Properdin-binding proteins and uses thereof

Abstract

The present disclosure provides isolated antigen-binding proteins capable of specifically binding to properdin. The present disclosure also provides anti-properdin antibodies, methods for preparing the same, and uses thereof.

Description

[Technical Field]

[0001] The present application relates to the field of biomedicine, and in particular to several isolated antigen-binding proteins that specifically bind to properdin and applications thereof. [Background technology]

[0002] The complement system, a crucial part of innate and adaptive immunity, plays a crucial role in the elimination of pathogens, cellular debris, and mutant cells. However, uncontrolled activation of this system plays a significant or critical role in the pathogenesis of human diseases, including eye disease, periodontal disease, cancer, autoimmune diseases, CNS / PNS diseases, kidney disease, and chronic hemolytic diseases. Complement inhibition has been successfully applied to the clinical or experimental treatment of a few human diseases, such as paroxysmal nocturnal hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), neuromyelitis optica spectrum disorder (NMOSD), thrombotic microangiopathy (TMA), age-related macular degeneration (AMD), IgA nephropathy (IgAN), and Alzheimer's disease (AD).

[0003] The complement system can be activated through three pathways: the classical (CP), lectin (LP), or alternative (AP) pathway. The AP pathway is a true defense system that is always active and accounts for approximately 80–90% of terminal pathway activation by forming a powerful amplification loop for the three complement pathways. C3 convertases have a short half-life of approximately 90 seconds under physiological conditions, either in the fluid phase or on cell surfaces. Properdin, a glycoprotein with low levels in plasma and high levels at inflammatory sites where it is released by activated neutrophils, is the only positive regulator of the complement system by binding and stabilizing surface-bound C3 convertase (C3bBb) and C5 convertase (C3bBbC3b), thereby extending the half-life of the nascent convertases by 5–10 fold, resulting in accelerated and efficient amplification of C3b deposition on target surfaces. Therapeutic inhibitors of properdin may block complement at an earlier stage by preventing uncontrolled amplification of AP and preserving physiological function of CP and LP activation, thus potentially offering a more effective and safer approach to ameliorating human disease when AP is involved in pathogenesis, particularly in diseases where properdin levels are elevated and properdin has been shown to play a key role in pathogenesis.

[0004] Over the past decade, targeting the complement system has gradually gained attention for the treatment of human diseases. Eculizumab (Soliris, Alexion Pharm), a humanized monoclonal antibody against the human complement C5 protein, was first approved by the FDA in 2007 for the treatment of PNH by interfering with terminal pathway effector production, and its indications were subsequently expanded to include aHUS, gMG, and NMOSD. Encouraged by this successful clinical application, C5 antagonists in various formats, such as modified peptides, aptamers, small molecule compounds (SMCs), siRNAs, or antisense oligonucleotides (ASOs), have been actively developed in clinical and preclinical settings. Molecular targets have been expanded to include the predominant complement proteins involved in complement activation via the classical, lectin, or alternative pathways, including C3, complement factor B, complement factor D, MASP-2 or MASP-3, C1, and complement factor H or I. In particular, OMS721 (Omeros), a human monoclonal antibody targeting mannose-binding lectin-associated serine protease-2 (MASP-2), significantly reduced the urinary albumin / creatinine ratio in patients in a phase 2 clinical trial for the treatment of IgA nephropathy. Its efficacy is unprecedented for other treatments and earned it FDA Breakthrough Drug Designation. Furthermore, other orally bioavailable drugs are currently undergoing phase 2 trials focusing on the amplification loop. LNP023 (Novartis) blocks CFB and is in clinical trials for several indications, including PNH and renal disease. Another potential target of convertase formation is properdin, and a fully human anti-properdin Fab (CLG561) has been developed by Novartis for use in AMD and evaluated as monotherapy or in combination with the anti-C5 mAb LFG316 in a phase 2 trial for geographic atrophy (NCT02515942).

[0005] Inhibition or modulation of properdin is an important therapeutic strategy for alleviating symptoms and slowing or preventing the progression of alternative pathway-related diseases. Blocking the alternative pathway without inhibiting the classical complement pathway by depleting, neutralizing, or inactivating properdin is a viable and promising therapeutic strategy. Summary of the Invention

[0006] The present disclosure provides an isolated antigen-binding protein that may have one or more of the following properties: 1) specifically binds to properdin; 2) inhibits the alternative pathway by binding properdin; 3) inhibits the interaction between properdin and C3; 4) selectively inhibits the alternative pathway but not the classical or lectin pathway; and 5) has cross-species properdin-binding and complement inhibitory activity in the AP-specific pathway in mammals. The isolated antigen-binding protein also exhibits serum stability in both plasma and formulation buffer. Multiple subcutaneous administrations of the isolated antigen-binding protein depleted properdin from the serum and consistently inhibited AP activity.

[0007] In one aspect, the application provides an isolated antigen binding protein comprising at least one CDR in a heavy chain variable region VH, wherein the VH comprises the amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17.

[0008] In some embodiments, the isolated antigen binding protein is capable of specifically binding to an epitope domain of properdin, hi some embodiments, the epitope comprises TSR5, TSR6, and / or TSR0 of properdin.

[0009] In some embodiments, the isolated antigen binding protein may have competitive target binding ability with a reference antibody, wherein the reference antibody comprises CDR1, CDR2, and CDR3, wherein the CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, the CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8, and the CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

[0010] In some embodiments, the reference antibody may comprise CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0011] In some embodiments, the reference antibody may comprise CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 2, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 7, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 13.

[0012] In some embodiments, the reference antibody may comprise CDR1, CDR2 and CDR3, wherein CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 3, CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 8, and CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 14.

[0013] In some embodiments, the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:66, and SEQ ID NO:70.

[0014] In some embodiments, the isolated antigen binding protein may comprise a CDR3, and the CDR3 may comprise the amino acid sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0015] In some embodiments, the isolated antigen binding protein may comprise CDR2, and the CDR2 may comprise the amino acid sequence set forth in SEQ ID NO:55.

[0016] In some embodiments, the isolated antigen binding protein may comprise a CDR2, and the CDR2 may comprise the amino acid sequence set forth in any one of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

[0017] In some embodiments, the isolated antigen binding protein may comprise a CDR1, which may comprise the amino acid sequence set forth in SEQ ID NO: 54, X1X2CMX5, where X1 is H or S or T or Y, X2 is G or Y, and X5 is A or G.

[0018] In some embodiments, the isolated antigen binding protein may comprise a CDR1, and the CDR1 may comprise the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

[0019] In some embodiments, the isolated antigen binding protein may comprise CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:54, X1X2CMX5, where X1 is H or S or T or Y, X2 is G or Y, and X5 is A or G; the CDR2 comprises the amino acid sequence set forth in SEQ ID NO:55; and the CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17.

[0020] In some embodiments, the isolated antigen binding protein may comprise CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, the CDR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11, and the CDR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17.

[0021] In some embodiments, the isolated antigen binding protein may comprise CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO: 1, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO: 6, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO: 12.

[0022] In some embodiments, the isolated antigen binding protein may comprise CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:2, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO:7, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO:13.

[0023] In some embodiments, the isolated antigen binding protein may comprise CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO:8, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO:14.

[0024] In some embodiments, the isolated antigen binding protein may comprise CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:4, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO:9, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO:15.

[0025] In some embodiments, the isolated antigen binding protein may comprise CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:5, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO:10, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO:16.

[0026] In some embodiments, the isolated antigen binding protein may comprise CDR1, CDR2 and CDR3, wherein the CDR1 comprises the amino acid sequence set forth in SEQ ID NO:3, the CDR2 comprises the amino acid sequence set forth in SEQ ID NO:11, and the CDR3 comprises the amino acid sequence set forth in SEQ ID NO:17.

[0027] In some embodiments, the isolated antigen binding protein may comprise FR1, wherein the C-terminus of said FR1 is linked directly or indirectly to the N-terminus of said CDR1, and said FR1 comprises the amino acid sequence set forth in SEQ ID NO:56.

[0028] In some embodiments, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30.

[0029] In some embodiments, the isolated antigen binding protein may comprise FR2, wherein said FR2 is positioned between said CDR1 and said CDR2, and wherein said FR2 comprises the amino acid sequence set forth in SEQ ID NO:57.

[0030] In some embodiments, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40.

[0031] In some embodiments, the isolated antigen binding protein may comprise a FR3, wherein the FR3 is positioned between the CDR2 and the CDR3, and wherein the FR3 comprises the amino acid sequence set forth in SEQ ID NO:58.

[0032] In some embodiments, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51.

[0033] In some embodiments, the isolated antigen binding protein may comprise a FR4, wherein the N-terminus of the FR4 is linked directly or indirectly to the C-terminus of the CDR3, and wherein the FR4 comprises the amino acid sequence set forth in SEQ ID NO:59.

[0034] In some embodiments, the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:52 and SEQ ID NO:53.

[0035] In some embodiments, the isolated antigen binding protein comprises FR1, FR2, FR3 and FR4, wherein said FR1 comprises the amino acid sequence set forth in SEQ ID NO:56, said FR2 comprises the amino acid sequence set forth in SEQ ID NO:57, said FR3 comprises the amino acid sequence set forth in SEQ ID NO:58, and said FR4 comprises the amino acid sequence set forth in SEQ ID NO:59.

[0036] In some embodiments, the isolated antigen binding protein comprises FR1, FR2, FR3, and FR4, wherein FR1 comprises the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; FR2 comprises the amino acid sequence set forth in any one of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40; FR3 comprises the amino acid sequence set forth in any one of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51; and FR4 comprises the amino acid sequence set forth in any one of SEQ ID NO:52 and SEQ ID NO:53.

[0037] In some embodiments, the isolated antigen binding protein comprises FR1, FR2, FR3 and FR4, and the isolated antigen binding protein comprises: FR1: SEQ ID NO: 18, FR2: SEQ ID NO: 31, FR3: SEQ ID NO: 41, FR4: SEQ ID NO: 52; FR1: SEQ ID NO: 19, FR2: SEQ ID NO: 32, FR3: SEQ ID NO: 42, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 19, FR2: SEQ ID NO: 32, FR3: SEQ ID NO: 43, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 20, FR2: SEQ ID NO: 32, FR3: SEQ ID NO: 43, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 20, FR2: SEQ ID NO: 31, FR3: SEQ ID NO: 43, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 21, FR2: SEQ ID NO: 33, FR3: SEQ ID NO: 44, FR4: SEQ ID NO: 52; FR1: SEQ ID NO: 22, FR2: SEQ ID NO: 34, FR3: SEQ ID NO: 42, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 22, FR2: SEQ ID NO: 34, FR3: SEQ ID NO: 45, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 22, FR2: SEQ ID NO: 33, FR3: SEQ ID NO: 45, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 23, FR2: SEQ ID NO: 35, FR3: SEQ ID NO: 46, FR4: SEQ ID NO: 52; FR1: SEQ ID NO: 24, FR2: SEQ ID NO: 36, FR3: SEQ ID NO: 47, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 24, FR2: SEQ ID NO: 36, FR3: SEQ ID NO: 48, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 25, FR2: SEQ ID NO: 36, FR3: SEQ ID NO: 48, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 25, FR2: SEQ ID NO: 35, FR3: SEQ ID NO: 48, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 26, FR2: SEQ ID NO: 37, FR3: SEQ ID NO: 49, FR4: SEQ ID NO: 52; FR1: SEQ ID NO: 27, FR2: SEQ ID NO: 38, FR3: SEQ ID NO: 42, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 27, FR2: SEQ ID NO: 39, FR3: SEQ ID NO: 42, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 28, FR2: SEQ ID NO: 39, FR3: SEQ ID NO: 42, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 28, FR2: SEQ ID NO: 39, FR3: SEQ ID NO: 43, FR4: SEQ ID NO: 53; FR1: SEQ ID NO: 28, FR2: SEQ ID NO: 37, FR3: SEQ ID NO: 43, FR4: SEQ ID NO: 53; FR1: SEQ ID NO:29, FR2: SEQ ID NO:31, FR3: SEQ ID NO:50, FR4: SEQ ID NO:52; and FR1: SEQ ID NO: 30, FR2: SEQ ID NO: 40, FR3: SEQ ID NO: 51, FR4: SEQ ID NO: 52 The amino acid sequence of any of the sets of amino acid sequences selected from the group consisting of:

[0038] In some embodiments, the isolated antigen binding protein comprises a heavy chain variable region VH comprising the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:92 and SEQ ID NO:93.

[0039] In some embodiments, the heavy chain variable region is a VHH.

[0040] In some embodiments, the isolated antigen binding protein may comprise an antibody heavy chain constant region.

[0041] In some embodiments, the heavy chain constant region may comprise a human Fc region.

[0042] In some embodiments, the heavy chain constant region can comprise the amino acid sequence set forth in SEQ ID NO:109.

[0043] In some embodiments, the isolated antigen binding protein may be linked directly or indirectly to a second antigen binding domain.

[0044] In some embodiments, the isolated antigen binding protein may be linked to a second antigen binding domain by a linker.

[0045] In some embodiments, the linker of the isolated antigen binding protein can be a polyglycine linker.

[0046] In some embodiments, the linker of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 108: GGGGSGGGSGGGGS.

[0047] In some embodiments, the second antigen-binding domain of the isolated antigen-binding protein is capable of binding to properdin.

[0048] In some embodiments, the second antigen-binding domain of the isolated antigen-binding protein may bind to a different epitope of properdin than the isolated antigen-binding protein.

[0049] In some embodiments, the second antigen-binding domain of the isolated antigen-binding protein is capable of binding to the same epitope of properdin as the isolated antigen-binding protein.

[0050] In some embodiments, the second antigen-binding domain of the isolated antigen-binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:92 and SEQ ID NO:93.

[0051] In some embodiments, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:81.

[0052] In some embodiments, the isolated antigen binding protein may comprise an antibody or antigen-binding fragment thereof.

[0053] In some embodiments, the isolated antigen-binding fragment may comprise a Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb.

[0054] In some embodiments, the antibody may be selected from the group consisting of a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0055] In some embodiments, the antibody may be a camelid antibody.

[0056] In another aspect, the present application provides a fusion protein comprising the isolated antigen binding protein.

[0057] In some embodiments, the fusion protein may comprise a functionally active protein.

[0058] In some embodiments, the isolated antigen binding protein may be directly or indirectly linked to the functionally active protein.

[0059] In some embodiments, the isolated antigen binding protein may be linked to the functionally active protein by a linker.

[0060] In some embodiments, the isolated antigen binding protein may be linked to the functionally active protein by more than one linker.

[0061] In some embodiments, the linker of the isolated antigen binding protein and the functionally active protein can be a polyglycine linker.

[0062] In some embodiments, the linker of the isolated antigen binding protein and the functionally active protein can be more than one polyglycine linker.

[0063] In some embodiments, the linker of the isolated antigen binding protein and the functionally active protein may comprise the amino acid sequence set forth in SEQ ID NO: 108: GGGGSGGGSGGGGS.

[0064] In some embodiments, the functionally active protein of the fusion protein can be Factor H.

[0065] In some embodiments, the Factor H of the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:110.

[0066] In some embodiments, the Factor H of the fusion protein may include functionally active fragments, orthologs, and variants thereof. In some embodiments, the sequence similarity may be at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to the corresponding sequence.

[0067] In some embodiments, the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:82.

[0068] In some embodiments, the functionally active protein of the fusion protein can be a VEGF inhibitory protein.

[0069] In some embodiments, the VEGF inhibitor protein of the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:113.

[0070] In some embodiments, the VEGF inhibitor protein of the fusion protein may include functionally active fragments, orthologs, and variants thereof. In some embodiments, the sequence similarity may be at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to the corresponding sequence.

[0071] In some embodiments, the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:114.

[0072] In some embodiments, the functionally active protein of the fusion protein can be a transferrin inhibitor protein.

[0073] In some embodiments, the transferrin inhibitor protein of the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:115.

[0074] In some embodiments, the transferrin inhibitor protein of the fusion protein can include functionally active fragments, orthologs, and variants thereof. In some embodiments, the sequence similarity can be at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to the corresponding sequence.

[0075] In some embodiments, the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:117.

[0076] In some embodiments, the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:118.

[0077] In another aspect, the present application provides a polypeptide comprising an isolated antigen binding protein.

[0078] In another aspect, the present application provides an immunoconjugate comprising the isolated antigen-binding protein or polypeptide.

[0079] In another aspect, the application provides an isolated nucleic acid molecule or a plurality of isolated nucleic acid molecules encoding an isolated antigen binding protein.

[0080] In another aspect, the present application further provides a vector comprising the nucleic acid molecule(s).

[0081] In another aspect, the application provides a cell comprising(s) said nucleic acid molecule(s), said polypeptide(s), said immunoconjugate(s), said isolated nucleic acid molecule(s), and / or said vector(s).

[0082] In another aspect, the present application provides a method of producing said isolated antigen binding protein or said polypeptide, said method comprising culturing a cell under conditions that allow expression of said isolated antigen binding protein or said polypeptide.

[0083] In another aspect, the present application provides a method for detecting properdin, said method comprising using said isolated antigen-binding protein or said polypeptide.

[0084] In some embodiments, the isolated antigen binding protein of the methods comprises the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118.

[0085] In another aspect, the present application provides a detection kit for properdin, comprising said isolated antigen-binding protein or said polypeptide.

[0086] In some embodiments, the isolated antigen binding protein of the detection kit comprises the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:116, SEQ ID NO:117, and SEQ ID NO:118.

[0087] In another aspect, the application provides the use of said isolated antigen binding protein or said polypeptide in the preparation of a kit.

[0088] In some embodiments, the isolated antigen binding protein of said use comprises the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:116, SEQ ID NO:117 and SEQ ID NO:118.

