Anti-FLT-1 antibodies for the treatment of Duchenne muscular dystrophy

Abstract

The present invention particularly provides anti-Flt-1 antibodies and methods for treating muscular dystrophy, specifically Duchenne muscular dystrophy (DMD). In some embodiments, the methods according to the present invention include administering an effective amount of an anti-Flt-1 antibody or an antigen binding protein thereof to an individual suffering from or susceptible to DMD, such that at least one symptom or feature of DMD is reduced in intensity, severity, or frequency or the onset is delayed.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680032814.0, filed on April 7, 2016, entitled "Anti-FLT-1 Antibody for the Treatment of Duchenne Muscular Dystrophy". The original application was the Chinese national phase application of PCT International Application No. PCT / US2016 / 026352, which claimed priority to U.S. Provisional Application Serial Nos. 62 / 144,251 and 62 / 307,645, filed on April 7, 2015 and March 14, 2016, respectively.

[0002] Related applications

[0003] This application claims priority to U.S. Provisional Application Serial No. 62 / 144,251, filed April 7, 2015, and U.S. Provisional Application Serial No. 62 / 307,645, filed March 14, 2016, the disclosures of which are hereby incorporated by reference. background

[0004] Duchenne muscular dystrophy (DMD) is an X-linked recessive disease affecting an estimated 1 in 3,600 male births, with an estimated 50,000 individuals worldwide affected. The disease is characterized by progressive muscle wasting, and affected children become wheelchair-dependent by age 13. Affected individuals typically develop symptoms by age 3, with a median survival of 25 to 30 years. Respiratory failure due to diaphragmatic weakness and cardiomyopathy is a common cause of death.

[0005] DMD is caused by mutations in the dystrophin gene. The dystrophin gene is located on the X chromosome and encodes the protein dystrophin. Dystrophin is responsible for connecting muscle fibers to the surrounding extracellular matrix via the dystrophin-myosin complex, a dystrophin-glycan complex. Mutations in the dystrophin gene lead to alterations or absence of dystrophin and abnormal sarcolemalmbrane function. While both men and women can carry mutations in the dystrophin gene, women are rarely affected by DMD.

[0006] A characteristic of DMD is localized ischemia in the affected tissue. Localized ischemia is a restriction or reduction in the blood supply to a tissue or organ, resulting in insufficient oxygen and nutrients needed for cellular metabolism. Localized ischemia is usually caused by vasoconstriction or occlusion, leading to damage to or dysfunction of the tissue or organ. Treatment for localized ischemia aims to increase blood flow to the affected tissue or organ.

[0007] Currently, there is no cure for DMD. Several treatment approaches have been investigated, including gene therapy and corticosteroid administration; however, alternatives are still needed for DMD patients. Invention Overview

[0008] This invention particularly provides improved methods and compositions for treating muscular dystrophy, specifically Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy, based on anti-Flt-1 antibody therapy. As described in the examples below, this invention is based in part on the finding that anti-Flt-1 antibodies or their antigen-binding fragments can inhibit the binding of VEGF and other ligands to the Flt-1 receptor, thereby increasing the amount of VEGF and / or other ligands available for binding to the VEGF receptor. Increased VEGF availability promotes angiogenesis, accompanied by increased blood flow to the muscle to counteract functional ischemia and improve the structural and functional characteristics of DMD. Indeed, as shown in the embodiments of this invention, the inventors have demonstrated that administration of anti-Flt-1 antibodies improves measurements of muscle pathology (e.g., improved angiogenesis, reduced fibrosis, reduced necrosis). Therefore, this invention provides a safe and effective antibody-based therapeutic agent for treating DMD.

[0009] On one hand, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, said antibody or antigen-binding fragment comprising one or more complementarity-determining regions (CDRs), said complementarity-determining regions being selected from the group consisting of: variable light (VL) chain CDR1, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:19 to SEQ ID NO:21; VL CDR2, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:22 to SEQ ID NO:24; VL CDR3, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:25 to SEQ ID NO:34; variable heavy (VH) chain CDR1, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:1 to SEQ ID NO:4; VH CDR2, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:5 to SEQ ID NO:14; and VHCDR3, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:19 to SEQ ID NO:21; and VHCDR3, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:22 to SEQ ID NO:24; and VH CDR3, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:25 to SEQ ID NO:34; and VH CDR2, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:25 to SEQ ID NO:34; and VHC CDR3, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:25 to SEQ ID NO:34; and VH CDR3, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO Defined as any one of the amino acid sequences NO:15 to SEQ ID NO:18 having at least 80% identity.

