Masp isoforms as inhibitors of complement activation

Abstract

This invention relates to MASP isotypes as inhibitors of complement activation. It also relates to novel ficolin-related peptides and peptides derived from these ficolin-related peptides for the treatment of conditions associated with inflammation, apoptosis, autoimmunity, coagulation, thrombosis, or coagulopathy, and for use as biomarkers. Furthermore, this invention relates to antibodies recognizing the novel ficolin-related peptides and peptides derived therefrom, nucleic acid molecules encoding the peptides, vectors for producing the peptides, and host cells.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201080041321.6, filed on July 16, 2010, entitled "MASP isotype as an inhibitor of complement activation". Invention Field

[0002] This invention relates to novel ficolin-related polypeptides and polypeptides derived therefrom for the treatment of conditions associated with inflammation, apoptosis, autoimmunity, coagulation, thrombosis, or coagulopathy, and for use as biomarkers. The invention also relates to antibodies recognizing the novel ficolin-related polypeptides and polypeptides derived therefrom, nucleic acid molecules encoding the polypeptides, vectors for producing the polypeptides, and host cells. Background of the Invention

[0003] Activation of the complement system (C) is accomplished via three distinct initiation pathways: the alternative pathway (AP), the classical pathway (CP), or the lectin pathway (LCP). AP activation occurs on exogenous surfaces and is initiated by the slow, spontaneous hydrolysis of C3 and the activity of factors properdin, factor B, and factor D to form the functional C3 convertase C3bBb. AP also functions as an amplification pathway (amplification loop) for the other two pathways. Recently, it has been shown that alternative convertase assembly can also be initiated by non-covalently attaching properdin to some target surfaces. On the other hand, CP activation is initiated when C1q binds to immunoglobulins complexed with antigens. The binding of C1q to immunoglobulins complexed with antigens triggers the activation of C1q-associated serine proteases C1r and C1s. C1s cleaves and activates C4 and C2, thereby forming the CP C3 convertase C4b2a. LCPs are activated when mannose-binding lectins (MBL) or ficolins bind to carbohydrates or acetylated compounds in a restricted pattern, for example, on the surface of microorganisms or when exposed to dying host cells. Upon binding with a ligand, the associated serine protease MASP-2 is activated and cleaves C4 and C2, forming the LCP C3 convertase C4b2a. The function of MASP-1 has been proposed to be involved in the stabilization of MASP-2 cleavage at C2 and also to direct lower-order cleavage at C3. However, other studies have linked the functions and activities of MASP-1 and MASP-2 to coagulation system cross-talk involving prothrombin, fibrinogen, and factor XIII. Using MASP1 / 3 knockout mice, MASP-1 has recently been shown to actually contribute to complement activity. The precise function of the recently discovered MBL-associated serine protease MASP-3 remains unclear. Several studies have been reported that show MASP-3 associates with a limited range of MBL oligomers, and that MASP-3 and small MBL-associated proteins (sMAPs) are involved in the regulation or inhibition of MBL-dependent LCP complement activation.

[0004] MASP-1 and -3 originate from the same MASP1 / 3 gene (located on chromosome 3q27-q28) via different splicing. They contain the same A chain, differing only in 15 C-terminal residues. The A chain includes two CUB (C1r / C1s, Urchin-EGF, bone morphogenetic protein) domains separated by an EGF (epidermal growth factor) domain, followed by two CCP (complement control protein) domains. The B chain, including the serine protease domain, differs from MASP-1 and MASP-3. MASP-2 and sMAP also originate from the same gene (located on chromosome 1p36-p36.2), with sMAP being a truncated form lacking the serine protease domain and the major portion of the A chain. While the MASP1 / 3 gene has been shown to be polymorphic, its functional importance remains poorly understood. However, there is some evidence suggesting that polymorphism in the MASP2 / sMAP gene is associated with an increased risk of infection. MASPs expression is localized in hepatocytes of the liver, but recent studies have documented that human MASP-3 mRNA (the only MASP-mRNA) is expressed in a wide range of tissues.

[0005] Purpose of the invention

[0006] The purpose of embodiments of this invention is to provide peptides suitable for treating conditions associated with inflammation, apoptosis, autoimmunity, coagulation, and / or thrombosis or coagulopathy. The peptides of this invention can be further adapted as biomarkers for the diagnosis and / or prediction of these indications, as well as for malignant diseases such as cancer. Invention Overview

[0007] The inventors have discovered that novel polypeptides associated with recognition molecules of the lectin complement pathway, as well as polypeptide fragments such as those derived therefrom, can be used to treat specific medical conditions related to diseases associated with inflammation, apoptosis, autoimmunity, coagulation, and / or thrombosis or coagulopathy.

[0008] Therefore, in a first aspect, the present invention relates to isolated ficolin-related polypeptides.

[0009] In a second aspect, the present invention relates to a polypeptide comprising the amino acid sequence SEQ ID NO:4 or a variant thereof or an immunological fragment thereof.

[0010] In a third aspect, the present invention relates to antibodies that specifically bind to the polypeptides of the present invention.

[0011] In a fourth aspect, the present invention relates to isolated nucleic acid molecules encoding the polypeptides of the present invention.

[0012] In another aspect, the present invention relates to isolated nucleic acid molecules comprising at least 70% identical nucleotide sequences to the sequence of SEQ NO:2.

[0013] In another aspect, the present invention relates to a carrier comprising an isolated nucleic acid molecule encoding the polypeptide of the present invention.

[0014] In another aspect, the present invention relates to a host cell comprising a vector containing an isolated nucleic acid molecule encoding a polypeptide of the present invention.

[0015] In another aspect, the present invention relates to a method for producing the polypeptide of the invention, the method comprising culturing the cells of the invention in a suitable growth medium under conditions that allow expression of a polynucleotide construct, and recovering the obtained polypeptide from the culture medium.

[0016] In another aspect, the present invention relates to compositions comprising the polypeptides of the present invention.

[0017] In another aspect, the present invention relates to pharmaceutical compositions comprising the polypeptides of the present invention.

[0018] In another aspect, the present invention relates to a method for detecting the polypeptides of the present invention in biological samples, the method comprising:

[0019] a) Obtain biological samples;

[0020] b) Contact the biological sample with the antibody of the present invention; and

[0021] c) If available, detect the complex of the antibody and the peptide;

[0022] This serves as an indicator of the presence of the polypeptide in the sample.

[0023] In another aspect, the present invention relates to the polypeptides of the present invention, which are used as pharmaceuticals.

[0024] In another aspect, the present invention relates to the use of the polypeptides of the present invention in the preparation of pharmaceuticals.

[0025] In another aspect, the present invention relates to the polypeptides of the present invention for the treatment of any indication related to inflammation, apoptosis, and / or autoimmunity.

[0026] In another aspect, the present invention relates to the polypeptides of the present invention for any indication related to the treatment of diseases associated with coagulation, thrombosis or coagulopathy.

[0027] In another aspect, the present invention relates to polypeptides for the prevention of thromboembolic complications in identified high-risk patients, such as those undergoing surgery or those suffering from congestive heart failure.

[0028] In another aspect, the present invention relates to the polypeptides of the present invention for the treatment of heart-related medical conditions.

[0029] In another aspect, the present invention relates to polypeptides for the treatment of medical conditions associated with ficolin-related polypeptide deficiency.

[0030] In another aspect, the present invention relates to a method for treating any indication related to inflammation, apoptosis, and / or autoimmunity, the method comprising administering a therapeutically or preventively effective amount of the polypeptide of the invention to a subject in need of it.

[0031] In another aspect, the present invention relates to a method for treating any indication related to a disease associated with coagulation, thrombosis, or coagulopathy, the method comprising administering to a subject in need of the present invention a therapeutically or preventively effective amount of the polypeptide of the present invention.

[0032] In another aspect, the present invention relates to a method for preventing thromboembolic complications in identified high-risk patients, such as those undergoing surgery or suffering from congestive heart failure, the method comprising administering a therapeutically or preventively effective amount of the polypeptide of the invention to a subject in need of it.

[0033] In another aspect, the present invention relates to a method for treating a heart-related medical condition, the method comprising administering to a subject in need of the present invention a therapeutically or preventively effective amount of the polypeptide thereof.

[0034] In another aspect, the present invention relates to a method for treating a medical condition associated with a deficiency of ficolin-related polypeptides, the method comprising administering a therapeutically or preventively effective amount of the polypeptide of the invention to a subject in need of it.

[0035] In another aspect, the present invention relates to nucleic acid probes capable of hybridizing with nucleic acid sequences encoding polypeptides of the present invention under stringent conditions.

[0036] In another aspect, the present invention relates to a method for detecting the presence of nucleic acids encoding the polypeptides of the present invention in biological samples, the method comprising:

[0037] a) Obtain biological samples;

[0038] b) Contact the biological sample with the nucleic acid probe of the present invention; and

[0039] c) If applicable, detect the complex of the nucleic acid probe with the nucleic acid encoding the polypeptide;

[0040] This serves as an indicator of the presence of the nucleic acid encoding the polypeptide in the sample.

[0041] In another aspect, the present invention relates to a method for diagnosing a condition associated with abnormal expression of ficolin-related peptides, the method comprising obtaining a biological sample from a patient and measuring the expression of ficolin-related peptides in the biological sample, wherein an increase or decrease in the expression of ficolin-related peptides compared to a control indicates that the patient has a condition associated with abnormal expression of ficolin-related peptides.

[0042] In another aspect, the present invention relates to an isolated composition comprising the polypeptide of the present invention and a combination of one or more proteins selected from Ficolin-1, 2, 3, mannose-binding lectin (MBL), C1q, pulmonary surfactant protein SP-A and / or SP-D, and intracellular collagen-like defense molecules such as CLL-11.

[0043] In another aspect, the present invention relates to compositions comprising the polypeptides of the present invention, which are pharmaceutical compositions.

[0044] In another aspect, the present invention relates to the pharmaceutical compositions described herein, which are used as medicines.

[0045] In another aspect, the present invention relates to the use of the compositions of the present invention in the preparation of a medicament.

[0046] In another aspect, the present invention relates to pharmaceutical compositions for the treatment of any indication related to inflammation, apoptosis, and / or autoimmunity.

[0047] In another aspect, the present invention relates to pharmaceutical compositions for the treatment of any indication as defined herein.

[0048] In another aspect, the present invention relates to a method for treating any indication as defined herein, the method comprising administering simultaneously or sequentially a therapeutic or preventative effective amount of the polypeptide of the present invention and one or more proteins selected from Ficolin-1, 2, 3, and mannose-binding lectin (MBL), C1q, pulmonary surfactant protein SP-A and / or SP-D, and intracellular collagen-like defense molecules, such as CLL-11.

[0049] In another aspect, the present invention relates to the use of the polypeptides of the invention as biomarkers in blood and tissues for the diagnosis and / or prediction of malignant diseases, such as cancerous diseases, such as brain tumors, liver tumors and reproductive tract tumors.

[0050] In another aspect, the present invention relates to the use of the polypeptides of the invention as biomarkers in blood and tissues for the diagnosis and / or prediction of autoimmune, metabolic and / or inflammatory conditions as defined herein.

[0051] Attached illustrations

[0052] Figure 1 Alternative transcription of the MASP-1 gene was detected in liver cDNA. MASP-1, MASP3, and FAP transcripts were amplified using a universal forward primer at exon 6 and specific reverse primers at exons 12 (MASP1), 11 (MASP3), and 8a (FAP). MASP1 produced a 500 bp fragment, MASP3 produced a 506 bp fragment, and FAP produced a 309 bp fragment.

[0053] Figure 2 The MASP1 gene undergoes alternative splicing. MASP1 is produced by slicing out exon 8a and exon 11, both of which contain stop codon sequences (marked with a black box). The MASP1 sequence contains a stop codon in exon 17. MASP3 is produced by slicing out exon 8a; if exon 8a is not sliced ​​out, FAP is produced. The FAP protein contains two CUB domains, an EFG domain, and a first CCP1 domain.

[0054] Figure 3 Tissue expression of the FAP fragment. The tissue distribution of the MASP-1, MASP3, and FAP genes was investigated in a cDNA genome from Clontech. MASP-1, MASP-3, and FAP transcripts were amplified using universal forward primers and specific reverse primers. GADPH was used as a reference gene. All three genes were highly expressed in the liver; additionally, FAP was strongly expressed in cardiac tissue (indicated by black arrows). Lesser expression of the FAP gene was detected in the brain, colon, prostate, skeletal muscle, and small intestine (indicated by white arrows).

[0055] Figure 4Alignment of MASP-1, MASP-3, and FAP. The protein sequences of MASP-1, MASP-3, and FAP were aligned using BioEdit software. MASP-1 and MASP-3 contain distinct C-terminal serine protease domains, while FAP does not contain any serine protease domains. Instead, this protein contains 17 novel amino acids in its C-terminal region.

[0056] Figure 5 The image shows the cDNA sequence and corresponding protein sequence of FAP. The cDNA sequence is shown on the top line, and the corresponding protein sequence is shown below. Exon regions are separated by black vertical lines. Amino acids believed to be involved in MBL / ficolins binding are marked with light yellow boxes.

[0057] Figure 6 MASP-1 complement activation. Human MBL was incubated with an increased amount of MASP-1. MASP-1 can activate both C3 and C4 complement proteins.

[0058] Figure 7 MASP-2 complement activation. Human MBL was incubated with an increased amount of MASP-2. MASP-2 strongly activates both C3 and C4 complement proteins.

[0059] Figure 8 MASP-3 inhibition of complement. Human MBL was incubated with an increased amount of MASP-3. MASP-3 was able to inhibit the activation of two complement proteins, C3 and C4.

[0060] Figure 9 Immunoprecipitation. Serum ficolin / MBL was immunoprecipitated using mAbs of anti-MBL 131-11, anti-Ficolin-2 clone 219, and anti-Ficolin-3 clone 334. This was followed by Dynal magnetic bead separation, SDS-PAGE, Western blotting, and the use of biotin-labeled anti-MASP-1 / MASP-3 clone 8B3 as a signal antibody.

[0061] Figure 10 FAP interacts with ficolin when it binds to acetylated human serum albumin (AcHSA). Eluted serum ficolin bound to AcHSA. Western blotting was performed using biotin-labeled anti-MASP-1 / MASP-3 clone 8B3 as a signal antibody.

[0062] Figure 11 : Kinetics and dissociation constants of the interactions between MASP-1 and MASP-3 and rFicolin-2 T et al., Mol. Immunol. (Molecular Immunology), 2007.

[0063] Figure 12 Comparison of 17 unique amino acids in GULF and FAP.

[0064] Figure 13 In the mannan / MBL ELISA assay, complement activation occurs at C4. Mannan-coated wells are incubated with or without recombinant human MBL, followed by serial dilutions of homozygous MBL-deficient serum. C4 deposition is measured using a polyclonal anti-C4c antibody. Error bars represent twice the standard error determined doubly for each point on the curve.

[0065] Figure 14 In the acetylated BSA / Ficolin-3 ELISA assay, complement activation occurs at C4. AcBSA-coated wells are incubated with or without recombinant human Ficolin-3, followed by serial dilutions of homozygous deficient Ficolin-3 serum. C4 deposition is measured using a polyclonal anti-C4c antibody. Error bars represent twice the standard error determined doubly for each point on the curve.

[0066] Figure 15 In the mannan / MBL ELISA assay, complement activation of C4 is achieved. Mannan-coated wells are incubated with recombinant human MBL, followed by incubation in one dimension with serially diluted rMASP-1 medium as serum-free culture supernatant. Subsequently, incubation is performed in a second dimension with serially diluted homozygous deficient serum of MBL. C4 deposition is measured using a polyclonal anti-C4c antibody. Error bars represent twice the standard error determined doubly for each point on the curve.

[0067] Figure 16 In the AcBSA / Ficolin-3 ELISA assay, complement activation of C4 is observed. AcBSA-coated wells are incubated with recombinant human Ficolin-3, followed by incubation in one dimension with serially diluted rMASP-1 medium as serum-free culture supernatant. Subsequently, incubation is performed in a second dimension with serially diluted Ficolin-3 homozygous deficient serum. C4 deposition is measured using a polyclonal anti-C4c antibody. Error bars represent twice the standard error determined doubly for each point on the curve.

[0068] Figure 17In the mannan / MBL ELISA, complement activation of C4 is achieved. Mannan-coated wells are incubated with recombinant human MBL, followed by incubation in one dimension with serially diluted rMASP-2 medium as serum-free culture supernatant. Subsequently, incubation is performed in a second dimension with serially diluted homozygous deficient serum of MBL. C4 deposition is measured using a polyclonal anti-C4c antibody. Error bars represent twice the standard error determined doubly for each point on the curve.

[0069] Figure 18 In the AcBSA / Ficolin-3 ELISA assay, complement activation occurs at C4. AcBSA-coated wells are incubated with recombinant human Ficolin-3, followed by incubation in one dimension with serially diluted rMASP-2 medium as serum-free culture supernatant. Subsequently, incubation is performed in a second dimension with serially diluted Ficolin-3 homozygous deficient serum. C4 deposition is measured using a polyclonal anti-C4c antibody. Error bars represent twice the standard error determined doubly for each point on the curve.

[0070] Figure 19 In the mannan / MBL ELISA assay, complement activation of C4 is achieved. Mannan-coated wells are incubated with recombinant human MBL, followed by incubation in one dimension with serially diluted rMASP-3 medium as serum-free culture supernatant. Subsequently, incubation is performed in a second dimension with serially diluted homozygous deficient serum of MBL. C4 deposition is measured using a polyclonal anti-C4c antibody. Error bars represent twice the standard error of the double determination at each point on the curve.

[0071] Figure 20 In the AcBSA / Ficolin-3 ELISA assay, complement activation of C4 is observed. AcBSA-coated wells are incubated with recombinant human Ficolin-3, followed by incubation in one dimension with serially diluted rMASP-3 as a serum-free culture supernatant. Subsequently, incubation is performed in a second dimension with serially diluted Ficolin-3 homozygous deficient serum. C4 deposition is measured using a polyclonal anti-C4c antibody. Error bars represent twice the standard error determined doubly for each point on the curve.

[0072] Figure 21Tissue distribution of FAP, MASP1, and MASP3. In cardiac tissue, FAP expression was significantly higher than that of MASP1 and MASP3. FAP expression in cardiac tissue was three-fold higher than in liver tissue. Furthermore, higher FAP expression was observed in the liver compared to that of MASP1 and MASP3. Significant amounts of FAP expression were also detected in brain, skeletal muscle, and prostate tissues. Experiments were performed in triplicate. The standard error of the mean is shown.

[0073] Figure 22 Immunohistochemical localization of MAP-1 in the liver was performed using polyclonal mouse antiserum targeting 17 FAP-specific C-terminal residues of the protein. Control staining was negative. Several different polyclonal antibodies against FAP (rabbit and mouse) showed the same staining pattern.

[0074] Figure 23 Immunohistochemical analysis of MAP-1 tissue localization (OM X10). Left panel shows staining with mAb (12B11) targeting MAP-1. Right panel shows staining with an isotype control of unrelated IgG1k mAb. (AB): cardiac muscle, (CD): skeletal muscle, (EF): liver sample, (GH): aortic tissue. The graduation bar in the lower right corner of all slides indicates 50 μm.

[0075] Figure 24 Immunoprecipitation of MAP-1 and MASP-1 / 3 serum complexes. (A) MAP-1 and MASP-1 / 3 were immunoprecipitated from serum using mAb 20C4 (anti-MAP-1) and mAb 8B3 (anti-MASP-1 / 3, with epitopes on the universal heavy chain). The reduced samples were electroblotted and developed with pAbs against MAP-1 or biotinylated mAbs against Ficolin-3 (FCN334) and MBL (Hyb 131-1). (B) Immunoprecipitation was performed from 1 ml, 300 μl, and 100 μl of serum using mAbs against MBL (Hyb 131-11), Ficolin-2 (FCN219), and Ficolin-3 (FCN334), respectively (left side). The controls are MAP-1 (sMAP-1) precipitated from serum using anti-MAP-1 mAb 20C4 and rMAP-1 (rMAP-1) precipitated from culture supernatant (right). Samples were analyzed using Western blots probing MAP-1 with pAbs.

[0076] Figure 25Effects of MASP-2 and MAP-1 on MBL and Ficolin-3-mediated complement C4 deposition. C4 deposition was measured using a polyclonal antibody against C4 and as an OD. 490-650nm Values ​​are given. Error bars indicate twice the standard error of the double determination. Approximate concentrations of rMBL, rFicolin-3, rMAP-1, and rMASP-2 are given in the figure labels. (A) Reconstruction of C4 deposition on mannan-coated surfaces using MBL-deficient serum with 400 ng / ml rMBL. Control without rMBL. (B) Dose-dependent effect of rMASP-2 on rMBL-mediated C4 deposition. (C) Dose-dependent effect of rMAP-1 on rMBL-mediated C4 deposition. (D) Reconstruction of C4 deposition on AcBSA-coated surfaces using Ficolin-3-deficient serum with 400 ng / ml rFicolin-3. (E) Dose-dependent effect of rMASP-2 on rFicolin-3-mediated C4 deposition. (F) Dose-dependent effect of rMAP-1 on rFicolin-3-mediated C4 deposition.

[0077] Figure 26 Effects of MASP-2 and MAP-1 on complement C4 deposition in a pure system. rMBL on the mannan surface was pre-incubated with serially diluted rMASP-2 in the first dimension. Then, serially modified rMAP-1 was applied in the second dimension, followed by 1 μg / ml of purified C4. C4 deposition was measured using pAbs targeting C4 and used as OD. 490-650nm The values ​​are given. The error bars represent twice the standard error of the double determination. The approximate concentrations of rMAP-1 and rMASP-2 are given in the figure labels.

[0078] Figure 27 SDS-PAGE analysis of rMAP-1. The left-hand side shows the immunoblotting analysis with + / - N-glucosidase F treatment (ENDO-F). The right-hand side shows the corresponding Coomassie staining.

[0079] Figure 28(A, B): Calibration curves. A) Calibration curves generated by a two-sided mAb 20C4 / mAb-8B3 ELISA using either serially diluted rMAP-1 applied to a MAP-1-depleted normal human serum bank (pNHS) or serially diluted rMAP-1 in PBS / 0.05% Tween / 10mM EDTA. Error bars represent twice the standard error determined by eight trials. B) Immunoblots of MAP-1-depleted serum, normal human serum, and MAP-1-infused MAP-1-depleted serum.

[0080] Figure 29A-C: Serum MAP-1 concentration. A) Serum MAP-1 concentration and distribution in 100 Danish blood donors. Mean serum level: 240 ng / ml. Range: 115–466 ng / ml; B) Correlation between MASP-3 and MAP-1 serum levels; C) Effects of serum freezing and thawing. Serum was frozen and thawed eight times, with MAP-1 levels measured in each round. Error bars represent twice the standard error of the double determination.

[0081] Figure 30: A) Association levels of MAP-1 with MBL, Ficolin-2, and Ficolin-3 in 100 Danish blood donors (expressed in relative OD490-650 nm units). P-values ​​were obtained using a nonparametric two-tailed t-test. B) Correlation between serum MAP-1 levels and their relative association with MBL, Ficolin-2, and Ficolin-3 (left-handed). Correlation between serum MBL, Ficolin-2, and Ficolin-3 levels and their relative association with MAP-1 (right-handed). Correlation p- and r-values ​​were calculated using a nonparametric Spearman rank correlation test.

[0082] Figure 31A -C: Sucrose gradient ultracentrifugation. A) Fractions (1-27) collected from serum subjected to a 10-30% sucrose density gradient. Fractions were analyzed by specific ELISA against MAP-1, MASP-3, MBL, Ficolin-2, and -3. Peaks of serum IgM (19S) and IgG (7S) are shown at the top of the graph. B) Fractions numbered 8-23 were analyzed by immunoblotting against MAP-1, MASP-1, MASP-3, sMAP, MASP-2, MBL, Ficolin-2, and Ficolin-3. C) Fractions 1-27 were analyzed by their ability to activate exogenously applied human C4 on immobilized acetylated BSA (a Ficolin-3 ligand) or mannan (an MBL ligand).

[0083] Detailed disclosure of the invention

[0084] This invention has discovered a novel 40 kDa plasma protein associated with a recognition molecule of the lectin complement pathway and identified it as a novel variant of MASP-1 / MASP-3, which in turn corresponds to the newly discovered plasma protein.

[0085] The inventors have indicated that this novel protein (which they have named FAP (Ficolin-associated protein) is... F icolin A ssociated PMBL / Ficolin-associated protein-1 (MBL / Ficolin-associated protein-1) lacks an enzyme domain but contains a ficolin / MBL binding domain, and is therefore predicted to participate in the regulation and inhibition of complement and coagulation functions through competition and substitution of MASPs or alternatively, but not mutually exclusively, as a protein involved in clearance or signal transduction functions.

[0086] Uncontrolled activation of the complement system and / or coagulation cascade is strongly associated with lethal and severe consequences in a wide range of diseases, including systemic inflammation and sepsis, myocardial infarction, and autoimmune diseases.

[0087] It has been shown that inhibition of coagulation and complement activation is a promising therapeutic tool.

[0088] This invention describes a possible novel inhibitor of complement and coagulation functions. However, the peptides of this invention may possess other functions, such as scavenging and / or signal transduction. Furthermore, they may be used as novel biomarkers in several disease conditions, including malignant diseases, autoimmune diseases, metabolic and / or inflammatory conditions.

[0089] The inventors of this invention have discovered a plasma protein in vivo called ficollin-associated protein (FAP) and shown that it primarily associates with ficolins. Figure 9 However, it may also associate with mannose-binding lectins. By searching the NCBI nucleotide database, the inventors of this invention discovered a possible transcriptomorph corresponding to the truncated MASP-1. Based on this sequence, primers were designed to amplify the putative new gene transcript. Subsequently, using human liver cDNA, a novel variant of the MASP-1 gene was identified. Figure 1 This mRNA chain was sequenced, and the amino acid sequence was determined, corresponding to a protein with a molecular weight of 40 kDa observed in plasma / serum. Figure 5 This novel protein is partially identical to MASP-1 and MASP-3, but lacks the serine protease domain; instead, it contains a new exon encoding 17 amino acids before the stop codon. This exon is truncated in the MASP1 and MASP3 transcripts. Figure 2 By using a set of mRNA expression libraries, the inventors have found evidence of strong expression of this protein in the heart and liver, and subsequently in skeletal muscle. Figure 3 Weak expression was observed in the brain, digestive tract, prostate, and spleen. Figure 3TaqMann analysis confirmed FAP expression in heart and liver cells. FAP expression was significantly higher in heart tissue than MASP1 and MASP3. FAP expression in heart tissue was three times higher than in the liver. Furthermore, higher FAP expression was observed in the liver compared to MASP1 and MASP3. Significant amounts of FAP expression were also detected in brain, skeletal muscle, and prostate tissues. The experiment was performed three times in duplicate.

[0090] The high expression in the heart is very significant, and the inventors propose the use of the polypeptide of the present invention as a very useful protectant against tissue damage in autoimmune, metabolic and / or inflammatory conditions (such as heart-related medical conditions).

[0091] The inventors have established a assay to evaluate complement activity induced by ficolins and mannose-binding lectins, and have thus been able to demonstrate the potential functional complement inhibition of FAP.

[0092] The inventors have developed a real-time quantitative assay to measure the accurate relative expression levels in different tissues.

[0093] The polypeptides of this invention can be generated using recombinant technology. Rabbits or mice can be immunized with a unique 17-amino acid-long peptide to obtain FAP polyclonal and monoclonal specific antibodies, respectively.

[0094] Specific FAP antibodies can be used for quantitative measurement of FAP and immunohistochemical detection in different tissues.

[0095] The binding constants between FAP and the different binding partners described herein can be determined in ELISA and by using surface plasmon resonance (Biacore) technology.

[0096] FAP-specific acceptor proteins, such as receptors that bind specifically to the cell surface, can be identified by standard assays known to those skilled in the art, such as assays in which the protein binds directly to the cell.

[0097] The novel protein Ficolin-associated protein (FAP) is an alternative splice variant of MASP1. This protein lacks the serine protease domain but retains a domain involved in binding to the initiator of the lectin pathway in the complement system. Therefore, the inventors predict that this protein participates in the regulation and inhibition of MASP-1 and MASP-3 (complement, lectin function, and other enzyme substrates) function through competition and substitution with MASPs. Alternatively, but not mutually exclusive, FAP can act as a scavenger, promoting the removal of FAP / MBL / ficolin complexes bound to endogenous waste products or pathogens.

[0098] Uncontrolled activation of the complement system and coagulation cascade is associated with adverse consequences, and functional inhibitors, such as the peptides of this invention, can very effectively control the complement system and coagulation cascade. Furthermore, the peptides of this invention can be used in other situations. Another perspective is that the protein can be used as a biomarker in various disease conditions.

[0099] The protein is unique and can provide the basis for new drugs and / or new diagnostic tools.

[0100] The polypeptide of the present invention, comprising the amino acid sequence SEQ ID NO:4 or its immunological fragment or variant, may have a specific function associated with that amino acid sequence. The inventors propose that the polypeptide may have a function or activity corresponding to the activity of one or more proteins selected from: DNMT1 DNA (cytokine-5-)-methyltransferase 1 (DNMT1), Golgi protein subfamily B member 1 (GOLGB1), A-kinase anchoring protein 9 (AKAP9), B- and T-lymphocyte-associated proteins (CD272 antigen), phagocytosis adaptor protein 1 (GULP) containing a PTB domain, and protein 2 containing a MACRO domain.

