Polypeptides targeting complement c5 and uses and products thereof

Abstract

The application discloses a polypeptide targeting complement C5 and application and products thereof, and belongs to the technical field of polypeptide preparation. The application first provides a C5 targeting polypeptide (named as C548), which can significantly inhibit the assembly of a membrane attack complex (MAC) in an inflammatory state, effectively reduces inflammation-mediated tissue damage, and provides a potential alternative antibody treatment strategy for autoimmune diseases and other complement abnormality related diseases.

Description

Technical Field

[0001] This invention belongs to the field of peptide preparation technology, specifically relating to a peptide that targets complement C5 and its applications and products. Background Technology

[0002] The complement system is an important component of the body's innate immunity, composed of more than 30 soluble proteins and membrane-bound proteins. It is widely present in serum, tissue fluid, and on the cell membrane surface, playing a crucial role in anti-infection, defense against pathogen invasion, and clearance of immune complexes. In addition to participating in innate immunity, the complement system also plays a multi-level role in adaptive immune regulation through mechanisms such as regulating B cells, T cells, and antibody responses. Activation of the complement system includes the classical pathway, the alternative pathway, and the lectin pathway, all ultimately converging at the terminal pathway.

[0003] As a key component of the complement cascade, C5 plays a central role in this process. Under inflammatory or immune-activated conditions, C5 convertase cleaves C5 into C5a, which has strong chemotactic and inflammatory amplification effects, and C5b, which participates in subsequent cascade reactions. C5b further assembles with C6, C7, C8, and C9 to form the membrane attack complex (MAC), leading to altered target cell membrane permeability and even cell lysis. Under physiological conditions, the complement system is finely regulated by various regulatory proteins to maintain immune homeostasis. However, when complement is abnormally activated or its regulation is imbalanced, it can lead to tissue damage and participate in the occurrence and development of various diseases, including but not limited to systemic lupus erythematosus, rheumatoid arthritis, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and neuromyelitis optica spectrum disorders. Therefore, intervention strategies targeting C5 are considered important therapeutic approaches for regulating the complement terminal pathway and blocking the formation of the membrane attack complex.

[0004] Currently, monoclonal antibody drugs targeting C5 are used clinically to treat complement-related diseases. Although these drugs have shown some clinical efficacy, as large molecular weight protein preparations, they still pose risks such as immunogenicity, limited tissue distribution, and high cost. Furthermore, in some central nervous system-related diseases, large molecular weight antibodies have limited ability to cross the blood-brain barrier, which may affect their efficacy in central target tissues. In contrast, small molecular weight peptide drugs typically have smaller molecular weights, better tissue penetration, and lower potential immunogenicity, and have received widespread attention in the treatment of immune and inflammatory diseases in recent years. However, peptide inhibitors targeting the C5 protein have not been adequately reported in the literature.

[0005] Therefore, it is necessary to develop a novel polypeptide molecule that can specifically bind to C5 and inhibit its function, in order to provide new treatment options for diseases related to complement abnormal activation. Summary of the Invention

[0006] In order to overcome the shortcomings of the prior art, the present invention aims to provide a polypeptide that targets complement C5 and its applications and products, so as to solve the technical problem of the lack of polypeptide inhibitors targeting C5 protein in the prior art.

[0007] To achieve the above objectives, the present invention employs the following technical solution: This invention discloses a polypeptide that targets complement C5, wherein the amino acid sequence of the polypeptide is any one of the following: (a) The amino acid sequence shown in SEQ ID NO:1; (b) An amino acid sequence that is more than 80% identical to SEQ ID NO:1; (c) An amino acid sequence obtained by substitution, deletion or addition of one or more amino acids from the amino acid sequence shown in SEQ ID NO:1, which is capable of specifically binding complement C5 and inhibiting the formation of the membrane attack complex.

[0008] Preferably, the amino acid sequence has more than 90% identity with SEQ ID NO:1.

[0009] The present invention also discloses that the DNA sequence encoding the polypeptide targeting complement C5 is any one of the following: (a) The nucleotide sequence shown in SEQ ID NO:2; (b) A nucleotide sequence that is more than 80% identical to SEQ ID NO:2; (c) A nucleotide sequence that can hybridize with the sequence described in (a) or (b) under strict hybridization conditions; (d) A nucleotide sequence that encodes an amino acid sequence having the same biological function as the amino acid sequence shown in SEQ ID NO:2.

[0010] Preferably, the sequence has more than 90% homology with SEQ ID NO.2.

[0011] Preferably, the N-terminus or C-terminus of the amino acid sequence of the polypeptide further includes a tag sequence, a linker peptide, or a fusion protein. More preferably, it is a His tag, a FLAG tag, a HA tag, an Fc fusion protein, or an albumin fusion protein.

