient dilution was then added, 37°C incubated for 1 hour. After washing the plate with PBST, 1:7000 diluted HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, item number: HRP-66005) was added, 25°C incubated for 45 minutes. After PBST washing the plate, 100 μL TMB (purchased from: Tian Gen Biochemical, item number: PA107-01) was added per well, 37°C color development for 15 minutes, then 100 μL of ELISA stop solution (purchased from: Solaibao, item number: C1058) was added. The absorbance (OD450) at 450 nm was measured by a microplate reader (Thermo Scientific, model: Multiskan SkyHigh).

[0201] The ELISA results of the anti-complement protein C3 / C3b nanobodies binding to C3 or C3b are shown in FIG. 1A and FIG. 1B, and the binding curves were fitted by four parameters to obtain the EC 50 values are shown in Table 6. The results show that under the experimental conditions of this example, each different anti-complement protein C3 / C3b nanobody has a high affinity for C3 or C3b antigen, and the EC 50 values are between 0.07-0.54 nM.

[0202] Table 6 EC 50 values of anti-complement protein C3 / C3b nanobodies binding to C3

[0203] The binding kinetics of anti-C3 nanobody NB082 to human C3 and human C3b were detected by surface plasmon resonance (SPR) method. Specifically, using Biacore 8K instrument (Cytiva), human-C3 or C3b antigen proteins (Comptech) were captured by CM5 chip. The test antibody was diluted with flow phase buffer HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20). The antigen immobilized chip was combined with different concentrations of antibody molecules, the binding time was 120 seconds, and the dissociation time was 150 seconds. The binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of the complement protein C3 / C3b binding agent to human-C3 / C3b were obtained by fitting with “1:1 binding” model. D The results are shown in Figures 2A-2B and Table 7. The results show that the anti-complement protein C3 / C3b nanobody NB082 has high affinity to human C3 and C3b proteins, and the affinity (K -11 ) to human C3 protein is 7.73 x 10 D M, and the affinity (K -11 ) to human C3b protein is 5.79 x 10

[0204] Table 7. Results of anti-complement protein C3 / C3b nanobody binding kinetics detection

[0205] Example 5. Inhibition activity of anti-complement protein C3 / C3b nanobody on C3 activation

[0206] 5.1 Hemolysis inhibition effect on alternative pathway and classical pathway

[0207] In this example, in vitro red blood cell hemolysis experiment was used to test the hemolysis inhibition effect of anti-C3 / C3b nanobody on alternative pathway and classical pathway.

[0208] For alternative pathway hemolysis activity test, GVB 0GVB-Mg-EGTA for cell washing and sample preparation. Take the appropriate amount of rabbit red blood cells, wash three times with an equal volume of GVB-EGTA-Mg, then resuspend the cells with twice the volume of GVB-EGTA-Mg, and store at 4°C. Prepare a concentration gradient dilution of anti-complement protein C3 / C3b nanobodies with GVB-EGTA-Mg. Mix the antibody dilution with human serum (purchased from Gemini Bio, item number: 100-512) in equal volumes, then add the prepared rabbit red blood cells, invert and mix, and incubate at 37°C for 30 minutes. Set up negative control, blank control, and positive control groups for each experiment. The negative control group is the experimental system without adding antibody dilution, to detect the hemolysis induced by human serum; the blank control group is the experimental system with only rabbit red blood cells and GVB-Mg-EGTA, to detect the experimental background value; the APL-1 (purchased from MedChemExpress, item number: HY-P3204) treatment group is used as the positive control group, and APL-1 is the monovalent active ingredient of the marketed drug APL-2. Then, add EDTA to each sample to terminate the reaction, and centrifuge at 4°C for 5 minutes. Take 100 μL of supernatant from each sample into a 96-well enzyme plate, and use an enzyme labeler (Thermo Scientific, model: Multiskan SkyHigh) to measure the absorbance at 412 nm. The following calculation method is used to calculate the hemolysis inhibition rate:

[0209] Plot the antibody concentration and inhibition rate and perform four-parameter fitting to calculate the IC 50 value.

[0210] The results of the anti-complement protein C3 / C3b nanobody test for alternative pathway hemolytic activity are shown in Figure 3A and Table 8. Nearly 10 molecules such as NB472 and NB082 have good inhibitory activity in the alternative pathway activated hemolysis experiment, which is better than the positive control APL-1.

[0211] Table 8 Anti-complement protein C3 / C3b nanobody alternative pathway hemolytic activity test

[0212] For classical pathway-dependent hemolytic activity testing, take GVB ++ (purchased from Comptech, item number: B102) for cell washing and sample preparation. Take an appropriate amount of sheep red blood cells, centrifuge at 800 rcf for 5 min, and wash with an equal volume of GVB ++Resuspend the cells, repeating the process three times. Sensitize the resuspended sheep red blood cells with hemolysin (purchased from Yuanye Biotechnology, catalog number: S25861-10), then store at 4°C for later use. Use GVB ++ A gradient dilution buffer was prepared for the anti-complement protein C3 / C3b nanobody. The antibody dilution buffer was mixed with human serum (GeminiBio, catalog number: 100-512), followed by the addition of 100 μL of sensitized sheep erythrocytes. The mixture was inverted and incubated at 37°C for 60 minutes. The sample was inverted and mixed every 20 minutes. A negative control group and a blank control group were included in each experiment. The negative control group consisted of the experimental system without antibody dilution buffer to detect hemolysis induced by human serum, while the blank control group consisted of only sensitized sheep erythrocytes and GVB. ++ The experimental system was used to detect background values. After 60 minutes of incubation, EDTA was added to each sample to stop the reaction, and the samples were centrifuged at 4°C for 5 minutes. 100 μL of supernatant from each sample was transferred to a 96-well microplate, and the absorbance at 412 nm was measured using a Thermo Scientific Multiskan SkyHigh microplate reader. The hemolysis inhibition rate was calculated using the following method:

[0213] The antibody concentration versus inhibition rate was plotted as a curve and fitted with four parameters to calculate the IC50. 50 value.

[0214] Results of classical pathway CP hemolytic activity assay and IC50 of anticomplement protein C3 / C3b nanobodies 50 As shown in Figure 3B and Table 9, nearly 10 molecules, including NB472 and NB082, exhibited good inhibitory activity in the hemolysis experiment activated by the classical pathway, which was superior to the positive control APL-1.

[0215] Table 9. Hemolytic activity assay of anticomplement protein C3 / C3b nanobodies via the classical pathway CP.