[0101] In some particularly interesting embodiments, the polypeptides of the present invention have functions or activities corresponding to the activity of GULP (gastrointestinal endothelial protein 1) containing a PTB domain.

[0102] definition

[0103] The term "ficolin-associated polypeptide" as used herein refers to any protein or polypeptide comprising amino acid sequence 20-380 of natural human ficolin-associated polypeptide (FAP) (SEQ ID NO:1) or amino acid sequence 16-363 of SEQ ID NO:9, its functional variants, functionally truncated versions, and functional derivatives or conjugates, wherein the polypeptide does not have complement activity but has the ability to compete with MASP-1, MASP-2, or MASP-3 for binding to ficolin-3, MBL, C1q, pulmonary surfactant proteins SP-A and / or SP-D and / or CL-L1 (and other members of the collagen lectin family). This includes, but is not limited to, human ficolin-associated polypeptide (FAP) having SEQ ID NO:1 and its variants.

[0104] The term “ficolin-associated protein (FAP)” as used herein refers to a protein having the amino acid sequence 1-380 (with or without a signal peptide, such as amino acid sequence 20-380) of the natural human FAP (SEQ ID NO:1), its natural allelic variants, and homologs. It also includes proteins with slightly modified amino acid sequences, such as modified N- or C-terminal N- or C-terminal N- or C-terminal amino acid deletions or additions, provided that these proteins substantially retain FAP activity. The term “ficolin-associated protein (FAP)” is used interchangeably herein with the terms “MAP-1” or “MBL / Ficolin-associated protein-1”. “FAP” as defined above also includes natural allelic variants that may exist and occur between individuals. The term also includes proteins with homologous sequences and similar functions derived from species other than humans (such as cattle, pigs, dogs, horses, rats, and mice). Furthermore, the degree and location of glycosylation or other post-translational modifications may vary depending on the nature of the chosen host cell and host cell environment.

[0105] The terms “MBL-associated serine protease-1” or “MASP-1” as used herein refer to a protein having the amino acid sequence 1-699 (with or without a signal peptide, such as amino acid sequence 20-699) of natural human MASP-1 (SEQ ID NO:5), its natural allele variants, and homologs. It should be understood that said sequence may be present in one or more peptide chains, such as in two chains, i.e., in the heavy and light chains of a natural human protein.

[0106] The terms “MBL-associated serine protease-3” or “MASP-3” as used herein refer to a protein having the amino acid sequence 1-728 (with or without a signal peptide, such as amino acid sequence 20-728) of natural human MASP-3 (SEQ ID NO:7), its natural allele variants, and homologs. It should be understood that said sequence may be present in one or more peptide chains, such as in two chains, i.e., in the heavy and light chains of a natural human protein.

[0107] The terms “MBL-associated serine protease-2” or “MASP-2” as used herein refer to a protein having the amino acid sequence 1-686 (with or without a signal peptide, such as amino acid sequence 16-686) of natural human MASP-2 (SEQ ID NO:9), its natural allele variants, and homologs. It should be understood that said sequence may be present in one or more peptide chains, such as in two chains, i.e., in the heavy and light chains of the natural human protein.

[0108] The terms “small MBL-associated protein,” “sMAP,” “19kD MBL-associated plasma protein,” or “MAp19” are used herein to refer to a protein having the amino acid sequence 1-185 (with or without a signal peptide, such as amino acid sequence 16-185) of the natural human sMAP (SEQ ID NO:11), its natural allele variants, and homologs.

[0109] The term "variant" (or "variants") used herein is intended to designate a ficolin-related polypeptide having the sequence SEQ ID NO:1 or a polypeptide comprising the amino acid sequence SEQ ID NO:4, wherein one or more amino acids have been substituted by another amino acid and / or one or more amino acids have been deleted and / or one or more amino acids have been inserted into the polypeptide and / or one or more amino acids have been added to the polypeptide. The addition may occur at the N-terminus, the C-terminus, or both ends. The "variant" as defined retains its functional activity. In some embodiments, the variant has 70% sequence identity with the sequence SEQ ID NO:1. In some embodiments, the variant has 80% sequence identity with the sequence SEQ ID NO:1. In other embodiments, the variant has 90% sequence identity with the sequence SEQ ID NO:1. In yet another embodiment, the variant has 95% sequence identity with the sequence SEQ ID NO:1.

[0110] In some embodiments, the variant shares 70% sequence identity with sequence SEQ ID NO:4. In some embodiments, the variant shares 80% sequence identity with sequence SEQ ID NO:4. In other embodiments, the variant shares 90% sequence identity with sequence SEQ ID NO:4. In yet another embodiment, the variant shares 95% sequence identity with sequence SEQ ID NO:4.

[0111] The phrases “functional variant,” “functional truncated version,” and “functional derivative” as used herein refer to variants, truncated versions, and derivatives of SEQ ID NO:1, wherein the polypeptide comprises the basic sequence portion of SEQ ID NO:1 and has at least the ability to competitively bind ficolins or MBL to MASP-1 or MASP-3 without complement activity and / or serine protease activity. It should be understood that ficolin-related polypeptides may have two or three features selected from variants, and / or truncated versions, and / or derivatives.

[0112] Functional variants of ficolin-related peptides have been generated in the same cell type, including those that exhibit at least about 25%, such as at least about 50%, such as at least about 75%, such as at least about 90% of the specific activity of wild-type FAP when detected in the assays described herein.

[0113] The term "immunologic fragment" as used herein refers to a fragment of amino acid sequence that has substantially the same functional activity and is recognized by antibodies in the same spatial orientation. Accordingly, a specific antibody binds to both the polypeptide and its immunological fragment.

[0114] The term "another amino acid" is used herein to mean an amino acid that is different from the amino acid naturally present at that position. This includes, but is not limited to, amino acids that can be encoded by polynucleotides. In some embodiments, the different amino acid is in the natural L form and can be encoded by polynucleotides.

[0115] The term “derivative” as used herein is intended to refer to ficolin-related polypeptides that exhibit substantially the same or enhanced biological activity relative to wild-type human FAP, wherein one or more amino acids of the parent polypeptide have been chemically modified, for example by alkylation, PEGylation, acylation, esterification, or amide formation.

[0116] The term "complement activity" as used in this article refers to the activity that activates the complement system. Complement activity can be measured using the assays described in the section entitled "Assays" of this article.

[0117] The term “mannose-binding lectin (MBL)” as used herein also refers to mannose-binding lectin, mannose-binding protein (MBP1), and mannose-binding protein, and the terms are used interchangeably.

[0118] The term "capable of association" as used herein refers to the ability of the protein of the present invention to specifically bind in solution to one or more initiators of the lectin pathway of the complement system or other proteins that may be involved in the action of the polypeptide.

[0119] The term "construction" is intended to refer to a polynucleotide fragment that may be based on a complete or partial naturally occurring nucleotide sequence encoding a target polypeptide. Constructs may optionally contain other polynucleotide fragments. Similarly, the term "amino acid that can be encoded by a polynucleotide construct" includes amino acids that can be encoded by a polynucleotide construct as defined above, i.e., amino acids such as Ala, Val, Leu, Ile, Met, Phe, Trp, Pro, Gly, Ser, Thr, Cys, Tyr, Asn, Glu, Lys, Arg, His, Asp, and Gln.

[0120] The term "vector" as used herein refers to any nucleic acid entity capable of amplification within a host cell. Therefore, a vector can be a spontaneously replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid. Alternatively, a vector can be one that, upon introduction into a host cell, integrates into the host cell genome and replicates along with the chromosome into which it has already been integrated. The choice of vector typically depends on the host cell to which it will be introduced. Vectors include, but are not limited to, plasmid vectors, phage vectors, viral vectors, or sticky-terminal plasmid vectors. Vectors typically contain an origin of replication and at least one optional gene, i.e., a gene encoding an easily detectable product or a gene whose presence is essential for cell growth.

[0121] In another aspect, the present invention provides recombinant host cells comprising the said polynucleotide construct or vector. In one embodiment, the recombinant host cell is a eukaryotic cell. In other embodiments, the recombinant host cell is of mammalian origin. In yet another embodiment, the recombinant host cell is selected from the group consisting of CHO cells, HEK cells, and BHK cells.

[0122] The term "host cell" is used herein to refer to any cell, including hybrid cells, in which heterologous DNA can be expressed. Typical host cells include, but are not limited to, insect cells, yeast cells, and mammalian cells, including human cells such as BHK, CHO, HEK, and COS cells. When practicing this invention, the cultured host cells are preferably mammalian cells, more preferably established mammalian cell lines, including but not limited to, CHO (e.g., ATCC CCL 61), COS-1 (e.g., ATCC CRL 1650), juvenile hamster kidney (BHK), and HEK293 (e.g., ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. The preferred BHK cell line is the tk-ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America) 79:1106-1110, 1982), hereinafter referred to as BHK 570 cells. The BHK 570 cell line can be obtained from the American Type Culture Collection (12301 Parklawn Dr., Rockville, MD 20852), ATCC accession number CRL 10314. The tk-ts13 BHK cell line can also be obtained from ATCC, with accession number CRL 1632. Other suitable cell lines include, but are not limited to, rat Hep I (rat hepatocellular carcinoma; ATCC CRL 1600), rat Hep II (rat hepatocellular carcinoma; ATCC CRL 1548), TCMK (ATCC CCL 139), human lung (ATCC HB 8065), NCTC 1469 (ATCC CCL 9.1), and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America) 77:4216-4220, 1980). Fusions of 3T3 cells, Namalwa cells, myeloma cells, and myeloma cells with other cell lines may also be used.

[0123] In another aspect, the present invention provides transgenic animals that contain and express the said polynucleotide construct.

[0124] In another aspect, the present invention provides transgenic plants that contain and express the said polynucleotide construct.

[0125] In another aspect, the present invention relates to a method for producing the ficolin-related polypeptide of the present invention, the method comprising culturing cells containing the polynucleotide construct in a suitable growth medium under conditions allowing expression of the polynucleotide construct, and recovering the obtained polypeptide from the culture medium.

[0126] When used herein, the term “suitable growth medium” means a culture medium containing the nutrients and other components required for cell growth and expression of the nucleic acid sequence encoding the ficolin-related polypeptide of the present invention.

[0127] In another aspect, the present invention relates to a method for producing ficolin-related polypeptides, the method comprising recovering the polypeptides from milk produced by transgenic animals.

[0128] In another aspect, the present invention relates to a method for producing ficolin-related polypeptides, the method comprising culturing cells of a transgenic plant containing a polynucleotide construct and recovering the polypeptide from the resulting plant.

[0129] In the context of this invention, the term "treatment" is intended to include the prevention of conditions (such as inflammation and reperfusion injury) anticipated to involve inappropriate complement activation and the regulation of existing conditions (such as myocardial infarction and stroke) with the aim of suppressing or minimizing tissue damage. Therefore, the term "treatment" includes the prophylactic administration of the ficolin-related polypeptides of this invention.

[0130] The term “subject” as used in this text is intended to refer to any animal, particularly mammals such as humans, and may be used interchangeably with the term “patient” where appropriate.

[0131] The term "sequence identity," as known in the art, refers to a relationship between two or more polypeptide molecules or two or more nucleic acid molecules determined by comparing their sequences. In the art, "identity" also means the degree of sequence correlation between nucleic acid molecules or polypeptides, which, depending on the specific case, is determined by the number of matches between chains of two or more nucleotide residues or two or more amino acid residues. "Identity" measures the percentage of identical matches between the smaller of two or more sequences, where gap alignments (if present) are interpreted by a specific mathematical model or computer program (i.e., an "algorithm").

[0132] The term "similarity" is a related concept, but as opposed to "identity," it refers to the sequence relationship between two sequences that include both identical matches and conserved substitution matches. For example, if two polypeptide sequences have 10 / 20 identical amino acids, and the rest are all non-conserved substitutions, then both the identity and similarity percentages are 50%. If, in the same instance, there are 5 additional positions with conserved substitutions, the identity percentage is still 50%, but the similarity percentage is 75% (15 / 20). Therefore, in the presence of conserved substitutions, the degree of similarity between the two polypeptides is higher than the percentage of identity between them.

[0133] Conserved modifications to the amino acid sequence SEQ ID NO:1 (and corresponding modifications to the encoding nucleotides) will produce ficolin-related peptides with similar functional and chemical characteristics to those of naturally occurring FAPs. Conversely, substantial modifications to the functional and / or chemical characteristics of ficolin-related peptides can be achieved by selecting substitutions in the amino acid sequence SEQ ID NO:1 that significantly differ in their retention of (a) the structure of the molecular backbone in the substituted region, e.g., as a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the role of the side chain volume.

[0134] For example, “conservative amino acid substitution” can include replacing a native amino acid residue with a non-native residue so that the effect on the polarity or charge of the amino acid residue at that position is small or non-existent. Furthermore, any native residue in a polypeptide can also be substituted with alanine, as previously described regarding “alanine scan mutagenesis” (see, for example, MacLennan et al., 1998, Acta Physiol. Scand. Suppl. 643:55-67; ​​Sasaki et al., 1998, Adv. Biophys. 35:1-24, which discuss alanine scan mutagenesis).

[0135] Ideal amino acid substitutions (whether conserved or non-conserved) can be determined by those skilled in the art when such substitutions are required. For example, amino acid substitutions can be used to identify important residues in ficolin-related peptides, or to increase or decrease the affinity of ficolin-related peptides described herein.

[0136] Based on common side-chain characteristics, naturally occurring residues can be classified into the following categories:

[0137] 1) Hydrophobic: Leucine, Met, Ala, Val, Leu, Ile;

[0138] 2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0139] 3) Acidity: Asp, Glu;

[0140] 4) Alkaline: His, Lys, Arg;

[0141] 5) Residues affecting chain orientation: Gly, Pro; and

[0142] 6) Aromatic: Trp, Tyr, Phe.

[0143] For example, non-conservative substitutions can include exchanging a member of one class with a member of another class. Such substituted residues can be introduced into regions homologous to human ficolin-related peptides and non-human ficolin-related peptides, or into non-homologous regions of the molecule.

[0144] When making such changes, the hydropathic index of the amino acids can be considered. Based on the hydrophobicity and charge characteristics of amino acids, each amino acid has been assigned a hydropathic index as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0145] The importance of the aqueous amino acid index for conferring biological function of protein-protein interactions is known in the art. Kyte et al., J. Mol. Biol. (Journal of Molecular Biology), 157:105-131 (1982). It is known that certain amino acids can be replaced by other amino acids with similar aqueous indices or scores and still retain similar biological activities. When variations are made based on the aqueous index, amino acid replacements with an aqueous index within ±2 are preferred, particularly those within ±1, and even more particularly preferred those within ±0.5.

[0146] It is also known in the art that similar amino acid substitutions can be effectively performed based on hydrophilicity, particularly in cases where the resulting biologically equivalent protein or peptide is intended for use in immunological implementations, as is the case here. The maximum local average hydrophilicity of a protein is controlled by the hydrophilicity of its adjacent amino acids and is related to its immunogenicity and antigenicity, i.e., to the protein's biological characteristics.

[0147] The following hydrophilicity values ​​have been assigned to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). When varying other similar hydrophilicity values, substitutions of amino acids with hydrophilicity values ​​within ±2 are preferred, particularly those within ±1, and even more particularly preferred those within ±0.5. Epitopes can also be identified from the primary amino acid sequence based on hydrophilicity. These epitopes are also referred to as "epitope core regions".

[0148] Those skilled in the art will be able to identify appropriate variants of the polypeptide shown in SEQ ID NO:1 using known techniques. To identify appropriate regions of the molecule that can be altered without destroying its activity, those skilled in the art can target regions considered unimportant to activity. For example, when similar polypeptides with similar activities from the same or other species are known, those skilled in the art can compare the amino acid sequence of the ficolin-related polypeptide with that of the similar polypeptide. Using such a comparison, one can identify conserved residues and portions of the molecule among the similar polypeptides. It should be understood that changes in regions of the ficolin-related polypeptide that are not conserved relative to the similar polypeptide are less likely to adversely affect the biological activity and / or structure of the ficolin-related polypeptide. Those skilled in the art will also appreciate that even in relatively conserved regions, naturally occurring residues can be replaced with chemically similar amino acids while preserving activity (conservative amino acid residue substitution). Therefore, even regions important for biological activity or structure may be subject to conserved amino acid substitutions without destroying biological activity or adversely affecting the polypeptide structure.

[0149] In addition, those skilled in the art can refer to structure-function studies that identify activity- or structurally important residues in similar peptides. Through such comparisons, one can predict the importance of amino acid residues in ficolin-related peptides corresponding to activity- or structurally important amino acid residues in similar peptides. Those skilled in the art can select chemically similar amino acid substitutions for the predicted important amino acid residues in ficolin-related peptides and other peptides of the present invention.

[0150] Those skilled in the art can also perform three-dimensional structure and amino acid sequence analysis relative to the structure of similar peptides. Referring to the aforementioned information, those skilled in the art can predict the amino acid residue alignment of ficolin-related peptides based on their three-dimensional structure. Those skilled in the art can choose not to fundamentally alter the predicted amino acid residues on the protein surface, as these residues can participate in important interactions with other molecules. Furthermore, those skilled in the art can generate detection variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using the activity assays described herein. These variants can be used to gather information about suitable variants. For example, variants with such changes will be avoided if alterations to specific amino acid residues are found to result in disruptive, undesirably reduced, or inappropriate activity. In other words, based on information gathered from such routine experiments, those skilled in the art can readily determine other amino acid substitutions, either alone or in combination with other mutations, that should be avoided.

[0151] Many scientific publications have focused on predicting secondary structures. See Moult J., Curr. Op. in Biotech., 7(4):422-427 (1996); Chou et al., Biochemistry, 13(2):222-245 (1974); Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv. Enzymol. Relat. Areas Mol. Biol, 47:45-148 (1978); Chou et al., Ann. Rev. Biochem., 47:251-276; and Chou et al., Biophys. J., 26:367-384 (1979). Furthermore, computer programs can now be used to assist in predicting secondary structures. One method for predicting secondary structures is based on homology modeling. For example, two peptides or proteins with greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. Recent growth in protein structure databases (PDBs) has provided improved predictability of secondary structures, including the potential number of folds in a peptide or protein structure. See Holm et al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been proposed (Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) that a finite number of folds exist in a given peptide or protein, and that structural predictions gain significant accuracy once the key number of structures has been resolved.

[0152] Other methods for predicting secondary structures include threading (Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al., Structure, 4(1):15-9 (1996)), pattern analysis (Bowie et al., Science, 253:164-170 (1991); Gribskov et al., Meth. Enzymol., 183:146-159 (1990); Gribskov et al., Proc. Nat. Acad. Sci., 84(13):4355-4358 (1987)), and evolutionary linking (see Home, ibid., and Brenner, ibid.).

[0153] The identity and similarity of related peptides can be easily calculated using known methods. The methods described include, but are not limited to, *Computational Molecular Biology*, edited by Lesk, AM, Oxford University Press, New York, 1988; *Biocomputing: Informatics and Genome Projects*, edited by Smith, DW, Academic Press, New York, 1993; *Computer Analysis of Sequence Data*, Part 1, edited by Griffin, AM, and Griffin, HG, Humana Press, New Jersey, 1994; *Sequence Analysis in Molecular Biology*, von Heinje, G., Academic Press, 1987; *Sequence Analysis Primer*, edited by Gribskov, M. and Devereux, J., M. Stockton Press, New York, 1991; and Carillo et al., SIAM J. Applied Those described in Math., 48:1073 (1988).

[0154] Preferred methods for determining identity and / or similarity are designed to give the maximum match between the detected sequences. Methods for determining identity and similarity are described in publicly available computer programs. Preferred computer program methods for determining identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al., Nucle. Acid. Res., 12:387 (1984); Genetics Computer Group, University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., 215:403-410 (1990)). The BLASTX procedure is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (BLAST Manual, Altschul et al. NCB / NLM / NIH Bethesda, Md. 20894; Altschul et al., ibid.). The well-known Smith-Waterman algorithm can also be used to determine identity.

[0155] The specific alignment scheme used to align two amino acid sequences may result in matching only a shorter region of the two sequences, and this small alignment region may have very high sequence identity, even though there is no significant relationship between the two full-length sequences. Therefore, in a preferred embodiment, the selected alignment method (GAP procedure) will result in an alignment spanning at least 50 consecutive amino acids across the target polypeptide.

[0156] For example, the computer algorithm GAP (Genetics Computer Group, University of Wisconsin, Madison, Wisconsin) is used to align two peptides whose sequence identity percentages are to be determined to best match their respective amino acids (“matching span”, as determined by the algorithm). A gap-opening penalty (which is calculated as 3 times the average diagonal; “average diagonal” is the average of the diagonals of the comparison matrix used; “diagonal” is a score or number assigned to each best amino acid match through a specific comparison matrix) and a gap-extending penalty (which is typically {score(1 / 10)} times the gap-opening penalty), along with comparison matrices such as PAM 250 or BLOSUM 62, are used in conjunction with this algorithm. The algorithm also uses standard comparison matrices (for PAM 250 comparison matrices, see Dayhoff et al., Atlas of Protein Sequence and Structure, Vol. 5, Supp. 3 (1978); for BLOSUM 62 comparison matrices, see Henikoff et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America), 89:10915-10919 (1992)).

[0157] Preferred parameters for peptide sequence comparison include the following:

[0158] Algorithm: Needleman et al., J. Mol. Biol (Journal of Molecular Biology), 48:443-453 (1970); Comparison matrix: BLOSUM 62 from Henikoff et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America), 89:10915-10919 (1992); Gap penalty: 12, Gap length penalty: 4, Similarity threshold: 0.

[0159] The GAP program uses the parameters described above. These parameters are the default parameters for peptide comparison using the GAP algorithm (and together with them, there is no penalty for terminal gaps).

[0160] Preferred parameters for nucleic acid molecular sequence comparison include the following:

[0161] Algorithm: Needleman et al., J. Mol Biol. (Molecular Biology Journal), 48:443-453 (1970); Comparison matrix: Match = +10, Mismatch = 0, Gap penalty: 50, Gap length penalty: 3.

[0162] The GAP program also uses the above parameters. These parameters are the default parameters for nucleic acid molecule comparisons.

[0163] Other exemplary algorithms can be used, such as gap opening penalties, gap extension penalties, comparison matrices, similarity thresholds, etc., including those described in the Program Manual, Wisconsin Package, 9th edition, September 1997. The specific choices to be made will be apparent to those skilled in the art and will depend on the specific comparison to be performed, such as DNA to DNA comparison, protein to protein comparison, or protein to DNA comparison; furthermore, regardless of whether the comparison is between given sequence pairs (in which case GAP or BestFit is generally preferred) or between a sequence and a large sequence database (in which case FASTA or BLASTA is preferred).

[0164] Preparation of Ficolin-related peptides and other peptides of the present invention

[0165] This invention also relates to methods for preparing the human Ficolin-related peptides and other peptides of the present invention described above. The Ficolin-related peptides and other peptides of the present invention described herein can be generated using recombinant nucleic acid technology. Typically, a cloned wild-type FAP nucleic acid sequence is modified to encode the desired protein. This modified sequence is then inserted into an expression vector, which is subsequently transformed or transfected into host cells. Higher eukaryotic cells, particularly cultured mammalian cells, are preferred as host cells. The complete amino acid and nucleotide sequences of human FAP are given by SEQ ID NO:1 and SEQ ID NO:2.

[0166] Amino acid sequence alterations can be achieved using a variety of techniques. Modification of nucleic acid sequences can be performed through site-specific mutagenesis. Techniques for site-specific mutagenesis are well known in the art and are described, for example, in Zoller and Smith (DNA 3:479-488, 1984) or "Splicing by extension overlap," Horton et al., Gene 77, 1989, pp. 61-68. Therefore, selective alterations can be introduced using the nucleotide and amino acid sequences of FAPs. Similarly, methods for preparing DNA constructs using polymerase chain reaction with specific primers are well known to those skilled in the art (see PCR Protocols, 1990, Academic Press, San Diego, California, USA).

[0167] The polypeptides of the present invention may also comprise non-naturally occurring amino acid residues. Non-naturally occurring amino acids include, but are not limited to, β-alanine, deaminohistidine, trans-3-methylproline, 2,4-methylproline, cis-4-hydroxyproline, trans-4-hydroxyproline, N-methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, piperacillin, thiazolidinylcarboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, valine, 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into polypeptides are known in the art. For example, in vitro systems can be used in which chemically aminoacylated repressor tRNAs are used to suppress nonsense mutations. Methods for synthesizing amino acids and aminoacylated tRNAs are known in the art. Transcription and translation of plasmids containing nonsense mutations were performed in a cell-free system comprising E. coli S30 extract and commercially available enzymes and other reagents. The peptides were purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993. In the second method, translation is performed in Xenopus oocytes via microinjection of mutant mRNA and chemically aminoacylated repressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third method, *E. coli* cells are cultured in the absence of the native amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-native amino acid (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-native amino acid is bound to the polypeptide to replace its native counterpart. See Koide et al., Biochem. 33:7470-6, 1994. Native amino acid residues can be converted into non-native species through in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0168] The nucleic acid constructs encoding the Ficolin-related peptides and other peptides of the present invention may suitably be genomic or cDNA-derived, for example, by preparing a genomic or cDNA library and by screening for all or part of the DNA sequence encoding the peptide using hybridization with synthetic oligonucleotide probes according to standard techniques (see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York, 1989).

[0169] Nucleic acid constructs encoding ficolin-related peptides can also be synthesized using established standard methods, such as the phosphorimide method described in Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859–1869, or the method described in Mattes et al., EMBO Journal 3 (1984), 801–805. Following the phosphorimide method, oligonucleotides are synthesized, for example, in an automated DNA synthesizer, purified, annealed, ligated, and cloned into suitable vectors. DNA sequences encoding the human ficolin-related peptides and other peptides of the present invention can also be prepared by polymerase chain reaction using specific primers, for example, as described below: US 4,683,202, Saiki et al., Science 239 (1988), 487–491, or Sambrook et al., ibid.

[0170] In addition, the nucleic acid construct can be a mixture of synthetic and genomic, synthetic and cDNA, or genomic and cDNA-derived fragments (appropriately) prepared according to standard techniques by linking synthetic, genomic or cDNA-derived fragments, said fragments corresponding to different parts of the complete nucleic acid construct.

[0171] The nucleic acid construct is preferably a DNA construct. The DNA sequences used to generate the Ficolin-related peptides and other peptides of the present invention typically encode a pre-proto-peptide at the N-terminus of the FAP, thereby enabling proper post-translational processing and secretion from the host cell.

[0172] The DNA sequences encoding the human Ficolin-related polypeptides and other polypeptides of the present invention are typically inserted into a recombinant vector, which can be any vector that facilitates the recombinant DNA step, and the choice of vector generally depends on the host cell to which the vector is to be introduced. Therefore, the vector can be a spontaneously replicating vector, i.e., a vector existing as an extrachromosomal entity whose replication does not depend on chromosome replication, such as a plasmid. Alternatively, the vector can be a vector that, when introduced into a host cell, integrates into the host cell genome and replicates along with the chromosome already integrated therein.

[0173] The vector is preferably an expression vector in which the DNA sequence encoding the human Ficolin-related polypeptide and other polypeptides of the present invention is operatively linked to additional fragments required for transcription of the DNA. Typically, the expression vector is derived from plasmid or viral DNA, or may contain components of both. The term "operatively linked" means that the fragments are arranged such that they function for their intended purpose, for example, transcription initiated by a promoter and proceeding along the DNA sequence encoding the polypeptide.

[0174] Expression vectors used to express the Ficolin-related peptides and other peptides of the present invention contain a promoter capable of directing the transcription of a cloned gene or cDNA. The promoter can be any DNA sequence that exhibits transcriptional activity in a selected host cell and can be derived from a gene encoding a protein that is homologous or heterologous to the host cell.

[0175] Examples of suitable promoters that direct the transcription of DNA encoding human Ficolin-related polypeptides in mammalian cells are the SV40 promoter (Subramani et al., Mol. Cell Biol. (Molecular Cell Biology) 1 (1981), 854-864), the MT-1 (metallothionein gene) promoter (Palmiter et al., Science 222 (1983), 809-814), the CMV promoter (Boshart et al., Cell 41:521-530, 1985) or the adenovirus 2 major late promoter (Kaufman and Sharp, Mol. Cell Biol. (Molecular Cell Biology), 2:1304-1319, 1982).

[0176] Examples of suitable promoters for insect cells include the polyhedrosis protein promoter (US 4,745,051; Vasuvedan et al., FEBS Lett. 311, (1992) 7-11), the P10 promoter (JM Vlak et al., J. Gen. Virology 69, 1988, pp. 765-776), the basic protein promoter of the alfalfa silver-striped moth polyhedrosis virus (Autographa californica polyhedrosis virus basic protein promoter) (EP 397485), the baculovirus immediate early gene 1 promoter (US 5,155,037; US 5,162,222), or the baculovirus 39K delayed early gene promoter (US 5,155,037; US 5,162,222).

