Biomacromolecule-targeted specific complement inhibitor, its preparation method and application

Abstract

The present application relates to a fusion protein, which comprises: (i) the extracellular domain of CRIg, (ii) a complement inhibitory domain and (iii) a potentiating domain. The present application also relates to a method for preparing and using the fusion protein, and a pharmaceutical composition comprising the fusion protein. The fusion protein of the present application not only has an obvious targeted complement inhibitory effect, but also is beneficial to improving drugability and / or production, and can be used for the treatment and prevention of various human diseases related to abnormal complement activation.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and specifically relates to the design, preparation and clinical application of a biological macromolecule-targeted specific complement inhibitor. Background Art

[0002] The complement system is an important part of innate immunity, an important regulator of acquired immunity, and an important bridge connecting innate immunity and adaptive immunity. The complement system mainly exists in the blood circulation and is a highly complex cascade reaction system composed of more than 30 activation factors, inhibitory factors and complement receptors, which can self-regulate. Its main physiological function is to clear invading pathogenic microorganisms and host cell debris, and coordinate the entire immune and inflammatory processes, and it is a key system for immune surveillance and homeostasis (Ricklin et al., 2010).

[0003] The complement system can generally be activated through three pathways: the classical pathway can be activated by IgG and IgM antibodies; the lectin pathway can be activated by mannose on the surface of bacteria; and the alternative pathway can be activated by various components such as cell wall / cell membrane components of pathogens or complement C3b analogs such as cobra venom, and can also be automatically activated by the hydration of C3.

[0004] After complement activation, it mainly exerts physiological effects through three ways (Dunkelberger and Song, 2010). First, C3a, especially C5a, produced after the activation of complement components C3 and C5, can bind to their receptors C3aR or C5aR1 and C5L2 expressed on immune cells respectively, recruit various immune cells, and secrete pro-inflammatory cytokines (TNF-α, IL-1, IL-6, etc.) and chemokines (MCP-1, MIP-2, KC, CINC, etc.) (Riedemann et al., 2003), thus producing a strong pro-inflammatory effect locally at the site of complement activation, that is, the pro-inflammatory effect. Second, another product C3b of C3 activation and its further degradation product iC3b can be inserted into the surface of target cells attacked by complement, "mark" these foreign components, and bind to various receptors expressed on immune cells such as CR1 / CR2 / CR3 / CR4 / CRIg, and finally immune cells phagocytose or lyse these foreign components, that is, the opsonophagocytic effect. Third, another product C5b of C5 activation can also be inserted into the surface of target cells attacked by complement, bind to complement C6 and C7 to form a stable complex C5b-7, and this complex further binds to C8 and C9. Finally, C9 polymerizes to form a C5b-9n complex, also known as the Membrane Attack Complex (MAC), and finally forms a pore with an inner diameter of 5 nm, an outer diameter of 20 nm, and a height of 15 nm on the target cell membrane, causing the change of osmotic pressure inside and outside the cell and directly lysing the cell (Tegla et al., 2011), that is, the cell lysis effect.

[0005] To avoid the "collateral damage" effect of the above physiological effects of complement activation on normal body cells, the body has evolved more than 10 kinds of complement regulatory proteins, which are expressed on cell membranes or circulate in the blood system and inhibit complement activation at different stages of complement activation. For example, complement inhibitory proteins CR1, CD46, CD55, and CD59 expressed on cell membranes, and C1-INH, C4BP, FH, Vitronectin, and S protein free in the blood circulation, etc. It is precisely because of these complement regulatory proteins that the body cells can avoid the killing of complement when complement is activated.

[0006] The complement system is a self - protective immune mechanism under normal conditions in the human body. However, under certain abnormal conditions of the body, an increase in the level of complement activation products or a decrease or even absence of complement regulatory protein levels can lead to over - activation of the complement, resulting in the killing of autologous tissue cells and ultimately leading to the occurrence and development of diseases, including Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical Haemolytic Uraemic Syndrome (aHUS), generalized Myasthenia Gravis (gMG), Neuromyelitis Optica Spectrum Disorders (NMOSD), Age - related Macular Degeneration (AMD), autoimmune hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, atherosclerosis, Parkinson's disease, Alzheimer's disease (senile dementia), asthma, allergy, psoriasis, multiple sclerosis, Crohn's disease, etc. (Ricklin and Lambris, 2007). The over - activation of complement is closely related to the occurrence and development of autoimmune diseases, especially in the early stage of pathogenesis. Therefore, complement system inhibitors can intervene in the early stage of the progression of the above - mentioned diseases and have great advantages and demands in clinical practice.

[0007] Based on the importance of the complement system in the pathogenesis of many autoimmune diseases and acute and chronic infections and other diseases, nearly 30 pharmaceutical companies have nearly 50 complement inhibitors in different stages of research and development, which also indirectly indicates that there is still a need to develop more ideal complement inhibitors. Summary of the Invention

[0008] The present application provides a fusion protein, which comprises: (i) the extracellular domain of CRIg; (ii) a complement inhibitory domain, which comprises a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55 and CD59; and (iii) a potentiating domain, which comprises a protein or a functional fragment thereof selected from the group consisting of the IgG Fc domain and human serum albumin. The inventors of the present application unexpectedly found that CRIg is linked to other complement regulatory proteins FH, CD55 or CD59, and further linked to the IgG Fc segment, which not only has an obvious complement inhibitory effect, but also is beneficial to production purification and improve drugability, such as improving pharmacokinetics (PK) / pharmacodynamics (PD) effects. In some cases, the present application uses CRIg, which can bind to the fragments C3b and / or iC3b formed after the activation of complement component C3 to produce a targeting effect, and is further linked to another component with a complement inhibitory effect, such as FH, CD55, or CD59, and further linked to the IgG Fc segment or HSA, to prepare a fusion protein (for example, by genetic engineering methods), and transports the recombinantly linked complement regulatory protein to the local complement activation site, ultimately achieving the effect of targeted inhibition of complement activation. This type of drug can be applied to the treatment and prevention of a variety of human diseases related to abnormal complement activation.

[0009] On the one hand, the present application provides a fusion protein, which comprises: (i) the extracellular domain of CRIg; (ii) a complement inhibitory domain, which comprises a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46 and CD59; and (iii) a potentiating domain, which comprises a protein or a functional fragment thereof selected from the group consisting of the IgG Fc domain and human serum albumin.

[0010] In some embodiments, the extracellular domain of CRIg comprises the amino acid sequence shown in SEQ ID NO.6.

[0011] In some embodiments, the C-terminus of the extracellular domain of CRIg is directly or indirectly connected to the N-terminus of the complement inhibitory domain.

[0012] In some embodiments, the C-terminus of the complement inhibitory domain is directly or indirectly connected to the N-terminus of the potentiating domain.

[0013] In some embodiments, the indirect connection includes connection through a linker.

[0014] In some embodiments, the linker comprises the amino acid sequence shown in any one of SEQ ID NO.44, 46, 48 and 50.

[0015] In certain embodiments, the complement inhibitory domain comprises the amino acid sequence shown in any one of SEQ ID NO:8, 18, 20, and 22.

[0016] In certain embodiments, the potentiating domain is human serum albumin.

[0017] In certain embodiments, the human serum albumin comprises the amino acid sequence shown in SEQ ID NO.12.

[0018] In certain embodiments, the fusion protein is in a single-chain structure.

[0019] In certain embodiments, the fusion protein sequentially comprises the extracellular domain of CRIg, the complement inhibitory domain, and the potentiating domain in the order from the N-terminus to the C-terminus.

[0020] In certain embodiments, the fusion protein comprises the amino acid sequence shown in SEQ ID NO.14.

[0021] In certain embodiments, the potentiating domain comprises an IgG Fc domain.

[0022] In certain embodiments, the IgG comprises a protein selected from the group consisting of human IgG1 and human IgG4.

[0023] In certain embodiments, the IgG Fc domain comprises the amino acid sequence shown in any one of SEQ ID NO:10, 30, 32, and 34.

[0024] In certain embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the extracellular domain of CRIg, the first complement inhibitory domain, and the first IgG Fc domain, and the second polypeptide chain comprises the extracellular domain of CRIg, the second complement inhibitory domain, and the second IgG Fc domain.

[0025] Wherein the first IgG Fc domain and the second IgG Fc domain can interact to form a dimer.

[0026] In certain embodiments, the first complement inhibitory domain and the second complement inhibitory domain each independently comprise a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46, and CD59.

[0027] In certain embodiments, the first complement inhibitory domain is the same as the second complement inhibitory domain.

[0028] In certain embodiments, the first IgG Fc domain and the second IgG Fc domain are the same.

[0029] In certain embodiments, the first IgG Fc domain and the second IgG Fc domain comprise the amino acid sequence shown in any one of SEQ ID NO: 10 and 30.

[0030] In certain embodiments, the first polypeptide chain and the second polypeptide chain are the same.

[0031] In certain embodiments, the first polypeptide chain and / or the second polypeptide chain comprise the amino acid sequence shown in any one of SEQ ID NO.14, 24, 26, and 28.

[0032] In certain embodiments, the first complement inhibitory domain and the second complement inhibitory domain are different.

[0033] In certain embodiments, the first complement inhibitory domain and the second complement inhibitory domain each independently comprise a protein selected from the group consisting of CD59 and CD55 or a functional fragment thereof.

[0034] In certain embodiments, the first complement inhibitory domain and the second complement inhibitory domain each independently comprise a protein selected from the group consisting of FH and CD55 or a functional fragment thereof.

[0035] In certain embodiments, the first complement inhibitory domain and the second complement inhibitory domain each independently comprise a protein selected from the group consisting of CD46 and CD59 or a functional fragment thereof.

[0036] In certain embodiments, the first IgG Fc domain and the second IgG Fc domain are the same.

[0037] In certain embodiments, the first IgG Fc domain and the second IgG Fc domain are different.

[0038] In certain embodiments, the first IgG Fc domain comprises the amino acid sequence shown in any one of SEQ ID NO.32 and 34.

[0039] In certain embodiments, the second IgG Fc domain comprises the amino acid sequence shown in any one of SEQ ID NO.32 and 34.

[0040] In certain embodiments, the first polypeptide chain comprises the amino acid sequences shown in SEQ ID NO.36, 38, 40, and 42.

[0041] In certain embodiments, the second polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO.36, 38, 40, and 42.

[0042] In certain embodiments, in the fusion protein:

[0043] the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.38; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.40; or,

[0044] the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.36; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.42;

[0045] the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.38; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.42.

[0046] On the other hand, the present application provides one or more isolated nucleic acid molecules encoding the fusion protein or a fragment thereof.

[0047] On the other hand, the present application provides a vector comprising the nucleic acid molecule.

[0048] On the other hand, the present application provides a cell comprising the vector or expressing the fusion protein.

[0049] On the other hand, the present application provides a method for preparing the fusion protein, which comprises the following steps: synthesizing the fusion protein, and / or culturing the cell under conditions for expressing the fusion protein.

[0050] On the other hand, the present application provides a pharmaceutical composition comprising the fusion protein and optionally a pharmaceutically acceptable carrier.

[0051] On the other hand, the present application provides the use of the fusion protein or the pharmaceutical composition in the preparation of a drug for treating diseases related to targeted inhibition of complement activation.

[0052] In certain embodiments, the disease includes an autoimmune disease.

[0053] In certain embodiments, the disease includes autoimmune myasthenia gravis.

[0054] Those skilled in the art can easily gain insights into other aspects and advantages of this application from the following detailed description. Only exemplary embodiments of this application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Accordingly, the descriptions in the drawings and the specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and the drawings described in detail below. A brief description of the drawings is as follows:

[0056] Figure 1 Shows the design diagrams of (CRIg-FH-IgG4Fc)×2 and CRIg-FH-HSA.

[0057] Figure 2 Shows the identification of the recombinant proteins of (CRIg-FH-IgG4Fc)×2 and CRIg-FH-HSA.

[0058] Figure 3 Shows the inhibitory effect of (CRIg-FH-IgG4Fc)×2 on the classical pathway of human serum complement.

[0059] Figure 4 Shows the inhibitory effect of (CRIg-FH-IgG4Fc)×2 on the alternative pathway of human serum complement.

[0060] Figure 5 Shows the inhibitory effect of CRIg-FH-HSA on the classical pathway of human serum complement.

[0061] Figure 6 Shows the inhibitory effect of CRIg-FH-HSA on the alternative pathway of human serum complement.

[0062] Figure 7 Shows the pharmacokinetic detection of (CRIg-FH-IgG4Fc)×2 in rats.

[0063] Figure 8 Shows the pharmacokinetic detection of CRIg-FH-HSA in rats.

[0064] Figure 9Shown are the design schematics of (CRIg-FH-IgG4Fc)×2, (CRIg-CD55-IgG4Fc)×2, (CRIg-CD46-IgG4Fc)×2, and (CRIg-CD59-IgG4Fc)×2.

[0065] Figure 10 Shown are the identifications of four recombinant proteins, namely (CRIg-FH-IgG4Fc)×2, (CRIg-CD55-IgG4Fc)×2, (CRIg-CD46-IgG4Fc)×2, and (CRIg-CD59-IgG4Fc)×2.

[0066] Figure 11 Shown is the inhibitory effect of (CRIg-CD55-IgG4Fc)×2 on the classical pathway of human serum complement.

[0067] Figure 12 Shown is the inhibitory effect of (CRIg-CD55-IgG4Fc)×2 on the alternative pathway of human serum complement.

[0068] Figure 13 Shown is the inhibitory effect of (CRIg-CD46-IgG4Fc)×2 on the classical pathway of human serum complement.

[0069] Figure 14 Shown is the inhibitory effect of (CRIg-CD46-IgG4Fc)×2 on the alternative pathway of human serum complement.

[0070] Figure 15 Shown is the inhibitory effect of (CRIg-CD59-IgG4Fc)×2 on the classical pathway of human serum complement.

[0071] Figure 16 Shown is the inhibitory effect of (CRIg-CD59-IgG4Fc)×2 on the alternative pathway of human serum complement.

[0072] Figure 17 Shown are the design schematics of three recombinant proteins, namely (CRIg-CD55-IgG1 Fc)(CRIg-FH-IgG1 Fc), (CRIg-CD46-IgG1 Fc)(CRIg-CD59-IgG1 Fc), and (CRIg-CD55-IgG1 Fc)(CRIg-CD59-IgG1 Fc).

[0073] Figure 18Shown are the identifications of three recombinant proteins: (CRIg-CD55-IgG1 Fc)(CRIg-FH-IgG1 Fc), (CRIg-CD46-IgG1 Fc)(CRIg-CD59-IgG1 Fc), and (CRIg-CD55-IgG1 Fc)(CRIg-CD59-IgG1 Fc).

[0074] Figure 19 Shown is the inhibitory effect of (CRIg-CD55-IgG1 Fc)(CRIg-FH-IgG1 Fc) on the classical pathway of human serum complement.

[0075] Figure 20 Shown is the inhibitory effect of (CRIg-CD55-IgG1 Fc)(CRIg-FH-IgG1 Fc) on the alternative pathway of human serum complement.

[0076] Figure 21 Shown is the inhibitory effect of (CRIg-CD46-IgG1 Fc)(CRIg-CD59-IgG1 Fc) on the classical pathway of human serum complement.

[0077] Figure 22 Shown is the inhibitory effect of (CRIg-CD46-IgG1 Fc)(CRIg-CD59-IgG1 Fc) on the alternative pathway of human serum complement.

[0078] Figure 23 Shown is the inhibitory effect of (CRIg-CD55-IgG1 Fc)(CRIg-CD59-IgG1 Fc) on the classical pathway of human serum complement.

[0079] Figure 24 Shown is the inhibitory effect of (CRIg-CD55-IgG1 Fc)(CRIg-CD59-IgG1 Fc) on the alternative pathway of human serum complement.

[0080] Figure 25 Shown is the change in body weight of EAMG rats treated with the drug (CRIg-CD59-IgG4Fc)×2.

[0081] Figure 26 Shown is the change in clinical score of EAMG rats treated with the drug (CRIg-CD59-IgG4Fc)×2.

[0082] Figure 27 Shown is the change in mortality of EAMG rats treated with the drug (CRIg-CD59-IgG4Fc)×2. Detailed implementation methods

[0083] The following specific embodiments illustrate the implementation manners of the invention of the present application. Those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

[0084] Term Definition

[0085] In the present application, the term "complement inhibitory domain" generally refers to a substance that can inhibit the activation of the complement system. The complement system can generally be activated through three pathways: the classical pathway can be activated by IgG and IgM antibodies; the lectin pathway can be activated by mannose on the surface of bacteria; and the alternative pathway can be activated by various components such as pathogen cell wall / membrane components or complement C3b analogs such as cobra venom, and can also be automatically activated by the hydration of C3. The complement inhibitory domain can inhibit at different stages of activation. The complement inhibitory domain described in the present application can be derived from complement regulatory proteins or their functional fragments, covering complement inhibitors present in the blood circulation and complement membrane regulatory proteins on the cell membrane surface, including but not limited to, C1-INH (C1 inhibitor), C4BP (C4 binding protein), factor I (FI), factor H (FH), S-protein, Clusterin, CD35 (also known as CR1), CD46 (also known as MCP), CD55 (also known as DAF), and / or CD59, and even the full-length or partial sequences of CRIg itself, etc.

[0086] In the present application, the term "CD55" generally refers to a complement regulatory protein, also known as complement decay-accelerating factor or DAF. CD55 can regulate the complement system. It recognizes the C4b and C3b fragments generated during the activation of complement component C4 (classical or lectin pathway) or C3 (alternative pathway). CD55 can interact with cell-associated C4b in the classical and lectin pathways, interfering with the conversion of C2 to C2b, thereby preventing the formation of the C4b2b C3 convertase; CD55 can also interact with C3b in the alternative pathway, interfering with the conversion of factor B to Bb, thereby preventing the formation of the alternative pathway C3bBb C3 convertase. The CD55 described in the present application may include the full-length CD55 protein or its fragments, as well as various variants thereof (for example, mutants, isomers). For example, an exemplary nucleic acid molecule encoding the CD55 may include the nucleotide sequence shown in SEQ ID NO.17, and an exemplary CD55 protein may include the amino acid sequence shown in SEQ ID NO.18.

[0087] In the present application, the term "CD59" generally refers to a complement regulatory protein, which may also be referred to as "membrane attack complex (MAC) inhibitory protein" (MAC-inhibitory protein, MAC-IP), "membrane inhibitor of reactive lysis" (MIRL), "membrane attack complex inhibitory factor" (MACIF), or protectin, and belongs to the LY6 / uPAR / α-neurotoxin protein family. CD59 can be linked to host cells via a glycosylphosphatidylinositol (GPI) anchor. When complement activation leads to the deposition of the C5b678 complex on host cells, CD59 can prevent the polymerization of C9 and the formation of the complement membrane attack complex. The CD59 described in the present application may include the full-length CD59 protein or its fragments, as well as its various variants (e.g., mutants, isomers). For example, an exemplary nucleic acid molecule encoding the CD59 may include the nucleotide sequence shown in SEQ ID NO.21, and an exemplary CD59 protein may include the amino acid sequence shown in SEQ ID NO.22.

[0088] In the present application, the term "CD46" generally refers to a complement regulatory protein, which may also be referred to as membrane cofactor protein (MCP). Generally speaking, CD46 has cofactor activity and can inactivate (by cleavage) complement components C3b and C4b through serum factor I, thereby protecting host cells from complement damage. The CD46 described in the present application may include the full-length CD46 protein or its fragments, as well as its various variants (e.g., mutants, isomers). For example, an exemplary nucleic acid molecule encoding the CD46 may include the nucleotide sequence shown in SEQ ID NO.19, and an exemplary CD46 protein may include the amino acid sequence shown in SEQ ID NO.20.

[0089] In the present application, the term "factor H (FH)" generally refers to a complement regulatory protein and is a member of the regulatory factors of the complement activation family. The main function of FH is to regulate the alternative pathway of the complement system, ensuring that the complement system functions against pathogens or other dangerous substances without damaging host tissues. The sequence of FH usually has some highly conserved motifs called short consensus repeats (SCRs). Each SCR consists of approximately 60 amino acids and has two disulfide bonds, a highly substituted hypervariable loop, and a short SCR linker of 3-8 residues. FH usually consists of 20 SCRs, and the functional characteristics of FH are usually localized on the SCRs. The FH described in the present application may include the full-length FH protein or its fragments (e.g., one or more SCRs), as well as various variants thereof (e.g., mutants, isomers). For example, an exemplary nucleic acid molecule encoding the FH may include the nucleotide sequence shown in SEQ ID NO.3, and an exemplary FH protein may include the amino acid sequence shown in SEQ ID NO.4. For example, the FH described in the present application may contain the SCR1-5 domain, and an exemplary nucleic acid molecule encoding the FH SCR1-5 domain may include the nucleotide sequence shown in SEQ ID NO.7, and an exemplary FH SCR1-5 domain may include the amino acid sequence shown in SEQ ID NO.8. In some cases, the FH may contain other SCR fragments.

[0090] In the present application, the term "single-chain structure" generally refers to an amino acid chain connected by covalent bonds (e.g., peptide bonds). The single-chain structure can be produced by connecting polypeptide fragments, or by first connecting the nucleic acids encoding the polypeptide fragments and then expressing them. For example, multiple polypeptide chains from the same or different sources can form a fusion protein with a single-chain structure.

[0091] In the present application, the term "human serum albumin (HSA)" generally refers to a globular protein encoded by the human gene ALB and is usually present in plasma. Native HSA consists of a single polypeptide chain and contains three domains: Domain I, Domain II, and Domain III. Each domain can include two subdomains, A and B, which form a cylindrical structure with the notches facing each other, making the structure of HSA relatively flexible and hydrophilic. In this article, the term may include naturally sourced (e.g., plasma-sourced) HSA, synthetically produced (e.g., produced by recombinant DNA technology) HSA. The term encompasses full-length HSA or its functional fragments. An exemplary nucleic acid molecule encoding the HSA protein may contain the nucleotide sequence shown in SEQ ID NO.11, and an exemplary HSA protein may contain the amino acid sequence shown in SEQ ID NO.12.

[0092] In the present application, the term "potentiating domain" generally refers to a polypeptide domain capable of enhancing the complement inhibitory effect. The complement inhibitory effect may include enhancing the intensity of the complement inhibitory activity of a substance having complement inhibitory activity (e.g., a complement inhibitory domain), increasing the time of the complement inhibitory effect of a substance having complement inhibitory activity (e.g., a complement inhibitory domain), reducing the time of degradation of a substance having complement inhibitory activity (e.g., a complement inhibitory domain), and / or prolonging the drug half-life of a substance having complement inhibitory activity (e.g., a complement inhibitory domain). The potentiating domain may include domains from HAS, IgG1, IgG2, IgG3, and / or IgG4 Fc. In the present application, the potentiating domain and the complement inhibitory domain may be on the same polypeptide chain or on different polypeptide chains.

[0093] In the present application, the term "IgG Fc domain" generally refers to the Fc region of an immunoglobulin or its domain, which may include the Fc domains from IgG1, IgG2, IgG3, and / or IgG4.

[0094] In the present application, the term "CRIg" generally refers to a complement membrane regulatory protein belonging to the immunoglobulin superfamily. CRIg can specifically recognize iC3b (non-activated C3b) and inhibit the activation of C3 convertase, thereby producing an inhibitory effect at an early stage of the complement cascade reaction. The CRIg may include CRIg from different species sources, e.g., human or mouse. In certain cases, the CRIg may be from human CRIg. Human CRIg may include the long form of CRIg, which includes the V-type and C2-type terminal Ig domains. Human CRIg also includes the short form of CRIg, which includes the V-type terminal Ig domain. An exemplary nucleic acid sequence encoding CRIg may be as shown in SEQ ID NO:1, and an exemplary CRIg protein may contain the amino acid sequence as shown in SEQ ID NO:2. Generally speaking, the functional region of CRIg is located in its extracellular domain. The term "CRIg extracellular domain" generally includes the extracellular functional region of CRIg. An exemplary nucleic acid sequence encoding the CRIg extracellular domain may be as shown in SEQ ID NO:5, and an exemplary CRIg extracellular domain may contain the amino acid sequence as shown in SEQ ID NO:6.

[0095] In the present application, the term "vector" generally refers to a nucleic acid molecule capable of self-replicating in a suitable host, which transfers the inserted nucleic acid molecule into host cells and / or between host cells. The vector may include vectors mainly for inserting DNA or RNA into cells, vectors mainly for replicating DNA or RNA, and expression vectors mainly for transcription and / or translation of DNA or RNA. The vector also includes vectors having multiple of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, by culturing a suitable host cell containing the vector, the vector can produce a desired expression product.