[0216] 5.2 Inhibitory effect on C3 cleavage and C3a formation in vitro

[0217] This embodiment uses an in vitro assembly method of C3 convertase C3bBb to test the ability of anti-C3 nanobodies NB472, NB090, and NB071 to inhibit the in vitro cleavage of C3 to generate C3a. Specifically, GVB was used... 0(Comptech, Cat. No. B103) and Mg-EGTA (Comptech, Cat. No. B106) were used for the convertase and sample preparation. Human complement C3b (Comptech, Cat. No. A114) was diluted to 200 nM in GVB-Mg-EGTA and then the concentration gradient dilutions of Nanobodies were added and incubated for 30 min at 25 °C. The concentration gradient dilutions of anti-complement C3 / C3b Nanobodies were prepared in GVB-Mg-EGTA. 200 nM FB (Comptech, Cat. No. A135), 2 nM FD (Comptech, Cat. No. A136) prepared in GVB-Mg-EGTA were added to the C3-antibody system and incubated for 15 min at 25 °C. Human complement C3 (Comptech, Cat. No. A113c) was diluted to 200 nM in GVB-Mg-EGTA and then added to the reaction system and incubated for 30 min at 25 °C. Rabbit serum (Sambecqia Bio, Cat. No. SBJ-O0009) diluted in GVB-Mg-EGTA was added 50 μL and incubated for 15 min at 25 °C to convert C3a cleaved by C3 convertase to the more stable C3a-desArg. The reaction was terminated by the addition of EDTA and then the C3a-desArg concentration was measured by ELISA. Control groups included: 1) a blank control group containing only C3 to detect the background value of the experiment, and 2) a negative control group containing a mixture of C3 and C3 convertase to detect the cleavage ability of C3 convertase on C3 in the absence of an inhibitor.

[0218] For ELISA measurement of C3a-desArg concentration, C3a / C3a des-Arg antibody (purchased from: GeneTex, Cat#GTX78198) was diluted to 1 pg / mL with coating buffer (purchased from: YuanYe Biotech, Cat#R20934) and added to the enzyme-labeled plate, 100 pL / well, 4°C coated overnight. After blocking with 5% skim milk powder and PBST (0.1% Tween20 in PBS, pH=7.4) washing, 100 pL of samples from C3 / C3 convertase / antibody reaction were added, and incubated at 37°C for 60 minutes. After washing the plate with PBST, 100 pL of 0.5 pg / mL Biotin anti-human C3a / C3a(desArg) / C3 Antibody (purchased from: BioLegend, Cat#518002) was added, and incubated at 37°C for 60 minutes. After washing the plate with PBST, 100 pL of 0.5 pg / mL HRP-labeled streptavidin (purchased from: Shenguo Biotech, Cat#D111054-0001) was added, and incubated at 25°C for 45 minutes. After washing the plate, 100 pL of TMB (purchased from: Tiangeng Biochemical, Cat#PA107-01) was added to each well, and incubated at 37°C for 15 minutes. After that, 100 pL of ELISA stop solution (purchased from: Solabio, Cat#C1058) was added. The absorbance (OD450) at 450 nm was measured by a microplate reader (Thermo Scientific, Model: Multiskan SkyHigh). The inhibition rate was calculated using the following formula:

[0219] The antibody concentration and inhibition rate were plotted and fitted with a four-parameter curve to calculate the IC 50 value.

[0220] The results of anti-complement protein C3 / C3b nanobodies inhibiting C3 convertase C3bBb assembly and C3a generation are shown in Figure 4 and Table 10. The results show that nanobodies NB472, NB090 and NB071 exhibit a clear dose-effect relationship in inhibiting C3 convertase C3bBb assembly and C3a generation, with IC 50 values between 104.5-163.2 nM, which can completely or nearly completely inhibit C3a release (maximum inhibition rate >99%).

[0221] Table 10 Anti-complement protein C3 / C3b nanobodies inhibiting C3 convertase C3bBb assembly and C3a generation test

[0222] Example 6 Humanization of anti-complement protein C3 / C3b nanobodies

[0223] 6.1 Humanization of anti-complement protein C3 / C3b nanobodies

[0224] In this embodiment, the camel-derived nanobodies NB472, NB090 and NB071 screened in Example 2 were humanized by CDR grafting. Specifically, the homology of the candidate antibody sequences with human antibody sequences was compared by antibody database IGBLAST and IMGT, and then the high-homology human antibody framework region sequences were combined with the three CDR region sequences of the candidate antibodies to construct humanized antibodies. In some selected frameworks, back mutations to the parent sequences were introduced to design antibody sequences with different degrees of humanization.

[0225] Based on the respective parent sequences of NB472, NB090 and NB071, antibody sequences with different degrees of humanization were designed, and the amino acid and nucleotide sequence numbers are shown in Tables 11, 12 and 13.

[0226] After gene synthesis, plasmid construction and suspension 293F cell transient expression, humanized anti-complement protein C3 / C3b nanobodies were obtained.

[0227] Table 11 Summary of humanized anti-complement protein C3 / C3b antibody NB472 design

[0228] Table 12 Summary of humanized anti-complement protein C3 / C3b antibody NB090 design

[0229] Table 13 Summary of humanized anti-complement protein C3 / C3b antibody NB071 design

[0230] 6.2 Detection of the inhibitory activity of humanized nanobodies on complement pathway activation

[0231] In this embodiment, the classical pathway hemolysis inhibition activity and the alternative pathway hemolysis inhibition activity of the humanized nanobodies were evaluated using the method described in Example 5; the ELISA binding activity of the humanized antibodies to human C3 / C3b was evaluated using the ELISA method described in Example 4, and comparative analysis was performed with the parent antibodies.

[0232] The results showed that for the humanized NB472 antibody molecules, in the ELISA binding activity experiment, the binding activity of each humanized NB472 antibody molecule and the parent molecule to human C3 / C3b was comparable. The classical pathway hemolysis activity test results of the NB472 humanized antibody molecules are shown in Figure 5A and Table 14, and the alternative pathway hemolysis activity test results are shown in Figure 5B and Table 15. The results showed that the activity of the NB472 humanized nanobodies was basically comparable to that of the NB472 parent antibody, and the hemolysis inhibition activity of NB472-Hz5 was superior to that of the NB472 parent antibody.