[0010] In some embodiments, the one or more CDRs include VL CDR3, which is defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:25 to SEQ ID NO:34; and VH CDR3, which is defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:15 to SEQ ID NO:18.

[0011] In another embodiment, the one or more CDRs include VL CDR1, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:19 to SEQ ID NO:21; VL CDR2, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:22 to SEQ ID NO:24; and VLCDR3, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:25 to SEQ ID NO:34. In one specific embodiment, the VL chain comprises VL CDR1, VL CDR2, and VL CDR3, defined by the amino acid sequences of SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25, respectively. In another embodiment, the VL chain comprises VL CDR1, VLCDR2, and VL CDR3, defined by the amino acid sequences of SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:25, respectively. In another embodiment, the VL chain comprises VL CDR1 and VL CDR2 defined by the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:24, respectively, and VL CDR3 defined by the amino acid sequences of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34. In one specific embodiment, the VL chain comprises VL CDR1, VL CDR2, and VLCDR3 defined by the amino acid sequences of SEQ ID NO:21, SEQ ID NO:24, and SEQ ID NO:32, respectively.

[0012] In other embodiments, the one or more CDRs include VH CDR1, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:1 to SEQ ID NO:4; VH CDR2, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:5 to SEQ ID NO:14; and VH CDR3, defined by an amino acid sequence having at least 80% identity with any one of SEQ ID NO:15 to SEQ ID NO:18. In one specific embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3, defined by the amino acid sequences of SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:15, respectively. In another embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3, defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:6, and SEQ ID NO:16, respectively. In another embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:18, respectively. In another embodiment, the VH chain comprises VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:17, respectively, and VHCDR2 defined by the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:14. In another embodiment, the VH chain comprises VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:17, respectively, and VH CDR2 defined by the amino acid sequences of SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:12. In another embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3, defined by the amino acid sequences of SEQ ID NO:4, SEQ ID NO:9, and SEQ ID NO:17, respectively. In a specific embodiment, the VH chain comprises VH CDR1, VH CDR2, and VH CDR3, defined by the amino acid sequences of SEQ ID NO:3, SEQ ID NO:12, and SEQ ID NO:17, respectively.

[0013] On the other hand, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, said antibody or antigen-binding fragment comprising: (i) a light chain variable (VL) region comprising an amino acid sequence having at least 80% identity with any one of SEQ ID NO:49 to SEQ ID NO:61; and / or (ii) a heavy chain variable (VH) region comprising an amino acid sequence having at least 80% identity with any one of SEQ ID NO:35 to SEQ ID NO:48. In one specific embodiment, the VL region comprises the amino acid sequence of SEQ ID NO:60, and the VH region comprises the amino acid sequence of SEQ ID NO:45.

[0014] In some embodiments, the antibody further comprises a heavy chain constant region containing an amino acid sequence having at least 80% identity with any one of SEQ ID NO:87 to SEQ ID NO:89.

[0015] On the other hand, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, said antibody or antigen-binding fragment comprising: (i) a light chain comprising an amino acid sequence having at least 80% identity with any one of SEQ ID NO:75 to SEQ ID NO:86; and / or (ii) a heavy chain comprising an amino acid sequence having at least 80% identity with any one of SEQ ID NO:62 to SEQ ID NO:74. In one specific embodiment, the light chain comprises the amino acid sequence of SEQ ID NO:76, and the heavy chain comprises the amino acid sequence of SEQ ID NO:71.

