Bifunctional polypeptides, polypeptide conjugates targeting inhibition of c3 and c5 complement, and uses thereof

Abstract

The application discloses a bifunctional polypeptide, a polypeptide conjugate and application thereof for targeting inhibition of complement C3 and C5. The polypeptide in the bifunctional polypeptide and the polypeptide conjugate comprises a structure shown in formula (I): Z1-Z2-Z3 (I), wherein Z1 has the activity of inhibiting the C5 complement activation path; Z2 is a connecting peptide; and Z3 has the activity of inhibiting the C3 complement activation path. The polypeptide of the application can not only inhibit the complement C3 activation and hinder the complement cascade reaction participated by C3b, but also inhibit the complement C5 activation and hinder the complement cascade reaction participated by C5b, the two inhibition effects synergistically exert the drug efficacy, show a stronger complement path inhibition effect, and can effectively inhibit related diseases and autoimmune diseases caused by excessive activation of the complement.

Description

Technical Field

[0001] This application belongs to the field of complement inhibitors, specifically relating to a bifunctional polypeptide, a polypeptide conjugate, and their applications that target and inhibit complement C3 and C5, and more specifically to a bifunctional polypeptide or a pharmaceutically acceptable salt thereof, a polypeptide conjugate or a pharmaceutically acceptable salt thereof, a pharmaceutical composition, and their uses. Background Technology

[0002] The complement system comprises approximately 20 circulating proteins synthesized by the liver. These proteins, known as "complement," have been observed to play a role in supplementing antibody responses during bacterial disruption. Before bacterial activation, complement exists in an inactive form. Complement plays a vital role in defending against pathogens and mediating immune responses. The complement system is activated primarily through three pathways: the classical pathway, the lectin pathway, and the alternative pathway (see appendix). Figure 1 The first convergence point of these three pathways is complement C3 protein, and the second convergence point is complement C5 protein. Complement C3 protein is cleaved into C3a and C3b by C3 convertase. C3a can cause inflammation and thrombosis, while C3b can cause extravascular hemolysis and can also bind to C3 convertase to generate C5 convertase. C5 convertase cleaves complement C5 protein into C5a and C5b. C5a can also cause inflammation and thrombosis. C5b binds to C6, C7, and C8 to form C5b-8. C5b-8 catalyzes the polymerization of C9 complement to form the C5b-9 membrane attack complex (MAC). MAC inserts into the target cell membrane, causing cell lysis.

[0003] Inappropriate or excessive complement activation can cause host cell damage and lead to related diseases, such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration (AMD), C3 complement glomerulonephropathy (C3G), immunoglobulin A nephropathy (IgAN), and generalized myasthenia gravis (gMG). Extensive efforts over the past few decades have been made to explore different complement inhibitors as therapeutic agents. Currently, the types of complement inhibitors used clinically include antibodies, small molecules, peptides, and small nucleic acids. The first complement inhibitor to be developed into a drug was eculizumab, which has been approved for four indications. The first approved indication was polycystic neuropathy (PNH), a disease caused by a mutation in the class A gene (PIGA) on the X chromosome of hematopoietic stem cells. This mutation leads to a defect in the synthesis of glycosylphosphatidylinositol (GPI) anchoring protein, resulting in a lack of GPI anchoring protein on the surface of mature blood cells. CD55 and CD59 cannot bind to the cell membrane through the GPI anchoring protein to inhibit the complement cascade, causing a large accumulation of the membrane attack complex (MAC) on the cell membrane. Extracellular solutions enter the cell through the MAC pores, causing the cell to swell and rupture. Clinically, PNH presents with symptoms such as hemolytic anemia, bone marrow failure, and thrombosis. Patients with PNH treated with eculizumab experience some improvement, but some still require regular blood transfusions to maintain normal function. One reason for this may be that eculizumab is a single-target C5 complement inhibitor and cannot inhibit C3b-mediated extravascular hemolysis. Pegcetacoplan is the first peptide inhibitor targeting C3 complement, effectively inhibiting complement C3 / C3b activation and simultaneously suppressing both extravascular and intravascular hemolysis, thus overcoming the limitations of C5 complement inhibitors. Human plasma C3 complement levels reach 120 mg / dL; therefore, Pegcetacoplan requires high-frequency, high-dose administration to achieve its efficacy, a limitation of complement inhibitors developed solely targeting C3 or C5 complement. Therefore, developing a drug that simultaneously targets both C3 and C5 complement has urgent clinical need and application value.

[0004] The active ingredient in Pegcetacoplan is compstatin, whose natural sequence was obtained by Professor Lambris in 1996 through phage screening. The sequence was optimized using amino acid substitution, methylation, and acetylation techniques to obtain the second-generation compstatin (Analog Cp05), which showed tens to hundreds of times greater affinity for C3 complement and enhanced complement inhibitory activity. The earliest patent application related to compstatin in China was filed in 2006 (CN 106188239A). Pegcetacoplan, a drug developed from compstatin, was approved by the FDA in 2021 and 2023 for the treatment of PNH and GA, respectively.

[0005] In addition to monoclonal antibodies and nucleic acids, complement inhibitors targeting C5 also include cyclic peptides, such as Zilucoplan. Zilucoplan is a polypeptide inhibitor that specifically targets C5 complement. Structurally, it uses a condensation of lysine ε-NH2 and aspartic acid γ-COOH to form a ring, which can specifically bind to complement C5 and its activating fragment C5b, blocking the complement cascade reaction. Zilucoplan was acquired by UCB through the acquisition of RA Pharmaceuticals, and it applied for PCT patents and a Chinese patent (CN106456701A) in 2015. Zilucoplan was approved for marketing in Japan, the United States, the European Union, and other regions in 2023, all for the indication of generalized myasthenia gravis (gMG). Currently, the drug is undergoing marketing application for the gMG indication in China.

[0006] Pegcetacoplan, a peptide inhibitor targeting C3 complement, is administered subcutaneously twice weekly to treat PNH disease. Zilucoplan, a peptide inhibitor targeting C5 complement, is administered subcutaneously once daily to treat generalized myasthenia gravis (gMG). Both drugs are single-target cyclic peptide complement inhibitors. Developing bifunctional cyclic peptides that simultaneously target and inhibit complement C3 and C5, with longer half-lives, has broad clinical application value. Summary of the Invention

[0007] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a polypeptide that targets and inhibits complement C3 and C5. This polypeptide can inhibit complement C3 activation and block complement cascade reactions involving C3b, as well as complement C5 activation and block complement cascade reactions involving C5b. The two inhibitory effects work synergistically to exert a stronger complement pathway inhibitory effect.

[0008] In one aspect of this application, a polypeptide or a pharmaceutically acceptable salt thereof is provided. According to embodiments of this application, the polypeptide comprises the structure shown in formula (I):

[0009] Z1-Z2-Z3(I),

[0010] Z1 has the ability to inhibit the C5 complement activation pathway;

[0011] Z2 is a linker peptide;

[0012] Z3 has the ability to inhibit the C3 complement activation pathway.

[0013] The bifunctional peptide of this application can inhibit complement C3 activation and block complement cascade reactions involving C3b, as well as complement C5 activation and block complement cascade reactions involving C5b. The two inhibitory effects work synergistically to exert a stronger complement pathway inhibitory effect.

[0014] In another aspect of this application, a peptide conjugate is proposed. According to embodiments of this application, the peptide conjugate or a pharmaceutically acceptable salt thereof comprises: the aforementioned peptide or a pharmaceutically acceptable salt thereof, and a coupling moiety, wherein the peptide is linked to the coupling moiety. As is previously known, the aforementioned peptide or a pharmaceutically acceptable salt thereof can inhibit complement C3 activation and block complement cascade reactions involving C3b, as well as complement C5 activation and block complement cascade reactions involving C5b. These two inhibitory effects work synergistically, exhibiting a stronger complement pathway inhibitory effect. Therefore, peptide conjugates containing the aforementioned peptide or a pharmaceutically acceptable salt thereof can effectively inhibit diseases and autoimmune diseases caused by excessive complement activation.

[0015] In another aspect of this application, a pharmaceutical composition is proposed. According to embodiments of this application, the pharmaceutical composition comprises: the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, or the aforementioned polypeptide conjugate or a pharmaceutically acceptable salt thereof. As is known, the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can inhibit complement C3 activation and block complement cascade reactions involving C3b, as well as complement C5 activation and block complement cascade reactions involving C5b. These two inhibitory effects work synergistically, exhibiting a stronger complement pathway inhibitory effect. Therefore, a pharmaceutical composition containing the aforementioned polypeptide or a pharmaceutically acceptable salt thereof can effectively inhibit diseases and autoimmune diseases caused by excessive complement activation.

[0016] In another aspect of this application, the application proposes the use of the aforementioned polypeptide or a pharmaceutically acceptable salt thereof, the aforementioned polypeptide conjugate or a pharmaceutically acceptable salt thereof, or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of diseases and autoimmune diseases caused by complement overactivation.

