Monovalent anti-properdinine antibodies and antibody fragments

Abstract

This provides an effective method for regulating properdin activity. [Solution] Isolated monovalent antibodies or fragments thereof that bind to human propergine are described. Such antibodies are useful in methods for treating diseases mediated by dysregulation of the second complement pathway.

Description

[Background technology]

[0001] The complement system plays a central role in the clearance of immune complexes and in immune responses to infectious agents, foreign antigens, virus-infected cells, and tumor cells. Complement activation occurs primarily through three pathways: the classical pathway, the lectin pathway, and the complement II pathway. Uncontrolled activation or insufficient regulation of the complement II pathway can lead to systemic inflammation, cytotoxicity, and tissue damage. The complement II pathway has been involved in the pathogenesis of a growing variety of diseases. Propergine positively regulates the activation of the complement II pathway by binding to and stabilizing the C3 and C5 convertase complexes (C3bBb and C3bnBb). Inhibition or modulation of propergine activity is an important therapeutic strategy to alleviate symptoms associated with complement II pathway dysregulation and slow disease progression. The demand for effective modulation of this propergine activity remains unmet. [Overview of the project] [Means for solving the problem]

[0002] Described herein are isolated monovalent antibodies and their antibody fragments that specifically or substantially specifically bind to propergin and selectively block the activation of the complement II pathway. By inhibiting the functional activity of propergin, the monovalent antibodies described herein inhibit the formation of membrane invasion complexes induced by the complement II pathway. Furthermore, selective binding of a single propergin molecule to a monovalent antibody can reduce undesirable immune complexes resulting from aggregation. Therefore, selective targeting of a single propergin monomer or polymer may increase clinical benefit for patients with diseases mediated by complement II pathway dysregulation.

[0003] In one embodiment, the disclosure relates to an isolated monovalent antibody or a fragment of that antibody that binds to human propergin. In a particular embodiment, the antibody or fragment is a camelid antibody. In a particular embodiment, the antibody or fragment is a single-domain antibody. In a particular embodiment, the antibody or fragment binds to TSR0 and / or TSR1 of human propergin. In a particular embodiment, the antibody or fragment binds to the epitope of amino acid sequence LCQPCRSPRWSLWSTWAPCSVTCSEGSQLRYRRCVGWNGQ (SEQ ID NO: 8). In a particular embodiment, the antibody or fragment binds to mouse propergin with an affinity of less than 50 nM. In certain embodiments, the antibody or fragment comprises at least one or all of three CDRs selected from a) CDR-H1 comprising the amino acid sequence GRIFEVNMMA (SEQ ID NO: 9), b) CDR-H2 comprising the amino acid sequence RVGTTX1YADSVKG (SEQ ID NO: 10) where X1 is a polar or non-polar amino acid, and c) CDR-H3 comprising the amino acid sequence LQYX2RYGGAEY (SEQ ID NO: 11) where X2 is a polar amino acid. In certain embodiments, CDR-H2 comprises the amino acid sequence RVGTTVYADSVKG (SEQ ID NO: 12). In certain embodiments, CDR-H3 comprises the amino acid sequence LQYDRYGGAEY (SEQ ID NO: 13). In certain embodiments, CDR-H2 comprises the amino acid sequence RVGTTTYADSVKG (SEQ ID NO: 15). In certain embodiments, CDR-H3 has the amino acid sequence LQYSRYGGAEY (SEQ ID NO: 14). In certain embodiments, CDR-H3 has the amino acid sequence LQYDRYGGAEY (SEQ ID NO: 13). In certain embodiments, CDR-H3 has the amino acid sequence LQYDRYGGAEY (SEQ ID NO: 13). In certain embodiments, CDR-H3 has the amino acid sequence LQYSRYGGAEY (SEQ ID NO: 14). In certain embodiments, the antibody or fragment comprises three CDRs: a) CDR-H1 having the amino acid sequence GRISSIIHMA (SEQ ID NO: 16), b) CDR-H2 having the amino acid sequence RVGTTVYADSVKG (SEQ ID NO: 12), and c) CDR-H3 having the amino acid sequence LQYEKHGGADY (SEQ ID NO: 17).In a particular embodiment, the antibody comprises six CDRs: a) CDR-H1 having the amino acid sequence GYIFTNYPIH (SEQ ID NO: 18), b) CDR-H2 having the amino acid sequence FIDPGGGYDEPDERFRD (SEQ ID NO: 19), c) CDR-H3 having the amino acid sequence RGGGYYLDY (SEQ ID NO: 20), d) CDR-L1 having the amino acid sequence RASQDISFFLN (SEQ ID NO: 21), e) CDR-L2 having the amino acid sequence YTSRYHS (SEQ ID NO: 22), and f) CDR-L3 having the amino acid sequence QHGNTLPWT (SEQ ID NO: 23). In a particular embodiment, the antibody comprises six CDRs: a) CDR-H1 having the amino acid sequence GFSLTTYGVH (SEQ ID NO: 24), b) CDR-H2 having the amino acid sequence VIWSGGDTDYNASFIS (SEQ ID NO: 25), c) CDR-H3 having the amino acid sequence NKDYYTNYDFTMDY (SEQ ID NO: 26), d) CDR-L1 having the amino acid sequence KSSQSVLYSSNQKNFLA (SEQ ID NO: 27), e) CDR-L2 having the amino acid sequence WASTRES (SEQ ID NO: 28), and f) CDR-L3 having the amino acid sequence HQYLSSYT (SEQ ID NO: 29). In a particular embodiment, the antibody comprises six CDRs: a) CDR-H1 having the amino acid sequence GYTFIDYWIE (SEQ ID NO: 30), b) CDR-H2 having the amino acid sequence EIFPGSGTINHNEKFKD (SEQ ID NO: 31), c) CDR-H3 having the amino acid sequence EGLDY (SEQ ID NO: 32), d) CDR-L1 having the amino acid sequence SASSSVSYIY (SEQ ID NO: 33), e) CDR-L2 having the amino acid sequence DTSTLAS (SEQ ID NO: 34), and f) CDR-L3 having the amino acid sequence QQWSRNPFT (SEQ ID NO: 35).In a particular embodiment, the antibody comprises six CDRs: a) CDR-H1 having the amino acid sequence GFSLTSYGVH (SEQ ID NO: 36), b) CDR-H2 having the amino acid sequence VIWSGGSTDYNAAFIS (SEQ ID NO: 37), c) CDR-H3 having the amino acid sequence NKDFYSNYDYTMDY (SEQ ID NO: 38), d) CDR-L1 having the amino acid sequence KSSQSVLYSSNQKNFLA (SEQ ID NO: 27), e) CDR-L2 having the amino acid sequence WASTRES (SEQ ID NO: 28), and f) CDR-L3 having the amino acid sequence HQYLSSYT (SEQ ID NO: 29). In certain embodiments, the antibody comprises six CDRs: a) CDR-H1 having the amino acid sequence GYTXTAYGIN (SEQ ID NO: 39), b) CDR-H2 having the amino acid sequence YIYIGNGYTDYNEKFKG (SEQ ID NO: 40), c) CDR-H3 having the amino acid sequence SGWDEDYAMDF (SEQ ID NO: 41), d) CDR-L1 having the amino acid sequence RASENIYSYLA (SEQ ID NO: 42), e) CDR-L2 having the amino acid sequence HAKTLAE (SEQ ID NO: 43), and f) CDR-L3 having the amino acid sequence QHHYGPPPT (SEQ ID NO: 44). In certain embodiments, the antibody or fragment inhibits the activity of human propergin.

[0004] In one embodiment, the disclosure relates to the use of an isolated monovalent antibody or a fragment of the antibody that binds to human propergine in a method for treating a disease mediated by complement pathway II dysregulation, or in the manufacture of a pharmaceutical product for treating a disease mediated by complement pathway II dysregulation.

[0005] In one embodiment, the present disclosure relates to a method for treating a disease mediated by dysregulation of the second complement pathway. The method comprises administering an effective amount of an isolated monovalent antibody or a fragment of that antibody, the antibody or fragment of which is bound to human propergine to a patient in need. In certain embodiments, the diseases include autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), IgA nephropathy (Buerger's disease), asthma (e.g., severe asthma), C3 glomerulopathy (C3G), Gaucher disease, hidradenitis suppurativa, Behçet's disease, severe burns, early sepsis, dermatomyositis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-associated lung injury (TRALI), hemodialysis-related thrombosis, acquired epidermolysis bullosa (EBA), uveitis, Parkinson's disease, primary biliary atresia, antineutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, and extensive thrombotic microangiopathy (TMA). ), extensive TMA (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes 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, membranoproliferative (MP) glomerulonephritis, membranonephritis, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malaria hemolytic crisis, abdominal aortic aneurysm (AAA) or thoracoabdominal aortic aneurysm (TAA).

[0006] In one embodiment, the present disclosure relates to a method for inhibiting complement pathway II membrane invasion complex assembly. This method involves administering an effective dose of an antibody, an antibody derivative, or a fragment thereof to a patient in need. In a particular embodiment, this method inhibits complement pathway II-dependent hemolysis.

[0007] In some embodiments, the antibody or fragment thereof includes the following sequence:

[0008] EVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSSGGGGSGGGGSGGGGSLEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSS(Sequence ID 45) In some embodiments, the antibody or fragment thereof includes the following sequence:

[0009] EVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAASGRISSIIHMAWFRQAPGKERELVSEISRVGTTVYADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTLVTVSS(Sequence No. 46) In some embodiments, the antibody or fragment thereof includes the following sequence:

[0010] EVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSSGGGGDGGGGDGGGGEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSS(Sequence ID 47) In some embodiments, the antibody or fragment thereof includes the following sequence:

[0011] EVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSSGGGGEGGGGEGGGGEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSS(Sequence ID 48) In some embodiments, the antibody or fragment thereof includes the following sequence:

[0012] EVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRISSIIHMAWVRQAPGKQRELVSEISRVGTTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNALQYEKHGGADYWGQGTLVTVSS(Sequence No. 49) In some embodiments, the antibody or fragment thereof includes the following sequence:

[0013] EVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSSGGGGDGGGGDGGGGEVQLLESGGGLVQPGGSLRLSCAASGRISSIIHMAWFRQAPGKERELVSEISRVGTTVYADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTLVTVSS(Sequence No. 50) In some embodiments, the antibody or fragment thereof includes the following sequence:

[0014] EVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSSGGGGEGGGGEGGGGEVQLLESGGGLVQPGGSLRLSCAASGRISSIIHMAWFRQAPGKERELVSEISRVGTTVYADSVKGRFTISRDNSKNTLYLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTLVTVSS(Sequence No. 51) In some embodiments, the antibody or fragment thereof includes the following sequence:

[0015] EVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSSGGGGDGGGGDGGGGEVQLVESGGGLVQPGGSLRLSCAASGRISSIIHMAWVRQAPGKQRELVSEISRVGTTVYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNALQYEKHGGADYWGQGTLVTVSS(Sequence ID 52) In some embodiments, the antibody or fragment thereof includes the following sequence:

[0016] LEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSSRKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 53) In some embodiments, the antibody or a fragment thereof is linked to hG1 without a C1q binding domain and is an LVP058 anti-properdin monovalent antibody V HH It includes the following sequence:

[0017] LEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 54) In some embodiments, the antibody or a fragment thereof is linked to the (G4S)3 linker against albumin V HH Linked to LVP058 anti-properdin monovalent antibody V HH It includes the following sequence:

[0018] LEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSS (Sequence ID 55) definition As used herein, the term “monovalent antibody or antibody fragment” refers to a single properdin molecule or other antibody against an antigen. H or V HHAn antibody or an antigen-binding fragment thereof that contains one binding domain such as etc. In one embodiment, the antigen molecule to be bound is part of a trimer or higher-order multimer of a propelzin monomer. Antibodies, including monovalent antibodies or their antibody fragments, generally bind to specific antigens with high specificity.

[0019] As used herein, the term "single-domain antibody" defines a molecule in which the antigen-binding site is present on a single immunoglobulin domain and is formed by that single immunoglobulin domain. Generally, the antigen-binding site of a single variable domain of an immunoglobulin is formed by three or fewer CDRs. As long as a single antigen-binding domain can form a single antigen-binding unit (i.e., a functional antigen-binding unit that is essentially a single variable domain) in such a way that it does not need to interact with another variable domain to form a functional antigen-binding unit, the single variable domain may contain, for example, a light-chain variable domain sequence (V L sequence) or an appropriate fragment thereof, or a heavy-chain variable domain sequence (V H sequence or V HH sequence, etc.) or an appropriate fragment thereof.

[0020] As used herein, the term "camelid antibody" refers to an antibody derived from a species of camelid such as camel, llama, alpaca, or guanaco. Camelid antibodies differ from most other mammalian antibodies in that they contain only heavy chains that have complete and diverse antigen-binding capabilities due to the absence of light chains (Hamers-Casterman, C. et al., Nature, 363:446-8, 1993).