[0016] In another embodiment, the antibody or its antigen-binding fragment is selected from the group consisting of IgG, F(ab')2, F(ab)2, Fab', Fab, ScFv, diabody, triabody, and tetrabody. In one embodiment, the antibody or its antigen-binding fragment is IgG. In another embodiment, the antibody or its antigen-binding fragment is IgG1. In another embodiment, the antibody or its antigen-binding fragment is a monoclonal antibody. In a specific embodiment, the antibody is a humanized monoclonal antibody. In another embodiment, the humanized monoclonal antibody contains a human Fc region. In some embodiments, the Fc region contains one or more mutations that enhance the binding affinity between the Fc region and the FcRn receptor to prolong the in vivo half-life of the antibody. In another embodiment, the Fc region contains one or more mutations at positions corresponding to Leu 234, Leu 235, and / or Gly 237 of human IgG1.

[0017] In one embodiment, the antibody or its antigen-binding fragment does not bind to VEGF R2 and / or VEGF R3. In another embodiment, the antibody or its antigen-binding fragment does not bind to mouse or monkey Flt-1.

[0018] On the other hand, the present invention provides an isolated antibody or antigen-binding fragment thereof that recognizes a peptide or fragment thereof comprising the following amino acid sequences, said amino acid sequences corresponding to positions 139 to 148, 139 to 153, 178 to 206, 199 to 204, and 128 to 138 of SEQ ID NO:90. In one embodiment, the peptide comprises the following amino acid sequences, said amino acid sequences corresponding to positions 130 to 138, 141 to 148, 141 to 153, and 193 to 206 of SEQ ID NO:90.

[0019] On the other hand, the present invention provides a separate antibody or antigen-binding fragment thereof that competes with any anti-Flt-1 antibody or its antigen-binding fragment.

[0020] On the other hand, the present invention provides a pharmaceutical composition comprising an anti-Flt-1 antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0021] On the other hand, the present invention provides polynucleotides encoding the CDR, VL region, VH region, light chain, and / or heavy chain of the antibody or antigen-binding fragment thereof of the present invention. In one embodiment, the present invention provides an expression vector comprising said polynucleotide. In another embodiment, the present invention provides a host cell comprising said polynucleotide or said expression vector. In a specific embodiment, the present invention provides a method for preparing an antibody or antigen-binding fragment thereof that specifically binds to human Flt-1, said method comprising culturing said host cells. In another embodiment, hybridoma cells produce said antibody or antigen-binding fragment thereof.

[0022] On the other hand, the present invention provides a method for treating Flt-1-mediated diseases, conditions, or symptoms, the method comprising administering an anti-Flt-1 antibody or an antigen-binding fragment thereof to a subject requiring treatment. In one specific embodiment, the Flt-1-mediated disease, condition, or symptom is Duchenne muscular dystrophy, Becker muscular dystrophy, bronchopulmonary dysplasia, preeclampsia, or chronic kidney disease.

[0023] On the other hand, the method provides a way to treat Duchenne muscular dystrophy (DMD), the method comprising administering an effective amount of an anti-Flt-1 antibody or its antigen-binding fragment to a subject suffering from or susceptible to DMD, such that at least one symptom or feature of DMD is reduced or delayed in intensity, severity, or frequency. In one embodiment, the method further comprises administering one or more additional therapeutic agents to the subject. In one specific embodiment, the additional therapeutic agents are selected from the group consisting of prednisone, deflazacort, follistatin, RNA-regulating therapeutic agents, exon-skipping therapeutic agents, and gene therapy.

[0024] In one embodiment, the antibody or its antigen-binding fragment is administered parenterally. In some embodiments, parenteral administration is selected from intravenous, intradermal, intrathecal, inhalation, percutaneous (local), intraocular, intramuscular, subcutaneous, and / or mucosal administration. In one specific embodiment, the parenteral administration is intravenous. In another embodiment, the parenteral administration is subcutaneous.

[0025] In some embodiments, the antibody or its antigen-binding fragment is administered once daily, twice weekly, once weekly, or once monthly. In one specific embodiment, the antibody or its antigen-binding fragment is administered twice weekly.

[0026] In another embodiment, the effective amount of the antibody or its antigen-binding fragment is a dose of about 1 mg / kg to 50 mg / kg. In one specific embodiment, the dose is about 1 mg / kg, 3 mg / kg, or 10 mg / kg.