[0017] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0018] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0019] Figure 1 A schematic diagram of the complement activation pathway;

[0020] Figure 2 This is a mass spectrometry diagram of the IPO2 molecular weight and mass peptide in Example 1 of this application;

[0021] Figure 3 The inhibitory activity of the classical complement pathway activating C3 protein in Example 2 of this application (IC) 50 );

[0022] Figure 4 The inhibitory activity of the complement bypass pathway activating C3 protein in Example 3 of this application (IC) 50 );

[0023] Figure 5 The hemolytic inhibitory activity (CH50) of the classical complement activation pathway in Example 4 of this application;

[0024] Figure 6 The hemolytic inhibitory activity (AH50) of the complement bypass activation pathway in Example 5 of this application;

[0025] Figure 7 shows the bifunctional polypeptide conjugate targeting and inhibiting complement C3 and C5 prepared in Example 6 of this application, wherein (a) is the structural formula of CP01; and (b) is the electrophoresis and HPLC of CP01.

[0026] Figure 8 shows the PK and PD studies of CP01 in cynomolgus monkeys via different administration routes in Example 9 of this application. In the figure, (a) PK content, (b) CH50 content, (c) C3 content, and (d) C5 content.

[0027] Figure 9 shows CP01 and Aspaveli in Embodiment 10 of this application. TM Studies of PK and PD in non-human primates. The figure shows (a) PK content injected subcutaneously, (b) CH50 content injected subcutaneously, (c) C3 content injected subcutaneously, (d) C3a content injected subcutaneously, (e) CH50 content injected intravenously, and (f) C3 content injected intravenously.

[0028] Figure 10 shows the PK and PD study of CP01 and CP05 in cynomolgus monkeys in Example 11 of this application. In the figure, (a) PK content, (b) CH50 content, (c) C5 content, and (d) C3 content.

[0029] Figure 11 shows the dose-response relationship of subcutaneous injection of CP01 in cynomolgus monkeys in Example 12 of this application. In the figure, (a) PK content, (b) CH50 content, and (c) C3 content are shown.

[0030] Figure 12 shows the pharmacodynamic study of intravitreal injection of CP01 in New Zealand rabbits in Example 13 of this application. In the figure, (a) is the PK content in the vitreous body and (b) is the PK content in the aqueous humor. Detailed Implementation

[0031] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0032] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0033] This application details

[0034] Definitions and General Terms

[0035] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0036] To facilitate understanding of this application, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this application pertains. Abbreviations for amino acid residues are standard 3-letter and / or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids (i.e., amino acids with a levorotatory conformation).

[0037] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this application but do not exclude other contents.

[0038] In this document, the terms “optional,” “optional,” “alternatively,” “optional,” or “optional” generally refer to an event or condition that may or may not occur as described below, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0039] In this document, the term "peptide" generally refers to a sequence of peptides consisting of 10 to 40 amino acids linked by amide bonds. The amino acids include L-amino acids and D-amino acids; L-amino acids refer to naturally occurring levorotatory α-amino acids, and D-amino acids refer to dextrorotatory α-amino acids, such as y representing D-tyrosine. Unless otherwise specified, all amino acids described in this patent application are L-amino acids. The L-type amino acids are well known to those skilled in the art, and their names are represented by three letters followed by one letter: glycine (Gly-G); alanine (Ala-A); valine (Val-V); leucine (Leu-L); isoleucine (Ile-I); phenylalanine (Phe-F); tyrosine (Tyr-Y); tryptophan (Try-W); cysteine ​​(Cys-C); methionine (Met-M); aspartic acid (Asp-D); glutamic acid (Glu-E); asparagine (Asn-N); glutamine (Gln-Q); arginine (Arg-R); lysine (Lys-K); histidine (His-H); proline (Pro-P); serine (Ser-S); and threonine (Thr-T).

[0040] In this article, the term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a similar manner to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code and their modified forms, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Common natural amino acids include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V).

[0041] In this article, the term "amino acid analogue" generally refers to compounds that have the same basic chemical structure as naturally occurring amino acids (i.e., the α-carbon bound to hydrogen, carboxyl, amino, and R groups), such as amino acid analogues produced by various chemical reactions involving substitution, replacement, acetylation, and amidation of the α-NH2 or α-COOH or side chain functional groups of L-type amino acids. For example: W Me Both 1MeTrp and 1MeTrp are different manifestations of the same substance, representing 1-methyl-tryptophan, which is produced by replacing the hydrogen on the "-NH-" side chain of tryptophan with a methyl group; W 5Me Both W and 5MeTrp represent 5-methyl-tryptophan; 5F Both 5FTrp and Ac-X represent 5-fluoro-tryptophan. Ac-X indicates the acetylation of the α-NH2 of amino acid X (any amino acid abbreviation), for example, Ac-I indicates the acetylation of the α-NH2 of isoleucine; Ac-C indicates the acetylation of the α-NH2 of cysteine; Ac-K indicates the acetylation of the α-NH2 of lysine. X-NH2 indicates the amidation of the α-COOH of amino acid X (any amino acid abbreviation), for example, Thr-NH2 indicates the amidation of the α-COOH of threonine. Aib is 2-methylalanine, produced by the substitution of a hydrogen atom on the α-carbon of alanine with a methyl group; Sar is sarcosine, produced by the substitution of one hydrogen atom on the α-NH2 of glycine with a methyl group, and can also be represented as G. Me mIle is produced by replacing one of the hydrogen atoms on the α-NH2 of isoleucine with a methyl group, and can also be represented as I. Me ;D Me It is N-methylaspartic acid; Tbg is tert-butylglycine; yI refers to D-tyrosine-isoleucine, where y is D-tyrosine, I is isoleucine, and yI is a dipeptide formed by the two amino acids.

[0042] In this document, the term "pharmaceutically acceptable salt" refers to the organic and inorganic salts of the polypeptides or polypeptide conjugates of this application. Pharmaceutically acceptable salts are well known in the art and are not limited to any particular type.

[0043] In this document, the terms "short peptide containing one lysine," "short peptide," "linker peptide," "linker peptide sequence," "linker sequence," and "linker" are used interchangeably and all refer to an amino acid sequence between the target complement inhibitor C3 sequence and the target complement inhibitor C5 sequence. For example, the linker peptide contains a PKP amino acid sequence, which serves to separate the target complement inhibitor C3 and target complement inhibitor C5 sequences, ensuring that the two sequences do not interfere with each other and can independently exert their respective biological functions. The linker peptide is connected to the target complement inhibitor C3 and target complement inhibitor C5 sequences in two ways: (1) the N-terminus of the linker sequence is connected to the C-terminus of the target complement inhibitor C5 sequence, and the C-terminus of the linker sequence is connected to the N-terminus of the target complement inhibitor C3 sequence; (2) the N-terminus of the linker sequence is connected to the C-terminus of the target complement inhibitor C3 sequence, and the C-terminus of the linker sequence is connected to the N-terminus of the target complement inhibitor C5 sequence. Unless otherwise specified, the Linker described in this application is connected to the C3 and C5 sequences of the target-inhibitory complement in the manner described in the first (1) application.

[0044] In this article, the term "complement" refers to a group of heat-sensitive proteins present in the serum or tissue fluid of humans and animals. These proteins, upon activation, possess enzymatic activity and mediate immune responses and inflammatory reactions. The complement system comprises over 50 soluble proteins and membrane proteins. It consists of intrinsic components, complement regulatory proteins, and complement receptors. Classified by activation pathway, the classical activation pathway includes components such as the C1q, C1r, C1s, C2, C3, C4, C5, C6, C7, C8, C9, and the C5b-9 complex, as well as complement degradation fragments such as C3a, C3b, C4a, C4b, and C5a. The alternative activation pathway mainly includes components such as factor B, factor D, and factor P. The lectin pathway mainly includes components such as MBL2, MASP-1, and MASP-2. In addition, it includes various receptor proteins, such as: C5a receptor (C5aR), C3a receptor (C3aR), complement receptor 1 (CR1), complement receptor 2 (CR2), complement receptor 3 (CR3, also known as CD45), etc.

[0045] In this document, the term "pharmaceutical composition" generally refers to a composition in unit dose form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with a carrier constituting one or more adjunct components.

[0046] In this article, the term "pharmaceuticalally acceptable" refers to a substance that is suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a substance with a reasonable benefit / risk ratio.

[0047] In this document, the term "pharmaceuticalally acceptable excipient" may include any solvent, stabilizer, buffer, or other liquid excipient, etc., suitable for the specific target dosage form. The use of any conventional excipients, except for those incompatible with the fusion protein of this application, such as any adverse biological effects or harmful interactions with any other component of the pharmaceutically acceptable composition, is also within the scope of this application.

[0048] In this article, the term "diseases and autoimmune diseases caused by inhibition of complement overactivation" generally refers to diseases resulting from abnormal or excessive complement activation. "Overactivation" refers to a higher level of complement pathway activation compared to normal physiological conditions, which may be due to increased activity of complement activation products or decreased activity of complement regulatory proteins (e.g., CD55, CD59, and / or FH). For example, compared to normal conditions, decreased expression of complement regulatory proteins, decreased replication or transcription levels of complement regulatory protein genes, and abnormal translation of complement regulatory proteins can lead to an inability to regulate the complement activation pathway. Complement overactivation may induce pro-inflammatory responses, opsonization, or cell lysis. In some cases, the body produces autoantibodies against normal self-antigens. These autoantibodies, upon binding to the antigens, activate the complement pathway. Due to decreased activity of substances related to complement inhibition (e.g., complement regulatory proteins), immune damage occurs to the body's own tissues, organs, and cells, ultimately leading to autoimmune diseases. In some cases, diseases related to targeted inhibition of complement activation include autoimmune diseases. In some cases, diseases associated with targeted inhibition of complement activation may be caused by acute or chronic infections. In some cases, diseases associated with targeted inhibition of complement activation may be caused by gene mutations. Diseases associated with targeted inhibition of complement activation include, but are not limited to:

[0049] Hematologic disorders: paroxysmal nocturnal hemoglobinuria (PNH), cold agglutinin disease, microangiopathy (e.g., thrombotic microangiopathy), vasculitis, autoimmune hemolytic anemia, aplastic anemia, etc.