[0089] In another aspect, the present application provides a pharmaceutical composition comprising said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said cell, and / or a pharmaceutically acceptable adjuvant and / or excipient.

[0090] In another aspect, the present application provides a pharmaceutical combination comprising said isolated antigen binding protein.

[0091] In another aspect, the application provides a method of inhibiting the alternative complement pathway, comprising administering an effective amount of said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said cell, and / or said pharmaceutical composition, and / or a pharmaceutically acceptable therapeutic agent.

[0092] In another aspect, the present application provides a method of inhibiting the alternative complement pathway, comprising administering an effective amount of the pharmaceutical combination and / or a pharmaceutically acceptable therapeutic agent.

[0093] In another aspect, the present application provides said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition, and / or said pharmaceutical combination for use in the prevention and / or treatment of disease.

[0094] In some embodiments, the disease may be caused by properdin.

[0095] In some embodiments, the disease may be mediated by an alternative pathway.

[0096] In some embodiments, the disease is autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), asthma, Gaucher disease, hidradenitis suppurativa (Hidradentitis suppurativa), Behcet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-related lung injury (TRALI), hemodialysis-induced thrombosis, epidermolysis bullosa acquisita (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, diffuse thrombotic microangiopathy (TMA), diffuse thrombotic microangiopathy (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes, and Hyperlipidemia and Thrombocytopenia (HELLP) syndrome, multiple sclerosis, antiphospholipid syndrome (APS), relapsing polychondritis, ischemic injury, stroke, graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), emphysema, atherosclerosis, acute coronary syndrome, hemorrhagic shock, rheumatoid arthritis, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, antiphospholipid syndrome (APS) pregnancy loss, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malarial hemolytic crisis, abdominal aortic aneurysm (AAA), or thoracoabdominal aortic aneurysm (TAA).

[0097] In another aspect, the present application provides use of said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said cell and / or said pharmaceutical composition, and / or said pharmaceutical combination in the manufacture of a medicament for the prevention and / or treatment of a disease.

[0098] In some embodiments, the disease of the use may be caused by properdin.

[0099] In some embodiments, the disease of said use may be mediated by an alternative pathway.

[0100] In some embodiments, the disease of said use is autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), asthma, Gaucher disease, hidradentitis suppurativa (Hidradentitis suppurativa), or the like. suppurativa), Behcet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-related lung injury (TRALI), hemodialysis-induced thrombosis, epidermolysis bullosa acquisita (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, diffuse thrombotic microangiopathy (TMA), diffuse thrombotic microangiopathy (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes, and Hyperlipidemia and Thrombocytopenia (HELLP) syndrome, multiple sclerosis, antiphospholipid syndrome (APS), relapsing polychondritis, ischemic injury, stroke, graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), emphysema, atherosclerosis, acute coronary syndrome, hemorrhagic shock, rheumatoid arthritis, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, antiphospholipid syndrome (APS) pregnancy loss, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malarial hemolytic crisis, abdominal aortic aneurysm (AAA), or thoracoabdominal aortic aneurysm (TAA).

[0101] In another aspect, the present application provides a method of preventing and / or treating a disease, comprising administering to a human / animal patient in need thereof an effective amount of said isolated antigen binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, said cell, said pharmaceutical composition, and / or said pharmaceutical combination.

[0102] In some embodiments, the disease of the method may be caused by properdin.

[0103] In some embodiments, the disease of the method may be mediated by an alternative pathway.

[0104] In some embodiments, the disease of the method is selected from the group consisting of autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), asthma, Gaucher disease, and hidradentitis suppurativa. suppurativa), Behcet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-related lung injury (TRALI), hemodialysis-induced thrombosis, epidermolysis bullosa acquisita (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, diffuse thrombotic microangiopathy (TMA), diffuse thrombotic microangiopathy (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes, and Hyperlipidemia and Thrombocytopenia (HELLP) syndrome, multiple sclerosis, antiphospholipid syndrome (APS), relapsing polychondritis, ischemic injury, stroke, graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), emphysema, atherosclerosis, acute coronary syndrome, hemorrhagic shock, rheumatoid arthritis, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, antiphospholipid syndrome (APS) pregnancy loss, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malarial hemolytic crisis, abdominal aortic aneurysm (AAA), or thoracoabdominal aortic aneurysm (TAA).

[0105]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

[0106] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "figure" and "FIG."). [Brief explanation of the drawings]

[0107] [Figure 1] FIG. 1 shows the results of human, mouse, and cynomolgus properdin analysis on non-reducing and reducing SDS-PAGE. [Figure 2] FIG. 2 shows the generation of recombinant VHH-Fc analysis on non-reducing and reducing SDS-PAGE. [Figure 3A] FIG. 3A shows ELISA binding of recombinant VHH-Fc to human properdin. [Figure 3B] FIG. 3B shows ELISA binding of recombinant VHH-Fc to cynomolgus monkey properdin. [Figure 3C] FIG. 3C shows ELISA binding of recombinant VHH-Fc to mouse properdin. [Figure 4A] FIG. 4A shows the complement inhibitory activity of recombinant VHH-Fc in human serum. [Figure 4B] FIG. 4B shows the complement inhibitory activity of recombinant VHH-Fc in mouse serum. [Figure 5] Figure 5 shows epitope binning of properdin (full length) binding VHHs. [Figure 6] Figure 6 shows the generation of biparatopic VHH analysis on non-reducing and reducing SDS-PAGE. [Figure 7A] FIG. 7A shows the binding of biparatopic VHHs to human properdin. [Figure 7B]FIG. 7B shows the binding of biparatopic VHHs to mouse properdin. [Figure 7C] FIG. 7C shows the binding of biparatopic VHHs to human properdin. [Figure 7D] FIG. 7D shows binding of biparatopic VHHs to mouse properdin. [Figure 8A] FIG. 8A shows the complement inhibitory activity of biparatopic VHHs in human serum. [Figure 8B] FIG. 8B shows the complement inhibitory activity of biparatopic VHHs in mouse serum. [Figure 8C] FIG. 8C shows the complement inhibitory activity of biparatopic VHHs in human serum. [Figure 8D] FIG. 8D shows the complement inhibitory activity of biparatopic VHHs in mouse serum. [Figure 9A] Figure 9A shows humanization of selected properdin binding VHHs. Human properdin binding. [Figure 9B] Figure 9B shows humanization of selected properdin binding VHHs. Mouse properdin binding. [Figure 9C] Figure 9C shows humanization of selected properdin binding VHHs. Human properdin binding. [Figure 9D] Figure 9D shows humanization of selected properdin binding VHHs. Mouse properdin binding. [Figure 9E] Figure 9E shows humanization of selected properdin-binding VHHs. Human AP activity. [Figure 9F] Figure 9F shows humanization of selected properdin-binding VHHs. Human AP activity. [Figure 9G] Figure 9G shows humanization of selected properdin-binding VHHs. Mouse AP activity. [Figure 9H] Figure 9H shows humanization of selected properdin-binding VHHs. Mouse AP activity. [Figure 10] Figure 10 shows epitope mapping of properdin (truncated variant) binding VHHs. [Figure 11A]Figure 11A shows the effect of properdin inhibitors on the interaction between C3 and properdin. Properdin binding assay. [Figure 11B] Figure 11B shows the effect of properdin inhibitors on the interaction between C3 and properdin. Competition assay. [Figure 12] FIG. 12 shows the results of VHH-CFH analysis on non-reducing and reducing SDS-PAGE. [Figure 13A] FIG. 13A shows the properdin binding activity of SLN12140 and SLN7207 in human serum. [Figure 13B] Figure 13B shows the properdin binding activity of SLN12140 and SLN7207 in mouse serum. [Figure 13C] FIG. 13C shows the properdin binding activity of SLN12140 and SLN7207 in human serum. [Figure 13D] Figure 13D shows the properdin binding activity of SLN12140 and SLN7207 in mouse serum. [Figure 14A] FIG. 14A shows the AP inhibitory activity of SLN12140 and SLN7207 in human serum. [Figure 14B] FIG. 14B shows the AP inhibitory activity of SLN12140 and SLN7207 in mouse serum. [Figure 14C] FIG. 14C shows the AP inhibitory activity of SLN12140 and SLN7207 in human serum. [Figure 14D] FIG. 14D shows the AP inhibitory activity of SLN12140 and SLN7207 in mouse serum. [Figure 15A] Figure 15A shows the pathway selectivity of SLN12140 in complement inactivation. AP. [Figure 15B] Figure 15B shows the pathway selectivity of SLN12140 in complement inactivation. CP. [Figure 15C] Figure 15C shows the pathway selectivity of SLN12140 in complement inactivation. LP. [Figure 16A]Figure 16A shows the cross-species reactivity of SLN12140 in an AP activity assay. AP activity in human serum. [Figure 16B] Figure 16B shows the cross-species reactivity of SLN12140 in an AP activity assay. AP activity in cynomolgus monkey serum. [Figure 16C] Figure 16C shows the cross-species reactivity of SLN12140 in an AP activity assay. AP activity in mouse serum. [Figure 16D] Figure 16D shows the cross-species reactivity of SLN12140 in an AP activity assay. AP activity in rat serum. [Figure 17] FIG. 17 shows the serum stability of SLN12140 in both plasma and formulation buffer. [Figure 18] FIG. 18 shows subcutaneous and intravenous single-dose pharmacokinetic studies in mice. [Figure 19A] FIG. 19A shows the dose-dependent subcutaneous PK of SLN12140 at a dose of 3 mpk. [Figure 19B] FIG. 19B shows the dose-dependent subcutaneous PK of SLN12140 at a dose of 10 mpk. [Figure 19C] FIG. 19C shows the dose-dependent subcutaneous PK of SLN12140 at a dose of 30 mpk. [Figure 20] FIG. 20 shows that multiple subcutaneous dosing of SLN12140 once a week for 3 weeks sustainably reduces target concentrations to low levels and consistently inhibits AP activity. [Figure 21] FIG. 21 shows that multiple subcutaneous dosing of SLN12140 once a week for 7 weeks in hCD89 Tg mice persistently reduces target concentrations to low levels and demonstrates stable pharmacokinetic characteristics of SLN12140. [Figure 22] Figure 22 shows a representative SDS-PAGE gel of fusion proteins expressed in HEK293 cells: purified fusion proteins SLN6073, SLN8284, and SLN12140 under non-reducing (lanes 1, 3, and 5) and reducing (lanes 2, 4, and 6) conditions. [Figure 23A]Figure 23A shows representative binding of SLN8284, SLN12140, and SLN6073 to properdin from different species in vitro. Human properdin binding of SLN8284, SLN6073, and SLN12140. [Figure 23B] Figure 23B shows representative binding of SLN8284, SLN12140, and SLN6073 to properdin from different species in vitro. Cynomolgus monkey properdin binding of SLN8284, SLN6073, and SLN12140. [Figure 23C] Figure 23C shows representative binding of SLN8284, SLN12140, and SLN6073 to properdin from different species in vitro. Mouse properdin binding of SLN8284, SLN6073, and SLN12140. [Figure 24A] Figure 24A shows the inhibition of the alternative complement pathway in rabbit erythrocytes by various concentrations of the fusion proteins SLN8284, SLN6073, and SLN12140. Inhibition of the alternative complement pathway by SLN8284, SLN6073, and SLN12140 in human serum. [Figure 24B] Figure 24B shows the inhibition of the alternative complement pathway in rabbit erythrocytes by various concentrations of the fusion proteins SLN8284, SLN6073, and SLN12140. Inhibition of the alternative complement pathway by SLN8284, SLN6073, and SLN12140 in mouse serum. [Figure 25A] Figure 25A shows in vitro binding of VEGF by SLN8284, SLN6073, and SLN12140 as detected by ELISA. Human VEGF121 binding of SLN8284, SLN6073, and SLN12140. [Figure 25B] Figure 25B shows in vitro binding of VEGF by SLN8284, SLN6073, and SLN12140 as detected by ELISA. Mouse VEGF120 binding of SLN8284, SLN6073, and SLN12140. [Figure 26A]Figure 26A shows the effect of blocking the interaction between VEGFA and VEGFR2 by SLN8284, SLN6073, and SLN12140. SLN8284, SLN6073, and SLN12140 block the interaction between hVEGFA and hVEGFR2. [Figure 26B] Figure 26B shows the effect of blocking the interaction between VEGFA and VEGFR2 by SLN8284, SLN6073, and SLN12140. SLN8284, SLN6073, and SLN12140 block the interaction between mVEGFA and mVEGFR2. [Figure 27] 1 shows the inhibition of VEGF-induced proliferation of human umbilical vein endothelial cells (HUVECs) by SLN8284, SLN6073, and SLN12140. [Figure 28] Figure 28 shows a representative SDS-PAGE gel of fusion proteins expressed in HEK293 cells: purified fusion proteins SLN12147, SLN12149, and SLN12150 under non-reducing (lanes 1, 3, and 5) and reducing (lanes 2, 4, and 6) conditions. [Figure 29A] Figure 29A shows the in vitro Elisa binding results of SLN12140, SLN12147, SLN12149 and SLN12150 to properdin of different species. Human properdin binding. [Figure 29B] Figure 29B shows the in vitro Elisa binding results of SLN12140, SLN12147, SLN12149 and SLN12150 to properdin of different species. Cynomolgus monkey properdin binding. [Figure 29C] Figure 29C shows the in vitro Elisa binding results of SLN12140, SLN12147, SLN12149 and SLN12150 to properdin of different species. Mouse properdin binding. [Figure 30A]Figure 30A shows the in vitro ELISA binding results of SLN12140, SLN12147, SLN12149, SLN12150, and SLN9056 to human transferrin (hTf). Binding results of SLN12147, SLN12149, and SLN9056 with hTf at pH 7.4. [Figure 30B] Figure 30B shows the in vitro ELISA binding results of SLN12140, SLN12147, SLN12149, SLN12150, and SLN9056 to human transferrin (hTf). Binding results of SLN12147, SLN12149, and SLN9056 with hTf at pH 6.0. [Figure 30C] Figure 30C shows the in vitro ELISA binding results of SLN12140, SLN12147, SLN12149, SLN12150 and SLN9056 to human transferrin (hTf). Binding results of SLN12140, SLN12150 with hTf at pH 7.4. [Figure 30D] Figure 30D shows the in vitro ELISA binding results of SLN12140, SLN12147, SLN12149, SLN12150, and SLN9056 to human transferrin (hTf). Binding results of SLN12140, SLN12150 with hTf at pH 6.0. [Figure 31A] Figure 31A shows the inhibition of AP activity of different species by SLN12140, SLN12147, SLN12149 and SLN12150 in ELISA. Human AP activity by fusion proteins. [Figure 31B] Figure 31B shows the inhibition of AP activity of different species by SLN12140, SLN12147, SLN12149 and SLN12150 in ELISA. Cynomolgus monkey AP activity by fusion proteins. [Figure 31C] Figure 31C shows the inhibition of AP activity of different species by SLN12140, SLN12147, SLN12149 and SLN12150 in ELISA. Mouse AP activity by fusion proteins. [Figure 32A]Figure 32A shows the results of SLN12140 and SLN12150 PK in mice. Drug in mouse serum. [Figure 32B] Figure 32B shows the PK results of SLN12140 and SLN12150 in mice. Drug in mouse brain tissue. [Figure 32C] Figure 32C shows the results of SLN12140 and SLN12150 PK in mice. Drug in mouse CSF. [Figure 32D] Figure 32D shows the results of SLN12140 and SLN12150 PK in mice. Drug ID % in mouse brain tissue. DETAILED DESCRIPTION OF THE INVENTION

[0108] While various embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used.

[0109] Terms and Definitions The term "antibody" is used in the broadest sense and may include, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies containing two light chains and two heavy chains), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), murine antibodies, human antibodies (fully human antibodies), humanized antibodies, chimeric antibodies, single-chain antibodies (e.g., scFv), antibody derivatives, and antigen-binding antibody fragments (e.g., Fab', VHH, and (Fab)2 fragments). The term "antibody" also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, aglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described herein. An "antibody" generally includes a protein comprising at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain may be composed of a heavy chain variable region (VH) and a heavy chain constant region. The VH region can be further distinguished as hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH can be composed of three CDR and four FR regions, which can be arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable region of the heavy chain contains a binding domain that interacts with an antigen (e.g., properdin). In the art, CDRs of antibodies can be defined in various ways, for example, by the Kabat definition rules based on sequence variability (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institutes of Health, Bessemer, Maryland (1991)), the Chothia definition rules based on the location of structural loop regions (see A1-Lazikani et al., J Mol Biol 273:927-48, 1997), and the IMGT definition rules based on concepts in the IMGT-ONTOLOGY and IMGT Scientific Chart Rules. In the present application, CDRs can be defined by the Kabat definition rules.

[0110] The term "antigen-binding domain" herein generally refers to a domain capable of binding to a target. For example, binding may require some complementarity in the binding sequence. For example, binding may require a special structure. For example, in the present application, the antigen-binding domain of an antigen-binding protein may specifically bind to an antigen (e.g., properdin, VEGF family, transferrin). For example, the antigen-binding domain may belong to an antibody or antigen-binding fragment, and may allow the antibody or antigen-binding fragment to bind to the target with higher affinity, avidity, ease, and / or duration than binding to other targets. For example, the antigen-binding domain may have a measurable and reproducible interaction, such as the binding between an antigen and an antibody, thereby allowing the presence of the target to be determined in the presence of a heterogeneous population of molecules (including biomolecules). A "binding sequence" refers to a specific amino acid sequence on a target (e.g., an antigen) that is complementary to the antigen-binding protein.