[0012] The present invention also discloses a pharmaceutical composition comprising a therapeutically effective amount of the above-described polypeptide targeting complement C5, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0013] The present invention also discloses the use of the above-described peptide or pharmaceutical composition targeting complement C5 in the preparation of medicaments for treating autoimmune diseases and / or C5 complement-related diseases.

[0014] Preferably, the autoimmune diseases include neuromyelitis optica spectrum disorders, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, and Sjögren's syndrome.

[0015] The above scheme clarifies the applicability of peptides in antibody-mediated complement overactivation diseases by specifically defining the types of autoimmune diseases, especially their therapeutic potential for central nervous system autoimmune diseases.

[0016] Preferably, the C5 complement-related diseases include paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, inflammatory response caused by trauma or infection, ischemia-reperfusion injury, Alzheimer's disease, and Parkinson's disease.

[0017] The above scheme clarifies the applicability of peptides in complement-mediated hematologic disorders, inflammatory responses, and neurodegenerative diseases by specifically defining the types of C5 complement-related diseases, thus broadening the clinical application scenarios of peptides.

[0018] Preferably, the drug exerts its therapeutic effect by inhibiting the cleavage of complement component C5 into C5a and C5b, thereby reducing the formation of membrane attack complexes.

[0019] This invention discloses a kit containing the aforementioned peptide targeting complement C5.

[0020] Preferably, the kit also includes reagents for detecting complement C5 levels, C5a levels, or the formation of the membrane attack complex C5b-9.

[0021] Compared with the prior art, the present invention has the following beneficial effects: The primary innovation of this invention lies in providing a novel C5-targeting peptide (named C548). Compared to existing large-molecule monoclonal antibodies, this peptide has an extremely small molecular weight (approximately 1.32 kDa), exhibiting excellent tissue penetration capabilities. It can effectively cross the blood-brain barrier, increasing drug concentration at lesion sites in the central nervous system, while completely avoiding human anti-mouse antibody reactions (HAMA), significantly reducing the risk of immunogenicity. Simultaneously, this peptide can significantly inhibit the assembly of the membrane attack complex (MAC) under inflammatory conditions, effectively alleviating inflammation-mediated tissue damage and providing a potential alternative antibody therapy strategy for autoimmune diseases and other complement-related disorders. Technically, this peptide can specifically bind to the complement C5 protein, effectively blocking the terminal pathway of C5b assembling with C6, C7, C8, and C9 to form the membrane attack complex (MAC) by inhibiting the cleavage of complement component C5 into C5a and C5b.

[0022] Experimental data show that this peptide not only binds to C5 with high specificity and concentration dependence, but also significantly reduces C5b-9 deposition on the cell surface in cell models, lowering complement-mediated cell death and exhibiting a clear dose-response relationship. Specifically, the formation of membrane attack complexes was evaluated by detecting the level of C5b-9 deposition on the cell surface using immunofluorescence. The results showed that compared with the control group, membrane attack complex deposition was significantly reduced in the C548-treated group. Cell viability analysis using a live / dead cell double staining kit showed that the cell death rate was significantly reduced in the C548-treated group, demonstrating a significant protective effect against complement-mediated damage, and exhibiting a dose-response relationship. Simultaneously, the antibody C548 of this invention can effectively inhibit the activation of the complement terminal pathway and reduce the formation of membrane attack complexes, thereby protecting AQP4-positive cells from complement-dependent cytotoxic damage. Attached Figure Description

[0023] Figure 1 To improve the specificity of ELISA detection of peptide C548; Figure 2 The SPR sensor plot shows a concentration-dependent binding interaction between C548 and C5; Figure 3 To verify the function of the polypeptides in this invention, the leftmost image in Figure A is the control image; the other three images show the addition of different polypeptides, and it can be seen that C548 has a significant protective effect, with a significant reduction in dead cells (red); Figure B is a statistical graph.

[0024] Figure 4 The dose-response relationship for verifying the function of the invented polypeptide C548 showed that as the amount of the invented polypeptide added increased, the number of dead cells decreased accordingly. Figure 5 The images show immunofluorescence and statistical graphs of peptide C548 inhibiting the deposition of membrane attack complex C5b-9 in an embodiment of the present invention; wherein, A represents C548 reducing complement deposition, and B represents statistical graphs of reduced complement deposition; Detailed Implementation To enable those skilled in the art to understand the features and effects of the present invention, the following description and definitions are only general descriptions of the terms and expressions mentioned in the specification. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0025] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0026] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0027] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0028] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0029] In this article, phage display technology is used: by fusing the gene encoding a foreign peptide with the gene of a phage coat protein, the target protein is displayed on the surface of the viral particle. After multiple rounds of biological screening, functional peptides with high affinity for specific target molecules can be efficiently isolated.