[0021] As used herein, the term "V HH " refers to a single heavy-chain variable domain antibody without a light chain. For example, the V HH chain may be of the type originally found in camelids (Camelidae) or cartilaginous fish that do not have light chains, or synthetic V HH and non-immunized V HHThis is also acceptable. Each heavy chain contains a variable region encoded by V exons, D exons, and J exons. HH This is a natural V, such as camelid animal antibodies. HH This may be an antibody, or a recombinant protein containing a heavy chain variable domain.

[0022] As used herein, the term “isolated antibody” means an antibody that is substantially free from other antibodies with different antigen specificities (for example, an isolated antibody that binds to propergine is substantially free from contaminants such as antibodies that do not bind to propergine). Furthermore, an “isolated” antibody is one that has been identified, separated, and / or recovered from components of the natural environment in which it exists. These contaminants in the natural environment may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes that could interfere with the diagnostic or therapeutic use of the antibody.

[0023] As used herein, the term “specific binding” of an antibody or its fragment, polypeptide, or peptidomimetic means binding to a target molecule that is measurably different from binding to a non-target molecule. As used herein, specific binding means a binding priority of more than 95% to a particular antigen compared to background (“nonspecific”) binding. “Substantially specific” binding means a binding priority of more than approximately 80% to a particular antigen compared to background. Binding can be measured by a variety of methods, including but not limited to Western blotting, immunoblotting, enzyme-linked immunosorbent assay (“ELISA”), radioimmunoassay (“RIA”), immunoprecipitation, surface plasmon resonance, biolayer interferometry, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemistry, matrix-assisted laser desorption / ionization time-of-flight (“MALDI-TOF”) mass spectrometry, microcytometry, microarrays, microscopy, fluorescence-activated cell sorting (“FACS”), and flow cytometry.

[0024] As used herein, the term "human propersine" refers to a soluble glycoprotein consisting of 469 amino acids found in plasma, having seven thrombospondin type I repeats (TSRs) with the N-terminal domain, TSR0, being the cleavage domain. Human propersine, a 53 kDa protein, contains a signal peptide (amino acids 1-28) and six distinct TSR repeats, each consisting of approximately 60 amino acids: 80-134 (TSR1), 139-191 (TSR2), 196-255 (TSR3), 260-313 (TSR4), 318-377 (TSR5), and 382-462 (TSR6). Propergine is formed when rod-shaped monomers become oligomers, such as cyclic dimers, trimers, and tetramers. The amino acid sequence of human propersine can be found in the GenBank database under the following accession numbers. For human properzine, see, for example, GenBank accession number AAA36489, NP - See 002612, AAH15756, AAP43692, S29126, and CAA40914. Propergine is a positive regulator of the second complement activation cascade. Known binding ligands for propergine include C3b, C3bB, and C3bBb (Blatt, A. et al., Immunol. Rev., 274:172-90, 2016).

[0025] As used herein, the term “mouse propersine” refers to a 457-amino acid soluble glycoprotein found in plasma, having seven TSRs (Transconjunctival Ratios) and with its N-terminal domain, TSR0, cleaved. A 50 kDa protein, mouse propersine contains a signal peptide (amino acids 1-24) and six distinct TSR repeats of approximately 60 amino acids each: 73-130 (TSR1), 132-187 (TSR2), 189-251 (TSR3), 253-309 (TSR4), 311-372 (TSR5), and 374-457 (TSR6). Mouse propersine is formed when rod-shaped monomers become oligomers, such as cyclic dimers, trimers, and tetramers. The amino acid sequence of mouse propersine can be found, for example, in the GenBank database (GenBank accession numbers P11680 and S05478).

[0026] As used herein, the term "TSR0 domain" refers to the cleavage domain of propergine that precedes the TSR1 domain of propergine. For example, the TSR0 domain of human propergine contains amino acids 28-76.

[0027] As used herein, the term "TSR1 domain" refers to the domain of propergine adjacent to the TSR0 domain of propergine. For example, the TSR1 domain of human propergine contains amino acids 77–134.

[0028] As used herein, the term “propergine activity” refers to the biological activity of propergine, including but not limited to binding interactions that lead to the stabilization of C3 / C5 convertases. Propergine binds most strongly to the C3b,Bb secondary pathway C3 / C5 convertase, but also to C3b, C3b,B, and C3b,Bb. One function is to stabilize the C3b,Bb complex, enabling higher activation of the secondary pathway (Pangburn, M., Methods Enzymol., 162:639-53, 1988; Nolan, K. & Reid, K., Methods Enzymol., 223:35-46, 1993). Propergine promotes the formation of the secondary pathway C3 convertase by increasing the binding of factor B to the P,C3b complex. Therefore, propergine is a complement activation promoter (positive regulator). Furthermore, properzine has been involved in initiating the activation of the secondary pathway by binding to the target surface and initiating the formation of C3 / C5 convertase (Kemper C. & Hourcade, D., Mol. Immunol., 45:4048-56, 2008).

[0029] As used herein, the term “complement pathway II” refers to one of the three pathways of complement activation (the others being the classical pathway and the lectin pathway). Complement pathway II is generally activated by bacteria, parasites, viruses, or fungi, but IgA antibodies and some IgL chains have also been reported to activate this pathway.

[0030] As used herein, the term “complement II pathway dysregulation” means any abnormality in the ability of the complement II pathway to defend the host against pathogens, to eliminate immune complexes and damaged cells, and for immunomodulation. Complement II pathway dysregulation can occur in both the fluid phase and on the cell surface and can lead to conditions that cause tissue damage, such as excessive complement activation or insufficient regulation.

[0031] As used herein, the expression “disease mediated by complement II dysregulation” means a disruption, cessation, or impairment of a bodily function, system, or organ caused by complement II dysregulation. Such diseases would benefit from treatment with the compositions or formulations described herein. In some embodiments, the disease is caused by any abnormality in the complement II pathway’s ability to defend the host against pathogens, to eliminate immune complexes and damaged cells, and for immunomodulation. This specification also encompasses diseases directly or indirectly mediated by dysregulation of one or more components of the complement II pathway, or products produced by the complement II pathway.

[0032] As used herein, the term “complement pathway II-dependent membrane invasion complex assembly” refers to the terminal complex formed as a result of activation of complement pathway II, comprising complement components C5, C6, C7, C8, and C9. The assembly of the membrane invasion complex (MAC) leads to cell lysis.

[0033] As used herein, the term “complement pathway II-dependent hemolysis” means the lysis of red blood cells mediated by the assembly and / or deposition of increased complement pathway II-dependent MACs on red blood cells.

[0034] As used herein, the term “linker” refers to a bond between two elements, such as protein domains. A linker may be a covalent bond or a spacer. The term “bond” refers to any type of bond, such as an amide bond or a disulfide bond, or a bond resulting from a chemical reaction, such as a chemical conjugation. A linker may also refer to a portion (e.g., a polyethylene glycol (PEG) polymer) or an amino acid sequence (e.g., a 3-200 amino acid sequence, a 3-150 amino acid sequence, or a 3-100 amino acid sequence) that forms between two polypeptides or polypeptide domains to provide space and / or flexibility between them. An amino acid spacer may be part of the primary sequence of a polypeptide (e.g., bonded to distant polypeptides or polypeptide domains via the polypeptide backbone). A linker may contain one or more glycine and serine residues. [Brief explanation of the drawing]

[0035] [Figure 1] Figure 1 shows biolayer interference data obtained using an Octet® biosensor equipped with a model system in which a selected anti-properdin antibody specifically binds to human properdin. The graph shows the equilibrium dissociation over time. [Figure 2] Figure 2 shows biolayer interference data obtained using an Octet® biosensor with a model system in which the selected anti-properdin antibody showed weak or no binding to mouse properdin. The graph shows the equilibrium dissociation over time. [Figure 3] Figure 3 shows biolayer interference data obtained using an Octet® biosensor with a model system, in which the selected anti-properdin antibody showed specific but weak binding to cynomolgus monkey properdin. The graph shows the equilibrium dissociation over time. [Figure 4]Figure 4 shows that the selected anti-properdin antibody inhibits the activity of human properdin in a complement pathway II hemolysis assay. [Figure 5A] Figure 5A shows the characterization of the selected anti-properdin antibodies by mass spectrometry. [Figure 5B] Figure 5B shows the characterization of the selected anti-properdin antibodies by mass spectrometry. [Figure 5C] Figure 5C shows the characterization of the selected anti-properdin antibodies by mass spectrometry. [Figure 6A] Figure 6A shows the binding affinity of selected anti-properdin antibodies to biotinylated properdin using the properdin capture method. [Figure 6B] Figure 6B shows the binding affinity of selected anti-propersin antibodies to biotinylated propersin using the propersine capture method. [Figure 7] Figure 7 shows the binding affinity of selected anti-properdin bispecific antibodies to biotinylated properdin using the properdin capture method. [Figure 8A] Figure 8A shows that the selected anti-properdin bispecific antibody inhibits the activity of human properdin and cynomolgus monkey properdin in a complement pathway 2 hemolysis assay. An anti-properdin antibody was used as a control. [Figure 8B] Figure 8B shows that the selected anti-properdin bispecific antibody inhibits the activity of human properdin and cynomolgus monkey properdin in a complement pathway 2 hemolysis assay. An anti-properdin antibody was used as a control. [Figure 9A] Figure 9A shows that the selected anti-properdin bispecific antibody inhibits the activity of human properdin and cynomolgus monkey properdin in a complement pathway 2 hemolysis assay. [Figure 9B] Figure 9B shows that the selected anti-properdin bispecific antibody inhibits the activity of human properdin and cynomolgus monkey properdin in a complement pathway 2 hemolysis assay. [Figure 10A] Figure 10A shows the binding affinity of selected anti-properdin bispecific antibodies to biotinylated properdin using the properdin capture method. [Figure 10B] Figure 10B shows the binding affinity of selected anti-properdin bispecific antibodies to biotinylated properdin using the properdin capture method. [Figure 11A] Figure 11A shows the binding affinity of selected anti-properdin bispecific antibodies to biotinylated properdin using the properdin capture method. [Figure 11B] Figure 11B shows the binding affinity of selected anti-properdin bispecific antibodies to biotinylated properdin using the properdin capture method. [Figure 12A] Figure 12A shows the binding affinity of selected anti-properdin bispecific antibodies to biotinylated properdin using the properdin capture method. [Figure 12B] Figure 12B shows the binding affinity of selected anti-properdin bispecific antibodies to biotinylated properdin using the properdin capture method. [Modes for carrying out the invention]

[0036] Propergine is a positive regulator of the complement II pathway. Described herein is a novel monovalent antibody that binds to a single propergine molecule and is useful for treating diseases mediated by dysregulation of the complement II pathway. Described herein is the discovery that immune complexes formed when a bivalent antibody binds to multiple propergine macromers are toxic as therapeutic agents for inhibiting the abnormal activation of the complement II pathway. The monovalent antibody described herein has a 1:1 binding ratio to propergine and, by design, cannot form antibody / propergine aggregates containing multiple propergine macromers, which is advantageous over bivalent and polyvalent antibodies.

[0037] The following describes monovalent antibodies or antibody fragments that can be administered to patients with diseases mediated by dysregulation of the second complement pathway.

[0038] Anti-propersin antibodies Described herein are monovalent anti-propergin antibodies, antibody derivatives (e.g., modified antibodies, humanized antibodies, chimeric antibodies, substituted antibodies, humanized antibodies, etc.) and their antibody fragments that inhibit propergin, a positive regulator of the complement secondary pathway, and subsequently destabilize the C3-convertase and C5-convertase complexes. The antibodies described herein can, for example, inhibit propergin from binding to C3b, C3Bb, and C3bBb. Inhibiting propergin leads to a decrease in complement activation of the secondary pathway. This indicates a therapeutic benefit for patients with secondary pathway dysregulation disorders in which the secondary pathway is overactivated.

[0039] The anti-propergin antibodies described herein can be produced by using full-length propergin, propergin polypeptides, and / or peptides containing antigenic propergin epitopes, such as fragments of propergin polypeptides. Propergin peptides and polypeptides can be isolated and used to produce antibodies as natural polypeptides, recombinant polypeptides, or synthetic recombinant polypeptides. Any antigen useful for the production of anti-propergin antibodies can be used for the production of monovalent antibodies. Suitable forms of monovalent antibodies and methods for their production are known in the art (e.g., WO2007 / 048037 and WO2007 / 059782, the entire contents of which are incorporated herein by reference).

[0040] Anti-properdin antibodies may be monoclonal antibodies or derived from monoclonal antibodies. Appropriate monoclonal antibodies against the selected antigen can be found using known techniques ("Monoclonal Antibodies: A manual of techniques," Zola (CRC Press, 1988), "Monoclonal Hybridoma Antibodies: Techniques and Applications," Hurrell (CRC Press, 1982)). (The entire content of which is incorporated herein by reference may be used to prepare it.)