[0177] Examples of suitable promoters for use in yeast host cells include promoters from yeast glycolysis genes (Hitzeman et al., J. Biol. Chem. 255 (1980), 12073-12080; Alber and Kawasaki, J. Mol. Appl. Gen. 1 (1982), 419-434) or alcohol dehydrogenase genes (Young et al., Genetic Engineering of Microorganisms for Chemicals (Hollaender et al., eds.), Plenum Press, New York, 1982), or TPI1 (US 4,599,311) or ADH2-4c (Russell et al., Nature 304 (1983), 652-654).

[0178] Examples of suitable promoters for filamentous fungal host cells include, for example, the ADH3 promoter (McKnight et al., The EMBO J. 4 (1985), 2093-2099) or the tpiA promoter. Other useful promoters are derived from genes encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic protease, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-amylase, Aspergillus niger or Aspergillus awamori gluA, Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, or Aspergillus nidulans acetamase. TAKA-amylase and gluA promoters are preferred. Suitable promoters are mentioned, for example, in EP 238 023 and EP 383 779.

[0179] If desired, the DNA sequences encoding the human Ficolin-related polypeptides and other polypeptides of the present invention can also be operatively ligated to suitable terminators, such as the human growth hormone terminator (Palmiter et al., Science 222, 1983, pp. 809-814) or the TPI1 terminator (Alber and Kawasaki, J. Mol. Appl. Gen. 1, 1982, pp. 419-434) or the ADH3 terminator (McKnight et al., The EMBO J. 4, 1985, pp. 2093-2099). The expression vector may also contain a set of RNA splicing sites located downstream of the promoter and upstream of the insertion site of the FAP sequence itself. Preferred RNA splicing sites may be obtained from adenovirus and / or immunoglobulin genes. The expression vector also contains a polyadenylation signal located downstream of the insertion site. Particularly preferred polyadenylation signals include early or late polyadenylation signals from SV40 (Kaufman and Sharp, ibid.), polyadenylation signals from the 5Elb region of adenovirus, human growth hormone gene terminators (DeNoto et al. Nucl. Acids Res. 9:3719-3730, 1981), or polyadenylation signals from the human FAP gene or bovine FAP gene. Expression vectors may also include non-coding viral leader sequences, such as the adenovirus 2 triplet leader sequence located between the promoter and the RNA splicing site; and enhancer sequences, such as the SV40 enhancer.

[0180] To direct the human Ficolin-related peptides and other peptides of the present invention into the secretion pathway of host cells, a secretion signal sequence (also known as a leader sequence, pre-origin sequence, or pre-sequence) can be provided in a recombinant vector. The secretion signal sequence is linked to the DNA sequence encoding the human Ficolin-related peptides and other peptides of the present invention in the correct reading frame. The secretion signal sequence is typically located at the 5' of the DNA sequence encoding the peptide. The secretion signal sequence may be generally associated with the protein or may originate from a gene encoding another secreted protein.

[0181] For secretion from yeast cells, the secretion signal sequence can encode any signal peptide that ensures efficient guidance of the expressed human Ficolin-related polypeptide and other polypeptides of the present invention to the cellular secretion pathway. The signal peptide can be a naturally occurring signal peptide, its functional portion, or a synthetic peptide. Suitable signal peptides have been identified as the α-factor signal peptide (see US 4,870,008), the mouse salivary amylase signal peptide (see O. Hagenbuchle et al., Nature 289, 1981, pp. 643-646), modified carboxypeptidase signal peptides (see LAValls et al., Cell 48, 1987, pp. 887-897), the yeast BAR1 signal peptide (see WO 87 / 02670), or the yeast aspartic protease 3 (YAP3) signal peptide (see M. Egel-Mitani et al., Yeast 6, 1990, pp. 127-137).

[0182] For efficient secretion in yeast, the sequence encoding the leader peptide can be inserted downstream of the signal sequence and upstream of the DNA sequence encoding the human Ficolin-related peptide and other peptides of the present invention. The leader peptide functions to allow the expressed peptide to be directed from the endoplasmic reticulum to the Golgi apparatus and further to secretory vesicles, thereby being secreted into the culture medium (i.e., exporting the human Ficolin-related peptide and other peptides of the present invention to the extracellular space or at least through the cell membrane into the periplasmic space of the yeast cell). The leader peptide can be a yeast α-factor leader (its applications are described, for example, in US 4,546,082, US 4,870,008, EP 16 201, EP 123 294, EP 123 544, and EP 163 529). Alternatively, the leader peptide can be a synthetic leader peptide, considered not to be a naturally occurring leader peptide. Synthetic leader peptides can be constructed, for example, as described in WO 89 / 02463 or WO 92 / 11378.

[0183] For applications in filamentous fungi, the signal peptide can be conveniently derived from genes encoding Aspergillus amylase or glucosylamylase, Mucor milhexa lipase or protease, or Humicola lanuginosa lipase. Preferably, the signal peptide is derived from genes encoding Aspergillus oryzae TAKA amylase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable amylase, or Aspergillus niger glucosylamylase. Suitable signal peptides are disclosed, for example, in EP 238 023 and EP215 594.

[0184] For applications in insect cells, signal peptides can be conveniently derived from insect genes (see WO 90 / 05783), such as the signal peptide of the lipomobilization hormone precursor in the lepidopteran tobacco hawk moth (Manduca sexta) (see US 5,023,328).

[0185] The steps for linking DNA sequences encoding the human ficolin-related polypeptide and other polypeptides of the present invention, promoters and optional terminators and / or secretion signal sequences, respectively, and inserting them into appropriate vectors containing information necessary for replication are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989).

[0186] Methods for transfecting mammalian cells and expressing the introduced DNA sequence are described in, for example, Kaufman and Sharp, J. Mol. Biol. (Journal of Molecular Biology) 159 (1982), 601-621; Southern and Berg, J. Mol. Appl. Genet. (Journal of Molecular Applied Genetics) 1 (1982), 327-341; Loyter et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America) 79 (1982), 422-426; Wigler et al., Cell 14 (1978), 725; Corsaro and Pearson, Somatic Cell Genetics 7 (1981), 603; Graham and van der Eb., Virology 52 (1973), 456; and Neumann et al., EMBO J. (EMBO Journal) 1 (1982), 841–845.

[0187] The cloned DNA sequence is introduced into cultured mammalian cells via, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-732, 1978; Corsaro and Pearson, Somatic Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) or electroporation (Neumann et al., EMBO J. 1:841-845, 1982). To identify and select cells expressing exogenous DNA, genes conferring selectable phenotypes (selectable markers) are typically introduced into the cells along with the target gene or cDNA. Preferred selectable markers include genes conferring resistance to drugs such as neomycin, hygromycin, and methotrexate. Selectable markers can be amplifiable selectable markers. A preferred amplifiable selectable marker is a dihydrofolate reductase (DHFR) sequence. The optional markers are described in the Thilly review (Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA, incorporated herein by reference). Those skilled in the art will readily be able to select the appropriate optional markers.

[0188] The optional marker can be introduced into the cell simultaneously with the target gene on a separate plasmid, or they can be introduced into the cell on the same plasmid. If on the same plasmid, the optional marker and the target gene can be under the control of different promoters or the same promoter, the latter arrangement producing a bicistronic messenger. This type of construct is known in the art (e.g., Levinson and Simonsen, US4,713,339). It is also advantageous to add additional DNA (known as "vector DNA") to the mixture introduced into the cell.

[0189] After cells have taken up the DNA, they are grown in an appropriate culture medium, typically for 1-2 days, and begin to express the target gene. When used herein, the term "appropriate growth medium" refers to a culture medium containing the nutrients and other components necessary for cell growth and expression of the target human Ficolin-related peptide. The culture medium typically includes a carbon source, a nitrogen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, proteins, and growth factors. Drug selection is then applied to select cells that stably express the selectable marker. For cells already transfected with an amplifiable selectable marker, the drug concentration can be increased to select clones with increased copy numbers, thereby increasing expression levels. Clones of stably transfected cells are then screened for expression of the target human Ficolin-related peptide.

[0190] The host cell into which the DNA sequence encoding the human Ficolin-related polypeptide and other polypeptides of the present invention is introduced can be any cell capable of producing post-translational modified human polypeptides, and includes yeast, fungi and higher eukaryotic cells.

[0191] Examples of mammalian cell lines used in this invention include COS-1 (ATCC CRL 1650), juvenile hamster kidney (BHK), and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977). A preferred BHK cell line is the tk-ts13 BHK cell line (Waechter and Baserga, Proc. Natl. Acad. Sci. USA 79:1106-1110, 1982, which is incorporated herein by reference), hereinafter referred to as BHK 570 cells. The BHK 570 cell line is deposited at the American Type Culture Collection (12301 Parklawn Dr., Rockville, Md. 20852) under ATCC accession number CRL 10314. The tk-ts13BHK cell line can also be obtained from ATCC, with accession number CRL 1632. In addition, several other cell lines can be used in this invention, including rat Hep I (rat hepatocellular carcinoma; ATCC CRL 1600), rat Hep II (rat hepatocellular carcinoma; ATCC CRL 1548), TCMK (ATCC CCL 139), human lung (ATCC HB 8065), NCTC1469 (ATCC CCL 9.1), CHO (ATCC CCL 61), and DUKX cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America) 77:4216-4220, 1980).

[0192] Examples of suitable yeast cells include cells of the genera *Saccharomyces* spp. or *Schizosaccharomyces* spp., particularly strains of *Saccharomyces cerevisiae* or *Saccharomyces kluyveri*. Methods for transforming yeast cells with heterologous DNA and producing heterologous peptides from them are described, for example, in US 4,599,311, US 4,931,373, US 4,870,008, 5,037,743, and US 4,845,075, all of which are incorporated herein by reference. Transformed cells are selected by a phenotype determined by selectable markers, typically drug resistance or the ability to grow under conditions lacking a specific nutrient (e.g., leucine). A preferred vector for use in yeast is the POT1 vector disclosed in US 4,931,373. The DNA sequence encoding the human ficolin-related polypeptide and other polypeptides of the present invention may follow the signal sequence and optionally follow the leader sequence, for example, as described above. Other examples of suitable yeast cells are strains of the genus Kluyveromyces, such as K. lactis, the genus Hansenula, such as H. polymorpha, or the genus Pichia, such as P. pastoris (see Gleeson et al., J. Gen. Microbiol. 132, 1986, pp. 3459-3465; US 4,882,279).

[0193] Other examples of fungal cells are filamentous fungal cells, such as those of the genera *Aspergillus*, *Neurospora*, *Fusarium*, or *Trichoderma*, particularly strains of *Aspergillus oryzae*, *Aspergillus nidus*, or *Aspergillus niger*. Protein expression using *Aspergillus* is documented, for example, in EP 272 277, EP 238 023, and EP 184 438. Transformation of *F. oxysporum* is performed, for example, as described in Malardier et al., 1989, Gene 78:147-156. Transformation of *Trichoderma*, for example, can be performed as described in EP 244 234.

[0194] When filamentous fungi are used as host cells, they can be transformed into the DNA constructs of the present invention, conveniently yielding recombinant host cells by integrating the DNA constructs into the host chromosome. This integration is generally considered advantageous because the DNA sequence is more likely to remain stably held in the cell. Integration of the DNA constructs into the host chromosome can be performed using conventional methods, for example, through homologous or heterologous recombination.

[0195] Transformation of insect cells and the production of heterologous polypeptides therein can be performed as described in US 4,745,051; US ​​4,879,236; US 5,155,037; 5,162,222; EP 397,485, all of which are incorporated herein by reference. Insect cell lines used as hosts can suitably be Lepidoptera cell lines, such as those of the fall armyworm (Spodopterafrugiperda) or the white armyworm (Trichoplusia ni) (see US 5,077,214). Culture conditions can suitably be as described, for example, in WO 89 / 01029 or WO 89 / 01028, or in any of the foregoing references.

[0196] The transformed or transfected host cells are then cultured in a suitable nutrient medium under conditions that allow for the expression of human Ficolin-related peptides. All or part of the peptides obtained can then be recovered from the culture. The culture medium used to culture the cells can be any conventional medium suitable for host cell growth, such as minimal medium or a complex medium containing appropriate supplements. Suitable media are available from commercial suppliers or can be prepared according to published formulations (e.g., in the catalogue of the American Type Culture Collection). The human Ficolin-related peptides produced by the cells can then be recovered from the culture medium by routine steps, including separating the host cells from the medium by centrifugation or filtration, precipitating the protein-like components of the supernatant or filtrate by salt (e.g., ammonium sulfate), and purifying them by various chromatographic methods, such as ion exchange chromatography, gel filtration chromatography, affinity chromatography, etc., depending on the type of peptide in question.

[0197] Transgenic animal technology can be used to produce the Ficolin-related polypeptides and other polypeptides of the present invention. Preferably, the proteins are produced in the mammary glands of female mammalian hosts. Expression in the mammary glands followed by secretion of the target protein into the milk overcomes many difficulties encountered when isolating proteins from other sources. The milk is easy to collect, can be obtained in large quantities, and is well-characterized biochemically. Furthermore, the main milk proteins are present in the milk at high concentrations (typically about 1 to 15 g / L).

[0198] From a commercial point of view, it is clear that species with high milk yields are preferred as hosts. While smaller animals, such as mice and rats, may be used (and are preferred at the evidence-gathering stage), livestock mammals, including but not limited to pigs, goats, sheep, and cattle, are preferred. Sheep are particularly preferred due to factors such as the species’ prior transgenic history, milk yield, cost, and availability of equipment for collecting sheep milk (see, for example, WO 88 / 00239, a comparison of factors influencing host species selection). Ideally, host animal breeds that have been bred for dairy use, such as East Friesland sheep, or cattle and sheep recently introduced into dairy farms through transgenic breeding, should be selected. In either case, known, healthy animals should be used.

[0199] To obtain expression in the mammary gland, transcription promoters from lactalbumin genes are used. Lactalbumin genes include those encoding casein (see US5,304,489), β-lactoglobulin, α-lactalbumin, and whey acidic proteins. β-lactoglobulin (BLG) promoters are preferred; in the case of sheep β-lactoglobulin genes, the region of at least the nearest 406 bp of the gene's 5' flanking sequence is typically used, although larger 5' flanking sequences are preferred, up to about 5 kbp, such as a ~4.25 kbp DNA fragment containing the 5' flanking promoter and the non-coding portion of the β-lactoglobulin gene (see Whitelaw et al., Biochem.J. 286:31 39 (1992)). Similar promoter DNA fragments from other species are also suitable.

[0200] Other regions of the β-lactoglobulin gene may also be incorporated into the construct, as may genomic regions of the gene to be expressed. It is generally accepted in the art that constructs lacking introns, for example, express poorly compared to those containing the stated DNA sequence (see Brinster et al., Proc. Natl. Acad. Sci. USA 85:836 840 (1988); Palmier et al., Proc. Natl. Acad. Sci. USA 88:478 482 (1991); Whitelaw et al., Transgenic Res. 1:3 13 (1991); WO89 / 01343; and WO 91 / 02318, which are respectively incorporated herein by reference). In this regard, it is generally preferred, where possible, to use all or some of the natural introns of a gene encoding the target protein or polypeptide, and therefore preferably to further include at least some introns from, for example, the β-lactoglobulin gene. One such region is a DNA sequence from the 3' uncoding region of the sheep β-lactoglobulin gene that provides intron splicing and RNA polyadenylation. When the natural 3' uncoding sequence of the gene is replaced, the sheep β-lactoglobulin fragment can enhance and stabilize the expression level of the target protein or polypeptide. In other embodiments, the region around the initiating ATG of the FAP sequence is replaced with a corresponding sequence from a milk-specific protein gene. Such a replacement provides a presumed tissue-specific initiation environment, thereby enhancing expression. Although smaller regions can be replaced, it is convenient to replace the entire FAP progenitor and 5' uncoding sequence with, for example, the BLG gene.

[0201] For the expression of the Ficolin-related polypeptides and other polypeptides of the present invention in transgenic animals, an expression unit is generated by operatively linking a DNA fragment encoding FAP to an additional DNA fragment required for its expression. The additional fragment includes the promoter mentioned above, as well as sequences providing for transcription termination and mRNA polyadenylation. The expression unit further includes a DNA fragment encoding a secretion signal sequence, which is operatively linked to a fragment encoding the modified FAP. The secretion signal sequence may be a natural FAP secretion signal sequence or may be a secretion signal sequence of another protein (such as a lactalbumin) (see, for example, von Heijne, Nucl. Acids Res. 14:4683 4690 (1986); and Meade et al., US4,873,316, which is incorporated herein by reference).

[0202] Although expression units can be constructed essentially by linking arbitrary sequences, the construction of expression units for transgenic units is conveniently carried out by inserting the FAP sequence into a plasmid or phage vector containing an additional DNA fragment. It is particularly convenient to provide a vector containing a DNA fragment encoding a milk protein, and to replace the coding region of the milk protein with the coding region of the FAP variant; this produces a gene fusion containing the expression control sequence of the milk protein gene. In any case, the expression unit is cloned into a plasmid or other vector to facilitate the amplification of the FAP sequence. Amplification is conveniently carried out in bacterial (e.g., *E. coli*) host cells, whereby the vector typically includes an origin of replication functioning in the bacterial host cell and optional markers. The expression unit is then introduced into the fertilized egg (including early-stage embryos) of the selected host species. The introduction of heterologous DNA can be accomplished through several pathways, including microinjection (e.g., U.S. Patent No. 4,873,191), retroviral infection (Jaenisch, Science 240:1468 1474 (1988)), or site-directed integration using embryonic stem (ES) cells (reviewed by Bradley et al., Bio / Technology 10:534 539 (1992)). The egg is then implanted into the fallopian tube or uterus of a pseudopregnant female and allowed to develop to full term. Offspring carrying the introduced DNA in their germline can pass that DNA on to their offspring in a normal, Mendelian manner, allowing for the development of a transgenic population.General procedures for producing transgenic animals are known in the art (see, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Bio / Technology 6:179-183 (1988); Wall et al., Biol. Reprod. 32:645-651 (1985); Buhler et al., Bio / Technology 8:140-143 (1990); Ebert et al., Bio / Technology 9:835-838 (1991); Krimpenfort et al., Bio / Technology 9:844-847 (1991); Wall et al., J. Cell. Biochem. 49:113 120 (1992); US4,873,191; US4,873,316; WO 88 / 00239, WO 90 / 05188, WO 92 / 11757; and GB87 / 00458). Techniques for introducing exogenous DNA sequences into mammals and their germline cells were initially developed in mice (see, for example, Gordon et al., Proc. Natl. Acad. Sci. USA 77:73807384 (1980); Gordon and Ruddle, Science 214:1244 1246 (1981); Palmiter and Brinster, Cell 41:343). 345 (1985); Brinster et al., Proc. Natl. Acad. Sci. USA (Proceedings of the National Academy of Sciences of the United States of America) 82:4438 4442 (1985); and Hogan et al. (ibid.)). These techniques were subsequently applied to larger animals, including livestock species (see, for example, WO 88 / 00239, WO 90 / 05188, and WO 92 / 11757; and Simons et al., Bio / Technology 6:179 183 (1988)).In summary, the most effective method currently used to produce transgenic mice or livestock involves injecting hundreds of linear target DNA molecules into a pronucleus of a fertilized egg, following established techniques. Alternatively, DNA can be injected into the cytoplasm of the zygote.

[0203] It can also be utilized in the production of transgenic plants. Expression can be generalized or directed to specific organs, such as tubers (see Hiatt, Nature 344:469 479 (1990); Edelbaum et al., J. Interferon Res. 12:449 453 (1992); Sijmons et al., Bio / Technology 8:217 221 (1990); and EP 0 255 378).

[0204] FAP purification

[0205] The Ficolin-related peptides and other peptides of the present invention can be recovered from cell culture media or milk. The Ficolin-related peptides and other peptides of the present invention can be purified by a variety of methods known in the art, including but not limited to, chromatography (e.g., ion exchange, affinity chromatography, hydrophobic chromatography, focusing chromatography, and size exclusion), electrophoresis (e.g., preparative isoelectric focusing (IEF), differential solubility (e.g., ammonium sulfate precipitation), or extraction (see, for example, *Protein Purification*, eds. J.-C. Janson and Lars Ryden, VCH Publishers, New York, 1989). Preferably, they can be purified by affinity chromatography on an anti-FAP antibody column. Additional purification can be achieved by conventional chemical purification methods, such as high-performance liquid chromatography. Other purification methods, including barium citrate precipitation, are known in the art and can be applied to purify the novel Ficolin-related peptides and other peptides described herein (see, for example, *Scopes, R., *Protein Purification*, Springer-Verlag, NY, 1982).

[0206] For therapeutic purposes, it is preferred that the Ficolin-related peptides and other peptides of the present invention be substantially pure. Therefore, in a preferred embodiment of the invention, the Ficolin-related peptides and other peptides of the present invention are purified to at least about 90-95% homogeneity, preferably at least about 98% homogeneity. Purity can be assessed, for example, by gel electrophoresis and N-terminal amino acid sequencing.

[0207] The term "isolated polypeptide" refers to the polypeptide (1) of the present invention having been isolated from at least about 50% of its naturally occurring polynucleotides, lipids, carbohydrates, or other substances (i.e., contaminants). Preferably, the isolated polypeptide is substantially free of any other contaminating polypeptides or other contaminants present in its natural environment that would interfere with its therapeutic, diagnostic, preventative, or research applications.

[0208] The term "microorganism" as used in this article refers to bacteria, fungi, archaea, protozoa; microscopic plants and animals (such as green algae or plankton), planarians, and amoebas. This definition includes pathogenic microorganisms.

[0209] Determination method

[0210] General steps for SDS-PAGE and Western blotting:

[0211] If recommended by the supplier, use Electrophoresis was performed on 10% or 4–12% (w / v) Bis-Tris polyacrylamide gels using a discontinuous buffer system (Invitrogen). Western blotting was performed using a polyvinylidene fluoride membrane (PVDF-HyBond, GE-healthcare, Hilleroed, Denmark, catalog number RPN303F), 2 μg / ml biotin-labeled primary monoclonal antibody, and secondary development using HRP-conjugated streptavidin (P0397, Dako, Glostrup, Denmark) diluted 1:1500 in PBS, 0.05% Tween 20. The membranes were developed with acetone and 0.04% 3-amino-9-ethylcarbazole (Sigma-aldrich, Broenby, Denmark, catalog number A5754-100G) in 0.015% H2O2 at pH 50 mM sodium citrate buffer.

[0212] Co-immunoprecipitation:

[0213] Immunoprecipitation of mannose-binding lectin (MBL) serum complex: 1 ml of normal human serum was diluted 1:1 in TBS (10 mM Tris, 140 mM NaCl, pH 7.5) and incubated at 4°C for 1 hour with 5 μg of MBL-specific mouse monoclonal antibody Hyb131-11 (Bioporto, Gentotte, Denmark).

[0214] Immunoprecipitation of Ficolin-2 serum complex: 0.5 ml of normal human serum was diluted 1:1 in TBS (10 mM Tris, 140 mM NaCl, pH 7.5) and incubated at 4°C for 1 hour with 5 μg of Ficolin-2 specific mouse monoclonal antibody Hyb219 (Munthe-Fog L, et al.).

[0215] Immunoprecipitation of Ficolin-3 serum complex: 0.2 ml of normal human serum was diluted 1:1 in TBS (10 mM Tris, 140 mM NaCl, pH 7.5) and incubated at 4°C for 1 hour with 5 μg of Ficolin-3 specific mouse monoclonal antibody Hyb334 (Munthe-Fog L, et al.).

[0216] Immune complex precipitation was performed using sheep anti-mouse IgG conjugated magnetic dynal beads (Dynal-Invitrogen, catalog number 112.02D): after incubation with serum and primary antibody (as described above), 5 x 10⁻⁶ beads were added. 7 Sheep anti-mouse conjugated magnetic dynal beads were incubated at 4°C for 30 minutes. The beads were then magnetically separated and treated with TBS-Tween-Ca. 2+ Washed three times with (10 mM Tris, 140 mM NaCl, 0.05% Tween, 5 mM CaCl2, pH 7.5), and finally boiled in SDS-PAGE and analyzed by Western blotting. Western blotting was performed using biotin-labeled monoclonal antibody mAb-8B3 (reacting with epitopes on the heavy chain / A chain shared by MASP-1 and -3).

[0217] Immunoaffinity purification of FAP: 10 mg mAb-8B3 (epitope reaction on the heavy chain / A chain common to FAP, MASP-1 and -3) or 10 mg rabbit polyclonal anti-FAP antibody was conjugated onto CNBr-activated agarose (GE-healthcare, Hilleroed, Denmark, catalog number 17-0430-01) and packed onto a column as recommended by the supplier.

[0218] Purification from serum: 150 ml of normal human serum pooling buffer was diluted 1:1 with TBS + 0.5 M NaCl + 10 mM EDTA (10 mM Tris, 640 mM NaCl, 10 mM EDTA, pH 7.5) and loaded onto the column described above. The column was washed with 1 μL of TBS + 0.5 M NaCl + 10 mM EDTA and eluted with 1 M glycine-HCl, pH 2.5, for a fraction of 1 ml. The fraction was analyzed by SDS-PAGE and Western blotting using biotin-labeled monoclonal antibody mAb-8B3.

[0219] Purification of recombinant FAP: 2-3 μL of culture supernatant from Chinese hamster ovary cells (CHO cells) expressing recombinant FAP (rFAP) (from CHO serum-free medium / Gibco-Invitrogen, catalog number 12651-014) was loaded onto the antibody column described above. The column was washed with 1.5 μL of TBS + 0.5 M NaCl + 10 mM EDTA, and eluted 1 mL of fraction with 1 M glycine-HCl, pH 2.5. The eluted fractions were analyzed by SDS-PAGE and Coomassie staining.

[0220] Recombinant expression of FAP: The pcDNA5 / FRT vector (Invitrogen, catalog number V6010-20) containing full-length cDNA was ordered from Genscript (Genscript, New Jersey, USA) and co-transfected with the pOG44 vector (Invitrogen, catalog number V6005-20) into the CHO Flp-In cell line (Invitrogen, catalog number R758-07). Cell selection and cloning were performed according to the supplier's recommendations (Invitrogen). Cells were grown in Freestyle CHO serum-free medium (Invitrogen, catalog number 12651-014), and the culture supernatant was collected and analyzed.

[0221] Production of monoclonal and polyclonal antibodies: As recommended by the supplier (Thermo Fisher Scientific / Pierce, Illinois, USA), a peptide construct of 17 C-terminal residues specific to FAP (ordered from Genscript, New Jersey, USA) was conjugated to the toxoid form of tetanus and diphtheria using a cysteine ​​conjugation method with m-maleimide benzoyl-N-hydroxysuccinimide ester.

[0222] Six mice and two rabbits were immunized three times (14 days apart) with 25 μg of antigen adsorbed onto Al(OH)3 and Freund's incomplete adjuvant. Polyclonal antibody titers were assessed using ELISA with different FAP peptides conjugated to protein carriers.

[0223] Polyclonal rabbit antiserum (≈10 ml) was collected 14 days after the first, second and third immunizations.

[0224] Two mice were used to produce monoclonal antibodies. Four days prior to fusion, the mice received an intravenous injection of 25 μg of antigen. Fusion was performed as described elsewhere (Kohler, G. and C. Milstein. 1975. Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256:495-497).

[0225] Clones are selected by differential ELISA screening of peptides coupled to different protein carriers.

[0226] Functional complement assay: FAP function was studied using serum homozygous deficient in Ficolin-3 and MBL.

[0227] Ficolin-3 assay: Maxisorp plates (NUNC, Roskilde, Denmark, catalog number 439454) were coated with 5 μg / ml acetylated bovine serum albumin in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.5) at 4°C for 12 hours. After blocking / washing four times in barbiturate / Tween buffer (4 mM barbiturate, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4 + 0.05% Tween), 500 ng / ml recombinant human Ficolin-3 in barbiturate / Tween buffer was added, and the plate was incubated at 20°C with shaking for 1.5 hours. After washing the plates twice in barbiturate / Tween buffer, serially diluted recombinant FAP, human MASP-1, -2, or -3 as serum-free culture supernatant was added to the first dimension of the separated plates, and the plates were incubated with shaking at 20°C for 1 hour. After washing the plates twice in barbiturate / Tween buffer, serially diluted Ficolin-3 or MASP-2 deficient serum was added to the second dimension of the plates, and the plates were incubated at 37°C for 30 minutes. After washing the plates four times in barbiturate / Tween buffer, complement factor C4 deposition was measured by incubation at 20°C for 1 hour with a 1:2000 diluted polyclonal rabbit antibody against human C4c (Dako, Glostrup, Danish catalog number Q0369), followed by four washes, and incubation at 20°C for 45 minutes with horseradish peroxidase-conjugated porcine anti-rabbit antibody (Dako, Glostrup, Danish catalog number P0399). The signal was obtained by dissolving o-phenylenediamine (OPD) (0.4 mg / ml) in citrate buffer (35 mM citrate, 65 mM Na2PO4, pH 5) with 0.12‰ (v / v) H2O2 in 100 μl / well for color development. The enzyme reaction was terminated with 1 M H2SO4, and the optical density (OD) levels were measured in the range of 490 nm–650 nm using a V-max kinetic-reader (molecular device).