[0030] This study used phage display technology to screen peptide sequences specifically targeting complement C5 from a random peptide library. This 12-peptide (molecular weight 1.32 kDa) demonstrated significant C5 inhibitory activity in vitro and completely avoided the human anti-mouse antibody reaction (HAMA). As the first reported C5-targeting peptide drug, its small molecule characteristics endow it with excellent tissue penetration ability, exhibiting good blood-brain barrier penetration efficiency in central nervous system disease models, and significantly increasing drug concentration at the lesion site.

[0031] The peptide targeting C5 is hereinafter referred to as C548, and the DNA sequence of C548 is shown below: GGGGAGAGTTCGGCGCGTGTGGATAAGGCTTTTTATG; The amino acid sequence of C548 is as follows: GESSARVDKAFM.

[0032] This treatment strategy is applicable to the following three categories of diseases: 1) autoimmune diseases, such as neuromyelitis optica spectrum disorders, multiple sclerosis, systemic lupus erythematosus, and myasthenia gravis; 2) complement system abnormality-related diseases, including paroxysmal nocturnal hemoglobinuria and atypical hemolytic uremic syndrome (aHUS); 3) traumatic inflammatory responses.

[0033] Example 1 This embodiment provides a polypeptide that targets complement C5, wherein the amino acid sequence of the polypeptide is any one of the following: (a) the amino acid sequence shown in SEQ ID NO:1; (b) an amino acid sequence having more than 80% identity with SEQ ID NO:1; (c) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids from the amino acid sequence shown in SEQ ID NO:1, and capable of specifically binding to complement C5 and inhibiting the formation of the membrane attack complex.

[0034] Specifically, in this embodiment, the amino acid sequence shown in SEQ ID NO:1 is GESSARVDKAFM, which is a small polypeptide composed of 12 amino acid residues with a molecular weight of approximately 1.32 kDa. The DNA sequence encoding this polypeptide is the nucleotide sequence shown in SEQ ID NO:2, specifically GGGGAGAGTTCGGCGCGTGTGGATAAGGCTTTTATG.

[0035] The targeting of complement C5 described in this application refers to the ability of the polypeptide to physically bind to the C5 protein, a key component in the complement cascade reaction, thereby interfering with the subsequent cleavage and assembly of C5. The inhibition of membrane attack complex formation described in this application refers to the polypeptide blocking the pathway of C5 cleavage into C5b by binding to C5, thus preventing C5b from assembling with C6, C7, C8, and C9 to form the membrane attack complex (MAC) that leads to cell lysis.

[0036] For the amino acid sequence described in item (b) above that has more than 80% identity with SEQ ID NO:1, the consideration for protection is that in protein engineering practice, in order to optimize the physicochemical properties of peptides such as stability, solubility, or affinity, it is often necessary to moderately modify the original sequence. Therefore, we understand that as long as the modified sequence achieves 80% or more of the identity threshold with the original SEQ ID NO:1 sequence in the identity comparison, such as 90%, 95%, or even 98%, and still retains the core biological function of specifically binding complement C5 and inhibiting MAC formation, it should be considered a sequence substantially equivalent to the present invention. At the same time, it should be understood that the selection of the identity percentage threshold should not limit the present invention to the specific values ​​listed. 80% is only a preferred minimum defense boundary, and higher identity percentages also fall within the scope of protection of the present invention.

[0037] Regarding the sequence obtained by substituting, deleting, or adding one or more amino acids from the amino acid sequence shown in SEQ ID NO:1 as described in item (c) above, this embodiment illustrates the following: Any substitution, deletion, or addition of amino acids must result in a new sequence that simultaneously satisfies the two core functional requirements of "specifically binding to complement C5" and "inhibiting the formation of the membrane attack complex." In other words, sequence variation is permitted, but variation cannot come at the expense of core functionality. For example, based on the SEQ ID NO:1 sequence, if a glycine residue is inserted at a specific position solely to increase the flexibility of the peptide, or if an amino acid is replaced with a naturally occurring homologous amino acid to reduce immunogenicity, as long as this modification does not disrupt the binding pocket conformation of the peptide and the C5 protein, and still exhibits the ability to inhibit MAC deposition during functional verification, such a derived sequence falls within the scope of protection of this invention.

[0038] The results of this invention are confirmed below by screening using phage display technology and verifying them with ELISA detection, specifically including the following steps: 1. Phage display technology (1) Antigen coating: Take 10 μL of C5 protein solution (1 mg / mL), mix it with 50 μL of 0.1 M sodium bicarbonate buffer (pH 8.6), and add it to a 96-well high-binding enzyme-linked immunosorbent assay (ELISA) plate. Coat at 4°C for 12 hours. In the first round of screening, the amount of antigen coated per well was 10 μg. In the second round of screening, the amount of antigen coated per well was reduced to 5 μg. In the third round of screening, 3 μg of antigen was coated per well to improve the screening pressure and specificity.

[0039] (2) Blocking treatment: Discard the coating solution and wash three times with PBS buffer for 5 minutes each time. Then add 100 μL of 5% BSA solution to each well and block at 4°C for 60 minutes to reduce nonspecific binding.