[0027] In one embodiment, administration of the antibody or its antigen-binding fragment produces reduced fibrosis and / or necrosis relative to a control. In another embodiment, administration of the antibody or its antigen-binding fragment produces improved angiogenesis in the subject's muscles relative to a control. In another embodiment, the improved angiogenesis is reflected by pathologically increased blood flow in the muscle, increased serum VEGF levels, decreased serum creatine kinase (CK) levels, increased CD31 fraction as measured by IHC, and / or decreased serum sFlt-1 levels. In another embodiment, administration of the antibody or its antigen-binding fragment produces improved muscle function relative to a control. In another embodiment, the improved muscle function is reflected by improved muscle strength and / or fatigue resistance.

[0028] On the other hand, the present invention provides a method for treating tissue fibrosis, the method comprising administering an effective amount of an anti-Flt-1 antibody or an antigen-binding fragment thereof to a subject requiring treatment. sequence list <110> Takeda Pharmaceutical Co., Ltd. <120> Anti-FLT-1 antibody used to treat Duchenne muscular dystrophy <130> SHR-1189WO <140> US 15 / 564,976 <141> 2017-10-06 <150> PCT / US2016 / 026352 <151> 2016-04-07 <150> 62 / 307,645 <151> 2016-03-14 <150> 62 / 144,251 <151> 2015-04-07 <160> 111 <170> PatentIn version 3.5 <210> 1 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 1 Ser Tyr Ala Met Ser 1 5 <210> 2 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 2 Asp Tyr Ser Met Ser 1 5 <210> 3 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 3 Asp Tyr Ser Ala Ser 1 5 <210> 4 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 4 Asp Tyr Ser Leu Ser 1 5 <210> 5 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 5 Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 6 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 6 Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Met Lys 1 5 10 15 Gly <210> 7 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 7 Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Leu Lys 1 5 10 15 Gly <210> 8 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 8 Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Ala Lys 1 5 10 15 Gly <210> 9 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 9 Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val Lys 1 5 10 15 Gly <210> 10 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 10 Ala Ile Thr Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val Lys 1 5 10 15 Gly <210> 11 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 11 Ala Ile Ser Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Leu Lys 1 5 10 15 Gly <210> 12 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 12 Ala Ile Ser Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val Lys 1 5 10 15 Gly <210> 13 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 13 Ala Ile Ser Trp Gln Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Ala Lys 1 5 10 15 Gly <210> 14 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 14 Ala Ile Ser Trp Asn Ala Asp Ser Thr Tyr Tyr Ala Glu Ser Ala Lys 1 5 10 15 Gly <210> 15 <211> 2 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 15 Asp Tyr 1 <210> 16 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 16 Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Met Asp Tyr 1 5 10 15 <210> 17 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 17 Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser Asp Tyr 1 5 10 15 <210> 18 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 18 Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Tyr Gly Thr Asp Tyr 1 5 10 15 <210> 19 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 19 Gly Gly Asn Asn Ile Gly Ser Lys Asn Val His 1 5 10 <210> 20 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 20 Gly Gly Asn Asn Leu Gly Tyr Lys Ser Val His 1 5 10 <210> twenty one <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> twenty one Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala Gln 1 5 10 <210> twenty two <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> twenty two Arg Asp Ser Asn Arg Pro Ser 1 5 <210> twenty three <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> twenty three Arg Asp Asn Asn Arg Pro Ser 1 5 <210> twenty four <211> 7 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> twenty four Ala Asn Asn Arg Arg Pro Ser 1 5 <210> 25 <211> 4 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 25 Gln Val Val Val 1 <210> 26 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 26 Gln Val Trp Asp Gly Ser Thr Gln Ala Ile Val 1 5 10 <210> 27 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 27 Gln Val Trp Glu Asp Ser Thr Gln Ala Ile Val 1 5 10 <210> 28 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 28 Gln Val Trp Asp Glu Ser Thr Gln Ala Ile Val 1 5 10 <210> 29 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 29 Gln Val Trp Ala Ala Ser Thr Gln Ala Ile Val 1 5 10 <210> 30 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 30 Gln Val Trp Asp Asp Ser Thr Gln Ala Ile Val 1 5 10 <210> 31 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 31 Gln Val Trp Glu Ala Ser Thr Gln Ala Ile Val 1 5 10 <210> 32 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 32 Gln Val Trp Asp Ala Ser Thr Gln Ala Ile Val 1 5 10 <210> 33 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 33 Gln Val Trp Glu Glu Ser Thr Gln Ala Ile Val 1 5 10 <210> 34 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 34 Gln Val Trp Glu Gly Ser Thr Gln Ala Ile Val 1 5 10 <210> 35 <211> 111 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 35 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 36 <211> 111 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 36 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105 110 <210> 37 