[0050] Kidney diseases: atypical hemolytic uremic syndrome (aHUS), IgA nephropathy, C3 glomerulonephropathy (C3G), nephritis (e.g., glomerulonephritis, immunoglobulin A nephropathy, membranoproliferative glomerulonephritis, membranous glomerulonephritis, lupus nephritis), and kidney injury, etc.

[0051] Immune system diseases: systemic lupus erythematosus, organ transplant rejection (e.g., kidney transplant rejection), autoimmune thrombocytopenic purpura, rheumatoid arthritis and other autoimmune diseases;

[0052] Ophthalmic diseases: Age-related macular degeneration (AMD), geographic atrophy (GA), Stargardt's disease, and keratoconjunctivitis, etc.

[0053] Neurological disorders: neuromyelitis optica spectrum disorders (NMOSD), myasthenia gravis (e.g., generalized myasthenia gravis (gMG)), amyotrophic lateral sclerosis, etc.

[0054] Respiratory diseases: acute respiratory distress syndrome, acute respiratory diseases caused by viral infections, etc.;

[0055] Digestive system diseases: protein-losing enteropathy;

[0056] Oral diseases: periodontitis and gingivitis, etc.;

[0057] Skin diseases: hidradenitis suppurativa, bullous pemphigoid, pyoderma gangrenosa, and systemic scleroderma, etc.;

[0058] Tumors: squamous cell carcinoma of the skin, lung cancer, and multiple myeloma, etc.

[0059] In this document, “treatment” means the provision of treatment, i.e., the provision of any type of medical or surgical treatment to a subject. Treatment may be used to reverse, alleviate, or inhibit the progression of a disease, prevent or reduce the likelihood of the disease, or to reverse, alleviate, inhibit, or prevent the progression of a disease, prevent or reduce one or more symptoms or manifestations of the disease. “Prevention” means preventing a disease or its symptoms or manifestations from occurring in at least some individuals for at least a period of time. Treatment may include the administration of a compound or composition to a patient after the development of one or more symptoms or manifestations of a disease. For example, to reverse, alleviate, reduce the severity of a disease, or inhibit one or more symptoms or manifestations of a disease. Peptides or drugs containing peptides may be administered to subjects who have developed a disease, or subjects who are at increased risk of developing the disease relative to members of the general population. Peptides or drugs containing peptides may be administered to subjects who have developed a disease and who have an increased risk of developing one or more specific symptoms or manifestations of the disease or disease exacerbation relative to other individuals diagnosed with the disease or relative to the typical or average risk of such symptoms or manifestations or exacerbations of the subject.

[0060] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The fusion protein or pharmaceutical composition of this application may be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are foreseeable, including peritoneal, intravenous, intramuscular, subcutaneous, etc., but this application is not limited to these exemplified routes of administration. Preferably, the fusion protein or pharmaceutical composition of this application is administered via intravenous or subcutaneous injection.

[0061] As used herein, the term “effective amount” or “effective dose” means an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals.

[0062] This application provides a detailed description of peptides, peptide conjugates, and their applications that target and inhibit C3 and C5 complement.

[0063] This application discloses a polypeptide or a pharmaceutically acceptable salt thereof, a polypeptide conjugate or a pharmaceutically acceptable salt thereof, a pharmaceutical composition thereof, and their uses, which will be described in detail below.

[0064] polypeptides or their pharmaceutically acceptable salts

[0065] In a first aspect of this application, a polypeptide or a pharmaceutically acceptable salt thereof is provided. According to embodiments of this application, the polypeptide comprises the structure shown in formula (I):

[0066] Z1-Z2-Z3(I),

[0067] Z1 has the ability to inhibit the C5 complement activation pathway;

[0068] Z2 is a linker peptide;

[0069] Z3 has the ability to inhibit the C3 complement activation pathway.

[0070] In an optional embodiment of this application, Z1 has the structure shown in formula (II):

[0071] Ac-X1VERFX2D Me VYWEY(II),

[0072] Among them, X1 and X2 form a ring through a disulfide bond or an amide bond;

[0073] Optionally, both X1 and X2 are selected from C;

[0074] Optionally, X1 is selected from K, and X2 is selected from D;

[0075] Optionally, X1 is selected from D, and X2 is selected from K.

[0076] In an optional embodiment of this application, Z3 has the structure shown in formula (III):

[0077] IX3VW Me QDWGAHRX4T-NH2(III),

[0078] Among them, X3 and X4 form a ring through disulfide bonds or amide bonds;

[0079] Optionally, both X3 and X4 are selected from C;

[0080] Optionally, X3 is selected from K, and X4 is selected from D;

[0081] Optionally, X3 is selected from D, and X4 is selected from K.

[0082] In an optional embodiment of this application, Z2 is a short peptide containing one lysine residue, wherein the number of amino acids in the short peptide is 2 to 9. The short peptide has the function of separating the complement-inhibiting C3 sequence (Z3) and the complement-inhibiting C5 sequence (Z1), so that the two sequences do not interfere with each other, and can not only independently exert their respective biological functions, but also coordinate the complement inhibition effect.

[0083] Optionally, Z2 is selected from PK, PKP, PKPSGG, PKPSGGSGG, GGSPKPSGG, PKPSGGGG.

[0084] In this application, the peptide Z1 inhibits complement C5 activation and blocks the complement cascade reaction involving C5b, Z3 inhibits complement C3 activation and blocks the complement cascade reaction involving C3b, and Z2 connects Z1 and Z3, enabling Z1 and Z3 to synergistically exert complement inhibition, exhibiting a stronger complement pathway inhibitory effect. Therefore, the above-mentioned peptide can effectively inhibit diseases and autoimmune diseases caused by excessive complement activation, and the peptide of this application can exhibit the advantages of lower dosage and / or longer-lasting efficacy.

[0085] In this paper, the aforementioned polypeptide may also be referred to as a bifunctional polypeptide, a bifunctional cyclic peptide, a bifunctional polypeptide that targets and inhibits complement C3 and C5, or a bifunctional cyclic peptide that targets and inhibits complement C3 and C5. The terms "bifunctional cyclic peptide that targets and inhibits complement C3 and C5" and "bifunctional cyclic peptide" are used interchangeably, referring to a polypeptide composed of a complement C3-targeting sequence and a complement C5-targeting sequence linked by a Z2 short peptide sequence, capable of simultaneously inhibiting complement C3 activation and preventing C3b from participating in the complement cascade reaction, and inhibiting complement C5 activation and preventing C5b from participating in the complement cascade reaction.

[0086] In an optional embodiment of this application, the polypeptide has the amino acid sequence described in any one of SEQ ID NO:1 to 9.

[0087] In an optional embodiment of this application, the polypeptide has two rings separated by a linker peptide Z2. Z3 is cyclic via an intrachain disulfide bond or an amide bond, and Z1 is cyclic via an intrachain disulfide bond or an amide bond.

[0088] peptide conjugates or their pharmaceutically acceptable salts

[0089] In a second aspect of this application, a polypeptide conjugate or a pharmaceutically acceptable salt thereof is provided. According to embodiments of this application, the polypeptide conjugate or a pharmaceutically acceptable salt thereof comprises: the polypeptide or a pharmaceutically acceptable salt thereof described in the first aspect, and a coupling moiety to which the polypeptide is linked.

[0090] As previously known, the aforementioned peptides or their pharmaceutically acceptable salts can inhibit complement C3 activation and block complement cascade reactions involving C3b, as well as complement C5 activation and block complement cascade reactions involving C5b. These two inhibitory effects work synergistically, resulting in a stronger complement pathway inhibitory effect. Therefore, peptide conjugates containing the aforementioned peptides or their pharmaceutically acceptable salts can effectively inhibit diseases and autoimmune diseases caused by excessive complement activation. Furthermore, the peptide conjugates of this application exhibit the advantages of lower dosage and / or longer-lasting efficacy.

[0091] In this paper, the aforementioned peptide conjugates may also be referred to as bifunctional peptide conjugates, or bifunctional peptide conjugates that target and inhibit complement C3 and C5. The terms "bifunctional peptide conjugate" and "bifunctional peptide conjugates that target and inhibit complement C3 and C5" are used interchangeably.

[0092] According to embodiments of this application, the above-mentioned polypeptide conjugate may further include at least one of the following technical features:

[0093] In an optional embodiment of this application, the polypeptide conjugate has the structure shown in formula (IV):

[0094] Peptide-coupled moiety-peptide (IV).

[0095] In one alternative embodiment of this application, the coupling portion includes, but is not limited to, polymers and carriers that bind to macromolecules in vivo.

[0096] In this article, the term "polymer" includes, but is not limited to, substances such as polyethylene glycol, immunoglobulins, and polylactic acid.

[0097] In this article, the term "carrier that binds to macromolecules in vivo" generally refers to carbohydrates, lipids, and proteins, with proteins specifically including albumin; the term "carrier that binds to macromolecules in vivo" usually refers to substances that can bind to albumin, including aliphatic compounds; the purpose of the carrier binding to macromolecules in vivo includes slowing the rate of drug release from the subcutaneous tissue into the bloodstream and reducing the rate of drug clearance by the glomeruli.