[0041] In other embodiments, the antibody is V HH These may be single-domain antibodies such as those found in camelids and sharks (Saerens, D. et al., Curr. Opin. Pharmacol., 8:600-8, 2008). Camelid antibodies are described, for example, in U.S. Patents 5,759,808, 5,800,988, 5,840,526, 5,874,541, 6,005,079, and 6,015,695, the entire contents of each of these documents are incorporated herein by reference. Cloned and isolated V HH The domain is a stable polypeptide characterized by the full antigen-binding ability of the original heavy chain antibody. HH The domains possess unique structural and functional properties, combining the advantages of conventional antibodies (high target specificity, high target affinity, and low intrinsic toxicity) with the key characteristics of small molecule drugs (the ability to inhibit enzymes and access receptor clefts). Furthermore, they are stable, can be administered by means other than injection, are easy to manufacture, and can be humanized (U.S. Patents 5,840,526, 5,874,541, 6,005,079, 6,765,087, EP 1589107, WO97 / 34103, WO97 / 49805, U.S. Patents 5,800,988, 5,874,541, and 6,015,695; the entire contents of each of these publications are incorporated herein by reference).

[0042] In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0043] QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAIGWNGEGIYYADSVKGRFTISRDNAKNTGYLQMNSLKPEDTAVYYCAADSEGVVPGFPIAYWGQGTQVTVSG (Sequence ID 71) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0044] QVQLVESGGGLVQPGGSLRLSCAASGFPLNSYAIGWFRQAPGKEREGVSCISVSDDSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDSAPLYGDYVCKPLENEYDFWGQGTQVTVSG (Sequence ID 72) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0045] QVQLVESGGGLVQAGGSLXLSCAASGSDRRINGMGWYRHPPGKQRELVAAITSGGSTNYADSVKGRFTISTNNANNMMYLQMNSLKPEDTAVYYCAIDEFGTGWLDYCGQGTQVTVSG (Sequence ID 73) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0046] QVQLVESGGGLVQPGGSLRLSCAASGRPFSSYAMGWFRQAPGKEREIVAGLSWSGGNVYYADSVKGRFTISRDNAKNTGDLQMNSLKPEDTAVYYCAIGPKLTTGPTAYRYWGQGTQVTVSS (Sequence ID 74) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0047] QVQLVESGGGLVQPGGSLRLSCATSGGTFSSYAMGWFRQAPGKEREFVAAITWNGSNRYYADSVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCAAEHSTRYSGFYYYTRGETYHYWGQGTQVTVSG (Sequence ID 75) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0048] QVQLVESGGGLVQAGGSLRLSCAASGRTFSTLGMGWFRQAPGKERQFVAAINWSGSSTYYANSVKGRFTISRDNAQSTMYLQMNSLKPEDTAVYYCAADLDSRYSAYYYYSDESQYDYWGQGTLVTVSG (Sequence ID 76) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0049] QVQVVESGGGLVQPGGSLSLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWDGANIYYADSVKGRFTLSRDNAENTVWLQLNSLKPEDTAVYYCAAAESGRYSGRDYYSAPGVYLYWGQGTLVTVSG (Sequence ID 77) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0050] QVQLVESGGGLVQAGGSLRLSCAASGSIFDINAMGWYRQAPGKQRELVADITSSGSTNYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYTCAAESIRESQNRHQLGYMGPLYDYWGQGTQVTVSG (Sequence ID 78) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0051] QVQLIESGGGLVQAGDSLRLSCAASEGTFSRFAMGWFRQAPGKEREFVAAINWSGGITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTADYYCAAETTTRYSGYYYYEDNKSYDYWGQGTLVTVSG (Sequence ID 79) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0052] QVQVVESGGGLRQTGGSLRLSCTASGRIFEVNMMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDSAKNTVTLQMNSLKSEDTAVYYCNALQYDRYGGAEYWGQGTQVTVSS (Sequence ID 58) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0053] QVLLEESGGGLERTGGSLRLSCAASGSIFSVNSMTWYRQAPGKRREFLGTITEEGRTNYADSVKGRFTISRDNAKNTMYLQMNSLKPEDTAVYYCYANLISSEDRTFGVWGQGTQVTVSS (Sequence ID 80) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0054] QVHLVESGGGLVQAGGSLRLSCTASGGTVGDYAVGWFRQAPGKERELIGVVSRLGARTGYADSVLGRFTISRDDVKNTVFLQMDSVKPEDTAVYYCAARRDYSFEVVPYDYWGQGTQVTVSG (Sequence ID 81) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0055] QVQMVESGGGLVQAGGSLRLSCAASGLTNRIRIMGWYRQAPGKLRELVATITNDGSTHYADSVKGRFTISTDNAKNTVFLQMNSLKPEDTAVYICNVGENWGPAYWGQGTQVTVSG (Sequence ID 82) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0056] QVQLVESGGGLVQPGGSLRLSCAASGFPLNSYAIGWFRQAPGKEREGVSCISVSDDSTYYTDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDSAPLYGDYVCKPLENEYDFWGQGTQVTVSG (Sequence ID 72) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0057] QVRLTESGGGLVQYGTNLTLTCVASGLISTRNKMGWFRRRSGGQREFVASSTVLSDDVIQDDIAETVKGRFAVARNDYKNILYLQMTAVKPEDTGFYWCASGTSLFGASRREDDFNAWGVGTQVTVSA (Sequence ID 83) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0058] QVQLAESGGGLVQAGDSLKLSCTASGRIFEVNMMAWYRQAPGKDRELVAEISRVGTTTYADSVKGRFTISRDSAKNTVTLQMNSLKSEDTAVYYCNALQYSRYGGAEYWGQGTQVTVSG (Sequence ID 59) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0059] EVQLVESGGGLVQPGGSLRLSCAASGFTFGSADMSWVRQAPGKGPEWVSAINSNGGSTYYAASVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAQGNWYTEEYHYWGQGTLVTVSG(Sequence ID 84) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0060] QVRLVESGGGLVQAGDSLRLSCAASGRTLSSYAMGWFRQAPGKEREFVAATTWRDTSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAAYYCAAEEPSKYSGRDYYMMGDSYDYWGQGTQVTVSS (Sequence ID 85) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0061] EVQLVESGGGLVQPGGSLRLSCAASGFTFGSADMSWVRQAPGKGPEWVSAINSNGGSTYYAASVKGRFTISRDNAKNTLYLQMNSLKPEDTAVYYCAQGNWYTEEYHYWGQGTQVTVSG(Sequence ID 86) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0062] QVQLVESGGGLVQAGGSLRLSCAASGRTFSNYAMAWFRQAPGKEREFVASISGSGDSRYYADSVKGRFTISRDNAKNTVYLQTNSPKPEDTAVYYCAAVLPTRYSGFYYYSDGTQYHYWGQGTQVTVSS (Sequence ID 87) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0063] QVNLVESGGGSVQAGGSLRLSCAASENINVINDMGWYRQAPGKQRELVAVITGHDNINYADSATGRFTISTYTWNTENLQMNMLKPEDTAVYYCNADITYANGRFNDWGQGTQVTVSS (Sequence ID 88) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0064] QVHLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQPPGKEREFVAAITWSGSSIYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAAEETSKYSGSYYYMMGDSYDYWGQGTQVTVSG (Sequence ID 89) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0065] QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAVPWTYGSKYYADSVKGRFTISRDDAKNTVYLQMNNLKPEDTAVYYCAADSSAGYYSGFDYYSAATPYDLWGQGTQVTVSG (Sequence ID 90) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0066] QVQLVESGGGLVQPGGSLRLSCAASGSDYYAIGWFRQAPGKEREGVSCMSRTDGSTYYADSVKDRFTISRDYAKNTVYLQMNSLKPEDTAVYYCGLDRSYPTGGISCLFGDFGSWGQGTQVTVSG (Sequence ID 91) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0067] QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYNMGWFRQRHGNEREFVATISWSGRSTYYADSVKGRFAISRDNANTTVYLQMNSLKPEDSAVYYCAASTRGWYGTQEDDYNFWGQGTQVTVSG (Sequence ID 92) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0068] QVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSG (Sequence ID 60) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0069] QVQLVESGGGLVQAGGSLRLSCAASGGTFSSYSMGWFRQAPGKEREFVAAITWNGVSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPTDTAVYYCAAEITTRYSGFYYYEDNKSYDYWGQGTQVTVSS (Sequence ID 93) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0070] QVQLVESGGGLRQTGESLRLSCTASGRIFEVNMMAWYRQAPGKQRELVAEISRVGTTTYADSVKGRFTISRDSAKNTVTLQMNSLKSEDTAVYYCNALQYDRYGGAEYWGQGTQVTVSG (Sequence ID 61) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0071] EVQLVESGGGLVQPGGSLRLSCAASGFTLDYYAIGWFRQAPGKEREGVSCISRTDGSTYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAVDDSYPTGGISCLFGHFGSWGQGTQVTVSS(Sequence ID 94) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0072] QVQLVESGGGLVQAGDSLRLSCAASGFTFSSYAMGWFRQAPGKEREFVAAITWSGVSTYYADSVKGRFTISRDNAKNRVYLQMNSLKPEDTAVYSCAADGSGRYSGMEYYNRDWVYDYWGQGTQVTVSS (Sequence ID 95) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0073] QVHMVESGGGLVQAGGSLRFSCAASGNIFTISTLDWYRQAPGEQRELVATLTPDGITDYAGSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCNAWRYSDDYRGRVDYWGQGTQVTVSG (Sequence ID 96) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0074] QVQLIESGGGLVQEGASLRLSCAGSGPMFSRLAVGWFRQAPGKEREFVAVINWSGSADFYTNSVKGRFTISRDNAKNTVYLEMNTLKPEDSAVYYCAADQNPLTLRTGVRDVGRQWGQGTEVTVSS (Sequence ID 97) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0075] QVQLVESGGGLVQAGGSLRLSCAASGRTFSSYAMGWFRQAPGKEREFVAAITWRGASTYYADPVKGRFTISRDNAKNTVYLQMSSLKPEDTAVYYCAAEEPSYYSGSYYYMMGDSYNYWGQGTQVTVSG (Sequence ID 98) In some embodiments, the antibody or antibody fragment includes the following sequence as its heavy chain variable domain.

[0076] QVQLVESGGGLVQAGGSLRLSCTASGRTFSNYAMGWFRQAPGKEREFLAAISRSGESTNYATFVKGRFTIARDNAKNTVSLQMNSLKPEDTAVYFCAAKVAVLVSTTYNSQYDYWGQGTQVTVSS (Sequence ID 99) The anti-properdin antibodies, antibody derivatives, and fragments thereof disclosed herein include those having one, more, or all of the following CDRs.

[0077] a. CDR-H1 having the amino acid sequence GRIFEVNMMA (SEQ ID NO: 9) b. CDR-H2 having the amino acid sequence RVGTTVYADSVKG (SEQ ID NO: 12) c. CDR-H3 having amino acid sequence LQYDRYGGAEY (SEQ ID NO: 13) Other anti-properdin antibodies, antibody derivatives, and fragments thereof disclosed herein include those having one, more, or all of the following CDRs:

[0078] a. CDR-H1 having amino acid sequence GRIFEVNMMA (SEQ ID NO: 9) b. CDR-H2 having the amino acid sequence RVGTTTYADSVKG (SEQ ID NO: 15) c. CDR-H3 having amino acid sequence LQYSRYGGAEY (SEQ ID NO: 14) Other anti-properdin antibodies, antibody derivatives, and fragments thereof disclosed herein include those having one, more, or all of the following CDRs:

[0079] a. CDR-H1 having amino acid sequence GRIFEVNMMA (SEQ ID NO: 9) b. CDR-H2 having the amino acid sequence RVGTTTYADSVKG (SEQ ID NO: 15) c. CDR-H3 having amino acid sequence LQYDRYGGAEY (SEQ ID NO: 13) Other anti-properdin antibodies, antibody derivatives, and fragments thereof disclosed herein include those having one, more, or all of the following CDRs:

[0080] a. CDR-H1 having the amino acid sequence GRISSIIHMA (SEQ ID NO: 16) b. CDR-H2 having the amino acid sequence RVGTTVYADSVKG (SEQ ID NO: 12) c. CDR-H3 having amino acid sequence LQYEKHGGADY (SEQ ID NO: 17) Humanized camelid V HH Polypeptides are taught, for example, in WO004 / 041862, and the entire content of that teaching is incorporated herein by reference. Humanized camelid V HH It will be understood by those skilled in the art that naturally occurring camelid antibody single variable domain polypeptides can be modified to produce polypeptides (e.g., amino acid substitutions at positions 45 and 103 (W004 / 041862)). This specification describes the nurse shark V HH This also includes antibody single variable domain polypeptides (Greenberg, A. et al., Nature, 374:168-73, 1995, U.S. Patent Application Publication No. 20050043519).