[0040] (3) Phage incubation: After blocking, wash three times with PBS, add 1×10¹¹ pfu phage display library to each well, and incubate at room temperature for 120 minutes to allow the phage displaying the peptide to fully bind to the coated C5 protein.

[0041] (4) Washing and elution: After incubation, wash 7 times with PBS to remove unbound or weakly bound phages. Increase the number of washes appropriately in the second and third rounds of screening. Then add Gly-HCl elution buffer at pH 2.2 and incubate at room temperature for 10 minutes to elute bound phages. Immediately add Tris-HCl buffer at pH 8.8 for neutralization.

[0042] (5) Phage amplification: The eluted phages were used to infect ER2738 Escherichia coli in the logarithmic growth phase and amplified by shaking at 37°C and 250 rpm for 12 hours.

[0043] (6) PEG precipitation enrichment: The amplification culture medium was centrifuged at 4℃ and 10,000 rpm for 20 minutes, and the supernatant was collected. A PEG / NaCl solution with a final concentration of 20% was added to the supernatant, mixed well, and allowed to stand at 4℃ for 12 hours to precipitate.

[0044] (7) Phage purification: The precipitate was centrifuged at 10,000 rpm for 15 minutes at 4°C, the supernatant was discarded, and the precipitate was resuspended in TBS buffer. PEG / NaCl was added again for secondary precipitation (ice bath for 60 minutes), the supernatant was discarded after centrifugation, and the phage particles were finally resuspended in 200 μL TBS.

[0045] (8) Titer determination and next round of screening: The phage titer was determined by gradient dilution. 1×10¹² Pfu phage was used for the next round of biological screening to gradually improve the screening specificity and affinity.

[0046] (9) Blue-white screening: Add the appropriately serially diluted bacteriophage infection solution to 5 mL of preheated upper agar medium, mix well and cover the surface of solid medium, incubate at 37℃ upside down for 12 hours, and perform blue-white screening.

[0047] (10) Positive clone analysis: Blue spot colonies were picked, template DNA was extracted, and the insert fragment was amplified by PCR using specific primers and sequence analysis was performed to determine the candidate polypeptide sequence.

[0048] Phage library next-generation sequencing PCR primers: Upstream primer F 5'-ATTCGCAATTCCTTTAGTGGTAC-3'; Downstream primer R 5'-CCTGTAGCATTCCACAGACA-3'.

[0049] 2. ELISA analysis and sequencing analysis (1) Antigen coating: The complement C5 protein was diluted with carbonate buffer (pH 9.6) and added to a 96-well high-binding enzyme-linked plate, 50 μL per well. The plate was incubated at 4°C for 12 hours to allow the antigen to be fully adsorbed onto the surface of the wells.

[0050] (2) Blocking treatment: After discarding the coating solution, wash three times with PBS buffer. Then add 200 μL of 2% BSA blocking solution to each well and incubate at room temperature for 1.5 hours to block non-specific binding sites.

[0051] (3) Peptide incubation: After blocking, wash three times with PBS. Add 50 μL of His-tagged candidate peptide solution to each well and incubate at room temperature for 1.5 hours to allow the peptide to bind to the immobilized C5 protein.

[0052] (4) Antibody binding reaction: After washing 3 times, add HRP-labeled anti-His antibody (diluted at a ratio of 1:1000), 50 μL per well, and incubate at room temperature for 1 hour.

[0053] (5) Color reaction: After washing 3 times, add TMB color substrate solution and react at room temperature in the dark for 5–10 minutes; after sufficient color development, add 2 mol / L H2SO4 to terminate the reaction.

[0054] (6) Results detection and analysis: The absorbance (OD450 value) of each well was measured at a wavelength of 450 nm using an ELISA reader. The binding ability of the peptide to C5 protein was evaluated by the OD value.

[0055] (7) Sequencing verification: High-affinity clones with strong binding signals (n=50) were screened and DNA sequencing analysis was performed to identify 9 unique polypeptide sequences.

[0056] Key results: Sequence C548 occurred most frequently (9 / 50), and ELISA analysis showed it had a significant binding signal and good specificity. From Figure 1 As can be seen from the results, peptide C548, which targets complement C5, was the most frequently detected peptide in the screening library (9 / 50) obtained through phage display technology. ELISA results showed that the binding signal of peptide C548 to complement C5 increased significantly with increasing concentration, and the goodness of fit R0 was [value missing]. 2 =0.9968, and the absorbance at 1000 ng / ml is close to 2.8, indicating that the peptide has a very strong binding signal to C5 and exhibits good concentration-dependent binding activity. At the same time, the peptide did not bind significantly to control proteins such as fetal bovine serum, complement C3, and complement C1Q, and the absorbance values ​​remained at the baseline level, demonstrating excellent binding specificity. This lays the foundation for the subsequent development of therapeutic drugs targeting complement C5.