[0098] In an optional embodiment of this application, Z2 in the polypeptide contains one lysine residue that can be used to directionally couple a polymer or a carrier that binds to macromolecules in vivo. The directional coupling method is that the lysine is combined with the polymer in the form of an amide bond, or the lysine is combined with a carrier that binds to macromolecules in vivo in any connectable manner.

[0099] In one optional embodiment of this application, the binding ratio of the polypeptide and the polymer is such that 1 mole of polymer molecule can bind 1 to 8 moles of bifunctional polypeptide.

[0100] In one alternative embodiment of this application, the coupling portion includes, but is not limited to, polyethylene glycol, fatty acid compounds, or carriers capable of binding to macromolecules in vivo.

[0101] In one alternative embodiment of this application, the coupling portion is selected from polyethylene glycol.

[0102] In one optional embodiment of this application, the polyethylene glycol is selected from double-ended activated polyethylene glycol.

[0103] In this paper, the term "double-ended activation" refers to the presence of arbitrary activated functional groups at both ends of polyethylene glycol. The activated functional groups at both ends may be the same or different.

[0104] In this document, the terms "reactive functional group," "activated functional group," and "functional group" are used interchangeably. The activated functional group includes, but is not limited to, alkenes, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, diazo compounds, nitro groups, nitrile groups, sulfides, sulfoxides, nitroso compounds, succinimidyl propionate (SPA), N-hydroxysuccinimidyl (NHS), succinimidyl carbonate (SC), succinimidyl acetate (SCM), maleimide, mercapto groups, acyl chlorides, acid anhydrides, etc. Methods for preparing polyethylene glycol containing the above functional groups can be obtained using techniques known in the art.

[0105] In one optional embodiment of this application, the dual-terminal activated groups of the polyethylene glycol are selected from N-hydroxysuccinimide, acyl chloride, or acid anhydride.

[0106] In one optional embodiment of this application, the dual-terminal activated groups of the dual-terminal activated polyethylene glycol are selected from N-hydroxysuccinimide.

[0107] In one optional embodiment of this application, the polyethylene glycol has a linear or branched structure.

[0108] In one optional embodiment of this application, the polyethylene glycol has a branched structure, and the number of branches does not exceed four.

[0109] In one optional embodiment of this application, the polyethylene glycol has a direct-connect structure.

[0110] In one optional embodiment of this application, the polyethylene glycol has 1 to 8 activating groups, for example, 1, 2, 3, 4, 5, 6, 7 or 8.

[0111] In one optional embodiment of this application, the polyethylene glycol has two activating groups.

[0112] In an optional embodiment of this application, the molecular weight of the dual-end activated polyethylene glycol is 2000 Da to 40000 Da, for example, 20000 Da, 21000 Da, 22000 Da, 23000 Da, 24000 Da, 25000 Da, 26000 Da, 27000 Da, 28000 Da, 29000 Da, 30000 Da, 31000 Da, 32000 Da, 33000 Da, 34000 Da, 35000 Da, 36000 Da, 37000 Da, 38000 Da, 39000 Da, 40000 Da, or any two of these values ​​as a range between the endpoint values.

[0113] In one optional embodiment of this application, the molecular weight of the dual-terminated activated polyethylene glycol is 20,000 Da to 40,000 Da. This allows both ends of the polyethylene glycol peptide to exert their respective biological effects, and by binding to different complement C3 (or C3b) and / or complement C5 (or C5b), further enhances the inhibitory effect of the complement activation pathway.

[0114] In one example of this application, 1 mole of double-ended activated polyethylene glycol in the polypeptide conjugate is coupled to 2 moles of polypeptide.

[0115] Pharmaceutical Composition

[0116] In a third aspect of this application, a pharmaceutical composition is provided. According to embodiments of this application, the pharmaceutical composition comprises: the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, or the polypeptide conjugate described in the second aspect or a pharmaceutically acceptable salt thereof.

[0117] As previously known, the aforementioned peptides or their pharmaceutically acceptable salts can inhibit complement C3 activation and block complement cascade reactions involving C3b, as well as complement C5 activation and block complement cascade reactions involving C5b. These two inhibitory effects work synergistically, resulting in a stronger complement pathway inhibitory effect. Therefore, pharmaceutical compositions containing the aforementioned peptides or their pharmaceutically acceptable salts can effectively inhibit diseases and autoimmune diseases caused by excessive complement activation.

[0118] In an optional embodiment of this application, the pharmaceutical composition further includes a pharmaceutically acceptable carrier, excipient, or mediator.

[0119] In one alternative embodiment of this application, pharmaceutically acceptable excipients refer to pharmaceutical excipients that are conventional in the pharmaceutical field, such as diluents, buffer solutions, osmotic pressure regulators, pH regulators, etc.

[0120] In one optional embodiment of this application, the buffer is selected from at least one of acetate, phosphate, borate, carbonate, and citrate, preferably acetate.

[0121] In one optional embodiment of this application, the osmotic pressure regulator is selected from at least one of mannitol, sucrose, maltose, sorbitol, and trehalose, preferably sorbitol.

[0122] In one optional embodiment of this application, the pH adjuster is selected from at least one of hydrochloric acid, glacial acetic acid, phosphoric acid, citric acid, sodium hydroxide, potassium hydroxide, and sodium carbonate, preferably glacial acetic acid and sodium hydroxide.

[0123] In one alternative embodiment of this application, a pharmaceutically acceptable carrier refers to a drug carrier conventional in the pharmaceutical field, such as a protectant.

[0124] In one alternative embodiment of this application, pharmaceutically acceptable mediators refer to pharmaceutical mediators conventional in the pharmaceutical field, such as solutions (e.g., water) and liposomes.

[0125] In one alternative embodiment of this application, examples of suitable pharmaceutically acceptable carriers, excipients, and mediators are well known in the art. Pharmaceutical compositions comprising such carriers, excipients, and mediators can be formulated using known conventional methods.

[0126] In some alternative embodiments, the pharmaceutical composition of this application may also contain other active ingredients for treatment.

[0127] The pharmaceutical composition of this application can be administered via various routes, such as enterically, orally (e.g., liquid solution), or by injection (e.g., intravenously, subcutaneously, intramuscularly, intraperitoneally, intradermally, or intravitreal). Preferably, the pharmaceutical composition of this application is in the form of a lyophilized formulation or an aqueous solution. The clinical dosing regimen will be determined by the attending physician and clinical factors. As is known in the medical field, the dosage for any given patient depends on many factors, including the patient's physique, weight, body surface area, age, the drug to be administered, sex, time and route of administration, general health, and other concurrently administered medications. The pharmaceutical composition of this application can be administered topically or systemically. Preferably, it can be administered intravenously or subcutaneously. The pharmaceutical composition of this application can also be administered directly to the target site, for example, by targeted administration to internal or external target sites.

[0128] use

[0129] In a fourth aspect of this application, the use of the polypeptide described in the first aspect, the polypeptide conjugate described in the second aspect, and the pharmaceutical composition described in the third aspect in the preparation of a medicament for the prevention and / or treatment of diseases related to complement overactivation and autoimmune diseases.

[0130] In an optional embodiment of this application, the related diseases and autoimmune diseases caused by complement overactivation include at least one of the following: paroxysmal nocturnal hemoglobinuria, cold agglutinin disease, thrombotic microangiopathy, autoimmune hemolytic anemia, atypical hemolytic uremic syndrome, C3 glomerulonephropathy, kidney transplant rejection, glomerulonephritis, immunoglobulin A nephropathy, membranoproliferative glomerulonephritis, membranous glomerulonephritis, lupus nephritis, hereditary angioedema, geographic atrophy, age-related macular degeneration, Stargardt disease, motor neuron disease, myasthenia gravis, hidradenitis suppurativa, antineutrophil cytoplasmic antibody-associated vasculitis, Guillain-Barré syndrome, and periodontitis.

[0131] method

[0132] In a fifth aspect of this application, a method for preventing and / or treating diseases and autoimmune diseases caused by complement overactivation is proposed. According to embodiments of this application, the method comprises administering to a subject a pharmaceutically acceptable dose of the polypeptide described in the first aspect or a pharmaceutically acceptable salt thereof, the polypeptide conjugate described in the second aspect or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the third aspect.

[0133] In one alternative embodiment of this application, the pharmaceutically acceptable dose may be selected from the effective dose (or effective amount).

[0134] The effective amount of the compound described in this application may vary depending on the administration method and the severity of the disease to be treated. A preferred effective amount can be determined by those skilled in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, and the route of administration. For example, due to the urgency of the treatment condition, several separate doses may be administered daily, or the dose may be reduced proportionally.

[0135] The polypeptides, polypeptide conjugates, or pharmaceutical compositions of this application may be incorporated into suitable pharmaceuticals, which may be prepared in various forms, such as liquids. Various routes of administration of the polypeptides, polypeptide conjugates, pharmaceutical compositions, or pharmaceuticals of this application are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, dermal, and oral administration, but this application is not limited to these exemplified routes of administration.