[0233] Table 14 Inhibition of classical pathway hemolysis by NB472 humanized antibody molecules

[0234] Table 15 Inhibition of alternative pathway hemolysis by NB472 humanized antibody molecules

[0235] For humanized NB090 antibody molecules, the binding activity of humanized NB090 to human C3 / C3b was comparable to the parent molecule in ELISA binding activity experiments. The results of the classical pathway hemolysis activity test of NB090 humanized antibodies are shown in Figure 6A and Table 16, and the results of the alternative pathway hemolysis activity test are shown in Figure 6B and Table 17. The results show that the activity of NB090 humanized nanobodies is essentially comparable to the activity of NB090 parent antibody, with the hemolysis inhibitory activity of NB090-Hz3b, NB090-Hz3d being superior to that of the NB090 parent antibody.

[0236] Table 16 Inhibition of classical pathway hemolysis by NB090 humanized antibodies

[0237] Table 17 Inhibition of alternative pathway hemolysis by NB090 humanized antibodies

[0238] For humanized NB071 antibody molecules, the binding activity of humanized NB071 antibody molecules to human C3 / C3b was comparable to the parent antibody in ELISA binding activity experiments. The results of the classical pathway hemolysis activity test of NB071 humanized antibodies are shown in Figure 7A and Table 18, and the results of the alternative pathway hemolysis activity test are shown in Figure 7B and Table 19. The results show that the activity of NB071 humanized nanobodies is essentially comparable to the activity of NB071 parent antibody, with the hemolysis inhibitory activity of NB071-Hz6a, NB071-Hz6b, NB071-Hz6d being superior to that of the NB071 parent antibody.

[0239] Table 18 Inhibition of classical pathway hemolysis by NB071 humanized antibodies

[0240] Table 19 Inhibition of alternative pathway hemolysis by NB071 humanized antibodies

[0241] 6.3 Affinity analysis of humanized nanobodies to human C3 / C3b

[0242] The affinity of humanized Nanobody NB071-Hz6a to human C3 / C3b was determined using surface plasmon resonance (SPR) method. Biacore 8K instrument (Cytiva) was used in the experiment, and CM5 chip was used to capture human C3 (purchased from Comptech, Cat. No. A113c) or C3b (purchased from Comptech, Cat. No. A114). The antibody to be tested was diluted with the running buffer HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.005% v / v Tween 20). The antigen immobilized chip was subjected to binding experiments with different concentrations of antibody molecules, with a binding time of 150 seconds and a dissociation time of 200 seconds. The binding rate (ka), dissociation rate (kd) and equilibrium dissociation constant (K D ) of humanized antibody NB071-Hz6a to human C3 / C3b were obtained by fitting the “1:1 binding” model, and the results are shown in Table 20 and Figures 8A, 8B. The results show that humanized antibody molecule NB071-Hz6a has high affinity to both human C3 and C3b proteins, with an affinity (K D ) of 1.29 x 10 -11 M to human C3 protein and an affinity (K D ) of 1.49 x 10 -11 M to human C3b protein.

[0243] Table 20. Results of binding kinetics detection of humanized Nanobody NB071-Hz6a to human C3 and C3b

[0244] Example 7. Epitope analysis of anti-complement protein C3 / C3b Nanobodies

[0245] 7.1. Formation of complex and epitope analysis

[0246] This example uses the method of chemical cross-linking mass spectrometry to deduce the binding epitope of anti-complement protein C3 / C3b Nanobodies or humanized Nanobodies NB090, NB071-Hz6a to C3, C3b. Specifically, the operation process is as follows:

[0247] 1) Mix the antibody with 0.5 mg / mL C3b or C3 at a molar ratio of 2:1, and incubate at 4°C for 1 h to obtain a complex.

[0248] 2) Add 12.5 mM BS3 (Cat. No. C15178241, Macklin) to cross-link the complex, and incubate at room temperature for 30 min.

[0249] 3) Add 50 mM tris-HCl pH 8.0 (item number T1150, Solarbio) to terminate cross-linking.

[0250] 4) The cross-linked sample is separated by 4%-20% SDS-PAGE (item number 36270ES10, Yeasen).

[0251] 5) The cross-linked protein complex is cut by trypsin (item number: T8658-1VL, manufacturer: Sigma).

[0252] 6) The obtained cut product is subjected to mass spectrometry analysis.

[0253] The results are shown in Table 21. The cross-linking sites of NB090 with C3 / C3b are mainly distributed in the MG6a domain, the MG7 domain, and the C345C domain. The cross-linking sites of NB071-Hz6a with C3 / C3b are mainly distributed in the MG6a domain, the MG6b domain, the MG7 domain, and the TED domain.

[0254] Table 21 Cross-linking information of anti-complement protein C3 / C3b antibody NB090, NB071-Hz6a with human complement protein C3 and C3b

[0255] According to the mass spectrometry analysis results, the C3b-NB090, C3-NB090, C3b-NB071-Hz6a, C3-NB071-Hz6a complex structures were simulated and calculated by molecular docking and molecular dynamics simulation. Specifically, using HADDOCK2.4, taking the cross-linking site and the C345C domain binding as the restriction condition, the antigens C3b and C3 were respectively docked with the antibodies NB090 and NB071-Hz6a, wherein the structures of C3b and C3 were from RCSB protein database (PDB:7BAG, PDB:2A73). The structure of NB090 and NB071-Hz6a was predicted by alphafold2. Molecular dynamics simulation (MD) was performed using GROMACS2020.05 software, taking the molecular docking result as the initial structure, which was placed in a periodic cubic box with a side length of 1.2 nm, and the concentration of NaCl was set to 0.15 mol / L to simulate the ion concentration under physiological conditions, while ensuring the electrical neutrality of the system. The maximum descent method was used for energy minimization, and a maximum of 5000 steps was performed to optimize the system structure, the V-rescale algorithm was used to maintain the system temperature at 310K, and the Parrinello-Rahman algorithm was used to keep the system pressure at 1bar. The formal simulation lasted for 100ns, and the time step was 2fs, and all simulations used the CHARMM36 force field. The RMSD value during simulation was calculated by the "gmx rms" module of GROMACS2020.05 software to evaluate the stability of C3-NB090, C3b-NB090, C3-NB071-Hz6a, C3b-NB071-Hz6a complex, and the calculation formula of RMSD is as follows:

[0256] Wherein, N is the number of atoms, m i is the atomic mass, r i (t1) is the spatial coordinate vector of the lambda h atom at time t1. r i (t2) is the coordinate vector of the atom in the reference structure, using the least squares fitting method. The first frame (0ns) of the simulation is defined as the reference structure, and the atomic coordinate vector is r i(t0). During the molecular dynamics simulation of C3b-NB090, C3-NB090, C3b-NB071-Hz6a, C3-NB071-Hz6a complex, there was no difference in the RMSD values of C3b and C3b-NB090, C3b-NB071-Hz6a complex, and the RMSD values of C3 and C3-NB090, C3-NB071-Hz6a complex, indicating that NB090 can be stably bound to the MG6a-MG7-C345C domain of C3b and C3; NB071-Hz6a can be stably bound to the MG6b-MG6a-MG7-TED domain of C3 and C3b, as shown in Figure 9.