[0111] The term "antigen-binding fragment" herein generally refers to one or more fragments of an antibody that specifically bind to an antigen. The antigen-binding function of an antibody can be achieved by a full-length antibody fragment. The antigen-binding function of an antibody can also be achieved by a heavy chain including an Fv, scFv, dsFv, Fab', or F(ab')2 fragment, or a light chain including an Fv, scFv, dsFv, Fab', or F(ab')2 fragment. The term "Fab" generally refers to a fragment containing a heavy chain variable domain and a light chain variable domain, and also containing a light chain constant domain and the first heavy chain constant domain (CH1). The term "Fab'" generally refers to a fragment that differs from Fab by the addition of several residues (including one or more cysteines from the antibody hinge region) to the carboxyl terminus of the heavy chain CH1. The term "F(ab')2" generally refers to a Fab' dimer, comprising an antibody fragment in which two Fab fragments are linked by disulfide bridges in the hinge region. The term "Fv" generally refers to the minimum antibody fragment that contains a complete antigen recognition and binding site. In some cases, this fragment may consist of a dimer in which one heavy-chain variable region and one light-chain variable region are tightly and non-covalently bound. The term "dsFv" generally refers to a disulfide-stabilized Fv fragment, which has a disulfide bond between a single light-chain variable region and a single heavy-chain variable region. The term "dAb fragment" generally refers to an antibody fragment consisting of a VH domain. The term "scFv" generally refers to a molecule produced by covalently linking and pairing one heavy-chain variable domain with one light-chain variable domain of an antibody via a flexible peptide linker. The term "Fd" generally refers to a fragment consisting of a VH and CH domain. For example, the term "antigen-binding fragment" may include a class of antibody VHHs that lack the antibody light chain and have only the heavy-chain variable region.

[0112] The term "antigen-binding protein" as used herein generally refers to a polypeptide molecule capable of specifically recognizing and / or neutralizing a particular antigen. For example, in the present application, the term "antigen-binding protein" may include an "antibody" or an "antigen-binding fragment," so long as it exhibits the desired antigen-binding activity. For example, the isolated antigen-binding protein may include a single-domain protein, e.g., the isolated antigen-binding protein may include any molecule comprising its antigen-binding portion. For example, the isolated antigen-binding protein may include a VHH-Fc protein or an Fc-VHH-VHH protein.

[0113] The term "camelid antibody" generally refers to an antibody derived from a camelid species, such as a camel, dromedary, llama, alpaca, or guanaco. Camelid antibodies lack light chains and therefore contain only heavy chains with complete and diverse antigen-binding capacity.

[0114] The term "VHH", also known as VHH domain, VHH antibody fragment, and VHH antibody, generally refers to the antigen-binding immunoglobulin (variable) domain of a "heavy chain antibody", e.g., having the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, which specifically binds to an epitope without requiring the presence of a second immunoglobulin variable domain.

[0115] The term "cell" generally refers to an individual cell, cell line, or cell culture that may contain or already contains a plasmid or vector comprising a nucleic acid molecule of the present application or that is capable of expressing an antibody or antigen-binding fragment thereof of the present application. A cell may include the progeny of a single host cell. Due to natural, accidental, or deliberate mutation, progeny cells and the original parent cell may not necessarily be identical in morphology or genome, so long as they are capable of expressing an antibody or antigen-binding fragment thereof of the present application. A cell may be obtained by transfecting a cell in vitro with a vector of the present application. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., yeast cell; e.g., COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell). In some cases, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell.

[0116] The term "chimeric antibody" generally refers to an antibody whose variable region is derived from one species and whose constant region is derived from another species. Generally, the variable region is derived from an antibody in an experimental animal such as a rodent (the "parent antibody"), and the constant region is derived from a human antibody, so that the resulting chimeric antibody is less likely to provoke an adverse immune response in an individual human compared to the parent (e.g., mouse-derived) antibody.

[0117] The terms "derivative," "variant," or "analog" are used interchangeably and generally refer to a polypeptide or polynucleotide of the present application that includes any substitution, variation, modification, substitution, deletion, and / or addition of one (or more) amino acid residues from / to a sequence, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions. For example, a derivative can have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to its corresponding sequence.

[0118] The term "epitope" generally refers to a domain or amino acid sequence that specifically binds to an antigen-binding protein. For example, the term "epitope" can include chemically active surface molecular groups (e.g., sugar side chains, phosphoryl groups, or sulfonyl groups). For example, the term "epitope" can have specific tertiary structural characteristics and / or specific charge characteristics.

[0119] The term "fully human antibody" generally refers to an antibody that contains only the sequence of human immunoglobulin protein. A fully human antibody can significantly reduce the adverse immune reactions caused by heterologous antibodies in the human body. Methods for obtaining fully human antibodies in the art may include phage display technology, transgenic mouse technology, ribosome display technology, RNA-polypeptide technology, etc.

[0120] The term "biparatopic antigen-binding protein" generally refers to an antigen-binding molecule that contains a first antigen-binding domain and a second antigen-binding domain. For example, the two antigen-binding domains bind to two different epitopes, e.g., non-overlapping epitopes of each antigen. For example, the first antigen-binding domain and the second antigen-binding domain may target the same antigen. For example, the first antigen-binding domain and the second antigen-binding domain target different epitopes of the same antigen. The portion of the antigen-binding protein that recognizes the epitope is called the paratope.

[0121] The term "fusion protein" generally refers to a protein composed of two or more polypeptides. The two or more polypeptide components may be linked directly or indirectly via a peptide linker / spacer. For example, the polypeptides are not naturally linked in their native state but are joined by a peptide bond via their respective amino and carboxyl termini to form a contiguous polypeptide. For example, the term "fusion protein" includes an antigen-binding protein prepared by the methods described herein and a functionally active protein. For example, the functionally active protein may be factor H. For example, the functionally active protein may be a VEGF inhibitory protein. For example, the functionally active protein may be a transferrin inhibitory protein. For example, a fusion protein may contain a prophylactic or therapeutic agent fused to a heterologous protein, polypeptide, or peptide. Here, the heterologous protein, polypeptide, or peptide may or may not be a different type or therapeutic agent. For example, a fusion protein may contain two different proteins, polypeptides, or peptides with immunomodulatory activity. For example, the fusion protein may retain or improve activity compared to the activity of the original polypeptide or protein. Typically, fusion protein can be produced by in vitro recombinant technology well known in the art.For example, fusion protein can contain antigen binding protein.For example, fusion protein can contain biological molecule.For example, fusion protein can be composed of properdin inhibitory protein and VEGF inhibitory protein.For example, fusion protein can be composed of properdin inhibitory protein and transferrin inhibitory protein.

[0122] The term "blood-brain barrier (BBB)" generally refers to the physiological barrier between the peripheral circulation and the brain and spinal cord. The blood-brain barrier is formed by tight junctions in the cell membranes of brain capillary endothelial cells, constituting a tight barrier that limits the transport of molecules, even very small molecules such as urea (60 daltons), into the brain. For example, brain capillary endothelial cells may have weaker pinocytosis. For example, the blood-brain barrier may include the BBB in the brain, the blood-spinal cord barrier in the spinal cord, and the blood-retinal barrier in the retina. For example, the BBB may also include the blood-CSF barrier (choroid plexus), in which case the barrier is composed of ependymal cells instead of capillary endothelial cells.

[0123] The term "humanized antibody" generally refers to an antibody in which some or all of the amino acids outside the CDRs of a non-human antibody (e.g., a murine antibody) have been replaced with corresponding amino acids from a human immunoglobulin. Small additions, deletions, insertions, substitutions, or modifications to these amino acids in the CDRs are permissible as long as the antibody's ability to bind to a specific antigen is still maintained. A humanized antibody may optionally contain at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigen specificity similar to that of the original antibody. "Humanized" forms of non-human (e.g., murine) antibodies may minimally comprise chimeric antibodies derived from non-human immunoglobulin sequences. In some cases, CDR residues in a human immunoglobulin (acceptor antibody) may be replaced with CDR residues (donor antibody) from a non-human species (e.g., mouse, rat, rabbit, or non-human primate) having desired properties, affinity, and / or capacity. In some cases, FR residues of a human immunoglobulin may be replaced with corresponding non-human residues. Furthermore, humanized antibodies may contain amino acid modifications that are not present in the recipient antibody or in the donor antibody. These modifications may be made to further improve properties, such as the binding affinity of the antibody.

[0124] The term "immunoconjugate" generally refers to a conjugate formed by conjugating (e.g., covalently linking via a linking molecule) an additional therapeutic agent to an isolated antigen binding protein, which conjugate is capable of delivering the additional therapeutic agent to a target cell via specific binding of the isolated antigen binding protein to an antigen on the target cell.

[0125] The term "isolated" antigen binding protein generally refers to an antigen binding protein that has been identified, isolated, and / or recovered from components of the environment (e.g., natural or recombinant) in which the antigen binding protein is produced. The contaminant components of the environment in which the antigen binding protein is produced are generally substances that would interfere with research, diagnostic, or therapeutic uses of the antigen binding protein, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. Isolated antigen binding proteins or antibodies are generally prepared by at least one purification step. The isolated antigen binding proteins of the present application generally specifically bind to properdin.

[0126] The term "isolated nucleic acid molecule" generally refers to DNA or RNA of genomic, mRNA, cDNA, or synthetic origin, or certain combinations thereof. An isolated nucleic acid molecule is free from all or part of the polynucleotides with which it is found in nature, or is linked to polynucleotides with which it is not naturally linked.

[0127] The term "monoclonal antibody" generally refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., a cluster in which some antibodies are identical except for some natural variants that may be present. Monoclonal antibodies are generally highly specific for a single antigenic site. Furthermore, unlike conventional polyclonal antibody preparations (which generally include different antibodies directed against different determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, an advantage of monoclonal antibodies resides in their ability to be synthesized by hybridoma culture, uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be prepared in hybridoma cells or by recombinant DNA methodologies.

[0128] The term "patient" generally refers to a human or non-human animal, including, but not limited to, a cat, dog, horse, pig, cow, sheep, rabbit, mouse, rat, or monkey.

[0129] The term "pharmaceutically acceptable adjuvant" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed to them at the dosages and concentrations used. Generally, physiologically acceptable carriers are pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides and proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0130] The terms "pharmaceutical combination" and "combination product" are used interchangeably and generally refer to a product resulting from the mixture or combination of more than one active ingredient, including both fixed and non-fixed combinations of active ingredients. The term "fixed combination" means that both the active ingredient and one or more combination partners are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that the active ingredient and one or more combination partners are administered to a patient simultaneously, together, or sequentially (without specific time restrictions) as separate entities, such administration providing the two compounds at therapeutically effective levels in the patient's body. For example, one active ingredient of a pharmaceutical combination can be an antigen-binding protein prepared by the methods described in this application.

[0131] The term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient. For example, the term "pharmaceutical composition" contains one or more antigen-binding proteins, typically prepared by the methods described in this application. A pharmaceutical composition may also contain one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, preservatives, and / or adjuvants. For example, acceptable components of a composition are non-toxic to a patient at the dosages and concentrations used. Pharmaceutical compositions in this application include, but are not limited to, liquid, frozen, and lyophilized compositions.

[0132] The terms "polypeptide," "peptide," and "protein" are used interchangeably and generally refer to polymers of amino acids of any length. Polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass modified amino acid polymers. These modifications may include disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (e.g., conjugation to a labeling component). The term "amino acid" includes natural and / or unnatural or synthetic amino acids, including glycine, D- and L-optical isomers, amino acid analogs, and peptidomimetics.

[0133] The terms "properdin," "factor P," and "Pillemer molecule" are used interchangeably and are positive regulators of alternative complement activation. For example, the term "properdin" can refer to the oligomerization of rod-shaped monomers into cyclic dimers, trimers, and tetramers. For example, the term "properdin" can refer to human properdin, a soluble glycoprotein of approximately 469 amino acids found in plasma and seven thrombospondin type I repeats (TSRs) with an N-terminal domain. For example, the term "properdin" can refer to mouse properdin, a soluble glycoprotein of approximately 457 amino acids found in plasma and seven TSRs with an N-terminal domain. The TSRs can be divided according to the common knowledge of those skilled in the art. For example, the term "properdin" can include full-length, truncated, and variant properdins.

[0134] The terms "VEGF" or "vascular endothelial growth factor" are used interchangeably and generally refer to a family of signaling proteins that can stimulate, for example, angiogenesis, vasculogenesis, and / or lymphangiogenesis. Members of the VEGF family include VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF (placental growth factor). In this application, the term "VEGF" can include functionally active fragments, orthologs, analogs, and variants thereof.

[0135] The term "transferrin" generally refers to a glycoprotein that can bind to and transport multivalent ions. For example, transferrin can be a single-chain glycoprotein. For example, transferrin can have a molecular weight of approximately 77,000 D. For example, transferrin can have a polysaccharide. For example, transferrin can have two ion-binding sites. For example, these ion-binding sites can have different affinities for iron ions. For example, the multivalent ions can be iron ions, chromium ions, manganese ions, cadmium ions, or nickel ions thereof. For example, each molecule of transferrin can bind two trivalent iron atoms. For example, transferrin can be iron-containing holotransferrin or iron-free apotransferrin. For example, transferrin can be mouse transferrin. For example, the amino acid sequence of mouse transferrin can be as set forth in GenBank: EDL21066.1, AAL34533.1, or AAL34533.1. For example, the transferrin can be human transferrin. For example, the amino acid sequence of human transferrin can be as set forth in GenBank: AAH59367.1, AAH59367.1, or AAB22049.1. In this application, the term "transferrin" can include functionally active fragments, orthologs, and variants thereof.

[0136] The term "transferrin receptor" generally refers to the carrier protein of transferrin. For example, the transferrin receptor may be a transmembrane glycoprotein. For example, the transferrin receptor may mediate the endocytosis of transferrin associated with two iron ions. For example, the transferrin receptor may maintain iron homeostasis in cells. For example, the transferrin receptor may be transferrin receptor 1 (TfR1) or transferrin receptor 2 (TfR2). For example, TfR1 and TfR2 may share approximately 45-66% homology in their extracellular domains but may have different expression patterns in the body. For example, TfR1 may have a higher affinity for transferrin than TfR2. For example, TfR2 may have a 25-fold lower affinity for transferrin compared to TfR1. For example, TfR2 may be primarily expressed in tissues involved in regulating iron metabolism, such as the liver and small intestine, while TfR1 is generally found on the surface of most body cells. The term "transferrin receptor 1" generally refers to a 97 kDa type 2 membrane protein expressed as a homodimer in the cell membrane. Transferrin internalization mediated by TfR1 has classically been described as the canonical iron uptake pathway. For example, the transferrin receptor can be a mouse transferrin receptor. For example, the amino acid sequence of the mouse transferrin receptor can be as set forth in GenBank: AAH54522.1, CAA40624.1, or NP_001344227.1. For example, the transferrin receptor can be a human transferrin receptor. For example, the amino acid sequence of the human transferrin receptor can be as set forth in GenBank: AAA61153.1, AAF04564.1, or AAB19499.1. In this application, the term "transferrin receptor" can include functionally active fragments, orthologs, and variants thereof.

[0137] The term "ortholog" generally refers to an amino acid sequence that shares a certain percentage of sequence identity and functional similarity with a reference amino acid sequence. For example, orthologs may include structurally similar sequences in different species due to evolution from a common ancestor. Orthologs can be identified using any method known in the art, preferably by using a BLAST tool to compare a reference sequence with a separate second sequence or sequence fragment or sequence database. For example, an ortholog may have at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity with its corresponding sequence.

[0138] The term "treatment" generally refers to the administration of an internal or external therapeutic agent to a patient having one or more disease symptoms, and further, the therapeutic agent is known to have a therapeutic effect on those symptoms. Generally, the therapeutic agent is administered to the patient in an amount sufficient to effectively alleviate one or more disease symptoms (a therapeutically effective amount). Desired therapeutic effects include slowing the rate of disease progression, ameliorating or alleviating the disease state, and reversing or improving the prognosis.

[0139] The term "vector" generally refers to a nucleic acid molecule capable of autonomous replication in a suitable host. A vector introduces an inserted nucleic acid molecule into and / or between host cells. Vectors may include vectors primarily for inserting DNA or RNA into cells, vectors primarily for replicating DNA or RNA, and vectors primarily for expressing DNA or RNA transcriptionally and / or translating. Vectors also include vectors with various functions as defined above. A vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Generally, a vector can produce a desired expression product by culturing a suitable host cell containing the vector.

[0140] The term "optional" or "optionally" means that the subsequently described event or circumstance may occur, but need not occur.

[0141] The term "comprise" generally refers to including, including, containing, or encompassing. In some instances, the term "comprise" also means "is / are" and "consisting of."

[0142] The term "about" generally refers to a variation within 0.5% to 10% above or below a specified value, for example, variations within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, and 10% above or below a specified value.

[0143] Detailed Description of the Invention Isolated antigen-binding proteins In one aspect, the present application provides an isolated antigen-binding protein capable of specifically binding to properdin in an ELISA binding assay using an isolated antigen-binding protein concentration of about 100 ng / ml or less (e.g., the concentration is about 50 ng / ml or less, about 55 ng / ml or less, about 60 ng / ml or less, about 65 ng / ml or less, about 70 ng / ml or less, about 75 ng / ml or less, about 80 ng / ml or less, about 85 ng / ml or less, about 90 ng / ml or less, or about 95 ng / ml or less). For example, the properdin may include human properdin, cynomolgus monkey properdin, and mouse properdin.

[0144] In the present application, the isolated antigen-binding protein can inhibit the alternative pathway by binding the protein and inducing hemolysis. For example, the percentage of hemolysis in an alternative pathway experiment can be determined by co-incubating complement-preserved serum and red blood cells. For example, the complement-preserved serum can be derived from a human or mouse. For example, the percentage of hemolysis can be about 60% or less at an isolated antigen-binding protein concentration of 500 nM (e.g., the percentage of hemolysis is about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, or about 5% or less).