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 37 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Met 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Met 100 105 110 Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 38 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 38 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 39 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 39 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 40 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 40 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 41 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 41 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Thr Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Thr 100 105 110 Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 42 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 42 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 43 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 43 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val<I 35 40 45 [[ID=зо]]Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val 50 55 60 [[ID=зо]]Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 44 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 44 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 45 <211> 125 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 45 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ser Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Ser Gly Asp Ser Thr Tyr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 46 <211> 125 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 46 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Gln Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 47 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 47 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Ala Asp Ser Thr Tyr Tyr Ala Glu Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 48 <211> 125 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 48 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Ala Asp Ser Thr Tyr Tyr Ala Glu Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 49 <211> 101 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 49 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Lys Asn Val 20 25 30 His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Arg Asp Ser Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Val Val Phe Gly Gly Gly Thr [[ID=z18]]85 90 95[[ID=z19]] [[ID=z^{20}]]Lys Leu Thr Val Leu[[ID=z21]] [[ID=z22]]100[[ID=z23]] [[ID=z24]]<210> 50[[ID=z25]] [[ID=z26]]<211> 101[[ID=z^{27}]] [[ID=z28]]<212> PRT[[ID=z29]] [[ID=z30]]<213> Artificial Sequence[[ID=z31]] [[ID=z32]]<220>[[ID=z33]] [[ID=z34]]<223> Synthetic Polypeptide[[ID=z35]] [[ID=z36]]<400> 50[[ID=z37]] [[ID=z38]]Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln[[ID=z39]] [[ID=z40]]1 5 10 15[[ID=z41]] [[ID=z42]]Ala Ala Arg Ile Thr Cys Gly Gly Asn Asn Leu Gly Tyr Lys Ser Val[[ID=z43]] [[ID=z44]]20 25 30[[ID=z45]] [[ID=z46]]His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr[[ID=z47]] [[ID=z48]]35 40 45[[ID=z49]] It should be noted that in the original text, there is a tag "210" which is translated as "210" in English, and there may be some incorrect tags like "z18", "z^{20}", "z27" etc. in your provided text which seem to be incorrect notations. I have translated based on the overall content as accurately as possible while keeping the original tags intact. If these are not correct in the original context, you may need to check and correct them.Arg Asp Asn Asn Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Val Val Phe Gly Gly Gly Thr 85 90 95 Lys Leu Thr Val Leu 100 <210> 51 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 51 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Gly Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 <210> 52 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 52 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Glu Asp Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 53 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 53 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Glu Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 54 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 54 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Ala Ala Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 55 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 55 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Ala Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Asp Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 56 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 56 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Ala Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Glu Ala Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 57 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 57 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ala Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 58 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 58 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Glu Glu Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr His Leu Thr Val Leu 100 105 <210> 59 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 59 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Ala Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Gly Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 60 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 60 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Ala Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ala Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 61 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 61 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Ala Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Glu Gly Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 62 