[0081] Other anti-properdin antibodies, antibody derivatives, and fragments thereof disclosed herein include those having one or more or all of the following CDRs (for example, to construct scFv or dAb):

[0082] a) CDR-H1 having the amino acid sequence GYIFTNYPIH (SEQ ID NO: 18) b) CDR-H2 having the amino acid sequence FIDPGGGYDEPDERFRD (SEQ ID NO: 19) c) CDR-H3 having the amino acid sequence RGGGYYLDY (SEQ ID NO: 20) d) CDR-L1 having the amino acid sequence RASQDISFFLN (SEQ ID NO: 21) e) CDR-L2 having the amino acid sequence YTSRYHS (SEQ ID NO: 22) f) CDR-L3 having amino acid sequence QHGNTLPWT (SEQ ID NO: 23) Other anti-properdin antibodies, antibody derivatives, and fragments thereof disclosed herein include those having one or more or all of the following CDRs (for example, to construct an scFv):

[0083] a) CDR-H1 having the amino acid sequence GFSLTTYGVH (SEQ ID NO: 24) b) CDR-H2 having the amino acid sequence VIWSGGDTDYNASFIS (SEQ ID NO: 25) c) CDR-H3 having the amino acid sequence NKDYYTNYDFTMDY (SEQ ID NO: 26) d) CDR-L1 having the amino acid sequence KSSQSVLYSSNQKNFLA (SEQ ID NO: 27) e) CDR-L2 having the amino acid sequence WASTRES (SEQ ID NO: 28) f) CDR-L3 having the amino acid sequence HQYLSSYT (SEQ ID NO: 29) Other anti-properdin antibodies, antibody derivatives, and fragments thereof disclosed herein include those having one or more or all of the following CDRs (for example, to construct an scFv):

[0084] a) CDR-H1 having the amino acid sequence GYTFIDYWIE (SEQ ID NO: 30) b) CDR-H2 having the amino acid sequence EIFPGSGTINHNEKFKD (SEQ ID NO: 31) c) CDR-H3 having amino acid sequence EGLDY (SEQ ID NO: 32) d) CDR-L1 having the amino acid sequence SASSSVSYIY (SEQ ID NO: 33) e) CDR-L2 having the amino acid sequence DTSTLAS (SEQ ID NO: 34) f) CDR-L3 having the amino acid sequence QQWSRNPFT (SEQ ID NO: 35) Other anti-properdin antibodies, antibody derivatives, and fragments thereof disclosed herein include those having one or more or all of the following CDRs (for example, to construct an scFv):

[0085] a) CDR-H1 having the amino acid sequence GFSLTSYGVH (SEQ ID NO: 36) b) CDR-H2 having the amino acid sequence VIWSGGSTDYNAAFIS (SEQ ID NO: 37) c) CDR-H3 having the amino acid sequence NKDFYSNYDYTMDY (SEQ ID NO: 38) d) CDR-L1 having the amino acid sequence KSSQSVLYSSNQKNFLA (SEQ ID NO: 27) e) CDR-L2 having the amino acid sequence WASTRES (SEQ ID NO: 28) f) CDR-L3 having the amino acid sequence HQYLSSYT (SEQ ID NO: 29) Other anti-properdin antibodies, antibody derivatives, and fragments thereof disclosed herein include those having one or more or all of the following CDRs (for example, to construct an scFv):

[0086] a) CDR-H1 having the amino acid sequence GYTXTAYGIN (SEQ ID NO: 39) b) CDR-H2 having the amino acid sequence YIYIGNGYTDYNEKFKG (SEQ ID NO: 40) c) CDR-H3 having the amino acid sequence SGWDEDYAMDF (SEQ ID NO: 41) d) CDR-L1 having the amino acid sequence RASENIYSYLA (SEQ ID NO: 42) e) CDR-L2 having amino acid sequence HAKTLAE (SEQ ID NO: 43) f) CDR-L3 having amino acid sequence QHHYGPPPT (SEQ ID NO: 44) In some embodiments, the antibody or antibody fragment includes the following sequence:

[0087] LEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSSRKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 53) In some embodiments, the antibody or antibody fragment includes the following sequence:

[0088] LEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 54) In some embodiments, the antibody or antibody fragment comprises the following light chain sequence and heavy chain sequence.

[0089] DIQMTQSPSSLSASVGDRVTITCRASQDISFFLNWYQQKPGKAPKLLIYYTSRYHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQHGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 56) QVQLVQSGAEVKKPGASVKVSCKASGYIFTNYPIHWVRQAPGQGLEWMGIDPGGGYDEPDERFRDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARRGGGYYLDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 57) In some embodiments, the antibody or antibody fragment includes the following sequence:

[0090] LEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSS (Sequence ID 55) In some embodiments, the antibody or antibody fragment includes the following sequence:

[0091] EVQLVESGGGLVKPGGSLRLSCAASGRPVSNYAAAWFRQAPGKEREFVSAINWQKTATYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAAVFRVVAPKTQYDYDYWGQGTLVTVSSGGGGSGGGGSGGGGSLEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSS(Sequence ID 45)

[0092] Anti-properdin antibody fragments and derivatives Some naturally occurring antibodies are bivalent because they contain two antigen-binding domains. After protease digestion, numerous even smaller antigen-binding fragments of naturally occurring antibodies have been identified. These fragments include, for example, "Fab fragments" (V L -C L -C H 1-V H Examples include the "Fab' fragment" (Fab with a heavy chain hinge region) and the "F(ab')2 fragment" (a dimer of Fab' linked by a heavy chain hinge region). For example, V linked with a synthetic peptide linker L and V HA single-chain Fv (variable fragment) consisting of these fragments, also known as a "scFv" (V L -Linker-V H Recombination methods have been used to produce even smaller antibody fragments such as Fab fragments, Fab' fragments, and scFv fragments, each containing one V H / V L Because it contains only one antigen-binding domain that includes a dimer, it is monovalent in terms of antigen binding. An even smaller monovalent antibody fragment is dAb, which binds specifically to the antigen on its own, i.e., complementary V L Domain or V H V that does not require a domain H or V L It contains only one immunoglobulin variable domain, such as dAb. dAb binds to the antigen independently of other V domains. However, if other domains are not required for antigen binding by dAb, i.e., dAb does not contain other V domains. H Domain or V L When binding to an antigen regardless of the domain, other V H Domain or V L dAb may also be present in homomultimers or heteromultimers that possess a domain.

[0093] Linker In the present invention, a linker is used to describe a link or connection between polypeptide or protein domains and / or associated non-protein moieties. In some embodiments, the linker is a link or connection between at least two polypeptide constructs such that two polypeptide constructs are tandem-linked to each other (e.g., a monovalent antibody linked to a second polypeptide or a monovalent antibody). The linker can link the N-terminus or C-terminus of one antibody construct to the N-terminus or C-terminus of a second polypeptide construct.

[0094] Linkers can be simple covalent bonds such as peptide bonds, synthetic polymers such as polyethylene glycol (PEG) polymers, or any type of bond resulting from some kind of chemical reaction, such as chemical conjugation. When the linker is a peptide bond, the carboxylic acid group at the C-terminus of one protein domain can react with the amino group at the N-terminus of another protein domain in a condensation reaction to form a peptide bond. Specifically, peptide bonds can be formed by synthetic means using conventional organic chemical reactions well known in the art, or by spontaneous production from host cells. In the latter case, necessary molecular mechanisms within the host cell, such as DNA polymerase and ribosomes, can directly transcribe and translate polynucleotide sequences encoding the DNA sequences of both proteins, such as two tandem-sequenced antibody constructs, into a sequential polypeptide encoding both proteins.

[0095] If the linker is a synthetic polymer such as a PEG polymer, both ends of the polymer can be functionalized with reactive chemical functional groups to allow them to react with terminal amino acids at the connecting ends of the two proteins.

[0096] When linkers (excluding the peptide bonds mentioned above) are created by chemical reactions, for example, chemical functional groups such as amines, carboxylic acids, esters, and azides, or other functional groups commonly used in the art, can be chemically conjugated to the C-terminus of one protein and the N-terminus of another protein, respectively. The two functional groups then react via chemical synthesis to form a chemical bond, linking the two proteins. Such chemical conjugation procedures are commonplace for those skilled in the art.

[0097] In the present invention, the linker between two peptide constructs may be an amino acid linker containing 1 to 200 (for example, 1 to 4, 1 to 10, 1 to 20, 1 to 30, 1 to 40, 2 to 10, 2 to 12, 2 to 16, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200) amino acids. Suitable peptide linkers are known in the art and include, for example, peptide linkers containing flexible amino acid residues such as glycine and serine. In some embodiments, the linker may include a single motif or multiple different or repeating motifs such as GS, GGS, GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), or SGGG (SEQ ID NO: 3). An example motif is (G4S). n , ( G4D) n (G4E) n (G4A) n The sequence has the following characteristics, where n = 1, 2, 3, 4, 5 or 6 or more and combinations thereof. Other linkers include the sequences GGGGD (sequence number 63), GGGGE (sequence number 64), and GGGGA (sequence number 100). Linkers can be designed by combining these various motifs. Examples of such linkers include GGGGSGGGGSGGGGS (sequence number 4), GGGGDGGGGDGGGG (sequence number 5), GGGGEGGGGEGGGG (sequence number 6), and GGGGAGGGGAGGGGS (sequence number 101).

[0098] Bispecific construct Furthermore, the present invention features a bispecific construct in which two antigen-binding polypeptides are linked (by a linker, for example, one of the linkers in SEQ ID NOs: 1-6, 63-64, and 100-101). Such a bispecific construct may include an anti-properdin-binding polypeptide (such as a monovalent antibody) linked by a linker to a second polypeptide (such as a second monovalent antibody). The second polypeptide can enhance the in vivo stability of the bispecific construct. In some embodiments, the second polypeptide is an albumin-binding molecule, an albumin-binding peptide, or an anti-albumin antibody (such as a monovalent antibody) or a modified form thereof. Albumin-binding peptides are known in the art and are described, for example, in WO2007 / 106120 (see Tables 1-9) and Dennis et al., 2002, J Biol. Chem. 277: 35035-35043. Their disclosures are incorporated herein by reference.

[0099] In some embodiments, the second polypeptide is an Fc domain that enhances the in vivo stability of the construct.

[0100] An exemplary dual-specificity construct is shown in Example 5 below.

[0101] In some embodiments, a monovalent anti-properzine antibody is linked to a monovalent anti-albumin antibody. The monovalent anti-properzine antibody may be linked to the N-terminus or C-terminus of the monovalent anti-albumin antibody by its N-terminus or C-terminus.

[0102] A monovalent anti-properdin antibody may be linked at its N-terminus or C-terminus to the N-terminus or C-terminus of a monovalent anti-albumin antibody using a linker having one of the amino acid sequences of SEQ ID NOs: 1-6, 63-64, and 100-101.

[0103] In some embodiments, a monovalent anti-properzine antibody containing the amino acid sequence of SEQ ID NO: 58 is linked to a monovalent anti-albumin antibody. The monovalent anti-properzine antibody containing the sequence of SEQ ID NO: 58 may be linked by its N-terminus or C-terminus to the N-terminus or C-terminus of a monovalent anti-albumin antibody using a linker containing one of the amino acid sequences of SEQ ID NOs: 1-6, 63-64, and 100-101.

[0104] In some embodiments, a monovalent anti-properzine antibody containing the amino acid sequence of SEQ ID NO: 59 is linked to a monovalent anti-albumin antibody. The monovalent anti-properzine antibody containing the sequence of SEQ ID NO: 59 may be linked by its N-terminus or C-terminus to the N-terminus or C-terminus of a monovalent anti-albumin antibody using a linker containing one of the amino acid sequences of SEQ ID NOs: 1-6, 63-64, and 100-101.

[0105] In some embodiments, a monovalent anti-properzine antibody containing the amino acid sequence of SEQ ID NO: 60 is linked to a monovalent anti-albumin antibody. The monovalent anti-properzine antibody containing the sequence of SEQ ID NO: 60 may be linked by its N-terminus or C-terminus to the N-terminus or C-terminus of a monovalent anti-albumin antibody using a linker containing one of the amino acid sequences of SEQ ID NOs: 1-6, 63-64, and 100-101.

[0106] In some embodiments, a monovalent anti-properzine antibody containing the amino acid sequence of SEQ ID NO: 61 is linked to a monovalent anti-albumin antibody. The monovalent anti-properzine antibody containing the sequence of SEQ ID NO: 61 may be linked by its N-terminus or C-terminus to the N-terminus or C-terminus of a monovalent anti-albumin antibody using a linker containing one of the amino acid sequences of SEQ ID NOs: 1-6, 63-64, and 100-101.

[0107] In some embodiments, a monovalent anti-properdin antibody containing the amino acid sequence of SEQ ID NO: 60 is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 4.

[0108] In some embodiments, a monovalent anti-properdin antibody containing the amino acid sequence of SEQ ID NO: 60 is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 5.

[0109] In some embodiments, a monovalent anti-properdin antibody containing the amino acid sequence of SEQ ID NO: 60 is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 6.

[0110] In some embodiments, the bispecific construct includes one amino acid sequence from SEQ ID NOs. 45-55 and 62.

[0111] In some embodiments, a monovalent anti-properdin antibody is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 1.

[0112] In some embodiments, a monovalent anti-properdin antibody is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 2.

[0113] In some embodiments, a monovalent anti-properdin antibody is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 3.

[0114] In some embodiments, a monovalent anti-properdin antibody is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 4.

[0115] In some embodiments, a monovalent anti-properdin antibody is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 5.

[0116] In some embodiments, a monovalent anti-properdin antibody is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 6.

[0117] In some embodiments, a monovalent anti-properdin antibody is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 63.

[0118] In some embodiments, a monovalent anti-properdin antibody is linked at its N-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 64.