[0057] 3. Surface Plasmon Resonance (SPR) Detection Method (1) To detect the binding kinetic parameters between the antibody and the antigen, a surface plasmon resonance (SPR) experiment was performed using a Biacore T200 instrument (GE Healthcare, Chicago, Illinois, USA).

[0058] (2) The C5 protein was diluted with 10 mM sodium acetate buffer (NaAC, pH 5.5). The diluted C5 protein was then immobilized on the surface of the carboxymethyl dextran matrix CM5 sensor chip. During the experiment, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES) buffer (pH 7.4) was used as the running buffer, and detection was performed at a flow rate of 20 μL / min at 25°C.

[0059] (3) The C5-peptide to be tested was injected into the surface of the sensor chip at the same flow rate for 90 seconds; then it was rinsed with running buffer for 180 seconds to record the dissociation process signal. After each sample injection, the flow cell was regenerated with 10 mM glycine-hydrochloric acid solution (pH 1.5) for 30 seconds.

[0060] (4) The binding curve data obtained from the experiment were processed using Biacore's supporting analysis software to calculate the binding kinetic constants between the antibody and the antigen, including the binding rate constant (ka), the dissociation rate constant (kd), and the equilibrium dissociation constant (KD).

[0061] See key results Figure 2 The sensor data showed that with increasing C548 concentration, the binding response value (RU) increased in a concentration-dependent manner, while the dissociation response value gradually decreased and stabilized, exhibiting typical specific binding-dissociation characteristics. Kinetic fitting revealed an equilibrium dissociation constant (KD) of 1.560 μM, further confirming the molecular basis of its targeting binding to complement C5.

[0062] 4. Functional verification To verify the functional effects of the antibody of this invention, an AQP4-positive cell complement-dependent injury model was established for in vitro experiments. CHO cells stably expressing M23-AQP4 were seeded at a density of 20,000 cells / well in 96-well culture plates and cultured overnight at 37°C and 5% CO2. After discarding the culture medium, the cells were washed with PBS, and 20% heat-inactivated serum from neuromyelitis optica (NMO) patients was added and incubated at room temperature for 30 minutes to allow the anti-AQP4 antibody to bind to the cells. Subsequently, 2% normal human complement serum and the test antibody were pre-incubated separately for 30 minutes before being added to the cells, and incubated at 27°C for 60 minutes to induce complement-dependent cytotoxicity.

[0063] See Figure 3Cell viability analysis was performed using a live / dead cell double staining kit. The C513, C536, and control peptide treatment groups showed no significant cell protection, while the C548 treatment group showed a significant reduction in cell death rate. Specifically, in an AQP4-positive cell complement-dependent injury model, the addition of peptide C548 with the SEQ ID NO:1 sequence significantly reduced the number of dead cells (red fluorescence) and the cell death rate significantly decreased to below approximately 20%, while the control group had a cell death rate as high as approximately 90%. This indicates that peptide C548 can effectively protect cells from complement system overactivation-mediated lysis damage and possesses good in vitro and in vivo therapeutic potential, thus providing direct cellular-level evidence for the functional limitation of inhibiting MAC formation.

[0064] See Figure 4 The dose-dependent cytoprotective effect of peptide C548 was further investigated. The results showed that complement-mediated cell death rate decreased significantly with increasing peptide C548 concentration. At a concentration of 200 μg / ml, the cell death rate was only about 5%, while it increased to about 90% when the concentration was reduced to 10 μg / ml. This indicates that the protective effect of peptide C548 against complement-mediated cell damage is clearly concentration-dependent, with a more significant protective effect at higher concentrations. This provides experimental evidence for subsequent dosage optimization and clinical application.

[0065] As can be seen, this embodiment establishes the absolute protection range of the core sequence of the complement C5 peptide and sets an anchor point for subsequent functional constraints. This specific amino acid sequence constitutes a spatial conformation that binds to the C5 protein with high affinity. This binding occupies the action site of the C5 convertase or alters the conformational dynamics of C5, preventing C5 from being effectively cleaved into C5a and C5b. This, in turn, blocks the supply of essential components for MAC assembly from the source, achieving the technical effect of reducing inflammation-mediated tissue damage.

[0066] Example 2 Based on Example 1, in order to further verify the core function of the polypeptide in specifically binding to complement C5 and inhibiting the formation of the membrane attack complex, this example establishes a homologous comparison example to demonstrate the irreplaceability of the sequence shown in SEQ ID NO:1.