[0257] To further confirm the binding epitope, this embodiment confirms the binding of anti-complement protein C3 / C3b nanobody NB090 to C345C domain by ELISA binding experiment. Referring to the method of Example 3, C345C domain protein (SEQ ID NO: 49) was expressed and purified. Referring to the method of Example 4, the ELISA binding activity detection was carried out, and the results are shown in Figure 10. Anti-complement protein C3 / C3b nanobody NB090 has high binding activity with C345C domain, and the EC50 is 0.19 nM.

[0258] 7.2 Functional verification of binding epitope

[0259] It is known that the MG6a-MG7-C345C domain of C3b is the main action region of C3b binding to factor B and factor H. This embodiment verifies the binding of NB090 to the MG6a-MG7-C345C domain by factor B competition experiment, factor B lysis experiment and H factor induced I factor degradation experiment.

[0260] A. Factor B competition experiment: competitive ELISA experiment, using factor B as a competitive agent to detect the effect of different concentration gradients of factor B on the binding of NB090 to C3b. Specifically, the operation process is as follows:

[0261] 1) Dilute human C3b (purchased from Comptech, item number: A114) to 1 μg / ml with coating buffer (purchased from Yuanye Biotechnology, item number: R20934) and add to the enzyme-labeled plate, 100 μL / well, 4°C coating overnight.

[0262] 2) After blocking with 5% skim milk powder and PBST (0.1% Tween20 in PBS, pH=7.4) washing, mix different concentration gradients of factor B diluent with 1 nM of NB090 mixture, total volume 100 μL, incubate at 37°C for 1 hour.

[0263] 3) After washing the plate with PBST, add HRP-labeled mouse anti-His tag monoclonal antibody (purchased from: Proteintech, item number: HRP-66005) diluted 1:7000, incubate at 25°C for 45 minutes.

[0264] 4) After washing the plate with PBST, add 100 μL of TMB (purchased from: Tiangen, item number: PA107-01) to each well, and develop color at 37°C for 15 minutes.

[0265] 5) Add 100 μL of ELISA stop solution (purchased from: Solarbio, item number: C1058).

[0266] 6) Measure the absorbance (OD450) at 450 nm using an enzyme-labeled instrument (Thermo Scientific, model: Multiskan SkyHigh).

[0267] The results are shown in FIG. 11A. As the concentration of the competitive agent B factor increases, the NB090 binding signal value to C3b gradually decreases, and at 10 μM B factor, nearly 60% of the NB090 binding to C3b can be competed.

[0268] B. B factor cleavage experiment: B factor can be cleaved into Ba and Bb by D factor after binding to C3b. In this embodiment, the degree of B factor cleavage under different conditions is detected by SDS-PAGE under non-reducing conditions. It is known that APL-2 (trade name: SYFOVRE, purchased from: Apellis, batch number: 1980264) and APL-1030 (SEQ ID NO: 50) bind to the MG4-MG5 domain of C3 and do not compete with B factor for binding to C3b, and are used as negative controls in this embodiment. Specifically, the operation process is as follows:

[0269] 1) Take GVB 0 (purchased from: Comptech, item number: B103) and Mg-EGTA (purchased from: Comptech, item number: B106) mixed at a ratio of 100: 1, ready for use.

[0270] 2) Take GVB-Mg-EGTA to prepare a 2 nM C3b dilution, and incubate C3b with the antibody to be tested at room temperature for 30 minutes.

[0271] 3) Add B factor and D factor to the reaction system, and continue to incubate at room temperature for 15 minutes.

[0272] 4) The sample is treated with non-reducing SDS-PAGE loading buffer, and analyzed by 4%-20% SDS-PAGE.

[0273] The results are shown in Figure 11B. In the NB090 experimental group, C3b was combined with NB090, and B factor was in a free state and was not cleaved by D factor. No Ba or Bb was produced in the reaction system, further confirming that NB090 competes with B factor to combine with C3b. Under the same experimental conditions, APL-2 and APL-1030 combined with the MG4-MG5 domain had no significant effect on the cleavage of B factor.

[0274] C. Factor H-mediated I factor degradation experiment: It is known that the main region of H factor combined with C3b is the MG7 domain, and the combination of H factor and C3b can provide action for I factor to cleave C3b. In this embodiment, the degree of C3b degradation under different experimental conditions is detected by SDS-PAGE under non-reducing conditions. Specifically, the operation process is as follows:

[0275] 1) Take GVB 0 (available from Comptech, item number: B103) and Mg-EGTA (available from Comptech, item number: B106) are mixed at a ratio of 100:1 for standby.

[0276] 2) Take GVB-Mg-EGTA to prepare a 2nM C3b diluent, and incubate C3b with the antibody to be tested at room temperature for 30 minutes.

[0277] 3) Add H factor and I factor to the reaction system, and incubate at 37°C for 2 hours.

[0278] 4) The sample is treated with reducing SDS-PAGE loading buffer, boiled at 95°C for 30 seconds, and then analyzed by 4%-20% SDS-PAGE.

[0279] The results are shown in Figure 11C. In the presence of NB090, C3b was not degraded to iC3b by I factor, i.e., NB090 can inhibit H factor-induced I factor degradation of C3b. Under the same experimental conditions, APL-2 and APL-1030 combined with the MG4-MG5 domain had no significant effect on the cleavage of C3b by I factor. In summary, the NB090 binding epitope is in the MG6a-MG7-C345C domain of C3b and C3.

[0280] The preferred embodiments of the application are described in detail above, but the application is not limited to the specific details in the above-described embodiments. Within the technical concept of the application, various simple modifications can be made to the technical solutions of the application, and these simple modifications all belong to the protection scope of the application.

[0281] It should be further noted that various specific technical features described in the above specific embodiments can be combined in any suitable manner, and in order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. An antigenic epitope peptide of complement protein C3 / C3b, characterized in that, The antigenic epitope peptide comprises an antigenic epitope peptide derived from the C345C domain, the MG7 domain, the MG6a domain, the MG6b and / or the TED domain of the complement protein C3 / C3b antigen.