[0145] In the present application, the isolated antigen-binding protein can specifically bind to the TSR5, TSR6, and / or TSR0 domains of properdin. For example, the binding epitope can be determined by combining a truncated variant of human properdin-biotin with thrombospondin repeats (TSR).

[0146] In the present application, the isolated antigen-binding protein can inhibit the interaction between properdin and C3. For example, the inhibitory activity of the isolated antigen-binding protein can be determined by competitive binding assay. The isolated binding protein can competitively bind to properdin so as to inhibit the combination of properdin and C3. For example, the isolated antigen-binding protein exhibits inhibitory activity against properdin binding to C3 in a dose-dependent manner.

[0147] In the present application, the isolated antigen-binding protein may selectively inhibit the alternative pathway but not the classical pathway or the lectin pathway. For example, pathway selectivity can be determined by the percent of hemolysis. For example, the isolated antigen-binding protein may inhibit the alternative pathway with an IC50 of about 50 nM or less (e.g., the IC50 is about 45 nM or less, about 40 nM or less, about 35 nM or less, about 30 nM or less, about 25 nM or less, about 20 nM or less, about 15 nM or less, about 10 nM or less, or about 5 nM or less). For example, the isolated antigen-binding protein does not exhibit inhibitory activity in the classical pathway, but a control exhibits inhibitory ability with an IC50 of 57 nM. For example, the isolated antigen-binding protein does not exhibit inhibitory activity in the lectin pathway, but a control exhibits inhibitory ability with an IC50 of 45 nM.

[0148] In the present application, the isolated antigen-binding protein may have cross-species properdin-binding activity and complement inhibitory activity in the AP-specific pathway in mammals. For example, the cross-species complement inhibitory activity can be determined by detecting hemolysis in different species. For example, the species may be human, cynomolgus monkey, mouse, and rat.

[0149] In one aspect, the application provides an isolated antigen binding protein that may comprise at least one CDR in a heavy chain variable region VH. The VH may comprise the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:92, and SEQ ID NO:93.

[0150] In the present application, the CDR of the isolated antigen-binding protein may be split in any form, and any form of split CDR may be included within the scope of the present application, as long as the VH is identical to the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:92 and SEQ ID NO:93.

[0151] The CDRs of an antibody, also known as complementarity-determining regions, are part of the variable region. The amino acid residues in this region may contact the antigen or antigenic epitope. CDRs can be determined using various coding systems, such as CCG, Kabat, Chothia, IMGT, AbM, and consensus Kabat / Chothia. These coding systems are known in the art, and those skilled in the art can determine CDR regions using different coding systems depending on the sequence and structure of the antibody. The CDR regions may differ when different coding systems are used. In this application, CDRs encompass CDR sequences divided according to any CDR division pattern, and variants thereof are also contemplated. Such variants include CDR amino acid sequences with one or more amino acid substitutions, deletions, and / or additions (e.g., 1 to 30, 1 to 20, or 1 to 10, or even, for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions, deletions, and / or insertions). Also encompassed are homologs comprising an amino acid sequence having at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence identity with the amino acid sequence of the CDR. In some embodiments, the isolated antigen binding proteins described herein are defined by the Kabat coding system.

[0152] In the present application, the isolated antigen-binding protein can bind to properdin, such as human properdin, cynomolgus monkey properdin, mouse properdin, or rat properdin.

[0153] In the present application, the isolated antigen binding protein may comprise a heavy chain variable region VH, which may comprise at least one, two or three of CDR3, CDR2 and CDR1.

[0154] In the present application, the CDR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16 and SEQ ID NO: 17. For example, the CDR3 sequence of the isolated antigen binding protein may be defined according to the Kabat coding system.

[0155] In the present application, the CDR2 of the isolated antigen binding protein has the amino acid sequence set forth in SEQ ID NO: 55: X1X2X3X4X5X6X7X8X9YX 11 X1 is F or I or absent, X2 is D or I, X3 is D or N or R or T, X4 is G or R or S or T, X5 is D or E, X6 is G or R, X7 is G or R or S or V or W, X8 is E or K or T, X9 is R or S or W or Y, and X 11 is A or T. For example, the CDR2 sequence of the isolated antigen binding protein may be defined according to the Kabat coding system.

[0156] In the present application, the CDR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11.

[0157] In the present application, the CDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 54: X1X2CMX5, where X1 is H or S or T or Y, X2 is G or Y, and X5 is A or G. For example, the CDR1 sequence of the isolated antigen binding protein may be defined according to the Kabat coding system.

[0158] In the present application, the CDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

[0159] For example, the CDR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16 and SEQ ID NO:17; the CDR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQ ID NO:11; and the CDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

[0160] For example, the CDR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 12, the CDR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 6, and the CDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 1. For example, the CDR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 13, the CDR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 7, and the CDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 2. For example, the CDR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 14, the CDR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 8, and the CDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 3. For example, the CDR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 15, the CDR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 9, and the CDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 4. For example, the CDR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 16, the CDR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 10, and the CDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 5. For example, the CDR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 17, the CDR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 11, and the CDR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 3.For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:92 and SEQ ID NO:93, or an antibody having the same CDRs (e.g., CDR1, CDR2 or CDR3).

[0161] In the present application, said isolated antigen binding protein may further comprise framework regions FR1, FR2, FR3 and FR4.

[0162] In the present application, the FR1 of the isolated antigen binding protein has the amino acid sequence set forth in SEQ ID NO: 56: X1VQLVESGGGX 11 VX 13 X 14 GGSLRLSCX 23 X 24 X 25 X 26 YX 28 X 29 X 30 X1 may comprise D or E or H or Q, and X 11 is L or S or V, and X 13 is H or Q, and X 14 is A or P or S or V, and X 23 is A or E or V, and X 24 is A or D or H or V, and X 25 is F or P or S, and X 26 is A or E or G, and X 28 is I or T or non-existent, and X 29 is H or S or Y or absent, and X 30 is G or S or T or absent. For example, the FR1 sequence of the isolated antigen binding protein may be defined according to the Kabat coding system.

[0163] In the present application, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29 and SEQ ID NO:30.

[0164] In the present application, the FR2 of the isolated antigen binding protein has the amino acid sequence set forth in SEQ ID NO: 57: WX2RQAPGX8X9X 10 EX 12 VX 14 X 15 wherein X2 is F or I, X8 is E or K, X9 is E or G, and X 10 is L or R, and X 12 is G or R, and X 14 is A or S, and X 15 is A or S or V. For example, the FR2 sequence of the isolated antigen binding protein may be defined according to the Kabat coding system.

[0165] In the present application, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40.

[0166] In the present application, the FR3 of the isolated antigen binding protein has the amino acid sequence set forth in SEQ ID NO: 58: RFTISX6DX8X9X 10 X 11 TLYLX 16 MNX 19 LX 21 X 22 EDTAX 27 YYCAX 32 wherein X6 is K or L or Q or R, X8 is I or N, X9 is A or S, and X 10 is E or K or T, and X 11is N or S, and X 16 is E or Q, and X 19 is I or N or S, and X 21 is K or Q or R, and X 22 is A or P or S, and X 27 is M or V, and X 32 is A or T. For example, the FR3 sequence of the isolated antigen binding protein may be defined according to the Kabat coding system.

[0167] In the present application, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51.

[0168] In the present application, the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 59:WGQGTX6VTVSS, wherein X6 is L or Q. For example, the FR4 sequence of the isolated antigen binding protein may be defined according to the Kabat coding system.

[0169] In the present application, the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:52 and SEQ ID NO:53.

[0170] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30; the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40; the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, and SEQ ID NO:51; and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:52 and SEQ ID NO:53.

[0171] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 18, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 31, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 41, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 60, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0172] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 19, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 32, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 42, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 61, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0173] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 19, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 32, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 42, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 62, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0174] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 19, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 32, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 43, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 63, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0175] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 20, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 32, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 43, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 64, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0176] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 20, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 31, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 43, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 65, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0177] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 21, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 33, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 44, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 66, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0178] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 22, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 34, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 42, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 67, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0179] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 22, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 34, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 45, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 68, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0180] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 22, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 33, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 45, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 69, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0181] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 23, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 35, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 46, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 70, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0182] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 24, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 36, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 47, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 71, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0183] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 24, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 36, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 48, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 72, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0184] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 25, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 36, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 48, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 73, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0185] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 25, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 35, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 48, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 74, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0186] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 26, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 37, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 49, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 75, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0187] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 27, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 38, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 42, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 76, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0188] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 27, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 39, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 42, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 77, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0189] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 28, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 39, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 42, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 78, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0190] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 28, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 39, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 43, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 79, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0191] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 28, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 37, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 43, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 53. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 80, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0192] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 29, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 31, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 50, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 92, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0193] For example, the FR1 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 30, the FR2 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 40, the FR3 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 51, and the FR4 of the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 52. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 93, or an antibody having the same FR (e.g., FR1, FR2, FR3, or FR4).

[0194] In the present application, the heavy chain variable region may comprise a VHH. The VHH may comprise any one of the amino acid sequences set forth in SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:92, and SEQ ID NO:93.

[0195] In the present application, the isolated antigen binding protein may comprise a heavy chain constant region.

[0196] For example, the Fc region of the isolated antigen-binding protein can be a human Fc region. For example, the human Fc region can be modified (e.g., by amino acid mutation) to achieve desired properties. For example, the human Fc region can comprise the amino acid sequence set forth in SEQ ID NO: 109.

[0197] In the present application, the isolated antigen-binding protein may be directly or indirectly linked to a second antigen-binding domain.

[0198] For example, the isolated antigen-binding protein may be linked via its N-terminus or C-terminus to the N-terminus or C-terminus of the second antigen-binding domain. For example, the isolated antigen-binding protein may be linked via its N-terminus or C-terminus to the N-terminus or C-terminus of the second antigen-binding domain using a linker. For example, the linker of the isolated antigen-binding protein may be a simple covalent bond (e.g., a peptide bond), a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer), or any type of bond created by a chemical reaction. For example, the linker of the isolated antigen-binding protein may be a polyglycine linker. For example, the linker of the isolated antigen-binding protein may comprise the amino acid sequence set forth in SEQ ID NO: 108: GGGSGGGGSGGGGGS.

[0199] In the present application, the second antigen-binding domain of the isolated antigen-binding protein may bind to a different target than the isolated antigen-binding protein.

[0200] In the present application, the second antigen-binding domain of the isolated antigen-binding protein may bind to the same target as the isolated antigen-binding protein.

[0201] For example, the second antigen-binding domain of the isolated antigen-binding protein may bind to properdin. For example, the second antigen-binding domain of the isolated antigen-binding protein may bind to a different epitope of properdin than the isolated antigen-binding protein. For example, the second antigen-binding domain of the isolated antigen-binding protein may bind to the same epitope of properdin as the isolated antigen-binding protein. For example, the second antigen-binding domain of the isolated antigen-binding protein may bind to the TSR5, TSR6, and / or TSR0 domain of properdin. For example, the second antigen-binding domain of the isolated antigen-binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:92 and SEQ ID NO:93.

[0202] For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:81.

[0203] For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in SEQ ID NO:116.

[0204] For example, the isolated antigen-binding protein may comprise an antibody or an antigen-binding fragment thereof. For example, the isolated antigen-binding protein may comprise a Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, VHH, and / or dAb. For example, the isolated antigen-binding protein may be selected from the group consisting of a monoclonal antibody, a single-chain antibody, a chimeric antibody, a humanized antibody, and a fully human antibody.

[0205] For example, the isolated antigen binding protein can be a camelid antibody.

[0206] In the present application, the isolated antigen-binding protein may have competitive target-binding ability with a reference antibody, and the reference antibody may comprise a heavy chain variable region VH, which may comprise at least one, two or three of CDR3, CDR2 and CDR1.

[0207] In the present application, the CDR3 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13, and SEQ ID NO: 14. For example, the CDR3 sequence of the reference antibody may be defined according to the Kabat coding system.

[0208] In the present application, the CDR2 of said reference antibody has the amino acid sequence set forth in SEQ ID NO: 55: X1X2X3X4X5X6X7X8X9YX 11 X1 is F or I or absent, X2 is D or I, X3 is D or N or R or T, X4 is G or R or S or T, X5 is D or E, X6 is G or R, X7 is G or R or S or V or W, X8 is E or K or T, X9 is R or S or W or Y, and X 11 is A or T. For example, the CDR2 sequence of the reference antibody may be defined according to the Kabat coding system.

[0209] In the present application, the CDR2 of said reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

[0210] In the present application, the CDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 54: X1X2CMX5, where X1 is H or S or T or Y, X2 is G or Y, and X5 is A or G. For example, the CDR1 sequence of the reference antibody may be defined according to the Kabat coding system.

[0211] In the present application, the CDR1 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3.

[0212] For example, the CDR3 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, the CDR2 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, and the CDR1 of the reference antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3.

[0213] For example, CDR3 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 12, CDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 6, and CDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 1. For example, CDR3 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 13, CDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 7, and CDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 2. For example, CDR3 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 14, CDR2 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 8, and CDR1 of the reference antibody may comprise the amino acid sequence set forth in SEQ ID NO: 3. For example, the isolated antigen binding protein may comprise the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73 and SEQ ID NO:74, or an antibody having the same CDR (e.g., CDR1, CDR2 or CDR3).

[0214] For example, an isolated antigen-binding protein can include functionally active fragments, orthologs, and variants thereof that maintain similar biological activity. For example, the sequence similarity can be at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to the corresponding sequence.

[0215] fusion proteins In another aspect, the present application provides a fusion protein that can include the isolated antigen-binding protein of the present application.

[0216] In the present application, the fusion protein may comprise a functionally active protein.

[0217] In the present application, the functionally active protein of the fusion protein can be directly or indirectly linked to the isolated antigen binding protein.

[0218] For example, the functionally active protein can be linked by its N-terminus or C-terminus to the N-terminus or C-terminus of the isolated antigen-binding protein. For example, the functionally active protein can be linked by its N-terminus or C-terminus to the N-terminus or C-terminus of the isolated antigen-binding protein using a linker. For example, the linker can be a simple covalent bond (e.g., a peptide bond), a synthetic polymer (e.g., a polyethylene glycol (PEG) polymer), or any type of bond created by a chemical reaction. For example, the linker can be a polyglycine linker. For example, the linker can comprise the amino acid sequence set forth in SEQ ID NO: 108: GGGSGGGGSGGGGGS.

[0219] For example, the functionally active protein can be Factor H. For example, the Factor H can comprise the amino acid sequence set forth in SEQ ID NO: 110. For example, the Factor H of the fusion protein can include functionally active fragments, orthologs, and variants thereof. For example, the sequence similarity can be at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to the corresponding sequence.

[0220] For example, the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:82.

[0221] For example, the functionally active protein of the fusion protein can be a VEGF inhibitory protein. For example, the VEGF inhibitory protein of the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO: 113. For example, the VEGF inhibitory protein of the fusion protein can comprise functionally active fragments, orthologs, and variants thereof. In some embodiments, the sequence similarity can be at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similarity to the corresponding sequence.

[0222] For example, the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:114.

[0223] For example, the functionally active protein of the fusion protein can be a transferrin inhibitor protein. For example, the factor H of the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO: 115. For example, the transferrin inhibitor protein of the fusion protein can include functionally active fragments, orthologs, and variants thereof. In some embodiments, the sequence similarity can be at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to the corresponding sequence.

[0224] For example, the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:117.

[0225] For example, the fusion protein can comprise the amino acid sequence set forth in SEQ ID NO:118.

[0226] For example, a fusion protein can comprise a prophylactic or therapeutic agent fused to a heterologous protein, polypeptide, or peptide, which may or may not be a different type or therapeutic agent.

[0227] For example, a fusion protein may contain two or more different proteins, polypeptides, or peptides with immunomodulatory activity.For example, a fusion protein may retain or improve activity compared to the activity of the original polypeptide or protein.Typically, a fusion protein can be produced by in vitro recombinant technology well known in the art.For example, a fusion protein may contain an antigen-binding protein.

[0228] Polypeptides and Immunoconjugates In another aspect, the application provides one or more polypeptides that may comprise the isolated antigen binding proteins of the application.

[0229] In another aspect, the present application provides one or more immunoconjugates that may comprise the isolated antigen-binding proteins of the present application. In certain embodiments, the immunoconjugates may further comprise a pharmaceutically acceptable therapeutic agent.

[0230] Nucleic acids, vectors and cells In another aspect, the present application further provides an isolated nucleic acid molecule or a plurality of isolated nucleic acid molecules. The nucleic acid molecule(s) may encode an antigen-binding protein of the present application. For example, each of the nucleic acid molecules may encode the entire antigen-binding protein or a portion thereof (e.g., one or more of CDR1-3, FR1-4, VH, VHH, or heavy chain).

[0231] The nucleic acid molecule(s) of the present application can be isolated. For example, the nucleic acid molecule can be produced or synthesized by the following methods: (i) in vitro amplification, e.g., by polymerase chain reaction (PCR) amplification; (ii) clonal recombination; (iii) purification, e.g., by restriction digestion and fractionation via gel electrophoresis; or (iv) synthetically, e.g., by chemical synthesis. In some embodiments, the isolated nucleic acid(s) are nucleic acid molecule(s) prepared by recombinant DNA technology.