[0067] This embodiment focuses on using negative control sequences selected from the same library as comparative examples. In the AQP4-positive cell complement-dependent injury model experiment, CHO cells stably expressing M23-AQP4 were selected. Heat-inactivated serum from patients with neuromyelitis optica spectrum disorder was added to allow anti-AQP4 antibodies to bind to the cells. Subsequently, normal human complement serum was added to induce complement-dependent cytotoxicity. Figure 3 and Figure 4As shown, under the same experimental conditions, although the C513 and C536 sequences screened from the same library are both 12-peptides and originate from the same phage display library as C548, at the same concentration, the cell death rates of the C513 and C536 treatment groups were almost the same as those of the control group, both reaching approximately 85% to 90%, showing no significant cell protective effect; while the cell death rate of the C548 treatment group, containing the SEQ ID NO:1 sequence, was significantly reduced to below approximately 20%, and the red fluorescence of dead cells was significantly reduced. Further combined... Figure 4 The dose-response curve showed that as the concentration of peptide C548 increased from 10 μg / ml to 200 μg / ml, the complement-mediated cell death rate decreased significantly, with the cell death rate at 200 μg / ml being only about 5%, demonstrating a clear concentration-dependent protective effect.

[0068] Also see Figure 5 Immunofluorescence was used to detect the inhibitory effect of antibody C548 on the formation of the membrane attack complex (C5b-9). The results showed that compared with the control group, the C548-treated group exhibited significantly reduced C5b-9 deposition on the cell surface and a markedly weakened red fluorescence signal. Quantitative analysis indicated that the proportion of C5b-9 positive cells was approximately 86% in the control group, while it was only about 15% in the experimental group, a highly significant difference (***P<0.001). These results confirm that antibody C548 can effectively inhibit the activation of the complement terminal pathway, reduce the formation of the membrane attack complex, and thus protect AQP4 positive cells from complement-dependent cytotoxic damage.

[0069] Combination Figure 5 The immunofluorescence assay results were used to compare and verify the inhibitory effect on the membrane attack complex. The results showed that C5b-9 deposition on the cell surface was significantly reduced in the C548 treatment group, the red fluorescence signal was significantly weakened, and the proportion of C5b-9 positive cells was only about 15%; while the proportion of C5b-9 positive cells in the control group was as high as about 86%. This comparative experiment profoundly reveals that simply having a 12-peptide length or originating solely from the same phage display screening system is insufficient to endow the peptide with the function of inhibiting MAC formation. The specific amino acid sequence shown in SEQ ID NO:1 constitutes the key code for binding to C5 and blocking its cleavage. Any deviation from this precise sequence, such as C513 or C536, will lead to a change in spatial conformation, thereby losing the ability to functionally bind to C5 and failing to block the generation of C5b and subsequent MAC assembly. This strongly demonstrates the irreplaceable nature of the SEQ ID NO:1 sequence in targeting C5 and inhibiting MAC formation, proving that the technical effect of this invention is not random or obvious, but rather a surprising effect brought about by a specific sequence.

[0070] It should be understood that C513 and C536 selected in the above comparative examples are merely illustrative examples and not restrictive. This embodiment verifies the results of Example 1 through explicit comparative data, that is, it establishes that only sequences that satisfy specific sequence identity or retain core functional variations can fall within the protection scope of this invention, thereby effectively preventing invalid sequences from eroding the scope of protection.

[0071] Example 3 Based on Example 1, this example further describes the encoding nucleic acid sequence and terminal modification of the polypeptide targeting complement C5. The DNA sequence encoding the polypeptide is any one of the following: (a) the nucleotide sequence shown in SEQ ID NO:2; (b) a nucleotide sequence having more than 80% identity with SEQ ID NO:2; (c) a nucleotide sequence that can hybridize with or to the sequence under strict hybridization conditions; (d) a nucleotide sequence encoding an amino acid sequence that has the same biological function as the amino acid sequence shown in SEQ ID NO:2.

[0072] Specifically, the nucleotide sequence shown in SEQ ID NO:2 is: GGGGAGAGTTCGGCGCGTGTGGATAAGGCTTTTATG, which precisely encodes the GESSARVDKAFM amino acid sequence shown in SEQ ID NO:1. For the identity sequences described above, nucleotide substitutions due to codon degeneracy or species expression bias are permitted, as long as the substitution does not alter the amino acid sequence of the final translated product or only produces an amino acid sequence highly homologous to SEQ ID NO:1 and retaining its core function. Even if its identity with SEQ ID NO:2 is only between 80% and 90%, but the final expressed polypeptide can still specifically bind to C5 and inhibit MAC formation, such nucleotide variants should be considered equivalent substitutions.

[0073] As one implementation, the N-terminus or C-terminus of the polypeptide's amino acid sequence further includes a tag sequence, a linker peptide, or a fusion protein. By introducing a modifying sequence at the end of the polypeptide, the workability and pharmacokinetic properties of the polypeptide are significantly improved without interfering with the C5 binding pocket. Specifically, the tag sequence may include a His tag, a FLAG tag, or a HA tag.