2. The antigenic epitope peptide according to claim 1, characterized in that, The antigenic epitope peptide has a length of 5-20 aa.

3. The antigenic epitope peptide according to claim 1 or 2, characterized in that, The antigenic epitope peptide comprises an amino acid sequence as shown in any one of SEQ ID NOs: 100-104, 114-117.

4. A mimetic of complement protein C3 / C3b antigen, characterized in that, The mimetic comprises the antigenic epitope peptide according to any one of claims 1-3.

5. A fusion protein, characterized in that, The fusion protein comprises the antigenic epitope peptide according to any one of claims 1-3 and / or the mimetic according to claim 4.

6. A biomaterial, characterized by, The biological material comprises: 1) a gene encoding the antigenic epitope peptide according to any one of claims 1-3, the mimetic according to claim 4 and / or the fusion protein according to claim 5; 2) a vector comprising the gene of 1); 3) a host cell comprising the gene of 1) and / or the vector of 2).

7. An antigen-presenting cell, characterized in that, The antigen presenting cell comprises the antigenic epitope peptide according to any one of claims 1-3, the mimetic according to claim 4, the fusion protein according to claim 5, the gene according to claim 6 and / or the vector according to claim 6.

8. Use of the antigenic epitope peptide according to any one of claims 1 to 3, the mimetic according to claim 4, the fusion protein according to claim 5, the biomaterial according to claim 6, or the antigen-presenting cell according to claim 7, characterized in that, The application comprises: 1) use in screening, detecting, preparing and / or purifying an anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof; 2) use in preparing an antigen-antibody complex, which comprises 2-1) the complement protein C3 / C3b antigenic epitope peptide according to any one of claims 1-3, the mimetic according to claim 4 or the fusion protein according to claim 5, and 2-2) an anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof; 3) use in preparing a medicament and / or a diagnostic reagent for treating and / or preventing a complement protein C3 / C3b antigen-related disease.

9. An anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof, characterized in that, The anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof can bind to the antigenic epitope peptide according to any one of claims 1-3 and / or the mimetic according to claim 4.

10. The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof of claim 9, wherein, The antibody or an antigen-binding fragment thereof can inhibit or compete with the binding of an endogenous factor to the complement protein C3 / C3b.

11. An anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof, characterized in that, The anti-complement protein C3 / C3b antibody or an antigen-binding fragment thereof comprises CDR-H1, CDR-H2 and CDR-H3 of a heavy chain variable region; wherein, 1) the amino acid sequence of CDR-H1 comprises any one of TFSSWHM (SEQ ID NO: 21), SDDAVG (SEQ ID NO: 22), SLPMG (SEQ ID NO: 24), VYTMA (SEQ ID NO: 28), X1YX2X3G (SEQ ID NO: 96) or an amino acid sequence having at least 80% identity to any one of TFSSWHM (SEQ ID NO: 21), SDDAVG (SEQ ID NO: 22), SLPMG (SEQ ID NO: 24), VYTMA (SEQ ID NO: 28), X1YX2X3G (SEQ ID NO: 96); 2) the amino acid sequence of CDR-H2 comprises AIYSX4X5X6TNYX7DSVKG (SEQ ID NO: 97), X8ITSGX9RIX 10 YVDSVKG (SEQ ID NO: 98), GFTSGGRTSYVDSVKG (SEQ ID NO: 29), AIYGSGRTNYVDSVKG (SEQ ID NO: 30), AITIGGSTYYADSVKG (SEQ ID NO: 33), SSGSSTSYADSVKG (SEQ ID NO: 37) or an amino acid sequence that is at least 80% identical to any one of AIYSX4X5X6TNYX7DSVKG (SEQ ID NO: 97), X8ITSGX9RIX 10 YVDSVKG (SEQ ID NO: 98), GFTSGGRTSYVDSVKG (SEQ ID NO: 29), AIYGSGRTNYVDSVKG (SEQ ID NO: 30), AITIGGSTYYADSVKG (SEQ ID NO: 33), SSGSSTSYADSVKG (SEQ ID NO: 37) or an amino acid sequence that is at least 80% identical to any one of AIYSX4X5X6TNYX7DSVKG (SEQ ID NO: 97), X8ITSGX9RIX 3) the amino acid sequence of CDR-H3 comprises LGEFX 11 GRPP (SEQ ID NO: 99), LGSKSD (SEQ ID NO: 39), QRPPYNTNSLRAAPRYDY (SEQ ID NO: 42), LGSLVNREVLP (SEQ ID NO: 43), LGLPLSHG (SEQ ID NO: 44), LGPKKS (SEQ ID NO: 45), ALWTGGRMGNY (SEQ ID NO: 46) or an amino acid sequence that is at least 80% identical to LGEFX 11 GRPP (SEQ ID NO: 99), LGSKSD (SEQ ID NO: 39), QRPPYNTNSLRAAPRYDY (SEQ ID NO: 42), LGSLVNREVLP (SEQ ID NO: 43), LGLPLSHG (SEQ ID NO: 44), LGPKKS (SEQ ID NO: 45), ALWTGGRMGNY (SEQ ID NO: 46) or an amino acid sequence that is at least 80% identical to LGEFX 12. The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof of claim 11, wherein, X1in SEQ ID NO: 96 represents T or M; X2represents H or A; X3represents M or K; X4in SEQ ID NO: 97 represents D or G; X5represents S or G; X6represents S or R; X7represents A or V; X8in SEQ ID NO: 98 represents A or G; X9represents L or G; X 10 represents S or N; X 11 represents P, A or S.

13. The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof of claim 11 or 12, characterized in that, The amino acid sequences of the CDR-H1, CDR-H2 and CDR-H3 include any one of the following groups: A) SEQ ID NO: 21, 29, 38; B) SEQ ID NO: 22, 30, 39; C) SEQ ID NO: 23, 31, 40; D) SEQ ID NO: 23, 32, 41; E) SEQ ID NO: 24, 33, 42; F) SEQ ID NO: 23, 34, 43; G) SEQ ID NO: 25, 35, 44; H) SEQ ID NO: 26, 35, 44; I) SEQ ID NO: 27, 36, 45; J) SEQ ID NO: 28, 37, 46; K) SEQ ID NO: 25, 93, 44; L) SEQ ID NO: 25, 94, 44; M) SEQ ID NO: 25, 95, 44.