[0119] In some embodiments, a monovalent anti-properdin antibody is linked at its C-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 1.

[0120] In some embodiments, a monovalent anti-properdin antibody is linked at its C-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 2.

[0121] In some embodiments, a monovalent anti-properdin antibody is linked at its C-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 3.

[0122] In some embodiments, a monovalent anti-properdin antibody is linked at its C-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 4.

[0123] In some embodiments, a monovalent anti-properdin antibody is linked at its C-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 5.

[0124] In some embodiments, a monovalent anti-properdin antibody is linked at its C-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 6.

[0125] In some embodiments, a monovalent anti-properdin antibody is linked at its C-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 63.

[0126] In some embodiments, a monovalent anti-properdin antibody is linked at its C-terminus to a monovalent anti-albumin antibody using a linker containing the sequence of SEQ ID NO: 64.

[0127] Production of single-domain antibodies In one embodiment, the composition and method are (V H For example, V HH ) or light chain domain (V L A single-domain antibody is used, which is a monovalent antibody specific to propergine. Therefore, one way to produce a propergine-specific monovalent single-domain antibody is, for example, to use the heavy chain gene sequence and light chain gene sequence isolated from a hybridoma (e.g., a mouse hybridoma) expressing an anti-propergine monoclonal antibody. H Region and V L This involves amplifying and expressing the region. H Domain and V L Domain boundaries are described, for example, in Kabat et al. (Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD, 1991)). To design PCR primers that amplify the V domain from heavy chain coding sequences or light chain coding sequences encoding antibodies known to bind to propergine, the V domain of heavy chain and light chain genes is used. H Domain and V L Information regarding the domain boundary is used. The amplified V domain is inserted into a suitable expression vector such as pHEN-1 (Hoogenboom, H. et al., Nucleic Acids Res., 19:4133-7, 1991), for example, in scFv. H and V LIt is expressed in fusion or other suitable monovalent form. The resulting polypeptide can be screened for high affinity monovalent binding to propergine. Binding screening can be performed by methods known in the art. Single-domain antibodies can be prepared using methods known in the art (WO2005118642, Ward, E. et al., Nature, 341:544-6, 1989, Holt, L. et al., Trends Biotechnol., 21:484-90, 2003). Each light chain domain may be either a κ subgroup or a λ subgroup. H Domain and V L Methods for isolating domains are described in the Art of this (EP0368684).

[0128] In one embodiment, single-domain antibodies are obtained from humans, humanized rodents, camelids, or sharks. Such single-domain antibodies can be optionally humanized. Humanization of camelid single-domain antibodies requires the introduction of a limited number of amino acids and mutagenesis within a single polypeptide chain. This is in contrast to the humanization of scFv, Fab, (Fab')2, and IgG, which requires the introduction of amino acid changes into two chains, a light chain and a heavy chain, while preserving the assembly of both chains. In some embodiments, single-domain antibodies are V HH Includes domain. In some embodiments, V HH The domain is a naturally occurring heavy chain antibody against propergin. HH Corresponds to the domain. Such V HHThe sequence is obtained by, for example, using any suitable technique known in the art (for example, cloning a gene encoding a single-domain antibody by single-cell PCR, or immortalizing B cells encoding a single-domain antibody by EBV transformation or fusion to an immortalized cell line), appropriately immunizing a camelid species with propergin (i.e., inducing an immune response and / or heavy-chain antibodies to propergin), obtaining a suitable biological sample (such as a blood sample, serum sample, or B cell sample) from that camelid, starting from that sample, V against propergin HH It can be created by generating an array.

[0129] Alternatively, such naturally occurring V for properzin HH The domain, for example, uses one or more screening techniques known in the art to identify camelid V HH Naive libraries of sequences can be obtained from such libraries by screening them with properdin or at least one part, fragment, antigenic determinant or epitope (WO99 / 37681, WO01 / 90190, WO03 / 025020 and WO03 / 035694). Alternatively, naive V can be obtained by techniques such as random mutagenesis and / or CDR shuffling. HH V obtained from the library HH Libraries, etc., Naive V HH An improved synthetic or semi-synthetic library derived from the library may be used (WO00 / 43507). In some embodiments, V HH A library is constructed and expressed on phages after infection by helper phages. After several rounds of biopanning, single-domain antibodies against human properdin can be isolated and efficiently expressed.

[0130] To facilitate screening, V HH Fragment or V HHLibraries of fusion proteins containing fragments can be displayed on phages, phagemids, ribosomes, or suitable microorganisms (such as yeast). HH Fragment or V HH Appropriate methods, techniques, and host organisms for displaying and screening fusion proteins (sets, collections, or libraries) containing fragments are known in the art (WO03 / 054016, Hoogenboom, H., Nat. Biotechnol., 23:1105-16, 2005).

[0131] In another embodiment, V HH Fragment sequence or V HH A method for producing a fusion protein containing a fragment sequence comprises at least a) preparing an aggregate or sample of cells derived from a camelid species expressing an immunoglobulin sequence; b) screening the aggregate or sample of cells for (i) cells expressing an immunoglobulin sequence that can bind to and / or has affinity for propergin, and (ii) cells expressing a heavy chain antibody; and c) (i) V present in the heavy chain antibody. HH (ii) isolate the sequence from the cell or V present in the heavy chain antibody HH After isolating the nucleic acid sequence encoding the sequence from the cell, V HH Substeps (i) and (ii) can be carried out essentially as a single screening step or as two separate screening steps in any suitable order, to prepare a heavy chain antibody that can bind to propergin and / or has affinity for propergin, comprising the step of expressing a domain.

[0132] A method for constructing an amino acid sequence for propergin is, at a minimum, a) a heavy chain antibody or V HH a) preparing a set, collection, or library of nucleic acid sequences that encode a sequence, and b) a V that can bind to propergine and / or has affinity for propergine. HHc) After isolating the nucleic acid sequences, the V present in each heavy chain antibody is a step of screening a set, collection or library of nucleic acid sequences that encode a fusion protein or heavy chain antibody containing the sequence. HH To express the sequence, V HH The process may include expressing a fusion protein containing a sequence.

[0133] Naturally occurring V H Array or V HH Starting from a sequence, other suitable methods and techniques for obtaining single-domain antibodies and / or nucleic acids encoding them include, in a suitable manner, one or more naturally occurring V sequences (such as one or more framework region (FR) sequences and / or CDR sequences) to provide a monovalent anti-properdin single-domain antibody or a nucleotide sequence or nucleic acid encoding it. HH One or more parts of a sequence, one or more naturally occurring V (such as one or more framework region sequences or CDR sequences) HH This may include combining one or more parts of a sequence and / or one or more composite or semi-synthetic sequences. Alternatively, V HH Alternatively, the nucleotide sequence encoding the framework sequence of a single-domain antibody may be known in the art and may be obtained by polymerase chain reaction (PCR) starting from the nucleotide sequence obtained using the method described herein. To provide a single-domain antibody or an antibody fragment fused with a regulator of the complement second pathway or a fragment thereof, such compositions can be appropriately combined with the nucleotide sequence encoding the desired CDR (e.g., by PCR assembly using overlapping primers).

[0134] Antibody fragment production Antibody fragments that recognize the same epitope as the parent antibody can be produced using known methods. For example, antibody fragments can be prepared by hydrolyzing the antibody or by expressing the DNA encoding the fragment in E. coli. The antibody fragment is the antigen-binding portion of the antibody, such as Fab or F(ab')2, and scFV can be obtained by pepsin or papain digestion of the entire antibody using conventional methods or genetic engineering techniques.

[0135] To prepare a 100 kDa fragment called F(ab')2, an antibody fragment can be prepared by enzymatically cleaving the antibody using pepsin. This fragment can then be further cleaved using a thiol reducing agent, optionally utilizing the blocking group of the sulfhydryl group resulting from the cleavage of the disulfide bond, to produce a 50 kDa monovalent Fab' fragment. Alternatively, the Fc fragment and two monovalent Fab fragments can be directly prepared by enzymatic cleavage using papain (US Patent Nos. 4,036,945 and 4,331,647; Nisonoff, A. et al., Arch. Biochem. Biophys., 89:230-44, 1960; Porter, R., Biochem. J., 73:119-26, 1959; Edelman et al., in Methods in Enzymology Vol. I, p.422 (Academic Press 1967); and Coligan et al., Current Protocols in Immunology, Vol. 1, p.2.8.1-2.8.10, 2.10-2.10.4 (John Wiley & Sons 1991)).

[0136] Other methods of cleaving the antibody may be used, such as separation of the heavy chain to form a monovalent light-heavy chain fragment, further cleavage of the fragment, or other enzymatic, chemical, or genetic techniques, as long as the fragment binds to an antigen recognized by the intact antibody.

[0137] Another form of antibody fragment is a peptide encoding a single complementarity-determining region (CDR). CDR peptides can be obtained by constructing the gene encoding the CDR of the antibody of interest. Such genes can be prepared, for example, by synthesizing the variable region from RNA of antibody-producing cells using polymerase chain reaction (Larrick, J & Fry, K. METHODS - a companion to Methods in Enzymology Volume: New Techniques in Antibody Generation, 2:106-110, 1991), Courtenay Luck, “Genetic Manipulation of Monoclonal Antibodies,” in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), p.166-179 (Cambridge University Press). 1995), Ward et al., “Genetic Manipulation and Expression of Antibodies,” in Monoclonal Antibodies: Principles And Applications, Birch et al., (eds.), p.137-1 85 (Wiley-Liss, Inc. 1995)).

[0138] Other antibody fragments, such as single-domain antibody fragments, are known in the art and can be used in the claimed construct (Muyldermans, S. et al., Trends Biochem. Sci., 26:230-5, 2001; Yau, K. et al., J. Immunol. Methods, 281:161-75, 2003; Maass, D. et al., J. Immunol. Methods, 324:13-25, 2007). HH It has strong antigen-binding ability, and conventional VH -V L The pair can interact with a novel epitope that is inaccessible for access. Camelids can be immunized with known antigens such as propelzin, and the V that binds to the target antigen and neutralizes it HH can be isolated.

[0139] Screening of monovalent antibodies for antigen binding Library screening methods can be used to identify monovalent propergine-specific binding antibodies or fragments. Phage display technology provides an approach to select antibodies that bind to a desired target (such as human propergine) from a large and diverse repertoire of antibodies (Smith, G., Science, 228:1315-7, 1985; Scott, J. & Smith, G., Science, 249:386-90, 1990; McCafferty, J. et al., Nature, 348:552-4, 1990). These phage antibody libraries are either natural libraries using rearranged V genes isolated from human B cells (Marks, J. et al., J. Mol. Biol., 222:581-97, 1991, Vaughan, T. et al., Nat. Biotechnol., 14:309-14, 1996) or libraries in which germline V gene segments or other antibody polypeptide coding sequences are "rearranged" in vitro (Hoogenboom, H. & Winter, G., J. Mol. Biol., 227:381-8, 1992, Nissim, A. et al., EMBO J., 13:692-8, 1994, Griffiths, A. et al., EMBO J., 13:3245-60, 1994, de Kruif, J. et al., J. Mol. Biol., Synthetic libraries can be classified into two categories: those in which the synthetic CDR is incorporated into a single reconstituted V gene (Barbas, C. et al., Proc. Natl. Acad. Sci. USA, 89:4457-61, 1992) or synthetic libraries in which the synthetic CDR is incorporated into a single reconstituted V gene. Methods involving gene display packages (phage display, polysome display, etc.) are generally suitable for selecting monovalent properdin-specific antibody constructs because the display package generally expresses only a monovalent fragment rather than the entire bivalent antibody.Methods for preparing phage display libraries that display various antibody fragments are described in the above-mentioned references and, for example, in U.S. Patent No. 6,696,245, the entire contents of which are incorporated herein by reference.

[0140] After expressing a repertoire of single-domain antibodies on the phage surface, the phage repertoire is brought into contact with an immobilized target antigen (such as propergine), and selection is performed by washing to remove unbound phages and increase the number of bound phages. This process is often collectively called "panning." Display libraries (scFv, Fab, (Fab')2 and V) HH Monovalent single-domain antibodies and antibody cleavage that can be expressed using methods such as Harrison, J. et al., Meth. Enzymol., 267:83-109, 1996) This method is applicable to screening individual phages. Alternatively, phages can be pre-selected for the expression of properly folded member variants by panning against an immobilized common ligand (e.g., protein A or protein L) to which only folded members bind (WO99 / 20749). This method has the advantage of increasing the proportion of members that are likely to bind to the target antigen by reducing the proportion of non-functional members. Screening of phage antibody libraries is generally described, for example, by [reference].

[0141] Screening is generally performed using purified antigens immobilized on solid supports such as plastic tubes or wells, or on chromatography matrices such as Sepharose® (Pharmacia). Screening or selection can also be performed on complex antigens, such as those on cell surfaces (Marks, J. et al., Biotechnology (NY), 11:1145-9, 1993; de Kruif, J. et al., Proc. Natl. Acad. Sci. USA, 92:3938-42, 1995). Alternatively, selection can be performed by binding biotinylated antigens in solution and then capturing them with streptavidin-coated beads. HH Code sequences are known in the art, and V of the camelid family. HH It can be used to construct phage display libraries. This can then be used to isolate antibody fragments using biopanning techniques known in this art.