[0096] In the present application, the term "cell" generally refers to an individual cell, cell line or cell culture that can or already contains a plasmid or vector including the nucleic acid molecule described in the present application, or is capable of expressing the antibody or its antigen-binding fragment described in the present application. The cell may include the progeny of a single host cell. Due to natural, accidental or deliberate mutations, the progeny cells may not necessarily be identical morphologically or genomically to the original parental cell, but are capable of expressing the antibody or its antigen-binding fragment described in the present application. The cell can be obtained by in vitro transfection of cells using the vector described in the present application. The cell can be a prokaryotic cell (such as Escherichia coli), or a eukaryotic cell (such as a yeast cell, such as a COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell or myeloma cell). In certain cases, the cell can be a mammalian cell. For example, the mammalian cell can be a CHO-K1 cell. In the present application, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only a certain specific cell, but also the progeny of these cells.

[0097] In the present application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable adjuvants, excipients or stabilizers, which are non-toxic to the cells or mammals exposed thereto at the doses and concentrations employed. Generally, a physiologically acceptable carrier is an aqueous PH buffer solution. Examples of physiologically acceptable carriers may include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions for salt formation such as sodium; and / or nonionic surfactants such as TWEEN TM, polyethylene glycol (PEG) and PLURONICS TM .

[0098] In the present application, the term "targeted inhibition of complement activation-related diseases" generally refers to inhibiting diseases caused by abnormal complement activation or overactivation of complement, which may be caused by an increase in the level and / or activity of complement activation products and / or a decrease in the level and / or activity of complement regulatory proteins (e.g., CD55, CD59, and / or FH). For example, compared with the normal state, a decrease in the expression of complement regulatory proteins, a decrease in the gene replication and / or transcription level of complement regulatory proteins, an abnormal translation process of complement regulatory proteins, etc., lead to the inability to regulate complement activation. After overactivation of complement, it may cause abnormal (e.g., excessive) pro-inflammatory responses, opsonophagocytic effects, and / or cytolytic effects. In some cases, the body produces autoantibodies against normal self-antigens. After these autoantibodies bind to the antigens, they activate the complement pathway. Due to a decrease in the level and / or activity of substances related to complement inhibitory activation (e.g., complement regulatory proteins), immune damage is caused to the body's own tissues, organs, and cells, ultimately leading to autoimmune diseases. In some cases, targeted inhibition of complement activation-related diseases includes autoimmune diseases. In some cases, the targeted inhibition of complement activation-related diseases may be caused by acute or chronic infections. In some cases, the targeted inhibition of complement activation-related diseases may be caused by gene mutations. Targeted inhibition of complement activation-related diseases may be selected from: Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical Haemolytic Uraemic Syndrome (aHUS), generalized Myasthenia Gravis (gMG), Neuromyelitis Optica Spectrum Disorders (NMOSD), Age-related Macular Degeneration (AMD), autoimmune hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, atherosclerosis, Parkinson's disease, Alzheimer's disease (senile dementia), asthma, allergy, psoriasis, multiple sclerosis, and Crohn's disease. For example, the disease is autoimmune myasthenia gravis.

[0099] In the present application, the term "autoimmune myasthenia gravis" generally refers to a chronic autoimmune disease in which the transmission of nerve and muscle signals is blocked, thereby affecting the strength of skeletal muscles. This disease can be caused by overactivation of complement, and the immune response attacks the proteins on the postsynaptic membrane of the neuromuscular junction, namely, acetylcholine receptors or receptor-associated proteins. Over time, the symptoms of autoimmune myasthenia gravis will gradually spread from the eye muscles to the facial and neck muscles, causing weakness, slurred speech, difficulty chewing and swallowing, and / or breathing difficulties, and gradually spread from the head and neck to other parts of the body, eventually leading to generalized myasthenia gravis. Detailed Description of the Invention

[0101] Fusion protein

[0102] On the one hand, the present application provides a fusion protein, which may comprise (i) the extracellular domain of CRIg. The extracellular domain of CRIg in the present application may comprise the amino acid sequence shown in SEQ ID NO.6. In certain cases, the extracellular domain of CRIg may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.6, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0103] In the present application, the fusion protein may comprise (ii) a complement inhibitory domain.

[0104] In certain cases, the complement inhibitory domain may comprise a protein derived from FH or a functional fragment thereof, and the FH may comprise the amino acid sequence shown in SEQ ID NO.4. In certain cases, the FH may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.4, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous. In certain cases, the complement inhibitory domain may comprise one or more SCR domains of FH. For example, the complement inhibitory domain may comprise the SCR1-5 domains of FH, and the SCR1-5 domains of FH may comprise the amino acid sequence shown in SEQ ID NO.8. In certain cases, the FH may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.8, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0105] In certain cases, the complement inhibitory domain may comprise a protein derived from CD55 or a functional fragment thereof, and the CD55 may comprise the amino acid sequence shown in SEQ ID NO.18. In certain cases, the CD55 may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.18, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0106] In certain cases, the complement inhibitory domain may comprise a protein derived from CD59 or a functional fragment thereof, and the CD59 may comprise the amino acid sequence shown in SEQ ID NO.22. In certain cases, the CD59 may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.22, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0107] In certain cases, the complement inhibitory domain may comprise a protein derived from CD46 or a functional fragment thereof, and the CD46 may comprise the amino acid sequence shown in SEQ ID NO.20. In certain cases, the CD46 may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.20, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0108] The complement inhibitory domain of the present application may comprise a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46, and CD59. In certain cases, the complement inhibitory domain may comprise the amino acid sequence shown in any one of SEQ ID NO:8, 18, 20, and 22. In certain cases, the complement inhibitory domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO.8, 18, 20, and 22, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0109] The complement inhibitory domain of the present application may comprise a protein selected from the group consisting of or a functional fragment thereof: factor H (FH), CD55, and CD59. In certain cases, the complement inhibitory domain may comprise the amino acid sequence shown in any one of SEQ ID NO:8, 18, and 22. In certain cases, the complement inhibitory domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO.8, 18, and 22, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0110] In the present application, the fusion protein may comprise (iii) a potentiating domain.

[0111] In certain cases, the potentiating domain may comprise an IgG Fc domain or a functional fragment thereof. In certain cases, the IgG Fc domain may be an IgG Fc4 domain, and the IgG Fc4 domain may comprise the amino acid sequence shown in SEQ ID NO.10. In certain cases, the potentiating domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.10, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0112] In certain cases, the IgG Fc domain may be an IgG Fc1 domain, and the IgG Fc1 domain may include mutants thereof. For example, the IgG Fc domain may be mutated to obtain a desired spatial structure, such as forming a knob-hole structure to better form a dimer. For example, the IgG Fc1 may comprise the amino acid sequence shown in any one of SEQ ID NO.30, 32, and 34. In certain cases, the potentiating domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO.30, 32, and 34, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0113] In the present application, the potentiating domain may comprise human serum albumin or a functional fragment thereof, and the human serum albumin may comprise the amino acid sequence shown in SEQ ID NO.12. In certain cases, the potentiating domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO.12, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0114] In the present application, the fusion protein may comprise (i) the extracellular domain of CRIg, (ii) a complement inhibitory domain, and (iii) a potentiating domain. In the fusion protein, the C-terminus of the extracellular domain of CRIg may be directly or indirectly linked to the N-terminus of the complement inhibitory domain, and the C-terminus of the complement inhibitory domain may be directly or indirectly linked to the N-terminus of the potentiating domain. For example, the fusion protein may sequentially comprise the extracellular domain of CRIg, the complement inhibitory domain, and the potentiating domain from the N-terminus to the C-terminus.

[0115] In the present application, the indirect linkage may comprise linkage via a linker. For example, the linker may include a linking peptide.

[0116] In certain cases, the C-terminus of the extracellular domain of CRIg may be linked to the N-terminus of the complement inhibitory domain via a linker, and the linker may comprise the amino acid sequence shown in any one of SEQ ID NOs. 44, 46, 48, and 50. In certain cases, the nucleotide sequence encoding the linker may be as shown in any one of SEQ ID NOs. 43, 45, 47, and 49.

[0117] In certain cases, the C-terminus of the complement inhibitory domain may be linked to the N-terminus of the potentiating domain via a linker, and the linker may comprise the amino acid sequence shown in any one of SEQ ID NOs. 44, 46, 48, and 50. In certain cases, the nucleotide sequence encoding the linker may be as shown in any one of SEQ ID NOs. 43, 45, 47, and 49.

[0118] In the present application, the fusion protein may be in a single-chain structure.

[0119] For example, the fusion protein may sequentially comprise the extracellular domain of CRIg, FH SCR1-5, and human serum albumin from the N-terminus to the C-terminus. For example, the fusion protein may comprise the amino acid sequence shown in SEQ ID NO. 16. For example, the fusion protein may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in SEQ ID NO. 16, such as any one of at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% homologous.

[0120] In the present application, the fusion protein may include a first polypeptide chain and a second polypeptide chain.

[0121] In certain cases, the first polypeptide chain may comprise the extracellular domain of CRIg, the first complement inhibitory domain, and the first IgG Fc domain. In certain cases, the first complement inhibitory domain may comprise a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46, and CD59. In certain cases, the first complement inhibitory domain may comprise a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, and CD59. In certain cases, the first IgG Fc domain may comprise a protein or a functional fragment thereof selected from the group consisting of IgG1 Fc and IgG4 Fc.

[0122] In certain cases, the second polypeptide chain may comprise the extracellular domain of CRIg, the second complement inhibitory domain, and the second IgG Fc domain. In certain cases, the second complement inhibitory domain may comprise a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46, and CD59. In certain cases, the second complement inhibitory domain may comprise a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, and CD59. In certain cases, the second IgG Fc domain may comprise a protein or a functional fragment thereof selected from the group consisting of IgG1 Fc and IgG4 Fc.

[0123] In certain cases, the first polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NOs. 14, 24, 26, 28, 38, 40, and 42. For example, the first polypeptide chain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NOs. 14, 24, 26, 28, 38, 40, and 42, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0124] In certain cases, the first polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NOs. 14, 24, 28, 38, 40, and 42. For example, the first polypeptide chain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NOs. 14, 24, 28, 38, 40, and 42, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% homologous.

[0125] In certain cases, the second polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NOs. 14, 24, 26, 28, 38, 40, and 42. For example, the second polypeptide chain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NOs. 14, 24, 26, 28, 38, 40, and 42, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0126] In certain cases, the second polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NOs. 14, 24, 28, 38, 40, and 42. For example, the second polypeptide chain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NOs. 14, 24, 28, 38, 40, and 42, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0127] In the present application, the fusion protein may comprise a first polypeptide chain and a second polypeptide chain, wherein the first IgG Fc domain of the first polypeptide chain and the second IgG Fc domain of the second polypeptide chain are capable of interacting to form a dimer.

[0128] In the present application, the first complement inhibitory domain of the first polypeptide chain of the fusion protein and the second complement inhibitory domain of the second polypeptide chain may be the same. For example, the first complement inhibitory domain and the second complement inhibitory domain may comprise a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD46, CD55, and CD59. For example, the first complement inhibitory domain and the second complement inhibitory domain may comprise a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, and CD59.

[0129] In certain cases, the first IgG Fc domain of the first polypeptide chain of the fusion protein and the second IgG Fc domain of the second polypeptide chain may be the same. For example, the first IgG Fc domain and / or the second IgG Fc domain may comprise the amino acid sequence shown in any one of SEQ ID NOs. 10 and 30. For example, the first IgG Fc domain and / or the second IgG Fc domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NOs. 10 and 30, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0130] For example, the fusion protein may comprise a first polypeptide chain and a second polypeptide chain, and the first polypeptide chain and the second polypeptide chain may be the same. For example, the first polypeptide chain and / or the second polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NO.14, 24, 26, and 28. For example, the first polypeptide chain and / or the second polypeptide chain may comprise the amino acid sequence shown in any one of SEQ ID NO.14, 24, and 28. For example, the first polypeptide chain and / or the second polypeptide chain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence shown in any one of SEQ ID NO.14, 24, 26, and 28, such as any one of the amino acid sequences that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0131] In the present application, the first complement inhibitory domain of the first polypeptide chain of the fusion protein and the second complement inhibitory domain of the second polypeptide chain may be different. For example, each of the first complement inhibitory domain and the second complement inhibitory domain independently comprises a protein or a functional fragment thereof selected from the group consisting of CD59 and CD55. For example, each of the first complement inhibitory domain and the second complement inhibitory domain independently comprises a protein or a functional fragment thereof selected from the group consisting of FH and CD55. For example, each of the first complement inhibitory domain and the second complement inhibitory domain independently comprises a protein or a functional fragment thereof selected from the group consisting of CD46 and CD59.

[0132] In some cases, the first IgG Fc domain of the first polypeptide chain of the fusion protein and the second IgG Fc domain of the second polypeptide chain may be the same. For example, the first IgG Fc domain and / or the second IgG Fc domain may comprise the amino acid sequence shown in any one of SEQ ID NO.10 and 30.

[0133] In some cases, the first IgG Fc domain of the first polypeptide chain of the fusion protein and the second IgG Fc domain of the second polypeptide chain may be different. For example, the first IgG Fc domain may comprise the amino acid sequence shown in SEQ ID NO.32, and the second IgG Fc domain may comprise the amino acid sequence shown in SEQ ID NO.34.

[0134] For example, from the N-terminus to the C-terminus, the first polypeptide chain or the second polypeptide chain of the fusion protein may sequentially comprise the extracellular domain of CRIg, CD46, and IgG1 Fc. For example, the first polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO.36.

[0135] For example, the first polypeptide chain or the second polypeptide chain of the fusion protein may sequentially include the extracellular domain of CRIg, CD55, and IgG1 Fc from the N-terminus to the C-terminus. For example, the first polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 38.

[0136] For example, the second polypeptide chain or the second polypeptide chain of the fusion protein may sequentially include the extracellular domain of CRIg, FH, and IgG1 Fc from the N-terminus to the C-terminus. The second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 40.

[0137] For example, the second polypeptide chain or the second polypeptide chain of the fusion protein may sequentially include the extracellular domain of CRIg, CD59, and IgG1 Fc from the N-terminus to the C-terminus. The second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 42.

[0138] For example, the first polypeptide chain may include the amino acid sequence shown in any one of SEQ ID NO. 36, 38, 40, and 42.

[0139] For example, the first polypeptide chain may include the amino acid sequence shown in any one of SEQ ID NO. 38, 40, and 42.

[0140] For example, the second polypeptide chain may include the amino acid sequence shown in any one of SEQ ID NO. 36, 38, 40, and 42.

[0141] For example, the second polypeptide chain may include the amino acid sequence shown in any one of SEQ ID NO. 38, 40, and 42.

[0142] For example, the first polypeptide chain of the fusion protein may include the amino acid sequence shown in SEQ ID NO. 38, and the second polypeptide chain may include the amino acid sequence shown in any one of SEQ ID NO. 40 and 42.

[0143] For example, the first polypeptide chain of the fusion protein may include the amino acid sequence shown in SEQ ID NO. 38; and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 40.

[0144] For example, the first polypeptide chain of the fusion protein may include the amino acid sequence shown in SEQ ID NO. 38; and the second polypeptide chain may include the amino acid sequence shown in SEQ ID NO. 42.

[0145] For example, the first polypeptide chain of the fusion protein may comprise the amino acid sequence shown in SEQ ID NO. 36; and the second polypeptide chain may comprise the amino acid sequence shown in SEQ ID NO. 42.

[0146] In the present application, the first polypeptide chain, the second polypeptide chain, the extracellular domain of CRIg, the complement inhibitory domain (e.g., factor H (FH), CD46, CD55, and CD59), and the enhancing domain (e.g., the IgGFc domain and / or human serum albumin and / or the fusion protein) not only include the respective amino acid sequences as described above, but may also include variants of the respective amino acid sequences.

[0147] In the present application, variants of the amino acid sequence may include: 1) an amino acid sequence having at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence homology with the corresponding amino acid sequence; and / or,

[0148] 2) an amino acid sequence obtained by substituting, deleting, or adding one or several (e.g., 1 - 2, 1 - 3, 1 - 4, 1 - 5, 1 - 6, 1 - 7, 1 - 8, 1 - 9, 1 - 10, 1 - 11, 1 - 12, or more) amino acids in the corresponding amino acid sequence.

[0149] In the present application, the term "homology" generally refers to sequence similarity or interchangeability between two or more polynucleotide sequences or between two or more polypeptide sequences. When using a computer program or software (e.g., EmbossNeedle or BestFit) to determine sequence identity, similarity, or homology between different amino acid sequences, default parameter settings can be used. An appropriate scoring matrix, such as blosum45 or blosum80, can also be selected to optimize the identity, similarity, or homology score. In certain embodiments, homologous polynucleotides include the following polynucleotides: those that can hybridize with a control polynucleotide sequence under stringent conditions and have at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even at least 100% sequence identity compared to the control polynucleotide sequence. Homologous polypeptides can be the following polypeptides: when sequence alignment is performed under optimized conditions, they have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or even at least 100% sequence identity with the control polypeptide sequence.

[0150] To determine sequence identity, sequence alignment can be performed, which can be carried out in various ways known to those skilled in the art, for example, using software such as BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR). Those skilled in the art can determine the appropriate parameters for alignment, including any algorithms required to achieve optimal alignment in the full-length sequences being compared.

[0151] In the present application, the amino acid substitutions can be conservative amino acid substitutions or non-conservative amino acid substitutions. The first polypeptide chain, the second polypeptide chain, the extracellular domain of CRIg, the complement inhibitory domain (e.g., factor H (FH), CD55, and CD59), and / or the enhancing domain (e.g., the IgG Fc domain and / or human serum albumin) after substitution still have the same or similar functional activities as the first polypeptide chain, the second polypeptide chain, the extracellular domain of CRIg, the complement inhibitory domain (e.g., factor H (FH), CD55, and CD59), and / or the enhancing domain (e.g., the IgG Fc domain and / or human serum albumin) before substitution.

[0152] For example, the amino acid substitution can be a non-conservative substitution. The non-conservative substitution can include changing an amino acid residue in the target protein or polypeptide in a non-conservative form, such as changing an amino acid residue with a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue with a different side chain size or a different property (e.g., hydrophobicity).

[0153] The amino acid substitution can also be a conservative substitution. The conservative substitution can include changing an amino acid residue in the target protein or polypeptide in a conservative form, such as changing an amino acid residue with a certain side chain size or a certain property (e.g., hydrophilicity) to an amino acid residue with the same or similar side chain size or the same or similar property (e.g., still hydrophilic). Such conservative substitutions generally do not have a great impact on the structure or function of the resulting protein. In the present application, amino acid sequence variants of the first polypeptide chain, the second polypeptide chain, the extracellular domain of CRIg, the complement inhibitory domain (e.g., factor H (FH), CD46, CD55, and CD59), and / or the enhancing domain (e.g., the IgG Fc domain and / or human serum albumin) can include conservative amino acid substitutions that do not significantly change the protein structure or its function.

[0154] As an example, the mutual substitution between each amino acid within each of the following groups can be considered a conservative substitution in the present application: the group of amino acids with non-polar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine.

[0155] Amino acid groups without charge and with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0156] Amino acid groups with negative charge and with polar side chains: aspartic acid and glutamic acid.

[0157] Positively charged basic amino acids: lysine, arginine, and histidine.

[0158] Amino acids with phenyl groups: phenylalanine, tryptophan, and tyrosine.

[0159] Nucleic acid molecule, vector and cell

[0160] In another aspect, the present application also provides one or more isolated nucleic acid molecules. The one or more nucleic acid molecules can encode the fusion protein or fragment thereof described in the present application. For example, each nucleic acid molecule among the one or more nucleic acid molecules can encode the complete fusion protein (for example, when the fusion protein is single-chain), or can encode a part of the fusion protein, for example, the first polypeptide chain, the second polypeptide chain, the extracellular domain of CRIg, the complement inhibitory domain (for example, factor H (FH), CD46, CD55, and CD59), and / or the enhancing domain (for example, the IgG Fc domain and / or human serum albumin, or one or more of them).

[0161] The nucleic acid molecule described in the present application can be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, for example, produced by polymerase chain reaction (PCR) amplification, (ii) produced by recombinant cloning, (iii) purified, for example, by digestion with enzymes and fractionation by gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis. In certain embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.

[0162] In the present application, nucleic acids encoding the fusion protein or its fragments can be prepared by a variety of methods known in the art, including but not limited to, using restriction fragment manipulation or overlap extension PCR with synthetic oligonucleotides. For specific operations, reference can be made to Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York N.Y., 1993.

[0163] In another aspect, the present application provides one or more vectors that contain one or more nucleic acid molecules described in the present application. Each vector may contain one or more of the said nucleic acid molecules. In addition, other genes may also be contained in the vector, such as marker genes that allow selection of the vector in a suitable host cell and under suitable conditions. In addition, the vector may also contain expression control elements that allow correct expression of the coding region in a suitable host. Such control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In certain embodiments, the expression control sequence is an adjustable element. The specific structure of the expression control sequence may vary according to the function of the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' untranslated sequences that are respectively involved in the initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, and the promoter region may contain a promoter sequence for functionally linking nucleic acids for transcriptional control. The expression control sequence may also include enhancer sequences or upstream activator sequences. In the present application, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerases, the human U6 RNA promoter, the CMV promoter, and their artificial hybrid promoters (such as CMV), where a part of the promoter may be fused with a part of the promoter of other cellular proteins (such as human GAPDH, glyceraldehyde-3-phosphate dehydrogenase) gene, and it may or may not contain additional introns. One or more nucleic acid molecules described in the present application can be operably linked to the expression control element. The vector may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in genetic engineering. For example, the vector is an expression vector.

[0164] In another aspect, the present application provides host cells, which may comprise one or more nucleic acid molecules and / or one or more vectors described in the present application. In certain embodiments, each host cell may comprise one or a nucleic acid molecule or vector described in the present application. In certain embodiments, each host cell may comprise multiple (e.g., 2 or more) or multiple types (e.g., 2 or more types) of nucleic acid molecules or vectors described in the present application. For example, the vectors described in the present application can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, etc. The vectors described in the present application can be introduced into the host cells by methods known in the art, such as electroporation, lipofectine transfection, lipofectamin transfection, etc.

[0165] Pharmaceutical composition

[0166] In another aspect, the present application provides a pharmaceutical composition, which comprises the fusion protein described above and optionally a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier generally refers to a carrier that can be used to prepare a pharmaceutical composition or formulation, which is usually safe, non-toxic, and neither biologically nor otherwise undesirable. The carrier used is usually a carrier suitable for administration to humans or other mammals. When preparing the composition, the active ingredient is usually mixed with the carrier, diluted or enclosed by the carrier. When the carrier is used as a diluent, it can be a solid, semi-solid or liquid material, which serves as a vehicle, carrier or medium for the antibody active ingredient. The pharmaceutically acceptable carrier may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes, and / or non-ionic surfactants, etc.

[0167] In the present application, the pharmaceutical composition can be formulated for oral administration, intravenous administration, intramuscular administration, in-situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration through a subcutaneous depot. Solutions or suspensions for transdermal administration or through a subcutaneous depot may include the following components: sterile diluents such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetate, citrate, or phosphate; and substances for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or a base, such as hydrochloric acid or sodium hydroxide.

[0168] Method and use

[0169] In another aspect, the present application provides a method for preparing the fusion protein or its fragment. The method may include the following steps: synthesizing the fusion protein or its fragment, and / or culturing the cells under conditions for expressing the fusion protein or its fragment. For example, by using an appropriate culture medium, appropriate temperature, culture time, etc., these methods are known to those of ordinary skill in the art.

[0170] The present application can utilize genetic engineering techniques to link the fusion protein or its fragment described in the present application. For example, the first polypeptide chain, the second polypeptide chain, the extracellular domain of CRIg, the complement inhibitory domain (such as factor H (FH), CD46, CD55, and CD59), and / or the enhancing domain (such as the IgG Fc domain and / or human serum albumin), or amino acid residues can be sequentially linked according to the sequence of the protein.

[0171] The present application can also utilize genetic engineering techniques to link the coding sequences of the fusion protein or its fragment described in the present application. For example, the coding sequences of the first polypeptide chain, the second polypeptide chain, the extracellular domain of CRIg, the complement inhibitory domain (such as factor H (FH), CD46, CD55, and CD59), and / or the enhancing domain (such as the IgG Fc domain and / or human serum albumin); or bases can be sequentially linked according to the sequence of the nucleic acid.