[0136] In an optional embodiment of this application, the related diseases and autoimmune diseases caused by the inhibition of complement overactivation include at least one of the following: paroxysmal nocturnal hemoglobinuria, cold agglutinin disease, thrombotic microangiopathy, autoimmune hemolytic anemia, atypical hemolytic uremic syndrome, C3 glomerulonephropathy, kidney transplant rejection, glomerulonephritis, immunoglobulin A nephropathy, membranoproliferative glomerulonephritis, membranous glomerulonephritis, lupus nephritis, hereditary angioedema, geographic atrophy, age-related macular degeneration, Stargardt disease, motor neuron disease, myasthenia gravis, hidradenitis suppurativa, antineutrophil cytoplasmic antibody-associated vasculitis, Guillain-Barré syndrome, and periodontitis.

[0137] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0138] Example 1: Synthesis of a bifunctional polypeptide targeting and inhibiting complement C3 and C5

[0139] Preparation of bifunctional peptides:

[0140] Based on the Fmoc solid-phase method, the bifunctional peptides in Table 2 were synthesized using the Fmoc-protected amino acids listed in Table 1. Taking IPO2 and IPO3 as examples, an appropriate amount of Rink amide MBHA resin was used, with DIC and HATU as coupling agents, and 20% PIP / DMF, TFA, and other solvents as deprotection solutions. Synthesis proceeded from the carboxyl terminus of the IPO2 peptide towards the amino terminus. When the peptide chain contained two cysteine ​​residues, 1% iodoformic acid solution was used to induce the cysteine ​​sulfhydryl groups to form a ring, and vitamin C solution was used to react with the excess iodine. After washing the column with DIC, deprotection and activation were repeated to continue coupling the next amino acid until two more cysteine ​​residues were coupled. The same method was used to induce the cysteine ​​sulfhydryl groups to form a ring. The amino acids were coupled until a bifunctional peptide was formed, which was then cleaved from the resin. The crude peptide was subjected to N-terminal acetylation in acetic acid solution, purified by refining or high-performance liquid chromatography, and lyophilized to obtain IPO2 peptide powder. Thin-layer chromatography and HPLC were used to monitor the reaction process, and LC-MS was used to identify the structure of the synthesized sequence. In the synthesis of IP03, the amino acid raw materials that form cyclization with amide bonds are tert-butyl disulfide and Fmoc-protected Fmoc-Lys(Tbeoc)-OH and Fmoc-Asp(Tbe)-OH. The synthesis method is a process well-known to practitioners in this industry, and the synthesis method of IP02 can also be referred to. After the synthesis is completed, the protecting groups are removed by a mixed solvent of 2-mercaptoethanol / DIPEA / 40% H2O / DMF. The peptide is cut off from the resin and then subjected to acetylation treatment, purification, freeze drying and other processes to finally obtain IP03 peptide powder.

[0141] The IPO1 and IPO4-IP09 peptides were synthesized using the method described above. This example demonstrates the mass spectrometry molecular weight and peptide mapping molecular weight identification results of the IPO2 peptide. (See attached image for details.) Figure 2 .

[0142] Table 1: Fmoc-protected amino acids

[0143] serial number Protect amino acids serial number Protect amino acids 1 Fmoc-Ile-OH 12 Fmoc-Thr(tBu)-OH 2 Fmoc-Cys(Trt) 13 Fmoc-Ser(tBu)-OH 3 Fmoc-Val-OH 14 Fmoc-Pro-OH 4 Fmoc-Trp(Me)-OH 15 Fmoc-Lys(Boc)-OH 5 Fmoc-Gln(Trt)-OH 16 Fmoc-Aib-OH 6 Fmoc-Asp(OtBu)-OH 17 Fmoc-Sar-OH 7 Fmoc-Trp(Boc)-OH 18 Fmoc-Ile(Me)-OH 8 Fmoc-Gly-OH 19 Fmoc-D-Tyr(tBu)-OH 9 Fmoc-Ala-OH 20 Fmoc-Lys(Tbeoc)-OH 10 Fmoc-His(Trt)-OH 21 Fmoc-Asp(Tbe)-OH 11 Fmoc-Arg(Pbf)-OH 22 Fmoc-Glu(Tbe)-OH

[0144] Table 2: Amino acid sequences of bifunctional peptides targeting complement C3 and C5

[0145] code name amino acid sequence Serial Number IP01 <![CDATA[[CVERFC]-D Me VYWEY-PKP-I-[CVW Me QDWGAHRC]-T]]> SEQ ID NO:1 IP02 <![CDATA[Ac-[CVERFC]-D Me VYWEY-PKPSGG-I-[CVW Me QDWGAHRC]-T-NH2]]> SEQ ID NO:2 IP03 <![CDATA[Ac-[KVERFD]-D Me VYWEY-GGSPKPSGG-I-[CVW Me QDWGAHRC]T]]> SEQ ID NO:3 IP04 <![CDATA[Ac-[CVERFC]-D Me VYWEY-GGSPKPSGG-I-[CVW Me QDWGAHRC]T-NH2]]> SEQ ID NO:4 IP05 <![CDATA[Ac-[CVERFC]-D Me VYWEY-PK-I-[CVW Me QDWGAHRC]-T-NH2]]> SEQ ID NO:5 IP06 <![CDATA[Ac-[CVERFC]-D Me VYWEY-PKPSGGSGG-I-[CVW Me QDWGAHRC]-T-NH2]]> SEQ ID NO:6 IP07 <![CDATA[Ac-I-[CVW Me QDWGAHRC]-T-PKPSGG-[CVERFC]-D Me VYWEY-NH2]]> SEQ ID NO:7 IP08 <![CDATA[Ac-I-[CVW Me QDWGAHRC]-T-PKPSGG-I-[CVW Me QDWGAHRC]-T-NH2]]> SEQ ID NO:8 IP09 <![CDATA[Ac-[CVERFC]-D Me VYWEY-PKPSGG-[CVERFC]-D Me VYWEYP-NH2]]> SEQ ID NO:9

[0146] In the table above, [] indicates that the amino acids form a ring, and "-" between amino acids indicates a peptide bond; "-" between amino acids and groups (-Ac or -NH2) indicates a chemical bond.

[0147] Example 2: C3 complement inhibition activity via the classical pathway

[0148] The complement inhibitory activity of the bifunctional peptide in Example 1 was determined using the classical complement protein activation pathway method. The detection steps are summarized as follows:

[0149] 1) Coat 96-well plates with chicken OVA antibody, incubate overnight at 2-8℃, and block the plates with PBS;

[0150] 2) Add rabbit anti-chicken OVA antibody, incubate at room temperature for 1 hour, wash and set aside;

[0151] 3) Add barbiturate buffer (VB++ buffer), candidate drug, and hirudin anticoagulated plasma sequentially, and incubate at room temperature for 1 hour;

[0152] 4) Add goat anti-human C3-HRP (manufacturer: Solarbio; product number: SE109) ​​and incubate at room temperature for 1 hour;

[0153] 5) After adding TMB for color development, measure the absorbance at 450nm / 630nm;

[0154] 6) Use software to fit the absorbance data to obtain IC. 50 value.

[0155] Table 3: C3 complement inhibition activity via classical pathway (IC50) 50 )

[0156] Code Name <![CDATA[IC 50 ]]> Code Name <![CDATA[IC 50 ]]> IP01 1.382μM IP07 0.357μM IP02 0.370μM IP08 0.132μM IP03 0.564μM IP09 >500μM IP04 0.552μM <![CDATA[ ① P14 peptide]]> 238.2μM IP05 3.425μM <![CDATA[ ② POT-4]]> 0.254μM IP06 0.574μM / /

[0157] Note: ① P14 peptide sequence: Ac-[CVERFC]-D Me VYWEYPK. IPO2 was obtained by enzymatic digestion with Lys-C enzyme (purchased from Chongqing Aimeidi Biotechnology Co., Ltd.);

[0158] ② POT-4 sequence: Ac-I-[CVW] Me QDWGAHRC]-T-NH2, purchased from

[0159] As shown in Table 3, the inhibitory effects of IPO2 and IPO7 on complement C3 / C3b were not significantly different from those of POT-4. P14 peptide and IPO9 had no or very low inhibitory effects on complement C3 / C3b, indicating that Z1, whether placed before or after Z3, does not interfere with Z3's C3 complement inhibitory effect. The inhibitory effects of IPO3, IPO4, and IPO6 on complement C3 / C3b were essentially the same and all superior to IPO1 and IPO5, indicating that the number of amino acids in the Z2 linker peptide is less than 3, and Z1 interferes with Z3's C3 complement inhibitory effect. The inhibitory effects of IPO2 and IPO7 on complement C3 / C3b were superior to those of IPO3, IPO4, and IPO6, indicating that a number of amino acids in the Z2 linker peptide is more suitable at 6. This embodiment exemplarily demonstrates the IC50 inhibitory activity (IC50) of some candidate peptides against complement C3 / C3b. 50 For data, please refer to Table 3. For complement inhibition activity curves, please refer to... Figure 3 .

[0160] Example 3: C3 complement inhibition activity via the bypass pathway

[0161] The complement inhibitory activity of the bifunctional peptide in Example 1 was determined using the complement protein bypass activation pathway method. The detection steps are summarized as follows:

[0162] 1) Coat 96-well plates with lipopolysaccharide (LPS), incubate at room temperature for 2 hours, and then block the microplate with 2% BSA;

[0163] 2) Add the candidate drug and hirudin anticoagulated plasma sequentially, and incubate at 37°C for 1 hour;

[0164] 3) Add mouse anti-C3b / iC3b monoclonal antibody and incubate at 37°C for 1 hour;

[0165] 4) Add goat anti-mouse IgG-HRP and incubate at 37°C for 1 hour;

[0166] 5) After adding TMB for color development, measure the absorbance at 450nm / 630nm;

[0167] 6) Use software to fit the absorbance data to obtain IC. 50 value.