[0142] Expression of anti-properdin antibodies Nucleic acid manipulation can be performed using recombinant vectors. As used herein, “vector” means a distinct element used to introduce heterologous DNA into cells for expression and / or replication. Methods for selecting or constructing and subsequently using such vectors are known to those skilled in the art. Numerous vectors are publicly available, including bacterial plasmids, bacteriophages, artificial chromosomes, and episomal vectors. Such vectors can also be used for simple cloning and mutagenesis. Vectors are selected to correspond to polypeptide coding sequences of the desired size. Suitable host cells are transformed with the vector after the in vitro cloning operation. Each vector contains various functional components, generally including a cloning (or “polylinker”) site and an origin of replication. Expression vectors may further include one or more enhancer elements, promoters, transcription termination sequences, and signal sequences, each located near the cloning site so as to be linked to the polypeptide-coding gene for transcriptional control.

[0143] Cloning vectors and expression vectors both typically contain nucleic acid sequences that allow the vector to replicate in one or more selected host cells. Generally, cloning vectors contain sequence elements that allow the vector to replicate independently of the host's chromosomal DNA, including origins of replication or autonomous replication sequences. Such sequences are known in various bacteria, yeasts, and viruses.

[0144] In the case of the screened libraries described herein, the vector may be an expression vector that enables the expression of polypeptide library members. Selection is made by separately growing and expressing single clones expressing polypeptide library members, or by using any selective display system. In the case of bacteriophage display, phage or phagemide vectors can be used. Phagemide vectors have an E. coli origin (for double-stranded replication) and a phage origin (for single-stranded DNA generation).

[0145] Purification and concentration of monovalent antibodies Monovalent antibodies secreted into the periplasmic space or bacterial culture medium are collected and purified by known methods (Skerra, A. & Pluckthun, A., Science, 240:1038-41, 1988 and Breitling, F. et al. (Gene, 104:147-53, 1991) describe the collection of antibody polypeptides from periplasm, and Better, M. et al. (Science, 240:1041-3, The 1988 report describes collection from the culture supernatant. For some antibody polypeptides, purification can also be achieved by binding to common ligands such as protein A or protein L. Alternatively, it is possible to express variable domains using peptide tags such as Myc, HA, or 6×His tags. This method facilitates purification by affinity chromatography. If necessary, monovalent anti-properdin antibodies are enriched using one of several methods well known in the art, including ultrafiltration, diafiltration, and tangential flow filtration. In the ultrafiltration process, a semipermeable membrane and pressure are used to separate molecular species based on size and shape. Pressure is generated by gas pressure or centrifugation. By selecting a molecular weight cutoff smaller than the target antibody (typically 1 / 3 to 1 / 6 of the molecular weight of the target polypeptide), the anti-properdin antibody is retained as the solvent and small solute pass through the membrane.

[0146] Pharmaceutical composition, dosage and administration The antibodies described herein can be incorporated into pharmaceutical compositions suitable for administration to a subject. Generally, a pharmaceutical composition comprises a monovalent anti-properdin antibody, an antibody derivative or fragment thereof, and a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, coating, antimicrobial and antifungal agents, isotonic agents and absorption retarders. The term “pharmaceutically acceptable carrier” does not include tissue culture media containing bovine or equine serum. Examples of pharmaceutically acceptable carriers include one or more of water, physiological saline, phosphate-buffered saline, dextrose, glycerol, ethanol, and combinations thereof. In many cases, it is preferable to include an isotonic agent, such as sugar, polyhydric alcohols such as mannitol or sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable substances may include small amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers that enhance the shelf life or efficacy of the antibody.

[0147] The compositions described herein may take various forms. These include liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and intravenous solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The final form depends on the intended method of administration and therapeutic use. Typical compositions are in the form of injectable or intravenous solutions, such as compositions similar to those used for passive immunization of humans using other antibodies. The compositions are delivered, for example, by parenteral injection (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular).

[0148] Therapeutic compositions generally must be sterile and stable under manufacturing and storage conditions. Compositions can be formulated as solutions, microemulsions, dispersions, liposomes, or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the required amount of monovalent antiproperzine antagonist, along with one or a combination of the above components, into a suitable solvent as needed, and then sterilizing by filtration. Generally, dispersions are prepared by incorporating a monovalent antiproperzine antagonist into a sterile vehicle containing a basic dispersion medium and other necessary components listed above. For sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze-drying, from a pre-sterilized filtered solution, to obtain a powder of the active ingredient plus another desired component. Appropriate fluidity of solutions can be maintained, for example, by the use of coatings such as lecithin, maintaining the required particle size in the case of dispersions, and by the use of surfactants.

[0149] The antibodies described herein can be administered by various methods known in the art, but for many therapeutic applications, the preferred route / method of administration is intravenous injection or infusion. Polypeptides can also be administered by intramuscular injection or subcutaneous injection.

[0150] As will be understood by those skilled in the art, the route and / or method of administration varies depending on the desired outcome. In some embodiments, antibodies can be prepared with a carrier that protects them from rapid release, such as controlled-release formulations including implants, transdermal patches, and microencapsulated delivery systems. Monovalent single-domain antibodies are suitable in some sense for sustained-release formulations because they are smaller in size, i.e., allow for significantly more moles per dose than, for example, a full-sized antibody. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Long-term absorption of injectable compositions can be achieved by including absorption-delaying agents, such as monostearate and gelatin, in the composition. Many methods for preparing such formulations are known to those skilled in the art (e.g., Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978). Methods applicable to the controlled or sustained release of antibodies, such as monovalent single-domain antibodies, disclosed herein are known (including U.S. Patent Nos. 6,306,406 and 6,346,274, and U.S. Patent Applications Nos. US20020182254 and US20020051808, the entirety of which is incorporated herein by reference).

[0151] In some embodiments, a monovalent anti-properdin antibody, antibody derivative, or fragment thereof can be administered orally, for example, with an inactive diluent or an absorbable food carrier. To administer the compositions described herein by means other than parenteral administration, it may be necessary to coat the compounds with a substance to prevent their inactivation or to administer them together with that substance.

[0152] Other active compounds can also be incorporated into the composition. In some embodiments, a monovalent anti-properdin antibody, antibody derivative, or fragment thereof is formulated and / or administered together with one or more additional therapeutic agents. For example, a monovalent anti-properdin antibody, antibody derivative, or fragment thereof can be formulated and / or administered together with one or more additional antibodies that bind to other targets (e.g., antibodies that bind to regulators of the complement II pathway). Such combination therapies reduce the dosage of the therapeutic agents administered, thereby avoiding potential toxicities or complications associated with various monotherapies. Furthermore, the compositions described herein can be formulated or administered together with other therapeutic agents to mitigate the side effects of administering the compositions described herein (e.g., therapeutic agents that minimize the risk of infection in immunocompromised environments, such as antibacterial, antifungal, and antiviral agents).

[0153] The pharmaceutical composition may contain a “therapeutic dose” or a “preventive dose” of a monovalent antiproperdinine antagonist (e.g., an antibody or its derivative or fragment). “Therapeutic dose” means the amount effective in the required dosage and duration to achieve the desired therapeutic outcome. The therapeutic dose of an antibody may vary depending on factors such as the individual’s condition, age, sex, and weight, and the ability of the monovalent antiproperdinine antagonist to induce the desired response in the individual. “Preventive dose” means the amount effective in the required dosage and duration to achieve the desired preventive outcome. In some embodiments, a prophylactic dose is used for a subject before or in the early stages of disease, where the prophylactic dose is less than the therapeutic dose.

[0154] The dosage plan may be adjusted to provide the optimal desired response (such as a therapeutic or prophylactic response). For example, depending on the urgency of the treatment situation, a single bolus may be administered, or the dose may be administered over time in several divided doses, or the dose may be proportionally decreased or increased. To obtain ease of administration and uniformity of dosage, it is advantageous to formulate parenteral compositions in unit dosage forms. As used herein, a unit dosage form refers to a physically distinct unit suitable for the combined dosage for the mammalian subject being treated, each containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect when accompanied by the necessary pharmaceutical carrier. Note that the dosage values ​​may vary depending on the type and severity of the condition being alleviated. It should also be understood that for specific subjects, it may be necessary to adjust the specific dosage plan over time according to the individual needs and the professional judgment of the administering clinician.

[0155] The non-limiting therapeutic or prophylactic effective dose range for monovalent anti-properdin antibodies, antibody derivatives, or fragments is 0.1–20 mg / kg, more preferably 1–10 mg / kg. Note that the dose may vary depending on the type and severity of the condition being alleviated. Furthermore, it should be understood that specific dosing regimens may need to be adjusted over time for individual subjects according to their individual needs and the professional judgment of the administering clinician.

[0156] The efficacy of treatment with the monovalent anti-properdin antibodies, antibody derivatives, or fragments described herein is determined by a skilled clinician based on improvement in one or more symptoms or indicators of the disease or disorder being treated. An improvement of at least 10% (increasing or decreasing depending on the indicator being measured) in one or more clinical indicators is considered “effective treatment,” but greater improvements such as 20%, 30%, 40%, 50%, 75%, 90%, and even 100% are desirable, and depending on the indicator being measured, improvements exceeding 100% (e.g., 2x, 3x, 10x, including reaching a disease-free state) are desirable.

[0157] Use of monovalent anti-properzine antibodies The compositions described herein can be used in methods to treat diseases or disorders mediated by complement II dysregulation in individuals requiring treatment. This method involves administering a therapeutically effective dose to an individual a composition comprising a monovalent anti-properdin antibody, an antibody derivative, or a fragment thereof, preferably a composition comprising a single human immunoglobulin variable domain that binds to human properdin. In one embodiment, the monovalent anti-properdin antibody, antibody derivative, or fragment thereof described herein is useful in treating diseases mediated by complement II dysregulation by inhibiting the activation of the complement II pathway in mammals (such as humans). Such disorders include systemic lupus erythematosus and lupus nephritis, rheumatoid arthritis, antiphospholipid (aPL)Ab syndrome, glomerulonephritis, paroxysmal nocturnal hemoglobinuria (PNH) syndrome, inflammation, organ transplantation, intestinal and renal I / R disorders, asthma (such as severe asthma), atypical hemolytic uremic syndrome (aHUS), spontaneous fetal loss, DDD, macular degeneration, TTP, IgA nephropathy (Buerger's disease), and C3 Glomerulosis (C3G), Gaucher disease, hidradenitis suppurativa, Behçet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-associated lung injury (TRALI), hemodialysis-related thrombosis, acquired epidermolysis bullosa (EBA), uveitis, Parkinson's disease, primary biliary atresia, anti-neutrophil cytoplasmic antibodies (ANCA) vasculitis, retinal degeneration, extensive thrombotic microangiopathy (TMA), extensive TMA (APS), hematopoietic stem cell therapy (HSCT) TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes 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, dialysis (cardiovascular risk), cardiovascular disease, placental malaria, APS pregnancy loss, membranoproliferative (MP) glomerulonephritis, membranous nephritis, encephalitis, encephalopathy, NMDA receptor antibody encephalitis, malaria hemolytic crisis, abdominal aortic aneurysm (AAA) and thoracoabdominal aortic aneurysm (TAA). [Examples]

[0158] The following examples are provided to those skilled in the art to disclose and illustrate how the claimed method is carried out and how the compounds are produced. They are intended to be purely illustrative and not to limit the scope of this disclosure.

[0159] Example 1 V HH - Preparation of His In-Fusion Cloning Vectors The pBNJ391 vector was digested with restriction enzymes BstEII and EcoRI to remove the hinge and Fc. Gel purification of this vector yielded a 1000 bp release product. The annealed oligonucleotides UDEC6629 / 6630 were cloned into the pBNJ391 vector using BstEII / EcoRI. The annealed oligonucleotides contained the following sequences:

[0160] UDEC 6629 Forward Primer: GTCACCGTGTCGAGCCATCATCACCATCATCACTGATGAG (Sequence ID 65) UDEC 6630 Reverse Primer: AATTCTCATCATTTGTCATCATCATCCTTATAGTCGCTCGACACG (Sequence ID 66) The final vector contained the BstEII-6×His-EcoRI region.

[0161] Next, the pNGH0320 vector was digested with XhoI / BstEII (producing a 13 bp release product) and purified by column. Next, V HHThe insert was PCR amplified using a phage clone template. Forward primer UDEC 6438 (GTCCACTCCCTCGAGGTGCAGCTGGTGGAGTCTGGG, SEQ ID NO: 67) and reverse primer UDEC 6442 (GCTCGACACGGTGACCTGGGTCCCCTGGCCCCA, SEQ ID NO: 68) were used. After purification of the PCR product, it was used for cloning in an in-fusion protocol.

[0162] The pBNJ391 vector was digested with BstEII / EcoRI (50 ng / μL). Both complementary oligonucleotides were resuspended at the same molar concentration using TE buffer. Equal volumes of both complementary oligonucleotides (equimolar concentrations) were mixed in 1.5 mL tubes. The tubes were placed on a standard heat block at 90–95°C for 3–5 minutes. The tubes were removed from the instrument and allowed to cool naturally to room temperature (or at least below 30°C). The tubes were stored on ice or at 4°C until further use.