[0172] In another aspect, the present application provides the use of the fusion protein or the pharmaceutical composition in the preparation of a medicament, which can be used to treat diseases related to targeted inhibition of complement activation. The diseases related to targeted inhibition of complement activation may be selected from: Paroxysmal Nocturnal Hemoglobinuria (PNH), atypical Haemolytic Uraemic Syndrome (aHUS), generalized Myasthenia Gravis (gMG), Neuromyelitis Optica Spectrum Disorders (NMOSD), Age-related Macular Degeneration (AMD), autoimmune hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, atherosclerosis, Parkinson's disease, Alzheimer's disease (senile dementia), asthma, allergy, psoriasis, multiple sclerosis, and Crohn's disease. The medicament of the present application can inhibit complement activation and / or protect cells from complement attack. In certain cases, the diseases may include autoimmune diseases. For example, the diseases may include autoimmune myasthenia gravis.

[0173] The present application also relates to the following embodiments:

[0174] 1. A fusion protein, comprising:

[0175] (i) the extracellular domain of CRIg;

[0176] (ii) a complement inhibitory domain, the complement inhibitory domain comprising a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46, and CD59; and

[0177] (iii) a potentiating domain, the potentiating domain comprising a protein or a functional fragment thereof selected from the group consisting of the IgG Fc domain and human serum albumin.

[0178] 2. The fusion protein according to embodiment 1, wherein the extracellular domain of CRIg comprises the amino acid sequence shown in SEQ ID NO.6.

[0179] 3. The fusion protein according to any one of embodiments 1-2, wherein the C-terminus of the extracellular domain of CRIg is directly or indirectly connected to the N-terminus of the complement inhibitory domain.

[0180] 4. The fusion protein according to any one of Embodiments 1-3, wherein the C-terminus of the complement inhibitory domain is directly or indirectly linked to the N-terminus of the potentiating domain.

[0181] 5. The fusion protein according to any one of Embodiments 1-4, wherein the indirect linkage includes linkage via a linker.

[0182] 6. The fusion protein according to Embodiment 5, wherein the linker comprises the amino acid sequence shown in any one of SEQ ID NOs. 44, 46, 48, and 50.

[0183] 7. The fusion protein according to any one of Embodiments 1-6, wherein the complement inhibitory domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 8, 18, 20, and 22.

[0184] 8. The fusion protein according to any one of Embodiments 1-7, wherein the potentiating domain is human serum albumin.

[0185] 9. The fusion protein according to any one of Embodiments 1-8, wherein the human serum albumin comprises the amino acid sequence shown in SEQ ID NO. 12.

[0186] 10. The fusion protein according to any one of Embodiments 1-9, which is a single-chain structure.

[0187] 11. The fusion protein according to any one of Embodiments 1-10, which sequentially includes the extracellular domain of CRIg, the complement inhibitory domain, and the potentiating domain in the order from the N-terminus to the C-terminus.

[0188] 12. The fusion protein according to any one of Embodiments 1-11, which comprises the amino acid sequence shown in SEQ ID NO. 14.

[0189] 13. The fusion protein according to any one of Embodiments 1-7, wherein the potentiating domain comprises an IgG Fc domain.

[0190] 14. The fusion protein according to any one of Embodiments 1-13, wherein the IgG includes proteins selected from the group consisting of human IgG1 and human IgG4.

[0191] 15. The fusion protein according to any one of Embodiments 1-14, wherein the IgG Fc domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 10, 30, 32, and 34.

[0192] 16. The fusion protein according to any one of embodiments 13 - 14, which comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the extracellular domain of CRIg, the first complement inhibitory domain, and the first IgG Fc domain, and the second polypeptide chain comprises the extracellular domain of CRIg, the second complement inhibitory domain, and the second IgG Fc domain,

[0193] wherein the first IgG Fc domain and the second IgG Fc domain are capable of interacting to form a dimer.

[0194] 17. The fusion protein according to embodiment 16, wherein the first complement inhibitory domain and the second complement inhibitory domain each independently comprise a protein or a functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46, and CD59.

[0195] 18. The fusion protein according to any one of embodiments 16 - 17, wherein the first complement inhibitory domain is the same as the second complement inhibitory domain.

[0196] 19. The fusion protein according to embodiment 18, wherein the first IgG Fc domain and the second IgG Fc domain are the same.

[0197] 20. The fusion protein according to any one of embodiments 18 - 19, wherein the first IgG Fc domain and the second IgG Fc domain comprise the amino acid sequence shown in any one of SEQ ID NO.10 and 30.

[0198] 21. The fusion protein according to any one of embodiments 18 - 20, wherein the first polypeptide chain and the second polypeptide chain are the same.

[0199] 22. The fusion protein according to any one of embodiments 18 - 21, wherein the first polypeptide chain and / or the second polypeptide chain comprise the amino acid sequence shown in any one of SEQ ID NO.14, 24, 26, and 28.

[0200] 23. The fusion protein according to any one of embodiments 16 - 17, wherein the first complement inhibitory domain is different from the second complement inhibitory domain.

[0201] 24. The fusion protein according to embodiment 23, wherein the first complement inhibitory domain and the second complement inhibitory domain each independently comprise a protein or a functional fragment thereof selected from the group consisting of CD59 and CD55.

[0202] 25. The fusion protein according to any one of embodiments 23-24, wherein each of the first complement inhibitory domain and the second complement inhibitory domain independently comprises a protein selected from the group consisting of FH and CD55 or a functional fragment thereof.

[0203] 26. The fusion protein according to any one of embodiments 23-25, wherein each of the first complement inhibitory domain and the second complement inhibitory domain independently comprises a protein selected from the group consisting of CD46 and CD59 or a functional fragment thereof.

[0204] 27. The fusion protein according to any one of embodiments 23-26, wherein the first IgG Fc domain and the second IgG Fc domain are the same.

[0205] 28. The fusion protein according to any one of embodiments 23-26, wherein the first IgG Fc domain and the second IgG Fc domain are different.

[0206] 29. The fusion protein according to embodiment 28, wherein the first IgG Fc domain comprises the amino acid sequence shown in any one of SEQ ID NO.32 and 34.

[0207] 30. The fusion protein according to any one of embodiments 28-29, wherein the second IgG Fc domain comprises the amino acid sequence shown in any one of SEQ ID NO.32 and 34.

[0208] 31. The fusion protein according to any one of embodiments 28-30, wherein the first polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO.36, 38, 40 and 42.

[0209] 32. The fusion protein according to any one of embodiments 28-31, wherein the second polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NO.36, 38, 40 and 42.

[0210] 33. The fusion protein according to any one of embodiments 23-32, wherein:

[0211] the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.38; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.40;

[0212] the first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.36; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO.42; or,

[0213] The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 38; and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO. 42.

[0214] 34. One or more isolated nucleic acid molecules encoding the fusion protein or fragment thereof according to any one of embodiments 1-33.

[0215] 35. A vector comprising the nucleic acid molecule according to embodiment 34.

[0216] 36. A cell comprising the vector according to embodiment 35, or expressing the fusion protein according to any one of embodiments 1-33.

[0217] 37. A method for preparing the fusion protein according to any one of embodiments 1-33, comprising the steps of: synthesizing the fusion protein according to any one of embodiments 1-33, and / or culturing the cell according to embodiment 36 under conditions for expressing the fusion protein according to any one of embodiments 1-33.

[0218] 38. A pharmaceutical composition comprising the fusion protein according to any one of embodiments 1-33 and optionally a pharmaceutically acceptable carrier.

[0219] 39. Use of the fusion protein according to any one of embodiments 1-33 or the pharmaceutical composition according to embodiment 37 in the preparation of a medicament for treating a disease related to targeted inhibition of complement activation.

[0220] 40. The use according to embodiment 39, wherein the disease includes autoimmune diseases.

[0221] 41. The use according to any one of embodiments 39-40, wherein the disease includes autoimmune myasthenia gravis.

[0222] Without being limited by any theory, the following examples are merely for illustrating the fusion protein, preparation method, and uses of the present application, and are not intended to limit the scope of the invention of the present application. ns: no significance, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

[0223] Examples

[0224] Example 1 The half-life of the CRIg-FH drug can be extended by increasing the IgG4 Fc fragment

[0225] Experimental purpose: Introduce human IgG Fc or HSA into the CRIg-FH complement inhibitor, construct expression vectors of two recombinant proteins, (CRIg-FH-IgG4Fc)×2 and His×6-CRIg-FH-HSA, perform eukaryotic expression and purification, detect their complement inhibitory activity and in vivo half-life, and finally screen and determine the protein fragment that can extend the half-life of CRIg-FH without affecting its complement inhibitory effect.

[0226] 1. Instruments and materials:

[0227] Electrically heated constant temperature water bath (DK-8D type, Shanghai Jinghong Experimental Equipment Co., Ltd.), PCR instrument (Mastercyclerpro-Eppendorf, Eppendorf, Germany), RNA / DNA concentration / purity detector (NANODROP 2000c, Thermo Scientific, USA), gel imager (Tanon 1600, Shanghai Tianneng Technology Co., Ltd.), CO2 cell incubator (240i, Thermo Scientific, USA), BioRAD Mini protein Tera system (BioRAD, USA), low-temperature horizontal centrifuge (Allegra X-15R Centrifuge, Beckman Coulter, USA).

[0228] 2. Experimental methods:

[0229] 2.1 Gene cloning and vector construction

[0230] Extract total RNA from human hepatoma cell line Hep3B using NucleoZOL (Macherey-Nagel, Germany), and then reverse transcribe the total RNA into cDNA using reverse transcriptase (PrimeScript TM RT Master Mix, Takara, Japan). The above cDNA is used to amplify the gene sequences of the SCR1-5 domains (E19-K323) encoding the FH complement inhibitory functional fragment and human serum albumin HSA by PCR with corresponding primers; in addition, total RNA is extracted from lymphoma cell U937 cells using a similar method, reverse transcribed into cDNA, and then the gene sequence encoding the extracellular domain (G19-K137) of the CRIg gene is amplified by PCR.

[0231] The nucleic acid sequence encoding the FH protein is shown as SEQ ID NO:3, the amino acid sequence of the FH protein is shown as SEQ ID NO:4, the nucleic acid sequence encoding the SCR1-5 domain of FH is shown as SEQ ID NO:7, and the amino acid sequence of the SCR1-5 domain of FH is shown as SEQ ID NO:8.

[0232] The nucleic acid sequence encoding the HAS protein is shown as SEQ ID NO:11, and the amino acid sequence of the HAS protein is shown as SEQ ID NO:12.

[0233] The nucleic acid sequence encoding the CRIg protein is shown as SEQ ID NO:1, and the amino acid sequence of the CRIg protein is shown as SEQ ID NO:2. The nucleic acid sequence encoding the extracellular domain of CRIg is shown as SEQ ID NO:5, and the amino acid sequence of the extracellular domain of CRIg is shown as SEQ ID NO:6.

[0234] Primers containing the sequences of the extracellular domain of CRIg and the FH SCR1-5 domain were designed, and the extracellular domain gene of CRIg and the FH SCR1-5 gene were ligated by the method of Overlapping PCR and inserted into the eukaryotic expression vector pFUSE-hIgG4-Fc1 (InvivoGen). Among them, the nucleic acid sequence encoding hIgG4-Fc is shown as SEQ ID NO:9, and the amino acid sequence of the hIgG4-Fc protein is shown as SEQ ID NO:10. In addition, after the extracellular domain gene of CRIg and FH SCR1-5 were ligated, they were ligated with the HSA gene by the method of Overlapping PCR and inserted into the expression vector pcDNA3.1 / His A (Invitrogen). After the above vectors were identified by bidirectional sequencing to confirm that the inserted sequences were correct, subsequent experiments were carried out. The vectors were named pFUSE-CRIg-FH-hIgG4Fc1 and pCDNA / His-CRIg-FH-HSA respectively.

[0235] 2.2 Protein expression

[0236] According to 2.0×10 7293FT cells at a density of

[0237] 2.3 Protein affinity purification, buffer replacement and concentration

[0238] Apply the Protein A antibody purification magnetic bead kit (China Beaver Nano-Technology (Suzhou) Co., Ltd.) to purify and elute the (CRIg-FH-IgG4Fc)×2 recombinant protein in the cell supernatant into the elution buffer, transfer it to an ultrafiltration tube (Millipore, USA), and replace the buffer with PBS and concentrate the protein by low-temperature centrifugation. Or apply the His·Bind affinity Purification Kit (Novagen / MerckMillipore) to purify the expressed His×6-CRIg-FH-HSA fusion protein according to the production instructions, and also replace the buffer with PBS and concentrate and store it in a similar way. The obtained recombinant proteins are named (CRIg-FH-IgG4Fc)×2 and CRIg-FH-HSA( Figure 1 )

[0239] 2.4 Detection of complement inhibitory activity

[0240] Use a commercially available kit Complement System Classical Pathway (COMPLCP310) and Complement Alternative Pathway (COMPLAP330) to detect the inhibitory effects of the above two recombinant proteins on the classical pathway and alternative pathway of complement, respectively.

[0241] 2.5 Detection of the in vivo drug half-life of the recombinant protein

[0242] SD rats were selected, with an equal number of males and females. (CRIg-FH-IgG4Fc)×2 was injected intravenously. Blood samples were collected at different time points before administration (D-1), 5 min after the start of administration on D1 (i.e., 2 min after the end of administration), 30 min, 4 h, 24 h, 2 d, 3 d, 5 d, 7 d, 10 d, 14 d, 21 d, and 28 d, and the sera were separated. The concentration of the recombinant protein in each sample was analyzed by ELISA. Or CRIg-FH-HSA (1 mg / kg) was injected intravenously. Blood samples were collected before administration, 5 min, 30 min, 1 h, 3 h, 6 h, 12 h, 24 h, 2 d, 3 d, 5 d, 7 d, 10 d, 14 d, 21 d, and 28 d after the start of administration, and the sera were separated.

[0243] The ELISA detection method was as follows: Rabbit anti-human CRIg monoclonal antibody (clone number 202, Sino Biological, Catalog#12163-H08H) was coated. After binding to the standard recombinant protein or the recombinant protein in the serum, (CRIg-FH-IgG4Fc)×2 was detected by mouse anti-human IgG4 fragment Secondary (5c7)[HRP] (NOVUS, Catalog#NB110-7081H) enzyme-labeled secondary antibody, or CRIg-FH-HSA was detected by polyclonal antibody to human serum albumin, HRP (ThermoFisher, Catalog#PA1-72058) enzyme-labeled secondary antibody. The plasma drug concentration of the recombinant protein in the serum was calculated through the standard curve, the time-drug serum concentration curve was plotted, and the half-life was calculated.

[0244] 3. Experimental results:

[0245] 3.1 Two recombinant proteins, (CRIg-FH-IgG4Fc)×2 and His×6-CRIg-FH-HSA, were induced, expressed, and purified using the eukaryotic system by the above method. After PAGE electrophoresis detection, the purity was greater than 95%. Their theoretical molecular weights were 146 kDa and 114 kDa respectively, and the actual sizes were consistent with the theoretical values ( Figure 2 ). The nucleic acid sequence encoding CRIg-FH-IgG4 Fc is shown in SEQ ID NO:13, and the amino acid sequence of CRIg-FH-IgG4 Fc protein is shown in SEQ ID NO:14; the nucleic acid sequence encoding CRIg-FH-HSA is shown in SEQ ID NO:15, and the amino acid sequence of CRIg-FH-HSA protein is shown in SEQ ID NO:16.

[0246] 3.2 We detected the inhibitory activities of (CRIg-FH-IgG4 Fc)×2 and CRIg-FH-HSA against the classical pathway and alternative pathway of human serum complement. The results showed that both (CRIg-FH-IgG4 Fc)×2 and CRIg-FH-HSA had inhibitory effects on complement. The IC50 of the former for inhibiting the classical pathway of complement was 91.38 nM( Figure 3 ), and the IC50 for inhibiting the alternative pathway of complement was 1.04 nM( Figure 4 ). The IC50 of the latter for inhibiting the classical pathway of complement was 1355 nM( Figure 5 ), and the IC50 for inhibiting the alternative pathway of complement was 10.39 nM( Figure 6 ).

[0247] 3.3 (CRIg-FH-IgG4 Fc)×2 and CRIg-FH-HSA

[0248] Detected the concentrations of (CRIg-FH-IgG4 Fc)×2 or CRIg-FH-HSA in sera collected at different time points, and plotted the pharmacokinetic curves of time-concentration. The pharmacokinetic curve of (CRIg-FH-IgG4 Fc)×2 is shown in Figure 7 , with a half-life of 142.2 hours; while the pharmacokinetic curve of CRIg-FH-HSA is shown in Figure 8 , with a half-life of 32.8 hours.

[0249] Example 2 FH is a complement inhibitory fragment linked to CRIg with excellent complement inhibitory effect

[0250] Experimental purpose: By constructing expression vectors of four recombinant proteins (CRIg-FH-IgG4Fc)×2, (CRIg-CD55-IgG4Fc)×2, (CRIg-CD46-IgG4Fc)×2 and (CRIg-CD59-IgG4Fc)×2 for eukaryotic expression and purification, and detecting their complement activities, to determine which inhibitory protein has the optimal complement inhibitory effect after being linked to CRIg among the four important complement inhibitory proteins FH, CD55, CD46 and CD59.

[0251] 1. Instruments and materials:

[0252] Electric thermostatic water bath (DK-8D type, Shanghai Jinghong Experimental Equipment Co., Ltd.), PCR instrument (Mastercycler pro-Eppendorf, Eppendorf Company, Germany), RNA / DNA concentration / purity detector (NANODROP 2000c, Thermo Scientific, USA), gel imager (Tanon 1600, Shanghai Tianneng Technology Co., Ltd.), CO2 cell incubator (240i, Thermo Scientific Company, USA), BioRAD Mini protein Tera system (BioRAD Company, USA), low-temperature horizontal centrifuge (Allegra X-15R Centrifuge, Beckman Coulter Company, USA).

[0253] 2. Experimental methods:

[0254] 2.1 Gene cloning and vector construction

[0255] Total RNA was extracted from human normal pancreatic duct epithelial cells HPDE6-C7 using NucleoZOL (Macherey-Nagel Company, Germany), and then the total RNA was reverse transcribed into cDNA using reverse transcriptase (PrimeScript TM RT Master Mix, Takara Company, Japan). The above cDNA was used to amplify the DNA sequences encoding CD55, CD46, and CD59 (all without the DNA sequence encoding the signal peptide) by PCR with the corresponding primers. Total RNA was extracted from human hepatoma cell line Hep3B cells using NucleoZOL in the same way. After reverse transcription into cDNA, the SCR1-5 domain (E19-K323) of the FH complement inhibitory functional fragment was amplified by PCR. Total RNA was extracted from lymphoma cells U937 cells, and after reverse transcription into cDNA, the gene sequence encoding the extracellular domain (G19-K137) of the CRIg gene was amplified by PCR. Among them, the nucleic acid sequence encoding CD55 is shown in SEQ ID NO:17, and the amino acid sequence of the CD55 protein is shown in SEQ ID NO:18; the nucleic acid sequence encoding CD46 is shown in SEQ ID NO:19, and the amino acid sequence of the CD46 protein is shown in SEQ ID NO:20; the nucleic acid sequence encoding CD59 is shown in SEQ ID NO:21, and the amino acid sequence of the CD59 protein is shown in SEQ ID NO:22.

[0256] Design primers that simultaneously contain the extracellular domain of CRIg and the coding sequences of FH SCR1-5 domain, CD55, CD46, or CD59. Use the method of overlapping PCR to ligate the extracellular domain gene of CRIg and the genes of FH SCR1-5, CD46, CD55, or CD59, and insert them into the eukaryotic expression vector pFUSE-hIgG4-Fc1 (InvivoGen). After the above vectors are confirmed by bidirectional sequencing to have the correct inserted sequences, subsequent experiments are carried out. The vectors are named pFUSE-CRIg-FH-IgG4Fc, pFUSE-CRIg-CD55-IgG4Fc, pFUSE-CRIg-CD46-IgG4Fc, and pFUSE-CRIg-CD59-IgG4Fc respectively.

[0257] 2.2 Protein expression

[0258] According to the density of 293FT cells at 2.0×10 7 cells / dish, evenly spread them in a 15-cm-diameter cell culture dish. When the cells grow to the logarithmic phase, use Lipofectamine2000 (Invitrogen, USA) reagent to transfect the large-scale plasmids of pFUSE-CRIg-FH-IgG4Fc, pFUSE-CRIg-CD55-IgG4Fc, pFUSE-CRIg-CD46-IgG4Fc, or pFUSE-CRIg-CD59-IgG4Fc constructed above. After culturing in a 37°C, 5% CO2 incubator for 6 hours, replace it with 293 protein expression serum-free medium (Gibco, USA), and continue to culture for three days. Then collect the cell culture supernatant, centrifuge to remove cells and cell debris, and concentrate the supernatant using an ultrafiltration tube for purification.

[0259] 2.3 Protein affinity purification, buffer replacement, and concentration

[0260] Apply the Protein A antibody purification magnetic bead kit (BeaverNano Technology (Suzhou) Co., Ltd., China) to purify and elute the recombinant proteins (CRIg-FH-IgG4Fc)×2, (CRIg-CD55-IgG4Fc)×2, (CRIg-CD46-IgG4Fc)×2, (CRIg-CD59-IgG4Fc)×2 in the cell supernatant into the elution buffer, transfer them to an ultrafiltration tube (Millipore, USA), and replace the buffer with PBS by low-temperature centrifugation, and concentrate the protein for storage.

[0261] 2.4 Complement inhibition activity detection

[0262] Use a commercially available kit Complement System Classical Pathway (COMPLCP310) and Complement Alternative Pathway (COMPLAP330) were respectively used to detect the inhibitory effects of the above-mentioned recombinant proteins on the classical pathway and alternative pathway of complement.

[0263] 3. Experimental results:

[0264] 3.1 Four recombinant proteins, (CRIg-FH-IgG4Fc)×2, (CRIg-CD55-IgG4Fc)×2, (CRIg-CD46-IgG4Fc)×2, and (CRIg-CD59-IgG4Fc)×2, were induced, expressed, and purified using the eukaryotic system by the above method Figure 9 ). PAGE electrophoresis was performed for detection, and the purity was greater than 95%. Their theoretical molecular weights were 146, 153, 157, and 101 kDa respectively, and the actual sizes were consistent with the theoretical values Figure 10 ). The nucleic acid sequence encoding CRIg-CD55-IgG4Fc is shown in SEQ ID NO:23, and the amino acid sequence of CRIg-CD55-IgG4Fc protein is shown in SEQ ID NO:24; the nucleic acid sequence encoding CRIg-CD46-IgG4Fc is shown in SEQ ID NO:25, and the amino acid sequence of CRIg-CD46-IgG4Fc protein is shown in SEQ ID NO:26; the nucleic acid sequence encoding CRIg-CD59-IgG4Fc is shown in SEQ ID NO:27, and the amino acid sequence of CRIg-CD59-IgG4Fc protein is shown in SEQ ID NO:28.

[0265] 3.2 We detected the inhibitory activities of four recombinant proteins, (CRIg-FH-IgG4Fc)×2, (CRIg-CD55-IgG4Fc)×2, (CRIg-CD46-IgG4Fc)×2, and (CRIg-CD59-IgG4Fc)×2, on the classical pathway and alternative pathway of human serum complement. The inhibitory effects of (CRIg-FH-IgG4 Fc)×2 on the classical and alternative pathways of complement are shown in Figure 3 and Figure 4 , and the inhibitory effects of (CRIg-CD55-IgG4Fc)×2 on the classical and alternative pathways of complement are shown in Figure 11 and Figure 12 , and the inhibitory effects of (CRIg-CD46-IgG4Fc)×2 on the classical and alternative pathways of complement are shown in Figure 13 and Figure 14, the inhibitory effects of (CRIg-CD59-IgG4Fc)×2 on the classical and alternative pathways of complement are shown in Figure 15 and Figure 16 . The IC50 values for their inhibition of the classical pathway of complement are shown in Table 1 in detail. Specifically, the IC50 values of (CRIg-FH-IgG4 Fc)×2 for inhibiting the classical and alternative pathways of complement are 91.38 nM and 1.04 nM, respectively; the IC50 values of (CRIg-CD55-IgG4Fc)×2 for inhibiting the classical and alternative pathways of complement are 23.25 nM and 19.66 nM, respectively; the IC50 value of (CRIg-CD46-IgG4Fc)×2 for inhibiting the classical pathway of complement is 44.06 nM; the IC50 value of (CRIg-CD59-IgG4Fc)×2 for inhibiting the classical pathway of complement is 44.84 nM. All four fusion proteins have inhibitory effects on the classical pathway and / or alternative pathway.