14. The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof according to any one of claims 11-13, characterized in that, The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof is humanized.

15. The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof according to any one of claims 11-14, characterized in that, The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof is a single domain antibody, a chimeric antibody, a Fab fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a dAb fragment, a F(ab')2 fragment, a single chain antibody or a linear antibody.

16. The anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof according to any one of claims 11-15, characterized in that, The amino acid sequence of the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof comprises any one of the amino acid sequences of SEQ ID NO: 11-20, 51-71, or has at least 80% identity to any one of the amino acid sequences of SEQ ID NO: 11-20, 51-71.

17. A fusion construct, comprising, The fusion construct comprises the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof of any one of claims 9-16.

18. The fusion construct of claim 17, wherein, The fusion construct further comprises other biologically active effector molecules, which include antibodies or antigen-binding fragments thereof, receptor domains or other functional components of other targets other than the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof of any one of claims 9-16, Preferably, the other functional components include, but are not limited to, one or more of the following combinations of serum albumin, cytokines, transferrin, scaffold proteins, oligopeptides, oligopeptide polymers, polypeptides, polypeptide polymers, polysaccharides, fatty chains, avidin, biotin, streptavidin, toxins, drugs, nucleic acids, radionuclides and their markers, PEG, complement factors or Fc fragments; Further preferably, the complement factor comprises Complement Factor H (FH), Complement Factor I (FI), Complement Decay-Accelerating Factor (CD55), Membrane Cofactor Protein (MCP), Complement Regulatory Protein CD59 (CD59), C4b-Binding Protein (C4BP), or C1 Inhibitor (C1INH); Preferably, the receptor domain comprises Complement Receptor 1 (CR1), Complement Receptor 2 (CR2), Complement Receptor 3 (CR3), Complement Receptor 4 (CR4), or Complement Receptor of the Immunoglobulin Family (CRIg). Preferably, the other target is selected from Vascular endothelial growth factor A (VEGFA), Vascular endothelial growth factor B (VEGFB), Vascular endothelial growth factor C (VEGFC), Vascular endothelial growth factor D (VEGFD), Vascular endothelial growth factor receptor (VEGFR), Fibroblast growth factor (FGF), Fibroblast growth factor receptor (FGFR), PLacental growth factor (PlGF), Platelet-derived growth factor (PDGF), Angiopoietins 2 (ANG2), Transforming growth factor (TGF), Integrin, Integrin Receptor, Interleukin (such as IL-1β, IL-2, IL-3, IL-4, IL-6, IL-10, IL-12, IL-15, IL-17, IL-23, etc.), Interleukin Receptor (such as IL1R1, IL2Rα, IL3R, IL4Rα, IL6R, IL10R, IL12R, IL15Rα, IL17R, IL23R, etc.), Proprotein convertase subtilisin / kexin type 9 (PCSK9), Tumor necrosis factor alpha (TNFα), Tumor necrosis factor receptor (TNFR), Receptor Activator of Nuclear Factor-κB Ligand (RANKL), Complement Component 5 (C5), Complement Component 5a (C5a), Complement Component 5b (C5b), Complement Component 5a Receptor (C5aR), Complement Component 5b Receptor (C5bR), Complement Component 5a Receptor 1 (C5aR1), Complement Component 5b Receptor 1 (C5bR1), Complement Component 5a Receptor 2 (C5aR2), Complement Component 5b Receptor 2 (C5bR2), Complement Component 5a Receptor 3 (C5aR3), Complement Component 5b Receptor 3 (C5bR3), Complement Component 5a Receptor 4 (C5aR4), Complement Component 5b Receptor 4 (C5bR4), Complement Component 5a Receptor 5 (C5aR5), Complement Component 5b Receptor 5 (C5bR5), Complement Component 5a Receptor 6 (C5aR6), Complement Component 5b Receptor 6 (C5bR6), Complement Component 5a Receptor 7 (C5aR7), Complement Component 5b Receptor 7 (C5bR7), Complement Component 5a Receptor 8 (C5aR8), Complement Component 5b Receptor 8 (C5bR8), Complement Component 5a Receptor 9 (C5aR9), Complement Component 5b Receptor 9 (C5bR9), Complement Component 5a Receptor 10 (C5aR10), Complement Component 5b Receptor 10 (C5bR10), Complement Component 5a Receptor 11 (C5aR11), Complement Component 5b Receptor 11 (C5bR11), Complement Component 5a Receptor 12 (C5aR12), Complement Component 5b Receptor 12 (C5bR12), Complement Component 5a Receptor 13 (C5aR13), Complement Component 5b Receptor 13 (C5bR13), Complement Component 5a Receptor 14 (C5aR14), Complement Component 5b Receptor 14 (C5bR14), Complement Component 5a Receptor 15 (C5aR15), Complement Component 5b Receptor 15 (C5bR15), Complement Component 5a Receptor 16 (C5aR16), Complement Component 5b Receptor 16 (C5bR16), Complement Component 5a Receptor 17 (C5aR17), Complement Component 5b Receptor 17 (C5bR17),C5), Complement Factor B (FB), Complement Factor D (FD), Complement Factor P (FP), Complement Component 1q (C1q), Complement Component 1s (C1s), Complement Component 4b (C4b), Complement Component 2 (C2), Mannan-Binding Lectin Serine Protease 2 (MASP2), Mannan-Binding Lectin Serine Protease 3 (MASP3), Complement Component 3a Receptor (C3aR), Complement Component 5a Receptor 1 (C5aR1), Complement Component 5a Receptor 2 (C5aR2), G Protein-Coupled Receptor (GPCR), glucagon-like peptide-1 receptor (GLP1R), Cluster of Differentiation 3 (CD3), Cluster of Differentiation 105 (CD105), Cluster of Differentiation 19 (CD19), Cluster of Differentiation 20 (CD20), Cluster of Differentiation 22 (CD22), Cluster of Differentiation 25 (CD25), Cluster of Differentiation 27 (CD27), Cluster of Differentiation 28 (CD28), Cluster of Differentiation 30 (CD30), Cluster of Differentiation 31 (CD31), Cluster of Differentiation 34 (CD34), Cluster of Differentiation 35 (CD35), Cluster of Differentiation 37 (CD37), Cluster of Differentiation 38 (CD38), Cluster of Differentiation 40 (CD40), Cluster of Differentiation 41 (CD41), Cluster of Differentiation 42 (CD42), Cluster of