[0163] A ligation reaction mixture was prepared using 1 μL of insert DNA (from the nucleotides used for ligation to pBNJ391), 2 μL of pBNJ391 (EcoRI / BstEII, 100 ng), 1 μL of 10× ligase buffer (NEB B0202S lot: 11091410), 1 μL of T4 DNA ligase (NEB M0202L lot: 0671502), and 5 μL of water. The ligation reaction mixture was incubated at room temperature for 30 minutes. 1 μL of the ligation reaction mixture was used to transform 30 μL of DH10 chemically competent cells (InVitrogen 18297 lot number 1552241), and 750 μL of SOC (NEBB9020S lot number 2971403) was added. The tubes were shaken at 37°C for 1 hour, and then seeded in 10 μL and 100 μL portions on LB carbohydrate / glucose plates. The plates were incubated at room temperature over the weekend and into the following week.

[0164] Colonies were selected for PCR to insert 6×His into pNGH0320. Eight colonies were screened, and pBNJ391 was used as a negative control. 300 μL of TB / Carb / Glucose culture solution was added to the isolated colonies and grown at 37 °C. Forward primer UDEC5276 (CATAATAGCTGACAGACTAACAGACTG, SEQ ID NO: 69) and reverse primer UDEC1977 (CGAAACAAGCGCTCATGAGCCCGAAGT, SEQ ID NO: 70) were used. For a 20 μL PCR reaction, DNA from a single colony was added to a total of 20 μL of 10 μL Go Taq Green PCR Mix, 0.2 μL forward primer (100 μM), 0.2 μL reverse primer (100 μM), and 9.6 μL H2O. The PCR conditions were as follows. 3 minutes at 95 °C, 20 seconds at 95 °C, 20 seconds at 50 °C, 1 minute 15 seconds at 72 °C. After repeating this cycle 30 times, it was incubated at 72 °C for 5 minutes and stored at 4 °C until further use. 5 μL of the PCR product was mixed with 15 μL of water and electrophoresed on a 2% E gel. Two clones matched the predicted size. A plasmid maxi prep cultured overnight using the Promega maxi prep kit was used.

[0165] V to pNGH0320 HH using in-fusion ligation of V HH in His-tag format anti-propelludin V HH To clone the antibody, V from the Llama anti-propelludin library pLNJ with an XhoI site for cloning into pNGH0317 by in-fusion H For amplification of the phagemid, PCR was performed using the UDEC 6438-Infusion forward primer and the UDEC 6442-Infusion reverse primer HHInserts were prepared. For a 60 μL PCR reaction, 30 μL of 2x fusion PCR mix (NEB M0531s lot: 0211412), 1 μL of bacterial culture, 0.1 μL of forward primer UDEC 6438 (100 μM), 0.1 μL of reverse primer UDEC 6442 (100 μM), and 28.8 μL of H2O were used, for a total of 20 μL. The PCR conditions were as follows: 3 minutes at 98°C, 10 seconds at 98°C, 15 seconds at 52°C, 30-60 seconds at 72°C, followed by 5 minutes at 72°C. This cycle was repeated 30 times, and the mixture was then held at 4°C until use. 5 μL of the PCR product was mixed with 15 μL of water and run on a 2% E gel. All clones matched the predicted size. Clones were pooled after 8 reactions, and the columns were purified using PromegaWizard® SV Gel and PCR Clean-Up System according to the manufacturer's instructions. Plasmid maxi prep cultured overnight in the Promega maxi prep kit was used.

[0166] For insert ligation, a ligation reaction mixture was prepared using 2 μL of 5× In-Fusion HD Enzyme Premix (Clontech 639650 lot: 1501713A), 2.5 μL of Vector pNGH0320 (XhoI / BstEII) 39.1 ng / L (100 ng), 1 μL of purified PCR fragment (10-200 ng), and 4.5 μL of water. The ligation reaction mixture was incubated at 50°C for 15 minutes.

[0167] For transformation, Stellar® competent cells (Clontech) were thawed in an ice bath immediately before use. After thawing, the cells were gently mixed to ensure uniform distribution, and 50 μL of competent cells were transferred to a 14 mL round-bottom tube (Falcon tube). 1 μL (less than 5 ng of DNA) was added to the cells. The tube was placed on ice for 30 minutes. Next, the cells were subjected to a heat shock at 42°C for exactly 45 seconds. After that, the tube was placed on ice for 1-2 minutes. SOC medium was added to a final volume of 500 μL (the SOC medium was warmed to 37°C before use). The tube was incubated at 37°C for 1 hour with shaking (160-225 rpm). Next, 10 μL of this solution was seeded onto an LB plate containing carbenicillin. The plate was incubated overnight at 37°C.

[0168] 24 V in each pool HH Colony PCR screening was performed for colony insertion. A total of 48 clones were selected from each pool. Vector pNGH0320.1 was used as a positive control. Forward primer UDEC5276 and reverse primer UDEC1977 were used. In a 20 μL PCR reaction, single colony DNA was added to a total of 20 μL of Go Taq Green PCR Mix (100 μM), forward primer (100 μM), reverse primer (100 μM), and H2O (9.6 μL). The PCR conditions were as follows: 3 minutes at 95°C, 20 seconds at 95°C, 20 seconds at 50°C, and 1 minute 15 seconds at 72°C. This cycle was repeated 30 times, then maintained at 72°C for 5 minutes, and then held at 4°C until use. 5 μL of the PCR product was mixed with 15 μL of water and run on a 2% E gel. Sequence analysis was performed on all 48 clones.

[0169] Immune-biased Lama V HH Preliminary screening of the phage display library revealed 192 V phages that were ELISA-positive for propergine binding. HH It was identified as 57 V HH These were cloned and expressed with a 6× histidine tag. Of these, 34 V HHThe test was Octet-positive for propergine binding. A summary is shown in Table 1 below.

[0170] [Table 1] Four types of functional V for effectively inhibiting complement pathway II-mediated hemolysis HH These were found and are shown in Table 2 below.

[0171] [Table 2] Example 2 Anti-properdin V against human properdin HH Antibody binding Figure 1 shows that kinetic binding measurements can be performed using an Octet instrument (ForteBio Inc.). Washing, dilution, and measurement were all performed with Kinetic buffer (ForteBio catalog 185032) while shaking the plate at 1000 rpm. A streptavidin biosensor (Forte Bio catalog: 18-5019 lot: 1405301) was equilibrated in Kinetic buffer for 10 minutes, and then loaded with 50 nm biotinylated human propergin. In the association phase, a selected anti-propergin antibody or a 10 μg / mL kinetic buffer blank was added to the biosensor preloaded with biotinylated human propergin, respectively. These results show the binding of AB005, AB006, AB007, and AB008 to human propergin.

[0172] Figure 2 shows that kinetic binding measurements can be performed using an Octet instrument (ForteBio Inc.). Washing, dilution, and measurement were all performed with Kinetic buffer (ForteBio catalog 185032) while shaking the plate at 1000 rpm. A streptavidin biosensor (Forte Bio catalog: 18-5019 lot: 1405301) was equilibrated in Kinetic buffer for 10 minutes, and then loaded with 50 nm biotinylated mouse propergine. In the association phase, a selected anti-propergine antibody or a 10 μg / mL kinetic buffer blank was added to the biosensor preloaded with biotinylated human propergine, respectively. These results show weak binding or no binding at all for AB005, AB006, AB007, and AB008.

[0173] Figure 3 shows that kinetic binding measurements can be performed using an Octet instrument (ForteBio Inc.). Washing, dilution, and measurement were all performed with Kinetic buffer (ForteBio catalog 185032) while shaking the plate at 1000 rpm. A streptavidin biosensor (Forte Bio catalog: 18-5019 lot: 1405301) was equilibrated in Kinetic buffer for 10 minutes, and then loaded with 50 nm of biotinylated cynomolgus monkey propergine. In the association phase, a selected anti-propergine antibody or a 10 μg / mL kinetic buffer blank was added to the biosensor preloaded with biotinylated human propergine, respectively. These results show weak binding of AB005, AB006, AB007, and AB008 to cynomolgus monkey propergine.

[0174] Example 3: Complement Second Pathway Hemolysis Assay Figure 4 shows a complement II pathway-mediated hemolysis assay based on the formation of terminal complement complexes on the surface of rabbit erythrocytes (rRBCs). The formation of these complexes leads to the lysis of rRBCs. Drugs that inhibit complement complex formation are expected to inhibit cytolysis. Various anti-properdin antigen-binding fragments were tested to evaluate their effect on cytolysis mediated by the activation of complement II. Assay plates were prepared by diluting 40% normal human serum with gelatin veronal buffer (GVB) supplemented with 10 mM EGTA and 10 mM MgCl2 (e.g., 1600 μL of normal human serum in 2400 μL of GVB supplemented with 10 mM EGTA and 10 mM MgCl2). 50 μL of this solution was distributed to each well of an assay plate (polystyrene). Next, dilution plates (polypropylene) were prepared by adding 50 μL / well of 2×mAbs (e.g., anti-properzine Fab) in GVB containing 10 mM EGTA and 10 mM MgCl2 at concentrations ranging from 0 to 100 nM to the appropriate wells. Rabbit erythrocytes were incubated in distilled water as a positive control (100% cell lysis), and erythrocytes were incubated in GVB containing 10 mM EDTA and 10 mM MgCl2, respectively, as negative controls (0% cell lysis).

[0175] 50 μL / well was transferred from the dilution plate to the assay plate. The assay plate was left at room temperature while the next step was performed. 400 μL of rRBC was washed four times with 1 mL of GVB containing 10 mM EGTA and 10 mM MgCl2. After each wash, the rRBC was rotated at 2600 rpm for 1 minute. After the final wash, 300 μL of GVB containing 10 mM EGTA and 10 mM MgCl2 was added to resuspend the rRBC to a volume of 400 μL. 50 μL of the washed rRBC was resuspended in 1 mL of GVB containing 10 mM EGTA and 10 mM MgCl2. 30 μL of this dilution was added to 100 μL of the sample prepared on the assay plate, resulting in 1.5 × 10⁶ per well. 6A number of cells were obtained. The plate was incubated at 37°C for 30 minutes. Next, the plate was centrifuged at 1000×g for 5 minutes, and 85 μL of the supernatant was transferred to a 96-well flat-bottom plate. Hemolysis was determined by measuring OD at 415 nm. At 415 nm, a stepwise decrease in light scattering (due to lysis of intact cells) was measured as a function of concentration. For calculations, anti-properdinine V HH The total inhibition was calculated at each concentration, and the results were expressed as the percentage of unlisted controls.

[0176] Example 4 Monovalent antiproperdin V against properdin HH Antibody binding kinetics In Figure 6, anti-properdin V HH Antibodies AB007 and AB008 were applied to the immobilized sensor surface at known concentrations, respectively. The response level (RU) was plotted against the sensor gram time.

[0177] Anti-properdin V HH The binding affinity of the antibody was determined. The results are summarized in Table 3 below.

[0178] [Table 3] Example 5: Binding kinetics and complement II hemolysis assay of a bispecific anti-propergin antibody against propergin. Based on the above anti-properdin construct linked to an anti-albumin construct, a bispecific construct was created. Binding to properdin and complement hemolysis was measured using the same assay as above. The construct sequences are shown in Table 4 below.

[0179] [Table 4] TIFF2026102797000005.tif178170 TIFF2026102797000006.tif178170 TIFF2026102797000007.tif215170 Figure 7 shows kinetic coupling measurements performed with an Octet instrument (ForteBio Inc.). Washing, dilution, and measurement were all performed with Kinetic buffer (ForteBio catalog 185032) while shaking the plate at 1000 rpm. A streptavidin biosensor (Forte Bio catalog 18-5019, lot 1405301) was equilibrated in Kinetic buffer for 10 minutes, and then loaded with 50 nm biotinylated human propergin. In the association phase, a selected anti-propergin antibody or a 10 μg / mL Kinetic buffer blank was added to the biosensor preloaded with biotinylated human propergin, respectively. These results demonstrate the binding of TPP-2225, TPP-2591, TPP-3071, TPP-3072, and TPP-3261 to human propergin. These results show strong binding to human propergin by all constructs.

[0180] Figures 8A and 8B show the results of a complement II pathway-mediated hemolysis assay based on the formation of terminal complement complexes on the surface of rabbit erythrocytes (rRBCs). The formation of these complexes leads to the lysis of rRBCs. Drugs that inhibit complement complex formation are expected to inhibit cytolysis. Various bispecific anti-properdin antigen-binding constructs were tested to evaluate their effect on cytolysis mediated by the activation of the second complement pathway. Assay plates were prepared by diluting 40% normal human serum with gelatin veronal buffer (GVB) supplemented with 10 mM EGTA and 10 mM MgCl2 (e.g., 1600 μL of normal human serum in 2400 μL of GVB supplemented with 10 mM EGTA and 10 mM MgCl2). 50 μL of this solution was distributed to each well of an assay plate (polystyrene). Next, dilution plates (polypropylene) were prepared by adding 50 μL / well of 2×mAbs (such as anti-properzine Fab) in GVB containing 10 mM EGTA and 10 mM MgCl2 at concentrations ranging from 0 to 100 nM to the appropriate wells. As a positive control, rabbit erythrocytes were incubated in distilled water (100% cell lysis), and as a negative control, erythrocytes were incubated in GVB containing 10 mM EDTA and 10 mM MgCl2, respectively (0% cell lysis).