[0168] Table 4: Complement inhibition activity of the bypass activation pathway (IC50) 50 )

[0169] Code Name <![CDATA[IC 50 ]]> Code Name <![CDATA[IC 50 ]]> IP01 3.192μM IP07 1.217μM IP02 1.016μM IP08 0.241μM IP03 1.943μM IP09 193.611μM IP04 2.137μM P14 peptide 73.984μM IP05 8.784μM POT-4 0.495μM IP06 1.993μM / /

[0170] As shown in Table 4, the inhibitory effects of IPO2 and IPO7 on complement C3 / C3b are about half that of POT-4, which is presumably related to the increased molar molecular weight of IPO2 and IPO7. In reality, the C3 / C3b complement inhibitory effect of the Z3 sequence on IPO2 and IPO7 is similar to that of POT-4. P14 peptide and IPO9 have no or very low inhibitory effects on complement C3 / C3b, indicating that Z1, whether before or after Z3, does not interfere with Z3's C3 complement inhibitory effect. The inhibitory effects of IPO3, IPO4, and IPO6 on complement C3 / C3b are basically consistent and all superior to IPO1 and IPO5, indicating that the number of amino acids in the Z2 linker peptide is less than 3, and Z1 interferes with Z3's C3 complement inhibitory effect. The inhibitory effects of IPO2 and IPO7 on complement C3 / C3b are superior to those of IPO3, IPO4, and IPO6, indicating that a number of amino acids in the Z2 linker peptide is more suitable. This embodiment exemplifies the inhibitory activity of some candidate peptides on complement C3 / C3b. 50 For data, please refer to Table 4. For complement inhibition activity curves, please refer to... Figure 4 Among them, P14 peptide and POT-4 served as control groups, and their specific sources are detailed in Example 2.

[0171] Example 4: Classical pathway hemolysis inhibition effect (CH50)

[0172] The hemolytic activity (CH50) of the bifunctional peptide in Example 1 against the classical complement activation pathway was determined using sheep erythrocytes. The detection procedure is summarized as follows:

[0173] ① Resuspension: GVB++ buffer (containing Mg 2+ Ca 2+ Dilute 4% sheep red blood cells to a red blood cell concentration of approximately 1×10⁻⁶. 9 cells / ml;

[0174] ② Sensitization: Add hemolysin, mix well, and incubate at 37°C for 20 minutes;

[0175] ③ Dilute sheep red blood cells to approximately 1×10⁻⁶. 9 cells / ml;

[0176] ④ Reaction: Sensitized sheep red blood cells were added to a 96-well plate, followed by plasma and candidate drugs. After mixing, the mixture was incubated at 37°C for 1 hour.

[0177] ⑤ Measure absorbance: Centrifuge the microplate at 4℃ (×1250g) for 7 min, collect the supernatant and measure the absorbance at 412nm.

[0178] Table 5: Data on the hemolytic inhibition effect (CH50) of the classical complement activation pathway

[0179] Code Name CH50 Code Name CH50 IP01 0.871μM IP07 2.303μM IP02 0.246μM IP08 1.498μM IP03 0.337μM IP09 0.311μM IP04 0.293μM P14 peptide 0.066μM IP05 1.198μM POT-4 1.837μM IP06 0.315μM / /

[0180] As shown in Table 5, the P14 peptide exhibits approximately 28 times stronger hemolytic inhibition against the classical complement activation pathway than POT-4, indicating that the Z1 ring in the peptide sequence has a stronger inhibitory effect on complement-mediated hemolysis than the Z3 ring. The CH50 data for IP03 and IP04 are consistent, indicating that the hemolytic inhibition effect of the peptide's Z1 sequence, whether cyclic with disulfide or amide bonds, is comparable to that of the classical complement activation pathway. IP02 and IP09 show comparable hemolytic inhibition effects against the classical complement activation pathway, and both are approximately 10 times stronger than IP07, suggesting that designing the Z1 sequence at the carboxyl terminus does not fully exert the hemolytic inhibition effect. IP01 and IP05 showed comparable and lower hemolytic inhibitory effects on the classical complement activation pathway than IP02, indicating that when the Z2 linker peptide is too short, the Z1 inhibitory effect on complement-mediated hemolysis is interfered with by the Z3 linker peptide. Comparison of IP03, IP04, and IP06 revealed that approximately six amino acids in the Z2 linker peptide is more suitable. Hemolytic inhibitory activity curves for some peptides are shown in [reference needed]. Figure 5 Among them, P14 peptide and POT-4 served as control groups, and their specific sources are detailed in Example 2.

[0181] Example 5: Hemolysis inhibition effect via bypass pathway (AH50)

[0182] The inhibitory hemolytic activity (AH50) of the bifunctional peptide in Example 1 against the complement bypass activation pathway was determined using rabbit erythrocytes. The AH50 detection procedure is summarized as follows:

[0183] ① Resuspension: Rabbit erythrocytes (RRBCs) were diluted to a concentration of approximately 2 × 10⁻⁶ in GVB++ buffer (containing 40 mmol / L MgCl₂ and 40 mmol / L EGTA). 8 cells / ml;

[0184] ②Reaction: Take 2×10 7 RRBCs at a concentration of cells / ml were added to a 96-well plate, followed by the addition of plasma and candidate drugs. After mixing, the plate was incubated at 37°C for 1 hour.

[0185] ③Measure absorbance: Centrifuge the microplate at 4℃ (1250G) for 7 min, collect the supernatant and measure the absorbance at 412 nm.

[0186] Table 6. Data on the hemolytic inhibition effect (AH50) of the complement alternative pathway activation.

[0187] Code Name AH50 Code Name AH50 IP01 1.716μM IP07 7.297μM IP02 0.643μM IP08 3.701μM IP03 0.807μM IP09 0.514μM IP04 1.014μM P14 peptide 0.119μM IP05 2.759μM POT-4 4.837μM IP06 0.964μM / /

[0188] Table 6 shows that the P14 peptide exhibits approximately 40 times stronger hemolytic inhibition against complement bypass activation pathways than POT-4, indicating that the Z1 ring in the peptide sequence has a stronger inhibitory effect on complement-mediated hemolysis than the Z3 ring. The AH50 data for IP03 and IP04 are consistent, indicating that the Z1 sequence of the peptide, whether cyclically formed with disulfide or amide bonds, has comparable hemolytic inhibition effects against complement bypass activation pathways. IP02 and IP09 show comparable hemolytic inhibition effects against complement bypass activation pathways, and both are approximately 11 times stronger than IP07, suggesting that designing the Z1 sequence at the carboxyl terminus does not fully exert the hemolytic inhibition effect. IP01 and IP05 showed comparable and lower hemolytic inhibitory effects on the complement bypass activation pathway than IP02, indicating that when the Z2 linker peptide is too short, the Z1 inhibitory effect on complement-mediated hemolysis is interfered with by the Z3 sequence in the peptide. Comparison of IP03, IP04, and IP06 revealed that approximately six amino acids in the Z2 linker peptide is more suitable. Hemolytic inhibitory activity curves for some peptides are shown in [reference needed]. Figure 6 Among them, P14 peptide and POT-4 served as control groups, and their specific sources are detailed in Example 2.

[0189] Example 6: Preparation of bifunctional peptide conjugates targeting complement C3 and C5

[0190] Preparation of bifunctional peptide conjugates:

[0191] To prepare the bifunctional peptide conjugates listed in Table 7, taking CP01 as an example: Weigh an appropriate amount of IPO2 peptide sample into a clean beaker, add a mixture of buffer salt (carbonate is selected in this preparation method, but phosphate and borate can also be used) and organic solvent (acetonitrile is selected in this preparation method, but ethanol, tetrahydrofuran, DMF, and NMP can also be used) and stir to dissolve; adjust the pH to the range of 8-12 using sodium hydroxide, and add a 40K molecular weight double-ended activated polyethylene glycol conjugate (purchased from Beijing Jiankai Technology Co., Ltd., specifically polyethylene glycol disuccinimide propionate, i.e., succinimide propionate-polyethylene glycol-succinimide propionate, SPA-PEG) at a molar ratio of IPO2 peptide to double-ended activated polymer of 2:1.5±0.5. 40K -SPA), the reaction was stirred at room temperature for 1.5 ± 0.5 h, diluted 3-5 times with an equal volume of purified water, and the pH was adjusted to 1-5 using an acidic aqueous solution (hydrochloric acid is selected in this preparation method, but sulfuric acid, phosphoric acid, and glacial acetic acid can also be used) to terminate the reaction. After filtration through a filter membrane, the sample was purified using cation exchange chromatography medium. Related substances and purity were monitored using RP-HPLC and SDS-PAGE. Ultrafiltration was used to remove salt to obtain the CP01 stock solution (see Figure 7a ~b).

[0192] The CP02-CP05 stock solutions were prepared according to the method described above. The structures of the bifunctional peptide conjugates in this example are shown in Table 3.

[0193] Table 7: Structures of bifunctional peptide conjugates targeting complement C3 and C5

[0194] name sequence structure CP01 <![CDATA[IP02-PEG 40K -IP02]]> CP02 <![CDATA[IP02-PEG 20K -IP02]]> CP03 <![CDATA[IP08-PEG 40K -IP08]]> CP04 <![CDATA[IP09-PEG 40K -IP09]]> CP05 <![CDATA[P14 peptide - PEG 40K - P14 peptide]]>

[0195] PEG 20K PEG 40K These are polyethylene glycols with molecular weights of 20K and 40K, respectively.