[0228] Mannose-binding lectin assay: Maxisorp plates (NUNC, Roskilde, Denmark, catalog number 439454) were coated with 10 μg / ml mannan (Sigma-Aldrich, Broenby, Denmark, catalog number M7504-1G) in coating buffer (15 mM Na2CO3, 35 mM NaHCO3, pH 9.5) for 12 hours at 4°C. After blocking / washing four times in barbiturate / Tween buffer (4 mM barbiturate, 145 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, pH 7.4 + 0.05% Tween), 0.5 μg / ml recombinant human mannose-binding lectin in barbiturate / Tween buffer was added, and the plate was incubated at 20°C with shaking for 1.5 hours. After washing the plates twice in barbiturate / Tween buffer, serially diluted recombinant FAP, human MASP-1, -2, or -3 as serum-free culture supernatant was added to the first dimension of the separated plates, and the plates were incubated with shaking at 20°C for 1 hour. After washing the plates twice in barbiturate / Tween buffer, serially diluted MBL or MASP-2 deficient serum was added to the second dimension of the plates, and the plates were incubated at 37°C for 45 minutes. After washing the plates four times in barbiturate / Tween buffer, complement factor C4 deposition was measured by incubation at 20°C for 1 hour with a 1:2000 diluted polyclonal rabbit antibody against human C4c (Dako, Glostrup, Danish catalog number Q0369), followed by four washes, and incubation at 20°C for 45 minutes with horseradish peroxidase-conjugated porcine anti-rabbit antibody (Dako, Glostrup, Danish catalog number P0399). The signal was obtained by colorimetric development of o-phenylenediamine (OPD) (0.4 mg / ml) dissolved in 100 μl / well of citrate buffer (35 mM citrate, 65 mM Na2PO4, pH 5) with 0.12‰ (v / v) H2O2. The enzyme reaction was terminated with 1 M H2SO4, and the optical density (OD) levels were measured in the 490 nm–650 nm range using a V-max kinetic-reading instrument (molecular devices).

[0229] Genotyping: Different genotyping methods can be used, among which biological assays are employed to determine the genotype of an individual. Various types of assays can be used, such as:

[0230] Hybridization-based methods

[0231] ο Dynamic allele-specific hybridization

[0232] ο Molecular indicator

[0233] οSNP microarray

[0234] Enzyme-based methods

[0235] Restricted fragment length polymorphism

[0236] ο PCR-based methods

[0237] οFlap endonuclease

[0238] primer extension

[0239] ο5'-nuclease

[0240] Oligonucleotide ligase assay

[0241] Other post-amplification methods based on the physical properties of DNA

[0242] ο Single-chain verification of polymorphism

[0243] Temperature gradient gel electrophoresis

[0244] denaturing high performance liquid chromatography

[0245] ο High-resolution unwinding of complete amplicon

[0246] οSNPlex

[0247] Sequencing

[0248] Application and pharmaceutical composition

[0249] Combination therapy

[0250] The ficolin-related peptides defined in this specification can be administered simultaneously or sequentially with one or more proteins selected from Ficolin-1, 2, 3, and mannose-binding lectin (MBL). The factor can be provided in a single dosage form comprising two compounds, or in the form of a component kit comprising a ficolin-related peptide formulation as a first unit dosage form and a formulation of the one or more other compounds as a second unit dosage form. Whenever the first, second, or third unit dosage, etc., is mentioned throughout this specification, it does not indicate a preferred order of administration, but is mentioned so for convenience only.

[0251] "Simultaneous" administration of ficolin-related peptide formulations and formulations of one or more other compounds means administration of the compound in a single dosage form, or administration of a first agent followed by a second agent, with an interval not exceeding 15 minutes, preferably 10 minutes, more preferably 5 minutes, and even more preferably 2 minutes. Either factor may be administered first.

[0252] "Sequential" administration means administering the first dose followed by the second dose, with an interval of more than 15 minutes. Either of the two unit dosage forms may be administered first. Preferably, both products are injected via the same intravenous route.

[0253] Another object of the present invention is to provide a pharmaceutical formulation comprising a ficolin-related polypeptide present at a serum / plasma concentration of 0 mg / ml to 1 mg / ml, and wherein said formulation has a pH of 2.0-10.0. The formulation may further comprise a buffer system, preservatives, isotonic agents, chelating agents, stabilizers, and surfactants. In some embodiments of the invention, the pharmaceutical formulation is an aqueous formulation, i.e., a formulation containing water. The formulation is typically a solution or suspension. In another embodiment of the invention, the pharmaceutical formulation is an aqueous solution. The term "aqueous formulation" is defined as a formulation containing at least 50% w / w water. Similarly, the term "aqueous solution" is defined as a solution containing at least 50% w / w water, and the term "aqueous suspension" is defined as a suspension containing at least 50% w / w water.

[0254] In other embodiments, the pharmaceutical preparation is a freeze-dried preparation to which a physician or patient adds solvent and / or diluent before use.

[0255] In other embodiments, the pharmaceutical formulation is a dry formulation that does not require pre-dissolution and is ready for immediate use (e.g., freeze-dried or spray-dried). In another aspect, the present invention relates to pharmaceutical formulations comprising an aqueous solution of a ficolin-related polypeptide and a buffer, wherein the ficolin-related polypeptide is present at a serum / plasma concentration of 0-1 mg / ml or higher, and wherein the formulation has a pH of about 2.0 to about 10.0.

[0256] In other embodiments of the invention, the pH of the formulation is selected from the list of the following: 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6. 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, and 10.0.

[0257] In another embodiment of the invention, the buffer is selected from the group consisting of: sodium acetate, sodium carbonate, citrate, glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium phosphate, and tris(hydroxymethyl)aminomethane, N-di(hydroxyethyl)glycine, tris(hydroxymethyl)methylglycine, malic acid, succinate, maleic acid, fumaric acid, tartaric acid, aspartic acid, or mixtures thereof. Each of these specific buffers constitutes an alternative embodiment of the invention.

[0258] In another embodiment of the invention, the formulation further comprises a pharmaceutical preservative. In another embodiment of the invention, the preservative is selected from the group consisting of phenol, o-cresol, m-cresol, p-cresol, methylparaben, propylparaben, 2-phenoxyethanol, butylparaben, 2-phenylethanol, benzyl alcohol, chlorobutanol, and thiomerosal, bromonitrile glycol, benzoic acid, imidureus, chlorhexidine, sodium dehydroacetate, chlorocresol, ethylparaben, benzyl chloride, chlorphenesine (3p-(chlorophenoxypropane)-1,2-diol), or mixtures thereof. In another embodiment of the invention, the preservative is present at a concentration of 0.1 mg / ml to 20 mg / ml. In another embodiment of the invention, the preservative is present at a concentration of 0.1 mg / ml to 5 mg / ml. In another embodiment of the invention, the preservative is present at a concentration of 5 mg / ml to 10 mg / ml. In another embodiment of the invention, the preservative is present at a concentration of 10 mg / ml to 20 mg / ml. Each of these specific preservatives constitutes an alternative embodiment of the invention. The use of preservatives in pharmaceutical compositions is well known to those skilled in the art. For convenience, refer to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0259] In another embodiment of the invention, the formulation further comprises an isotonic agent. In another embodiment of the invention, the isotonic agent is selected from the group consisting of: salts (e.g., sodium chloride), sugars or sugar alcohols, amino acids (e.g., L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine), sugar alcohols (e.g., glycerol, 1,2-propanediol, propylene glycol, 1,3-propanediol, 1,3-butanediol), polyethylene glycol (e.g., PEG400), or mixtures thereof. Any sugar, such as monosaccharides, disaccharides, or polysaccharides, or water-soluble dextran, can be used, including, for example, fructose, glucose, mannose, sorbitol, xylose, maltose, lactose, sucrose, trehalose, dextran, mycosaccharides, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, and sodium carboxymethyl cellulose. In some embodiments, the sugar additive is sucrose. Sugar alcohols are defined as C4-C8 carbohydrates (hydrocarbons) having at least one -OH group, and include, for example, mannitol, sorbitol, inositol, galactitol, euonymol, xylitol, and arabinitol. In some embodiments, the sugar alcohol is mannitol. The sugars or sugar alcohols mentioned above can be used alone or in combination. There is no fixed limitation on the amount used, provided that the sugar or sugar alcohol is soluble in the liquid formulation and does not adversely affect the stabilizing effect obtained by using the method of the present invention. In some embodiments, the sugar or sugar alcohol concentration is from about 1 mg / ml to about 150 mg / ml. In another embodiment of the invention, the isotonic agent is present at a concentration of 1 mg / ml to 50 mg / ml. In another embodiment of the invention, the isotonic agent is present at a concentration of 1 mg / ml to 7 mg / ml. In another embodiment of the invention, the isotonic agent is present at a concentration of 8 mg / ml to 24 mg / ml. In another embodiment of the invention, the isotonic agent is present at a concentration of 25 mg / ml to 50 mg / ml. Each of these specific isotonic agents constitutes an alternative embodiment of the present invention. The use of isotonic agents in pharmaceutical compositions is well known to those skilled in the art. For convenience, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0260] In another embodiment of the invention, the formulation further comprises a chelating agent. In another embodiment of the invention, the chelating agent is selected from salts of ethylenediaminetetraacetic acid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In another embodiment of the invention, the chelating agent is present at a concentration of 0.1 mg / ml to 5 mg / ml. In another embodiment of the invention, the chelating agent is present at a concentration of 0.1 mg / ml to 2 mg / ml. In another embodiment of the invention, the chelating agent is present at a concentration of 2 mg / ml to 5 mg / ml. Each of these specific chelating agents constitutes an alternative embodiment of the invention. The use of chelating agents in pharmaceutical compositions is well known to those skilled in the art. For convenience, refer to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0261] In another embodiment of the invention, the formulation further comprises a stabilizer. The use of stabilizers in pharmaceutical compositions is well known to those skilled in the art. For convenience, refer to Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0262] More specifically, the compositions of the present invention are stable liquid pharmaceutical compositions whose therapeutically active ingredients include polypeptides that may exhibit aggregate formation during storage as liquid pharmaceutical formulations. "Aggregate formation" means physical interactions between polypeptide molecules that result in the formation of oligomers that may remain soluble, or the formation of large, visible aggregates that precipitate from the solution. "During storage" means that the liquid pharmaceutical composition or formulation is not administered to a subject immediately after preparation. Instead, after preparation, it is packaged and stored, either in liquid form, in a frozen state, or in a dry state, and subsequently reformed into a liquid form or other form suitable for administration to a subject. "Dried form" means that liquid pharmaceutical compositions or preparations are dried by freeze-drying (i.e., lyophilization; see, for example, Williams and Polli (1984) J. Parenteral Sci. Technol. 38:48-59) or spray-drying (see Masters (1991) Spray-Drying Handbook (5th edition; Longman Scientific and Technical, Essez, UK), pp.491-676; Broadhead et al. (1992) Drug Devel. Ind. Pharm. 18:1169-1206; Drying can be performed using methods such as Mumenthaler et al. (1994) Pharm. Res. (Pharmaceutical Research) 11:12-20, or air drying (Carpenter and Crowe (1988) Cryobiology (Cryobiology) 25:459-470; and Roser (1991) Biopharm. (Biopharmaceutical) 4:47-53). During storage of liquid pharmaceutical compositions, peptide aggregation can adversely affect the biological activity of the peptides, leading to a loss of therapeutic efficacy. Furthermore, aggregation formation can cause other problems, such as blockage of infusion tubing, membranes, or pumps when administering peptide-containing pharmaceutical compositions using infusion systems.

[0263] The pharmaceutical compositions of the present invention may further comprise an amino acid base sufficient to reduce the formation of polypeptide aggregates during storage. "Amino acid base" means an amino acid or combination of amino acids, wherein any given amino acid is present in its free base form or in its salt form. When using a combination of amino acids, all amino acids may be present in their free base form, all may be present in their salt form, or some may be present in their free base form while others are present in their salt form. In some embodiments, the amino acids used to prepare the compositions of the present invention are those carrying charged side chains, such as arginine, lysine, aspartic acid, and glutamic acid. Any stereoisomer (i.e., L, D, or DL ​​isomers) or combinations of these stereoisomers of a specific amino acid (e.g., glycine, methionine, histidine, imidazole, arginine, lysine, isoleucine, aspartic acid, tryptophan, threonine, and mixtures thereof) may be present in the pharmaceutical compositions of the present invention, provided that the specific amino acid is present in its free base form or in its salt form. In some embodiments, the L-stereoisomer is used. The compositions of the present invention may also be formulated with analogs of these amino acids. "Amino acid analogue" means a naturally occurring amino acid derivative that results in a reduction of polypeptide aggregate formation during storage of the liquid pharmaceutical composition of the present invention. Suitable arginine analogues include, for example, aminoarginine, ornithine, and N-mono- and L-arginine; suitable methionine analogues include ethionine and butionine; and suitable cysteine ​​analogues include S-methyl-L-cysteine. As with other amino acids, the amino acid analogue is incorporated into the composition in its free base form or its salt form. In another embodiment of the invention, the amino acid or amino acid analogue is used in combination to prevent or delay protein aggregation.

[0264] In another embodiment of the invention, to inhibit the oxidation of methionine residues to methionine sulfoxides, methionine (or other sulfur-containing amino acids or amino acid analogs) may be added when the polypeptide acting as a therapeutic agent contains at least one methionine residue that is susceptible to such oxidation. "Inhibition" means minimal accumulation of the type of methionine oxidation over time. Inhibition of methionine oxidation results in the polypeptide retaining more of its correct molecular form. Any stereoisomer of methionine (L, D, or DL ​​isomers) or combinations thereof may be used. The amount added should be sufficient to inhibit the oxidation of methionine residues so that the amount of methionine sulfoxides is acceptable for the therapeutic structure. Typically, this means that the composition contains no more than about 10% to about 30% methionine sulfoxides. This is typically achieved by adding methionine such that the ratio of added methionine to methionine residues is in the range of about 1:1 to about 1000:1, such as from 10:1 to about 100:1.

[0265] In another embodiment of the invention, the formulation further comprises a stabilizer selected from the group consisting of high molecular weight polymers or low molecular weight compounds. In another embodiment of the invention, the stabilizer is selected from polyethylene glycol (e.g., PEG 3350), polyvinyl alcohol (PVA), polyvinylpyrrolidone, carboxy- / hydroxycellulose or derivatives thereof (e.g., HPC, HPC-SL, HPC-L, and HPMC), cyclodextrin, sulfur-containing substances such as monothioglycerol, mercaptoacetic acid, and 2-methylthioethanol, and various salts (e.g., sodium chloride). Each of these specific stabilizers constitutes an alternative embodiment of the invention.

[0266] The pharmaceutical composition may also contain additional stabilizing agents that further enhance the stability of the therapeutically active peptide therein. Stabilizing agents of particular interest to this invention include, but are not limited to, methionine and EDTA, which protect the peptide against methionine oxidation, and nonionic surfactants, which protect the peptide against aggregation associated with freeze-thaw cycles or mechanical shearing.

[0267] In another embodiment of the invention, the formulation further comprises a surfactant. In yet another embodiment of the invention, the surfactant is selected from detergents, ethoxylated castor oil, polyethylene glycol-modified glycerol esters, acetylated glycerol monoesters, dehydrated sorbitol fatty acid esters, polyoxypropylene-polyoxyethylene block copolymers (e.g., poloxamer, etc.). F68, poloxamer 188 and 407, Triton X-100), polyoxyethylene sorbitan fatty acid esters, polyoxyethylene and polyethylene derivatives, such as alkylated and alkoxylated derivatives (Tween, e.g., Tween-20, Tween-40, ... Tween-80 and Brij-35), monoglycerides or their ethoxylated derivatives, diglycerides or their polyoxyethylene derivatives, alcohols, glycerol, lectins and phospholipids (e.g., phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, diphosphatidylglycerol and sphingomyelin), phospholipid derivatives (e.g., dipalmitoylphosphatidic acid) and lysophospholipids (e.g., palmitoyl lysophosphatidyl-L-serine and ethanolamine, choline, serine or threonine 1-acyl-sn-glycerol-3-phosphate) and alkyl, alkoxy (alkyl ester), alkoxy (alkyl ether) derivatives of lysophosphatidyl and phosphatidylcholine, such as lauroyl and myristoyl derivatives of lysophosphatidylcholine, dipalmitoylphosphatidylcholine, Modification with polar head groups, such as choline, ethanolamine, phosphatidic acid, serine, threonine, glycerol, inositol, and positively charged DODAC, DOTMA, DCP, BISHOP, lysophosphatidylserine and lysophosphatidylthreonine, and glycerophospholipids (e.g., cephalin), glyceroglycolipids (e.g., galactopyransoide), sphingomyelin (e.g., ceramide, gangliosides), dodecylphosphocholine, lecithin, fusobionic acid derivatives (e.g., sodium taurine-dihydrofusobionic acid), long-chain fatty acids and their C6-C12 salts (e.g., oleic acid and caprylic acid), acylcarnitine and its derivatives, N-type lysine, arginine or histidine. α - An acylated derivative, or a side-chain acylated derivative of lysine or arginine, comprising an N-terminal dipeptide of any combination of lysine, arginine, or histidine and neutral or acidic amino acids. α -Acylated derivatives, N-terminated tripeptides comprising any combination of a neutral amino acid and two charged amino acids. α-Acylated derivatives, DSS (sodium docusate, CAS Registry No. [577-11-7]), calcium docusate, CAS Registry No. [128-49-4]), potassium docusate, CAS Registry No. [7491-09-0], SDS (sodium dodecyl sulfate or sodium lauryl sulfate), sodium caprylate, cholic acids or their derivatives, bile acids and their salts and glycine or taurine conjugates, ursodeoxycholic acid, sodium cholate, sodium deoxycholate, sodium taurocholate, sodium glycocholate, N-hexadecyl-N,N- Dimethyl-3-amino-1-propane sulfonate, anionic (alkyl-aryl-sulfonate) monovalent surfactants, zwitterionic surfactants (e.g., N-alkyl-N,N-dimethylammonio-1-propanesulfonates, 3-cholamido-1-propyldimethylammonio-1-propanesulfonate), cationic surfactants (quaternary ammonium bases) (e.g., hexadecyltrimethylammonium bromide, hexadecylpyridine chloride). Nonionic surfactants (e.g., dodecyl β-D-glucopyranoside), poloxamines (e.g., Tetronic's), which are tetrafunctional block copolymers derived from propylene oxide and ethylene oxide through the continuous addition of ethylenediamine, or said surfactants may be selected from the group consisting of imidazole derivatives or mixtures thereof. Each of these specific surfactants constitutes an alternative embodiment of the invention.

[0268] The use of surfactants in pharmaceutical compositions is well known to those skilled in the art. For convenience, see Remington: The Science and Practice of Pharmacy, 19th edition, 1995.

[0269] It is possible that other components may be present in the peptide pharmaceutical formulation of the present invention. These additional components may include wetting agents, emulsifiers, antioxidants, fillers, tension modifiers, chelating agents, metal ions, oily excipients, proteins (e.g., human serum albumin, gelatin, or protein) and zwitterions (e.g., amino acids such as betaine, taurine, arginine, glycine, lysine, and histidine). Of course, these additional components should not adversely affect the overall stability of the pharmaceutical formulation of the present invention.

[0270] The pharmaceutical composition of the present invention comprising ficolin-related polypeptides can be administered to a patient requiring the treatment at several sites, such as at local sites, for example, skin and mucous membrane sites, at sites of bypass absorption, for example, in arteries, veins, or the heart, and at sites involving absorption, for example, in the skin, under the skin, in the muscle, or in the abdomen.

[0271] Topical application can be particularly beneficial for treating conditions associated with local inflammation, such as burn-related inflammation or other skin-related conditions. Therefore, in some implementations, application is carried out through topical application.

[0272] In some specific implementations, eye drops can be used for eye-related conditions such as keratitis, such as diffuse lamellar keratitis (DLK).

[0273] The pharmaceutical compositions of the present invention can be administered via several routes of administration, such as to the tongue, sublingually, sublingually, in the mouth, orally, in the stomach and intestines, in the nose, in the lungs, for example, through the bronchioles and alveoli or combinations thereof, through the epidermis, skin, percutaneously, through the vagina, rectum, eyes, for example, through the conjunctiva, ureter, and parenterally to patients requiring the treatment.

[0274] The compositions of the present invention can be administered in several dosage forms, for example, as solutions, suspensions, emulsions, microemulsions, multilayer emulsions, foams, ointments, pastes, plasters, greases, tablets, coated tablets, rinses, capsules, such as hard gelatin capsules and soft gelatin capsules, suppositories, rectal capsules, drops, gels, sprays, powders, aerosols, inhalers, ophthalmic drops, ophthalmic ointments, ophthalmic cleansers, vaginal pessaries, vaginal rings, vaginal ointments, injections, in-situ conversion solutions, such as in-situ gelation, in-situ fixation, in-situ precipitation, in-situ crystallization, infusion solutions, and implants.

[0275] The compositions of the present invention can be further mixed in or attached to drug carriers, drug delivery systems and advanced drug delivery systems, for example, by covalent, hydrophobic and electrostatic interactions, thereby further increasing the stability of ficolin-related peptides, increasing bioavailability, increasing solubility, reducing side effects, enabling elective therapies known to those skilled in the art, and increasing patient compliance or any combination thereof. Examples of carriers, drug delivery systems, and advanced drug delivery systems include, but are not limited to, polymers such as cellulose and its derivatives, polysaccharides such as dextran and its derivatives, starch and its derivatives, poly(vinyl alcohol), acrylic acid and methacrylic acid polymers, polylactic acid and polyglycolic acid and their block copolymers, polyethylene glycol, carrier proteins such as albumin, gels such as thermogelation systems, such as block copolymerization systems known to those skilled in the art, micelles, liposomes, microspheres, nanoparticles, liquid crystals and their dispersions, L2 phases and their dispersions, which exhibit phase transition behavior in lipid-water systems known to those skilled in the art, polymeric micelles, multilayer emulsions, self-emulsifying, self-microemulsifying, cyclodextrins and their derivatives, and dendritic polymers.

[0276] The compositions of the present invention are used in solid, semi-solid, powder and solution formulations suitable for pulmonary administration of ficolin-related polypeptides, for example, by using a measured dose inhaler, dry powder inhaler and nebulizer, all of which are devices known to those skilled in the art.

[0277] The compositions of the present invention are particularly useful in formulations of controlled-release, sustained-release, extended-release, delayed-release, and slow-release drug delivery systems. More specifically, but not limited to, the compositions are used in formulations of parenteral controlled-release and sustained-release systems (which result in a multiple reduction in the number of doses), as is well known to those skilled in the art. Even more preferred are subcutaneously administered controlled-release and sustained-release systems. Without limiting the scope of the invention, examples of useful controlled-release systems and compositions include hydrogels, oil gels, liquid crystals, polymeric micelles, microspheres, and nanoparticles.

[0278] Methods for producing controlled-release systems for the compositions of the present invention include, but are not limited to, crystallization, condensation, co-crystallization, precipitation, co-precipitation, emulsification, dispersion, high-pressure homogenization, encapsulation, spray drying, microencapsulation, coagulation, solvent evaporation to produce microspheres, extrusion, and supercritical fluid processes. General References: See Handbook of Pharmaceutical Controlled Release (Wise, DL, ed. Marcel Dekker, New York, 2000) and Drug and the Pharmaceutical Sciences, Vol. 99: Protein Formulation and Delivery (MacNally, EJ, ed. Marcel Dekker, New York, 2000).

[0279] Drug administration can be performed via subcutaneous, intramuscular, intraperitoneal, or intravenous injection using a syringe, optionally a pen syringe. Alternatively, parenteral administration can be performed via an infusion pump. Another option is a composition of a solution or suspension of ficolin-related peptides for nasal or pulmonary spray administration. Yet another option is that the pharmaceutical compositions of the present invention containing ficolin-related peptides can also be adapted for transdermal administration, for example, by needle-free injection or from a patch, optionally an iontophoresis patch, or via mucosal administration, such as oral administration.

[0280] The term "stable formulation" refers to a formulation that has increased physical stability, increased chemical stability, or increased physical and chemical stability.

[0281] The term "physical stability" of a protein formulation, as used herein, refers to the tendency of a protein to form biologically inactive and / or insoluble aggregates due to exposure to thermo-mechanical stress and / or interaction with unstable interfaces and surfaces (such as hydrophobic surfaces and interfaces). The physical stability of an aqueous protein formulation is assessed by visual inspection and / or turbidity measurement after the formulation, filled in a suitable container (e.g., a canister or vial), has been exposed to mechanical / physical stress (e.g., agitation) at different temperatures for varying durations. Visual inspection of the formulation is performed with clear, focused light against a dark background. Turbidity of the formulation is characterized by a visual score ranking the degree of turbidity, e.g., on a scale of 0-3 (a formulation showing no turbidity corresponds to a visual score of 0, and a formulation showing visible turbidity under sunlight corresponds to a visual score of 3). When a formulation exhibits visible turbidity under sunlight relative to protein aggregation, it is classified as physically unstable. Alternatively, the turbidity of the formulation can be assessed using simple turbidity measurements known to those skilled in the art. The physical stability of an aqueous protein formulation can also be assessed using spectroscopic reagents or probes of protein conformational state. Probes are preferably small molecules that preferentially bind to non-native conformational isomers of proteins. An example of a small-molecule spectroscopic probe for protein structures is Thioflavin T. Thioflavin T is a fluorescent dye widely used for the detection of amyloid fibrils. In the presence of fibrils and possibly other protein conformations, Thioflavin T produces a novel excitation maxima at approximately 450 nm and enhanced emission at approximately 482 nm upon binding to the fibril form. Unbound Thioflavin T exhibits essentially no fluorescence at these wavelengths.

[0282] Other small molecules can be used as probes to detect changes in protein structure from native to non-native states. For example, "hydrophobic patch" probes preferentially bind to the exposed hydrophobic patch of a protein. The hydrophobic patch is typically embedded in the tertiary structure of a native protein but is exposed when the protein begins to unfold or denature. Examples of these small molecules and spectroscopic probes are aromatic, hydrophobic dyes such as anthraquinone, acridine, and phenanthroline. Other spectroscopic probes are metal-amino acid complexes, such as cobalt metal complexes of hydrophobic amino acids (e.g., phenylalanine, leucine, isoleucine, methionine, and valine).

[0283] The term “chemical stability” in this context refers to a covalent chemical change in the protein structure that, compared to the natural protein structure, results in the formation of chemically degraded products with potentially lower biological efficacy and / or potentially increased immunogenic properties. Depending on the type and nature of the natural protein and the environment in which it is exposed, a variety of chemically degraded products can form. Elimination of chemical degradation is perhaps the least likely to be completely avoided, and it is known to those skilled in the art that an increased amount of chemically degraded products is commonly observed during the storage and use of protein formulations. Most proteins tend to deamidinate, a process in which the side-chain amide group in a glutamyl or asparagine acyl residue is hydrolyzed to form a free carboxylic acid. Other degradation pathways involve the formation of high molecular weight transconvertants, in which two or more protein molecules covalently bind to each other through transamidation and / or disulfide interactions, resulting in the formation of covalently bound dimers, oligomers, and polymers (Stability of Protein Pharmaceuticals, Ahern TJ & Manning M.C., Plenum Press, New York 1992). Oxidation (e.g., oxidation of methionine residues) is another protein degradation change mentioned. The chemical stability of protein formulations can be assessed by measuring the amount of chemical degradation products at different time points after exposure to different environmental conditions (e.g., typically accelerated degradation product formation by increasing temperature). The amount of each individual degradation product is usually determined by isolating that degradation product, which depends on molecular size and / or charge using various chromatographic techniques (e.g., SEC-HPLC and / or RP-HPLC).

[0284] Therefore, as mentioned above, a "stable formulation" refers to a formulation with increased physical stability, increased chemical stability, or increased physical and chemical stability. Generally, a formulation must be stable during use and storage until the product's expiration date (under recommended use and storage conditions).

[0285] In some embodiments of the invention, pharmaceutical formulations containing ficolin-related peptides are stable after use for more than 6 weeks and storage for more than 3 years. In other embodiments of the invention, pharmaceutical formulations containing ficolin-related peptides are stable after use for more than 4 weeks and storage for more than 3 years. In yet another embodiment of the invention, pharmaceutical formulations containing ficolin-related peptides are stable after use for more than 4 weeks and storage for more than 2 years. In yet another embodiment of the invention, pharmaceutical formulations containing ficolin-related peptides are stable after use for more than 2 weeks and storage for more than 2 years.

[0286] Specific embodiments of the present invention

[0287] As described above, the present invention relates to isolated ficolin-related polypeptides and polypeptides containing the amino acid sequence SEQ ID NO:4, or variants thereof or immunological fragments thereof.

[0288] In some embodiments, the polypeptides of the present invention are substantially pure.

[0289] In some embodiments, the polypeptide of the present invention is capable of associating with mannose-binding lectin (MBL).

[0290] In some embodiments, the polypeptide of the present invention can associate with any one of ficolin-1, ficolin-2, or ficolin-3.

[0291] In some embodiments, the peptides of the present invention can associate with any of C1q, pulmonary surfactant proteins SP-A and / or SP-D, and intracellular collagen-like defense molecules such as CLL-11.