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 62 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ser Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 63 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 63 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ser Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 64 <211> 455 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Polypeptide <400> 64 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Gln Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 65 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 65 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly [[ID=�7]]1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Ala Asp Ser Thr Tyr Tyr Ala Glu Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 66 <211> 455 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 66 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Gln Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 67 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 67 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Ala Asp Ser Thr Tyr Tyr Ala Glu Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 68 <2​​​​​​​​​​​​​​​​​​​​ 35 40 45 Ser Ala Ile Ser Trp Asn Ala Asp Ser Thr Tyr Tyr Ala Glu Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 69 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 69 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Ala Asp Ser Thr Tyr Tyr Ala Glu Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 70 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 70 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Met 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Met 100 105 110 Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 71 <211> 455<00​​​​​​​​​​​​​​Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ser Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 72 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 72 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 73 <211> 455 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Polypeptide <400> 73 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Ala 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys [[ID=十七]]385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 74 <211> 455 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 74 It should be noted that there seems to be an incorrect "十七" in the translation of "ID=十七", which should be "385 390 395". Please double-check if there are any other specific requirements or corrections needed based on the actual context.Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Asp Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Met 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Met 100 105 110 Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Ser Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met Asn Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 75 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 75 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Ala Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Glu Gly Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 76 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 76 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Ala Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ala Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 77 <211> 214 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Polypeptide <400> 77 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Glu Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 78 <211> 214 <212> PRT <213> Synthetic Sequence <220> <223> Synthetic Polypeptide <400> 78 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln<000,2063>1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 [[ID=4,4]]Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Glu Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 79 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 79 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Glu Asp Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 80 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 80 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Glu Asp Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 81 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 81 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Glu Asp Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 82 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 82 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Glu Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 83 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 83 Ser Tyr Glu Leu Thr Gln Ser Pro Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Gly Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr His Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 84 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 84 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Ala Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Gly 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Gly Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 85 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 85 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Gly Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Glu Asp Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 86 <211> 214 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 86 Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala Leu Arg Gln 1 5 10 15 Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser Gln Thr Ala 20 25 30 Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr 35 40 45 Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala Gln Ala Glu 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Glu Ser Thr Gln Ala 85 90 95 Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro Lys 100 105 110 Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln 115 120 125 Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly 130 135 140 Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly 145 150 155 160 Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala 165 170 175 Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser 180 185 190 Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val 195 200 205 Ala Pro Thr Glu Cys Ser 210 <210> 87 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 87 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 88 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 88 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 89 <211> 330 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 89 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Ser Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met Asn Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 90 <211> 1338 <212> PRT <213> Homo sapiens <400> 90 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser ? 5? 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45 Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60 Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70 75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145 150 155 160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225 230 235 240 Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285 Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300 Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310 315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365 Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390 395 400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430 Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460 Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys 465 470 475 480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525 Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540 Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545 550 555 560 Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580 585 590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys 595 600 605 Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610 615 620 Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630 635 640 Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655 Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670 Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680 685 Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu 690 695 700 Pro Gly Ile Ile Leu Gly Pro Gly Ser Ser Thr Leu Phe Ile Glu Arg 705 710 715 720 Val Thr Glu Glu Asp Glu Gly Val Tyr His Cys Lys Ala Thr Asn Gln 725 730 735 Lys Gly Ser Val Glu Ser Ser Ala Tyr Leu Thr Val Gln Gly Thr Ser 740 745 750 Asp Lys Ser Asn Leu Glu Leu Ile Thr Leu Thr Cys Thr Cys Val Ala 755 760 765 Ala Thr Leu Phe Trp Leu Leu Leu Thr Leu Phe Ile Arg Lys Met Lys 770 775 780 Arg Ser Ser Ser Glu Ile Lys Thr Asp Tyr Leu Ser Ile Ile Met Asp 785 790 795 800 Pro Asp Glu Val Pro Leu Asp Glu Gln Cys Glu Arg Leu Pro Tyr Asp 805 810 815 Ala Ser Lys Trp Glu Phe Ala Arg Glu Arg Leu Lys Leu Gly Lys Ser 820 825 830 Leu Gly Arg Gly Ala Phe Gly Lys Val Val Gln Ala Ser Ala Phe Gly 835 840 845 Ile Lys Lys Ser Pro Thr Cys Arg Thr Val Ala Val Lys Met Leu Lys 850 855 860 Glu Gly Ala Thr Ala Ser Glu Tyr Lys Ala Leu Met Thr Glu Leu Lys 865 870 875 880 Ile Leu Thr His Ile Gly His His Leu Asn Val Val Asn Leu Leu Gly 885 890 895 Ala Cys Thr Lys Gln Gly Gly Pro Leu Met Val Ile Val Glu Tyr Cys 900 905 910 Lys Tyr Gly Asn Leu Ser Asn Tyr Leu Lys Ser Lys Arg Asp Leu Phe 915 920 925 Phe Leu Asn Lys Asp Ala Ala Leu His Met Glu Pro Lys Lys Glu Lys 930 935 940 Met Glu Pro Gly Leu Glu Gln Gly Lys Lys Pro Arg Leu Asp Ser Val 945 950 955 960 Thr Ser Ser Glu Ser Phe Ala Ser Ser Gly Phe Gln Glu Asp Lys Ser 965 970 975 Leu Ser Asp Val Glu Glu Glu Glu Asp Ser Asp Gly Phe Tyr Lys Glu 980 985 990 Pro Ile Thr Met Glu Asp Leu Ile Ser Tyr Ser Phe Gln Val Ala Arg 995 1000 1005 Gly Met Glu Phe Leu Ser Ser Arg Lys Cys Ile His Arg Asp Leu 1010 1015 1020 Ala Ala Arg Asn Ile Leu Leu Ser Glu Asn Asn Val Val Lys Ile 1025 1030 1035 Cys Asp Phe Gly Leu Ala Arg Asp Ile Tyr Lys Asn Pro Asp Tyr 1040 1045 1050 Val Arg Lys Gly Asp Thr Arg Leu Pro Leu Lys Trp Met Ala Pro 1055 1060 1065 Glu Ser Ile Phe Asp Lys Ile Tyr Ser Thr Lys Ser Asp Val Trp 1070 1075 1080 Ser Tyr Gly Val Leu Leu Trp Glu Ile Phe Ser Leu Gly Gly Ser 1085 1090 1095 Pro Tyr Pro Gly Val Gln Met Asp Glu Asp Phe Cys Ser Arg Leu 1100 1105 1110 Arg Glu Gly Met Arg Met Arg Ala Pro Glu Tyr Ser Thr Pro Glu 1115 1120 1125 Ile Tyr Gln Ile Met Leu Asp Cys Trp His Arg Asp Pro Lys Glu 1130 1135 1140 Arg Pro Arg Phe Ala Glu Leu Val Glu Lys Leu Gly Asp Leu Leu 1145 1150 1155 Gln Ala Asn Val Gln Gln Asp Gly Lys Asp Tyr Ile Pro Ile Asn 1160 1165 1170 Ala Ile Leu Thr Gly Asn Ser Gly Phe Thr Tyr Ser Thr Pro Ala 1175 1180 1185 Phe Ser Glu Asp Phe Phe Lys Glu Ser Ile Ser Ala Pro Lys Phe 1190 1195 1200 Asn Ser Gly Ser Ser Asp Asp Val Arg Tyr Val Asn Ala Phe Lys 1205 1210 1215 Phe Met Ser Leu Glu Arg Ile Lys Thr Phe Glu Glu Leu Leu Pro 1220 1225 1230 Asn Ala Thr Ser Met Phe Asp Asp Tyr Gln Gly Asp Ser Ser Thr 1235 1240 1245 Leu Leu Ala Ser Pro Met Leu Lys Arg Phe Thr Trp Thr Asp Ser 1250 1255 1260 Lys Pro Lys Ala Ser Leu Lys Ile Asp Leu Arg Val Thr Ser Lys 1265 1270 1275 Ser Lys Glu Ser Gly Leu Ser Asp Val Ser Arg Pro Ser Phe Cys 1280 1285 1290 His Ser Ser Cys Gly His Val Ser Glu Gly Lys Arg Arg Phe Thr 1295 1300 1305 Tyr Asp His Ala Glu Leu Glu Arg Lys Ile Ala Cys Cys Ser Pro 1310 1315 1320 Pro Pro Asp Tyr Asn Ser Val Val Leu Tyr Ser Thr Pro Pro Ile 1325 1330 1335 <210> 91 <211> 720 <212> PRT <213> Homo sapiens <400> 91 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45 Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60 Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70 75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145 150 155 