[0196] Example 7: Preparation of a bifunctional peptide conjugate formulation targeting complement C3 and C5 inhibition

[0197] Preparation of bifunctional peptide conjugate formulations:

[0198] Prepare an acetate buffer solution and add sorbitol excipient, stirring to dissolve. The acetate concentration is approximately 1 mg / ml, and the sorbitol concentration is approximately 82 mg / ml. Take an appropriate amount of the bifunctional polypeptide conjugate stock solution prepared in Example 6 (200 mg protein), add it to the sorbitol-containing acetate solution, stir to mix well, and adjust the pH to 5.0 ± 0.2 with glacial acetic acid or sodium hydroxide solution. Determine the protein content, aseptically filter, dispense into vials, cap and label, and store at 2℃~8℃ for later use. CP01~CP05 formulations are obtained.

[0199] Example 8: Affinity of candidate structures with C3 and C5 complement.

[0200] Binding affinity and kinetics were determined using a multi-cycle kinetic method. First, the C3 protein was directly immobilized onto the detection channel of the CM5 chip via amino-linked coupling. Then, serially diluted mobile phase peptide molecules were detected, with one cycle performed for each concentration. Kinetics, steady-state binding signals, and affinity were measured. The C5 protein was immobilized using the same method, and binding detection was performed on the bifunctional peptide from Example 1 and the bifunctional peptide conjugate from Example 2 (referred to as candidate drugs).

[0201] The results showed that both the bifunctional peptides and bifunctional peptide conjugates of this application possessed C3 complement affinity and C5 complement affinity. This embodiment provides illustrative examples of the affinity kinetic data for some peptides and peptide conjugates, as shown in Table 8. P14 peptide and POT-4 served as control groups; their specific sources are detailed in Example 4.

[0202] Table 8: Affinity test results of some candidate structures with complement C3 and complement C5

[0203]

[0204]

[0205] Note: Aspaveli TM It is the product name of Pegcetacoplan in the European market, and it was purchased from the European market (lot number: 3961601K);

[0206] As shown in Table 8, the affinity of IPO2 peptide to complement C3 is comparable to that of POT-4 peptide, and the affinity of IPO2 peptide to complement C5 is comparable to that of P14 peptide. The CP04 candidate drug, which is formed by coupling with 40 kDa polyethylene glycol, still shows good affinity to C3 complement and C5 complement.

[0207] Example 9: PK and PD studies of CP01 administered via different routes in cynomolgus monkeys

[0208] This embodiment studies the pharmacokinetics of the polypeptide conjugate CP01 in cynomolgus monkeys via subcutaneous and intravenous injections. The specific steps are as follows:

[0209] Four adult cynomolgus monkeys were divided into two groups and administered a single subcutaneous injection and a single intravenous injection, respectively, at a dose of 2 mg / kg.

[0210] Sampling points for subcutaneous administration: Before administration, and at 2h, 8h, 24h, 48h, 72h, 96h, 120h, 168h, and 336h after administration (one additional sampling point is added for the intravenous administration group: 5min after administration). Approximately 1ml of whole blood was collected from the subcutaneous veins of the forelimbs and / or hindlimbs of each animal. After blood collection, the blood samples were transferred to blood collection tubes containing EDTA-K2 anticoagulant, manually inverted at least 5 times, and then temporarily stored in an ice box. The plasma was centrifuged at 1500g for 10min at 2-8℃ and stored at -60℃ for later use.

[0211] PD sampling points for subcutaneous administration: before administration, and at 2h, 8h, 24h, 72h, 120h, 168h, and 336h after administration (one additional sampling point is added for the intravenous administration group: 5min after administration). Approximately 3ml of whole blood was collected from the subcutaneous veins of the forelimbs and / or hindlimbs of each animal. After blood collection, the samples were transferred to centrifuge tubes containing separating gel and coagulant, centrifuged at 1500g for 15min at room temperature, and the serum was collected and stored at -60℃ for later use.

[0212] Plasma drug concentrations were detected using a self-developed LC-MS / MS method, and serum C3, C3a, C5, C5a, and CH50 levels were detected using commercial kits. Some PK / PD chromatograms are shown below. Figure 8a ~d.

[0213] CP01 was administered to cynomolgus monkeys via single injection, either subcutaneously or intravenously. PK testing results showed that the average T in the subcutaneous group was... 1 / 2The mean duration of action was 50.4 hours, significantly better than the intravenous administration group (mean T). 1 / 2 =14.2h), indicating that CP04 can exert a longer-lasting effect through subcutaneous administration, potentially allowing for lower dosing frequencies. PD test results showed that CH50 14 days after subcutaneous administration was equivalent to 29.3% of the pre-administration level, while CH50 14 days after intravenous administration was equivalent to 77.4% of the pre-administration level, further demonstrating that CP04 can exert a longer-lasting effect through subcutaneous administration. Other PD indicators (C3, C3a, C5, C5a) also showed that subcutaneous injection was superior to intravenous injection for CP01.

[0214] Example 10: CP01 and Aspaveli TM PK and PD studies in non-human primates

[0215] This embodiment describes the peptide conjugate CP01 and Aspaveli. TM Pharmacokinetic studies in cynomolgus monkeys. Specific steps are as follows:

[0216] Four adult cynomolgus monkeys were divided into two groups. CP01 and Aspaveli were administered intravenously to each group, respectively. After a 28-day washout period, the two groups were cross-administered via subcutaneous injection.

[0217]

[0218] PK sampling points for the intravenous administration group: Before administration, and at 5 min, 2 h, 8 h, 24 h, 48 h, 72 h, 96 h, 120 h, and 168 h after administration, approximately 1 ml of whole blood was collected from the subcutaneous veins of the forelimbs and / or hindlimbs of each animal. After collection, the blood samples were transferred to blood collection tubes containing EDTA-K2 anticoagulant, manually inverted at least 5 times, and temporarily stored in an ice box. The blood samples were then centrifuged at 1500g for 10 min at 2–8°C, and the plasma was collected and stored at -60°C for later use. PD sampling points for the intravenous administration group: Before administration, and at 2 h, 8 h, 24 h, 72 h, 120 h, and 168 h after administration, approximately 3 ml of whole blood was collected from the subcutaneous veins of the forelimbs and / or hindlimbs of each animal. After collection, the blood samples were transferred to centrifuge tubes containing separating gel and coagulant, centrifuged at 1500g for 15 min at room temperature, and the serum was collected and stored at -60°C for later use.

[0219] PK sampling points for the subcutaneous drug administration group: Before administration, and at 2h, 8h, 24h, 48h, 72h, 96h, 120h, 168h, 240h, and 336h after administration, approximately 1ml of whole blood was collected from the subcutaneous veins of each animal's forelimb and / or hindlimb. After collection, the blood samples were transferred to blood collection tubes containing EDTA-K2 anticoagulant, manually inverted at least 5 times, and temporarily stored in an ice box. The blood samples were then centrifuged at 1500g for 10min at 2–8℃, and the plasma was collected and stored at -60℃ for later use. PD sampling points for the subcutaneous drug administration group: Before administration, and at 2h, 8h, 24h, 72h, 120h, 168h, and 336h after administration, approximately 3ml of whole blood was collected from the subcutaneous veins of each animal's forelimb and / or hindlimb. After collection, the blood samples were transferred to centrifuge tubes containing separating gel and coagulant, centrifuged at 1500g for 15min at room temperature, and the serum was collected and stored at -60℃ for later use.

[0220] The drug concentration in plasma was detected using a self-developed LC-MS / MS method, and the levels of CH50, C3, and C3a in serum were detected using commercial kits. Some PK / PD chromatograms are shown below. Figure 9a ~f.

[0221] CP01 and Aspaveli TM The cynomolgus monkeys were administered a single dose via subcutaneous injection. PK test results showed that the average T of CP01 was... 1 / 2 For 50.4h, Aspaveli TM Average T 1 / 2 The duration of action of CP01 subcutaneously injected was 50.8 h, and its long-term efficacy was comparable to that of Aspaveli. PD test results showed that the CH50 of the CP01 group at 14 days was 29.3% of the pre-dose level, while that of Aspaveli was... TM Five days after administration, CH50 in the group recovered to 74.5% of the pre-administration level, indicating that subcutaneous injection of CP01 significantly inhibited the activation of the classical complement pathway compared to Aspaveli. TM It demonstrates the advantages of lower dosage and longer-lasting efficacy.

[0222] CP01 and Aspaveli TM The cynomolgus monkeys were administered the drug via a single intravenous injection. PK testing results showed that CP01 and Aspaveli... TM Average T 1 / 2 Both had significantly shorter half-lives than those for subcutaneous injection, indicating that both drugs are more suitable for subcutaneous administration. PD test results showed that the CH50 of the CP01 group at 7 days was equivalent to 17.1% of the pre-administration level, while Aspaveli... TMAfter 3 and 5 days of administration, CH50 levels in the CP01 group recovered to 21.8% and 68.9% of pre-administration levels, respectively, indicating that the inhibitory effect of intravenous CP01 on the classical complement pathway activation was significantly stronger than that of Aspaveli. TM It demonstrates the advantages of lower dosage and longer-lasting efficacy.