[0232] In the present application, the nucleic acid(s) encoding the antibody and its antigen-binding fragments can be prepared by various methods known in the art. These methods include, but are not limited to, using restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. For specific procedures, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989 and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0233] In another aspect, the present application provides a vector or vectors, each containing one or more nucleic acid molecules of the present application. Each vector may contain one or more of the nucleic acid molecules. Furthermore, the vector may also contain other genes, such as marker genes that allow the vector to be selected in an appropriate host cell under appropriate conditions. Furthermore, the vector may also contain expression control elements that enable the correct expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that control gene transcription or mRNA translation. In some embodiments, the expression control sequence is a controllable element. The specific structure of the expression control sequence may vary depending on the species or cell type, but generally includes 5' non-transcribed sequences and 5' and 3' non-translated sequences involved in transcription and translation initiation, respectively, such as a TATA box, capping sequence, CAAT sequence, etc. For example, the 5' non-transcribed expression control sequence may include a promoter region, which may include a promoter sequence operably linked to a nucleic acid for transcriptional regulation. The expression regulatory sequence may further include an enhancer sequence or an upstream activation sequence. In the present application, suitable promoters may include, for example, promoters for SP6, T3, and T7 polymerase, human U6 RNA promoter, CMV promoter, and artificial hybrid promoters thereof (such as CMV), and a portion of the promoter may be fused with a portion of the promoter of an additional cellular protein gene (such as human GAPDH and glyceraldehyde-3-phosphate dehydrogenase), and the promoter may or may not contain additional introns. The nucleic acid molecule(s) of the present application may be operably linked to an expression regulatory element. The vector may include, for example, a plasmid, cosmid, virus, bacteriophage, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector.

[0234] In another aspect, the present application provides host cells that can include the nucleic acid molecule(s) of the present application and / or one or more vectors of the present application. In some embodiments, each or each type of host cell can include one or more types of nucleic acid molecules or vectors of the present application. In some embodiments, each or each type of cell can include multiple (e.g., two or more) or multiple types (e.g., two or more) of vectors of the present application. For example, the vectors of the present application can be introduced into host cells, e.g., eukaryotic cells such as cells of plant origin, fungal cells, or yeast cells. The vectors of the present application can be introduced into host cells by methods known in the art, such as electroporation, lipofectin transfection, lipofectamine transfection, etc.

[0235] Preparation method In another aspect, the present application provides a preparation method for the isolated antigen-binding protein. This method may include culturing the host cell of the present application under conditions such that the isolated antigen-binding protein is expressed. For example, an appropriate medium, an appropriate temperature, an appropriate culture time, etc. may be used, and these methods will be understood by those skilled in the art.

[0236] Any method suitable for producing monoclonal antibodies can be used to produce the isolated antigen-binding proteins (e.g., anti-properdin antibodies) of the present application. For example, an animal can be immunized with linked or naturally occurring properdin or a fragment thereof. Any suitable immunization method, including adjuvants, immunostimulants, and repeated booster immunizations, can be used, and one or more routes can be used.

[0237] Any suitable form of properdin can be used as an immunogen (antigen) to produce non-human antibodies specific to properdin and to screen the biological activity of the antibodies. The eliciting immunogen can be human properdin, recombinant mouse, or a peptide containing a single or multiple epitopes. The immunogen can be used alone or in combination with one or more immunogenic enhancers known in the art. The immunogen can be purified from a natural source or produced in a genetically modified cell. The DNA encoding the immunogen can be of genomic or non-genomic (e.g., cDNA) origin. An appropriate gene vector can be used to express the DNA encoding the immunogen, including, but not limited to, adenovirus vectors, adeno-associated virus vectors, baculovirus vectors, materials, and non-viral vectors.

[0238] An exemplary method for discovering isolated antigen binding proteins of the present application is described in Example 1.

[0239] Immunization can be performed using recombinant mouse properdin in healthy camels. The essential constant domain sequences can be optimized by screening antibodies in the biological assays described in the examples below to produce the desired biological activity.

[0240] An exemplary method for humanizing an isolated antigen binding protein of the present application is described in Example 2.

[0241] The sequence of the DNA molecule of the isolated antigen-binding protein or fragment thereof in the present application can be obtained by conventional techniques, such as methods using PCR amplification or genomic library screening.

[0242] Once the sequence is obtained, it can be obtained on a large scale by recombinant means, generally by cloning the sequence into a vector, introducing it into cells, and then isolating the sequence from the propagated host cells by conventional methods.

[0243] Furthermore, especially when the fragment is short, the sequence can be synthesized by artificial synthesis.Generally, a fragment with a very long sequence can be obtained by first synthesizing a number of small fragments, and then linking these small fragments.Then, the nucleic acid molecule can be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art.

[0244] The present application also relates to vectors containing the above-mentioned suitable nucleic acid molecules and suitable promoters or regulatory sequences. These vectors can be used to transform suitable host cells to enable the suitable host cells to express proteins. The host cells can be prokaryotic cells such as bacterial cells, or lower eukaryotic cells such as yeast cells, or higher eukaryotic cells such as mammalian cells. For example, animal cells can include, but are not limited to, CHO-S, CHO-K1, and HEK-293 cells.

[0245] The step of transforming host cells with recombinant DNA in the present application can be carried out using techniques well known in the art. The resulting transformants can be cultured by conventional methods to express the polypeptides encoded by the nucleic acid molecule(s) of the present application. Depending on the host cells used, the host cells are cultured in conventional media under appropriate conditions. Generally, the host cells are cultured and transformed under conditions suitable for the expression of the isolated antigen-binding proteins of the present application. The isolated antigen-binding proteins of the present application are then purified and obtained using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography, as well as other conventional separation and purification means well known to those skilled in the art.

[0246] The resulting monoclonal antibodies can be identified by conventional means, for example, the binding specificity of the monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays, such as fluorescence-activated cell sorting (FACS), radioimmunoassay (RIA), or enzyme-linked immunosorbent assay (ELISA).

[0247] Pharmaceutical Compositions and Pharmaceutical Combinations In another aspect, the present application provides pharmaceutical compositions. The pharmaceutical compositions may comprise the isolated antigen-binding proteins, polypeptides, immunoconjugates, isolated nucleic acid molecules, vectors, cells, and / or pharmaceutically acceptable adjuvants and / or excipients described herein. In the present application, pharmaceutically acceptable adjuvants may include buffers, antioxidants, preservatives, low-molecular-weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counterions, metal complexes, and / or non-ionic surfactants. Except where conventional media or agents are incompatible with the cells described herein, their use in the pharmaceutical compositions of the present application is contemplated. In the present application, pharmaceutically acceptable excipients may include additives other than the main drug in pharmaceutical preparations and may also be referred to as auxiliary materials. For example, excipients may include binders, fillers, disintegrants, and lubricants in tablets. For example, excipients may include wine, vinegar, medicinal juice, etc. in traditional Chinese medicine pills. For example, excipients may include the base portion of semi-solid preparations such as ointments and creams. For example, excipients may include preservatives, antioxidants, flavorings, perfumes, cosolvents, emulsifiers, solubilizers, isotonicity adjusting agents, and coloring agents in liquid formulations. Pharmaceutical preparations should be compatible with the mode of administration. The pharmaceutical compositions of the present application can be prepared into injection forms by conventional methods, for example, using normal saline or aqueous solutions containing glucose and other adjuvants. Pharmaceutical compositions such as injections and solutions should be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount. Furthermore, the isolated antigen-binding proteins of the present application may be used together with other therapeutic agents.

[0248] In another aspect, the present application provides a pharmaceutical combination comprising the isolated antigen binding protein and one or more active ingredients.

[0249] The isolated antigen-binding proteins, pharmaceutical compositions, or pharmaceutical combinations described herein may be formulated, dosed, and administered in accordance with the principles of good medical practice. Considerations in this regard include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the administration schedule, and other factors known to medical professionals. Therapeutic agents (e.g., anti-properdin antibodies) need not be formulated and / or administered together with one or more drugs currently used to prevent or treat the disorder in question, but may optionally be formulated and / or administered together. The effective amount of such other drugs will depend on the amount of therapeutic agent (e.g., anti-properdin antibodies) present in the formulation, the type of disorder or treatment, and the other factors discussed above. In general, these drugs may be used at any dose and via any route empirically / clinically determined to be appropriate. The dose of the antibody administered in combination therapy may be reduced compared to monotherapy. The progress of such therapy can be easily monitored by conventional techniques.

[0250] Kits, Uses and Methods In another aspect, the present application provides a method for detecting or determining properdin, which may include using said isolated antigen-binding protein or said polypeptide.

[0251] In the present application, the method may include an in vitro method, an ex vivo method, a method of non-diagnostic or non-therapeutic interest, for example, a method for detecting the presence and / or amount of properdin for non-diagnostic purposes, comprising: 1) contacting a sample with an antigen binding protein of the present application; 2) detecting the presence and / or amount of said antigen binding protein bound by said sample obtained from said subject to determine the presence and / or level of expression of properdin in said sample; The method may include:

[0252] For example, the isolated antigen binding protein of the method may comprise the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:116, SEQ ID NO:117 and SEQ ID NO:118.

[0253] For example, the isolated antigen binding protein of the method may comprise the amino acid sequence set forth in any one of SEQ ID NO:92 and SEQ ID NO:93.

[0254] For example, the isolated antigen binding protein of the method may comprise the amino acid sequence set forth in SEQ ID NO:92.

[0255] For example, the isolated antigen binding protein of the method may comprise the amino acid sequence set forth in SEQ ID NO:93.

[0256] In another aspect, the present application provides a kit for properdin, which may include the use of the isolated antigen-binding protein or the polypeptide. In the present application, the kit may further include instructions documenting a method for detecting the presence and / or amount of properdin. For example, the method may include an in vitro method, an ex vivo method, a non-diagnostic or non-therapeutic method of interest.

[0257] For example, the kit can be an ELISA kit comprising the isolated antigen-binding protein or the polypeptide. For example, the ELISA kit can detect properdin by direct ELISA, indirect ELISA, sandwich ELISA, or competitive ELISA.

[0258] For example, the isolated antigen-binding protein or the polypeptide can be used as a capture antibody.

[0259] For example, the isolated antigen-binding protein or the polypeptide can be used as a detection antibody. For example, the detection antibody can be linked to HRP (horseradish peroxidase). For example, the detection antibody can be linked to ALP (alkaline phosphatase).

[0260] For example, the capture antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 116, SEQ ID NO: 117, and SEQ ID NO: 118. For example, the capture antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO: 92 and SEQ ID NO: 93.

[0261] For example, the capture antibody may comprise the amino acid sequence set forth in SEQ ID NO: 92. For example, the capture antibody may comprise the amino acid sequence set forth in SEQ ID NO: 93.

[0262] For example, the detection antibody may comprise the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:116, SEQ ID NO:117 and SEQ ID NO:118.

[0263] In another aspect, the present application provides the use of said isolated antigen binding protein or said polypeptide in the preparation of a kit for use in a method of detecting the presence and / or amount of properdin, for example, said method may include an in vitro method, an ex vivo method, a non-diagnostic or non-therapeutic method of interest.

[0264] For example, the isolated antigen-binding protein of said use may comprise the amino acid sequence set forth in any one of SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63, SEQ ID NO:64, SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:116, SEQ ID NO:117 and SEQ ID NO:118. For example, the isolated antigen-binding protein of said use may comprise the amino acid sequence set forth in any one of SEQ ID NO:92 and SEQ ID NO:93. For example, the isolated antigen-binding protein of said use may comprise the amino acid sequence set forth in SEQ ID NO:92. For example, the isolated antigen-binding protein of said use may comprise the amino acid sequence set forth in SEQ ID NO:93.

[0265] In another aspect, the present application provides a method of inhibiting the alternative complement pathway, comprising administering to a subject in need thereof an effective amount of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition, and / or a pharmaceutically acceptable therapeutic agent. In the present application, methods of modulating an immune response may include in vitro methods, ex vivo methods, non-diagnostic or non-therapeutic methods of interest.

[0266] In another aspect, the present application provides a method of inhibiting the alternative complement pathway, comprising administering to a subject in need thereof an effective amount of the pharmaceutical composition, pharmaceutical combination, and / or pharmaceutically acceptable therapeutic agent. In the present application, the method of modulating an immune response can include in vitro methods, ex vivo methods, non-diagnostic or non-therapeutic methods of interest.

[0267] In another aspect, the present application provides a method of inhibiting properdin binding to C3, comprising administering to a subject in need thereof an effective amount of said isolated antigen-binding protein, said polypeptide, said immunoconjugate, said isolated nucleic acid molecule, said vector, and / or said cell. The method may be an ex vivo or an in vitro method.

[0268] In another aspect, the present application provides an isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, or the pharmaceutical composition for preventing and / or treating a disease. For example, the disease may be caused by properdin. For example, the disease may be mediated by an alternative pathway. For example, the disease may be autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), asthma, Gaucher disease, hidradentitis suppurativa, or the like. suppurativa), Behcet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-related lung injury (TRALI), hemodialysis-induced thrombosis, epidermolysis bullosa acquisita (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, diffuse thrombotic microangiopathy (TMA), diffuse thrombotic microangiopathy (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes, and Hyperlipidemia and Thrombocytopenia (HELLP) syndrome, multiple sclerosis, antiphospholipid syndrome (APS), relapsing polychondritis, ischemic injury, stroke, graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), emphysema, atherosclerosis, acute coronary syndrome, hemorrhagic shock, rheumatoid arthritis, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, antiphospholipid syndrome (APS) pregnancy loss, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malarial hemolytic crisis, abdominal aortic aneurysm (AAA), or thoracoabdominal aortic aneurysm (TAA).

[0269] In another embodiment, the kit, the pharmaceutical composition, and / or the pharmaceutical combination are used for the prevention and / or treatment of a disease in the present application. For example, the disease may be caused by properdin. For example, the disease may be mediated by an alternative pathway. For example, the disease may be autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), asthma, Gaucher disease, hidradenitis suppurativa (Hidradentitis suppurativa), or the like. suppurativa), Behcet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-related lung injury (TRALI), hemodialysis-induced thrombosis, epidermolysis bullosa acquisita (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, diffuse thrombotic microangiopathy (TMA), diffuse thrombotic microangiopathy (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes, and Hyperlipidemia and Thrombocytopenia (HELLP) syndrome, multiple sclerosis, antiphospholipid syndrome (APS), relapsing polychondritis, ischemic injury, stroke, graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), emphysema, atherosclerosis, acute coronary syndrome, hemorrhagic shock, rheumatoid arthritis, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, antiphospholipid syndrome (APS) pregnancy loss, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malarial hemolytic crisis, abdominal aortic aneurysm (AAA), or thoracoabdominal aortic aneurysm (TAA).

[0270] In another aspect, the present application provides use of the isolated antigen-binding protein, the polypeptide, the immunoconjugate, the isolated nucleic acid molecule, the vector, the cell, and / or the pharmaceutical composition for the preparation of a medicament for the prevention and / or treatment of a disease in the present application. For example, the disease may be caused by properdin. For example, the disease may be mediated by an alternative pathway. For example, the disease may be autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), asthma, Gaucher disease, hidradentitis suppurativa, or the like. suppurativa), Behcet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-related lung injury (TRALI), hemodialysis-induced thrombosis, epidermolysis bullosa acquisita (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, diffuse thrombotic microangiopathy (TMA), diffuse thrombotic microangiopathy (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes, and Hyperlipidemia and Thrombocytopenia (HELLP) syndrome, multiple sclerosis, antiphospholipid syndrome (APS), relapsing polychondritis, ischemic injury, stroke, graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), emphysema, atherosclerosis, acute coronary syndrome, hemorrhagic shock, rheumatoid arthritis, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, antiphospholipid syndrome (APS) pregnancy loss, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malarial hemolytic crisis, abdominal aortic aneurysm (AAA), or thoracoabdominal aortic aneurysm (TAA).

[0271] In another aspect, the present application provides use of a pharmaceutical combination for the manufacture of a medicament for the prevention and / or treatment of a disease in the present application. For example, the disease may be caused by properdin. For example, the disease may be mediated by an alternative pathway. For example, the disease may be autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), asthma, Gaucher disease, hidradenitis suppurativa (Hidradentitis), and the like. suppurativa), Behcet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-related lung injury (TRALI), hemodialysis-induced thrombosis, epidermolysis bullosa acquisita (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, diffuse thrombotic microangiopathy (TMA), diffuse thrombotic microangiopathy (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes, and Hyperlipidemia and Thrombocytopenia (HELLP) syndrome, multiple sclerosis, antiphospholipid syndrome (APS), relapsing polychondritis, ischemic injury, stroke, graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), emphysema, atherosclerosis, acute coronary syndrome, hemorrhagic shock, rheumatoid arthritis, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, antiphospholipid syndrome (APS) pregnancy loss, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malarial hemolytic crisis, abdominal aortic aneurysm (AAA), or thoracoabdominal aortic aneurysm (TAA).

[0272] In another aspect, the present application provides a method for preventing and / or treating a disease or disorder, the method comprising administering the isolated antigen-binding protein, the isolated nucleic acid molecule, the vector, the cell, or the pharmaceutical composition to a subject in need of such prevention and / or treatment. For example, the disease may be caused by properdin. For example, the disease may be mediated by an alternative pathway. For example, the disease may be autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), asthma, Gaucher disease, hidradentitis suppurativa, or the like. suppurativa), Behcet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-related lung injury (TRALI), hemodialysis-induced thrombosis, epidermolysis bullosa acquisita (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, diffuse thrombotic microangiopathy (TMA), diffuse thrombotic microangiopathy (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes, and Hyperlipidemia and Thrombocytopenia (HELLP) syndrome, multiple sclerosis, antiphospholipid syndrome (APS), relapsing polychondritis, ischemic injury, stroke, graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), emphysema, atherosclerosis, acute coronary syndrome, hemorrhagic shock, rheumatoid arthritis, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, antiphospholipid syndrome (APS) pregnancy loss, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malarial hemolytic crisis, abdominal aortic aneurysm (AAA), or thoracoabdominal aortic aneurysm (TAA).