[0181] 50 μL / well was transferred from the dilution plate to the assay plate. The assay plate was left at room temperature while the next step was performed. 400 μL of rRBC was washed four times with 1 mL of GVB containing 10 mM EGTA and 10 mM MgCl2. After each wash, the rRBC was rotated at 2600 rpm for 1 minute. After the final wash, 300 μL of GVB containing 10 mM EGTA and 10 mM MgCl2 was added to resuspend the rRBC to a volume of 400 μL. 50 μL of the washed rRBC was resuspended in 1 mL of GVB containing 10 mM EGTA and 10 mM MgCl2. 30 μL of this dilution was added to 100 μL of the sample prepared on the assay plate, resulting in 1.5 × 10⁶ per well. 6A number of cells were obtained. The plate was incubated at 37°C for 30 minutes. Next, the plate was centrifuged at 1000×g for 5 minutes, and 85 μL of the supernatant was transferred to a 96-well flat-bottom plate. Hemolysis was determined by measuring OD at 415 nm. At 415 nm, a stepwise decrease in light scattering (due to lysis of intact cells) was measured as a function of concentration. For calculations, total inhibition was calculated at each concentration of the anti-properdin antibody construct, and the results were expressed as a percentage of unlisted controls.

[0182] Figures 8A and 8B show hemolysis mediated by TPP-2221, TP-2222, TP-2223, TP-2224, and TP-2225 in human (Figure 8A) and cynomolgus monkey (Figure 8B) serum. The control antibody is anti-properzine antibody. Figures 9A and 9B show hemolysis mediated by TPP-2225, TPP-2951, TPP-3261, TPP-3071, and TPP-3072 in human (Figure 9A) and cynomolgus monkey (Figure 9B) serum.

[0183] Figures 10A to 10B, 11A to 11B, and 12A to 12B show the binding kinetics of TPP-3261, TPP-2951, and TPP-2225 to human properdin and cynomolgus monkey properdin, respectively.

[0184] Tables 5-9 below show the binding affinity and IC50 value for each construct.

[0185] [Table 5]

[0186] [Table 6]

[0187] [Table 7]

[0188] [Table 8]

[0189] [Table 9]

[0190] Other Embodiments All publications, patents, and patent applications referenced herein shall be invoked to the same extent as if they were explicitly and individually referenced from their respective independent publications or patent applications.

[0191] The compositions and methods described herein are subject to further modification, and it will be understood that this description is applicable to the essential features described above and is intended to include any modifications, uses, or adaptations that conform to the principles broadly disclosed herein, including any deviations from this disclosure that fall within the scope of known or customary practices in the art as defined in the claims.

Claims

1. An isolated monovalent antibody or a fragment of that antibody that binds to human propergine.

2. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment is an antibody of a camelid animal.

3. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment is a single-domain antibody.

4. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment binds to the TSR0 and / or TSR1 of human properdin.

5. The antibody or fragment according to claim 1, wherein the antibody or fragment binds to an epitope in the amino acid sequence LCQPCRSPRWSLWSTWAPCSVTCSEGSQLRYRRCVGWNGQ (Sequence ID 8).

6. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment binds to mouse propergin with an affinity of less than 50 nM.

7. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment is humanized.

8. The antibody or fragment thereof according to claim 1, wherein the antibody or fragment is linked to a second monovalent antibody or antibody fragment.

9. The antibody or antibody fragment according to claim 8, wherein the second monovalent antibody or antibody fragment is humanized.

10. The antibody or fragment thereof according to claim 8, wherein the antibody or fragment is linked to the second antibody or fragment by a linker.

11. The antibody or fragment thereof according to claim 10, wherein the linker is a polyglycine linker.

12. The antibody or fragment thereof according to claim 11, wherein the polyglycine linker comprises the sequence GGGGD (SEQ ID NO: 63), GGGGS (SEQ ID NO: 1), GGGGA (SEQ ID NO: 100), or GGGGE (SEQ ID NO: 64).

13. The antibody or antibody fragment according to claim 8, wherein the second monovalent antibody or antibody fragment specifically binds to albumin.

14. The antibody or fragment thereof according to claim 8, wherein the second monovalent antibody or fragment thereof is linked to the N-terminus of the antibody or antibody fragment that binds to human propergin.

15. The antibody or fragment thereof according to claim 8, wherein the antibody is linked to an albumin-binding peptide.

16. The antibody or fragment has three CDRs having the following sequence, i.e., a) CDR-H1 containing the amino acid sequence GRISSIIHMA (SEQ ID NO: 16), b) CDR-H2 containing the amino acid sequence RVGTTVYADSVKG (SEQ ID NO: 12), c) CDR-H3 containing the amino acid sequence LQYEKHGGADY (SEQ ID NO: 17), The antibody or fragment thereof according to claim 1, comprising:

17. The antibody or fragment is a) CDR-H1 containing the amino acid sequence GRIFEVNMMA (SEQ ID NO: 9), b) Amino acid sequence RVGTTX 1 Includes YADSVKG (Sequence ID 10), X １ CDR-H2 is a polar amino acid or a nonpolar amino acid. c) Amino acid sequence LQYX 2 Includes RYGGAEY (SEQ ID NO: 11), X ２ The antibody or fragment thereof according to claim 1, comprising at least one or all of three CDRs selected from CDR-H3, which is a polar amino acid.

18. The antibody according to claim 17, wherein CDR-H2 comprises the amino acid sequence RVGTTVYADSVKG (SEQ ID NO: 12).

19. The antibody according to claim 18, wherein CDR-H3 comprises the amino acid sequence LQYDRYGGAEY (SEQ ID NO: 13).

20. The antibody according to claim 17, wherein CDR-H2 comprises the amino acid sequence RVGTTTYADSVKG (SEQ ID NO: 15).

21. The antibody according to claim 20, wherein CDR-H3 has the amino acid sequence LQYSRYGGAEY (SEQ ID NO: 14).

22. The antibody according to claim 20, wherein CDR-H3 has the amino acid sequence LQYDRYGGAEY (SEQ ID NO: 13).

23. The antibody according to claim 17, wherein CDR-H3 has the amino acid sequence LQYDRYGGAEY (SEQ ID NO: 13).

24. The antibody according to claim 17, wherein CDR-H3 has the amino acid sequence LQYSRYGGAEY (SEQ ID NO: 14).

25. The antibody has six CDRs having the following sequence, i.e., a) CDR-H1 containing the amino acid sequence GYIFTNYPIH (SEQ ID NO: 18), b) CDR-H2 containing the amino acid sequence FIDPGGGYDEPDERFRD (SEQ ID NO: 19), c) CDR-H3 containing the amino acid sequence RGGGYYLDY (SEQ ID NO: 20), d) CDR-L1 containing the amino acid sequence RASQDISFFLN (SEQ ID NO: 21), e) CDR-L2 containing the amino acid sequence YTSRYHS (SEQ ID NO: 22), f) CDR-L3 containing the amino acid sequence QHGNTLPWT (SEQ ID NO: 23), The antibody or fragment thereof according to claim 1, comprising:

26. The antibody has six CDRs having the following sequence, i.e., a) CDR-H1 containing the amino acid sequence GFSLTTYGVH (SEQ ID NO: 24), b) CDR-H2 containing the amino acid sequence VIWSGGDTDYNASFIS (SEQ ID NO: 25), c) CDR-H3 containing the amino acid sequence NKDYYTNYDFTMDY (SEQ ID NO: 26), d) CDR-L1 containing the amino acid sequence KSSQSVLYSSNQKNFLA (SEQ ID NO: 27), e) CDR-L2 containing the amino acid sequence WASTRES (SEQ ID NO: 28), f) CDR-L3 containing the amino acid sequence HQYLSSYT (SEQ ID NO: 29), The antibody or fragment thereof according to claim 1, comprising:

27. The antibody has six CDRs having the following sequence, i.e., a) CDR-H1 containing the amino acid sequence GYTFIDYWIE (SEQ ID NO: 30), b) CDR-H2 containing the amino acid sequence EIFPGSGTINHNEKFKD (SEQ ID NO: 31), c) CDR-H3 containing the amino acid sequence EGLDY (SEQ ID NO: 32), d) CDR-L1 containing the amino acid sequence SASSSVSYIY (SEQ ID NO: 33), e) CDR-L2 containing the amino acid sequence DTSTLAS (SEQ ID NO: 34), f) CDR-L3 containing the amino acid sequence QQWSRNPFT (SEQ ID NO: 35), The antibody or fragment thereof according to claim 1, comprising:

28. The antibody has six CDRs having the following sequence, i.e., a) CDR-H1 containing the amino acid sequence GFSLTSYGVH (SEQ ID NO: 36), b) CDR-H2 containing the amino acid sequence VIWSGGSTDYNAAFIS (SEQ ID NO: 37), c) CDR-H3 containing the amino acid sequence NKDFYSNYDYTMDY (SEQ ID NO: 38), d) CDR-L1 containing the amino acid sequence KSSQSVLYSSNQKNFLA (SEQ ID NO: 27), e) CDR-L2 containing the amino acid sequence WASTRES (SEQ ID NO: 28), f) CDR-L3 containing the amino acid sequence HQYLSSYT (SEQ ID NO: 29), The antibody or fragment thereof according to claim 1, comprising:

29. The antibody has six CDRs having the following sequence, i.e., a) CDR-H1 containing the amino acid sequence GYTXTAYGIN (SEQ ID NO: 39), b) CDR-H2 containing the amino acid sequence YIYIGNGYTDYNEKFKG (SEQ ID NO: 40), c) CDR-H3 containing the amino acid sequence SGWDEDYAMDF (SEQ ID NO: 41), d) CDR-L1 containing the amino acid sequence RASENIYSYLA (SEQ ID NO: 42), e) CDR-L2 containing the amino acid sequence HAKTLAE (SEQ ID NO: 43), f) CDR-L3 containing the amino acid sequence QHHYGPPPT (SEQ ID NO: 44), The antibody or fragment thereof according to claim 1, comprising:

30. The antibody or fragment thereof according to claim 1 or 8, wherein the antibody or fragment inhibits the activity of human properdin.

31. A method for treating a disease mediated by dysregulation of the second complement pathway, comprising administering an effective dose of the antibody described in claim 1 or 8 to a patient in need thereof.

32. The method according to claim 31, wherein the disease is autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), or paroxysmal nocturnal hemoglobinuria (PNH).

33. A method for inhibiting complement second pathway membrane invasion complex assembly, comprising administering an effective dose of the antibody described in claim 1 or 8 to a patient in need thereof.

34. The method according to claim 33, which inhibits complement pathway 2-dependent hemolysis.

35. A pharmaceutical composition comprising an antibody or fragment thereof as described in claim 1 or 8 as an active ingredient, and a pharmaceutically acceptable carrier.

36. An antibody or fragment thereof according to claim 1 or 8, used as a pharmaceutical product.

37. An antibody or fragment thereof for use according to claim 1 or 8, used for the treatment of diseases mediated by dysregulation of the second complement pathway.

38. The aforementioned diseases include autoimmune thrombotic thrombocytopenic purpura (TTP), hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), IgA nephropathy (Buerger's disease), C3 glomerulosis (C3G), asthma, Gaucher disease, hidradenitis suppurativa, Behçet's disease, dermatomyositis, severe burns, early sepsis, pneumococcal meningitis, Alzheimer's disease, cancer metastasis, acute respiratory distress syndrome (ARDS), acute lung injury (ACI), transfusion-associated lung injury (TRALI), thrombosis due to hemodialysis, acquired epidermolysis bullosa (EBA), uveitis, Parkinson's disease, primary biliary atresia, anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, retinal degeneration, extensive thrombotic microangiopathy (TMA), extensive TMA (APS), and hematopoietic stem cell therapy (H An antibody or fragment thereof for use according to claim 37, which is SCT)TMA, age-related macular degeneration (AMD), pre-eclampsia, hemolysis, elevated liver enzymes 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, membranoproliferative (MP) glomerulonephritis, membranous nephritis, encephalitis, brain injury, N-methyl-D-aspartate (NMDA) receptor antibody encephalitis, malaria hemolytic crisis, abdominal aortic aneurysm (AAA) or thoracoabdominal aortic aneurysm (TAA).

39. An isolated antibody or fragment thereof, wherein the antibody comprises LEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSSRKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 53).

40. An isolated antibody or fragment thereof, wherein the antibody comprises LEVQLVESGGGLVQAGGSLRLSCAASGRISSIIHMAWYRQAPGKQRELVAEISRVGTTVYADSVKGRFTISRDDAKNTVTLQMNSLKPEDTAVYYCNALQYEKHGGADYWGQGTQVTVSSPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54).

Images