[0074] In addition to the tag sequence, the peptide can also be fused to an Fc fusion protein or an albumin fusion protein at its terminal. It should be understood that although the peptide C548 shown in SEQ ID NO:1 has the advantages of small molecular weight and strong tissue penetration, small peptides often face the challenge of short half-life and rapid clearance in vivo. By fusing the Fc fragment of human IgG1 or human serum albumin (HSA) to the C-terminus or N-terminus of C548, the hydrodynamic radius of the peptide can be significantly increased, thereby reducing the rapid clearance caused by glomerular filtration. Simultaneously, the Fc fragment can also be recycled in vivo by binding to the neonatal Fc receptor (FcRn), while the albumin fusion protein utilizes the long half-life of albumin itself to play a similar role.

[0075] It should be understood that the His tag, Fc fusion protein, and albumin fusion protein described above are merely illustrative examples and not limiting. In other embodiments, other types of tags or other half-life extension strategies (such as PEGylation) may be selected according to specific purification requirements or administration routes. As long as these modifications do not disrupt the core binary function of peptide-specific binding to C5 and inhibiting MAC formation, they should be considered reasonable extensions of the technical solutions of this invention.

[0076] Example 4 This embodiment further illustrates the use of peptides or pharmaceutical compositions targeting complement C5 in the preparation of drugs, which can be used to prepare drugs for the treatment of autoimmune diseases and / or C5 complement-related diseases.

[0077] The autoimmune diseases described in this application refer to diseases in which the body's immune system produces abnormal immune responses to its own tissues, leading to overactivation of the complement system and mediating damage to its own tissues.

[0078] The C5 complement-related diseases described in this application refer to non-autoimmune diseases that involve abnormal activation of the complement terminal pathway, massive deposition of MAC, and subsequent cell lysis or amplified inflammation during the pathological process. The reason for requesting protection for these two types of diseases is that they share the common feature of abnormal cleavage of complement C5 and subsequent uncontrolled assembly of MAC. Therefore, the core mechanism remains as described above: the drug exerts its therapeutic effect by inhibiting the cleavage of complement component C5 into C5a and C5b, thereby reducing the formation of membrane attack complexes. Specifically, the polypeptide C548 of this invention specifically binds to the C5 protein through its specific amino acid sequence (SEQ ID NO:1). This binding physically occupies the action site of C5 convertase or induces a conformational change in C5, preventing C5 convertase from effectively recognizing and cleaving C5. This blocking effect cuts off the generation of C5a at its source, thereby inhibiting the amplification of inflammatory chemotactic signals; simultaneously, because the generation of C5b is blocked, the subsequent assembly pathways with C6 to C9 lose their necessary initiating components, and the formation of MAC is thus comprehensively inhibited.

[0079] Combination Figure 5 Experimental evidence can directly confirm the above-mentioned mechanism of action. For example... Figure 5 The immunofluorescence results showed that in the AQP4-positive cell complement-dependent injury model, the control group had a large amount of C5b-9 deposits on the cell surface, with a strong red fluorescence signal and a C5b-9 positive cell ratio of approximately 86%. This indicates that the complement terminal pathway was fully activated, and C5 was cleaved into C5b and assembled into MAC. In contrast, the experimental group treated with peptide C548 showed a significant reduction in C5b-9 deposition on the cell surface, a marked decrease in the red fluorescence signal, and a C5b-9 positive cell ratio of only about 15%. This data directly demonstrates that peptide C548 can effectively inhibit C5 cleavage in the complex complement environment in vitro, resulting in a sharp decrease in C5b production, which in turn prevents MAC from effectively assembling and depositing on the target cell surface.

[0080] Specifically, the autoimmune diseases include neuromyelitis optica spectrum disorders, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, and Sjögren's syndrome.

[0081] Furthermore, the drugs prepared for the above-mentioned uses are not limited to treating disease states with obvious pathological damage, but can also be extended to early intervention scenarios to prevent abnormal complement activation, such as in the prevention of ischemia-reperfusion injury after organ transplantation, where the peptide drug can be administered in advance to inhibit the massive formation of MAC that is about to occur.

[0082] Example 5 This embodiment further provides a kit, the core active component of which is the polypeptide C548 or its homologous derivative having the sequence SEQ ID NO:1 described in detail in the foregoing embodiments.

[0083] In addition, the kit typically includes a buffer system to maintain peptide activity, instructions for use, and necessary consumables. For example, peptide C548 can be pre-dissolved in phosphate-buffered saline (PBS) containing a stabilizer and packaged as a lyophilized powder or ready-to-use aqueous solution in sterile glass ampoules or plastic centrifuge tubes.