[0273] In another aspect, the present application provides a method for preventing and / or treating a disease or disorder, comprising administering the pharmaceutical combination to a subject in need thereof. For example, the disease may be caused by properdin. For example, the disease may be mediated by an alternative pathway. For example, the disease may be autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), C3 glomerulopathy (C3G), asthma, Gaucher disease, hidradenitis suppurativa (Hidradentitis suppurativa), or the like. suppurativa), Behcet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-related lung injury (TRALI), hemodialysis-induced thrombosis, epidermolysis bullosa acquisita (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, diffuse thrombotic microangiopathy (TMA), diffuse thrombotic microangiopathy (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes, and Hyperlipidemia and Thrombocytopenia (HELLP) syndrome, multiple sclerosis, antiphospholipid syndrome (APS), relapsing polychondritis, ischemic injury, stroke, graft-versus-host disease (GvHD), chronic obstructive pulmonary disease (COPD), emphysema, atherosclerosis, acute coronary syndrome, hemorrhagic shock, rheumatoid arthritis, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, antiphospholipid syndrome (APS) pregnancy loss, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malarial hemolytic crisis, abdominal aortic aneurysm (AAA), or thoracoabdominal aortic aneurysm (TAA).

[0274] The pharmaceutical compositions, pharmaceutical combinations and methods described herein can be used in combination with other types of treatment, such as chemotherapy, surgery, radiation, gene therapy, and the like.

[0275] In this application, the subject and / or patient / animal patient may include a human or a non-human animal. For example, the non-human animal may be selected from the group consisting of monkeys, chickens, geese, cats, dogs, mice, and rats. Furthermore, the non-human animal may also include any animal species other than humans, such as livestock animals, rodents, primates, domestic animals, or poultry. The human may be Caucasian, African, Asian, amphibian, or of other ethnic groups, or a mixture of various ethnic groups. As another example, the person may be an elderly person, an adult, a teenager, a child, or an infant.

[0276] The effective dose for humans can be estimated from the effective dose for experimental animals.For example, Freiich et al. describe the dose relationship between animals and humans (based on milligrams per square meter of body surface) (Freirich et al., Cancer Chemother. Rep. 50, 219 (1966)).Body surface area can be approximately determined from the height and weight of the patient / animal.See, for example, Scientific Tables, Geigy Pharmaceuticals, Ardsley, NY, 537 (1970).

[0277] example The following examples are given to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the following experiments are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, such as bp, base pairs; kb, kilobase; pl, picoliter; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acid; nt, nucleotide; im, intramuscular; ip, intraperitoneal; sc, subcutaneous, etc.

[0278] Example 1. Discovery of properdin-binding antibody fragments 1.1 Preparation of human, mouse, and cynomolgus monkey properdin Human, mouse, and cynomolgus properdin sequences from the Uniprot database were subcloned into expression vectors synthesized by GENEWIZ. Protein expression was performed using transient expression in HEK293 cells transfected with PEI (Polysciences, catalog number 24765-1). Cultures were grown for 5-7 days in shake flasks at scales ranging from 100 to 200 ml. Cells were removed by centrifugation, and the culture supernatant was used for protein purification by Ni-Sepharose with elution using PBS buffer at pH 7.4. The purified proteins were analyzed by 4-12% SDS-PAGE under non-reducing and reducing conditions (Figure 1).

[0279] 1.2 VHH immune library construction Immunization was performed using recombinant mouse properdin in two healthy camels. On day 60, after four rounds of immunization, a phage-displayed VHH library was constructed from PMBCs from the immunized camels by following a standardized protocol. The final phage-displayed VHH library contained 2.1 x 10 9 There were independent clones, 92% of which encoded VHH-gp3 fusion proteins.

[0280] 1.3 Phage panning The immune VHH library, as previously prepared, was used for VHH selection and subjected to four rounds of panning in 1.5 ml Eppendorf tubes. 12 CFU phage were incubated with 10 μg of biotinylated properdin in 1 ml of blocking buffer (1% BSA in PBS) at room temperature for 1 hour to create a phage / target mixture. Simultaneously, 100 μl of streptavidin-coated Dynabeads M-280 (Invitrogen, 11206D) was washed five times with 1 ml of blocking buffer in an Eppendorf microtube. The phage / target mixture was then incubated with Mag beads prepared as described above for 30 minutes at room temperature on a rotator. To recover the phage bound to the Mag beads, the reaction tube was placed on a magnetic rack for 30 seconds. After removing the supernatant, the beads were washed 10 times with PBS containing 0.5% Tween 20 (0.5% PBST), followed by three washes with PBS. The phage were eluted with 1 ml of trypsin (10 μg / ml in PBS) at 37°C for 30 minutes. After each round of selection, 100 μl of eluted phage was used to culture mid-logarithmic phase E. coli TG1 (OD ) grown at 37°C for phage titer determination. 600 = 0.6). To monitor the progress of the selection process, the enrichment value of properdin-specific VHHs was also evaluated. The remaining eluted phages were used for subsequent amplification in E. coli TG1 (OD 600The bacteria were then infected with M13KO7 helper phage at a ratio of 20:1 (phage:bacteria) to rescue the phage particles. A mixture of kanamycin (50 μg / mL) and ampicillin (100 μg / mL) was added to the culture, and the bacteria were further grown for 4 hours at 30°C with shaking at 220 rpm. The culture was centrifuged at 4,000 g for 20 minutes, and the supernatant was added to 20% (w / v) polyethylene glycol 6000 / 2.5 M NaCl (PEG / NaCl) to precipitate the phage. The sample was incubated overnight on ice and then centrifuged at 8,000 g for 20 minutes at 4°C. The pellet was resuspended in PBS, and PEG precipitation was repeated once as described above. The final phage pellet was resuspended in 1 ml of PBS and diluted with 10 12 The phages were used in subsequent rounds of panning. The general panning procedure was repeated for three more rounds. The modification was antigens from different species in order to have cross-reactive and affinity-matched phage clones. The panning summary is listed in Table 1. [Table 1]

[0281] 1.4 Phage screening methods For phage-based Elisa screening, individual bacterial colonies were picked and inoculated into 200 μl of 2xYT-GA medium and grown at 37°C with shaking (250 rpm) for 4-5 hours. 10 μl of the culture was then transferred into a new deep 96-well plate containing 200 μl of 2xYT-GA medium, and the OD 600 The cultures were incubated as described above until the ΔΨ reached approximately 0.6. VHH expression was induced with 1 mM IPTG (Sangon Biotech) for 16 hours at 30°C with shaking (250 rpm). The overnight cultures were spun at 4,000 rpm at 4°C for 30 minutes, after which the supernatants were collected for phage ELISA.

[0282] After four rounds of panning, the outputs from the second, third, and fourth rounds were screened by phage enzyme-linked immunosorbent assay (ELISA). The wells of a MaxiSorp 96-well plate were coated with 1 μg / ml streptavidin in coating buffer overnight at 4°C. An equal concentration of BSA was used as a control for nonspecific binding. After washing with PBST, remaining protein binding sites in the wells were blocked with 1% BSA for 1 hour at 37°C. The blocking reagent was discarded, and the wells were washed three times with 0.05% PBST. 5 μg / ml biotin-human / mouse / cynomolgus lutropin was added to the wells for 1 hour at 37°C. The supernatant was discarded, and the wells were washed three times with 0.05% PBST. 100 μl of the supernatant prepared above was added to the appropriate wells and incubated with shaking at 37°C for 1 hour. The supernatant was discarded, and nonspecific phages were removed by washing three times with 0.05% PBST. Interactions between the antigen and phage-VHH were detected using a 5000-fold diluted solution of anti-Myc-HRP (Abcam, ab62928). After 1 h of incubation at 37 °C, the plate was washed as before, and 100 μl of TMB substrate solution was added and incubated at room temperature for 15 min. The reaction was stopped by adding 100 μl / well of stop solution, and the plate was scanned at 450 nm using a microplate reader. ELISA-positive clones were defined as those showing an ELISA signal at least three times stronger on the antigen-coated plate than on the BSA-coated plate. In parallel, the genetic diversity of the ELISA-positive clones was determined using DNA sequencing, and phages with different amino acid sequences of the VHH were considered unique clones. In total, 76 unique clones with different CDR sequences were identified as positive in target binding assays using phage ELISA, and 37 were selected for expression and purification in HEK293 cells. [Table 2]

[0283] The properdin-binding VHH sequences were as follows: >SLN7150 (SEQ ID NO: 60); SLN12066 (SEQ ID NO: 61); SLN12067 (SEQ ID NO: 62); >SLN12068 (SEQ ID NO: 63); SLN12069 (SEQ ID NO: 64); SLN12070 (SEQ ID NO: 65); >SLN7160 (SEQ ID NO: 66); SLN12075 (SEQ ID NO: 67); SLN12076 (SEQ ID NO: 68); >SLN12077 (SEQ ID NO: 69); SLN7162 (SEQ ID NO: 70); SLN12078 (SEQ ID NO: 71); >SLN12079 (SEQ ID NO: 72); SLN12080 (SEQ ID NO: 73); SLN12081 (SEQ ID NO: 74); >SLN12036 (SEQ ID NO: 75); SLN12082 (SEQ ID NO: 76); SLN12083 (SEQ ID NO: 77); >SLN12084 (SEQ ID NO: 78); SLN12085 (SEQ ID NO: 79); SLN12086 (SEQ ID NO: 80); >SLN7151 (SEQ ID NO: 83); SLN7152 (SEQ ID NO: 84); SLN7153 (SEQ ID NO: 85); >SLN7154 (SEQ ID NO: 86); SLN7155 (SEQ ID NO: 87); SLN7156 (SEQ ID NO: 88); >SLN7159 (SEQ ID NO: 89); SLN7161 (SEQ ID NO: 90); SLN12027 (SEQ ID NO: 91); >SLN12030 (SEQ ID NO: 92); SLN12039 (SEQ ID NO: 93); SLN12041 (SEQ ID NO: 94); >SLN12042 (SEQ ID NO: 95); SLN12044 (SEQ ID NO: 96); SLN12045 (SEQ ID NO: 97).

[0284] Example 2 Identification of properdin-binding antibodies with inhibitory effects on complement activation via the alternative pathway 2.1 Expression and purification of VHH-FC Unique VHH clones were selected for subcloning to generate recombinant plasmids for producing VHH-FC proteins using degenerate primers (forward: SEQ ID NO: 111, reverse: SEQ ID NO: 112). After confirming the DNA sequence by DNA sequencing, recombinant plasmids were prepared and the fusion proteins were expressed and purified by following standard protocols.

[0285] To express the recombinant VHH-Fc protein, 100 ml of Expi293F™ cells were cultured in OPM-CD05 Medium (OPM, Cat. No. 81075-001) to produce approximately 3×10 cells with a viability of over 95%. 6 A cell density of 1000 viable cells / ml was reached. The plasmid was diluted in OPM-CD05 Medium to a concentration of 1.5 μg / ml in a total volume of 5 ml. The transfection reagent PEI was diluted to the same volume of 5 ml with OPM-CD05 Medium so that when the diluted DNA and PEI were mixed together, the DNA:PEI ratio was 1:4 (m / m). After 15 minutes of incubation at room temperature, the DNA / PEI complex was added onto the prepared Expi293F™ cells by gentle swirling. The cell culture was then placed in an incubator at 37°C with orbital shaking, 80% or greater relative humidity, and 5% CO2. Twenty-four hours after transfection, 5% peptone (1 mg / ml) and 2% glucose (330 g / l) were slowly added to the culture. After several days of cultivation, the cell culture supernatant was collected by sequential centrifugation at 1,200 rpm for 10 min and 3,900 rpm for 20 min before being used for protein A purification.

[0286] VHH-Fc was purified using Protein A (BIOON, HZ1011-2). 1 ml of Protein A slurry was loaded onto a 20 ml column (G-bios, C006197-0025). After equilibrating the column with 10 column volumes (CV) of PBS, the cell culture supernatant prepared as described above was loaded and allowed to pass through the Protein A column twice by gravity. After washing the column with 10 CV of PBS, the VHH-Fc protein was eluted using 10 ml of 0.1 M glycine-HCl buffer (pH 3.0). The eluted protein was neutralized with 100 μl of 1 M (pH 8.5) Tris-HCl buffer, and the pH was adjusted to 7.4. The Protein A affinity column was regenerated by sequentially washing with PBS, ddH2O, and 20% ethanol, and then stored. The eluted protein was desalted through an Amicon UltraCel 30K centrifuge (Milipore, UFC903016). Briefly, the eluted protein was diluted with 10 ml of PBS and concentrated to 1.5 ml by centrifugation at least three times. The final protein solution was made up to less than 1 ml in PBS and filtered through a 0.22 μm filter.

[0287] The purity of VHH-Fc was analyzed by SDS-PAGE. Briefly, 2 µg of protein in 4x LDS Sample buffer was loaded and analyzed using a SurePAGE gel in Tris-MOPS SDS buffer (Genscript, M00138) at a constant voltage of 160 V for 50 minutes. Proteins were visualized with Coomassie stain (TIANGEN, catalog no. PA101) according to the manufacturer's instructions. Purified proteins were analyzed using a 4-12% gradient SDS-PAGE gel under non-reducing or reducing conditions (Figure 2).

[0288] 2.2 Elisa join For binding ELISA, 96-well immunoplates were coated with 100 μl / well of 1 μg / ml streptavidin and incubated overnight at 4°C. The wells were washed three times with PBST and blocked with 200 μl of 1% BSA / PBS for 1 hour at room temperature. After washing three times with PBST, 100 μl / well of human properdin-biotin, mouse properdin-biotin, or cynomolgus monkey properdin-biotin (5 μg / ml) was added and incubated for 1 hour at room temperature. The plates were washed three times with PBST, and 100 μl / well of VHH-Fc diluted 5-fold starting at 10 μg / ml was added and incubated for 1 hour at room temperature. The plates were washed three times with PBST, and 100 μl of goat anti-human Fc-HRP (Sigma, A0170) diluted 1 / 5000 in 1% BSA / PBST was added to each well and incubated for 1 hour at room temperature. The plates were then washed as above, and 100 μl of TMB substrate was added and incubated for 15 minutes at room temperature. The reaction was stopped by adding 100 μl / well of stop solution, and the plates were read at 450 nm using a microplate reader. Thirty-seven recombinant VHH-Fc clones exhibited human properdin binding activity (FIG. 3A), cynomolgus monkey properdin binding activity (FIG. 3B), and mouse properdin binding activity (FIG. 3C), and all clones exhibited cross-binding and affinity-matched activity.

[0289] 2.3 AP activity For the human alternative pathway experiments, all test samples were diluted with PBS and added in duplicate to a U-bottom 96-well microtiter plate (50 μl / well). Simultaneously, human complement-preserved serum (Quidel, A113) was diluted to 20% vol / vol with GVBS-EGTA (1×AP buffer: 10 mM Mg / EGTA, 0.1% gelatin, 145 mM NaCl, 2.5 mM sodium barbital, pH 7.4), incubated on ice for 30 minutes, and added to the same 96-well column (50 μl / well) so that the final concentration of human serum in each well was 10%. Then, prepared rabbit red blood cells (4×10 8 / ml) was washed three times with 1 ml of 1x AP buffer and 5x10 7The rabbit erythrocytes (2.5 × 10 6 A 50 μl aliquot of the rabbit red blood cells (containing 50 μl of PBS + 50 μl of 1×AP buffer only (negative control) was added to the above plates, mixed thoroughly, and incubated at 37°C for 30 minutes. Each plate contained two wells of 50 μl of identically prepared rabbit red blood cells incubated with 50 μl of PBS + 50 μl of 1×AP buffer only (negative control) as a control for spontaneous hemolysis, two wells containing 100 μl of ddH2O to serve as a control for 100% lysis, and two wells containing 10 mM EDTA (Thermo 15575-038) as a serum blank control. After incubation, the plates were then centrifuged at 600 rpm for 2 minutes, and 100 μl of the supernatant was transferred to a new flat-bottom 96-well plate. Hemoglobin release was determined at OD 405 nm using a microplate reader, and the percent hemolysis was determined using the following formula: Percent hemolysis (%): 100 x (OD of sample - OD of EDTA) / (OD of 100% lysis - OD of negative control)

[0290] For the mouse alternative pathway experiments, the process was essentially the same as above, except that the final concentration of mouse serum was 30% and the final concentration of human serum was 10%, and the incubation time was 30 min instead of 1 h in the mouse alternative pathway assay (Figure 4). Some clones showed complement inhibitory activity in human (Figure 4A) and mouse (Figure 4B) serum at 500 nM.

[0291] Example 3. Biparatopic engineering of properdin-binding antibody fragments 3.1 Epitope binning assay using full-length human / mouse properdin A 96-well immunoplate was coated with 100 μl / well of 5 μg / ml VHH-Fc fusion protein and incubated overnight at 4°C. The wells were washed three times with PBST and blocked with 200 μl of 1% BSA / PBS for 1 hour at room temperature. 60 μl of human properdin-biotin or mouse properdin-biotin (0.5 μg / ml) and 60 μl of VHH-Fc fusion protein (20 μg / ml) were premixed, and 100 μl was transferred to each VHH-Fc-coated, BSA-blocked well, and incubation continued for 1 hour at room temperature. The plate was washed three times with PBST, and 100 μl of SA-HRP (Sigma, S5512) diluted 1 / 5000 in 1% BSA / PBST was added to each well and incubated for 1 hour at room temperature. The plate was then washed as described above, and 100 μl of TMB substrate was added and incubated for 15 minutes at room temperature. The reaction was stopped by adding 100 μl / well of stop solution, and the plate was read at 450 nm using a microplate reader. VHHs with competitive target binding ability were grouped into the same bin. The results showed that SLN7150, SLN12036, and SLN12042 belonged to bin #1; SLN7150, SLN12036, and SLN12027 belonged to bin #2; SLN7150, SLN12036, SLN12041, SLN12044, and SLN12045 belonged to bin #3; SLN7160 and SLN7155 belonged to bin #4; SLN7162 may have a different epitope; and most of the other VHH-Fc belonged to bin #5 (Figure 5).