[0292] In some embodiments, the polypeptides of the present invention are capable of associating with specific receptor proteins, such as specific receptors.

[0293] In some embodiments, the polypeptide of the present invention comprises amino acid sequence 20-297 of SEQ NO:3, or a functional variant thereof.

[0294] In some embodiments, the polypeptide of the present invention comprises amino acid sequence 20-380 of SEQ NO:1, or a functional variant thereof.

[0295] In some embodiments, the polypeptide of the present invention comprises amino acid sequence 16-296 of SEQ ID NO:9 or a functional variant thereof.

[0296] In some embodiments, the polypeptide of the present invention has a molecular weight of about 40 kDa on SDS-PAGE under non-reducing conditions.

[0297] In some embodiments, the polypeptide of the present invention is N-linked glycosylated at one or two amino acids corresponding to positions selected from SEQ NO:1 49 and 178.

[0298] In some embodiments, the polypeptide of the present invention is a recombinant protein.

[0299] In some embodiments, the polypeptide of the present invention is in the form of a homodimer.

[0300] In some embodiments, the polypeptide of the present invention consists of amino acid sequence 20-380 of SEQ ID NO 1.

[0301] In some embodiments, the polypeptide of the present invention comprises the amino acid sequence of SEQ ID NO:4 or a variant thereof or an immunological fragment.

[0302] In some embodiments, the polypeptide of the present invention consists of SEQ ID NO:4, or a variant thereof, or an immunological fragment.

[0303] In some embodiments, the polypeptides of the present invention mediate the phagocytosis of dying cells or dead cells, and / or cell debris, such as apoptotic cells.

[0304] In some embodiments, the peptides of the present invention mediate the phagocytosis of microorganisms.

[0305] In some embodiments, the antibody that specifically binds to the polypeptide of the present invention is a monoclonal antibody.

[0306] In some embodiments, the antibody that specifically binds to the polypeptide of the present invention is a polyclonal antibody.

[0307] In some embodiments, the polypeptides of the present invention have similar activities to other proteins with sequence homology, such as engulfing adaptor proteins (GULPs).

[0308] In some embodiments, the isolated nucleic acid molecule encoding the polypeptide of the present invention comprises a nucleotide sequence having at least 70% identity with the sequence of SEQ NO:2.

[0309] In some embodiments, the host cell of the present invention is a eukaryotic cell.

[0310] In some embodiments, the host cell of the present invention is of mammalian origin.

[0311] In some embodiments, the host cells of the present invention are selected from the group consisting of CHO cells, HEK cells and BHK cells.

[0312] In some embodiments, the peptides of the present invention are used to treat any indication related to inflammation, apoptosis, and / or autoimmunity.

[0313] In some embodiments, the peptides of the present invention are used to treat any autoimmune disease, such as Addison's disease, autoimmune hemolytic anemia, autoimmune thyroiditis, Crohn's disease, Graves' disease, Guillain-Barré syndrome, systemic lupus erythematosus (SLE), lupus nephritis, multiple sclerosis, myasthenia gravis, psoriasis, primary biliary cirrhosis, and rheumatoid arthritis. Arthritis and uveitis, asthma, atherosclerosis, type I diabetes, psoriasis, and various allergies.

[0314] In some embodiments, the polypeptides of the present invention are used to treat any inflammatory condition selected from the group consisting of: appendicitis, peptic ulcer, gastric ulcer, duodenal ulcer, peritonitis, pancreatitis, ulcerative colitis, pseudomembranous colitis, acute colitis, ischemic colitis, diverticulitis, epiglottitis, achalasia, cholangitis, cholecystitis, hepatitis, Crohn's disease, enteritis, Whipple's disease, allergy, and immune complex disease. Diseases including organ ischemia, reperfusion injury, organ necrosis, hay fever, sepsis, septicemia, endotoxic shock, cachexia, hyperpyrexia, eosinophilic granuloma, granulomatosis, sarcoidosis, septic abortion, epididymitis, vaginitis, prostatitis, urethritis, bronchitis, emphysema, and rhinitis. Pneumonia, pneumoconiosis, alveolitis, bronchiolitis, pharyngitis, pleurisy, sinusitis.Influenza, respiratory syncytial virus infection, HIV infection, hepatitis B virus infection, hepatitis C virus infection, disseminated bacteremia, dengue fever, candidiasis, malaria, filariasis, amebiasis, hydatid cysts, burns, dermatitis, dermatomyositis, sunburn, urticaria, warts. Wheals, vasculitis, angiitis, endocarditis, arteritis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, Alzheimer's disease, coeliac disease, congestive heart failure, adult respiratory distress syndrome, meningitis, encephalitis, multiple sclerosis, cerebral infarction, cerebral embolism. Embolism, Guillame-Barre syndrome, neuritis, neuralgia, spinal cord injury, paralysis, uveitis, arthritis, arthralgia, osteomyelitis, fasciitis.Paget's disease, gout, periodontal disease, rheumatoid arthritis, synovitis, myasthenia gravis, thyroiditis, systemic lupus erythematosus, Goodpasture's syndrome, Behcet's syndrome, allograft rejection, graft-versus-host disease, type 1 diabetes, ankylosing spondylitis, Berger's disease, Reiter's syndrome and Hodgkin's disease, keratitis, type 2 diabetes, cystic fibrosis, myocardial infarction. Reperfusion injury, stroke, dermatomyositis, metabolic syndrome, systemic inflammatory response syndrome, sepsis, multiple organ failure, disseminated intravascular coagulation, anaphylactic shock, vascular complication and nephropathy associated with type 1 and / or type 2 diabetes, meningitis, bacterial septicemia, complicated malaria, atypical hemolytic uremic syndrome, hemolytic uremic syndrome, age-related macular degeneration.Paroxysmal nocturnal hemoglobinuria, snake venombite bites, burns, and organ transplant complications.

[0315] In some embodiments, the polypeptides of the present invention are used to treat any inflammatory condition selected from the group consisting of: organ ischemia, reperfusion injury, organ necrosis, vasculitis, endocarditis, atherosclerosis, thrombophlebitis, pericarditis, myocarditis, myocardial ischemia, periarteritis nodosa, rheumatic fever, congestive heart failure, adult respiratory distress syndrome, cerebral infarction, cerebral embolism, vascular complications, and nephropathy associated with type 1 and / or type 2 diabetes.

[0316] In some embodiments, the polypeptides of the present invention are used for any indication related to the treatment of diseases associated with coagulation, thrombosis, or coagulopathy.

[0317] In some embodiments, the peptides of the present invention are indicated for treatment of diseases or conditions related to coagulation, thrombosis, or coagulopathy, including inflammatory responses and chronic thromboembolic diseases or conditions related to fibrin formation, including vascular conditions such as thrombosis, such as deep vein thrombosis, arterial thrombosis, postoperative thrombosis, coronary artery bypass grafting (CABG), percutaneous transdermal coronary angioplasty (PTCA), platelet deposition stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, atherosclerosis, restenosis, such as atherosclerosis and / or restenosis after angioplasty, acute and chronic indications such as inflammation, sepsis, septic shock, septicemia, hypotension, adult respiratory distress syndrome (ARDS), and systemic inflammatory response syndrome. This medication is indicated for the prevention and treatment of atherosclerotic arteries in mammals at risk of thrombosis, including: septicemia syndrome (SIRS), disseminated intravascular coagulopathy (DIC), pulmonary embolism, pathological platelet deposition, myocardial infarction, or for the prophylactic treatment of atherosclerotic vessels in mammals at risk of thrombosis, venous occlusive disease following peripheral blood progenitor cell (PBPC) transplantation, hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP), and rheumatic fever.

[0318] In some embodiments, the peptides of the present invention are used for indications related to diseases or conditions associated with coagulation, thrombosis, or coagulopathy, including inflammatory responses and chronic thromboembolic diseases or conditions related to fibrin formation, including vascular conditions such as thrombosis, such as deep vein thrombosis, arterial thrombosis, postoperative thrombosis, coronary artery bypass grafting (CABG), percutaneous transdermal coronary angioplasty (PTCA), platelet deposition stroke, tumor growth, tumor metastasis, angiogenesis, thrombolysis, atherosclerosis, restenosis, such as atherosclerosis and / or restenosis after angioplasty, acute and chronic indications such as inflammation, pathological platelet deposition, myocardial infarction, or for prophylactic treatment of mammals with atherosclerotic vessels at risk of thrombosis, venous occlusive disease after peripheral blood progenitor cell (PBPC) transplantation, hemolytic uremic syndrome (HUS), and thrombotic thrombocytopenic purpura (TTP), and rheumatic fever.

[0319] In some embodiments, the polypeptides of the present invention are used to prevent thromboembolic complications in identified high-risk patients, such as those who have undergone surgery or suffer from congestive heart failure.

[0320] In some embodiments, the polypeptides of the present invention are used to treat heart-related medical conditions.

[0321] In some embodiments, the polypeptides of the present invention are used to treat medical conditions associated with ficolin-related polypeptide deficiency.

[0322] Example 1

[0323] Detection of alternative splicing transcripts of the MASP1 gene

[0324] Methods: To detect the three transcripts of MASP1: MASP1, MASP3, and FAP, specific primers were designed for each variant. PCR was performed using a universal forward primer in exon 6 (5′-gcacccagagccacagtg-3′) and specific reverse primers: MASP1 in exon 12 (5′-gccttccagtgtgtgggc-3′), MASP3 in exon 11 (5′-gccttccagagtgtggtca-3′), and FAP in exon 8a (5′-cgatctggagagcgaactc-3′). Figure 1PCR amplification was performed in 20 μl volumes containing: 50 ng liver cDNA (Clontech), 0.25 μM of each primer, 2.5 mM MgCl2, 0.2 mM dNTPs, 50 mM KCl, 10 mM Tris·HCl, pH 8.4, and 0.4 units of PlatinumTaq DNA polymerase (Invitrogen). The PCR reaction was performed with the following cycling parameters: 10 min at 94 °C, 30 or 40 cycles (30 sec at 94 °C, 50 sec at 58 °C, 90 sec at 72 °C), 10 min at 72 °C. Samples were analyzed on 2% agarose gels.

[0325] Results: Variable transcription of the MASP1 gene was detected in liver cDNA. MASP1, MASP3, and FAP transcripts were amplified using a universal forward primer at exon 6 and specific reverse primers at exons 12 (MASP1), 11 (MASP3), and 8a (FAP). MASP1 produced a 500 bp fragment, MASP3 produced a 506 bp fragment, and FAP produced a 309 bp fragment.

[0326] FAP fragment organization and expression

[0327] Methods: The expression of MASP1, MASP3, and FAP in commercially available human tissue cDNAs (Clontech) was investigated using the same PCR assays described above. Samples were analyzed on 2% agarose gels.

[0328] Results: Tissue distribution of MASP1, MASP3, and FAP genes was investigated in the cDNA genome from Clontech. Figure 2 MASP1, MASP3, and FAP transcripts were amplified using universal forward primers and specific reverse primers. GADPH was used as a reference gene. All three genes were highly expressed in the liver, and FAP was also strongly expressed in cardiac tissue (indicated by black arrows). Lesser expression of the FAP gene was detected in the brain, colon, prostate, skeletal muscle, and small intestine (indicated by white arrows).

[0329] DNA sequencing of FAP exon 8a from 100 individuals

[0330] Methods: Direct sequencing of exon 8a was performed on genomic DNA templates from 100 healthy Caucasian individuals, encompassing the intron-exon boundaries of the MASP1 / MASP3 / FAP genes spanning positions +44,083 to +44,431 relative to the translation ATG origin. This fragment was amplified using a single primer set (forward: 5′-ctgttcttcacactggctg-3′, reverse: 5′-ctgctgagatcatgttgttc-3′), where the forward primer contained the 5′-T7 sequence (5′-ttatacgactcacta-3′). PCR amplification was performed in 20 μl volumes containing: 50 ng genomic DNA, 0.25 μM of each primer, 2.5 mM MgCl2, 0.2 mM dNTPs, 50 mM KCl, 10 mM Tris·HCl, pH 8.4, and 0.4 units of Platinum Taq DNA polymerase (Invitrogen). The PCR reaction was performed according to the following cycling parameters: 2 min 94 °C, 15 cycles (30 sec 94 °C, 60 sec 64 °C, 60 sec 72 °C), 15 cycles (30 sec 94 °C, 60 sec 58 °C, 60 sec 72 °C), 5 min 72 °C, and sequencing was performed in the forward direction using 5′-biotinylated sequencing primers according to the protocol of the ABI BigDye Cyclic Sequencing Terminator Kit (Applied Biosystems, Foster City, CA). Sequencing reactions were performed using streptavidin beads (GenoVision) purified on a PyroMark VacuumPrep Workstation (Biotage). Sequence analysis was performed on an ABI Prism 3100 gene analyzer (Applied Biosystems). The obtained DNA sequences were aligned using BioEdit software, and DNA polymorphisms were visually verified from sequence electrophoresis images.

[0331] Results: All sequences were aligned using BioEdit software. No genetic variations were observed in exon 8a or exon-intron regions in 100 healthy individuals.

[0332] Example 2

[0333] Immunoprecipitation

[0334] MAP-1-specific immunoprecipitation from serum was performed using either MAP-1-specific mAb 20C4 (generated against the 17 MAP-1-specific C-terminal peptide) or mAb 8B3, a monoclonal antibody against the common heavy chain reaction of MASP-1 / 3, which served as a control precipitation antibody. A total of 10 μg of anti-MAP-1 or MASP-1 / 3 antibody was allowed to bind to sheep, mouse, or rabbit IgG Dyna beads (M-280, catalog number 112.02D / 112.04D, Dynal / Invitrogen). After a washing step, the beads were loaded with normal human serum pooling buffer (1:1 diluted in TBS) and incubated inverted at 4°C for 1 hour. After a final washing step and magnetic separation, the beads were boiled in SDS loading buffer and subjected to SDS-PAGE and Western blotting, with Western blotting using antibodies against MAP-1, MBL, and Ficolin-3 for detection.

[0335] The same precipitation steps described above were performed using mAbs targeting MBL (Hyb 131-11, Bioporto, Denmark), Ficolin-2 (FCN219), and Ficolin-3 (FCN334). To compensate for differences in serum concentrations of MBL, Ficolin-2, and Ficolin-3, they were precipitated from 1 ml, 300 μl, and 100 μl of serum, respectively. Samples were analyzed by SDS-PAGE and Western blotting, with Western blotting performed using a pAb targeting MAP-1.

[0336] Immunohistochemistry

[0337] CHO cells expressing rMAP-1 were produced in culture flasks at RPMI +10%. Cells were collected at 80-90% confluence, fixed in 4% formaldehyde-PBS for 24 hours, and then embedded in paraffin. The same fixation and paraffin embedding were also performed on six different human liver tissues, two different myocardial tissues, two skeletal muscle tissues, and two samples from the human aorta. Sections of 5 μm thickness were obtained using a Leitz-Wetzlar microtome and placed on glass slides, stored at 4°C until analysis. Pretreatment and analysis were performed as described above. The primary antibody was either the MAP-1-specific monoclonal antibody mAb 12B11 or affinity-purified, monospecific rabbit anti-MAP-1, all diluted to 5 μg / ml. Isotype antibody controls were applied to tissues at the same concentration. The secondary antibody was EnVision. TM Antibodies (HRP-anti-mouse or HRP-anti-rabbit, Dako, Glostrup, Denmark). Staining pattern analysis was performed under a Leica DMLB2 microscope.

[0338] SDS-PAGE and Western blot

[0339] Basically, as the supplier stated, use Electrophoresis was performed on 10% or 4–12% (w / v) Bis-Tris polyacrylamide gels using a discontinuous buffer system (Invitrogen). Western blotting was performed using polyvinylidene fluoride membranes (PVDF-HyBond, Amersham Bioscience), 2 μg / ml primary mAbs, and secondary development was performed using HRP-conjugated streptavidin (PO397, Dako) diluted 1:1500 in PBS, 0.05% Tween 20, or HRP-rabbit anti-mouse IgG (PO260, Dako) diluted 1:1000. The membranes were developed using 3-amino-9-ethylcarbazole (Sigma) (0.04% in acetone) in 50 mM sodium acetate buffer, pH 5, and 0.015% H2O2.

[0340] complement activation assay

[0341] Essentially, the effect of MAP-1 on MBL and Ficolin-3-mediated complement factor C4 deposition was evaluated as described above. Briefly, mannan (MBL ligand) (Sigma-Aldrich M7504) or acetylated bovine serum albumin (Ficolin-3 ligand) was immobilized at 10 μg / ml onto Maxisorp ELISA plates (Nunc, Denmark). After washing, rMBL or rFicolin-3 (0.4 μg / ml) was added and incubated for 1.5 h. rMAP-1 or rMASP-2 was applied at a 2-fold serial dilution for 1 h in the first dimension, followed by incubation at 37°C for 45 min with a serial dilution of serum lacking MBL or Ficolin-3 in the second dimension. C4 deposition was measured using pAb (Q0369, Dako, Glostrup / Denmark) against C4c.

[0342] In addition, we evaluated the substitution of MASP-2 by MAP-1 using a pure system. As previously described, in the rMBL / mannan matrix, rMASP-2 was pre-incubated in serially diluted buffer at 20°C for 45 min in the first dimension, and then incubated with rMAP-1 diluted buffer at 20°C for 45 min in the second dimension. Purified C4 (from Quidel, CA, USA) was added at a concentration of 1 μg / mL and incubated at 37°C for 45 min. Detection was performed as described above.

[0343] result

[0344] MAP-1 coprecipitated with Ficolin-2, Ficolin-3 and MBL

[0345] To investigate the potential association of MAP-1 with MBL and Ficolin-3, we precipitated serum complexes using both anti-MAP-1 mAb20C4 and mAb targeting the common heavy chain of MASP-1 and MASP-3 (mAb8B3). The precipitates were then analyzed by Western blotting, with the blots detected by antibodies against MAP-1, MBL, and Ficolin-3, respectively. We observed a co-precipitated band of Ficolin-3, but also a weaker band containing MBL. Figure 24 A). Samples were not detected using antibodies against Ficolin-2 because they do not function in Western blots. Immunoprecipitation was then reversed using mAbs against MBL, Ficolin-2, and Ficolin-3 to precipitate 1 ml, 300 μl, and 100 μl of serum, respectively (to adjust for differences in serum concentrations of MBL (2 μg / ml), Ficolin-2 (5 μg / ml), and Ficolin-3 (20 μg / ml), respectively). Samples were subsequently analyzed by Western blot with an antibody against MAP-1. A distinct MAP-1 band was observed in the precipitates from Ficolin-2 and -3, while a much weaker band was observed in the MBL precipitate, with immunoprecipitated rMAP-1 and serum MAP-1 serving as controls. Figure 24 B).

[0346] MAP-1 inhibits complement activity in the lectin pathway.

[0347] Serum deficient in MBL and Ficolin-3 was combined with rMBL and rFicolin-3 to reconstruct the complement C4 activation activity of MBL and Ficolin-3. Mannan and acetylated BSA served as ligands for MBL and Ficolin-3, respectively. Both rMBL and rFicolin-3 were able to initiate C4 deposition in MBL- and Ficolin-3-deficient serum, respectively. Figure 25 A and 25D). Application of rMASP-2 resulted in a strongly positive dose-dependent increase in C4 deposition via both Ficolin-3 and MBL activation pathways (A and 25D). Figure 25 B and 25E), while the application of rMAP-1 resulted in a significant dose-dependent inhibition of C4 deposition via both pathways (B and 25E). Figure 25 C and 25F).

[0348] Additionally, we addressed the potential substitution of MASP-2 by MAP-1 using a pure fraction system containing only rMBL, rMASP-2, rMAP-1, and purified C4. rMASP-2 was pre-incubated with serially diluted mannan / rMBL complexes. Then, varying concentrations of rMAP-1 were added, followed by purified C4. Application of rMAP-1 to this system significantly resulted in a dose-dependent inhibition of C4 deposition. Figure 26 ).

[0349] Example 3

[0350] Determining the serum concentration and association specificity of a novel MBL / Ficolin-associated protein 1 (MAP-1).

[0351] A full-length, label-free recombinant construct of MAP-1 was generated and stably expressed in CHO-DG44 cells. A specific monoclonal antibody against MAP-1 was produced. A quantitative ELISA for serum MAP-1 measurement was also established, and the association between serum MAP-1 and Ficolin-2, -3, and MBL was examined by ELISA and density gradient grading.

[0352] Recombinant protein

[0353] As described elsewhere (Hummelshoj et al., Mol Immunol 44, 401-11, 2007; Larsen et al., J Biol Chem 279, 21302-11, 2004; Ma et al., J Biol Chem 2009, Oct 9; 284(41)), the improvement lies in the use of PowerCHO1 serum-free medium (Lonza, Vallensbaek / Denmark, www.lonza.com The label-free full-length construct of human MAP-1 was expressed in CHO-DG44 cells using [a specific medium]. As previously mentioned, we purified rMAP-1 using antibody affinity purification (Skjoedt et al., 2009; Immunobiology, Nov 23). Briefly, essentially following the method described by Pfeiffer et al. (Pfeiffer et al., J Immunol Methods 97, 1-9, 1987), 15 mg of anti-MAP-1 antibody (mAb 20C4) was covalently coupled to CNBr-activated agarose and used as the purification matrix. An anti-MAP-1 column was also used to deplete MAP-1 in serum.

[0354] Monoclonal antibody production proceeded as described above (Skjoedt et al., J Biol Chem 285, 8234-43, 2010).

[0355] use The system (Invitrogen), as recommended, was used with discontinuous buffer for electrophoresis on 10% or 4–12% (w / v) Bis-Tris polyacrylamide gels. Western blotting was performed using a polyvinylidene fluoride membrane (PVDF-HyBond, GE Healthcare). The membrane was developed with 2 μg / ml primary antibody, and secondary development was performed using 0.04% 3-amino-9-ethylcarbazole (Sigma-Aldrich, Broendby / Denmark, www.sigmaaldrich.com) + 0.015% H2O2 in 50 mM sodium acetate buffer at pH 5 as a substrate, with HRP-conjugated streptavidin or HRP-rabbit anti-mouse IgG (P0397 / PO260, Dako, Glostrup / Denmark) diluted 1:1500. www.dako.com ) conduct.

[0356] As recommended and previously described (Skjoedt et al., 2009), rMAP-1 was treated with N-glycosidase-F / ENDO-F (N-glycosidase-F deglycosylation kit, Roche, Mannheim / Germany). www.roche.com Processing. The products were analyzed by SDS-PAGE under reducing conditions followed by Coomassie staining or Western blotting.

[0357] The specificity of anti-MAP-1 mAb 20C4 has been previously demonstrated (Skjoedt et al., 2010). In quantitative MAP-1 ELISA, mAb 20C4 is used as a capture antibody, which is immobilized at 6 μg / ml on Maxisorb ELISA plates (NUNC). TM Roskilde / Denmark www.nuncbrand.com The corrector (rMAP-1 or rMAP-1 incorporated into MAP-1-depleted serum) was serially diluted or donor serum samples were applied in PBS + 0.05% Tween 20 + 0.5% bovine serum and 10 mM EDTA. The detection antibody was biotin-labeled mAb 8B3 used at 3 μg / ml, which reacted with MASP-1, -3 and MAP-1 in a co-chain reaction as described above (Skjoedt et al., 2010; Skjoedt et al., 2009).

[0358] Serum concentrations of Ficolin-2 and -3 were determined as described by Munthe-Fog et al. and Hummelshoj et al. (Hummelshoj et al., HumMol Genet (Human Molecular Genetics) 14, 1651-8, 2005; Munthe-Fog et al., Scand J Immunol 65, 383-92, 2007; Munthe-Fog et al., Mol Immunol (Molecular Immunology) 45, 2660-6, 2008), and serum concentrations of MBL and MASP-3 were determined as previously described (Skjoedt et al., 2009).

[0359] Colorimetric analysis was performed using o-phenylenediamine (Dako, Glostrup / Denmark), and the enzyme reaction was terminated with 1M H₂SO₄ as recommended. Optical density (OD 490nm–650nm) levels were measured using a V-max kinetic-reader (Molecular Devices, Sunnyvale / CA / US).

[0360] The relative association between MAP-1 and MBL, Ficolin-2, and -3 was essentially as described above (Skjoedt et al., 2009), with the improvement of using MAP-1-specific mAb 20C4 as the capture antibody (coated at 6 μg / ml). The detection mAbs were biotin-labeled FCN-219 (Ficolin-2 specific) or FCN-334 (Ficolin-3 specific) (24-25), or Hyb 131-11, all applied at 2 μg / ml. Serum samples from the same 100 Danish blood donors as described above were analyzed.

[0361] Normal human serum was subjected to sucrose gradient separation. 0.75 ml of serum was loaded onto a 40 ml centrifuge column composed of a 10-30% sucrose gradient buffered in 10 mM Tris, 145 mM NaCl, 3 mM CaCl2, and 30 μg / ml human serum albumin. The loaded column was centrifuged at 4°C and 150,000 x g for 24 hours using an SW28 rotor in an L70 Beckmann ultracentrifuge. A 1.5 ml fraction was collected from the bottom and analyzed by specific ELISA or Western blotting against the following antigens: MAP-1, MASP-1, MASP-2, MASP-3, sMAP, MBL, Ficolin-2, and Ficolin-3. The peaks of serum IgM (19S) and IgG (7S) were also evaluated, indicating the ratio of molecular surface area to mass. Additionally, the ability of the fraction to activate exogenously applied C4 was analyzed. In short, as described above (Skjoedt et al., 2010), fractions were applied in serial dilutions to ELISA plates coated with acetylated BSA (Ficolin-3 ligand) or mannan (MBL ligand), and then incubated with shaking at 4°C for 1 hour. The plates were then washed and incubated at 37°C for 1 hour with 1 μg / ml purified C4. C4 deposition was subsequently measured using a polyclonal antibody against C4c (Q 0369, Dako, Glostrup, Denmark).

[0362] Statistical analysis

[0363] Using Prism4 software (GraphPad Software, Inc., La Jolla / CA / US), www.graphpad.com ), computational statistics (Spearman non-parametric correlation, non-parametric two-tailed t-test) and serum levels of MAP-1, MBL, Ficolin-2 and -3.

[0364] result

[0365] Purification and characterization of rMAP-1

[0366] Expression of rMAP-1 in CHO DG44 resulted in high yields in the presence of 150 nM methotrexate (yield in serum-free medium: 10–20 μg / ml). After purification, rMAP-1 was analyzed by SDS-PAGE followed by Coomassie brilliant blue staining or Western blotting. SDS-PAGE / Coomassie staining analysis revealed bands with an estimated reduced molecular weight of ~45 kDa. Figure 27Deglycosylation of rMAP-1 with N-glycosidase F resulted in a molecular weight change of ~40 kDa, corresponding to the theoretical mass without a signal peptide. This pattern was also observed in immunoblotting using a specific antibody against MAP-1.

[0367] MAP-1 serum levels

[0368] We developed a quantitative ELISA to determine serum MAP-1 levels. This assay is based on MAP-1-specific mAb 20C4 as a capture antibody and detection antibody (mAb 8B3) recognizing the common heavy chain of MASP-1, -3, and MAP-1. Excellent parallelism was observed between purified rMAP-1 corrector and MAP-1-depleted serum doped with purified MAP-1 at known concentrations according to a standard curve. Figure 28 We analyzed serum MAP-1 levels in 100 Danish blood donors and found a mean of 240 ng / ml, ranging from 115 to 466 ng / ml. Figure 29A As mentioned earlier, we measured serum MASP-3 levels in the same group (Skjoedt et al., 2009) and plotted MAP-1 and MASP-3 concentrations. Figure 29B We found no correlation between serum concentrations of MAP-1 and MASP-3, even though they represent variable transcripts from the same gene.

[0369] We evaluated the stability of the antigen and assay in serum and in freeze-thaw cycles. Figure 29C We observed that the assessment of MAP-1 was very robust and independent of freeze-thaw cycles.

[0370] Association between MAP-1 and Ficolin-2, -3 and MBL

[0371] To measure the interactions between MAP-1 and MBL, Ficolin-2, and -3, we developed three different ELISAs using mAb 20C4 as the capture antibody and probed with the following biotin-labeled mAbs: FCN-219 (Ficolin-2 specific), FCN-334 (Ficolin-3 specific), or Hyb 131-11 (MBL specific). We analyzed the same 100 donor serum samples used for MAP-1 determination and assessed serum association levels between MAP-1 and Ficolin-2, -3, and MBL, given as relative OD490–650 nm. Figure 30A In addition, we measured serum concentrations of MBL, Ficolin-2, and -3 as described above (Skjoedt et al., 2009).

[0372] We found that MAP-1 exists in complexes with MBL, Ficolin-2, and -3. However, it appears that the major portion of MAP-1 associates with ficolins, and especially Ficolin-3 (p<0.0001), a pattern previously observed with MASP-3 (Skjoedt et al., 2009).

[0373] We plotted serum concentrations of MAP-1, MBL, Ficolin-2, and -3 at relative association levels and found that the association between MAP-1 and MBL was highly correlated with MBL levels (Spearman r: 0.92, p < 0.0001). Figure 30B (Top right side). Conversely, relative MAP-1 association with Ficolin-2 and -3 was correlated with serum MAP-1 levels (Spearman r: 0.45 and 0.61, respectively, p < 0.0001). Figure 30B (Left hand side). Although we observed some correlation between MAP-1 concentration and relative association with MBL and Ficolin-3 concentration, this trend was not very pronounced.