[0266] Example 3 Research and Development of Bispecific Targeted Complement Inhibitors

[0267] Experimental Purpose: By constructing three bispecific targeted recombinant protein expression vectors, namely (CRIg-CD55-IgG1 Fc)(CRIg-FH-IgG1Fc), (CRIg-CD46-IgG1Fc)(CRIg-CD59-IgG1 Fc), and (CRIg-CD55-IgG1 Fc)(CRIg-CD59-IgG1 Fc), eukaryotic expressing and purifying these fusion proteins, and detecting their complement inhibitory activities, to detect and screen more effective complement inhibitors.

[0268] 1. Instruments and Materials:

[0269] Electrically heated constant temperature water bath (DK-8D type, Shanghai Jinghong Experimental Equipment Co., Ltd.), PCR instrument (Mastercyclerpro-Eppendorf, Eppendorf Company, Germany), RNA / DNA concentration / purity detector (NANODROP 2000c, Thermo Scientific Company, USA), gel imaging system (Tanon 1600, Shanghai Tianneng Technology Co., Ltd.), CO2 cell incubator (240i, Thermo Scientific Company, USA), BioRAD Mini protein Tera system (BioRAD Company, USA), low-temperature horizontal centrifuge (Allegra X-15R Centrifuge, Beckman Coulter Company, USA).

[0270] 2. Experimental Methods:

[0271] 2.1 Gene Cloning and Vector Construction

[0272] By designing mutant primers, the primer mutation sites of the eukaryotic expression vector pFUSE-hIgG1-Fc1 were mutated (Alegre et al., 1992; Carter, 2001; Merchant et al., 1998; Ridgway et al., 1996; Xu et al., 2000) using a site-directed mutagenesis kit (TOYOBO, Japan) to generate two mutant vectors, pFUSE-hIgG1-Fc1 knob mutant and pFUSE-hIgG1-Fc1 hole mutant.

[0273] Among them, the nucleic acid sequence encoding IgG1 Fc is shown in SEQ ID NO:29, and the amino acid sequence of the IgG1 Fc protein is shown in SEQ ID NO:30; the nucleic acid sequence encoding Knob mutant is shown in SEQ ID NO:31, and the amino acid sequence of the Knob mutant protein is shown in SEQ ID NO:32; the nucleic acid sequence encoding Hole mutant is shown in SEQ ID NO:33, and the amino acid sequence of the Hole mutant protein is shown in SEQ ID NO:34.

[0274] Using the CRIg-FH, CRIg-CD46, CRIg-CD55, and CRIg-CD59 gene sequences constructed in Examples 1 and 2, the above fusion fragments CRIg-CD46 and CRIg-CD55 were inserted into the pFUSE-hIgG1-Fc knob mutant vector using a one-step rapid cloning kit (Yeasen Biotech Co., Ltd., Shanghai), and the plasmids were named pFUSE-CRIg-CD46-hIgG1 Fc knob and pFUSE-CRIg-CD55-hIgG1 Fc knob, respectively. Then, the fusion fragments CRIg-FH and CRIg-CD59 were inserted into the pFUSE-IgG1-Fc hole mutant vector using the one-step rapid cloning kit, and the plasmids were named pFUSE-CRIg-FH-IgG1 Fc hole and pFUSE-CRIg-CD59-IgG1 Fc hole, respectively.

[0275] Among them, the nucleic acid sequence encoding CRIg-CD46-IgG1 Fc knob is shown in SEQ ID NO: 35, and the amino acid sequence of CRIg-CD46-IgG1 Fc knob protein is shown in SEQ ID NO: 36; the nucleic acid sequence encoding CRIg-CD55-IgG1 Fc knob is shown in SEQ ID NO: 37, and the amino acid sequence of CRIg-CD55-IgG1 Fc knob protein is shown in SEQ ID NO: 38; the nucleic acid sequence encoding CRIg-FH-IgG1 Fc hole is shown in SEQ ID NO: 39, and the amino acid sequence of CRIg-FH-IgG1 Fc hole protein is shown in SEQ ID NO: 40; the nucleic acid sequence encoding CRIg-CD59-IgG1 Fc hole is shown in SEQ ID NO: 41, and the amino acid sequence of CRIg-CD59-IgG1 Fc hole protein is shown in SEQ ID NO: 42.

[0276] 2.2 Protein expression

[0277] According to the 293FT cell density of 2.0×10 7 cells / dish, evenly spread them in a 15-cm-diameter cell culture dish. When the cells grow to the logarithmic phase, use Lipofectamine 2000 (Invitrogen, USA) reagent to co-transfect the large-scale extracted plasmids of pFUSE-CRIg-CD46-hIgG1 Fc knob and pFUSE-CRIg-CD59-hIgG1 Fc hole, pFUSE-CRIg-CD55-hIgG1 Fc knob and pFUSE-CRIg-CD59-hIgG1 Fc hole, pFUSE-CRIg-CD55-hIgG1 Fc knob and pFUSE-CRIg-FH-hIgG1 Fc hole. After culturing in a 37°C, 5% CO2 incubator for 6 hours, replace it with 293 protein expression serum-free medium (Gibco, USA), and continue to culture for three days. Then collect the cell culture supernatant, centrifuge to remove cells and cell debris, and concentrate the supernatant using an ultrafiltration tube for purification.

[0278] 2.3 Protein affinity purification, buffer replacement and protein concentration

[0279] Apply the Protein A antibody purification magnetic bead kit (Beaver Nano-Technology (Suzhou) Co., Ltd.) to purify and elute the recombinant proteins (CRIg-CD46-IgG1 Fc)(CRIg-CD59-IgG1 Fc), (CRIg-CD55-IgG1 Fc)(CRIg-CD59-IgG1Fc), and (CRIg-CD55-IgG1 Fc)(CRIg-FH-IgG1 Fc) in the cell supernatant into the elution buffer, transfer them into an ultrafiltration tube (Millipore Corporation, USA), and replace the buffer with PBS and concentrate the proteins by low-temperature centrifugation.

[0280] 2.4 Complement inhibition activity detection

[0281] Use a commercially available kit Complement System Classical Pathway (COMPLCP310) and Complement Alternative Pathway (COMPLAP330) were used to detect the inhibitory effects of the above two recombinant proteins on the classical and alternative pathways of complement, respectively. The data were analyzed using Graphpad Prism 7.0 software.

[0282] 3. Experimental results:

[0283] 3.1 Through the above methods, three types of bispecific targeting complement inhibitor recombinant proteins (CRIg-CD55-IgG1 Fc)(CRIg-FH-IgG1 Fc), (CRIg-CD46-IgG1 Fc)(CRIg-CD59-IgG1 Fc), and (CRIg-CD55-IgG1 Fc)(CRIg-CD59-IgG1 Fc) were induced, expressed, and purified using the eukaryotic system Figure 17 ), and PAGE electrophoresis was performed for detection. The purity was greater than 95%. Their theoretical molecular weights were 151, 130, and 128 kDa respectively, and the actual sizes were consistent with the theoretical values Figure 18 .

[0284] 3.2 The IC50 inhibition activities of the three bispecific targeting complement inhibitors on the classical and alternative pathways of complement are shown in Table 1. The inhibitory effects of (CRIg-CD55-IgG1 Fc)(CRIg-FH-IgG1 Fc) on the classical and alternative pathways of complement are shown respectively as Figure 19 and Figure 20 shown; the inhibitory effects of (CRIg-CD46-IgG1 Fc)(CRIg-CD59-IgG1 Fc) on the classical and alternative pathways of complement are shown respectively as Figure 21 and Figure 22As shown; the effects of (CRIg - CD55 - IgG1 Fc) and (CRIg - CD59 - IgG1 Fc) on inhibiting the classical and alternative complement pathways are respectively as Figure 23 and Figure 24 shown. All three bispecific targeted complement inhibitors can inhibit either the classical or alternative complement pathway.

[0285] Table 1 Comparison of in vitro complement inhibitory effects of complement inhibitors

[0286]

[0287]

[0288] Pharmacodynamic effects of (CRIg - FH - IgG4Fc)×2 in treating a rat model of myasthenia gravis in Example 4

[0289] Experimental purpose: Among the above - mentioned fusion proteins, we selected the (CRIg - FH - IgG4Fc)×2 complement inhibitor to detect its pharmacodynamic effects on diseases with excessive complement activation in vivo. Autoantibodies against the acetylcholine receptor (AChR) bind to AChR at the neuromuscular junction, directly down - regulating the AChR expression level, or indirectly causing AChR dysfunction or a decrease in expression level by activating the classical complement pathway, both of which lead to neuromuscular transmission disorders and ultimately present clinical manifestations of MG such as muscle weakness (Gomez et al., 2010; Phillips and Vincent, 2016). We detected the therapeutic effect of the (CRIg - FH - IgG4Fc)×2 complement inhibitor in a rat model of myasthenia gravis - experimental autoimmune myasthenia gravis (EAMG) to lay a foundation for subsequent potential clinical applications.

[0290] 1. Instruments and materials:

[0291] Animal weighing balance (Shanghai Precision Scientific Instrument Co., Ltd., YP2001N), Lewis rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.), InVivo MAb anti - human / rat / fish AChR (Bio X Cell, West Lebanon, NH).

[0292] 2. Experimental methods:

[0293] 2.1 Induction of EAMG

[0294] Intraperitoneal injection of anti-AChR antibody mAb35 (1 - 3 mg / kg) to female rats around four weeks old is a common method internationally for inducing a systemic myasthenia gravis-like experimental autoimmune myasthenia gravis (EAMG) model (Hepburn et al., 2007; Liu et al., 2007; Papanastasiou et al., 2000; Poulas et al., 2000). The rats were divided into a normal group without injection of anti-AChR antibody, a group intraperitoneally injected with anti-AChR antibody (1.5 mg / kg) and injected with PBS, and five groups intraperitoneally injected with anti-AChR antibody and injected with five different doses (0.5, 1, 2, 5, and 10 mg / kg) of (CRIg-FH-IgG4Fc)×2 drug treatment groups. There were a total of 7 groups above, and the experiments were completed in two times. There were 40 rats in the first experiment and 50 rats in the second experiment. The grouping is shown in Table 2.

[0295] Table 2 Grouping for observing the efficacy of (CRIg-FH-IgG4Fc)×2 drug against AChR antibody-induced EAMG in rats

[0296]

[0297]

[0298] 2.2 Monitoring of EAMG phenotype

[0299] The rats were experimented after at least a 5 - 7-day adaptation period. They were weighed every 24 hours before and after the experiment and clinically scored. The specific scoring criteria were: can grasp and lift the cage lid, 0 points; can grasp but not lift the cage lid, 1 point; cannot grasp the cage lid, 2 points; cannot grasp objects and hindlimb paralysis, 3 points; death or near-death state, 4 points (Piddlesden et al., 1996; Soltys et al., 2009). It was found in the experiment that most rats in the PBS control group of the model group died or were on the verge of death at 48 hours. Therefore, according to ethics, the observation of this group ended at the 48-hour time point, while the clinical scores of the (CRIg-FH-IgG4Fc)×2 drug treatment group had basically recovered at 96 hours. Therefore, the observation of this group ended at the 96-hour time point, and the animal mortality rates at 24 and 48 hours were counted.

[0300] 2.3 Statistical analysis

[0301] Experimental data were expressed as Mean±SEM. Body weight was analyzed by two tails t-test, clinical scores were analyzed by Two-way ANOVA, and mortality was analyzed by Chi-Square and Fisher’s Exact Test. ns: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0302] 3. Experimental results:

[0303] The body weights of normal rats without injection of anti-AChR antibody and (CRIg-FH-IgG4Fc)×2 gradually increased. However, the body weights of rats injected with anti-AChR antibody without drug treatment decreased by 11.2% at 24 hours and 15.5% at 48 hours. The proportion of rats with clinical scores of 3-4 at 24 hours reached 82% (14 / 17), and at 48 hours it reached 88% (15 / 17). Among the 17 rats, 2 rats were on the verge of death or had died at 24 hours, and by 48 hours, a total of 9 rats had died, with a mortality rate of 52.9% (9 / 17). Among the remaining 8 surviving animals, 6 were also close to the dying state, indicating that the EAMG rat model was successfully established ( Figures 25 to 27 ).

[0304] Pretreatment with (CRIg-FH-IgG4Fc)×2 could significantly improve the symptoms of EAMG. After treatment with the lowest drug dose of 0.5mg / kg, the body weight only decreased by 1.6% at 24 hours, the clinical score was 1.1 points, at 48 hours the body weight only decreased by 2.3%, the clinical score was 1.3 points, and the animal mortality rate was 14.3% (1 / 7), with obvious curative effects. After treatment with higher drug doses, the body weight did not decrease significantly at 24 hours, and the clinical scores gradually decreased in a dose-dependent manner; at 48 hours, the body weight gradually recovered, and the clinical scores also gradually decreased, and there was a certain dose-effect relationship with the dose, that is, the higher the dose, the more the body weight increased, while the clinical score was lower ( Figures 25 to 27 ). In addition, all animals in the drug treatment groups were observed until 96 hours. The body weights of the surviving rats at 48 hours showed a gradually increasing trend, and the clinical scores gradually decreased to normal values, indicating that the EAMG symptoms gradually recovered to normal.

[0305] 4. Discussion:

[0306] Female rats were intraperitoneally injected with anti-AChR antibody mAb35 (1.5 mg / kg) around four weeks to induce the EAMG phenotype, which was manifested as a significant decrease in rat body weight within 48 hours, a rapid loss of motor ability, a decrease in the grasping force of the forelimbs, and animal death, indicating successful modeling. Monitoring of rat body weight, clinical scoring, and animal death showed that LM007 could exert an obvious therapeutic effect on EAMG at a drug dose of 0.5 mg / kg. With the increase in dose, there was a certain dose-dependent relationship between the efficacy and the dose, and the disease process was almost completely blocked at a dose of 5 mg / kg and above. In the rat EAMG model induced by anti-AChR antibody, the overactivation of complement played a major role, but in very few rats, the pathogenic effect might not depend on the activation of complement. Sequence Listing <110> Shanghai Kangjing Biomedical Technology Co., Ltd. <120> Biomacromolecule Targeting Specific Complement Inhibitor and Its Preparation Method and Application <130> 0175-PA-001 <160> 50 <170> PatentIn version 3.5 <210> 1 <211> 1200 <212> DNA <213> Homo sapiens <400> 1 atggggatct tactgggcct gctactcctg gggcacctaa cagtggacac ttatggccgt 60 cccatcctgg aagtgccaga gagtgtaaca ggaccttgga aaggggatgt gaatcttccc 120 tgcacctatg accccctgca aggctacacc caagtcttgg tgaagtggct ggtacaacgt 180 ggctcagacc ctgtcaccat ctttctacgt gactcttctg gagaccatat ccagcaggca 240 aagtaccagg gccgcctgca tgtgagccac aaggttccag gagatgtatc cctccaattg 300 agcaccctgg agatggatga ccggagccac tacacgtgtg aagtcacctg gcagactcct 360 gatggcaacc aagtcgtgag agataagatt actgagctcc gtgtccagaa actctctgtc 420 tccaagccca cagtgacaac tggcagcggt tatggcttca cggtgcccca gggaatgagg 480 attagccttc aatgccaggc tcggggttct cctcccatca gttatatttg gtataagcaa 540 cagactaata accaggaacc catcaaagta gcaaccctaa gtaccttact cttcaagcct 600 gcggtgatag ccgactcagg ctcctatttc tgcactgcca agggccaggt tggctctgag 660 cagcacagcg acattgtgaa gtttgtggtc aaagactcct caaagctact caagaccaag 720 actgaggcac ctacaaccat gacatacccc ttgaaagcaa catctacagt gaagcagtcc 780 [[ID=!6]]tgggactgga ccactgacat ggatggctac cttggagaga ccagtgctgg gccaggaaag 840 agcctgcctg tctttgccat catcctcatc atctccttgt gctgtatggt ggtttttacc 900 atggcctata tcatgctctg tcggaagaca tcccaacaag agcatgtcta cgaagcagcc 960 agggcacatg ccagagaggc caacgactct ggagaaacca tgagggtggc catcttcgca 1020 It should be noted that there seems to be a typo in the original text where "actgaggcac ctacaaccat gacatacccc ttgaaagcaa catctacagt gaagcagtcc 780" is mislabeled as "actgaggcac ctacaaccat gacatacccc ttgaaagcaa catctacagt gaagcagtcc 780" in the translation. It should be "actgaggcac ctacaaccat gacatacccc ttgaaagcaa catctacagt gaagcagtcc 780" without the "!" in the translation of ID=16. Also, the "agcaccctgg agatggatga ccggagccac tacacgtgtg aagtcacctg gcagactcct 360" part in the translation of ID=0 should be "agcaccctgg agatggatga ccggagccac tacacgtgtg aagtcacctg gcagactcct 360" without the "!" at the end. These are likely just display or input errors in the provided text for translation.agtggctgct ccagtgatga gccaacttcc cagaatctgg gcaacaacta ctctgatgag 1080 ccctgcatag gacaggagta ccagatcatc gcccagatca atggcaacta cgcccgcctg 1140 ctggacacag ttcctctgga ttatgagttt ctggccactg agggcaaaag tgtctgttaa 1200 <210> 2 <211> 399 <212> PRT <213> Homo sapiens <400> 2 Met Gly Ile Leu Leu Gly Leu Leu Leu Leu Gly His Leu Thr Val Asp 1 5 10 15 Thr Tyr Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro 20 25 30 Trp Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly 35 40 45 Tyr Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro 50 55 60 Val Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala 65 70 75 80 Lys Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val 85 90 95 Ser Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr 100 105 110 Cys Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp 115 120 125 Lys Ile Thr Glu Leu Arg Val Gln Lys Leu Ser Val Ser Lys Pro Thr 130 135 140 Val Thr Thr Gly Ser Gly Tyr Gly Phe Thr Val Pro Gln Gly Met Arg 145 150 155 160 Ile Ser Leu Gln Cys Gln Ala Arg Gly Ser Pro Pro Ile Ser Tyr Ile 165 170 175 Trp Tyr Lys Gln Gln Thr Asn Asn Gln Glu Pro Ile Lys Val Ala Thr 180 185 190 Leu Ser Thr Leu Leu Phe Lys Pro Ala Val Ile Ala Asp Ser Gly Ser 195 200 205 Tyr Phe Cys Thr Ala Lys Gly Gln Val Gly Ser Glu Gln His Ser Asp 210 215 220 Ile Val Lys Phe Val Val Lys Asp Ser Ser Lys Leu Leu Lys Thr Lys 225 230 235 240 Thr Glu Ala Pro Thr Thr Met Thr Tyr Pro Leu Lys Ala Thr Ser Thr 245 250 255 Val Lys Gln Ser Trp Asp Trp Thr Thr Asp Met Asp Gly Tyr Leu Gly 260 265 270 Glu Thr Ser Ala Gly Pro Gly Lys Ser Leu Pro Val Phe Ala Ile Ile 275 280 285 Leu Ile Ile Ser Leu Cys Cys Met Val Val Phe Thr Met Ala Tyr Ile 290 295 300 Met Leu Cys Arg Lys Thr Ser Gln Gln Glu His Val Tyr Glu Ala Ala 305 310 315 320 Arg Ala His Ala Arg Glu Ala Asn Asp Ser Gly Glu Thr Met Arg Val 325 330 335 Ala Ile Phe Ala Ser Gly Cys Ser Ser Asp Glu Pro Thr Ser Gln Asn 340 345 350 Leu Gly Asn Asn Tyr Ser Asp Glu Pro Cys Ile Gly Gln Glu Tyr Gln 355 360 365 Ile Ile Ala Gln Ile Asn Gly Asn Tyr Ala Arg Leu Leu Asp Thr Val 370 375 380 Pro Leu Asp Tyr Glu Phe Leu Ala Thr Glu Gly Lys Ser Val Cys 385 390 395 <210> 3 <211> 3696 <212> DNA <213> Homo sapiens <400> 3 atgagacttc tagcaaagat tatttgcctt atgttatggg ctatttgtgt agcagaagat 60 tgcaatgaac ttcctccaag aagaaataca gaaattctga caggttcctg gtctgaccaa 120 acatatccag aaggcaccca ggctatctat aaatgccgcc ctggatatag atctcttgga 180 aatgtaataa tggtatgcag gaagggagaa tgggttgctc ttaatccatt aaggaaatgt 240 cagaaaaggc cctgtggaca tcctggagat actccttttg gtacttttac ccttacagga 300 ggaaatgtgt ttgaatatgg tgtaaaagct gtgtatacat gtaatgaggg gtatcaattg 360 ctaggtgaga ttaattaccg tgaatgtgac acagatggat ggaccaatga tattcctata 420 tgtgaagttg tgaagtgttt accagtgaca gcaccagaga atggaaaaat tgtcagtagt 480 gcaatggaac cagatcggga ataccatttt ggacaagcag tacggtttgt atgtaactca 540 ggctacaaga ttgaaggaga tgaagaaatg cattgttcag acgatggttt ttggagtaaa 600 gagaaaccaa agtgtgtgga aatttcatgc aaatccccag atgttataaa tggatctcct 660 atatctcaga agattattta taaggagaat gaacgatttc aatataaatg taacatgggt 720 tatgaataca gtgaaagagg agatgctgta tgcactgaat ctggatggcg tccgttgcct 780 tatgaataca gtgaaagagg agatgctgta tgcactgaat ctggatggcg tccgttgcct 780 tcatgtgaag aaaaatcatg tgataatcct tatattccaa atggtgacta ctcaccttta 840 tcatgtgaag aaaaatcatg tgataatcct tatattccaa atggtgacta ctcaccttta 840 aggattaaac acagaactgg agatgaaatc acgtaccagt gtagaaatgg tttttatcct 900 aggattaaac acagaactgg agatgaaatc acgtaccagt gtagaaatgg tttttatcct 900 gcaacccggg gaaatacagc aaaatgcaca agtactggct ggatacctgc tccgagatgt 960 gcaacccggg gaaatacagc aaaatgcaca agtactggct ggatacctgc tccgagatgt 960 accttgaaac cttgtgatta tccagacatt aaacatggag gtctatatca tgagaatatg 1020 accttgaaac cttgtgatta tccagacatt aaacatggag gtctatatca tgagaatatg 1020 cgtagaccat actttccagt agctgtagga aaatattact cctattactg tgatgaacat 1080 cgtagaccat actttccagt agctgtagga aaatattact cctattactg tgatgaacat 1080 tttgagactc cgtcaggaag ttactgggat cacattcatt gcacacaaga tggatggtcg 1140 tttgagactc cgtcaggaag ttactgggat cacattcatt gcacacaaga tggatggtcg 1140 ccagcagtac catgcctcag aaaatgttat tttccttatt tggaaaatgg atataatcaa 1200 ccagcagtac catgcctcag aaaatgttat tttccttatt tggaaaatgg atataatcaa 1200 aatcatggaa gaaagtttgt acagggtaaa tctatagacg ttgcctgcca tcctggctac 1260 aatcatggaa gaaagtttgt acagggtaaa tctatagacg ttgcctgcca tcctggctac 1260 gctcttccaa aagcgcagac cacagttaca tgtatggaga atggctggtc tcctactccc 1320 gctcttccaa aagcgcagac cacagttaca tgtatggaga atggctggtc tcctactccc 1320 agatgcatcc gtgtcaaaac atgttccaaa tcaagtatag atattgagaa tgggtttatt 1380 agatgcatcc gtgtcaaaac atgttccaaa tcaagtatag atattgagaa tgggtttatt 1380 tctgaatctc agtatacata tgccttaaaa gaaaaagcga aatatcaatg caaactagga 1440 tctgaatctc agtatacata tgccttaaaa gaaaaagcga aatatcaatg caaactagga 1440 tatgtaacag cagatggtga aacatcagga tcaattacat gtgggaaaga tggatggtca 1500 gctcaaccca cgtgcattaa atcttgtgat atcccagtat ttatgaatgc cagaactaaa 1560 aatgacttca catggtttaa gctgaatgac acattggact atgaatgcca tgatggttat 1620 gaaagcaata ctggaagcac cactggttcc atagtgtgtg gttacaatgg ttggtctgat 1680 ttacccatat gttatgaaag agaatgcgaa cttcctaaaa tagatgtaca cttagttcct 1740 gatcgcaaga aagaccagta taaagttgga gaggtgttga aattctcctg caaaccagga 1800 tttacaatag ttggacctaa ttccgttcag tgctaccact ttggattgtc tcctgacctc 1860 ccaatatgta aagagcaagt acaatcatgt ggtccacctc ctgaactcct caatgggaat 1920 gttaaggaaa aaacgaaaga agaatatgga cacagtgaag tggtggaata ttattgcaat 1980 cctagatttc taatgaaggg acctaataaa attcaatgtg ttgatggaga gtggacaact 2040 ttaccagtgt gtattgtgga ggagagtacc tgtggagata tacctgaact tgaacatggc 2100 tgggcccagc tttcttcccc tccttattac tatggagatt cagtggaatt caattgctca 2160 gaatcattta caatgattgg acacagatca attacgtgta ttcatggagt atggacccaa 2220 cttccccagt gtgtggcaat agataaactt aagaagtgca aatcatcaaa tttaattata 2280 cttgaggaac atttaaaaaa caagaaggaa ttcgatcata attctaacat aaggtacaga 2340 tgtagaggaa aagaaggatg gatacacaca gtctgcataa atggaagatg ggatccagaa 2400 gtgaactgct caatggcaca aatacaatta tgcccacctc cacctcagat tcccaattct 2460 cacaatatga caaccacact gaattatcgg gatggagaaa aagtatctgt tctttgccaa 2520 gaaaattatc taattcagga aggagaagaa attacatgca aagatggaag atggcagtca 2580 ataccactct gtgttgaaaa aattccatgt tcacaaccac ctcagataga acacggaacc 2640 attaattcat ccaggtcttc acaagaaagt tatgcacatg ggactaaatt gagttatact 2700 tgtgagggtg gtttcaggat atctgaagaa aatgaaacaa catgctacat gggaaaatgg 2760 agttctccac ctcagtgtga aggccttcct tgtaaatctc cacctgagat ttctcatggt 2820 gttgtagctc acatgtcaga cagttatcag tatggagaag aagttacgta caaatgtttt 2880 gaaggttttg gaattgatgg gcctgcaatt gcaaaatgct taggagaaaa atggtctcac 2940 cctccatcat gcataaaaac agattgtctc agtttaccta gctttgaaaa tgccataccc 3000 atgggagaga agaaggatgt gtataaggcg ggtgagcaag tgacttacac ttgtgcaaca 3060 tattacaaaa tggatggagc cagtaatgta acatgcatta atagcagatg gacaggaagg 3120 ccaacatgca gagacacctc ctgtgtgaat ccgcccacag tacaaaatgc ttatatagtg 3180 tcgagacaga tgagtaaata tccatctggt gagagagtac gttatcaatg taggagccct 3240 tatgaaatgt ttggggatga agaagtgatg tgtttaaatg gaaactggac ggaaccacct 3300 caatgcaaag attctacagg aaaatgtggg ccccctccac ctattgacaa tggggacatt 3360 acttcattcc cgttgtcagt atatgctcca gcttcatcag ttgagtacca atgccagaac 3420 ttgtatcaac ttgagggtaa caagcgaata acatgtagaa atggacaatg gtcagaacca 3480 ccaaaatgct tacatccgtg tgtaatatcc cgagaaatta tggaaaatta taacatagca 3540 ttaaggtgga cagccaaaca gaagctttat tcgagaacag gtgaatcagt tgaatttgtg 3600 tgtaaacggg gatatcgtct ttcatcacgt tctcacacat tgcgaacaac atgttgggat 3660 gggaaactgg agtatccaac ttgtgcaaaa agatag 3696 <210> 4 <211> 1231 <212> PRT <213> Homo sapiens <400> 4 Met Arg Leu Leu Ala Lys Ile Ile Cys Leu Met Leu Trp Ala Ile Cys 1 5 10 15 Val Ala Glu Asp Cys Asn Glu Leu Pro Pro Arg Arg Asn Thr Glu Ile 20 25 30 Leu Thr Gly Ser Trp Ser Asp Gln Thr Tyr Pro Glu Gly Thr Gln Ala 35 40 45 Ile Tyr Lys Cys Arg Pro Gly Tyr Arg Ser Leu Gly Asn Val Ile Met 50 55 60 Val Cys Arg Lys Gly Glu Trp Val Ala Leu Asn Pro Leu Arg Lys Cys 65 70 75 80 Gln Lys Arg Pro Cys Gly His Pro Gly Asp Thr Pro Phe Gly Thr Phe 85 90 95 Thr Leu Thr Gly Gly Asn Val Phe Glu Tyr Gly Val Lys Ala Val Tyr 100 105 110 Thr Cys Asn Glu Gly Tyr Gln Leu Leu Gly Glu Ile Asn Tyr Arg Glu 115 120 125 Cys Asp Thr Asp Gly Trp Thr Asn Asp Ile Pro Ile Cys Glu Val Val 130 135 140 Lys Cys Leu Pro Val Thr Ala Pro Glu Asn Gly Lys Ile Val Ser Ser 145 150 155 160 Ala Met Glu Pro Asp Arg Glu Tyr His Phe Gly Gln Ala Val Arg Phe 165 170 175 Val Cys Asn Ser Gly Tyr Lys Ile Glu Gly Asp Glu Glu Met His Cys 180 185 190 Ser Asp Asp Gly Phe Trp Ser Lys Glu Lys Pro Lys Cys Val Glu Ile 195 200 205 Ser Cys Lys Ser Pro Asp Val Ile Asn Gly Ser Pro Ile Ser Gln Lys 210 215 220 Ile Ile Tyr Lys Glu Asn Glu Arg Phe Gln Tyr Lys Cys Asn Met Gly 225 230 235 240 Tyr Glu Tyr Ser Glu Arg Gly Asp Ala Val Cys Thr Glu Ser Gly Trp 245 250 255 Arg Pro Leu Pro Ser Cys Glu Glu Lys Ser Cys Asp Asn Pro Tyr Ile 260 265 270 Pro Asn Gly Asp Tyr Ser Pro Leu Arg Ile Lys His Arg Thr Gly Asp 275 280 285 Glu Ile Thr Tyr Gln Cys Arg Asn Gly Phe Tyr Pro Ala Thr Arg Gly 290 295 300 Asn Thr Ala Lys Cys Thr Ser Thr Gly Trp Ile Pro Ala Pro Arg Cys 305 310 315 320 Thr Leu Lys Pro Cys Asp Tyr Pro Asp Ile Lys His Gly Gly Leu Tyr 325 330 335 His Glu Asn Met Arg Arg Pro Tyr Phe Pro Val Ala Val Gly Lys Tyr 340 345 350 Tyr Ser Tyr Tyr Cys Asp Glu His Phe Glu Thr Pro Ser Gly Ser Tyr 355 360 365 Trp Asp His Ile His Cys Thr Gln Asp Gly Trp Ser Pro Ala Val Pro 370 375 380 Cys Leu Arg Lys Cys Tyr Phe Pro Tyr Leu Glu Asn Gly Tyr Asn Gln 385 390 395 400 Asn His Gly Arg Lys Phe Val Gln Gly Lys Ser Ile Asp Val Ala Cys 405 410 415 His Pro Gly Tyr Ala Leu Pro Lys Ala Gln Thr Thr Val Thr Cys Met 420 425 430 Glu Asn Gly Trp Ser Pro Thr Pro Arg Cys Ile Arg Val Lys Thr Cys 435 440 445 Ser Lys Ser Ser Ile Asp Ile Glu Asn Gly Phe Ile Ser Glu Ser Gln 450 455 460 Tyr Thr Tyr Ala Leu Lys Glu Lys Ala Lys Tyr Gln Cys Lys Leu Gly 465 470 475 480 Tyr Val Thr Ala Asp Gly Glu Thr Ser Gly Ser Ile Thr Cys Gly Lys 485 490 495 Asp Gly Trp Ser Ala Gln Pro Thr Cys Ile Lys Ser Cys Asp Ile Pro 500 505 510 Val Phe Met Asn Ala Arg Thr Lys Asn Asp Phe Thr Trp Phe Lys Leu 515 520 525 Asn Asp Thr Leu Asp Tyr Glu Cys His Asp Gly Tyr Glu Ser Asn Thr 530 535 540 Gly Ser Thr Thr Gly Ser Ile Val Cys Gly Tyr Asn Gly Trp Ser Asp 545 550 555 560 Leu Pro Ile Cys Tyr Glu Arg Glu Cys Glu Leu Pro Lys Ile Asp Val 565 570 575 His Leu Val Pro Asp Arg Lys Lys Asp Gln Tyr Lys Val Gly Glu Val 580 585 590 Leu Lys Phe Ser Cys Lys Pro Gly Phe Thr Ile Val Gly Pro Asn Ser 595 600 605 Val Gln Cys Tyr His Phe Gly Leu Ser Pro Asp Leu Pro Ile Cys Lys 610 615 620 Glu Gln Val Gln Ser Cys Gly Pro Pro Pro Glu Leu Leu Asn Gly Asn 625 630 635 640 Val Lys Glu Lys Thr Lys Glu Glu Tyr Gly His Ser Glu Val Val Glu 645 650 655 Tyr Tyr Cys Asn Pro Arg Phe Leu Met Lys Gly Pro Asn Lys Ile Gln 660 665 670 Cys Val Asp Gly Glu Trp Thr Thr Leu Pro Val Cys Ile Val Glu Glu 675 680 685 Ser Thr Cys Gly Asp Ile Pro Glu Leu Glu His Gly Trp Ala Gln Leu 690 695 700 Ser Ser Pro Pro Tyr Tyr Tyr Gly Asp Ser Val Glu Phe Asn Cys Ser 705 710 715 720 Glu Ser Phe Thr Met Ile Gly His Arg Ser Ile Thr Cys Ile His Gly 725 730 735 Val Trp Thr Gln Leu Pro Gln Cys Val Ala Ile Asp Lys Leu Lys Lys 740 745 750 Cys Lys Ser Ser Asn Leu Ile Ile Leu Glu Glu His Leu Lys Asn Lys 755 760 765 Lys Glu Phe Asp His Asn Ser Asn Ile Arg Tyr Arg Cys Arg Gly Lys 770 775 780 Glu Gly Trp Ile His Thr Val Cys Ile Asn Gly Arg Trp Asp Pro Glu 785 790 795 800 Val Asn Cys Ser Met Ala Gln Ile Gln Leu Cys Pro Pro Pro Pro Gln 805 810 815 Ile Pro Asn Ser His Asn Met Thr Thr Thr Leu Asn Tyr Arg Asp Gly 820 825 830 Glu Lys Val Ser Val Leu Cys Gln Glu Asn Tyr Leu Ile Gln Glu Gly 835 840 845 Glu Glu Ile Thr Cys Lys Asp Gly Arg Trp Gln Ser Ile Pro Leu Cys Val Glu Lys Ile Pro Cys Ser Gln Pro Pro Gln Ile Glu His Gly Thr 865 870 875 880 Ile Asn Ser Ser Arg Ser Ser Gln Glu Ser Tyr Ala His Gly Thr Lys 885 890 895​ Leu Ser Tyr Thr Cys Glu Gly Gly Phe Arg Ile Ser Glu Glu Asn Glu 900 905 910 Thr Thr Cys Tyr Met Gly Lys Trp Ser Ser Pro Pro Gln Cys Glu Gly 915 920 925 Leu Pro Cys Lys Ser Pro Pro Glu Ile Ser His Gly Val Val Ala His 930 935 940 Met Ser Asp Ser Tyr Gln Tyr Gly Glu Glu Val Thr Tyr Lys Cys Phe 945 950 955 960 Glu Gly Phe Gly Ile Asp Gly Pro Ala Ile Ala Lys Cys Leu Gly Glu 965 970 975 Lys Trp Ser His Pro Pro Ser Cys Ile Lys Thr Asp Cys Leu Ser Leu 980 985 990 Pro Ser Phe Glu Asn Ala Ile Pro Met Gly Glu Lys Lys Asp Val Tyr 995 1000 1005 Lys Ala Gly Glu Gln Val Thr Tyr Thr Cys Ala Thr Tyr Tyr Lys Met 1010 1015 1020 Asp Gly Ala Ser Asn Val Thr Cys Ile Asn Ser Arg Trp Thr Gly Arg 1025 1030 1035 1040 Pro Thr Cys Arg Asp Thr Ser Cys Val Asn Pro Pro Thr Val Gln Asn 1045 1050 1055 Ala Tyr Ile Val Ser Arg Gln Met Ser Lys Tyr Pro Ser Gly Glu Arg 1060 1065 1070 Val Arg Tyr Gln Cys Arg Ser Pro Tyr Glu Met Phe Gly Asp Glu Glu 1075 1080 1085 Val Met Cys Leu Asn Gly Asn Trp Thr Glu Pro Pro Gln Cys Lys Asp 1090 1095 1100 Ser Thr Gly Lys Cys Gly Pro Pro Pro Pro Ile Asp Asn Gly Asp Ile 1105 1110 1115 1120 Thr Ser Phe Pro Leu Ser Val Tyr Ala Pro Ala Ser Ser Val Glu Tyr 1125 1130 1135 Gln Cys Gln Asn Leu Tyr Gln Leu Glu Gly Asn Lys Arg Ile Thr Cys 1140 1145 1150 Arg Asn Gly Gln Trp Ser Glu Pro Pro Lys Cys Leu His Pro Cys Val 1155 1160 1165 Ile Ser Arg Glu Ile Met Glu Asn Tyr Asn Ile Ala Leu Arg Trp Thr 1170 1175 1180 Ala Lys Gln Lys Leu Tyr Ser Arg Thr Gly Glu Ser Val Glu Phe Val 1185 1190 1195 1200 Cys Lys Arg Gly Tyr Arg Leu Ser Ser Arg Ser His Thr Leu Arg Thr 1205 1210 1215 Thr Cys Trp Asp Gly Lys Leu Glu Tyr Pro Thr Cys Ala Lys Arg 1220 1225 1230 <210> 5 <211> 357 <212> DNA <213> Homo sapiens <400> 5 ggccgtccca tcctggaagt gccagagagt gtaacaggac cttggaaagg ggatgtgaat 60 cttccctgca cctatgaccc cctgcaaggc tacacccaag tcttggtgaa gtggctggta 120 caacgtggct cagaccctgt caccatcttt ctacgtgact cttctggaga ccatatccag 180 caggcaaagt accagggccg cctgcatgtg agccacaagg ttccaggaga tgtatccctc 240 caattgagca ccctggagat ggatgaccgg agccactaca cgtgtgaagt cacctggcag 300 actcctgatg gcaaccaagt cgtgagagat aagattactg agctccgtgt ccagaaa 357 <210> 6 <211> 119 <212> PRT <213> Homo sapiens <400> 6 Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp Lys 1 5 10 15 Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr Thr 20 25 30 Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val Thr 35 40 45 Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys Tyr 50 55 60 Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser Leu 65 70 75 80 Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys Glu 85 90 95 Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys Ile 100 105 110 Thr Glu Leu Arg Val Gln Lys 115 <210> 7 <211> 915 <212> DNA <213> Homo sapiens <400> 7 gaagattgca atgaacttcc tccaagaaga aatacagaaa ttctgacagg ttcctggtct 60 gaccaaacat atccagaagg cacccaggct atctataaat gccgccctgg atatagatct 120 cttggaaatg taataatggt atgcaggaag ggagaatggg ttgctcttaa tccattaagg 180 aaatgtcaga aaaggccctg tggacatcct ggagatactc cttttggtac ttttaccctt 240 acaggaggaa atgtgtttga atatggtgta aaagctgtgt atacatgtaa tgaggggtat 300 caattgctag gtgagattaa ttaccgtgaa tgtgacacag atggatggac caatgatatt 360 cctatatgtg aagttgtgaa gtgtttacca gtgacagcac cagagaatgg aaaaattgtc 420 agtagtgcaa tggaaccaga tcgggaatac cattttggac aagcagtacg gtttgtatgt 480 aactcaggct acaagattga aggagatgaa gaaatgcatt gttcagacga tggtttttgg 540 agtaaagaga aaccaaagtg tgtggaaatt tcatgcaaat ccccagatgt tataaatgga 600 tctcctatat ctcagaagat tatttataag gagaatgaac gatttcaata taaatgtaac 660 atgggttatg aatacagtga aagaggagat gctgtatgca ctgaatctgg atggcgtccg 720 ttgccttcat gtgaagaaaa atcatgtgat aatccttata ttccaaatgg tgactactca 780 cctttaagga ttaaacacag aactggagat gaaatcacgt accagtgtag aaatggtttt 840 tatcctgcaa cccggggaaa tacagcaaaa tgcacaagta ctggctggat acctgctccg 900 agatgtacct tgaaa 915 <210> 8 <211> 305 <212> PRT <213> Homo sapiens <400> 8 Glu Asp Cys Asn Glu Leu Pro Pro Arg Arg Asn Thr Glu Ile Leu Thr 1 5 10 15 Gly Ser Trp Ser Asp Gln Thr Tyr Pro Glu Gly Thr Gln Ala Ile Tyr 20 25 30 Lys Cys Arg Pro Gly Tyr Arg Ser Leu Gly Asn Val Ile Met Val Cys 35 40 45 Arg Lys Gly Glu Trp Val Ala Leu Asn Pro Leu Arg Lys Cys Gln Lys 50 55 60 Arg Pro Cys Gly His Pro Gly Asp Thr Pro Phe Gly Thr Phe Thr Leu 65 70 75 80 Thr Gly Gly Asn Val Phe Glu Tyr Gly Val Lys Ala Val Tyr Thr Cys 85 90 95 Asn Glu Gly Tyr Gln Leu Leu Gly Glu Ile Asn Tyr Arg Glu Cys Asp 100 105 110 Thr Asp Gly Trp Thr Asn Asp Ile Pro Ile Cys Glu Val Val Lys Cys 115 120 125 Leu Pro Val Thr Ala Pro Glu Asn Gly Lys Ile Val Ser Ser Ala Met 130 135 140 Glu Pro Asp Arg Glu Tyr His Phe Gly Gln Ala Val Arg Phe Val Cys 145 150 155 160 Asn Ser Gly Tyr Lys Ile Glu Gly Asp Glu Glu Met His Cys Ser Asp 165 170 175 Asp Gly Phe Trp Ser Lys Glu Lys Pro Lys Cys Val Glu Ile Ser Cys 180 185 190 Lys Ser Pro Asp Val Ile Asn Gly Ser Pro Ile Ser Gln Lys Ile Ile 195 200 205 Tyr Lys Glu Asn Glu Arg Phe Gln Tyr Lys Cys Asn Met Gly Tyr Glu 210 215 220 Tyr Ser Glu Arg Gly Asp Ala Val Cys Thr Glu Ser Gly Trp Arg Pro 225 230 235 240 Leu Pro Ser Cys Glu Glu Lys Ser Cys Asp Asn Pro Tyr Ile Pro Asn 245 250 255 Gly Asp Tyr Ser Pro Leu Arg Ile Lys His Arg Thr Gly Asp Glu Ile 260 265 270 Thr Tyr Gln Cys Arg Asn Gly Phe Tyr Pro Ala Thr Arg Gly Asn Thr 275 280 285 Ala Lys Cys Thr Ser Thr Gly Trp Ile Pro Ala Pro Arg Cys Thr Leu 290 295 300 Lys 305 <210> 9 <211> 675 <212> DNA <213> Homo sapiens <400> 9 cccccatgcc catcatgccc agcacctgag ttcctggggg gaccatcagt cttcctgttc 60 cccccaaaac ccaaggacac tctcatgatc tcccggaccc ctgaggtcac gtgcgtggtg 120 gtggacgtga gccaggaaga ccccgaggtc cagttcaact ggtacgtgga tggcgtggag 180 gtgcataatg ccaagacaaa gccgcgggag gagcagttca acagcacgta ccgtgtggtc 240 agcgtcctca ccgtcctgca ccaggactgg ctgaacggca aggagtacaa gtgcaaggtc 300 tccaacaaag gcctcccgtc ctccatcgag aaaaccatct ccaaagccaa agggcagccc 360 cgagagccac aggtgtacac cctgccccca tcccaggagg agatgaccaa gaaccaggtc 420 agcctgacct gcctggtcaa aggcttctac cccagcgaca tcgccgtgga gtgggagagc 480 aatgggcagc cggagaacaa ctacaagacc acgcctcccg tgctggactc cgacggctcc 540 ttcttcctct acagcaggct aaccgtggac aagagcaggt ggcaggaggg gaatgtcttc 600 tcatgctccg tgatgcatga ggctctgcac aaccactaca cacagaagag cctctccctg 660 tctctgggta aataa 675 <210> 10 <211> 224 <212> PRT <213> Homo sapiens <400> 10 Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser 1 5 10 15 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 20 25 30 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 35 40 45 Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 50 55 60 Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 65 70 75 80 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 85 90 95 Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr 100 105 110 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 115 120 125 Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 130 135 140 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 145 150 155 160 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 165 170 175 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 180 185 190 Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 195 200 205 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 210 215 220 <210> 11 <211> 1758 <212> DNA <213> Homo sapiens <400> 11 gatgcacaca agagtgaggt tgctcatcgg tttaaagatt tgggagaaga aaatttcaaa 60 gccttggtgt tgattgcctt tgctcagtat cttcagcagt gtccatttga agatcatgta 120 aaattagtga atgaagtaac tgaatttgca aaaacatgtg ttgctgatga gtcagctgaa 180 aattgtgaca aatcacttca tacccttttt ggagacaaat tatgcacagt tgcaactctt 240 cgtgaaacct atggtgaaat ggctgactgc tgtgcaaaac aagaacctga gagaaatgaa 300 tgcttcttgc aacacaaaga tgacaaccca aacctccccc gattggtgag accagaggtt 360 gatgtgatgt gcactgcttt tcatgacaat gaagagacat ttttgaaaaa atacttatat 420 gaaattgcca gaagacatcc ttacttttat gccccggaac tccttttctt tgctaaaagg 480 tataaagctg cttttacaga atgttgccaa gctgctgata aagctgcctg cctgttgcca 540 aagctcgatg aacttcggga tgaagggaag gcttcgtctg ccaaacagag actcaagtgt 600 gccagtctcc aaaaatttgg agaaagagct ttcaaagcat gggcagtagc tcgcctgagc 660 cagagatttc ccaaagctga gtttgcagaa gtttccaagt tagtgacaga tcttaccaaa 720 gtccacacgg aatgctgcca tggagatctg cttgaatgtg ctgatgacag ggcggacctt 780 gccaagtata tctgtgaaaa tcaagattcg atctccagta aactgaagga atgctgtgaa 840 aaacctctgt tggaaaaatc ccactgcatt gccgaagtgg aaaatgatga gatgcctgct 900 gacttgcctt cattagctgc tgattttgtt gaaagtaagg atgtttgcaa aaactatgct 960 gaggcaaagg atgtcttcct gggcatgttt ttgtatgaat atgcaagaag gcatcctgat 1020 tactctgtcg tgctgctgct gagacttgcc aagacatatg aaaccactct agagaagtgc 1080 tgtgccgctg cagatcctca tgaatgctat gccaaagtgt tcgatgaatt taaacctctt 1140 gtggaagagc ctcagaattt aatcaaacaa aattgtgagc tttttgagca gcttggagag 1200 tacaaattcc agaatgcgct attagttcgt tacaccaaga aagtacccca agtgtcaact 1260 ccaactcttg tagaggtctc aagaaaccta ggaaaagtgg gcagcaaatg ttgtaaacat 1320 cctgaagcaa aaagaatgcc ctgtgcagaa gactatctat ccgtggtcct gaaccagtta 1380 tgtgtgttgc atgagaaaac gccagtaagt gacagagtca ccaaatgctg cacagaatcc 1440 ttggtgaaca ggcgaccatg cttttcagct ctggaagtcg atgaaacata cgttcccaaa 1500 gagtttaatg ctgaaacatt caccttccat gcagatatat gcacactttc tgagaaggag 1560 agacaaatca agaaacaaac tgcacttgtt gagctcgtga aacacaagcc caaggcaaca 1620 aaagagcaac tgaaagctgt tatggatgat ttcgcagctt ttgtagagaa gtgctgcaag 1680 gctgacgata aggagacctg ctttgccgag gagggtaaaa aacttgttgc tgcaagtcaa 1740 gctgccttag gcttataa 1758 <210> 12 <211> 585 <212> PRT <213> Homo sapiens <400> 12 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro 210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His 450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 <210> 13 <211> 1950 <212> DNA <213> Artificial Sequence <220> <223> CRIg-FH-IgG4 Fc nucleic acid sequence <400> 13 atgggccgtc ccatcctgga agtgccagag agtgtaacag gaccttggaa aggggatgtg 60 aatcttccct gcacctatga ccccctgcaa ggctacaccc aagtcttggt gaagtggctg 120 gtacaacgtg gctcagaccc tgtcaccatc tttctacgtg actcttctgg agaccatatc 180 cagcaggcaa agtaccaggg ccgcctgcat gtgagccaca aggttccagg agatgtatcc 240 ctccaattga gcaccctgga gatggatgac cggagccact acacgtgtga agtcacctgg 300 cagactcctg atggcaacca agtcgtgaga gataagatta ctgagctccg tgtccagaaa 360 gaagattgca atgaacttcc tccaagaaga aatacagaaa ttctgacagg ttcctggtct 420 gaccaaacat atccagaagg cacccaggct atctataaat gccgccctgg atatagatct 480 cttggaaatg taataatggt atgcaggaag ggagaatggg ttgctcttaa tccattaagg 540 aaatgtcaga aaaggccctg tggacatcct ggagatactc cttttggtac ttttaccctt 600 acaggaggaa atgtgtttga atatggtgta aaagctgtgt atacatgtaa tgaggggtat 660 caattgctag gtgagattaa ttaccgtgaa tgtgacacag atggatggac caatgatatt 720 cctatatgtg aagttgtgaa gtgtttacca gtgacagcac cagagaatgg aaaaattgtc 780 agtagtgcaa tggaaccaga tcgggaatac cattttggac aagcagtacg gtttgtatgt 840 aactcaggct acaagattga aggagatgaa gaaatgcatt gttcagacga tggtttttgg 900 agtaaagaga aaccaaagtg tgtggaaatt tcatgcaaat ccccagatgt tataaatgga 960 tctcctatat ctcagaagat tatttataag gagaatgaac gatttcaata taaatgtaac 1020 atgggttatg aatacagtga aagaggagat gctgtatgca ctgaatctgg atggcgtccg 1080 ttgccttcat gtgaagaaaa atcatgtgat aatccttata ttccaaatgg tgactactca 1140 cctttaagga ttaaacacag aactggagat gaaatcacgt accagtgtag aaatggtttt 1200 tatcctgcaa cccggggaaa tacagcaaaa tgcacaagta ctggctggat acctgctccg 1260 agatgtacct tgaaaccccc atgcccatca tgcccagcac ctgagttcct ggggggacca 1320 tcagtcttcc tgttcccccc aaaacccaag gacactctca tgatctcccg gacccctgag 1380 gtcacgtgcg tggtggtgga cgtgagccag gaagaccccg aggtccagtt caactggtac 1440 gtggatggcg tggaggtgca taatgccaag acaaagccgc gggaggagca gttcaacagc 1500 acgtaccgtg tggtcagcgt cctcaccgtc ctgcaccagg actggctgaa cggcaaggag 1560 tacaagtgca aggtctccaa caaaggcctc ccgtcctcca tcgagaaaac catctccaaa 1620 gccaaagggc agccccgaga gccacaggtg tacaccctgc ccccatccca ggaggagatg 1680 accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctaccccag cgacatcgcc 1740 gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 1800 gactccgacg gctccttctt cctctacagc aggctaaccg tggacaagag caggtggcag 1860 gaggggaatg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacacag 1920 aagagcctct ccctgtctct gggtaaataa 1950 <210> 14 <211> 649 <212> PRT <213> Artificial Sequence <220> <223> CRIg-FH-IgG4 Fc protein sequence <400> 14 Met Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp 1 5 10 15 Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr 20 25 30 Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val 35 40 45 Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys 50 55 60 Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser 65 70 75 80 Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys 85 90 95 Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys 100 105 110 Ile Thr Glu Leu Arg Val Gln Lys Glu Asp Cys Asn Glu Leu Pro Pro 115 120 125 Arg Arg Asn Thr Glu Ile Leu Thr Gly Ser Trp Ser Asp Gln Thr Tyr 130 135 140 Pro Glu Gly Thr Gln Ala Ile Tyr Lys Cys Arg Pro Gly Tyr Arg Ser 145 150 155 160 Leu Gly Asn Val Ile Met Val Cys Arg Lys Gly Glu Trp Val Ala Leu 165 170 175 Asn Pro Leu Arg Lys Cys Gln Lys Arg Pro Cys Gly His Pro Gly Asp 180 185 190 Thr Pro Phe Gly Thr Phe Thr Leu Thr Gly Gly Asn Val Phe Glu Tyr 195 200 205 Gly Val Lys Ala Val Tyr Thr Cys Asn Glu Gly Tyr Gln Leu Leu Gly 210 215 220 Glu Ile Asn Tyr Arg Glu Cys Asp Thr Asp Gly Trp Thr Asn Asp Ile 225 230 235 240 Pro Ile Cys Glu Val Val Lys Cys Leu Pro Val Thr Ala Pro Glu Asn 245 250 255 Gly Lys Ile Val Ser Ser Ala Met Glu Pro Asp Arg Glu Tyr His Phe 260 265 270 Gly Gln Ala Val Arg Phe Val Cys Asn Ser Gly Tyr Lys Ile Glu Gly 275 280 285 Asp Glu Glu Met His Cys Ser Asp Asp Gly Phe Trp Ser Lys Glu Lys 290 295 300 Pro Lys Cys Val Glu Ile Ser Cys Lys Ser Pro Asp Val Ile Asn Gly 305 310 315 320 Ser Pro Ile Ser Gln Lys Ile Ile Tyr Lys Glu Asn Glu Arg Phe Gln 325 330 335 Tyr Lys Cys Asn Met Gly Tyr Glu Tyr Ser Glu Arg Gly Asp Ala Val 340 345 350 Cys Thr Glu Ser Gly Trp Arg Pro Leu Pro Ser Cys Glu Glu Lys Ser 355 360 365 Cys Asp Asn Pro Tyr Ile Pro Asn Gly Asp Tyr Ser Pro Leu Arg Ile 370 375 380 Lys His Arg Thr Gly Asp Glu Ile Thr Tyr Gln Cys Arg Asn Gly Phe 385 390 395 400 Tyr Pro Ala Thr Arg Gly Asn Thr Ala Lys Cys Thr Ser Thr Gly Trp 405 410 415 Ile Pro Ala Pro Arg Cys Thr Leu Lys Pro Pro Cys Pro Ser Cys Pro 420 425 430 Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 435 440 445 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 450 455 460 Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr 465 470 475 480 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 485 490 495 Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 500 505 510 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 515 520 525 Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 530 535 540 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met 545 550 555 560 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 565 570 575 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 580 585 590 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 595 600 605 Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val 610 615 620 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 625 630 635 640 Lys Ser Leu Ser Leu Ser Leu Gly Lys 645 <210> 15 <211> 3030 <212> DNA <213> Artificial Sequence <220> <223> CRIg-FH-HSA nucleic acid sequence <400> 15 ggccgtccca tcctggaagt gccagagagt gtaacaggac cttggaaagg ggatgtgaat 60 cttccctgca cctatgaccc cctgcaaggc tacacccaag tcttggtgaa gtggctggta 120 caacgtggct cagaccctgt caccatcttt ctacgtgact cttctggaga ccatatccag 180 caggcaaagt accagggccg cctgcatgtg agccacaagg ttccaggaga tgtatccctc 240 caattgagca ccctggagat ggatgaccgg agccactaca cgtgtgaagt cacctggcag 300 actcctgatg gcaaccaagt cgtgagagat aagattactg agctccgtgt ccagaaagaa 360 gattgcaatg aacttcctcc aagaagaaat acagaaattc tgacaggttc ctggtctgac 420 caaacatatc cagaaggcac ccaggctatc tataaatgcc gccctggata tagatctctt 480 ggaaatgtaa taatggtatg caggaaggga gaatgggttg ctcttaatcc attaaggaaa 540 tgtcagaaaa ggccctgtgg acatcctgga gatactcctt ttggtacttt tacccttaca 600 ggaggaaatg tgtttgaata tggtgtaaaa gctgtgtata catgtaatga ggggtatcaa 660 ttgctaggtg agattaatta ccgtgaatgt gacacagatg gatggaccaa tgatattcct 720 atatgtgaag ttgtgaagtg tttaccagtg acagcaccag agaatggaaa aattgtcagt 780 agtgcaatgg aaccagatcg ggaataccat tttggacaag cagtacggtt tgtatgtaac 840 tcaggctaca agattgaagg agatgaagaa atgcattgtt cagacgatgg tttttggagt 900 aaagagaaac caaagtgtgt ggaaatttca tgcaaatccc cagatgttat aaatggatct 960 cctatatctc agaagattat ttataaggag aatgaacgat ttcaatataa atgtaacatg 1020 ggttatgaat acagtgaaag aggagatgct gtatgcactg aatctggatg gcgtccgttg 1080 ccttcatgtg aagaaaaatc atgtgataat ccttatattc caaatggtga ctactcacct 1140 ttaaggatta aacacagaac tggagatgaa atcacgtacc agtgtagaaa tggtttttat 1200 cctgcaaccc ggggaaatac agcaaaatgc acaagtactg gctggatacc tgctccgaga 1260 tgtaccttga aagatgcaca caagagtgag gttgctcatc ggtttaaaga tttgggagaa 1320 gaaaatttca aagccttggt gttgattgcc tttgctcagt atcttcagca gtgtccattt 1380 gaagatcatg taaaattagt gaatgaagta actgaatttg caaaaacatg tgttgctgat 1440 gagtcagctg aaaattgtga caaatcactt catacccttt ttggagacaa attatgcaca 1500 gttgcaactc ttcgtgaaac ctatggtgaa atggctgact gctgtgcaaa acaagaacct 1560 gagagaaatg aatgcttctt gcaacacaaa gatgacaacc caaacctccc ccgattggtg 1620 agaccagagg ttgatgtgat gtgcactgct tttcatgaca atgaagagac atttttgaaa 1680 aaatacttat atgaaattgc cagaagacat ccttactttt atgccccgga actccttttc 1740 tttgctaaaa ggtataaagc tgcttttaca gaatgttgcc aagctgctga taaagctgcc 1800 tgcctgttgc caaagctcga tgaacttcgg gatgaaggga aggcttcgtc tgccaaacag 1860 agactcaagt gtgccagtct ccaaaaattt ggagaaagag ctttcaaagc atgggcagta 1920 gctcgcctga gccagagatt tcccaaagct gagtttgcag aagtttccaa gttagtgaca 1980 gatcttacca aagtccacac ggaatgctgc catggagatc tgcttgaatg tgctgatgac 2040 agggcggacc ttgccaagta tatctgtgaa aatcaagatt cgatctccag taaactgaag 2100 gaatgctgtg aaaaacctct gttggaaaaa tcccactgca ttgccgaagt ggaaaatgat 2160 gagatgcctg ctgacttgcc ttcattagct gctgattttg ttgaaagtaa ggatgtttgc 2220 aaaaactatg ctgaggcaaa ggatgtcttc ctgggcatgt ttttgtatga atatgcaaga 2280 aggcatcctg attactctgt cgtgctgctg ctgagacttg ccaagacata tgaaaccact 2340 ctagagaagt gctgtgccgc tgcagatcct catgaatgct atgccaaagt gttcgatgaa 2400 tttaaacctc ttgtggaaga gcctcagaat ttaatcaaac aaaattgtga gctttttgag 2460 cagcttggag agtacaaatt ccagaatgcg ctattagttc gttacaccaa gaaagtaccc 2520 caagtgtcaa ctccaactct tgtagaggtc tcaagaaacc taggaaaagt gggcagcaaa 2580 tgttgtaaac atcctgaagc aaaaagaatg ccctgtgcag aagactatct atccgtggtc 2640 ctgaaccagt tatgtgtgtt gcatgagaaa acgccagtaa gtgacagagt caccaaatgc 2700 tgcacagaat ccttggtgaa caggcgacca tgcttttcag ctctggaagt cgatgaaaca 2760 tacgttccca aagagtttaa tgctgaaaca ttcaccttcc atgcagatat atgcacactt 2820 tctgagaagg agagacaaat caagaaacaa actgcacttg ttgagctcgt gaaacacaag 2880 cccaaggcaa caaaagagca actgaaagct gttatggatg atttcgcagc ttttgtagag 2940 aagtgctgca aggctgacga taaggagacc tgctttgccg aggagggtaa aaaacttgtt 3000 gctgcaagtc aagctgcctt aggcttataa 3030 <210> 16 <211> 1009 <212> PRT <213> Artificial Sequence <220> <223> CRIg-FH-HSA protein sequence <400> 16 Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp Lys 1 5 10 15 Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr Thr 20 25 30 Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val Thr 35 40 45 Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys Tyr 50 55 60 Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser Leu 65 70 75 80 Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys Glu 85 90 95 Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys Ile 100 105 110 Thr Glu Leu Arg Val Gln Lys Glu Asp Cys Asn Glu Leu Pro Pro Arg 115 120 125 Arg Asn Thr Glu Ile Leu Thr Gly Ser Trp Ser Asp Gln Thr Tyr Pro 130 135 140 Glu Gly Thr Gln Ala Ile Tyr Lys Cys Arg Pro Gly Tyr Arg Ser Leu 145 150 155 160 Gly Asn Val Ile Met Val Cys Arg Lys Gly Glu Trp Val Ala Leu Asn 165 170 175 Pro Leu Arg Lys Cys Gln Lys Arg Pro Cys Gly His Pro Gly Asp Thr 180 185 190 Pro Phe Gly Thr Phe Thr Leu Thr Gly Gly Asn Val Phe Glu Tyr Gly 195 200 205 Val Lys Ala Val Tyr Thr Cys Asn Glu Gly Tyr Gln Leu Leu Gly Glu 210 215 220 Ile Asn Tyr Arg Glu Cys Asp Thr Asp Gly Trp Thr Asn Asp Ile Pro 225 230 235 240 Ile Cys Glu Val Val Lys Cys Leu Pro Val Thr Ala Pro Glu Asn Gly 245 250 255 Lys Ile Val Ser Ser Ala Met Glu Pro Asp Arg Glu Tyr His Phe Gly 260 265 270 Gln Ala Val Arg Phe Val Cys Asn Ser Gly Tyr Lys Ile Glu Gly Asp 275 280 285 Glu Glu Met His Cys Ser Asp Asp Gly Phe Trp Ser Lys Glu Lys Pro 290 295 300 Lys Cys Val Glu Ile Ser Cys Lys Ser Pro Asp Val Ile Asn Gly Ser 305 310 315 320 Pro Ile Ser Gln Lys Ile Ile Tyr Lys Glu Asn Glu Arg Phe Gln Tyr 325 330 335 Lys Cys Asn Met Gly Tyr Glu Tyr Ser Glu Arg Gly Asp Ala Val Cys 340 345 350 Thr Glu Ser Gly Trp Arg Pro Leu Pro Ser Cys Glu Glu Lys Ser Cys 355 360 365 Asp Asn Pro Tyr Ile Pro Asn Gly Asp Tyr Ser Pro Leu Arg Ile Lys 370 375 380 His Arg Thr Gly Asp Glu Ile Thr Tyr Gln Cys Arg Asn Gly Phe Tyr 385 390 395 400 Pro Ala Thr Arg Gly Asn Thr Ala Lys Cys Thr Ser Thr Gly Trp Ile 405 410 415 Pro Ala Pro Arg Cys Thr Leu Lys Asp Ala His Lys Ser Glu Val Ala 420 425 430 His Arg Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu 435 440 445 Ile Ala Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val 450 455 460 Lys Leu Val Asn Glu Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp 465 470 475 480 Glu Ser Ala Glu Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp 485 490 495 Lys Leu Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala 500 505 510 Asp Cys Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln 515 520 525 His Lys Asp Asp Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val 530 535 540 Asp Val Met Cys Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys 545 550 555 560 Lys Tyr Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro 565 570 575 Glu Leu Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys 580 585 590 Cys Gln Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu 595 600 605 Leu Arg Asp Glu Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys 610 615 620 Ala Ser Leu Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val 625 630 635 640 Ala Arg Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser 645 650 655 Lys Leu Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly 660 665 670 Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile 675 680 685 Cys Glu Asn Gln Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu 690 695 700 Lys Pro Leu Leu Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp 705 710 715 720 Glu Met Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser 725 730 735 Lys Asp Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly 740 745 750 Met Phe Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val 755 760 765 Leu Leu Leu Arg Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys 770 775 780 Cys Ala Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu 785 790 795 800 Phe Lys Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys 805 810 815 Glu Leu Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu 820 825 830 Val Arg Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val 835 840 845 Glu Val Ser Arg Asn Leu Gly Lys Val Gly Ser Lys Cys Cys Lys His 850 855 860 Pro Glu Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val 865 870 875 880 Leu Asn Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg 885 890 895 Val Thr Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe 900 905 910 Ser Ala Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala 915 920 925 Glu Thr Phe Thr Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu 930 935 940 Arg Gln Ile Lys Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys 945 950 955 960 Pro Lys Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala 965 970 975 Ala Phe Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe 980 985 990 Ala Glu Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly 995 1000 1005 Leu <210> 17 <211> 1041 <212> DNA <213> Artificial Sequence <220> <223> CD55 nucleic acid sequence <400> 17 gactgtggcc ttcccccaga tgtacctaat gcccagccag ctttggaagg ccgtacaagt 60 tttcccgagg atactgtaat aacgtacaaa tgtgaagaaa gctttgtgaa aattcctggc 120 gagaaggact cagtgatctg ccttaagggc agtcaatggt cagatattga agagttctgc 180 aatcgtagct gcgaggtgcc aacaaggcta aattctgcat ccctcaaaca gccttatatc 240 actcagaatt attttccagt cggtactgtt gtggaatatg agtgccgtcc aggttacaga 300 agagaacctt ctctatcacc aaaactaact tgccttcaga atttaaaatg gtccacagca 360 gtcgaatttt gtaaaaagaa atcatgccct aatccgggag aaatacgaaa tggtcagatt 420 gatgtaccag gtggcatatt atttggtgca accatctcct tctcatgtaa cacagggtac 480 aaattatttg gctcgacttc tagtttttgt cttatttcag gcagctctgt ccagtggagt 540 gacccgttgc cagagtgcag agaaatttat tgtccagcac caccacaaat tgacaatgga 600 ataattcaag gggaacgtga ccattatgga tatagacagt ctgtaacgta tgcatgtaat 660 aaaggattca ccatgattgg agagcactct atttattgta ctgtgaataa tgatgaagga 720 gagtggagtg gcccaccacc tgaatgcaga ggaaaatctc taacttccaa ggtcccacca 780 acagttcaga aacctaccac agtaaatgtt ccaactacag aagtctcacc aacttctcag 840 aaaaccacca caaaaaccac cacaccaaat gctcaagcaa cacggagtac acctgtttcc 900 aggacaacca agcattttca tgaaacaacc ccaaataaag gaagtggaac cacttcaggt 960 actacccgtc ttctatctgg gcacacgtgt ttcacgttga caggtttgct tgggacgcta 1020 gtaaccatgg gcttgctgac t 1041 <210> 18 <211> 347 <212> PRT <213> Artificial Sequence <220> <223> CD55 protein sequence <400> 18 Asp Cys Gly Leu Pro Pro Asp Val Pro Asn Ala Gln Pro Ala Leu Glu 1 5 10 15 Gly Arg Thr Ser Phe Pro Glu Asp Thr Val Ile Thr Tyr Lys Cys Glu 20 25 30 Glu Ser Phe Val Lys Ile Pro Gly Glu Lys Asp Ser Val Ile Cys Leu 35 40 45 Lys Gly Ser Gln Trp Ser Asp Ile Glu Glu Phe Cys Asn Arg Ser Cys 50 55 60 Glu Val Pro Thr Arg Leu Asn Ser Ala Ser Leu Lys Gln Pro Tyr Ile 65 70 75 80 Thr Gln Asn Tyr Phe Pro Val Gly Thr Val Val Glu Tyr Glu Cys Arg 85 90 95 Pro Gly Tyr Arg Arg Glu Pro Ser Leu Ser Pro Lys Leu Thr Cys Leu 100 105 110 Gln Asn Leu Lys Trp Ser Thr Ala Val Glu Phe Cys Lys Lys Lys Ser 115 120 125 Cys Pro Asn Pro Gly Glu Ile Arg Asn Gly Gln Ile Asp Val Pro Gly 130 135 140 Gly Ile Leu Phe Gly Ala Thr Ile Ser Phe Ser Cys Asn Thr Gly Tyr 145 150 155 160 Lys Leu Phe Gly Ser Thr Ser Ser Phe Cys Leu Ile Ser Gly Ser Ser 165 170 175 Val Gln Trp Ser Asp Pro Leu Pro Glu Cys Arg Glu Ile Tyr Cys Pro 180 185 190 Ala Pro Pro Gln Ile Asp Asn Gly Ile Ile Gln Gly Glu Arg Asp His 195 200 205 Tyr Gly Tyr Arg Gln Ser Val Thr Tyr Ala Cys Asn Lys Gly Phe Thr 210 215 220 Met Ile Gly Glu His Ser Ile Tyr Cys Thr Val Asn Asn Asp Glu Gly 225 230 235 240 Glu Trp Ser Gly Pro Pro Pro Glu Cys Arg Gly Lys Ser Leu Thr Ser 245 250 255 Lys Val Pro Pro Thr Val Gln Lys Pro Thr Thr Val Asn Val Pro Thr 260 265 270 Thr Glu Val Ser Pro Thr Ser Gln Lys Thr Thr Thr Lys Thr Thr Thr 275 280 285 Pro Asn Ala Gln Ala Thr Arg Ser Thr Pro Val Ser Arg Thr Thr Lys 290 295 300 His Phe His Glu Thr Thr Pro Asn Lys Gly Ser Gly Thr Thr Ser Gly 305 310 315 320 Thr Thr Arg Leu Leu Ser Gly His Thr Cys Phe Thr Leu Thr Gly Leu 325 330 335 Leu Gly Thr Leu Val Thr Met Gly Leu Leu Thr 340 345 <210> 19 <211> 1074 <212> DNA <213> Artificial Sequence <220> <223> CD46 nucleic acid sequence <400> 19 tgtgaggagc caccaacatt tgaagctatg gagctcattg gtaaaccaaa accctactat 60 gagattggtg aacgagtaga ttataagtgt aaaaaaggat acttctatat acctcctctt 120 gccacccata ctatttgtga tcggaatcat acatggctac ctgtctcaga tgacgcctgt 180 tatagagaaa catgtccata tatacgggat cctttaaatg gccaagcagt ccctgcaaat 240 gggacttacg agtttggtta tcagatgcac tttatttgta atgagggtta ttacttaatt 300 ggtgaagaaa ttctatattg tgaacttaaa ggatcagtag caatttggag cggtaagccc 360 ccaatatgtg aaaaggtttt gtgtacacca cctccaaaaa taaaaaatgg aaaacacacc 420 tttagtgaag tagaagtatt tgagtatctt gatgcagtaa cttatagttg tgatcctgca 480 cctggaccag atccattttc acttattgga gagagcacga tttattgtgg tgacaattca 540 gtgtggagtc gtgctgctcc agagtgtaaa gtggtcaaat gtcgatttcc agtagtcgaa 600 aatggaaaac agatatcagg atttggaaaa aaattttact acaaagcaac agttatgttt 660 gaatgcgata agggttttta cctcgatggc agcgacacaa ttgtctgtga cagtaacagt 720 acttgggatc ccccagttcc aaagtgtctt aaagtgctgc ctccatctag tacaaaacct 780 ccagctttga gtcattcagt gtcgacttct tccactacaa aatctccagc gtccagtgcc 840 tcaggtccta ggcctactta caagcctcca gtctcaaatt atccaggata tcctaaacct 900 gaggaaggaa tacttgacag tttggatgtt tgggtcattg ctgtgattgt tattgccata 960 gttgttggag ttgcagtaat ttgtgttgtc ccgtacagat atcttcaaag gaggaagaag 1020 aaaggcacat acctaactga tgagacccac agagaagtaa aatttacttc tctc 1074 <210> 20 <211> 358 <212> PRT <213> Artificial Sequence <220> <223> CD46 protein sequence <400> 20 Cys Glu Glu Pro Pro Thr Phe Glu Ala Met Glu Leu Ile Gly Lys Pro 1 5 10 