[0292] 3.2 Preparation of biparatopic VHHs VHHs from different bins were combined with G4S linkers to generate biparatopic Fc-VHH-VHH fusion proteins, as listed in Table 3. Plasmid construction and protein purification procedures can be found above. SDS-PAGE analysis and characterization results are shown in Figure 6. [Table 3]

[0293] 3.3 Characteristics of biparatopic VHH In target binding assays, the 10 biparatopic VHHs showed negligible effects compared to the effects of single VHHs in either humans (Figures 7A and 7C) or mice (Figures 7B and 7D), as shown in Figure 8. In contrast, in alternative pathway activity assays, the biparatopic VHHs exhibited superior complement inhibitory activity to monovalent VHHs, especially in human serum, as shown in Figure 8.

[0294] Example 4 Humanization of properdin-binding VHH VHH humanization was performed using standard procedures of CDR grafting and structural refinement. For the humanization design, recombinant DNA constructs were generated to generate the above-mentioned recombinant constructs. Humanized sequences with comparable or superior affinity to properdin as the original VHH, with acceptable expression and stability levels, were selected for further development. Figure 9 shows that the humanized VHH variant SLN7160 has a similar profile to the parent VHH in terms of its binding to human (Figure 9A) and mouse properdin (Figure 9B). The VHH variant SLN12036 has a similar profile to the parent VHH in terms of its binding to human (Figure 9C) and mouse properdin (Figure 9D). On the other hand, SLN7160 had negligible complement inhibitory activity in human (Figure 9E) and mouse serum (Figure 9F), and it is noteworthy that humanized SLN12083 exhibited superior human alternative pathway activity to the original VHH SLN12036 (Figures 9G and 9H).

[0295] Example 5 Epitope mapping assay using truncated human properdin A 96-well immunoplate was coated with 100 μl / well of 1 μg / ml streptavidin and incubated overnight at 4°C. The wells were washed three times with PBST and blocked with 200 μl of 1% BSA / PBS for 1 hour at room temperature. After washing three times with PBST, 100 μl / well of a truncated variant of human properdin-biotin (50 μg / ml) was added and incubated for 1 hour at room temperature. The plate was washed three times with PBST, and 100 μl / well of 10 μg / ml VHH-Fc was added and incubated for 1 hour at room temperature. The plate was washed three times with PBST, and 100 μl of goat anti-human Fc-HRP (Sigma, A0170) diluted 1 / 5000 in 1% BSA / PBST was added to each well and incubated for 1 hour at room temperature. The plate was then washed as described above, and 100 μl of TMB substrate was added and incubated for 15 minutes at room temperature. The reaction was stopped by adding 100 μl / well of stop solution, and the plate was read at 450 nm using a microplate reader. The results are shown in Figure 10, which shows the binding of SLN12068, SLN12075, and SLN12083 to distinct thrombospondin repeats (TSRs) for TSR0, TSR1, TSR2, TSR3, TSR4, TSR5, TSR6, TSR0-6, TSR1-6, TSR2-6, TSR3-6, TSR4-6, TSR5-6, and TSR6, respectively. The biologically active TSR5-6 and TSR0 domains play a key role in properdin binding to SLN12068, SLN12075, and SLN12083.

[0296] Example 6. Effect of properdin inhibitors on the interaction between C3 and properdin 6.1 C3 binding assay A Maxisorp 96-well plate was coated with 100 μl / well of 2 μg / ml human C3 (Sigma, C2910-.1MG) in PBS, pH 7.4, and left overnight at 4°C. After washing three times with PBST, the wells were blocked with 2% BSA in PBS for 1 hour at 37°C. Serial three-fold dilutions of biotin-human properdin (starting at 90 μg / ml) (100 μl / well) were added to the wells and incubated for 1 hour at 37°C. The wells were washed three times with PBST, and HRP-labeled streptavidin (1 / 5000 dilution) (Sigma, s5512) was added to the wells. The plate was then incubated for 1 hour at 37°C. The plate was then washed as above, and 100 μl of TMB substrate was added and incubated for 15 minutes at room temperature. The reaction was stopped by adding 100 μl / well of stop solution and the plate was read at 450 nm by a microplate reader.

[0297] 6.2 Competitive Binding Assays A Maxisorp 96-well plate was coated with C3 (see Binding Assay), blocked with 1% BSA in PBST, and washed three times with PBST. 100 μl / well of a 5-fold serial dilution of FP inhibitor (starting at 50 nM) and 100 μl / well of a fixed amount of properdin (20 μg / ml) in PBS were added to each well and incubated at 37°C for 1 hour. The wells were washed three times again with PBST, and HRP-labeled streptavidin (1 / 5000 dilution) (Sigma, s5512) was added to the wells. The plate was then incubated at 37°C for 1 hour. The plate was then washed as above, and 100 μl of TMB substrate was added and incubated at room temperature for 15 minutes. The reaction was stopped by adding 100 μl / well of stop solution, and the plate was read at 450 nm using a microplate reader. As shown in Figure 11A, properdin bound to C3 in a dose-dependent manner. Furthermore, SLN12068, SLN12075, and SLN12083 also exhibited inhibitory activity against properdin binding to C3 (Figure 11B).

[0298] Example 7: Engineered Factor H to VHH fusion proteins as dual inhibitors of the alternative pathway 7.1 Biparatopic engineering of humanized VHH sequences Using the above procedure, biparatopic VHHs with humanized sequences via G4S linkers were generated. The purified proteins were analyzed by 4-12% SDS-PAGE under non-reducing and reducing conditions (Figure 12).

[0299] Target binding ELISAs shown in Figure 13 indicated that such humanized biparatopic VHHs had negligible effects on single VHHs by human properdin binding (Figure 13A) and mouse properdin binding assays (Figure 13B). Alternative pathway activity indicated that biparatopic SLN12140 AP activity was superior to that of single VHHs (Figures 14A and 14B).

[0300] 7.2 Fusion proteins of engineered VHH to factor H Truncated CFH (domains 1-4) was fused to the C-terminus of SLN12140 to form SLN7207, creating a bifunctional recombinant protein that inhibits complement activation. The results are shown in Figure 13. Panels (C) and (D) show the binding activity of SLN7207 and SLN12140 to human properdin-biotin and mouse properdin-biotin. Panels (C) and (D) of Figure 14 show the human and mouse alternative pathway activity of SLN12140 and SLN7207. Fusion of CFH (domains 1-4) at the C-terminus of SLN12140 had negligible effect on the efficacy of SLN12140 in human serum but increased biological activity by approximately threefold in mouse serum. In both cases, both SLN12140 and SLN7207 demonstrated alternative pathway inhibitory activity at the nM level in both humans and mice, with an IC50 of 17 nM in humans. Such functional proteins offer a potential therapeutic strategy for complement hyperactivation diseases.

[0301] Example 8 Pathway Selectivity and Cross-Species Activity of SLN12140 8.1 Pathway selectivity in complement inactivation 8.1.1 Complement inactivation in the alternative pathway For the alternative pathway assay, see 2.3 for procedures. The inhibition curves of the single VHHs SLN12075, SLN12083, and biparatopic SLN12140 demonstrated consistent alternative pathway complement inhibitory activity, with SLN12140 exhibiting superior activity compared to the single VHHs, with an IC50 of 17 nM. As a control, eculizumab (Targetmol, T9915), a recombinant humanized monoclonal antibody against complement protein C5, also exhibited inhibitory activity in the AP pathway, with an IC50 of 50 nM.

[0302] 8.1.2 Classical pathway complement inactivation For the classical pathway assay, all test samples were serially diluted 1:3 with PBS and added in duplicate to a U-bottom 96-well microtiter plate (50 μl / well). Human complement-preserved serum (Quidel A113) was diluted to 20% vol / vol with GVB2+ buffer (0.1% gelatin, 141 mM NaCl, 0.5 mM MgCl2, 0.15 mM CaCl2, 1.8 mM sodium barbital) (Comp Tech B100) and added to the same 96-well plate row (50 μl / well) so that the final concentration of human serum in each well was 10%. The plate was then incubated at room temperature for 30 minutes. Chicken red blood cells (1–4 × 10 cells) based on the sample were then added to the same 96-well plate. 8 ) were washed three times with 1 ml of GVBS2+ buffer and 1 × 10 8 The cells were resuspended in GVBS2+ buffer to a final concentration of 1 × 10 / ml. Subsequently, 1–6 ml of chicken erythrocytes were sensitized by adding 3% anti-chicken erythrocyte polyclonal antibody (Rockland, 103-4139) and incubated on ice for 15 min with frequent mixing. The cells were then washed twice with 1 ml of GVBS2+ buffer and diluted to 1 × 10 8 Chicken erythrocytes (3 × 10 6A 30 μl aliquot of GVBS2+ cells was added to the plate as above, mixed thoroughly, and incubated at 37°C for 30 minutes. Each plate then contained two wells of 50 μl of identically prepared chicken red blood cells (one incubated with 50 μl of PBS + 50 μl of GVBS2+ buffer only (negative control) as a control for spontaneous hemolysis), two wells containing 10 mM EDTA (Thermo 15575-038) as a serum blank, and two wells of normal NHS as 100% lysis. The plates were then centrifuged at 600 rpm for 2 minutes, and 100 μl of the supernatant was transferred to a new flat-bottom 96-well plate. Hemoglobin release was determined at OD405nm using a microplate reader, and percent hemolysis was determined using the following formula: Hemolysis (%): 100 x (OD sample - OD EDTA blank) / (OD 100% lysis - OD EDTA blank)

[0303] As shown in Figure 15B, neither single nor biparatopic VHHs of alternative pathway-specific antibodies showed inhibitory activity in the classical pathway. As a control, eculizumab (Targetmol, T9915), a recombinant humanized monoclonal antibody against complement protein C5, also showed inhibitory activity in the CP pathway with an IC50 of 57 nM.

[0304] 8.1.3 Complement inactivation in the lectin pathway For the lectin pathway assay: A 0.3 ml aliquot of mannan solution (0.5 mg / ml) was mixed with an equal volume of CrCl solution (0.5 mg / ml) (Sigma 27096-100G-F, lot no. BCCB5331) and an equal volume of chicken red blood cell suspension (1 × 10 95 × 10 cells) were added, and the mixture was incubated at 25°C for 15 minutes with occasional mixing. Then, mannan (ME) (Sigma M7604-100MG, Lot No. SLOF4977)-coated erythrocytes were washed three times by centrifugation in GVBS2+ (Gelatin-Veronal Buffered Saline, 5 mM Veronal buffer, pH 7.4, containing 0.145 M NaCl, 0.1% gelatin, 0.15 M CaCl, and 0.5 mM MgCl) (Comp Tech, B100). 7 The cells were resuspended in GVBS2+ to a final concentration of 100 cells / ml and stored on ice. All test samples were serially diluted 1:3 in PBS (from 500 nM to 0.2 nM) and added in duplicate to a U-bottom 96-well microtiter plate (50 μl / well). Human complement-preserved serum was diluted to 20% vol / vol in GVBS2+ and added to the same 96-well plate row (50 μl / well) so that the final concentration of human serum in each well was 10%. 100 μl of ddH2O or serum alone and 50 μl of PBS + 50 μl of GVBS2+ were used as 100% lysis and 0% controls, respectively, and 10 mM EDTA was used as a serum blank. Chicken red blood cells (2.5 × 10 6 A 50 μl aliquot of the culture medium (cells) was added to the plate as above, mixed thoroughly, and incubated at 37°C for 60 minutes. The plate was then centrifuged at 1,000g for 2 minutes, and 100 μl of the supernatant was transferred to a new flat-bottom 96-well plate. Hemoglobin release was determined at OD405nm using a microplate reader, and percent hemolysis was determined using the following formula: Percent hemolysis (%): 100 x (OD of sample - OD of EDTA) / (OD of 100% lysis - OD of EDTA)

[0305] The inhibition curves of VHHs are shown in Figure 15C. Neither monotopic nor biparatopic VHHs exhibited inhibitory activity in the lectin pathway, demonstrating their specificity in the alternative pathway. As a control, eculizumab (Targetmol, T9915), a recombinant humanized monoclonal antibody against complement protein C5, also exhibited inhibitory activity in the LP pathway with an IC50 of 45 nM.

[0306] 8.2 Human, cynomolgus monkey, mouse, and rat cross-species activity of SLN12140 For the alternative pathway assay, see section 2.3 for the procedure. The VHH inhibition curves shown in panels (A), (B), (C), and (D) of Figure 16 and Figure 15 demonstrate the alternative pathway activity of SLN12075, SLN12083, and SLN12140 in humans, cynomolgus monkeys, mice, and rats, respectively. SLN12140 exhibits excellent cross-species complement inhibitory activity in the AP-specific pathway, including humans, mice, monkeys, and rats. Furthermore, all of these VHHs exhibited bivalent activity, which is higher than that of single VHHs.

[0307] Example 9 Serum stability of SLN12140 and its PK profile in mice 9.1 Serum stability of SLN12140 in mouse plasma Test compound SLN12140 was prepared at a concentration of 10 μg / mL using C57BL / 6 mouse plasma, human plasma, and protein formulation buffer. Samples were prepared and stored at -80°C. Samples were then incubated at 37°C under constant temperature and humidified conditions and collected at each time point (96, 72, 48, 24, 6, 2, and 0 hours). Supernatants from each sample were obtained and analyzed for quantification by analytical ELISA, as described below. All assays were performed in triplicate. Figure 17 shows buffer stability and plasma stability in human and mouse plasma. SLN12140 (SEQ ID NO: 81) maintained stable concentrations in the formulation buffer within 96 hours and maintained stable concentrations up to 96 hours, with 70% of SLN12140 still detectable.

[0308] 9.2 Establishment of sample analysis methods 9.2.1 Total Drug PK Assay Development and QC Validation Goat F(ab')2 anti-human IgG-Fc (Abeam, catalog no. Ab98587) was coated onto a 96-well enzyme-binding plate, washed three times with PBST (Tween-20, 0.1%), and blocked for 1 hour with 200 μl of 1% BSA in PBST at 37°C. After washing three times with PBST, serial dilutions of SLN12140 standard, serum samples, and quality control samples in PBST containing 1% BSA were added. After incubation for 1 hour at 37°C, the cells were washed three times with PBST, and horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Fc-specific) antibody (Sigma, catalog no. A0170) was added and incubated for 1 hour at 37°C. After washing three times with PBST, 100 μl of TMB substrate solution was added and incubated for 15 minutes. The absorbance was read at 450 nm after adding stop solution. The precision and accuracy of the standard curve and quality control materials were verified by SoftMax software, and sample concentrations were calculated. Validation of the PK method using three standard concentrations (high, medium, and low) demonstrated the precision (CV%<20%) and accuracy (RE%+ / -25%) of this method meets the sample testing requirements.

[0309] 9.2.2 Total mFP Assay Development and QC Validation SLN12039 (SEQ ID NO: 93) was coated onto a 96-well enzyme-binding plate, washed three times with PBST (Tween-20, 0.1%), and blocked with 200 μl of 1% BSA in PBST at 37° C. for 1 hour. After washing three times with PBST, serially diluted mFP (Linno) in 1% BSA in PBST was added and incubated at 37° C. for 1 hour. After washing three times with PBST, biotinylated SLN12030 antibody (Linno) was added and incubated at 37° C. for 1 hour. After washing three times with PBST, horseradish peroxidase (HRP)-labeled streptavidin antibody (Sigma, catalog no. S5512) was added and incubated at 37° C. for 1 hour. After washing three times with PBST, 100 μl of TMB substrate solution was added and incubated for 15 minutes. After adding stop solution, absorbance was read at 450 nm. The precision and accuracy of the standard curve and quality control materials were verified by SoftMax software, and sample concentrations were calculated. Validation of the PK method using three standard concentrations (high, medium, and low) demonstrated the precision (CV%<20%) and accuracy (RE%+ / -25%) of this method meets the sample testing requirements.

[0310] 9.3 Subcutaneous and intravenous single-dose pharmacokinetic studies in mice A PK study was conducted in 8-week-old male C57BL / 6 mice. The study consisted of two groups, four animals per group, each receiving a single dose of 2.21 mpg of SLN12140 via subcutaneous or intravenous injection. Serum samples were collected at pre-dose (D0), 0.5 hours, 2 hours, 6 hours, 24 hours (D1), 48 hours (D2), 72 hours (D3), 120 hours (D5), 144 hours (D7), and 240 hours (D10). Serum was separated from the serum samples and the PK method described above was used for quantitative analysis of the drug in serum. PK parameters were calculated using PK Solver software. As shown in Figure 18, the terminal elimination half-life of SLN12140 in mice after iv and sc administration was 2.5 days. SLN12140 was rapidly absorbed after subcutaneous administration, with the AUC 0~tThe bioavailability based on SLN12140 (SEQ ID NO: 81) was 40%. These results demonstrated that SLN12140 (SEQ ID NO: 81) exhibits an adequate half-life and favorable bioavailability.