[0374] Density gradient gradation

[0375] To investigate the distribution of MAP-1 with associated molecules and to test the proportion of non-associated molecules, we fractionated normal human serum using a 10–30% sucrose gradient and ultracentrifugation. Subsequently, the collected fractions of MAP-1, MASP-3, MBL, Ficolin-2, and -3 were analyzed by ELISA. Figure 31A Fractions of MAP-1, MASP-1, -2 and -3, sMAP, MBL, Ficolin-2 and -3 were collected by Western blot analysis. Figure 31B The results showed that serum MAP-1 was present only in fractions containing ficolins and MBL, indicating that MAP-1 does not exist as an unassociated molecule. The same pattern was observed for sMAP, MASP-1, -2, and -3. Furthermore, the data indicated that MAP-1, sMAP, and most of MASP-1, -2, and -3 were co-localized in the peak fraction of ficolin-3. This distribution was also analyzed by size exclusion chromatography on a Sephadex-200 column. Equivalent distribution patterns of the molecules were observed (data not shown).

[0376] Finally, we evaluated the ability of sucrose gradient fractionation to activate exogenously applied C4. Solid-phase mannan and acetylated BSA were used as ligands for MBL and Ficolin-3, respectively. We observed two distinct C4 deposition profiles, reflecting the peaks of the Ficolin-3 and MBL complexes separated by the sucrose gradient. Figure 31C ).

[0377] discuss

[0378] To investigate structural aspects and determine serum levels of a novel MBL / Ficolin-associated protein 1 (MAP-1), we expressed label-free recombinant MAP-1 and generated a specific antibody against it. N-glycosidase F treatment and SDS-PAGE analysis showed that MAP-1 was glycosylated, yielding a molecular weight of ~45 kDa with N-glycans, and a molecular weight of ~40 kDa after deglycosylation, equivalent to the molecular weight calculated from the putative amino acid sequence without a signal peptide.

[0379] We constructed a quantitative MAP-1 ELISA using a monoclonal antibody targeting the C-terminus of MAP-1 and determined the serum concentration range in 100 healthy Danish blood donors. In the donor group, we found relatively lower serum concentrations (mean: 240 ng / ml, range 115–466 ng / ml) than MASP-3 concentrations (mean: 6500 ng / ml). Furthermore, there was no correlation between the serum concentrations of the two proteins, suggesting that although these two molecules are different splice variants of the same gene, their expression regulation is different. Recently, significant differences in tissue distribution of MASP-1, -3, and MAP-1 have been documented (Degn et al., 2009; Skjoedt et al., 2010). The discovery of the major difference in serum concentrations between MASP-3 and MAP-1 further supports the idea that the regulatory mechanisms of transcriptomorphs derived from the MASP1 gene differ.

[0380] We developed an ELISA-based assay to assess the relative association between serum MAP-1 and MBL, Ficolin-2, and -3, respectively. Additionally, we determined the serum concentrations of Ficolin-2, -3, and MBL, thus correlating them with relative association levels. Results showed that MAP-1 primarily associated with Ficolin-3 and Ficolin-2, with relatively weak association with MBL. It can be argued that this distribution reflects differences in mean serum concentrations of MBL, Ficolin-2, and -3. However, while MBL-MAP-1 association correlated with MBL concentration, the correlation for Ficolin-2, where serum MAP-1 concentration correlated with Ficolin-2 association levels, was similarly weak. The relative association between Ficolin-3 and MAP-1 was highly correlated with serum MAP-1 concentration, but with a weak positive correlation to serum Ficolin-3 levels. These findings suggest that the primary association between MAP-1 and Ficolin-2 and -3 is not solely due to relatively high Ficolin-2 and -3 concentrations. This distribution pattern was further confirmed by analyzing serum samples subjected to density gradient separation. We found a clear trend: not only MAP-1, but also sMAP, MASP-1, -2, and -3 co-localized with the Ficolin-3 peak fraction. This is a phenomenon we previously observed with MASP-3 (Skjoedt et al., 2009). The separation of the Ficolin-3 and MBL peak fractions was also assessed by their ability to activate exogenously added C4 on acetylated BSA (Ficolin-3 ligand) and mannan (MBL ligand). C4 deposition on these two distinct activation surfaces clearly indicated different peak fractions containing either the MBL or Ficolin-3 complex.

[0381] Data from sucrose gradient density analysis also indicate that the surface-to-mass ratio of MBL is higher than that of Ficolin-2 and Ficolin-3, supporting observations from recent studies suggesting that MBL has a very loose and open conformation in its quaternary structure (Jensenius et al., 2009). However, the smaller surface-to-mass ratio of ficolins may also reflect the molecular distribution of associated molecules such as MAP-1, sMAP, and MASPs. In this respect, greater association with MAP-1 / sMAP / MASPs may lead to higher mass and further migration through the density gradient.

[0382] In summary, we have shown that MAP-1 exists at lower serum concentrations compared to MASP-3, and that MAP-1 primarily circulates as a complex with ficolins, but also to some extent with a complex with MBL. Furthermore, we may demonstrate that ficolins-3 appears to be the predominant MAP-1 conjugate among LCP recognition molecules.

[0383] SEQ ID NO: 1. The complete 380-amino acid sequence of human FAP. (Two potential glycosylation sites identified at amino acid positions 49 and 178 are highlighted).

[0384]

[0385] SEQ ID NO: 2. Complete cDNA nucleotide sequence of human FAP.

[0386] atgaggtggctgcttctctattatgctctgtgcttctccctgtcaaaggcttcagcccacaccgtggagctaaacaata tgtttggccagatccagtcgcctggttatccagactctatcccagtgattcagaggtgacttggaatatcactgtcccagatgggtttcggatcaagctttatcatgcacttcaacttggaatcctcctaccttgtgaatatgactatgtgaag gtagaaactgaggaccaggtgctggcaaccttctgtggcagggagaccacagacacagacagactcccggccaggagggggtcctcccctggctcttcatgtccatcactttccggtcagatttctccaatgaggagcgtttcacaggcttga tgcccactacatggctgtggatgtggacgagtgcaaggaggagaggaggagctgtcctgtgaccaactactgccaaactacattggcggctactgctcctcggctacatcctccacacagacaacaggacctgccgagtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggagctactgccacaactacattggcggagcttactgctcctcggctacatcctccacacagacaacaggacctgccgagtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggaggtggagctactgccacaactacattggcggagctactgctcctcggctacatcctccacacacagacaacaggagctgccgagtggagctactgccacaact gcagtgacaacctcttcactcaaggactggggtgatcaccagccctgacttcccaaacccttaccccaagagctctgaatgcctgtataccatcgagctggaggagggtttcatggtcaacctgcagtttgaggacatatttgacattgaggaccat cctgaggtgccctgcccctatgactacatcaagatcaaagttggtccaaaagttttggggcctttctgtggagagaaagccccagaacccatcagcacccagagccacagtgtcctgatcctgttccatagtgacaactcgggagagaaccggggctggaggctctcatacagggctgcaggaaatgagtgcccaggctacagcctcctgtccatgggaaaatcgagccctcccaagccaagtatttcttcaaagaccaagtgctcgtcagctgtgacacaggctacaaagtgctgaaggataatgtggagatgg acacattccagattgagtgtctgaaggatgggacgtggagtaacaagattcccacctgtaaaaaaaatgaaatcgatctggagagcgaactcaagtcagagcaagtgacagagtga

[0387] SEQ NO: 3. Minimal sequence of a ficolin-related polypeptide containing the CUB1-EGF-CUB2 domain, comprising a signal peptide of amino acids 1-19. This sequence corresponds to exons 2 to 6.

[0388]

[0389] SEQ ID NO: 4. FAP's unique terminal 17 amino acids

[0390] KNEIDLESELKSEQVTE

[0391] SEQ ID NO: 5 Protein sequence of human MASP-1.

[0392] MRWLLLYYALCFSLSKASAHTVELNNMFGQIQSPGYPDSYPSDSEVTWNITVPDGFRIKLYFMHFNLESSYL CEYDYVKVETEDQVLATFCGRETTDTEQTPGQEVVLSPGSFMSITFRSDFSNEERFTGFDAHYMAVDVDECKEREDEELSCDHYCHNYIGGYYCSCRFGYILHTDNRTCRVECSDNLFTQRTGVITSPDFPNPYPKSSECLYTI ELEEGFMVNLQFEDIFDIEDHPEVPCPYDYIKIKVGPKVLGPFCGEKAPEPISTQSHSVLILFHSDNSGENRGWRLSYRAAGNECPELQPPVHGKIEPSQAKYFFKDQVLVSCDTGYKVLKDNVEMDTFQIECLKDGTWSNKIP TCKIVDCRAPGELEHGLITFSTRNNLTTYKSEIKYSCQEPYYKMLNNNTGIYTCSAQGVWMNKVLGRSLPTCLPVCGLPKFSRKLMARIFNGRPAQKGTTPWIAMLSHLNGQPFCGGSLLGSSWIVTAAHCLHQSLDPEDPTLR DSDLLSPSDFKIILGKHWRLRSDENEQHLGVKHTTLHPQYDPNTFENDVALVELLESPVLNAFVMPICLPEGPQQEGAMVIVSGWGKQFLQRFPETLMEIEIPIVDHSTCQKAYAPLKKKVTRDMICAGEKEGGKDACAGDSGG PMVTLNRERGQWYLVGTVSWGDDCGKKDRYGVYSYIHHNKDWIQRVTGVRN

[0393] SEQ ID NO: 6 cDNA sequence of human MASP-1

[0394] GAAGTCAGCCACACAGGATAAAGGAGGGAAGGGAAGGAGCAGATCTTTTCGGTAGGAAGACAGATTTTGT TGTCAGGTTCCTGGGAGTGCAAGAGCAAGTCAAAGGAGAGAGAGAGGAGAGAGGAAAAGCCAGAGGGAGAGAGGGGGAGAGGGGATCTGTTGCAGGCAGGGGAAGGCGTGACCTGAATGGAGAATGCCAGCCAATTCCAG AGACACACAGGGACCTCAGAACAAAGATAAGGCATCACGGACACCACACCGGGCACGAGCTCACAGGCAAGTCAAGCTGGGAGGACCAAGGCCGGGCAGCCGGGAGCACCCAAGGCAGGAAAATGAGGTGGCTGCTTCTC TATTATGCTCTGTGCTTCTCCCTGTCAAAGGCTTCAGCCCACACCGTGGAGCTAAACAATATGTTTGGCC AGATCCAGTCGCCTGGTTATCCAGACTCCTATCCCAGTGATTCAGAGGTGACTTGGAATATCACTGTCCCAGATGGGTTTCGGATCAAGCTTTACTTCATGCACTTCAACTTGGAATCCTCCTACCTTTGTGAATATGAC TATGTGAAGGTAGAAACTGAGGACCAGGTGCTGGCAACCTTCTGTGGCAGGGAGACCACAGACACAGAGCAGACTCCCGGCCAGGAGGTGGTCCTCTCCCCTGGCTCCTTCATGTCCATCACTTTCCGGTCAGATTTCTC CAATGAGGAGCGTTTCACAGGCTTTGATGCCCACTACATGGCTGTGGATGTGGACGAGTGCAAGGAGAGGGAGGACGAGGAGCTGTCCTGTGACCACTACTGCCACAACTACATTGGCGGCTACTACTGCTCCTGCCGCT TCGGCTACATCCTCCACACAGACAACAGGACCTGCCGAGTGGAGTGCAGTGACAACCTCTTCACTCAAAGGACTGGGGTGATCACCAGCCCTGACTTCCCAAACCCTTACCCCAAGAGCTCTGAATGCCTGTATACCATCGAGCTGGAGGAGGGTTTCATGGTCAACCTGCAGTTTGAGGACATATTTGACATTGAGGACCATCCTGAGGTGCCCTGCCCCTATGACTACATCAAGATCAAAGTTGGTCCAAAAGTTTTGGGGCCTTTCTGTGGAGAGAA AGCCCCAGAACCCATCAGCACCCAGAGCCACAGTGTCCTGATCCTGTTCCATAGTGACAACTCGGGAGAGAACCGGGGCTGGAGGCTCTCATACAGGGCTGCAGGAAATGAGTGCCCAGAGCTACAGCCTCCTGTCCATG GGAAAATCGAGCCCTCCCAAGCCAAGTATTTCTTCAAAGACCAAGTGCTCGTCAGCTGTGACACAGGCTACAAAGTGCTGAAGGATAATGTGGAGATGGACACATTCCAGATTGAGTGTCTGAAGGATGGGACGTGGAGT AACAAGATTCCCACCTGTAAAATTGTAGACTGTAGAGCCCCAGGAGAGCTGGAACACGGGCTGATCACCTTCTCTACAAGGAACAACCTCACCACATACAAGTCTGAGATCAAATACTCCTGTCAGGAGCCCTATTACAA GATGCTCAACAATAACACAGGTATATATACCTGTTCTGCCCAAGGAGTCTGGATGAATAAAGTATTGGGGAGAAGCCTACCCACCTGCCTTCCAGTGTGTGGGCTCCCCAAGTTCTCCCGGAAGCTGATGGCCAGGATCT TCAATGGACGCCCAGCCCAGAAAGGCACCACTCCCTGGATTGCCATGCTGTCACACCTGAATGGGCAGCC CTTCTGCGGAGGCTCCCTTCTAGGCTCCAGCTGGATCGTGACCGCCGCACACTGCCTCCACCAGTCACTCGATCCGGAAGATCCGACCCTACGTGATTCAGACTTGCTCAGCCCTTCTGACTTCAAAATCATCCTGGGCAAGCATTGGAGGCTCCGGTCAGATGAAAATGAACAGCATCTCGGCGTCAAACACACCACTCTCCACCCCCAGTATGATCCCAACACATTCGAGAATGACGTGGCTCTGGTGGAGCTGTTGGAGAGCCCAGTGCTGAATGCCTTCGTGATGCCCATCTGTCTGCCTGAGGGACCCCAGCAGGAAGGAGCCATGGTCATCGTCAGCGGCTGGGGGAAGCAGTTCTTGCAAAGGTTCCCAGAGACCCTGATGGAGATTGAAATCCCGATTGTTGACCACAGCAC CTGCCAGAAGGCTTATGCCCCGCTGAAGAAGAAAGTGACCAGGGACATGATCTGTGCTGGGGAGAAGGAAGGGGGAAAGGACGCCTGTGCGGGTGACTCTGGAGGCCCCATGGTGACCCTGAATAGAGAAAGAGGCCAGT GGTACCTGGTGGGCACTGTGTCCTGGGGTGATGACTGTGGGAAGAAGGACCGCTACGGAGTATACTCTTACATCCACCACAACAAGGACTGGATCCAGAGGGTCACCGGAGTGAGGAACTGAATTTGGCTCCTCAGCCCC AGCACCACCAGCTGTGGGCAGTCAGTAGCAGAGGACGATCCTCCGATGAAAGCAGCCATTTCTCCTTTCCTTCCTCCCATCCCCCCTCCTTCGGCCTATCCATTACTGGGCAATAGAGCAGGTATCTTCACCCCCTTTTC ACTCTCTTTAAAGAGATGGAGCAAGAGAGTGGTCAGAACACAGGCCGAATCCAGGCTCTATCACTTACTA GTTTGCAGTGCTGGGCAGGTGACTTCATCTCTTCGAACTTCAGTTTCTTCATAAGATGGAAATGCTATACCTTACCTACCTCGTAAAAGTCTGATGAGGAAAAGATTAACTAATAGATGCATAGCACTTAACAGAGTGCATAGCATACACTGTTTTCAATAAATGCACCTTAGCAGAAGGTCGATGTGTCTACCAGGCAGACGAAGCTCTCTTACAAACCCCTGCCTGGGTCTTAGCATTGATCAGTGACACACCTCTCCCCTCAACCTTGACCATCTCC ATCTGCCCTTAAATGCTGTATGCTTTTTTGCCACCGTGCAACTTGCCCAACATCAATCTTCACCCTCATCCCTAAAAAAGTAAAACAGACAAGGTTCTGAGTCCTGTGGTATGTCCCCTAGCAAATGTAACTAGGAACATGCACTAGATGACAGATTGCGGGAGGGCCTGAGAGAAGCAGGGACAGGAGGGAGCCTGGGGATTGTGGTTT GGGAAGGCAGACACCTGGTTCTAGAACTAGCTCTGCCCTTAGCCCCCTGTATGACCCTATGCAAGTCCTCCTCCCTCATCTCAAAGGGTCCTCAAAGCTCTGACGATCTAAGATACAATGAAGCCATTTTCCCCCTGATA AGATGAGGTAAAGCCAATGTAACCAAAAGGCAAAAATTACAATCGGTTCAAAGGAACTTTGATGCAGACAAAATGCTGCTGCTGCTGCTCCTGAAATACCCACCCCTTTCCACTACGGGTGGGTTCCCAAGGACATGGGA CAGGCAAAGTGTGAGCCAAAGGATCCTTCCTTATTCCTAAGCAGAGCATCTGCTCTGGGCCCTGGCCTCCTTCCCTTCTTGGGAAACTGGGCTGCATGAGGTGGGCCCTGGTAGTTTGTACCCCAGGCCCCTATACTCTT CCTTCCTATGTCCACAGCTGACCCCAAGCAGCCGTTCCCCGACTCCTCACCCCTGAGCCTCACCCTGAACTCCCTCATCTTGCAAGGCCATAAGTGTTTTCCAAGCAAAATGCCTCTCCCATCCTCTCTCAGGAAGCTTCTAGAGACTTTATGCCCTCCAGAGCTCCAAGATATAAGCCCTCCAAGGGATCAGAAGCTCCAAGTTCCTGTCTTCTGTTTTATAGAAATTGATCTTCCCTGGGGGACTTTAACTCTTGACCTGTATGCAGCTGTTGGAGTA ATTCCAGGTCTCTTGAAAAAAAAGAGGAAGATAATGGAGAATGAGAACATATATATATATATATTAAGCCCCAGGCTGAATACTCAGGGACAGCAATTCACAGCCTGCCTCTGGTTCTATAAACAAGTCATTCTACCTCT TTGTGCCCTGCTGTTTATTCTGTAAGGGGAAGGTGGCAATGGGACCCAGCTCCATCAGACACTTGTCAAGCTAGCAGAAACTCCATTTTCAATGCCAAAGAAGAACTGTAATGCTGTTTTGGAATCATCCCAAGGCATCC CAAGACACCATATCTTCCCATTTCAAGCACTGCCTGGGCACACCCCAACATCCCAGGCTGTGGTGGCTCCTGTGGGAACTACCTAGATGAAGAGAGTATCATTTATACCTTCTAGGAGCTCCTATTGGGAGACATGAAAC ATATGTAATTGACTACCATGTAATAGAACAAACCCTGCCAAGTGCTGCTTTGGAAAGTCATGGAGGTAAAAGAAAGACCATTC

[0395] SEQ ID NO: 7 Amino acid sequence of human MASP-3.

[0396] MRWLLLYYALCFSLSKASAHTVELNNMFGQIQSPGYPDSYPSDSEVTWNITVPDGFRIKLYFMHFNLESSYLCEYDYVKVETEDQ VLATFCGRETTDTEQTPGQEVVLSPGSFMSITFRSDFSNEERFTGFDAHYMAVDVDECKEREDEELSCDHYCHNYIGGYYCSCRFGYILHTDNRTCRVECSDNLFTQRTGVITSPDFPNPYPKSSECLYTIELEEGFMVNLQFEDIFDIEDHPEVPCPYDYIKIKVGPKV LGPFCGEKAPEPISTQSHSVLILFHSDNSGENRGWRLSYRAAGNECPELQPPVHGKIEPSQAKYFFKDQVLVSCDTGYKVLKDNVEMDTFQIECLKDGTWSNKIPTCKIVDCRAPGELEHGLITFSTRNNLTTYKSEIKYSCQEPYYKMLNNNTGIYTCSAQGVWMNKVL GRSLPTCLPECGQPSRSLPSLVKRIIGGRNAEPGLFPWQALIVVEDTSRVPNDKWFGSGALLSASWILTAAHVLRSQRRDTTVIP VSKEHVTVYLGLHDVRDKSGAVNSSAARVVLHPDFNIQNYNHDIALVQLQEPVPLGPHVMPVCLPRLEPEGPAPHMLGLVAGWGISNPNVTVDEIISSGTRTLSDVLQYVKLPVVPHAECKTSYESRSGNYSVTENMFCAGYYEGGKDTCLGDSGGAFVIFDDLSQRWVV QGLVSWGGPEECGSKQVYGVYTKVSNYVDWVWEQMGLPQSVVEPQVER <0开000875>SEQ ID NO: 8 cDNA sequence of human MASP-3

[0398] It should be noted that there seems to be a mistake in the original text where "<0开000875>" is written. It should probably be "

[0397] ". The translation is done as accurately as possible based on the provided text.GAAGTCAGCCACACAGGATAAAGGAGGGAAGGGAAGGAGCAGATCTTTTCGGTAGGAAGACAGATTTTGT TGTCAGGTTCCTGGGAGTGCAAGAGCAAGTCAAAGGAGAGAGAGAGGAGAGAGGAAAAGCCAGAGGGAGAGAGGGGGAGAGGGGATCTGTTGCAGGCAGGGGAAGGCGTGACCTGAATGGAGAATGCCAGCCAATTCCAG AGACACACAGGGACCTCAGAACAAAGATAAGGCATCACGGACACCACACCGGGCACGAGCTCACAGGCAAGTCAAGCTGGGAGGACCAAGGCCGGGCAGCCGGGAGCACCCAAGGCAGGAAAATGAGGTGGCTGCTTCTC TATTATGCTCTGTGCTTCTCCCTGTCAAAGGCTTCAGCCCACACCGTGGAGCTAAACAATATGTTTGGCCAGATCCAGTCGCCTGGTTATCCAGACTCCTATCCCAGTGATTCAGAGGTGACTTGGAATATCACTGTCCC AGATGGGTTTCGGATCAAGCTTTACTTCATGCACTTCAACTTGGAATCCTCCTACCTTTGTGAATATGAC TATGTGAAGGTAGAAACTGAGGACCAGGTGCTGGCAACCTTCTGTGGCAGGGAGACCACAGACACAGAGCAGACTCCCGGCCAGGAGGTGGTCCTCTCCCCTGGCTCCTTCATGTCCATCACTTTCCGGTCAGATTTCTC CAATGAGGAGCGTTTCACAGGCTTTGATGCCCACTACATGGCTGTGGATGTGGACGAGTGCAAGGAGAGG GAGGACGAGGAGCTGTCCTGTGACCACTACTGCCACAACTACATTGGCGGCTACTACTGCTCCTGCCGCTTCGGCTACATCCTCCACACAGACAACAGGACCTGCCGAGTGGAGTGCAGTGACAACCTCTTCACTCAAAGGACTGGGGTGATCACCAGCCCTGACTTCCCAAACCCTTACCCCAAGAGCTCTGAATGCCTGTATACCATCGAGCTGGAGGAGGGTTTCATGGTCAACCTGCAGTTTGAGGACATATTTGACATTGAGGACCATCCTGAGG TGCCCTGCCCCTATGACTACATCAAGATCAAAGTTGGTCCAAAAGTTTTGGGGCCTTTCTGTGGAGAGAAAGCCCCAGAACCCATCAGCACCCAGAGCCACAGTGTCCTGATCCTGTTCCATAGTGACAACTCGGGAGAG AACCGGGGCTGGAGGCTCTCATACAGGGCTGCAGGAAATGAGTGCCCAGAGCTACAGCCTCCTGTCCATGGGAAAATCGAGCCCTCCCAAGCCAAGTATTTCTTCAAAGACCAAGTGCTCGTCAGCTGTGACACAGGCTA CAAAGTGCTGAAGGATAATGTGGAGATGGACACATTCCAGATTGAGTGTCTGAAGGATGGGACGTGGAGTAACAAGATTCCCACCTGTAAAATTGTAGACTGTAGAGCCCCAGGAGAGCTGGAACACGGGCTGATCACCT TCTCTACAAGGAACAACCTCACCACATACAAGTCTGAGATCAAATACTCCTGTCAGGAGCCCTATTACAAGATGCTCAACAATAACACAGGTATATATACCTGTTCTGCCCAAGGAGTCTGGATGAATAAAGTATTGGGG AGAAGCCTACCCACCTGCCTTCCAGAGTGTGGTCAGCCCTCCCGCTCCCTGCCAAGCCTGGTCAAGAGGATCATTGGGGGCCGAAATGCTGAGCCTGGCCTCTTCCCGTGGCAGGCCCTGATAGTGGTGGAGGACACTTC GAGAGTGCCAAATGACAAGTGGTTTGGGAGTGGGGCCCTGCTCTCTGCGTCCTGGATCCTCACAGCAGCTCATGTGCTGCGCTCCCAGCGTAGAGACACCACGGTGATACCAGTCTCCAAGGAGCATGTCACCGTCTACCTGGGCTTGCATGATGTGCGAGACAAATCGGGGGCAGTCAACAGCTCAGCTGCCCGAGTGGTGCTCCACCC AGACTTCAACATCCAAAACTACAACCACGATATAGCTCTGGTGCAGCTGCAGGAGCCTGTGCCCCTGGGACCCCACGTTATGCCTGTCTGCCTGCCAAGGCTTGAGCCTGAAGGCCCGGCCCCCCACATGCTGGGCCTGG TGGCCGGCTGGGGCATCTCCAATCCCAATGTGACAGTGGATGAGATCATCAGCAGTGGCACACGGACCTTGTCAGATGTCCTGCAGTATGTCAAGTTACCCGTGGTGCCTCACGCTGAGTGCAAAACTAGCTATGAGTCC CGCTCGGGCAATTACAGCGTCACGGAGAACATGTTCTGTGCTGGCTACTACGAGGGCGGCAAAGACACGTGCCTTGGAGATAGCGGTGGGGCCTTTGTCATCTTTGATGACTTGAGCCAGCGCTGGGTGGTGCAAGGCCT GGTGTCCTGGGGGGGACCTGAAGAATGCGGCAGCAAGCAGGTCTATGGAGTCTACACAAAGGTCTCCAATTACGTGGACTGGGTGTGGGAGCAGATGGGCTTACCACAAAGTGTTGTGGAGCCCCAGGTGGAACGGTGAG CTGACTTACTTCCTCGGGGCCTGCCTCCCCTGAGCGAAGCTACACCGCACTTCCGACAGCACACTCCACATTACTTATCAGACCATATGGAATGGAACACACTGACCTAGCGGTGGCTTCTCCTACCGAGACAGCCCCCA GGACCCTGAGAGGCAGAGTGTGGTATAGGGAAAAGGCTCCAGGCAGGAGACCTGTGTTCCTGAGCTTGTCCAAGTCTCTTTCCCTGTCTGGGCCTCACTCTACCGAGTAATACAATGCAGGAGCTCAACCAAGGCCTCTGTGCCAATCCCAGCACTCCTTTCCAGGCCATGCTTCTTACCCCAGTGGCCTTTATTCACTCCTGACCACTTATCAAACCCATCGGTCCTACTGTTGGTATAACTGAGCTTGGACCTGACTATTAGAAAATGGTTTCTAACA TTGAACTGAATGCCGCATCTGTATATTTTCCTGCTCTGCCTTCTGGGACTAGCCTTGGCCTAATCCTTCCTCTAGGAGAAGAGCATTCAGGTTTTGGGAGATGGCTCATAGCCAAGCCCCTCTCTCTTAGTGTGATCCCT TGGAGCACCTTCATGCCTGGGGTTTCTCTCCCAAAAGCTTCTTGCAGTCTAAGCCTTATCCCTTATGTTC CCCATTAAAGGAATTTCAAAAGACATGGAGAAAGTTGGGAAGGTTTGTGCTGACTGCTGGGAGCAGAATAGCCGTGGGAGGCCCACCAAGCCCTTAAATTCCCATTGTCAACTCAGAACACATTTGGGCCCATATGCCAC CCTGGAACACCAGCTGACACCATGGGCGTCCACACCTGCTGCTCCAGACAAGCACAAAGCAATCTTTCAGCCTTGAAATGTATTATCTGAAAGGCTACCTGAAGCCCAGGCCCGAATATGGGGACTTAGTCGATTACCTG GAAAAAGAAAAGACCCACACTGTGTCCTGCTGTGCTTTTGGGCAGGAAAATGGAAGAAAGAGTGGGGTGGGCACATTAGAAGTCACCCAAATCCTGCCAGGCTGCCTGGCATCCCTGGGGCATGAGCTGGGCGGAGAATC CACCCCGCAGGATGTTCAGAGGGACCCACTCCTTCATTTTTCAGAGTCAAAGGAATCAGAGGCTCACCCATGGCAGGCAGTGAAAAGAGCCAGGAGTCCTGGGTTCTAGTCCCTGCTCTGCCCCCAACTGGCTGTATAACCTTTGAAAAATCATTTTCTTTGTCTGAGTCTCTGGTTCTCCGTCAGCAACAGGCTGGCATAAGGTCCCCTGCAGGTTCCTTCTAGCTGGAGCACTCAGAGCTTCCCTGACTGCTAGCAGCCTCTCTGGCCCTCACAGGGC TGATTGTTCTCCTTCTCCCTGGAGCTCTCTCTCCTGAAAATCTCCATCAGAGCAAGGCAGCCAGAGAAGCCCCTGAGAGGGAATGATTGGGAAGTGTCCACTTTCTCAACCGGCTCATCAAACACACTCCTTTGTCTATGAATGGCACATGTAAATGATGTTATATTTTGTATCTTTTATATCATATGCTTCACCATTCTGTAAAGGGCCTCTGCATTGTTGCTCCCATCAGGGGTCTCAAGTGGAAATAAACCCTCGTGGATAACCAAAAAAAAAAAAA AAAAAAA