15 Lys Pro Tyr Tyr Glu Ile Gly Glu Arg Val Asp Tyr Lys Cys Lys Lys 20 25 30 Gly Tyr Phe Tyr Ile Pro Pro Leu Ala Thr His Thr Ile Cys Asp Arg 35 40 45 Asn His Thr Trp Leu Pro Val Ser Asp Asp Ala Cys Tyr Arg Glu Thr 50 55 60 Cys Pro Tyr Ile Arg Asp Pro Leu Asn Gly Gln Ala Val Pro Ala Asn 65 70 75 80 Gly Thr Tyr Glu Phe Gly Tyr Gln Met His Phe Ile Cys Asn Glu Gly 85 90 95 Tyr Tyr Leu Ile Gly Glu Glu Ile Leu Tyr Cys Glu Leu Lys Gly Ser 100 105 110 Val Ala Ile Trp Ser Gly Lys Pro Pro Ile Cys Glu Lys Val Leu Cys 115 120 125 Thr Pro Pro Pro Lys Ile Lys Asn Gly Lys His Thr Phe Ser Glu Val 130 135 140 Glu Val Phe Glu Tyr Leu Asp Ala Val Thr Tyr Ser Cys Asp Pro Ala 145 150 155 160 Pro Gly Pro Asp Pro Phe Ser Leu Ile Gly Glu Ser Thr Ile Tyr Cys 165 170 175 Gly Asp Asn Ser Val Trp Ser Arg Ala Ala Pro Glu Cys Lys Val Val 180 185 190 Lys Cys Arg Phe Pro Val Val Glu Asn Gly Lys Gln Ile Ser Gly Phe 195 200 205 Gly Lys Lys Phe Tyr Tyr Lys Ala Thr Val Met Phe Glu Cys Asp Lys 210 215 220 Gly Phe Tyr Leu Asp Gly Ser Asp Thr Ile Val Cys Asp Ser Asn Ser 225 230 235 240 Thr Trp Asp Pro Pro Val Pro Lys Cys Leu Lys Val Leu Pro Pro Ser 245 250 255 Ser Thr Lys Pro Pro Ala Leu Ser His Ser Val Ser Thr Ser Ser Thr 260 265 270 Thr Lys Ser Pro Ala Ser Ser Ala Ser Gly Pro Arg Pro Thr Tyr Lys 275 280 285 Pro Pro Val Ser Asn Tyr Pro Gly Tyr Pro Lys Pro Glu Glu Gly Ile 290 295 300 Leu Asp Ser Leu Asp Val Trp Val Ile Ala Val Ile Val Ile Ala Ile 305 310 315 320 Val Val Gly Val Ala Val Ile Cys Val Val Pro Tyr Arg Tyr Leu Gln 325 330 335 Arg Arg Lys Lys Lys Gly Thr Tyr Leu Thr Asp Glu Thr His Arg Glu 340 345 350 Val Lys Phe Thr Ser Leu 355 <210> 21 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> CD59 nucleic acid sequence <400> 21 ctgcagtgct acaactgtcc taacccaact gctgactgca aaacagccgt caattgttca 60 tctgattttg atgcgtgtct cattaccaaa gctgggttac aagtgtataa caagtgttgg 120 aagtttgagc attgcaattt caacgacgtc acaacccgct tgagggaaaa tgagctaacg 180 tactactgct gcaagaagga cctgtgtaac tttaacgaac agcttgaaaa tggtgggaca 240 tccttatcag agaaaacagt tcttctgctg gtgactccat ttctggcagc agcctggagc 300 cttcatccc 309 <210> 22 <211> 103 <212> PRT <213> Artificial Sequence <220> <223> CD59 protein sequence <400> 22 Leu Gln Cys Tyr Asn Cys Pro Asn Pro Thr Ala Asp Cys Lys Thr Ala 1 5 10 15 Val Asn Cys Ser Ser Asp Phe Asp Ala Cys Leu Ile Thr Lys Ala Gly 20 25 30 Leu Gln Val Tyr Asn Lys Cys Trp Lys Phe Glu His Cys Asn Phe Asn 35 40 45 Asp Val Thr Thr Arg Leu Arg Glu Asn Glu Leu Thr Tyr Tyr Cys Cys 50 55 60 Lys Lys Asp Leu Cys Asn Phe Asn Glu Gln Leu Glu Asn Gly Gly Thr 65 70 75 80 Ser Leu Ser Glu Lys Thr Val Leu Leu Leu Val Thr Pro Phe Leu Ala 85 90 95 Ala Ala Trp Ser Leu His Pro 100 <210> 23 <211> 2076 <212> DNA <213> Artificial Sequence <220> <223> CRIg - CD55 - IgG4Fc nucleic acid sequence <400> 23 atgggccgtc ccatcctgga agtgccagag agtgtaacag gaccttggaa aggggatgtg 60 aatcttccct gcacctatga ccccctgcaa ggctacaccc aagtcttggt gaagtggctg 120 gtacaacgtg gctcagaccc tgtcaccatc tttctacgtg actcttctgg agaccatatc 180 cagcaggcaa agtaccaggg ccgcctgcat gtgagccaca aggttccagg agatgtatcc 240 ctccaattga gcaccctgga gatggatgac cggagccact acacgtgtga agtcacctgg 300 cagactcctg atggcaacca agtcgtgaga gataagatta ctgagctccg tgtccagaaa 360 gactgtggcc ttcccccaga tgtacctaat gcccagccag ctttggaagg ccgtacaagt 420 tttcccgagg atactgtaat aacgtacaaa tgtgaagaaa gctttgtgaa aattcctggc 480 gagaaggact cagtgatctg ccttaagggc agtcaatggt cagatattga agagttctgc 540 aatcgtagct gcgaggtgcc aacaaggcta aattctgcat ccctcaaaca gccttatatc 600 actcagaatt attttccagt cggtactgtt gtggaatatg agtgccgtcc aggttacaga 660 agagaacctt ctctatcacc aaaactaact tgccttcaga atttaaaatg gtccacagca 720 gtcgaatttt gtaaaaagaa atcatgccct aatccgggag aaatacgaaa tggtcagatt 780 gatgtaccag gtggcatatt atttggtgca accatctcct tctcatgtaa cacagggtac 840 aaattatttg gctcgacttc tagtttttgt cttatttcag gcagctctgt ccagtggagt 900 gacccgttgc cagagtgcag agaaatttat tgtccagcac caccacaaat tgacaatgga 960 ataattcaag gggaacgtga ccattatgga tatagacagt ctgtaacgta tgcatgtaat 1020 aaaggattca ccatgattgg agagcactct atttattgta ctgtgaataa tgatgaagga 1080 gagtggagtg gcccaccacc tgaatgcaga ggaaaatctc taacttccaa ggtcccacca 1140 acagttcaga aacctaccac agtaaatgtt ccaactacag aagtctcacc aacttctcag 1200 aaaaccacca caaaaaccac cacaccaaat gctcaagcaa cacggagtac acctgtttcc 1260 aggacaacca agcattttca tgaaacaacc ccaaataaag gaagtggaac cacttcaggt 1320 actacccgtc ttctatctgg gcacacgtgt ttcacgttga caggtttgct tgggacgcta 1380 gtaaccatgg gcttgctgac tcccccatgc ccatcatgcc cagcacctga gttcctgggg 1440 ggaccatcag tcttcctgtt ccccccaaaa cccaaggaca ctctcatgat ctcccggacc 1500 cctgaggtca cgtgcgtggt ggtggacgtg agccaggaag accccgaggt ccagttcaac 1560 tggtacgtgg atggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagttc 1620 aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaacggc 1680 aaggagtaca agtgcaaggt ctccaacaaa ggcctcccgt cctccatcga gaaaaccatc 1740 tccaaagcca aagggcagcc ccgagagcca caggtgtaca ccctgccccc atcccaggag 1800 gagatgacca agaaccaggt cagcctgacc tgcctggtca aaggcttcta ccccagcgac 1860 atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1920 gtgctggact ccgacggctc cttcttcctc tacagcaggc taaccgtgga caagagcagg 1980 tggcaggagg ggaatgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 2040 acacagaaga gcctctccct gtctctgggt aaataa 2076 <210> 24 <211> 691 <212> PRT <213> Artificial Sequence <220> <223> CRIg-CD55-IgG4Fc <400> 24 Met Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp 1 5 10 15 Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr 20 25 30 Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val 35 40 45 Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys 50 55 60 Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser 65 70 75 80 Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys 85 90 95 Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys 100 105 110 Ile Thr Glu Leu Arg Val Gln Lys Asp Cys Gly Leu Pro Pro Asp Val 115 120 125 Pro Asn Ala Gln Pro Ala Leu Glu Gly Arg Thr Ser Phe Pro Glu Asp 130 135 140 Thr Val Ile Thr Tyr Lys Cys Glu Glu Ser Phe Val Lys Ile Pro Gly 145 150 155 160 Glu Lys Asp Ser Val Ile Cys Leu Lys Gly Ser Gln Trp Ser Asp Ile 165 170 175 Glu Glu Phe Cys Asn Arg Ser Cys Glu Val Pro Thr Arg Leu Asn Ser 180 185 190 Ala Ser Leu Lys Gln Pro Tyr Ile Thr Gln Asn Tyr Phe Pro Val Gly 195 200 205 Thr Val Val Glu Tyr Glu Cys Arg Pro Gly Tyr Arg Arg Glu Pro Ser 210 215 220 Leu Ser Pro Lys Leu Thr Cys Leu Gln Asn Leu Lys Trp Ser Thr Ala 225 230 235 240 Val Glu Phe Cys Lys Lys Lys Ser Cys Pro Asn Pro Gly Glu Ile Arg 245 250 255 Asn Gly Gln Ile Asp Val Pro Gly Gly Ile Leu Phe Gly Ala Thr Ile 260 265 270 Ser Phe Ser Cys Asn Thr Gly Tyr Lys Leu Phe Gly Ser Thr Ser Ser 275 280 285 Phe Cys Leu Ile Ser Gly Ser Ser Val Gln Trp Ser Asp Pro Leu Pro 290 295 300 Glu Cys Arg Glu Ile Tyr Cys Pro Ala Pro Pro Gln Ile Asp Asn Gly 305 310 315 320 Ile Ile Gln Gly Glu Arg Asp His Tyr Gly Tyr Arg Gln Ser Val Thr 325 330 335 Tyr Ala Cys Asn Lys Gly Phe Thr Met Ile Gly Glu His Ser Ile Tyr 340 345 350 Cys Thr Val Asn Asn Asp Glu Gly Glu Trp Ser Gly Pro Pro Pro Glu 355 360 365 Cys Arg Gly Lys Ser Leu Thr Ser Lys Val Pro Pro Thr Val Gln Lys 370 375 380 Pro Thr Thr Val Asn Val Pro Thr Thr Glu Val Ser Pro Thr Ser Gln 385 390 395 400 Lys Thr Thr Thr Lys Thr Thr Thr Pro Asn Ala Gln Ala Thr Arg Ser 405 410 415 Thr Pro Val Ser Arg Thr Thr Lys His Phe His Glu Thr Thr Pro Asn 420 425 430 Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser Gly His 435 440 445 Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr Met Gly 450 455 460 Leu Leu Thr Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly 465 470 475 480 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 485 490 495 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 500 505 510 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 515 520 525 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 530 535 540 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 545 550 555 560 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 565 570 575 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 580 585 590 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 595 600 605 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 610 615 620 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 625 630 635 640 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 645 650 655 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 660 665 670 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 675 680 685 Leu Gly Lys 690 <210> 25 <211> 2109 <212> DNA <213> Artificial Sequence <220> <223> CRIg-CD46-IgG4Fc nucleic acid sequence<400> 25 atgggccgtc ccatcctgga agtgccagag agtgtaacag gaccttggaa aggggatgtg 60 aatcttccct gcacctatga ccccctgcaa ggctacaccc aagtcttggt gaagtggctg 120 gtacaacgtg gctcagaccc tgtcaccatc tttctacgtg actcttctgg agaccatatc 180 cagcaggcaa agtaccaggg ccgcctgcat gtgagccaca aggttccagg agatgtatcc 240 ctccaattga gcaccctgga gatggatgac cggagccact acacgtgtga agtcacctgg 300 cagactcctg atggcaacca agtcgtgaga gataagatta ctgagctccg tgtccagaaa 360 tgtgaggagc caccaacatt tgaagctatg gagctcattg gtaaaccaaa accctactat 420 gagattggtg aacgagtaga ttataagtgt aaaaaaggat acttctatat acctcctctt 480 gccacccata ctatttgtga tcggaatcat acatggctac ctgtctcaga tgacgcctgt 540 tatagagaaa catgtccata tatacgggat cctttaaatg gccaagcagt ccctgcaaat 600 gggacttacg agtttggtta tcagatgcac tttatttgta atgagggtta ttacttaatt 660 ggtgaagaaa ttctatattg tgaacttaaa ggatcagtag caatttggag cggtaagccc 720 ccaatatgtg aaaaggtttt gtgtacacca cctccaaaaa taaaaaatgg aaaacacacc 780 tttagtgaag tagaagtatt tgagtatctt gatgcagtaa cttatagttg tgatcctgca 840 cctggaccag atccattttc acttattgga gagagcacga tttattgtgg tgacaattca 900 gtgtggagtc gtgctgctcc agagtgtaaa gtggtcaaat gtcgatttcc agtagtcgaa 960 aatggaaaac agatatcagg atttggaaaa aaattttact acaaagcaac agttatgttt 1020 gaatgcgata agggttttta cctcgatggc agcgacacaa ttgtctgtga cagtaacagt 1080 acttgggatc ccccagttcc aaagtgtctt aaagtgctgc ctccatctag tacaaaacct 1140 ccagctttga gtcattcagt gtcgacttct tccactacaa aatctccagc gtccagtgcc 1200 tcaggtccta ggcctactta caagcctcca gtctcaaatt atccaggata tcctaaacct 1260 gaggaaggaa tacttgacag tttggatgtt tgggtcattg ctgtgattgt tattgccata 1320 [[ID=z0]]gttgttggag ttgcagtaat ttgtgttgtc ccgtacagat atcttcaaag gaggaagaag 1380 aaaggcacat acctaactga tgagacccac agagaagtaa aatttacttc tctcccccca 1440 It should be noted that there seems to be a mislabeling in your original text. The "z0" in the translated content should be "20" according to the sequence. Please check the original text for accuracy.tgcccatcat gcccagcacc tgagttcctg gggggaccat cagtcttcct gttcccccca 1500 tgcccatcat gcccagcacc tgagttcctg gggggaccat cagtcttcct gttcccccca 1500 aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 1560 aaacccaagg acactctcat gatctcccgg acccctgagg tcacgtgcgt ggtggtggac 1560 gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 1620 gtgagccagg aagaccccga ggtccagttc aactggtacg tggatggcgt ggaggtgcat 1620 aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 1680 aatgccaaga caaagccgcg ggaggagcag ttcaacagca cgtaccgtgt ggtcagcgtc 1680 ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 1740 ctcaccgtcc tgcaccagga ctggctgaac ggcaaggagt acaagtgcaa ggtctccaac 1740 aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1800 aaaggcctcc cgtcctccat cgagaaaacc atctccaaag ccaaagggca gccccgagag 1800 ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1860 ccacaggtgt acaccctgcc cccatcccag gaggagatga ccaagaacca ggtcagcctg 1860 acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1920 acctgcctgg tcaaaggctt ctaccccagc gacatcgccg tggagtggga gagcaatggg 1920 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1980 cagccggaga acaactacaa gaccacgcct cccgtgctgg actccgacgg ctccttcttc 1980 ctctacagca ggctaaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 2040 ctctacagca ggctaaccgt ggacaagagc aggtggcagg aggggaatgt cttctcatgc 2040 tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 2100 tccgtgatgc atgaggctct gcacaaccac tacacacaga agagcctctc cctgtctctg 2100 ggtaaataa 2109 ggtaaataa 2109 <210> 26 <210> 26 <211> 702 <211> 702 <212> PRT <212> PRT <213> Artificial Sequence <220> <223> CRIg-CD46-IgG4Fc Protein Sequence <400> 26 Met Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp 1 5 10 15 Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr 20 25 30 Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val 35 40 45 Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys 50 55 60 Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser 65 70 75 80 Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys 85 90 95 Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys 100 105 110 Ile Thr Glu Leu Arg Val Gln Lys Cys Glu Glu Pro Pro Thr Phe Glu 115 120 125 Ala Met Glu Leu Ile Gly Lys Pro Lys Pro Tyr Tyr Glu Ile Gly Glu 130 135 140 Arg Val Asp Tyr Lys Cys Lys Lys Gly Tyr Phe Tyr Ile Pro Pro Leu 145 150 155 160 Ala Thr His Thr Ile Cys Asp Arg Asn His Thr Trp Leu Pro Val Ser 165 170 175 Asp Asp Ala Cys Tyr Arg Glu Thr Cys Pro Tyr Ile Arg Asp Pro Leu 180 185 190 Asn Gly Gln Ala Val Pro Ala Asn Gly Thr Tyr Glu Phe Gly Tyr Gln 195 200 205 Met His Phe Ile Cys Asn Glu Gly Tyr Tyr Leu Ile Gly Glu Glu Ile 210 215 220 Leu Tyr Cys Glu Leu Lys Gly Ser Val Ala Ile Trp Ser Gly Lys Pro 225 230 235 240 Pro Ile Cys Glu Lys Val Leu Cys Thr Pro Pro Pro Lys Ile Lys Asn 245 250 255 Gly Lys His Thr Phe Ser Glu Val Glu Val Phe Glu Tyr Leu Asp Ala 260 265 270 Val Thr Tyr Ser Cys Asp Pro Ala Pro Gly Pro Asp Pro Phe Ser Leu 275 280 285 Ile Gly Glu Ser Thr Ile Tyr Cys Gly Asp Asn Ser Val Trp Ser Arg 290 295 300 Ala Ala Pro Glu Cys Lys Val Val Lys Cys Arg Phe Pro Val Val Glu 305 310 315 320 Asn Gly Lys Gln Ile Ser Gly Phe Gly Lys Lys Phe Tyr Tyr Lys Ala 325 330 335 Thr Val Met Phe Glu Cys Asp Lys Gly Phe Tyr Leu Asp Gly Ser Asp 340 345 350 Thr Ile Val Cys Asp Ser Asn Ser Thr Trp Asp Pro Pro Val Pro Lys 355 360 365 Cys Leu Lys Val Leu Pro Pro Ser Ser Thr Lys Pro Pro Ala Leu Ser 370 375 380 His Ser Val Ser Thr Ser Ser Thr Thr Lys Ser Pro Ala Ser Ser Ala 385 390 395 400 Ser Gly Pro Arg Pro Thr Tyr Lys Pro Pro Val Ser Asn Tyr Pro Gly 405 410 415 Tyr Pro Lys Pro Glu Glu Gly Ile Leu Asp Ser Leu Asp Val Trp Val 420 425 430 Ile Ala Val Ile Val Ile Ala Ile Val Val Gly Val Ala Val Ile Cys 435 440 445 Val Val Pro Tyr Arg Tyr Leu Gln Arg Arg Lys Lys Lys Gly Thr Tyr 450 455 460 Leu Thr Asp Glu Thr His Arg Glu Val Lys Phe Thr Ser Leu Pro Pro 465 470 475 480 Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 485 490 495 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 500 505 510 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 515 520 525 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 530 535 540 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 545 550 555 560 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 565 570 575 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 580 585 590 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 595 600 605 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 610 615 620 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 625 630 635 640 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 645 650 655 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 660 665 670 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 675 680 685 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 690 695 700 <210> 27 <211> 1344 <212> DNA <213> Artificial Sequence <220> <223> CRIg - CD59 - IgG4Fc nucleic acid sequence <400> 27 atgggccgtc ccatcctgga agtgccagag agtgtaacag gaccttggaa aggggatgtg 60 aatcttccct gcacctatga ccccctgcaa ggctacaccc aagtcttggt gaagtggctg 120 gtacaacgtg gctcagaccc tgtcaccatc tttctacgtg actcttctgg agaccatatc 180 cagcaggcaa agtaccaggg ccgcctgcat gtgagccaca aggttccagg agatgtatcc 240 ctccaattga gcaccctgga gatggatgac cggagccact acacgtgtga agtcacctgg 300 cagactcctg atggcaacca agtcgtgaga gataagatta ctgagctccg tgtccagaaa 360 ctgcagtgct acaactgtcc taacccaact gctgactgca aaacagccgt caattgttca 420 tctgattttg atgcgtgtct cattaccaaa gctgggttac aagtgtataa caagtgttgg 480 aagtttgagc attgcaattt caacgacgtc acaacccgct tgagggaaaa tgagctaacg 540 tactactgct gcaagaagga cctgtgtaac tttaacgaac agcttgaaaa tggtgggaca 600 tccttatcag agaaaacagt tcttctgctg gtgactccat ttctggcagc agcctggagc 660 cttcatcccc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 720 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 780 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 840 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 900 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaacggcaa ggagtacaag 960 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 1020 gggcagcccc gagagccaca ggtgtacacc ctgcccccat cccaggagga gatgaccaag 1080 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 1140 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 1200 gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 1260 aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 1320 ctctccctgt ctctgggtaa ataa 1344 <210> 28 <211> 447 <212> PRT <213> Artificial Sequence <220> <223> CRIg - CD59 - IgG4Fc Protein Sequence <400> 28 Met Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp 1 5 10 15 Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr 20 25 30 Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val 35 40 45 Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys 50 55 60 Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser 65 70 75 80 Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys 85 90 95 Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys 100 105 110 Ile Thr Glu Leu Arg Val Gln Lys Leu Gln Cys Tyr Asn Cys Pro Asn 115 120 125 Pro Thr Ala Asp Cys Lys Thr Ala Val Asn Cys Ser Ser Asp Phe Asp 130 135 140 Ala Cys Leu Ile Thr Lys Ala Gly Leu Gln Val Tyr Asn Lys Cys Trp 145 150 155 160 Lys Phe Glu His Cys Asn Phe Asn Asp Val Thr Thr Arg Leu Arg Glu 165 170 175 Asn Glu Leu Thr Tyr Tyr Cys Cys Lys Lys Asp Leu Cys Asn Phe Asn 180 185 190 Glu Gln Leu Glu Asn Gly Gly Thr Ser Leu Ser Glu Lys Thr Val Leu 195 200 205 Leu Leu Val Thr Pro Phe Leu Ala Ala Ala Trp Ser Leu His Pro Pro 210 215 220 Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 29 <211> 681 <212> DNA <213> Artificial Sequence <220> <223> IgG1 Fc nucleic acid sequence <400> 29 gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 30 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> IgG1 Fc protein sequence <400> 30 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 31 <211> 681 <212> DNA <213> Artificial Sequence <220> <223> Knob mutant nucleic acid sequence <400> 31 gacaaaactc acacatgccc accgtgccca gcacctgaac tcgagggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 420 aaccaggtca gcctgtactg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 32 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Knob mutant protein sequence <400> 32 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Glu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Tyr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 33 <211> 681 <212> DNA <213> Artificial Sequence <220> <223> Hole mutant nucleic acid sequence <400> 33 gacaaaactc acacatgccc accgtgccca gcacctgaac tcgagggggg accgtcagtc 60 ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120 tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240 cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300 tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360 gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggagga gatgaccaag 420 aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480 tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540 gacggctcct tcttcctcac cagcaagctc accgtggaca agagcaggtg gcagcagggg 600 aacgtcttct catgctccgt gatgcacgag gctctgcaca accactacac gcagaagagc 660 ctctccctgt ctccgggtaa a 681 <210> 34 <211> 227 <212> PRT <213> Artificial Sequence <220> <223> Hole mutant protein sequence <400> 34 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Glu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Lys 225 <210> 35 <211> 2118 <212> DNA <213> Artificial Sequence <220> <223> CRIg-CD46-IgG1 Fc knob nucleic acid sequence <400> 35 atgggccgtc ccatcctgga agtgccagag agtgtaacag gaccttggaa aggggatgtg 60 aatcttccct gcacctatga ccccctgcaa ggctacaccc aagtcttggt gaagtggctg 120 gtacaacgtg gctcagaccc tgtcaccatc tttctacgtg actcttctgg agaccatatc 180 cagcaggcaa agtaccaggg ccgcctgcat gtgagccaca aggttccagg agatgtatcc 240 ctccaattga gcaccctgga gatggatgac cggagccact acacgtgtga agtcacctgg 300 cagactcctg atggcaacca agtcgtgaga gataagatta ctgagctccg tgtccagaaa 360 tgtgaggagc caccaacatt tgaagctatg gagctcattg gtaaaccaaa accctactat 420 gagattggtg aacgagtaga ttataagtgt aaaaaaggat acttctatat acctcctctt 480 gccacccata ctatttgtga tcggaatcat acatggctac ctgtctcaga tgacgcctgt 540 tatagagaaa catgtccata tatacgggat cctttaaatg gccaagcagt ccctgcaaat 600 gggacttacg agtttggtta tcagatgcac tttatttgta atgagggtta ttacttaatt 660 ggtgaagaaa ttctatattg tgaacttaaa ggatcagtag caatttggag cggtaagccc 720 ccaatatgtg aaaaggtttt gtgtacacca cctccaaaaa taaaaaatgg aaaacacacc 780 tttagtgaag tagaagtatt tgagtatctt gatgcagtaa cttatagttg tgatcctgca 840 cctggaccag atccattttc acttattgga gagagcacga tttattgtgg tgacaattca 900 gtgtggagtc gtgctgctcc agagtgtaaa gtggtcaaat gtcgatttcc agtagtcgaa 960 aatggaaaac agatatcagg atttggaaaa aaattttact acaaagcaac agttatgttt 1020 gaatgcgata agggttttta cctcgatggc agcgacacaa ttgtctgtga cagtaacagt 1080 acttgggatc ccccagttcc aaagtgtctt aaagtgctgc ctccatctag tacaaaacct 1140 ccagctttga gtcattcagt gtcgacttct tccactacaa aatctccagc gtccagtgcc 1200 tcaggtccta ggcctactta caagcctcca gtctcaaatt atccaggata tcctaaacct 1260 gaggaaggaa tacttgacag tttggatgtt tgggtcattg ctgtgattgt