[0223] Example 11: PK and PD study of CP01 and CP05 in cynomolgus monkeys

[0224] This embodiment studies the pharmacokinetics of peptide conjugates CP01 and CP05 in cynomolgus monkeys. The specific steps are as follows:

[0225] Four adult cynomolgus monkeys were divided into two groups and administered CP01 and CP05 via single subcutaneous injection, respectively, at a dose of 1.0 mg / kg.

[0226] The PK sampling points were as follows: before drug administration, and 2h, 8h, 24h, 48h, 72h, 96h, 120h, 168h, and 336h after drug administration. Approximately 1ml of whole blood was collected from the subcutaneous veins of the forelimbs and / or hindlimbs of each animal. After blood collection, the blood samples were transferred to blood collection tubes containing EDTA-K2 anticoagulant, manually inverted at least 5 times, and then temporarily stored in an ice box. The blood samples were centrifuged at 1500g for 10min at 2-8℃, and the plasma was collected and stored at -60℃ for later use.

[0227] PD sampling points were as follows: before drug administration, and at 2h, 8h, 24h, 72h, 120h, 168h, and 336h after drug administration. Approximately 3ml of whole blood was collected from the subcutaneous veins of the forelimbs and / or hindlimbs of each animal. After collection, the blood samples were transferred to centrifuge tubes containing separating gel and coagulant, and centrifuged at 1500g for 15min at room temperature. Serum was then stored at -60℃ for later use. The drug concentration in plasma was detected using a self-developed LC-MS / MS method, and the C3, C5, and CH50 levels in serum were detected using commercial kits. The PK / PD chromatogram is shown below. Figure 10a ~d.

[0228] CP01 and CP05 were administered subcutaneously to cynomolgus monkeys via a single dose. PK assays showed that CP01 and CP05 had almost identical plasma elimination half-lives, a result of using the same polyethylene glycol conjugation. PD assays showed that CP01 had a slightly better inhibitory effect on CH50 and C5 than CP05, but its inhibitory effect on C3 was significantly better than CP05, indicating that CP01 can exert both C3 and C5 complement inhibitory effects in vivo.

[0229] Example 12: Dose-response study of subcutaneous injection of CP01 in cynomolgus monkeys

[0230] This embodiment studies the pharmacokinetics of the polypeptide conjugate CP01 in cynomolgus monkeys. The specific steps are as follows:

[0231] Four adult cynomolgus monkeys were divided into two groups and administered CP01 via single subcutaneous injection at doses of 1.0 mg / kg and 2.0 mg / kg, respectively.

[0232] The PK sampling points were as follows: before drug administration, and 2h, 8h, 24h, 48h, 72h, 96h, 120h, 168h, and 336h after drug administration. Approximately 1ml of whole blood was collected from the subcutaneous veins of the forelimbs and / or hindlimbs of each animal. After blood collection, the blood samples were transferred to blood collection tubes containing EDTA-K2 anticoagulant, manually inverted at least 5 times, and then temporarily stored in an ice box. The blood samples were centrifuged at 1500g for 10min at 2-8℃, and the plasma was collected and stored at -60℃ for later use.

[0233] PD sampling points were as follows: before drug administration, and at 2h, 8h, 24h, 72h, 120h, 168h, and 336h after drug administration. Approximately 3ml of whole blood was collected from the subcutaneous veins of the forelimbs and / or hindlimbs of each animal. After collection, the blood samples were transferred to centrifuge tubes containing separating gel and coagulant, and centrifuged at 1500g for 15min at room temperature. Serum was collected and stored at -60℃ for later use. The drug concentration in plasma was detected using a self-developed LC-MS / MS method, and the C3, C3a, C5, C5a, and CH50 levels in serum were detected using commercial kits. Some PK / PD chromatograms are shown below. Figure 11a ~c.

[0234] When CP01 was administered subcutaneously to cynomolgus monkeys at different doses, the PK / PD test results all showed a significant dose-dependent effect.

[0235] Example 13: Pharmacodynamic study of intravitreal injection of CP01 in New Zealand rabbits

[0236] This embodiment studies the pharmacokinetics of the polypeptide conjugate CP01 in the vitreous body of New Zealand rabbits. The specific steps are as follows:

[0237] Four New Zealand rabbits were randomly divided into two groups. CP01 was administered via intravitreal injection at a volume of 0.1 ml / eye. The dosages for the two groups were 0.3 mg / eye and 0.6 mg / eye, respectively.

[0238] Vitreous fluid and aqueous humor were collected from both groups of animals before drug administration and at 24h, 72h, 120h, 168h, 336h, 504h, and 672h after drug administration, with a collection volume of approximately 50–100 μl per sample. Left and right eyes were distinguished. Collected samples were temporarily stored in crushed ice and then transferred to -60℃ or below for storage as soon as possible. Drug concentrations in the vitreous fluid and aqueous humor were determined using a self-developed LC-MS / MS method. PK results are shown in [link to pharmacokinetic data]. Figure 12a~b.

[0239] CP01 was injected intravitreally into New Zealand rabbits at different doses. The results showed that the half-life of CP01 in the vitreous body was 5-7 days. The content of CP01 in the aqueous humor was significantly lower than that in the vitreous body, but its half-life was also about 5-7 days.

[0240] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0241] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A bifunctional polypeptide that targets and inhibits complement C3 and C5, characterized in that, The polypeptide comprises the structure shown in formula (I): Z1-Z2-Z3(I), Z1 has the ability to inhibit the C5 complement activation pathway; Z2 is a linker peptide; Z3 has the ability to inhibit the C3 complement activation pathway.

2. The bifunctional polypeptide according to claim 1, characterized in that, Z1 has the structure shown in equation (II): Ac-X1VERFX2D Me VYWEY(II), Among them, X1 and X2 form a ring through a disulfide bond or an amide bond; Optionally, both X1 and X2 are selected from C; Optionally, X1 is selected from K, and X2 is selected from D; Optionally, X1 is selected from D, and X2 is selected from K.

3. The bifunctional polypeptide according to claim 1, characterized in that, Z3 has the structure shown in equation (III): IX3VW Me QDWGAHRX4T-NH2(III), Among them, X3 and X4 form a ring through disulfide bonds or amide bonds; Optionally, X3 and X4 are selected from C; Optionally, X3 is selected from K, and X4 is selected from D; Optionally, X3 is selected from D, and X4 is selected from K.

4. The bifunctional polypeptide according to claim 1, characterized in that, Z2 is a short peptide containing one lysine residue, wherein the number of amino acids in the short peptide is 2 to 9. Optionally, Z2 is selected from PK, PKP, PKPSGG, PKPSGGSGG, GGSPKPSGG.

5. The bifunctional polypeptide according to claim 1, characterized in that, The polypeptide has an amino acid sequence shown in any one of SEQ ID NO:1 to 9.

6. A bifunctional polypeptide conjugate, characterized in that, The bifunctional polypeptide according to any one of claims 1 to 5, and the coupling portion thereof, wherein the polypeptide is linked to the coupling portion.

7. The polypeptide conjugate according to claim 6, characterized in that, The polypeptide conjugate has the structure shown in formula (IV): Peptide-coupled moiety-peptide (IV).

8. The polypeptide conjugate according to any one of claims 6 to 7, characterized in that, The coupling portion is selected from polyethylene glycol; Optionally, the polyethylene glycol is selected from double-ended activated polyethylene glycol; Optionally, the molecular weight of the double-ended activated polyethylene glycol is 20,000 Da to 40,000 Da; Optionally, the dual-terminal activated groups of the dual-terminal activated polyethylene glycol are selected from succinimide propionate (SPA), N-hydroxysuccinimide (NHS), succinimide carbonate (SC), succinimide acetate (SCM), acyl chloride, and acid anhydride.

9. A pharmaceutical composition, characterized in that, include: The bifunctional polypeptide or a pharmaceutically acceptable salt thereof as described in any one of claims 1 to 5, or the polypeptide conjugate or a pharmaceutically acceptable salt thereof as described in any one of claims 6 to 8; Optional, further including pharmaceutically acceptable carriers, excipients, and mediators; Optionally, the pharmaceutical composition may be administered subcutaneously, intravenously, or via the vitreous humor.

10. Use of the polypeptide of any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof, the polypeptide conjugate of any one of claims 6 to 8 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention and / or treatment of diseases related to complement hyperactivation and autoimmune diseases; Optionally, the related diseases and autoimmune diseases caused by complement overactivation include paroxysmal nocturnal hemoglobinuria, cold agglutinin disease, microangiopathy, vasculitis, autohemolytic anemia, aplastic anemia, atypical hemolytic uremic syndrome, immunoglobulin A nephropathy, C3 glomerulonephritis, glomerulonephritis, kidney injury, membranoproliferative glomerulonephritis, membranous glomerulonephritis, lupus nephritis, systemic lupus erythematosus, organ transplant rejection, autoimmune thrombocytopenic purpura, rheumatoid arthritis. Wet arthritis, geographic atrophy, age-related macular degeneration, Stargardt's disease, keratoconjunctivitis, hereditary angioedema, neuromyelitis optica spectrum disorders, motor neuron disease, myasthenia gravis, amyotrophic lateral sclerosis, acute respiratory distress syndrome, acute respiratory diseases caused by viral infections, protein-losing enteropathy, gingivitis, periodontitis, hidradenitis suppurativa, pemphigoid, pyoderma gangrenosa, systemic scleroderma, squamous cell carcinoma of the skin, lung cancer, multiple myeloma, etc.

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