Differentiation 43 (CD43), Cluster of Differentiation 44 (CD44), Cluster of Differentiation 45 (CD45), Cluster of Differentiation 46 (CD46), Cluster of Differentiation 47 (CD47), Cluster of Differentiation 48 (CD48), Cluster of Differentiation 49 (CD49), Cluster of Differentiation 50 (CD50), Cluster of Differentiation 51 (CD51), Cluster of Differentiation 52 (CD52), Cluster of Differentiation 53 (CD53), Cluster of Differentiation 54 (CD54), Cluster of Differentiation 55 (CD55), Cluster of Differentiation 56 (CD56), Cluster of Differentiation 57 (CD57), Cluster of Differentiation 58 (CD58), Cluster of Differentiation 59 (CD59), Cluster of Differentiation 60 (CD60), Cluster of Differentiation 61 (CD61), Cluster of Differentiation 62 (CD62), Cluster of Differentiation 63 (CD63), Cluster of Differentiation 64 (CD64), Cluster of Differentiation 65 (CD65), Cluster of Differentiation 66 (CD66), Cluster of Differentiation 67 (CD67), Cluster of Differentiation 68 (CD68), Cluster of Differentiation 69 (CD69), Cluster of Differentiation 70 (CD70), Cluster of Differentiation 71 (CD71), Cluster of Differentiation 72 (CD72), Cluster of Differentiation 73 (CD73), Cluster of Differentiation 74 (CD74), Cluster of Differentiation 75 (CD75), Cluster of Differentiation 76 (CD76), Cluster of Differentiation 77 (CD77), Cluster of Differentiation 78 (CD78), Cluster of Differentiation 79 (CD79), Cluster of Differentiation 80 (CD80), Cluster of Differentiation 81 (CD81), Cluster of Differentiation 82 (CD82), Cluster of Differentiation 83 (CD83), Cluster of Differentiation 84 (CD84), Cluster of Differentiation 85 (CD85), Cluster of Differentiation 86 (CD86), Cluster of Differentiation 87 (CD87), Cluster of Differentiation 88 (CD88), Cluster of Differentiation 89 (CD89), Cluster of Differentiation 90 (CD90), Cluster of Differentiation 91 (CD91), Cluster of Differentiation 92 (CD92), Cluster of Differentiation 93 (CD93), Cluster of Differentiation 94 (CD94), Cluster of Differentiation 95 (CD95), Cluster of Differentiation 96 (CD96), Cluster ofCD30), Cluster of Differentiation 33 (CD33), Cluster of Differentiation 38 (CD38), Cluster of Differentiation 40 (CD40), Cluster of Differentiation 47 (CD47), Cluster of Differentiation 80 (CD80 / B7-1), Cluster of Differentiation 86 (CD86 / B7-2), Cluster of Differentiation 96 (CD96), Cluster of Differentiation 99 (CD99), Cluster of Differentiation 111 (CD111), Cluster of Differentiation 112 (CD112), Cluster of Differentiation 123 (CD123), Cluster of Differentiation 133 (CD133), Cluster of Differentiation 138 (CD138), Cluster of Differentiation 155 (CD155), Cluster of Differentiation 171 (CD171), Claudin 18.2 (CLDN18.2), Tumor necrosis factor receptor superfamily, member 4 (TNFRSF4 / OX40 / CD134), Inducible T-Cell Co Stimulator (ICOS), Cytotoxic T-lymphocyte-associated protein 4 (CTLA4), Tumor necrosis factor receptor superfamily, member 9 (TNFRSF9 / 4-1BB / CD137), Tumor necrosis factor receptor superfamily, member 18 (TNFRSF18 / NTSR2 / CD358), Programmed cell death 1 (PD-1), Programmed cell death ligand 1 (PD-L1), Programmed cell death ligand 2 (PD-L2), Cytotoxic and regulatory T-lymphocyte molecule (CTLA4), Inducible T-Cell Co-Stimulator (ICOS), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin domain and mucin domain-3 (TIM-3), T-cell immunoglobulin domain and mucin domain-4 (TIM-4), T-cell immunoglobulin domain and mucin domain-1 (TIM-1), T-cell immunoglobulin domain and mucin domain-2 (TIM-2), T-cell immunoglobulin domain and mucin domain-3 (TIM-3), T-cell immunoglobulin domain and mucin domain-4 (TIM-4), T-cell immunoglobulin domain and mucin domain-5 (TIM-5), T-cell immunoglobulin domain and mucin domain-6 (TIM-6), T-cell immunoglobulin domain and mucin domain-7 (TIM-7), T-cell immunoglobulin domain and mucin domain-8 (TIM-8), T-cell immunoglobulin domain and mucin domain-9 (TIM-9), T-cell immunoglobulin domain and mucin domain-10 (TIM-10), T-cell immunoglobulin domain and mucin domain-11 (TIM-11), T-cell immunoglobulin domain and mucin domain-12 (TIM-12), T-cell immunoglobulin domain and mucin domain-13 (TIM-13), T-cell immunoglobulin domain and mucin domain-14 (TIM-14), T-cell immunoglobulin domain and mucin domain-15 (TIM-15), T-cell immunoglobulin domain and mucin domain-16 (TIM-16), T-cell immunoglobulin domain and mucin domain-17 (TIM-17), T-cell immunoglobulin domain and mucin domain-18 (TIM-18), T-cell immunoglobulin domain and mucin domain-19 (TIM-19), T-cell immunoglobulin domain and mucin domain-20 (TIM-20), T-cell immunoglobulin domain and mucin domain-21 (TIM-21), T-cell immunoglobulin domain and mucin domain-22 (TNFRSF9 / 4-1BB / CD137), T cell receptor (TCR), B-and T-Lymphocyte Attenuator (BTLA), T cell immunoglobulin domain and mucin domain-3 (TIM-3), Lymphocyte Activation Gene 3 (LAG3), Galectin-9 (GAL9), Programmed cell death 1 ligand 1 (PD-L1), Programmed cell death 1 ligand 2 (PD-L2), Programmed cell death protein 1 (PD-1), T cell immune receptor with Ig and ITIM domains (TIGIT), Epidermal growth factor receptor (EGFR), Human Epidermal Growth Factor Receptor 2 (HER2), Prostate Stem Cell Antigen (PSCA), Carcinoembryonic antigen (CEA), Familial adenomatous polyposis (FAP), Epidermal growth factor receptor variant type III (EGFRvIII), B cell maturation antigen (BCMA), Prostate Specific Membrane Antigen (PSMA), Cancer antigen 125 (CA125), Ephrin A Receptor 2 (EphA2), Cellular-mesenchymal epithelial transition factor (Twist), CD133, CD44, CD24, CD49f, CD90, CD271, CD271, CD200, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326, CD326,c-Met), L1 -Cell Adhesion Molecule (L1 CAM), Signaling Lymphocytic Activation Molecule Family, Member 7 (SLAMF7 / CS1), Receptor tyrosine kinase-like orphan receptor 1 (ROR1), Recombinant Mycobacterium Tuberculosis Fusion Protein, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), Mucin 1 (MUC1), Mucin 16 (MUC16), Mesothelin, Cluster of Differentiation 174 (Lewis Y / CD174), Glypican 3 (GPC3), Disialoganglioside-GD2 (GD2), Eukaryotic division factor-like and proliferation-associated protein (EPG), Delta-Like Ligand 3 (DLL3), or Trophoblast Glycoprotein (TPBG / 5T4).