tattgccata 1320 gttgttggag ttgcagtaat ttgtgttgtc ccgtacagat atcttcaaag gaggaagaag 1380 aaaggcacat acctaactga tgagacccac agagaagtaa aatttacttc tctcgacaaa 1440 actcacacat gcccaccgtg cccagcacct gaactcgagg ggggaccgtc agtcttcctc 1500 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 1560 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 1620 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 1680 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 1740 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 1800 ccccgagaac cacaggtgta caccctgccc ccatcccggg aggagatgac caagaaccag 1860 gtcagcctgt actgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 1920 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 1980 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 2040 ttctcatgct ccgtgatgca cgaggctctg cacaaccact acacgcagaa gagcctctcc 2100 ctgtctccgg gtaaataa 2118 <210> 36 <211> 705 <212> PRT <213> Artificial Sequence <220> <223> CRIg - CD46 - IgG1 Fc knob protein sequence <400> 36 Met Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp<00> 1 5 10 15 Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr 20 25 30 Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val 35 40 45 Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys 50 55 60 Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser 65 70 75 80 Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys 85 90 95 Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys 100 105 110 Ile Thr Glu Leu Arg Val Gln Lys Cys Glu Glu Pro Pro Thr Phe Glu 115 120 125 Ala Met Glu Leu Ile Gly Lys Pro Lys Pro Tyr Tyr Glu Ile Gly Glu 130 135 140 Arg Val Asp Tyr Lys Cys Lys Lys Gly Tyr Phe Tyr Ile Pro Pro Leu 145 150 155 160 Ala Thr His Thr Ile Cys Asp Arg Asn His Thr Trp Leu Pro Val Ser 165 170 175 Asp Asp Ala Cys Tyr Arg Glu Thr Cys Pro Tyr Ile Arg Asp Pro Leu 180 185 190 Asn Gly Gln Ala Val Pro Ala Asn Gly Thr Tyr Glu Phe Gly Tyr Gln 195 200 205 Met His Phe Ile Cys Asn Glu Gly Tyr Tyr Leu Ile Gly Glu Glu Ile 210 215 220 Leu Tyr Cys Glu Leu Lys Gly Ser Val Ala Ile Trp Ser Gly Lys Pro 225 230 235 240 Pro Ile Cys Glu Lys Val Leu Cys Thr Pro Pro Pro Lys Ile Lys Asn 245 250 255 Gly Lys His Thr Phe Ser Glu Val Glu Val Phe Glu Tyr Leu Asp Ala 260 265 270 Val Thr Tyr Ser Cys Asp Pro Ala Pro Gly Pro Asp Pro Phe Ser Leu 275 280 285 Ile Gly Glu Ser Thr Ile Tyr Cys Gly Asp Asn Ser Val Trp Ser Arg 290 295 300 Ala Ala Pro Glu Cys Lys Val Val Lys Cys Arg Phe Pro Val Val Glu 305 310 315 320 Asn Gly Lys Gln Ile Ser Gly Phe Gly Lys Lys Phe Tyr Tyr Lys Ala 325 330 335 Thr Val Met Phe Glu Cys Asp Lys Gly Phe Tyr Leu Asp Gly Ser Asp 340 345 350 Thr Ile Val Cys Asp Ser Asn Ser Thr Trp Asp Pro Pro Val Pro Lys 355 360 365 Cys Leu Lys Val Leu Pro Pro Ser Ser Thr Lys Pro Pro Ala Leu Ser 370 375 380 His Ser Val Ser Thr Ser Ser Thr Thr Lys Ser Pro Ala Ser Ser Ala 385 390 395 400 Ser Gly Pro Arg Pro Thr Tyr Lys Pro Pro Val Ser Asn Tyr Pro Gly 405 410 415 Tyr Pro Lys Pro Glu Glu Gly Ile Leu Asp Ser Leu Asp Val Trp Val 420 425 430 Ile Ala Val Ile Val Ile Ala Ile Val Val Gly Val Ala Val Ile Cys 435 440 445 Val Val Pro Tyr Arg Tyr Leu Gln Arg Arg Lys Lys Lys Gly Thr Tyr 450 455 460 Leu Thr Asp Glu Thr His Arg Glu Val Lys Phe Thr Ser Leu Asp Lys 465 470 475 480 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Glu Gly Gly Pro 485 490 495 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 500 505 510 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 515 520 525 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 530 535 540 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 545 550 555 560 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 565 570 575 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 580 585 590 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 595 600 605 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Tyr 610 615 620 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 625 630 635 640 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 645 650 655 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 660 665 670 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 675 680 685 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 690 695 700 Lys 705 <210> 37 <211> 2085 <212> DNA <213> Artificial Sequence <220> <223> CRIg-CD55-IgG1 Fc knob nucleic acid sequence <400> 37 atgggccgtc ccatcctgga agtgccagag agtgtaacag gaccttggaa aggggatgtg 60 aatcttccct gcacctatga ccccctgcaa ggctacaccc aagtcttggt gaagtggctg 120 gtacaacgtg gctcagaccc tgtcaccatc tttctacgtg actcttctgg agaccatatc 180 cagcaggcaa agtaccaggg ccgcctgcat gtgagccaca aggttccagg agatgtatcc 240 ctccaattga gcaccctgga gatggatgac cggagccact acacgtgtga agtcacctgg 300 cagactcctg atggcaacca agtcgtgaga gataagatta ctgagctccg tgtccagaaa 360 gactgtggcc ttcccccaga tgtacctaat gcccagccag ctttggaagg ccgtacaagt 420 tttcccgagg atactgtaat aacgtacaaa tgtgaagaaa gctttgtgaa aattcctggc 480 gagaaggact cagtgatctg ccttaagggc agtcaatggt cagatattga agagttctgc 540 aatcgtagct gcgaggtgcc aacaaggcta aattctgcat ccctcaaaca gccttatatc 6{00} actcagaatt attttccagt cggtactgtt gtggaatatg agtgccgtcc aggttacaga 660 agagaacctt ctctatcacc aaaactaact tgccttcaga atttaaaatg gtccacagca 720 gtcgaatttt gtaaaaagaa atcatgccct aatccgggag aaatacgaaa tggtcagatt 780 gatgtaccag gtggcatatt atttggtgca accatctcct tctcatgtaa cacagggtac 840 aaattatttg gctcgacttc tagtttttgt cttatttcag gcagctctgt ccagtggagt 900 gacccgttgc cagagtgcag agaaatttat tgtccagcac caccacaaat tgacaatgga 960 ataattcaag gggaacgtga ccattatgga tatagacagt ctgtaacgta tgcatgtaat 1020 aaaggattca ccatgattgg agagcactct atttattgta ctgtgaataa tgatgaagga 1080 gagtggagtg gcccaccacc tgaatgcaga ggaaaatctc taacttccaa ggtcccacca 1140 acagttcaga aacctaccac agtaaatgtt ccaactacag aagtctcacc aacttctcag 1200 aaaaccacca caaaaaccac cacaccaaat gctcaagcaa cacggagtac acctgtttcc 1260 aggacaacca agcattttca tgaaacaacc ccaaataaag gaagtggaac cacttcaggt 1320 actacccgtc ttctatctgg gcacacgtgt ttcacgttga caggtttgct tgggacgcta 1380 gtaaccatgg gcttgctgac tgacaaaact cacacatgcc caccgtgccc agcacctgaa 1440 ctcgaggggg gaccgtcagt cttcctcttc cccccaaaac ccaaggacac cctcatgatc 1500 tcccggaccc ctgaggtcac atgcgtggtg gtggacgtga gccacgaaga ccctgaggtc 1560 aagttcaact ggtacgtgga cggcgtggag gtgcataatg ccaagacaaa gccgcgggag 1620 gagcagtaca acagcacgta ccgtgtggtc agcgtcctca ccgtcctgca ccaggactgg 1680 ctgaatggca aggagtacaa gtgcaaggtc tccaacaaag ccctcccagc ccccatcgag 1740 aaaaccatct ccaaagccaa agggcagccc cgagaaccac aggtgtacac cctgccccca 1800 tcccgggagg agatgaccaa gaaccaggtc agcctgtact gcctggtcaa aggcttctat 1860 cccagcgaca tcgccgtgga gtgggagagc aatgggcagc cggagaacaa ctacaagacc 1920 acgcctcccg tgctggactc cgacggctcc ttcttcctct acagcaagct caccgtggac 1980 aagagcaggt ggcagcaggg gaacgtcttc tcatgctccg tgatgcacga ggctctgcac 2040 aaccactaca cgcagaagag cctctccctg tctccgggta aataa 2085 <210> 38 <211> 694 <212> PRT <213> Artificial Sequence <220> <223> CRIg-CD55-IgG1 Fc knob Protein Sequence <400> 38 Met Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp 1 5 10 15 Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr 20 25 30 Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val 35 40 45 Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys 50 55 60 Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser 65 70 75 80 Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys 85 90 95 Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys 100 105 110 Ile Thr Glu Leu Arg Val Gln Lys Asp Cys Gly Leu Pro Pro Asp Val 115 120 125 Pro Asn Ala Gln Pro Ala Leu Glu Gly Arg Thr Ser Phe Pro Glu Asp 130 135 140 Thr Val Ile Thr Tyr Lys Cys Glu Glu Ser Phe Val Lys Ile Pro Gly 145 150 155 160 Glu Lys Asp Ser Val Ile Cys Leu Lys Gly Ser Gln Trp Ser Asp Ile 165 170 175 Glu Glu Phe Cys Asn Arg Ser Cys Glu Val Pro Thr Arg Leu Asn Ser 180 185 190 Ala Ser Leu Lys Gln Pro Tyr Ile Thr Gln Asn Tyr Phe Pro Val Gly 195 200 205 Thr Val Val Glu Tyr Glu Cys Arg Pro Gly Tyr Arg Arg Glu Pro Ser 210 215 220 Leu Ser Pro Lys Leu Thr Cys Leu Gln Asn Leu Lys Trp Ser Thr Ala 225 230 235 240 Val Glu Phe Cys Lys Lys Lys Ser Cys Pro Asn Pro Gly Glu Ile Arg 245 250 255 Asn Gly Gln Ile Asp Val Pro Gly Gly Ile Leu Phe Gly Ala Thr Ile 260 265 270 Ser Phe Ser Cys Asn Thr Gly Tyr Lys Leu Phe Gly Ser Thr Ser Ser 275 280 285 Phe Cys Leu Ile Ser Gly Ser Ser Val Gln Trp Ser Asp Pro Leu Pro 290 295 300 Glu Cys Arg Glu Ile Tyr Cys Pro Ala Pro Pro Gln Ile Asp Asn Gly 305 310 315 320 Ile Ile Gln Gly Glu Arg Asp His Tyr Gly Tyr Arg Gln Ser Val Thr 325 330 335 Tyr Ala Cys Asn Lys Gly Phe Thr Met Ile Gly Glu His Ser Ile Tyr 340 345 350 Cys Thr Val Asn Asn Asp Glu Gly Glu Trp Ser Gly Pro Pro Pro Glu 355 360 365 Cys Arg Gly Lys Ser Leu Thr Ser Lys Val Pro Pro Thr Val Gln Lys 370 375 380 Pro Thr Thr Val Asn Val Pro Thr Thr Glu Val Ser Pro Thr Ser Gln 385 390 395 400 Lys Thr Thr Thr Lys Thr Thr Thr Pro Asn Ala Gln Ala Thr Arg Ser 405 410 415 Thr Pro Val Ser Arg Thr Thr Lys His Phe His Glu Thr Thr Pro Asn 420 425 430 Lys Gly Ser Gly Thr Thr Ser Gly Thr Thr Arg Leu Leu Ser Gly His 435 440 445 Thr Cys Phe Thr Leu Thr Gly Leu Leu Gly Thr Leu Val Thr Met Gly 450 455 460 Leu Leu Thr Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 465 470 475 480 Leu Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 485 490 495 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 500 505 510 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 515 520 525 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 530 535 540 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 545 550 555 560 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 565 570 575 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 580 585 590 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 595 600 605 Gln Val Ser Leu Tyr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 610 615 620 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 625 630 635 640 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 645 650 655 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 660 665 670 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 675 680 685 Ser Leu Ser Pro Gly Lys 690 <210> 39 <211> 1959 <212> DNA <213> Artificial Sequence <220> <223> CRIg-FH-IgG1 Fc hole nucleic acid sequence <400> 39 atgggccgtc ccatcctgga agtgccagag agtgtaacag gaccttggaa aggggatgtg 60 aatcttccct gcacctatga ccccctgcaa ggctacaccc aagtcttggt gaagtggctg 120 gtacaacgtg gctcagaccc tgtcaccatc tttctacgtg actcttctgg agaccatatc 180 cagcaggcaa agtaccaggg ccgcctgcat gtgagccaca aggttccagg agatgtatcc 240 ctccaattga gcaccctgga gatggatgac cggagccact acacgtgtga agtcacctgg 300 cagactcctg atggcaacca agtcgtgaga gataagatta ctgagctccg tgtccagaaa 360 gaagattgca atgaacttcc tccaagaaga aatacagaaa ttctgacagg ttcctggtct 420 gaccaaacat atccagaagg cacccaggct atctataaat gccgccctgg atatagatct 480 cttggaaatg taataatggt atgcaggaag ggagaatggg ttgctcttaa tccattaagg 540 aaatgtcaga aaaggccctg tggacatcct ggagatactc cttttggtac ttttaccctt 600 acaggaggaa atgtgtttga atatggtgta aaagctgtgt atacatgtaa tgaggggtat 660 caattgctag gtgagattaa ttaccgtgaa tgtgacacag atggatggac caatgatatt 720 cctatatgtg aagttgtgaa gtgtttacca gtgacagcac cagagaatgg aaaaattgtc 780 agtagtgcaa tggaaccaga tcgggaatac cattttggac aagcagtacg gtttgtatgt 840 aactcaggct acaagattga aggagatgaa gaaatgcatt gttcagacga tggtttttgg 900 agtaaagaga aaccaaagtg tgtggaaatt tcatgcaaat ccccagatgt tataaatgga 960 tctcctatat ctcagaagat tatttataag gagaatgaac gatttcaata taaatgtaac 1020 atgggttatg aatacagtga aagaggagat gctgtatgca ctgaatctgg atggcgtccg 1080 ttgccttcat gtgaagaaaa atcatgtgat aatccttata ttccaaatgg tgactactca 1140 cctttaagga ttaaacacag aactggagat gaaatcacgt accagtgtag aaatggtttt 1200 tatcctgcaa cccggggaaa tacagcaaaa tgcacaagta ctggctggat acctgctccg 1260 agatgtacct tgaaagacaa aactcacaca tgcccaccgt gcccagcacc tgaactcgag 1320 gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1380 acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1440 aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1500 tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1560 ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1620 atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1680 gaggagatga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1740 gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1800 cccgtgctgg actccgacgg ctccttcttc ctcaccagca agctcaccgt ggacaagagc 1860 aggtggcagc aggggaacgt cttctcatgc tccgtgatgc acgaggctct gcacaaccac 1920 tacacgcaga agagcctctc cctgtctccg ggtaaataa 1959 <210> 40 <211> 652 <212> PRT <213> Artificial Sequence <220> <223> CRIg-FH-IgG1 Fc hole protein sequence <400> 40 Met Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp 1 5 10 15 Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr 20 25 30 Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val 35 40 45 Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys 50 55 60 Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser 65 70 75 80 Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys 85 90 95 Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys 100 105 110 Ile Thr Glu Leu Arg Val Gln Lys Glu Asp Cys Asn Glu Leu Pro Pro 115 120 125 Arg Arg Asn Thr Glu Ile Leu Thr Gly Ser Trp Ser Asp Gln Thr Tyr 130 135 140 Pro Glu Gly Thr Gln Ala Ile Tyr Lys Cys Arg Pro Gly Tyr Arg Ser 145 150 155 160 Leu Gly Asn Val Ile Met Val Cys Arg Lys Gly Glu Trp Val Ala Leu 165 170 175 Asn Pro Leu Arg Lys Cys Gln Lys Arg Pro Cys Gly His Pro Gly Asp 180 185 190 Thr Pro Phe Gly Thr Phe Thr Leu Thr Gly Gly Asn Val Phe Glu Tyr 195 200 205 Gly Val Lys Ala Val Tyr Thr Cys Asn Glu Gly Tyr Gln Leu Leu Gly 210 215 220 Glu Ile Asn Tyr Arg Glu Cys Asp Thr Asp Gly Trp Thr Asn Asp Ile 225 230 235 240 Pro Ile Cys Glu Val Val Lys Cys Leu Pro Val Thr Ala Pro Glu Asn 245 250 255 Gly Lys Ile Val Ser Ser Ala Met Glu Pro Asp Arg Glu Tyr His Phe 260 265 270 Gly Gln Ala Val Arg Phe Val Cys Asn Ser Gly Tyr Lys Ile Glu Gly 275 280 285 Asp Glu Glu Met His Cys Ser Asp Asp Gly Phe Trp Ser Lys Glu Lys 290 295 300 Pro Lys Cys Val Glu Ile Ser Cys Lys Ser Pro Asp Val Ile Asn Gly 305 310 315 320 Ser Pro Ile Ser Gln Lys Ile Ile Tyr Lys Glu Asn Glu Arg Phe Gln 325 330 335 Tyr Lys Cys Asn Met Gly Tyr Glu Tyr Ser Glu Arg Gly Asp Ala Val 340 345 350 Cys Thr Glu Ser Gly Trp Arg Pro Leu Pro Ser Cys Glu Glu Lys Ser 355 360 365 Cys Asp Asn Pro Tyr Ile Pro Asn Gly Asp Tyr Ser Pro Leu Arg Ile 370 375 380 Lys His Arg Thr Gly Asp Glu Ile Thr Tyr Gln Cys Arg Asn Gly Phe 385 390 395 400 Tyr Pro Ala Thr Arg Gly Asn Thr Ala Lys Cys Thr Ser Thr Gly Trp 405 410 415 Ile Pro Ala Pro Arg Cys Thr Leu Lys Asp Lys Thr His Thr Cys Pro 420 425 430 Pro Cys Pro Ala Pro Glu Leu Glu Gly Gly Pro Ser Val Phe Leu Phe 435 440 445 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 450 455 460 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 465 470 475 480 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 485 490 495 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 500 505 510 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 515 520 525 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 530 535 540 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 545 550 555 560 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 565 570 575 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 580 585 590 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 595 600 605 Phe Phe Leu Thr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 610 615 620 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 625 630 635 640 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 645 650 <210> 41 <211> 1353 <212> DNA <213> Artificial Sequence <220> <223> CRIg-CD59-IgG1 Fc hole nucleic acid sequence <400> 41 atgggccgtc ccatcctgga agtgccagag agtgtaacag gaccttggaa aggggatgtg 60 aatcttccct gcacctatga ccccctgcaa ggctacaccc aagtcttggt gaagtggctg 120 gtacaacgtg gctcagaccc tgtcaccatc tttctacgtg actcttctgg agaccatatc 180 cagcaggcaa agtaccaggg ccgcctgcat gtgagccaca aggttccagg agatgtatcc 240 ctccaattga gcaccctgga gatggatgac cggagccact acacgtgtga agtcacctgg 300 cagactcctg atggcaacca agtcgtgaga gataagatta ctgagctccg tgtccagaaa 360 ctgcagtgct acaactgtcc taacccaact gctgactgca aaacagccgt caattgttca 420 tctgattttg atgcgtgtct cattaccaaa gctgggttac aagtgtataa caagtgttgg 480 aagtttgagc attgcaattt caacgacgtc acaacccgct tgagggaaaa tgagctaacg 540 tactactgct gcaagaagga cctgtgtaac tttaacgaac agcttgaaaa tggtgggaca 600 tccttatcag agaaaacagt tcttctgctg gtgactccat ttctggcagc agcctggagc 660 tccttatcag agaaaacagt tcttctgctg gtgactccat ttctggcagc agcctggagc 660 cttcatcccg acaaaactca cacatgccca ccgtgcccag cacctgaact cgagggggga 720 cttcatcccg acaaaactca cacatgccca ccgtgcccag cacctgaact cgagggggga 720 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 1080 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1140 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200 ctggactccg acggctcctt cttcctcacc agcaagctca ccgtggacaa gagcaggtgg 1260 ctggactccg acggctcctt cttcctcacc agcaagctca ccgtggacaa gagcaggtgg 1260 cagcagggga acgtcttctc atgctccgtg atgcacgagg ctctgcacaa ccactacacg 1320 cagcagggga acgtcttctc atgctccgtg atgcacgagg ctctgcacaa ccactacacg 1320 cagaagagcc tctccctgtc tccgggtaaa taa 1353 <210> 42 <211> 450 <212> PRT <213> Artificial Sequence <220> <223> CRIg - CD59 - IgG1 Fc hole protein sequence <400> 42 Met Gly Arg Pro Ile Leu Glu Val Pro Glu Ser Val Thr Gly Pro Trp 1 5 10 15 Lys Gly Asp Val Asn Leu Pro Cys Thr Tyr Asp Pro Leu Gln Gly Tyr 20 25 30 Thr Gln Val Leu Val Lys Trp Leu Val Gln Arg Gly Ser Asp Pro Val 35 40 45 Thr Ile Phe Leu Arg Asp Ser Ser Gly Asp His Ile Gln Gln Ala Lys 50 55 60 Tyr Gln Gly Arg Leu His Val Ser His Lys Val Pro Gly Asp Val Ser 65 70 75 80 Leu Gln Leu Ser Thr Leu Glu Met Asp Asp Arg Ser His Tyr Thr Cys 85 90 95 Glu Val Thr Trp Gln Thr Pro Asp Gly Asn Gln Val Val Arg Asp Lys 100 105 110 Ile Thr Glu Leu Arg Val Gln Lys Leu Gln Cys Tyr Asn Cys Pro Asn 115 120 125 Pro Thr Ala Asp Cys Lys Thr Ala Val Asn Cys Ser Ser Asp Phe Asp 130 135 140 Ala Cys Leu Ile Thr Lys Ala Gly Leu Gln Val Tyr Asn Lys Cys Trp 145 150 155 160 Lys Phe Glu His Cys Asn Phe Asn Asp Val Thr Thr Arg Leu Arg Glu 165 170 175 Asn Glu Leu Thr Tyr Tyr Cys Cys Lys Lys Asp Leu Cys Asn Phe Asn 180 185 190 Glu Gln Leu Glu Asn Gly Gly Thr Ser Leu Ser Glu Lys Thr Val Leu 195 200 205 Leu Leu Val Thr Pro Phe Leu Ala Ala Ala Trp Ser Leu His Pro Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Glu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450 <210> 43 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Linker 1 nucleic acid sequence <400> 43 ggcggaggtg gatctggcgg aggtggatct ggcggaggtg gatct 45 <210> 44 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker 1 protein sequence <400> 44 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Linker 2 nucleic acid sequence <400> 45 ggcggaggtg gaggcggagg tgga 24 <210> 46 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Linker 2 Protein Sequence <400> 46 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 47 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Linker 3 Nucleic Acid Sequence <400> 47 gaagctgccg caaaggaagc tgccgcaaag gaagctgccg caaag 45 <210> 48 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Linker 3 Protein Sequence <400> 48 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 <210> 49 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Linker 4 Nucleic Acid Sequence <400> 49 gttagtcaaa cttctaaatt aactcgtgcc gagacagttt tccctgatgt t 51 <210> 50 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Linker 4 Protein Sequence <400> 50 Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp 1 5 10 15 Val

Claims

1. A fusion protein, which is a dimer composed of a first polypeptide chain and a second polypeptide chain, wherein: (1) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 14, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 14; (2) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 24, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 24; (3) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 26, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 26; (4) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 28, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 28; (5) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 38, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 40; (6) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 36, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO: 42; or (7) The amino acid sequence of the first polypeptide chain is as shown in SEQ ID NO: 38, and the amino acid sequence of the second polypeptide chain is as shown in SEQ ID NO:

42.

2. An isolated nucleic acid molecule encoding the fusion protein according to claim 1.

3. A vector comprising the nucleic acid molecule according to claim 2.

4. A cell comprising the vector according to claim 3, or expressing the fusion protein according to claim 1.

5. A method for preparing the fusion protein according to claim 1, which comprises the following steps: synthesizing the fusion protein according to claim 1, and / or culturing the cell according to claim 4 under conditions for expressing the fusion protein according to claim 1.

6. A pharmaceutical composition comprising the fusion protein according to claim 1 and a pharmaceutically acceptable carrier.

7. Use of the fusion protein according to claim 1 or the pharmaceutical composition according to claim 6 in the preparation of a drug for treating an autoimmune disease.

8. The use according to claim 7, wherein the disease is myasthenia gravis with autoimmune disorder.

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