[0084] In summary, the antibodies of this invention can be used to prepare drugs for the prevention and / or treatment of complement-mediated diseases, including but not limited to neuromyelitis optica spectrum disorders, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and trauma-related inflammatory reactions. Specific applications are as follows: (1) Establishment of a complement-dependent cell injury model: CHO cells stably expressing M23-AQP4 were seeded at a density of approximately 20,000 cells / well in 96-well culture plates and cultured overnight at 37°C and 5% CO2 to allow the cells to adhere and grow. After discarding the culture medium, the cells were washed with PBS buffer and approximately 20% heat-inactivated serum from patients with neuromyelitis optica spectrum disorder (NMOSD) was added. The cells were incubated at room temperature for approximately 30 minutes to allow the anti-AQP4 antibody in the serum to bind to AQP4 on the cell surface. Approximately 2% normal human complement serum was pre-incubated with the test antibody for approximately 30 minutes and then added to the cells. The cells were incubated at approximately 27°C for approximately 60 minutes to induce complement-dependent cytotoxicity (CDC).

[0085] (2) Detection of membrane attack complex: The level of C5b-9 deposition on the cell surface was detected by immunofluorescence to evaluate the formation of membrane attack complex. The results showed that, compared with the control group, the deposition of C5b-9 in cells treated with the antibody C548 of this invention was significantly reduced, while the C513 and C536 treatment groups did not show significant inhibitory effects.

[0086] (3) Cell viability analysis: Cell viability was detected using a live / dead cell double staining kit. The results showed that the cell death rate was significantly reduced in the C548 treatment group, indicating a significant protective effect against complement-mediated cell damage; the C513 and C536 groups did not show significant protective effects.

[0087] The above results demonstrate that the antibody C548 of this invention can effectively inhibit the activation of the complement terminal pathway and reduce the formation of membrane attack complexes, thereby protecting AQP4-positive cells from complement-dependent cytotoxic damage. Based on its function, the antibody C548 of this invention can be used to prepare drugs for the prevention and / or treatment of complement-mediated diseases, including but not limited to: neuromyelitis optica spectrum disorders (NMOSD and related autoimmune diseases), multiple sclerosis (MS), systemic lupus erythematosus (SLE), myasthenia gravis (MG), paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), complement-mediated inflammatory responses related to trauma or infection, and other complement-related diseases.

[0088] The preferred embodiments of this disclosure have been described in detail above with reference to the accompanying drawings. However, this disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this disclosure, various simple modifications can be made to the technical solutions of this disclosure, and these simple modifications all fall within the protection scope of this disclosure.

[0089] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. To avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0090] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

[0091] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. A polypeptide targeting complement C5, characterized in that, The amino acid sequence of the polypeptide is any one of the following: (a) The amino acid sequence shown in SEQ ID NO:1; (b) An amino acid sequence that is more than 80% identical to SEQ ID NO:1; (c) An amino acid sequence obtained by substitution, deletion or addition of one or more amino acids from the amino acid sequence shown in SEQ ID NO:1, which is capable of specifically binding complement C5 and inhibiting the formation of the membrane attack complex.

2. The polypeptide targeting complement C5 according to claim 1, characterized in that, The DNA sequence encoding this polypeptide is any of the following: (a) The nucleotide sequence shown in SEQ ID NO:2; (b) A nucleotide sequence that is more than 80% identical to SEQ ID NO:2; (c) A nucleotide sequence that can hybridize with the sequence described in (a) or (b) under strict hybridization conditions; (d) A nucleotide sequence that encodes an amino acid sequence having the same biological function as the amino acid sequence shown in SEQ ID NO:

2.

3. The polypeptide targeting complement C5 according to claim 1, characterized in that, The N-terminus or C-terminus of the amino acid sequence of the polypeptide further includes a tag sequence, a linker peptide, or a fusion protein.

4. A pharmaceutical composition, characterized in that, The present invention comprises a therapeutically effective amount of the polypeptide targeting complement C5 as described in any one of claims 1-3, and one or more pharmaceutically acceptable carriers, diluents or excipients.

5. Use of the polypeptide targeting complement C5 according to any one of claims 1-3 or the pharmaceutical composition according to claim 4 in the preparation of a medicament for treating autoimmune diseases and / or C5 complement-related diseases.

6. The application according to claim 5, characterized in that, The autoimmune diseases mentioned include neuromyelitis optica spectrum disorders, multiple sclerosis, systemic lupus erythematosus, myasthenia gravis, rheumatoid arthritis, and Sjögren's syndrome.

7. The application according to claim 5, characterized in that, The C5 complement-related diseases include paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, inflammatory response caused by trauma or infection, ischemia-reperfusion injury, Alzheimer's disease, and Parkinson's disease.

8. The application according to any one of claims 5-7, characterized in that, The drug exerts its therapeutic effect by inhibiting the cleavage of complement component C5 into C5a and C5b, thereby reducing the formation of membrane attack complexes.

9. A reagent kit, characterized in that, The polypeptide targeting complement C5 as described in any one of claims 1-3.

10. A reagent kit according to claim 1, characterized in that, The kit also includes reagents for detecting complement C5 levels, C5a levels, or the formation of the membrane attack complex C5b-9.

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