19. A nucleic acid, comprising: The nucleic acid encodes the anti-complement protein C3 / C3b antibody or antigen binding fragment thereof of any one of claims 9-16 or the fusion construct of any one of claims 17-18.

20. The nucleic acid of claim 19, wherein The nucleic acid comprises any one of the nucleotide sequences of SEQ ID NOs: 1-10, 72-92, or a degenerate sequence thereof, or a nucleotide sequence having at least 80% identity to any one of the nucleotide sequences of SEQ ID NOs: 1-10, 72-92, and having the function of encoding the anti-complement protein C3 antibody or antigen binding fragment thereof.

21. A vector, comprising: The vector comprises the nucleic acid of any one of claims 19-20.

22. A host cell, characterized in that, The host cell comprises the nucleic acid of any one of claims 19-20 or the vector of claim 21.

23. A method of producing an anti-complement protein C3 / C3b antibody or antigen binding fragment thereof according to any one of claims 9 to 16 or a fusion construct according to any one of claims 17 to 18, characterized in that, The preparation method comprises culturing the host cell of claim 22 to express the anti-complement protein C3 / C3b antibody or antigen binding fragment thereof or the fusion construct.

24. A product for the treatment, prevention and / or diagnosis of a disease, characterized in that The product for treating, preventing, and / or diagnosing a disease comprises any one of: A) the anti-complement protein C3 / C3b antibody or antigen binding fragment thereof of any one of claims 9-16; B) the fusion construct of any one of claims 17-18; C) the nucleic acid of any one of claims 19-20; D) the vector of claim 21; or, E) the host cell of claim 22.

25. A product for treating, preventing, and / or diagnosing a disease, characterized in that, The product for treating, preventing, and / or diagnosing a disease comprises any one of: a) the antigenic epitope peptide of any one of claims 1-3; b) the mimetic of claim 4; c) the fusion protein of claim 5; d) the biomaterial of claim 6; or, e) the antigen presenting cell of claim 7.

26. Use of the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof of any one of claims 9-16, the fusion construct of any one of claims 17-18, the nucleic acid of any one of claims 19-20, the vector of claim 21, the host cell of claim 22, in the manufacture of a product for the treatment and / or prevention of a disease associated with complement activation, or in the manufacture of a diagnostic product or tracer for a disease associated with complement activation.

27. Use of the antigenic epitope peptide of any one of claims 1-3, the mimetic of claim 4, the fusion protein of claim 5, the biomaterial of claim 6, and / or the antigen presenting cell of claim 7, in the manufacture of a product for the treatment and / or prevention of a disease associated with complement activation, or in the manufacture of a diagnostic product or tracer for a disease associated with complement activation.

28. A method for detecting complement protein C3 / C3b, characterized by, The detection method comprises combining a sample to be detected with the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof of any one of claims 9-16, and then detecting the content of the complex formed by complement protein C3 / C3b and the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof.

29. A method for treating and / or preventing a disease, characterized in that, The method comprises administering to an individual the anti-complement protein C3 / C3b antibody or antigen-binding fragment thereof of any one of claims 9-16, the fusion construct of any one of claims 17-18, the nucleic acid of any one of claims 19-20, the vector of claim 21, the host cell of claim 22, or the product for the treatment and / or prevention of a disease of any one of claims 24-25.

30. A method for treating and / or preventing a disease, characterized in that, The method comprises administering to an individual the antigenic epitope peptide of any one of claims 1-3, the mimetic of claim 4, the fusion protein of claim 5, the biomaterial of claim 6, and / or the antigen presenting cell of claim 7.

31. The method of any one of claims 29-30, wherein, The disease is a disease associated with complement activation; preferably, the disease is selected from the group consisting of rheumatoid arthritis (RA); lupus nephritis (LN); IgA nephraopathy, C3 Glomerulopathy (C3G); ischemia-reperfusion injury (IRI); paroxysmal nocturnal hemoglobinuria (PNH); atypical haemolytic uraemic syndrome (aHUS); dense deposit disease (DDD); Age related Macular Degeneration (AMD); geographic atrophy (GA); hemolysis elevated liver enzymes low platelets (HELLP); Thrombotic Thrombocytopenic Purpura (TTP); Spontaneous Abortion (SAB); Pauci-immune vasculitis; epidermolysis bullosa (EB); Recurrent miscarriage (RM); multiple sclerosis (MS); traumatic brain injury (TBI); and injuries caused by myocardial infarction, cardiopulmonary bypass and hemodialysis; CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (CHAPLE disease). The disease is a disease associated with complement activation; preferably, the disease is selected from the group consisting of rheumatoid arthritis (RA); lupus nephritis (LN); IgA nephraopathy, C3 Glomerulopathy (C3G); ischemia-reperfusion injury (IRI); paroxysmal nocturnal hemoglobinuria (PNH); atypical haemolytic uraemic syndrome (aHUS); dense deposit disease (DDD); Age related Macular Degeneration (AMD); geographic atrophy (GA); hemolysis elevated liver enzymes low platelets (HELLP); Thrombotic Thrombocytopenic Purpura (TTP); Spontaneous Abortion (SAB); Pauci-immune vasculitis; epidermolysis bullosa (EB); Recurrent miscarriage (RM); multiple sclerosis (MS); traumatic brain injury (TBI); and injuries caused by myocardial infarction, cardiopulmonary bypass and hemodialysis; CD55 deficiency with hyperactivation of complement, angiopathic thrombosis, and protein-losing enteropathy (CHAPLE disease).

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