[0399] SEQ ID NO: 9 Protein sequence of human MASP-2

[0400] MRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFTHFDLELSHLCE YDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSLGSSLDITFRSDYSNEKPFTGFEAFYAAEDIDECQVAPGEAPTCDHHCHNHLGGFYCSCRAGYVLHRNKRTCSALCSGQVFTQRSGELSSPEYPRPYPKLSSCTYSISLEEGFSVILDFVESFDVETHPETLCPYDFLKIQTDREEHGPFCGKTLPHRIETKSNTVTITFVTDESGDHTGWKIHYTSTAQPCPYPMAPPNGHVSPVQAKYILKDSFSIFCETGYELLQGHLPLKSFTAVCQKDGSWDRPMPACSI VDCGPPDDLPSGRVEYITGPGVTTYKAVIQYSCEETFYTMKVNDGKYVCEADGFWTSSKGEKSLPVCEPVC GLSARTTGGRIYGGQKAKPGDFPWQVLILGGTTAAGALLYDNWVLTAAHAVYEQKHDASALDIRMGTLKRLSPHYTQAWSEAVFIHEGYTHDAGFDNDIALIKLNNKVVINSNITPICLPRKEAESFMRTDDIGTASGWGLTQ RGFLARNLMYVDIPIVDHQKCTAAYEKPPYPRGSVTANMLCAGLESGGKDSCRGDSGGALVFLDSETERWFVGGIVSWGSMNCGEAGQYGVYTKVINYIPWIENIISDF

[0401] SEQ ID NO: cDNA sequence of human MASP-2

[0402] GGCCAGCTGGACGGGCACACCATGAGGCTGCTGACCCTCCTGGGCCTTCTGTGTGGCTCGGTGGCCACCC CCTTGGGCCCGAAGTGGCCTGAACCTGTGTTCGGGCGCCTGGCATCCCCCGGCTTTCCAGGGGAGTATGCCAATGACCAGGAGCGGCGCTGGACCCTGACTGCACCCCCCGGCTACCGCCTGCGCCTCTACTTCACCCAC TTCGACCTGGAGCTCTCCCACCTCTGCGAGTACGACTTCGTCAAGCTGAGCTCGGGGGCCAAGGTGCTGGCCACGCTGTGCGGGCAGGAGAGCACAGACACGGAGCGGGCCCCTGGCAAGGACACTTTCTACTCGCTGGG CTCCAGCCTGGACATTACCTTCCGCTCCGACTACTCCAACGAGAAGCCGTTCACGGGGTTCGAGGCCTTCTATGCAGCCGAGGACATTGACGAGTGCCAGGTGGCCCCGGGAGAGGCGCCCACCTGCGACCACCACTGCC ACAACCACCTGGGCGGTTTCTACTGCTCCTGCCGCGCAGGCTACGTCCTGCACCGTAACAAGCGCACCTGCTCAGCCCTGTGCTCCGGCCAGGTCTTCACCCAGAGGTCTGGGGAGCTCAGCAGCCCTGAATACCCACGG CCGTATCCCAAACTCTCCAGTTGCACTTACAGCATCAGCCTGGAGGAGGGGTTCAGTGTCATTCTGGACT TTGTGGAGTCCTTCGATGTGGAGACACACCCTGAAACCCTGTGTCCCTACGACTTTCTCAAGATTCAAACAGACAGAGAAGAACATGGCCCATTCTGTGGGAAGACATTGCCCCACAGGATTGAAACAAAAAGCAACACG GTGACCATCACCTTTGTCACAGATGAATCAGGAGACCACACAGGCTGGAAGATCCACTACACGAGCACAGCGCAGCCTTGCCCTTATCCGATGGCGCCACCTAATGGCCACGTTTCACCTGTGCAAGCCAAATACATCCTGAAAGACAGCTTCTCCATCTTTTGCGAGACTGGCTATGAGCTTCTGCAAGGTCACTTGCCCCTGAAATCCTTTACTGCAGTTTGTCAGAAAGATGGATCTTGGGACCGGCCAATGCCCGCGTGCAGCATTGTTGACTGTG GCCCTCCTGATGATCTACCCAGTGGCCGAGTGGAGTACATCACAGGTCCTGGAGTGACCACCTACAAAGCTGTGATTCAGTACAGCTGTGAAGAGACCTTCTACACAATGAAAGTGAATGATGGTAAATATGTGTGTGAG GCTGATGGATTCTGGACGAGCTCCAAAGGAGAAAAATCACTCCCAGTCTGTGAGCCTGTTTGTGGACTATCAGCCCGCACAACAGGAGGGCGTATATATGGAGGGCAAAAGGCAAAACCTGGTGATTTTCCTTGGCAAGT CCTGATATTAGGTGGAACCACAGCAGCAGGTGCACTTTTATATGACAACTGGGTCCTAACAGCTGCTCATGCCGTCTATGAGCAAAAACATGATGCATCCGCCCTGGACATTCGAATGGGCACCCTGAAAAGACTATCAC CTCATTATACACAAGCCTGGTCTGAAGCTGTTTTTATACATGAAGGTTATACTCATGATGCTGGCTTTGACAATGACATAGCACTGATTAAATTGAATAACAAAGTTGTAATCAATAGCAACATCACGCCTATTTGTCTG CCAAGAAAAGAAGCTGAATCCTTTATGAGGACAGATGACATTGGAACTGCATCTGGATGGGGATTAACCCAAAGGGGTTTTCTTGCTAGAAATCTAATGTATGTCGACATACCGATTGTTGACCATCAAAAATGTACTGC TGCATATGAAAAGCCACCCTATCCAAGGGGAAGTGTAACTGCTAACATGCTTTGTGCTGGCTTAGAAAGTGGGGGCAAGGACAGCTGCAGAGGTGACAGCGGAGGGGCACTGGTGTTTCTAGATAGTGAAACAGAGAGGTGGTTTGTGGGAGGAATAGTGTCCTGGGGTTCCATGAATTGTGGGGAAGCAGGTCAGTATGGAGTCTACACAAAAGTTATTAACTATATTCCCTGGATCGAGAACATAATTAGTGATTTTTAACTTGCGTGTCTGCAGTCAAGGATTCTTCATTTTTAGAAATGCCTGTGAAGACCTTGGCAGCGACGTGGCTCGAGAAGCATTCATCATT ACTGTGGACATGGCAGTTGTTGCTCCACCCAAAAAAACAGACTCCAGGTGAGGCTGCTGTCATTTCTCCACTTGCCAGTTTAATTCCAGCCTTACCCATTGACTCAAGGGGACATAAACCACGAGAGTGACAGTCATCTT TGCCCACCCAGTGTAATGTCACTGCTCAAATTACATTTCATTACCTTAAAAAGCCAGTCTCTTTTCATACTGGCTGTTGGCATTTCTGTAAACTGCCTGTCCATGCTCTTTGTTTTTAAACTTGTTCTTATTGAAAAAAA AAAAAAAAAA

[0403] SEQ ID NO: 11 Amino acid sequence of human sMAP (MAp19)

[0404] MRLLTLLGLLCGSVATPLGPKWPEPVFGRLASPGFPGEYANDQERRWTLTAPPGYRLRLYFTHFDLELSHL CEYDFVKLSSGAKVLATLCGQESTDTERAPGKDTFYSLGSSLDITFRSDYSNEKPFTGFEAFYAAEDIDECQVAPGEAPTCDHHCHNHLGGFYCSCRAGYVLHRNKRTCSEQSL

[0405] SEQ ID NO: 12 cDNA sequence of human sMAP (MAp19)

[0406] GGCCAGCTGGACGGGCACACCATGAGGCTGCTGACCCTCCTGGGCCTTCTGTGTGGCTCGGTGGCCACCC CCTTGGGCCCGAAGTGGCCTGAACCTGTGTTCGGGCGCCTGGCATCCCCCGGCTTTCCAGGGGAGTATGCCAATGACCAGGAGCGGCGCTGGACCCTGACTGCACCCCCCGGCTACCGCCTGCGCCTCTACTTCACCCAC TTCGACCTGGAGCTCTCCCACCTCTGCGAGTACGACTTCGTCAAGCTGAGCTCGGGGGCCAAGGTGCTGG CCACGCTGTGCGGGCAGGAGAGCACAGACACGGAGCGGGCCCCTGGCAAGGACACTTTCTACTCGCTGGGCTCCAGCCTGGACATTACCTTCCGCTCCGACTACTCCAACGAGAAGCCGTTCACGGGGTTCGAGGCCTTC TATGCAGCCGAGGACATTGACGAGTGCCAGGTGGCCCCGGGAGAGGCGCCCACCTGCGACCACCACTGCCACAACCACCTGGGCGGTTTCTACTGCTCCTGCCGCGCAGGCTACGTCCTGCACCGTAACAAGCGCACCTG CTCAGAGCAGAGCCTCTAGCCTCCCCTGGAGCTCCGGCCTGCCCAGCAGGTCAGAAGCCAGAGCCAGCCTGCTGGCCTCAGCTCCGGGTTGGGCTGAGATGGCTGTGCCCCAACTCCCATTCACCCACCATGGACCCAAT AATAAACCTGGCCCCACCCCAAAAAAAAAAAAAAAAAA

[0407] DNA primer:

[0408] SEQ ID NO: 13: 5'-gcacccagagccacagtg-3'

[0409] SEQ ID NO: 14: 5'-gccttccagtgtgtgggc-3'

[0410] SEQ ID NO: 15: 5-gccttccagagtgtggtca-3'