[0311] 9.4 Subcutaneous dose-dependent pharmacokinetic studies in mice A dose-dependent pharmacokinetic (PK) study was conducted in 8-week-old male C57BL / 6 mice. The study consisted of three groups of five animals, each receiving a single subcutaneous dose of 3, 10, and 30 mpk, respectively. Blood samples were collected pre-dose (0 h) and at eight post-dose sampling times: 2 h, 6 h, 24 h (D1), 48 h (D2), 72 h (D3), 120 h (D5), 144 h (D7), and 240 h (D10). Once collected, blood samples were centrifuged at 1500 g for 10 min at 4°C and stored at -80°C prior to analysis. The PK method described above was used for quantitative analysis of the drug in serum, and PK parameters were calculated using PK Solver software. As shown in Figures 19A-19C, the exposure of SLN12140 (SEQ ID NO: 81) in terms of AUC increased more than linearly with dose throughout the dose range of 3, 10, and 30 mpk. The concentration of mFP also maintained a highly consistent correlation with drug concentration. Low levels of FP concentration could be maintained for 48 hours at 3 mpk, 120 hours at 10 mpk, and 168 hours at 30 mpk, respectively, demonstrating excellent target inhibitory activity and an excellent safety profile.

[0312] 9.5 Multiple-dose pharmacokinetic studies in mice A PK study was conducted in 8-week-old male C57BL / 6 mice. A subcutaneous dose of 30 mpk SLN12140 was administered once every 7 days for four consecutive doses. Blood samples were collected before each dose (time 0) and 24 hours after each dose. After collection, the blood samples were centrifuged at 1500 g for 10 minutes at 4°C after 2 hours of natural coagulation at room temperature and stored at -80°C before analysis. The above-described PK method was used for quantitative analysis of the drug in serum, and PK parameters were calculated using PK Solver software. Simultaneously, AP activity was measured at different sample collection time points using the above-described method. As shown in Figure 20, FP concentrations significantly decreased 24 hours after administration and remained below physiological levels 7 days after administration, and no risk of increased FP was observed after multiple doses. The AP activity detected by the erythrocyte lysis assay was highly consistent with the FP concentration, and as the FP concentration decreased, the AP activity significantly decreased, and SLN12140 (SEQ ID NO: 81) showed excellent and sustained AP pathway inhibitory activity after multiple administrations.

[0313] 9.6 Multiple-dose pharmacokinetic study in hCD89 Tg mice PK studies were performed in 13-14 week-old male hCD89 Tg C57BL / 6 mice. hCD89 transgenic (Tg) mice express human CD89 on macrophages / monocytes, and the central role of soluble CD89 in the pathogenesis of IgAN has been documented. A subcutaneous dose of 30 mpk SLN12140 was administered once every 7 days for seven consecutive doses. Blood samples were collected before each dose (time 0) and 24 hours after each dose. After collection, the blood samples were centrifuged at 1500 g for 10 minutes at 4°C after 2 hours of natural coagulation at room temperature and stored at -80°C before analysis. The PK method described above was used for quantitative analysis of the drug in serum, and PK parameters were calculated using PK Solver software. Simultaneously, AP activity was measured at different sample collection time points using the above method. As shown in Figure 21, after seven consecutive administrations, SLN12140 (SEQ ID NO: 81) serum concentration exhibits sustained and stable pharmacokinetic properties, and weekly administration can continuously reduce target concentration at a low level, and according to blood concentration analysis and target concentration analysis, there is no risk of immune complex formation. At the same time, the mice are observed to be in good overall health.

[0314] Example 10 SLN12140 Fusion with VEGF Inhibitors 10.1 Production of recombinant anti-complement protein VHH-G4Fc, anti-VEGF protein VHH-G4Fc, and bispecific proteins inhibiting both the complement pathway and the VEGF pathway Nucleotide sequences corresponding to the amino acids of a bispecific fusion protein (SLN8284, SEQ ID NO: 114) with a VEGF inhibitory domain fused to the N-terminus and a complement inhibitory domain fused to the C-terminus of the Fc domain, and a complement inhibitory protein (SLN12140, SEQ ID NO: 81) or a VEGF inhibitory protein (SLN6073, SEQ ID NO: 113) with a G4Fc domain fused to the C-terminus were constructed in the plasmids pCDNA3.4 or pCP. After confirming these plasmid sequences by sequencing, small-scale production of recombinant proteins was performed using transient transfection of HEK293 cells with the recombinant plasmids using lipofectamine or PEI as transfection reagents. Cultures were grown for 5 to 7 days in shake flasks at scales ranging from 30 ml to 100 ml. Cells were removed by centrifugation, and the culture supernatant was used for protein purification by protein A chromatography. The purified proteins were quantified by Coomassie Plus (Bradford) Assay Kit and analyzed on a 4-12% gradient SDS-PAGE gel under non-reducing (lanes 1, 3, and 5) or reducing (lanes 2, 4, and 6) conditions (Figure 22).

[0315] 10.2 In vitro binding of SLN8284, SLN12140, and SLN6073 to properdin from different species ELISA was performed to determine whether proteins directly bind to properdin. Briefly, the wells of a 96-well ELISA plate were coated with streptavidin (1 μg / ml in CBS, 100 μl / well) and incubated overnight at 4 ° C. After three washes with PBST, the plate was blocked with 1% BSA in PBST for 1 hour at room temperature. After three washes with PBST, biotinylated human, cynomolgus monkey, or mouse properdin protein (2 μg / ml in BSA, 100 μL / well) was added and incubated for 1 hour at room temperature. After three washes with PBST, purified SLN8284 (SEQ ID NO: 114), SLN12140 (SEQ ID NO: 81), and SLN6073 (SEQ ID NO: 113) (serial dilutions in BSA starting from 50 nM) were added and incubated for 1 hour at room temperature. After washing three times with PBST, anti-human IGG (FC specific) peroxide (Sigma catalog number A0170-1ML) was added to each well for 1 hour of incubation. After washing three times with PBST, a stop reagent for TMB substrate was added to each well, and the OD absorbance at 450 nm was measured. Data was analyzed by sigmoidal curve fitting using GraphPad Prism 8.0. As shown in Figure 23, SLN8284 and 12140 showed strong binding to human, cynomolgus monkey, or mouse properdin, respectively, while SLN6073 (SEQ ID NO: 113) failed to bind to human, cynomolgus monkey, or mouse properdin (Figures 23A, 23B, and 23C).

[0316] 10.3 Inhibition of the alternative complement pathway by SLN8284, SLN12140, and SLN6073 In contrast to the classical and lectin complement pathways, which require both magnesium and calcium ions for activation, activation of the alternative complement pathway requires only magnesium ions. Therefore, to quantify alternative complement activity in the presence of the fusion protein, we modified the above assay so that rabbit erythrocytes (ER) were incubated with serum, 0–1500 nM fusion protein, 5 mM Mg2+, and 5 mM EGTA, which preferentially chelates calcium ions.

[0317] For this assay, all fusion protein samples were serially diluted in PBS and added to a U-bottom 96-well microtiter plate. Normal human serum (Complement Technology catalog number NHS A113) or complement-preserved mouse serum was diluted to the correct concentration in assay buffer AP (20 mM Mg / EGTA in GVB0) and incubated on ice for 30 minutes. Then, 5 × 10 fusion protein samples were added to the assay buffer. 7 Rabbit red blood cells / ml (SenBeiJia Biological Technology Co., Ltd. Catalog No. SBJ-RBC-RAB003-10mL) were prepared. SLN8284, SLN6073, and SLN12140 at concentrations of 0–1500 nM were added to 2.5 × 10 6 Inhibition of the alternative complement pathway was initiated by mixing EA with dilutions of normal human or mouse serum containing 1000 μg of rabbit erythrocytes at 37°C for 30 minutes or 1 hour. Hemolysis of EA was then assayed by measuring absorbance at OD 405 nm. Data were analyzed by sigmoidal curve fitting using Prism 8. Analysis of the percentage of hemolysis of EA in the presence of the fusion proteins demonstrated that SLN8284 exhibited high inhibitory activity with an IC50 of 5.166 nM or 63.4 nM (Figures 24A and 24B, filled squares). SLN12140 (SEQ ID NO: 81) showed improved inhibitory activity (2.3- or 1.7-fold) with an IC50 of 2.293 nM or 37.83 nM (Figures 24A and 24B, inverted triangles). SLN6073 (SEQ ID NO: 113) fails to show an inhibitory effect (FIGS. 24A, 24B, filled circles).

[0318] 10.4 In vitro binding of SLN8284, SLN12140, and SLN6073 to human and mouse VEGF ELISA was performed to determine whether the protein directly binds to VEGF. Briefly, the wells of a 96-well ELISA plate were coated with streptavidin (1 μg / mL in CBS, 100 μL / well) and incubated overnight at 4 ° C. After three washes with PBST, the plate was blocked with 1% BSA in PBST for 1 hour at room temperature. After three washes with PBST, biotinylated human or mouse VEGF protein (0.5 μg / mL in BSA, 100 μL / well) was added and incubated for 1 hour at room temperature. After three washes with PBST, purified SLN8284 (SEQ ID NO: 114), SLN12140 (SEQ ID NO: 81), or SLN6073 (SEQ ID NO: 113) protein (serial dilutions in BSA starting from 50 nM) was added and incubated for 1 hour at room temperature. After washing three times with PBST, anti-human IGG (FC specific) peroxide (Sigma catalog number A0170-1ML) was added to each well for 1 hour of incubation. After washing three times with PBST, a stop reagent for TMB substrate was added to each well, and the OD absorbance at 450 nm was measured. Data was analyzed by sigmoidal curve fitting using GraphPad Prism 8.0. As shown in Figure 25, SLN8284 (SEQ ID NO: 114) and SLN6073 (SEQ ID NO: 113) showed strong binding to human or mouse VEGF, respectively, while SLN12140 (SEQ ID NO: 81) failed to show binding to human, cynomolgus monkey, or mouse properdin (Figures 25A and 25B). (Human VEGF121: Accession No.: P15692-9; VEGF120: Accession No.: Q00731-3)

[0319] 10.5 Blockade of the interaction between VEGFA and VEGFR2 by SLN8284, SLN6073, and SLN12140 ELISA was performed to determine whether proteins could block the interaction between VEGFA and VEGFR2. Briefly, wells of a 96-well ELISA plate were coated with streptavidin (1 μg / ml in CBS, 100 μl / well) and incubated overnight at 4°C. After three washes with PBST, the plate was blocked with 1% BSA in PBST for 1 hour at room temperature. After three washes with PBST, biotinylated human or mouse VEGF protein (0.07 μg / ml in BSA) was added and incubated for 1 hour at room temperature. After three washes with PBST, human or mouse VEGFR1 (0.14 μg / ml or 0.07 μg / ml in BSA) mixed with purified SLN8284, SLN12140, or SLN6073 (serial dilutions in BSA starting at 100 nM) was added and incubated for 1 hour at room temperature. After washing three times with PBST, anti-mouse IgG Fc-HRP (Abcam, lot number GR3396448-2) was added to each well for 1 hour of incubation. After washing three times with PBST, a stop reagent for TMB substrate was added to each well, and the OD absorbance at 450 nm was measured. Data were analyzed by sigmoidal curve fitting using GraphPad Prism 8.0. As shown in Figure 26, SLN8284 and 6073 can effectively block the interaction between human or mouse VEGFA and human or mouse VEGFR2. SLN12140 cannot block the interaction between human or mouse VEGFA and human or mouse VEGFR2 (Figures 26A and 26B). (hVEGFR2: Accession No.: P35968-1; mVEGFR2: Accession No.: P35918-1)

[0320] 10.6 Inhibition of VEGF-dependent HUVEC proliferation assay by SLN8284, SLN12140, and SLN6073 All proteins were tested for their ability to inhibit the VEGF signaling pathway (e.g., inhibit VEGF activity) in a cell-based assay. For example, SLN8284 was tested for its ability to inhibit the VEGF signaling pathway (e.g., inhibit VEGF activity) in this cell-based assay and compared to the VEGF inhibitory activity of SLN6073 and SLN12140. Human umbilical vein endothelial cells are often used to demonstrate VEGF-dependent cell proliferation, which can be inhibited by binding of fusion proteins to VEGF. In this assay, HUVECs were maintained in endothelial cell growth medium containing 1% FBS. 3,000 HUVEC cells per well were seeded in complete medium (1% FBS) in a 96-well flat-bottom microtiter plate and cultured overnight. The medium was discarded from the cell plate, and 100 μL of various concentrations of fusion protein (serial dilutions starting from 1,000 nM in PBS) mixed with 50 ng / mL VEGF-A was added to each well and incubated at 37°C for 72 hours. Cell proliferation was assayed by adding 10 μL of CCK-8 (Dojindo, Inc.) to each well and incubating at 37° C. for 2.5 hours. Cell proliferation was measured at OD absorbance of 450 nm. The results showed that SLN8284 and SLN6073 significantly inhibited VEGF-induced HUVEC proliferation compared to the control (SLN12140), and the inhibitory effect of SLN8284 was similar to that of SLN6073 ( FIG. 27 ). SLN8284 (SEQ ID NO: 114) SLN6073 (SEQ ID NO: 113)

[0321] Example 11 SLN12140 fusion with hTf-binding VHH (9056VHH) 11.1 Production of anti-CFP recombinant antibodies with / without the 9056 VHH domain Fusion proteins SLN12147 (12075-(G4S)3-12083-(G4S)3-His) and SLN12140 (12075-(G4S)3-12083-(G4S)3-Fc), SLN12149 (12075-(G4S)3-12083-(G4S)3-9056-G4S-His) and SLN12150 (12075-(G4S)3-12083-(G4S)3-Fc-(G4S)3-9056) were constructed from SLN12147 and SLN12140. Small-scale production of the recombinant proteins was performed using transient transfection of HEK293 cells with the recombinant plasmids using PEI as the transfection reagent. Cultures were grown in 100 ml shake flasks for 5–7 days. Cells were removed by centrifugation, and the culture supernatant was used for protein purification with Ni-NTA or Protein A Sepharose. Purified proteins were analyzed using 4-12% gradient SDS-PAGE gels under non-reducing or reducing conditions (Figure 28).

[0322] 11.2 CFP-binding assays using recombinant antibodies in different species To test the binding activity of recombinant antibodies against CFP, 96-well microplates were coated with streptavidin (1 μg / ml in PBS, 100 μl / well) and incubated overnight at 4°C. After three washes with PBST, the plates were blocked with 1% BSA in PBST for 1 hour at room temperature. After three washes with PBST, biotinylated human / rhesus / mouse CFP (10 / 5 / 2 μg / ml in 1% BSA, 100 μl / well) was added and incubated for 1 hour at room temperature. After three washes with PBST, recombinant antibodies (50 nM, 5-fold dilutions, serially diluted in 1% BSA, 100 μl / well) were added and incubated for 1 hour at room temperature. After sequential incubation with SLN12143 (1 μg / ml, conjugated 12075 / 12083 humanized framework) and secondary anti-mFc-HRP, plates were washed three times with PBST and incubated with substrate solution and stop solution as described above for the ELISA (Figure 29).

[0323] 11.3 Human Transferrin Binding Assay Using Recombinant Antibodies To test the binding activity of the recombinant antibodies to human transferrin, 96-well microplates were coated with streptavidin (1 μg / ml in PBS, 100 μl / well) and incubated overnight at 4°C. After three washes with PBST, the plates were blocked with 1% BSA in PBST for 1 hour at room temperature. After three washes with PBST, the recombinant antibodies (1000 nM, 5-fold dilutions, serially diluted with 1% BSA) were premixed with biotinylated human transferrin (2 μg / ml in 1% BSA), and the mixture (100 μl / well) was added to the plate and incubated for 1 hour at room temperature. Secondary anti-hFc-HRP / anti-6xhis-HRP was then sequentially incubated, after which the plates were washed three times with PBST and incubated with substrate solution and stop solution as described above for ELISA (Figure 30).

[0324] 11.4 Inhibition of human / cynomolgus / mouse AP activity with recombinant antibodies The recombinant antibodies were serially diluted in 1x PBS and then transferred to a U-bottom 96-well plate (50 μl / well). 20% human serum / 20% cynomolgus serum was prepared in 1x AP buffer, and 60% mouse serum was prepared in 2x AP buffer, and incubated on ice for 30 minutes. During serum incubation, rabbit RBCs were prepared: 0.5 ml of rabbit red blood cells (4x10 8 / ml) was washed three times with 1 ml of 1x AP buffer and 5x10 7 The cells were resuspended in 1x AP buffer to a final concentration of 1 / ml (4 ml). Activated serum (50 μl / well) and red blood cells (50 μl / well) were then added to the sample wells. The plates with human / cynomolgus serum were incubated at 37°C for 30 minutes, and the plates with mouse serum were incubated at 37°C for 60 minutes. The plates were centrifuged at 600 rpm for 2 minutes, and 100 μL of supernatant was transferred to a new flat-bottom 96-well plate. Hemoglobin release was determined at OD 405 nm using a microplate reader (Figure 31).

[0325] 11.5 Mouse BBB pK Assay in Mouse Tissues Using Recombinant Antibodies 11.5.1 PK Assay Method Principle (SLN12140 / SLN12150 / SLN12147 / SLN12149) This method utilizes an antibody sandwich mode ELISA to detect analytes in mouse serum / brain tissue homogenate / CSF. The SLN2102-coated assay plate is incubated overnight. The next day, the analyte in the sample and STD / QC are captured on the plate by SLN2102. After sample incubation is complete, a biotin-labeled detection antibody is added to the plate. The plate is washed to remove excess detection antibody (Bio-SLN2108). SA-HRP is added to bind biotin. Upon completion, TMB is added and the plate is read on a microplate reader. The resulting OD is proportional to the amount of analyte present in the sample and STD / QC (Figure 32). SLN12147 (SEQ ID NO: 116) SLN12149 (SEQ ID NO: 117) SLN12150 (SEQ ID NO: 118)

[0326] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. The present invention is not intended to be limited by the specific examples provided herein. While the present invention has been described with reference to the foregoing specification, the description and illustration of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it should be understood that all aspects of the present invention are not limited to the specific depictions, configurations, or relative proportions set forth herein, which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the present disclosure. Accordingly, it is contemplated that the present invention shall also cover any such alternatives, modifications, variations, or equivalents. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

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