[0411] SEQ ID NO: 16: 5′-cgatctggagagcgaactc-3′

[0412] SEQ ID NO: 17: 5′-ctgttcttcacactggctg-3′

[0413] SEQ ID NO: 18: 5′-ctgctgagatcatgttgttc-3′

[0414] SEQ ID NO: 19: 5′-TTATACGACTCACTA-3′ sequence list <110> Danish National Hospital University of Copenhagen University of Southern Denmark <120> MASP isotypes as inhibitors of complement activation <130> 17952PCT00 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 380 <212> PRT <213> Human (Homo sapiens) <400> 1 Met Arg Trp Leu Leu Leu Tyr Tyr Ala Leu Cys Phe Ser Leu Ser Lys 1 5 10 15 Ala Ser Ala His Thr Val Glu Leu Asn Asn Met Phe Gly Gln Ile Gln 20 25 30 Ser Pro Gly Tyr Pro Asp Ser Tyr Pro Ser Asp Ser Glu Val Thr Trp 35 40 45 Asn Ile Thr Val Pro Asp Gly Phe Arg Ile Lys Leu Tyr Phe Met His 50 55 60 Phe Asn Leu Glu Ser Ser Tyr Leu Cys Glu Tyr Asp Tyr Val Lys Val 65 70 75 80 Glu Thr Glu Asp Gln Val Leu Ala Thr Phe Cys Gly Arg Glu Thr Thr 85 90 95 Asp Thr Glu Gln Thr Pro Gly Gln Glu Val Val Leu Ser Pro Gly Ser 100 105 110 Phe Met Ser Ile Thr Phe Arg Ser Asp Phe Ser Asn Glu Glu Arg Phe 115 120 125 Thr Gly Phe Asp Ala His Tyr Met Ala Val Asp Val Asp Glu Cys Lys 130 135 140 Glu Arg Glu Asp Glu Glu Leu Ser Cys Asp His Tyr Cys His Asn Tyr 145 150 155 160 Ile Gly Gly Tyr Tyr Cys Ser Cys Arg Phe Gly Tyr Ile Leu His Thr 165 170 175 Asp Asn Arg Thr Cys Arg Val Glu Cys Ser Asp Asn Leu Phe Thr Gln 180 185 190 Arg Thr Gly Val Ile Thr Ser Pro Asp Phe Pro Asn Pro Tyr Pro Lys 195 200 205 Ser Ser Glu Cys Leu Tyr Thr Ile Glu Leu Glu Glu Gly Phe Met Val 210 215 220 Asn Leu Gln Phe Glu Asp Ile Phe Asp Ile Glu Asp His Pro Glu Val 225 230 235 240 Pro Cys Pro Tyr Asp Tyr Ile Lys Ile Lys Val Gly Pro Lys Val Leu 245 250 255 Gly Pro Phe Cys Gly Glu Lys Ala Pro Glu Pro Ile Ser Thr Gln Ser 260 265 270 His Ser Val Leu Ile Leu Phe His Ser Asp Asn Ser Gly Glu Asn Arg 275 280 285 Gly Trp Arg Leu Ser Tyr Arg Ala Ala Gly Asn Glu Cys Pro Glu Leu 290 295 300 Gln Pro Pro Val His Gly Lys Ile Glu Pro Ser Gln Ala Lys Tyr Phe 305 310 315 320 Phe Lys Asp Gln Val Leu Val Ser Cys Asp Thr Gly Tyr Lys Val Leu 325 330 335 Lys Asp Asn Val Glu Met Asp Thr Phe Gln Ile Glu Cys Leu Lys Asp 340 345 350 Gly Thr Trp Ser Asn Lys Ile Pro Thr Cys Lys Lys Asn Glu Ile Asp 355 360 365 Leu Glu Ser Glu Leu Lys Ser Glu Gln Val Thr Glu 370 375 380 <210> 2 <211> 1143 <212> DNA <213> Human <400> 2 atgaggtggc tgcttctcta ttatgctctg tgcttctccc tgtcaaaggc ttcagcccac 60 accgtggagc taaacaatat gtttggccag atccagtcgc ctggttatcc agactcctat 120 cccagtgatt cagaggtgac ttggaatatc actgtcccag atgggtttcg gatcaagctt 180 tacttcatgc acttcaactt ggaatcctcc tacctttgtg aatatgacta tgtgaaggta 240 gaaactgagg accaggtgct ggcaaccttc tgtggcaggg agaccacaga cacagagcag 300 actcccggcc aggaggtggt cctctcccct ggctccttca tgtccatcac tttccggtca 360 gatttctcca atgaggagcg tttcacaggc tttgatgccc actacatggc tgtggatgtg 420 gacgagtgca aggagaggga ggacgaggag ctgtcctgtg accactactg ccacaactac 480 attggcggct actactgctc ctgccgcttc ggctacatcc tccacacaga caacaggacc 540 tgccgagtgg agtgcagtga caacctcttc actcaaagga ctggggtgat caccagccct 600 gacttcccaa acccttaccc caagagctct gaatgcctgt ataccatcga gctggaggag 660 ggtttcatgg tcaacctgca gtttgaggac atatttgaca ttgaggacca tcctgaggtg 720 ccctgcccct atgactacat caagatcaaa gttggtccaa aagttttggg gcctttctgt 780 ggagagaaag ccccagaacc catcagcacc cagagccaca gtgtcctgat cctgttccat 840 agtgacaact cgggagagaa ccggggctgg aggctctcat acagggctgc aggaaatgag 900 tgcccagagc tacagcctcc tgtccatggg aaaatcgagc cctcccaagc caagtatttc 960 ttcaaagacc aagtgctcgt cagctgtgac acaggctaca aagtgctgaa ggataatgtg 1020 gagatggaca cattccagat tgagtgtctg aaggatggga cgtggagtaa caagattccc 1080 acctgtaaaa aaaatgaaat cgatctggag agcgaactca agtcagagca agtgacagag 1140 tga 1143 <210> 3 <211> 297 <212> PRT <213> Human <400> 3 Met Arg Trp Leu Leu Leu Tyr Tyr Ala Leu Cys Phe Ser Leu Ser Lys 1 5 10 15 Ala Ser Ala His Thr Val Glu Leu Asn Asn Met Phe Gly Gln Ile Gln 20 25 30 Ser Pro Gly Tyr Pro Asp Ser Tyr Pro Ser Asp Ser Glu Val Thr Trp 35 40 45 Asn Ile Thr Val Pro Asp Gly Phe Arg Ile Lys Leu Tyr Phe Met His 50 55 60 Phe Asn Leu Glu Ser Ser Tyr Leu Cys Glu Tyr Asp Tyr Val Lys Val 65 70 75 80 Glu Thr Glu Asp Gln Val Leu Ala Thr Phe Cys Gly Arg Glu Thr Thr 85 90 95 Asp Thr Glu Gln Thr Pro Gly Gln Glu Val Val Leu Ser Pro Gly Ser 100 105 110 Phe Met Ser Ile Thr Phe Arg Ser Asp Phe Ser Asn Glu Glu Arg Phe 115 120 125 Thr Gly Phe Asp Ala His Tyr Met Ala Val Asp Val Asp Glu Cys Lys 130 135 140 Glu Arg Glu Asp Glu Glu Leu Ser Cys Asp His Tyr Cys His Asn Tyr 145 150 155 160 Ile Gly Gly Tyr Tyr Cys Ser Cys Arg Phe Gly Tyr Ile Leu His Thr 165 170 175 Asp Asn Arg Thr Cys Arg Val Glu Cys Ser Asp Asn Leu Phe Thr Gln 180 185 190 Arg Thr Gly Val Ile Thr Ser Pro Asp Phe Pro Asn Pro Tyr Pro Lys 195 200 205 Ser Ser Glu Cys Leu Tyr Thr Ile Glu Leu Glu Glu Gly Phe Met Val 210 215 220 Asn Leu Gln Phe Glu Asp Ile Phe Asp Ile Glu Asp His Pro Glu Val 225 230 235 240 Pro Cys Pro Tyr Asp Tyr Ile Lys Ile Lys Val Gly Pro Lys Val Leu 245 250 255 Gly Pro Phe Cys Gly Glu Lys Ala Pro Glu Pro Ile Ser Thr Gln Ser 260 265 270 His Ser Val Leu Ile Leu Phe His Ser Asp Asn Ser Gly Glu Asn Arg 275 280 285 Gly Trp Arg Leu Ser Tyr Arg Ala Ala 290 295 <210> 4 <211> 17 <212> PRT <213> human <400> 4 Lys Asn Glu Ile Asp Leu Glu Ser Glu Leu Lys Ser Glu Gln Val Thr 1 5 10 15 Glu <210> 5 <211> 699 <212> PRT <213> human <400> 5 Met Arg Trp Leu Leu Leu Tyr Tyr Ala Leu Cys Phe Ser Leu Ser Lys 1 5 10 15 Ala Ser Ala His Thr Val Glu Leu Asn Asn Met Phe Gly Gln Ile Gln 20 25 30 Ser Pro Gly Tyr Pro Asp Ser Tyr Pro Ser Asp Ser Glu Val Thr Trp 35 40 45 Asn Ile Thr Val Pro Asp Gly Phe Arg Ile Lys Leu Tyr Phe Met His 50 55 60 Phe Asn Leu Glu Ser Ser Tyr Leu Cys Glu Tyr Asp Tyr Val Lys Val 65 70 75 80 Glu Thr Glu Asp Gln Val Leu Ala Thr Phe Cys Gly Arg Glu Thr Thr 85 90 95 Asp Thr Glu Gln Thr Pro Gly Gln Glu Val Val Leu Ser Pro Gly Ser 100 105 110 Phe Met Ser Ile Thr Phe Arg Ser Asp Phe Ser Asn Glu Glu Arg Phe 115 120 125 Thr Gly Phe Asp Ala His Tyr Met Ala Val Asp Val Asp Glu Cys Lys 130 135 140 Glu Arg Glu Asp Glu Glu Leu Ser Cys Asp His Tyr Cys His Asn Tyr 145 150 155 160 Ile Gly Gly Tyr Tyr Cys Ser Cys Arg Phe Gly Tyr Ile Leu His Thr 165 170 175 Asp Asn Arg Thr Cys Arg Val Glu Cys Ser Asp Asn Leu Phe Thr Gln 180 185 190 Arg Thr Gly Val Ile Thr Ser Pro Asp Phe Pro Asn Pro Tyr Pro Lys 195 200 205 Ser Ser Glu Cys Leu Tyr Thr Ile Glu Leu Glu Glu Gly Phe Met Val 210 215 220 Asn Leu Gln Phe Glu Asp Ile Phe Asp Ile Glu Asp His Pro Glu Val 225 230 235 240 Pro Cys Pro Tyr Asp Tyr Ile Lys Ile Lys Val Gly Pro Lys Val Leu 245 250 255 Gly Pro Phe Cys Gly Glu Lys Ala Pro Glu Pro Ile Ser Thr Gln Ser 260 265 270 His Ser Val Leu Ile Leu Phe His Ser Asp Asn Ser Gly Glu Asn Arg 275 280 285 Gly Trp Arg Leu Ser Tyr Arg Ala Ala Gly Asn Glu Cys Pro Glu Leu 290 295 300 Gln Pro Pro Val His Gly Lys Ile Glu Pro Ser Gln Ala Lys Tyr Phe 305 310 315 320 Phe Lys Asp Gln Val Leu Val Ser Cys Asp Thr Gly Tyr Lys Val Leu 325 330 335 Lys Asp Asn Val Glu Met Asp Thr Phe Gln Ile Glu Cys Leu Lys Asp 340 345 350 Gly Thr Trp Ser Asn Lys Ile Pro Thr Cys Lys Ile Val Asp Cys Arg 355 360 365 Ala Pro Gly Glu Leu Glu His Gly Leu Ile Thr Phe Ser Thr Arg Asn 370 375 380 Asn Leu Thr Thr Tyr Lys Ser Glu Ile Lys Tyr Ser Cys Gln Glu Pro 385 390 395 400 Tyr Tyr Lys Met Leu Asn Asn Asn Thr Gly Ile Tyr Thr Cys Ser Ala 405 410 415 Gln Gly Val Trp Met Asn Lys Val Leu Gly Arg Ser Leu Pro Thr Cys 420 425 430 Leu Pro Val Cys Gly Leu Pro Lys Phe Ser Arg Lys Leu Met Ala Arg 435 440 445 Ile Phe Asn Gly Arg Pro Ala Gln Lys Gly Thr Thr Pro Trp Ile Ala 450 455 460 Met Leu Ser His Leu Asn Gly Gln Pro Phe Cys Gly Gly Ser Leu Leu 465 470 475 480 Gly Ser Ser Trp Ile Val Thr Ala Ala His Cys Leu His Gln Ser Leu 485 490 495 Asp Pro Glu Asp Pro Thr Leu Arg Asp Ser Asp Leu Leu Ser Pro Ser 500 505 510 Asp Phe Lys Ile Ile Leu Gly Lys His Trp Arg Leu Arg Ser Asp Glu 515 520 525 Asn Glu Gln His Leu Gly Val Lys His Thr Thr Leu His Pro Gln Tyr 530 535 540 Asp Pro Asn Thr Phe Glu Asn Asp Val Ala Leu Val Glu Leu Leu Glu 545 550 555 560 Ser Pro Val Leu Asn Ala Phe Val Met Pro Ile Cys Leu Pro Glu Gly 565 570 575 Pro Gln Gln Glu Gly Ala Met Val Ile Val Ser Gly Trp Gly Lys Gln 580 585 590 Phe Leu Gln Arg Phe Pro Glu Thr Leu Met Glu Ile Glu Ile Pro Ile 595 600 605 Val Asp His Ser Thr Cys Gln Lys Ala Tyr Ala Pro Leu Lys Lys Lys 610 615 620 Val Thr Arg Asp Met Ile Cys Ala Gly Glu Lys Glu Gly Gly Lys Asp 625 630 635 640 Ala Cys Ala Gly Asp Ser Gly Gly Pro Met Val Thr Leu Asn Arg Glu 645 650 655 Arg Gly Gln Trp Tyr Leu Val Gly Thr Val Ser Trp Gly Asp Asp Cys 660 665 670 Gly Lys Lys Asp Arg Tyr Gly Val Tyr Ser Tyr Ile His His Asn Lys 675 680 685 Asp Trp Ile Gln Arg Val Thr Gly Val Arg Asn 690 695 <210> 6 <211> 4353 <212> DNA <213> Human <400> 6 gaagtcagcc acacaggata aaggagggaa gggaaggagc agatcttttc ggtaggaaga 60 cagattttgt tgtcaggttc ctgggagtgc aagagcaagt caaaggagag agagaggaga 120 gaggaaaagc cagagggaga gagggggaga ggggatctgt tgcaggcagg ggaaggcgtg 180 acctgaatgg agaatgccag ccaattccag agacacacag ggacctcaga acaaagataa 240 ggcatcacgg acaccacacc gggcacgagc tcacaggcaa gtcaagctgg gaggaccaag 300 gccgggcagc cgggagcacc caaggcagga aaatgaggtg gctgcttctc tattatgctc 360 tgtgcttctc cctgtcaaag gcttcagccc acaccgtgga gctaaacaat atgtttggcc 420 agatccagtc gcctggttat ccagactcct atcccagtga ttcagaggtg acttggaata 480 tcactgtccc agatgggttt cggatcaagc tttacttcat gcacttcaac ttggaatcct 540 cctacctttg tgaatatgac tatgtgaagg tagaaactga ggaccaggtg ctggcaacct 600 tctgtggcag ggagaccaca gacacagagc agactcccgg ccaggaggtg gtcctctccc 660 ctggctcctt catgtccatc actttccggt cagatttctc caatgaggag cgtttcacag 720 gctttgatgc ccactacatg gctgtggatg tggacgagtg caaggagagg gaggacgagg 780 agctgtcctg tgaccactac tgccacaact acattggcgg ctactactgc tcctgccgct 840 tcggctacat cctccacaca gacaacagga cctgccgagt ggagtgcagt gacaacctct 900 tcactcaaag gactggggtg atcaccagcc ctgacttccc aaacccttac cccaagagct 960 ctgaatgcct gtataccatc gagctggagg agggtttcat ggtcaacctg cagtttgagg 1020 acatatttga cattgaggac catcctgagg tgccctgccc ctatgactac atcaagatca 1080 aagttggtcc aaaagttttg gggcctttct gtggagagaa agccccagaa cccatcagca 1140 cccagagcca cagtgtcctg atcctgttcc atagtgacaa ctcgggagag aaccggggct 1200 ggaggctctc atacagggct gcaggaaatg agtgcccaga gctacagcct cctgtccatg 1260 ggaaaatcga gccctcccaa gccaagtatt tcttcaaaga ccaagtgctc gtcagctgtg 1320 acacaggcta caaagtgctg tggagatgga cacattccag attgagtgtc 1440 caggagagct ggaacacggg ctgatcacct tctctacaag gaacaacctc accacataca 1500 agtctgagat caaatactcc tgtcaggagc cctattacaa gatgctcaac ataaacag 1560 gtatatatac ctgttctgcc caaggagtct ggatgaataa agtattgggg agaagcctac 1620 ccacctgcct tccagtgtgt gggctcccca agttctcccg gaagctgatg gccaggatct 1680 tcaatggacg cccagcccag aaaggcacca ctccctggat tgccatgctg tcacacctga 1740 atgggcagcc cttctgcgga ggctcccttc taggctccag ctggatcgtg accgccgcac 1800 actgcctcca ccagtcactc gatccggaag atccgaccct acgtgattca gacttgctca 1860 gcccttctga cttcaaaatc atcctgggca agcattggag gctccggtca gatgaaaatg 1920 aacagcatct cggcgtcaaa cacaccactc tccaccccca gtatgatccc aacacattcg 1980 agaatgacgt ggctctggtg gagctgttgg agagcccagt gctgaatgcc ttcgtgatgc 2040 ccatctgtct gcctgaggga ccccagcagg aaggagccat ggtcatcgtc agcggctggg 2100 ggaagcagtt cttgcaaagg ttcccagaga ccctgatgga gattgaaatc ccgattgttg 2160 2220 tctgtgctgg ggagaaggaa gggggaaagg acgcctgtgc gggtgactct ggaggcccca 2280 tggtgaccct gatagaaga agaggccagt ggtacctggt gggcactgtg tcctggggtg 2340 atgactgtgg gaaaggac cgctacggag tatactctta catccaccac aacaaggact 2400 ggatccagag ggtcaccgga gtgaggaact gaatttggct cctcagcccc agcaccacca 2460 gctgtgggca gtcagtagca gaggacgatc ctccgatgaa agcagccatt tctcctttcc 2520 ttcctcccat cccccctcct tcggcctatc cattactggg caatagagca ggtatcttca 2580 cccccttttc actctcttta aagagatgga ccaagagagt ggtcagaaca caggccgaat 2640 ccaggctcta tcacttacta gtttgcagtg ctgggcaggt gacttcatct cttcgaactt 2700 cagtttcttc atagatgga aatgctatac cttacctacc tcgtaaaagt ctgatgagga 2760 aaaattaac taatagatgc atagcactta agagagtgca tagcatacac tgttttcaat 2820 aaatgcacct tagcagaagg tcgatgtgtc taccaggcag acgaagctct cttacaaacc 2880 cctgcctggg tcttagcatt gatcagtgac acacctctcc cctcaacctt gaccatctcc 2940 atctgccctt aaatgctgta tgcttttttg ccaccgtgca acttgcccaa catcaatctt 3000 caccctcatc cctaaaaaag taaaacagac aaggttctga gtcctgtggt atgtccccta 3060 ccaaatgtaa ctaggaacat gcaactagatg acagattgcg ggagggcctg agaagcag 3120 ggacaggagg gagcctgggg attgtggttt gggaaggcag acacctggtt ctagaactag 3180 ctctgccctt agccccctgt atgaccctat gcaagtcctc ctccctcatc tcaaagggtc 3240 ctcaaagctc tgacgatcta agatacaatg aagccatttt ccccctgata agatgaggta 3300 aagccaatgt aaccaaaagg caaaaattac aatcggttca aaggaacttt gatgcagaca 3360 aaatgctgct gctgctgctc ctgaaatacc cacccctttc cactacgggt gggttcccaa 3420 ggacatggga caggcaaagt gtgagccaaa ggatccttcc ttattcctaa gcagagcatc 3480 tgctctgggc cctggcctcc ttccctcttt gggaaactgg gctgcatgag gtgggccctg 3540 gtagttgta ccccaggccc ctatactctt ccttcctatg tccacagctg accccaagca 3600 gccgttcccc gactcctcac ccctgagcct caccctgaac tccctcatct tgcaaggcca 3660 taagtgtttt ccaagcaaaa tgcctctccc atcctctctc aggaagcttc tagagacttt 3720 atgccctcca gagctccaag atataagccc tccaagggat cagaagctcc aagttcctgt 3780 cttctgtttt atagaaattg atcttccctg ggggacttta actcttgacc tgtatgcagc 3840 tgttggagta attccaggtc tcttgaaaaa aaagaggaag ataatggaga atgagaacat 3900 atatatatat atattaagcc ccaggctgaa tactcaggga cagcaattca cagcctgcct 3960 ctggttctat aaacaagtca ttctacctct ttgtgccctg ctgtttattc tgtaagggga 4020 aggtggcaat gggacccagc tccatcagac acttgtcaag ctagcagaaa ctccattttc 4080 aatgccaaag aagaactgta atgctgtttt ggaatcatcc caaggcatcc caagacacca 4140 tatcttccca tttcaagcac tgcctgggca caccccaaca tcccaggctg tggtggctcc 4200 tgtgggaact acctagatga agagagtatc atttatacct tctaggagct cctattggga 4260 gacatgaaac atatgtaatt gactaccatg taatagaaca aaccctgcca agtgctgctt 4320 tggaaagtca tggaggtaaa agaaagacca ttc 4353 <210> 7 <211> 728 <212> PRT <213> Human <400> 7 Met Arg Trp Leu Leu Leu Tyr Tyr Ala Leu Cys Phe Ser Leu Ser Lys 1 5 10 15 Ala Ser Ala His Thr Val Glu Leu Asn Asn Met Phe Gly Gln Ile Gln 20 25 30 Ser Pro Gly Tyr Pro Asp Ser Tyr Pro Ser Asp Ser Glu Val Thr Trp 35 40 45 Asn Ile Thr Val Pro Asp Gly Phe Arg Ile Lys Leu Tyr Phe Met His 50 55 60 Phe Asn Leu Glu Ser Ser Tyr Leu Cys Glu Tyr Asp Tyr Val Lys Val 65 70 75 80 Glu Thr Glu Asp Gln Val Leu Ala Thr Phe Cys Gly Arg Glu Thr Thr 85 90 95 Asp Thr Glu Gln Thr Pro Gly Gln Glu Val Val Leu Ser Pro Gly Ser 100 105 110 Phe Met Ser Ile Thr Phe Arg Ser Asp Phe Ser Asn Glu Glu Arg Phe 115 120 125 Thr Gly Phe Asp Ala His Tyr Met Ala Val Asp Val Asp Glu Cys Lys 130 135 140 Glu Arg Glu Asp Glu Glu Leu Ser Cys Asp His Tyr Cys His Asn Tyr 145 150 155 160 Ile Gly Gly Tyr Tyr Cys Ser Cys Arg Phe Gly Tyr Ile Leu His Thr 165 170 175 Asp Asn Arg Thr Cys Arg Val Glu Cys Ser Asp Asn Leu Phe Thr Gln 180 185 190 Arg Thr Gly Val Ile Thr Ser Pro Asp Phe Pro Asn Pro Tyr Pro Lys 195 200 205 Ser Ser Glu Cys Leu Tyr Thr Ile Glu Leu Glu Glu Gly Phe Met Val 210 215 220 Asn Leu Gln Phe Glu Asp Ile Phe Asp Ile Glu Asp His Pro Glu Val 225 230 235 240 Pro Cys Pro Tyr Asp Tyr Ile Lys Ile Lys Val Gly Pro Lys Val Leu 245 250 255 Gly Pro Phe Cys Gly Glu Lys Ala Pro Glu Pro Ile Ser Thr Gln Ser 260 265 270 His Ser Val Leu Ile Leu Phe His Ser Asp Asn Ser Gly Glu Asn Arg 275 280 285 Gly Trp Arg Leu Ser Tyr Arg Ala Ala Gly Asn Glu Cys Pro Glu Leu 290 295 300 Gln Pro Pro Val His Gly Lys Ile Glu Pro Ser Gln Ala Lys Tyr Phe 305 310 315 320 Phe Lys Asp Gln Val Leu Val Ser Cys Asp Thr Gly Tyr Lys Val Leu 325 330 335 Lys Asp Asn Val Glu Met Asp Thr Phe Gln Ile Glu Cys Leu Lys Asp 340 345 350 Gly Thr Trp Ser Asn Lys Ile Pro Thr Cys Lys Ile Val Asp Cys Arg 355 360 365 Ala Pro Gly Glu Leu Glu His Gly Leu Ile Thr Phe Ser Thr Arg Asn 370 375 380 Asn Leu Thr Thr Tyr Lys Ser Glu Ile Lys Tyr Ser Cys Gln Glu Pro 385 390 395 400 Tyr Tyr Lys Met Leu Asn Asn Asn Thr Gly Ile Tyr Thr Cys Ser Ala 405 410 415 Gln Gly Val Trp Met Asn Lys Val Leu Gly Arg Ser Leu Pro Thr Cys 420 425 430 Leu Pro Glu Cys Gly Gln Pro Ser Arg Ser Leu Pro Ser Leu Val Lys 435 440 445 Arg Ile Ile Gly Gly Arg Asn Ala Glu Pro Gly Leu Phe Pro Trp Gln 450 455 460 Ala Leu Ile Val Val Glu Asp Thr Ser Arg Val Pro Asn Asp Lys Trp 465 470 475 480 Phe Gly Ser Gly Ala Leu Leu Ser Ala Ser Trp Ile Leu Thr Ala Ala 485 490 495 His Val Leu Arg Ser Gln Arg Arg Asp Thr Thr Val Ile Pro Val Ser 500 505 510 Lys Glu His Val Thr Val Tyr Leu Gly Leu His Asp Val Arg Asp Lys 515 520 525 Ser Gly Ala Val Asn Ser Ser Ala Ala Arg Val Val Leu His Pro Asp 530 535 540 Phe Asn Ile Gln Asn Tyr Asn His Asp Ile Ala Leu Val Gln Leu Gln 545 550 555 560 Glu Pro Val Pro Leu Gly Pro His Val Met Pro Val Cys Leu Pro Arg 565 570 575 Leu Glu Pro Glu Gly Pro Ala Pro His Met Leu Gly Leu Val Ala Gly 580 585 590 Trp Gly Ile Ser Asn Pro Asn Val Thr Val Asp Glu Ile Ile Ser Ser 595 600 605 Gly Thr Arg Thr Leu Ser Asp Val Leu Gln Tyr Val Lys Leu Pro Val 610 615 620 Val Pro His Ala Glu Cys Lys Thr Ser Tyr Glu Ser Arg Ser Gly Asn 625 630 635 640 Tyr Ser Val Thr Glu Asn Met Phe Cys Ala Gly Tyr Tyr Glu Gly Gly 645 650 655 Lys Asp Thr Cys Leu Gly Asp Ser Gly Gly Ala Phe Val Ile Phe Asp 660 665 670 Asp Leu Ser Gln Arg Trp Val Val Gln Gly Leu Val Ser Trp Gly Gly 675 680 685 Pro Glu Glu Cys Gly Ser Lys Gln Val Tyr Gly Val Tyr Thr Lys Val 690 695 700 Ser Asn Tyr Val Asp Trp Val Trp Glu Gln Met Gly Leu Pro Gln Ser 705 710 715 720 Val Val Glu Pro Gln Val Glu Arg 725 <210> 8 <211> 4137 <212> DNA <213> Human <400> 8 gaagtcagcc acacaggata aaggagggaa gggaaggagc agatcttttc ggtaggaaga 60 cagattttgt tgtcaggttc ctgggagtgc aagagcaagt caaaggagag agagaggaga 120 gaggaaaagc cagagggaga gagggggaga ggggatctgt tgcaggcagg ggaaggcgtg 180 acctgaatgg agaatgccag ccaattccag agacacacag ggacctcaga acaaagataa 240 ggcatcacgg acaccacacc gggcacgagc tcacaggcaa gtcaagctgg gaggaccaag 300 gccgggcagc cgggagcacc caaggcagga aaatgaggtg gctgcttctc tattatgctc 360 tgtgcttctc cctgtcaaag gcttcagccc acaccgtgga gctaaacaat atgtttggcc 420 agatccagtc gcctggttat ccagactcct atcccagtga ttcagaggtg acttggaata 480 tcactgtccc agatgggttt cggatcaagc tttacttcat gcacttcaac ttggaatcct 540 cctacctttg tgaatatgac tatgtgaagg tagaaactga ggaccaggtg ctggcaacct 600 tctgtggcag ggagaccaca gacacagagc agactcccgg ccaggaggtg gtcctctccc 660 ctggctcctt catgtccatc actttccggt cagatttctc caatgaggag cgtttcacag 720 gctttgatgc ccactacatg gctgtggatg tggacgagtg caaggagagg gaggacgagg 780 agctgtcctg tgaccactac tgccacaact acattggcgg ctactactgc tcctgccgct 840 tcggctacat cctccacaca gacaacagga cctgccgagt ggagtgcagt gacaacctct 900 tcactcaaag gactggggtg atcaccagcc ctgacttccc aaacccttac cccaagagct 960 ctgaatgcct gtataccatc gagctggagg agggtttcat ggtcaacctg cagtttgagg 1020 acatatttga cattgaggac catcctgagg tgccctgccc ctatgactac atcaagatca 1080 aagttggtcc aaaagttttg gggcctttct gtggagagaa agccccagaa cccatcagca 1140 cccagagcca cagtgtcctg atcctgttcc atagtgacaa ctcgggagag aaccggggct 1200 ggaggctctc atacagggct gcaggaaatg agtgcccaga gctacagcct cctgtccatg 1260 ggaaaatcga gccctcccaa gccaagtatt tcttcaaaga ccaagtgctc gtcagctgtg 1320 acacaggcta caaagtgctg tggagatgga cacattccag attgagtgtc 1440 caggagagct ggaacacggg ctgatcacct tctctacaag gaacaacctc accacataca 1500 agtctgagat caaatactcc tgtcaggagc cctattacaa gatgctcaac ataaacag 1560 gtatatatac ctgttctgcc caaggagtct ggatgaataa agtattgggg agaagcctac 1620 ccacctgcct tccagagtgt ggtcagccct cccgctccct gccaagcctg gtcaagga 1680 tcattgggg ccgaaatgct gagcctggcc tcttccccgtg gcaggccctg atagtggtgg 1740 aggacacttc gagagtgcca aatgacaagt ggtttgggag tggggccctg ctctctgcgt 1800 cctggatcct cacagcagct catgtgctgc gctcccagcg tagagacacc acggtgatac 1860 cagtctccaa ggagcatgtc accgtctacc tgggcttgca tgatgtgcga gacaaatcgg 1920 gggcagtcaa cagctcagct gcccgagtgg tgctccaccc agacttcaac atccaaaact 1980 acaaccacga tatagctctg gtgcagctgc aggagcctgt gcccctggga ccccacgtta 2040 tgcctgtctg cctgccaagg cttgagcctg aaggcccggc cccccacatg ctgggcctgg 2100 tggccggctg gggcatctcc aatcccaatg tgacagtgga tgagatcatc agcagtggca 2160 cacggacctt gtcagatgtc ctgcagtatg tcaagttacc cgtggtgcct cacgctgagt 2220 gcaaaactag ctatgagtcc cgctcgggca attacagcgt cacggagaac atgttctgtg 2280 ctggctacta cgagggcggc aaagacacgt gccttggaga tagcggtggg gcctttgtca 2340 tctttgatga cttgagccag cgctgggtgg tgcaaggcct ggtgtcctgg gggggacctg 2400 aagaatgcgg cagcaagcag gtctatggag tctacacaaa ggtctccaat tacgtggact 2460 gggtgtggga gcagatgggc ttaccacaaa gtgttgtgga gccccaggtg gaacggtgag 2520 ctgacttact tctcggggc ctgcctcccc tgagcgaagc tacaccgcac ttccgacagc 2580 acactccaca ttacttatca gaccatatgg aatggaacac actgacctag cggtggcttc 2640 tcctaccgag acagccccca ggaccctgag aggcagagtg tggtataggg aaaaggctcc 2700 aggcaggaga cctgtgttcc tgagcttgtc caagtctctt tccctgtctg ggcctcactc 2760 taccgagtaa tacaatgcag gagctcaacc aaggcctctg tgccaatccc agcactcctt 2820 tccaggccat gcttcttacc ccagtggcct ttattcactc ctgaccactt atcaaaccca 2880 tcggtcctac tgttggtata actgagcttg gacctgacta ttagaaaatg gtttctaaca 2940 ttgaactgaa tgccgcatct gtatattttc ctgctctgcc ttctgggact agccttggcc 3000 taatccttcc tctaggagaa gagcattcag gttttgggag atggctcata gccaagcccc 3060 tctctcttag tgtgatccct tggagcacct tcatgcctgg ggtttctctc ccaaaagctt 3120 cttgcagtct aagccttatc ccttatgttc cccattaaag gaatttcaaa agacatggag 3180 aaagttggga aggtttgtgc tgactgctgg gagcagaata gccgtgggag gcccaccaag 3240 cccttaaatt cccattgtca actcagaaca catttgggcc catatgccac cctggaacac 3300 cagctgacac catgggcgtc cacacctgct gctccagaca agcacaaagc aatctttcag 3360 ccttgaaatg tattatctga aaggctacct gaagcccagg cccgaatatg gggacttagt 3420 cgattacctg gaaaaagaaa agacccacac tgtgtcctgc tgtgcttttg ggcaggaaaa 3480 tggaagaaag agtggggtgg gcacattaga agtcacccaa atcctgccag gctgcctggc 3540 atccctgggg catgagctgg gcggagaatc caccccgcag gatgttcaga gggacccact 3600 ccttcatttt tcagagtcaa aggaatcaga ggctcaccca tggcaggcag tgaaaagagc 3660 caggagtcct gggttctagt ccctgctctg cccccaactg gctgtataac ctttgaaaaa 3720 tcattttctt tgtctgagtc tctggttctc cgtcagcaac aggctggcat aaggtcccct 3780 gcaggttcct tctagctgga gcactcagag cttccctgac tgctagcagc ctctctggcc 3840 ctcacagggc tgattgttct ccttctccct ggagctctct ctcctgaaaa tctccatcag 3900 agcaaggcag ccagagaagc ccctgagagg gaatgattgg gaagtgtcca ctttctcaac 3960 cggctcatca aacacactcc tttgtctatg aatggcacat gtaaatgatg ttatattttg 4020 tatcttttat atcatatgct tcaccattct gtaaagggcc tctgcattgt tgctcccatc 4080 aggggtctca agtggaaata aaccctcgtg gataaccaaa aaaaaaaaaa aaaaaaa 4137 <210> 9 <211> 686 <212> PRT <213> Human <400> 9 Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 1 5 10 15 Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser 20 25 30 Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45 Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60 Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80 Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95 Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110 Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val 130 135 140 Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160 Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn 165 170 175 Lys Arg Thr Cys Ser Ala Leu Cys Ser Gly Gln Val Phe Thr Gln Arg 180 185 190 Ser Gly Glu Leu Ser Ser Pro Glu Tyr Pro Arg Pro Tyr Pro Lys Leu 195 200 205 Ser Ser Cys Thr Tyr Ser Ile Ser Leu Glu Glu Gly Phe Ser Val Ile 210 215 220 Leu Asp Phe Val Glu Ser Phe Asp Val Glu Thr His Pro Glu Thr Leu 225 230 235 240 Cys Pro Tyr Asp Phe Leu Lys Ile Gln Thr Asp Arg Glu Glu His Gly 245 250 255 Pro Phe Cys Gly Lys Thr Leu Pro His Arg Ile Glu Thr Lys Ser Asn 260 265 270 Thr Val Thr Ile Thr Phe Val Thr Asp Glu Ser Gly Asp His Thr Gly 275 280 285 Trp Lys Ile His Tyr Thr Ser Thr Ala Gln Pro Cys Pro Tyr Pro Met 290 295 300 Ala Pro Pro Asn Gly His Val Ser Pro Val Gln Ala Lys Tyr Ile Leu 305 310 315 320 Lys Asp Ser Phe Ser Ile Phe Cys Glu Thr Gly Tyr Glu Leu Leu Gln 325 330 335 Gly His Leu Pro Leu Lys Ser Phe Thr Ala Val Cys Gln Lys Asp Gly 340 345 350 Ser Trp Asp Arg Pro Met Pro Ala Cys Ser Ile Val Asp Cys Gly Pro 355 360 365 Pro Asp Asp Leu Pro Ser Gly Arg Val Glu Tyr Ile Thr Gly Pro Gly 370 375 380 Val Thr Thr Tyr Lys Ala Val Ile Gln Tyr Ser Cys Glu Glu Thr Phe 385 390 395 400 Tyr Thr Met Lys Val Asn Asp Gly Lys Tyr Val Cys Glu Ala Asp Gly 405 410 415 Phe Trp Thr Ser Ser Lys Gly Glu Lys Ser Leu Pro Val Cys Glu Pro 420 425 430 Val Cys Gly Leu Ser Ala Arg Thr Thr Gly Gly Arg Ile Tyr Gly Gly 435 440 445 Gln Lys Ala Lys Pro Gly Asp Phe Pro Trp Gln Val Leu Ile Leu Gly 450 455 460 Gly Thr Thr Ala Ala Gly Ala Leu Leu Tyr Asp Asn Trp Val Leu Thr 465 470 475 480 Ala Ala His Ala Val Tyr Glu Gln Lys His Asp Ala Ser Ala Leu Asp 485 490 495 Ile Arg Met Gly Thr Leu Lys Arg Leu Ser Pro His Tyr Thr Gln Ala 500 505 510 Trp Ser Glu Ala Val Phe Ile His Glu Gly Tyr Thr His Asp Ala Gly 515 520 525 Phe Asp Asn Asp Ile Ala Leu Ile Lys Leu Asn Asn Lys Val Val Ile 530 535 540 Asn Ser Asn Ile Thr Pro Ile Cys Leu Pro Arg Lys Glu Ala Glu Ser 545 550 555 560 Phe Met Arg Thr Asp Asp Ile Gly Thr Ala Ser Gly Trp Gly Leu Thr 565 570 575 Gln Arg Gly Phe Leu Ala Arg Asn Leu Met Tyr Val Asp Ile Pro Ile 580 585 590 Val Asp His Gln Lys Cys Thr Ala Ala Tyr Glu Lys Pro Pro Tyr Pro 595 600 605 Arg Gly Ser Val Thr Ala Asn Met Leu Cys Ala Gly Leu Glu Ser Gly 610 615 620 Gly Lys Asp Ser Cys Arg Gly Asp Ser Gly Gly Ala Leu Val Phe Leu 625 630 635 640 Asp Ser Glu Thr Glu Arg Trp Phe Val Gly Gly Ile Val Ser Trp Gly 645 650 655 Ser Met Asn Cys Gly Glu Ala Gly Gln Tyr Gly Val Tyr Thr Lys Val 660 665 670 Ile Asn Tyr Ile Pro Trp Ile Glu Asn Ile Ile Ser Asp Phe 675 680 685 <210> 10 <211> 2460 <212> DNA <213> Human <400> 10 ggccagctgg acgggcacac catgaggctg ctgaccctcc tgggccttct gtgtggctcg 60 gtggccaccc ccttgggccc gaagtggcct gaacctgtgt tcgggcgcct ggcatccccc 120 ggctttccag gggagtatgc caatgaccag gagcggcgct ggaccctgac tgcacccccc 180 ggctaccgcc tgcgcctcta cttcacccac ttcgacctgg agctctccca cctctgcgag 240 tacgacttcg tcaagctgag ctcgggggcc aaggtgctgg ccacgctgtg cgggcaggag 300 agcacagaca cggagcgggc ccctggcaag actcgctggg ctccagcctg 360 420. gacattacct tccgctccga ctactccaac gagaagccgt tcacggggtt cgaggccttc tatgcagccg aggacattga cgagtgccag gtggccccgg gagaggcgcc cacctgcgac 480 caccactgcc acaaccacct gggcggtttc tactgctcct gccgcgcagg ctacgtcctg 540 caccgtaaca agcgcacctg ctcagccctg tgctccggcc aggtcttcac ccagaggtct ggggagctca gcagccctga atacccacgg ccgtatccca aactctccag ttgcacttac 660 agcatcagcc tggaggaggg gttcagtgtc attctggact ttgtggagtc cttcgatgtg 720 gagacacacc ctgaaaccct gtgtccctac gactttctca agattcaaac agacagagaa gacatggcc cattctgtgg gaagacattg ccccacagga ttgaaacaaa aagcaacacg gtgaccatca cctttgtcac agatgaatca ggagaccaca caggctgga gatccactac acgagcacag cgcagccttg cccttatccg atggcgccac ctaatggcca cgtttcacct 960 gtgcaagcca aatacatcct gaagacagc ttctccatct tttgcgagac tggctatgag 1020 cttctgcaag gtcacttgcc cctgaaatcc tttactgcag tttgtcagaa agatggatct 1080 tgggaccggc caatgcccgc gtgcagcatt gttgactgtg gccctcctga tgatctaccc 1140 agtggccgag tggagtacat cacaggtcct ggagtgacca cctacaaagc tgtgattcag 1200 1260 gctgatggat tctggacgag ctccaaaagga gaaaatcac tcccagtctg tgagcctgtt 1320 tgtggactat cagcccgcac aacaggaggg cgtatatatg gagggcaaaa ggcaaaacct 1380 ggtgattttc cttggcaagt cctgatatta ggtggaacca cagcagcagg tgcactttta 1440 tatgacaact gggtcctaac agctgctcat gccgtctatg agcaaaaaca tgatgcatcc 1500 gccctggaca ttcgaatggg caccctgaaa agactatcac ctcatattac acaagcctgg 1560 tctgaagctg tttttataca tgaaggttat actcatgatg ctggctttga caatgacata 1620 gcactgatta attgataa caaagttgta atcaatgca acatcacgcc tattgtctg 1680 ccaagaaaag aagctgaatc ctttatgagg acagatgaca ttggactgc atctggatgg 1740 ggattaaccc aaaggggtt tcttgctaga aatctaatgt atgtcgacat accgattgtt 1800 gaccatcaa aatgtactgc tgcatatgaa aagccaccct atccagggg aagtgtact 1860 gctaacatgc ttgtgctgg cttagaaagt gggggcaagg acagctgcag aggtgacagc 1920 ggaggggcac tggtgttct agatagtgaa acagagaggt ggtttgtggg aggaatagtg 1980 tcctggggtt ccatgaattg tgggaagca ggtcagtatg gagtctacac aaagttatt 2040 aactatattc cctggatcga gaacatatt agtgatttt aacttgcgtg tctgcagtca 2100 aggattcttc atttttagaa atgctgtga agaccttggc agcgacgtgg ctcgagaagc 2160 attcatcatt actgtggaca tggcagttgt tgctccaccc aaaaaaacag actcaggtg 2220 aggctgctgt cattctcca cttgccagtt taattccagc cttacccatt gactcaaggg 2280 gataaacc acgagagtga cagtcatctt tgccaccca gtgtaatgtc actgctcaa 2340 ttacatttca ttaccttaaa aagccagtct cttttcatac tggctgttgg catttctgta 2400 aactgcctgt ccatgctctt tgtttttaaa cttgttctta ttgaaaaaaa aaaaaaaaaa 2460 <210> 11 <211> 185 <212> PRT <213> Human <400> 11 Met Arg Leu Leu Thr Leu Leu Gly Leu Leu Cys Gly Ser Val Ala Thr 1 5 10 15 Pro Leu Gly Pro Lys Trp Pro Glu Pro Val Phe Gly Arg Leu Ala Ser 20 25 30 Pro Gly Phe Pro Gly Glu Tyr Ala Asn Asp Gln Glu Arg Arg Trp Thr 35 40 45 Leu Thr Ala Pro Pro Gly Tyr Arg Leu Arg Leu Tyr Phe Thr His Phe 50 55 60 Asp Leu Glu Leu Ser His Leu Cys Glu Tyr Asp Phe Val Lys Leu Ser 65 70 75 80 Ser Gly Ala Lys Val Leu Ala Thr Leu Cys Gly Gln Glu Ser Thr Asp 85 90 95 Thr Glu Arg Ala Pro Gly Lys Asp Thr Phe Tyr Ser Leu Gly Ser Ser 100 105 110 Leu Asp Ile Thr Phe Arg Ser Asp Tyr Ser Asn Glu Lys Pro Phe Thr 115 120 125 Gly Phe Glu Ala Phe Tyr Ala Ala Glu Asp Ile Asp Glu Cys Gln Val 130 135 140 Ala Pro Gly Glu Ala Pro Thr Cys Asp His His Cys His Asn His Leu 145 150 155 160 Gly Gly Phe Tyr Cys Ser Cys Arg Ala Gly Tyr Val Leu His Arg Asn 165 170 175 Lys Arg Thr Cys Ser Glu Gln Ser Leu 180 185 <210> 12 <211> 738 <212> DNA <213> Human <400> 12 ggccagctgg acgggcacac catgaggctg ctgaccctcc tgggccttct gtgtggctcg 60 gtggccaccc ccttgggccc gaagtggcct gaacctgtgt tcgggcgcct ggcatccccc 120 ggctttccag gggagtatgc caatgaccag gagcggcgct ggaccctgac tgcacccccc 180 ggctaccgcc tgcgcctcta cttcacccac ttcgacctgg agctctccca cctctgcgag 240 tacgacttcg tcaagctgag ctcgggggcc aaggtgctgg ccacgctgtg cgggcaggag 300 agcacagaca cggagcgggc ccctggcaag actcgctggg ctccagcctg 360 420. gacattacct tccgctccga ctactccaac gagaagccgt tcacggggtt cgaggccttc tatgcagccg aggacattga cgagtgccag gtggccccgg gagaggcgcc cacctgcgac 480 caccactgcc acaaccacct gggcggtttc tactgctcct gccgcgcagg ctacgtcctg 540 caccgtaaca agcgcacctg ctcagagcag agcctctagc ctcccctgga gctccggcct gcccagcagg tcagaagcca gagccagcct gctggcctca gctccgggtt gggctgagat 660 ggctgtgccc caactcccat tcacccacca tggacccat grandfatheracctg gccccacccc 720 aaaaaaaaa aaaaaaaaa <210> 13 <211> 18 <212> DNA <213> The snowstorm <220> <223> DNA based <400> 13 gcacccagag ccacagtg <210> 14 <211> 18 <212> DNA <213> The snowstorm <220> <223> DNA based <400> 14 gccttccagt gtgtgggc <210> 15 <211> 19 <212> DNA <213> Artificial sequence <220> <223> DNA primers <400> 15 gccttccaga gtgtggtca 19 <210> 16 <211> 19 <212> DNA <213> Artificial sequence <220> <223> DNA primers <400> 16 cgatctggag agcgaactc 19 <210> 17 <211> 19 <212> DNA <213> Artificial sequence <220> <223> DNA primers <400> 17 ctgttcttca cactggctg 19 <210> 18 <211> 20 <212> DNA <213> Artificial sequence <220> <223> DNA primers <400> 18 ctgctgagat catgttgttc 20 <210> 19 <211> 15 <212> DNA <213> Artificial sequence <220> <223> DNA primers <400> 19 ttatacgact cacta 15

Claims

1. A composition for inhibiting lectin pathway complement activation, the composition comprising a ficolin-related polypeptide consisting of amino acid residues at positions 20-380 of the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide does not have serine protease activity, and wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

4.

2. The composition according to claim 1, wherein the polypeptide does not have a signal peptide.

3. The composition according to claim 1, wherein the polypeptide is capable of associating with mannose-binding lectin (MBL).

4. The composition according to claim 1, wherein the polypeptide is capable of associating with any of ficolin-1, ficolin-2, or ficolin-3.

5. The composition according to claim 1, wherein the polypeptide is N-linked glycosylated at one or both amino acids corresponding to positions 49 and 178 of SEQ NO:

1.

6. The composition according to claim 1, wherein the polypeptide is a recombinant protein.