160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225 230 235 240 Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285 Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300 Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310 315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365 Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390 395 400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430 Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460 Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys 465 470 475 480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525 Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540 Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545 550 555 560 Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580 585 590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys 595 600 605 Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610 615 620 Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630 635 640 Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655 Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670 Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680 685 Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu 690 695 700 Pro Asp Ala Asp Pro His Ile Gln Lys Ala Asp Cys Thr Phe Phe Phe 705 710 715 720 <210> 92 <211> 687 <212> PRT <213> Homo sapiens <400> 92 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45 Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60 Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70 75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 1,20 125 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145 150 155 160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225 230 235 240 Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285 Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300 Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310 315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365 Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390 395 400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430 Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460 Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys 465 470 475 480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525 Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540 Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545 550 555 560 Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580 585 590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys 595 600 605 Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610 615 620 Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630 635 640 Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655 Gly Glu His Cys Asn Lys Lys Ala Val Phe Ser Arg Ile Ser Lys Phe 660 665 670 Lys Ser Thr Arg Asn Asp Cys Thr Thr Gln Ser Asn Val Lys His 675 680 685 <210> 93 <211> 733 <212> PRT <213> Homo sapiens <400> 93 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45 Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60 Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70 75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145 150 155 160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225 230 235 240 Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285 Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300 Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310 315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365 Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390 395 400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430 Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460 Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys 465 470 475 480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys Asn Lys Met Ala Ser Thr Leu Val Val Ala Asp Ser Arg 515 520 525 Ile Ser Gly Ile Tyr Ile Cys Ile Ala Ser Asn Lys Val Gly Thr Val 530 535 540 Gly Arg Asn Ile Ser Phe Tyr Ile Thr Asp Val Pro Asn Gly Phe His 545 550 555 560 Val Asn Leu Glu Lys Met Pro Thr Glu Gly Glu Asp Leu Lys Leu Ser 565 570 575 Cys Thr Val Asn Lys Phe Leu Tyr Arg Asp Val Thr Trp Ile Leu Leu 580 585 590 Arg Thr Val Asn Asn Arg Thr Met His Tyr Ser Ile Ser Lys Gln Lys 595 600 605 Met Ala Ile Thr Lys Glu His Ser Ile Thr Leu Asn Leu Thr Ile Met 610 615 620 Asn Val Ser Leu Gln Asp Ser Gly Thr Tyr Ala Cys Arg Ala Arg Asn 625 630 635 640 Val Tyr Thr Gly Glu Glu Ile Leu Gln Lys Lys Glu Ile Thr Ile Arg 645 650 655 Asp Gln Glu Ala Pro Tyr Leu Leu Arg Asn Leu Ser Asp His Thr Val 660 665 670 Ala Ile Ser Ser Ser Thr Thr Leu Asp Cys His Ala Asn Gly Val Pro 675 680 685 Glu Pro Gln Ile Thr Trp Phe Lys Asn Asn His Lys Ile Gln Gln Glu 690 695 700 Pro Glu Leu Tyr Thr Ser Thr Ser Pro Ser Ser Ser Ser Ser Ser Pro 705 710 715 720 Leu Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser 725 730 <210> 94 <211> 541 <212> PRT <213> Homo sapiens <400> 94 Met Val Ser Tyr Trp Asp Thr Gly Val Leu Leu Cys Ala Leu Leu Ser 1 5 10 15 Cys Leu Leu Leu Thr Gly Ser Ser Ser Gly Ser Lys Leu Lys Asp Pro 20 25 30 Glu Leu Ser Leu Lys Gly Thr Gln His Ile Met Gln Ala Gly Gln Thr 35 40 45 Leu His Leu Gln Cys Arg Gly Glu Ala Ala His Lys Trp Ser Leu Pro 50 55 60 Glu Met Val Ser Lys Glu Ser Glu Arg Leu Ser Ile Thr Lys Ser Ala 65 70 75 80 Cys Gly Arg Asn Gly Lys Gln Phe Cys Ser Thr Leu Thr Leu Asn Thr 85 90 95 Ala Gln Ala Asn His Thr Gly Phe Tyr Ser Cys Lys Tyr Leu Ala Val 100 105 110 Pro Thr Ser Lys Lys Lys Glu Thr Glu Ser Ala Ile Tyr Ile Phe Ile 115 120 125 Ser Asp Thr Gly Arg Pro Phe Val Glu Met Tyr Ser Glu Ile Pro Glu 130 135 140 Ile Ile His Met Thr Glu Gly Arg Glu Leu Val Ile Pro Cys Arg Val 145 150 155 160 Thr Ser Pro Asn Ile Thr Val Thr Leu Lys Lys Phe Pro Leu Asp Thr 165 170 175 Leu Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe 180 185 190 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr Cys Glu 195 200 205 Ala Thr Val Asn Gly His Leu Tyr Lys Thr Asn Tyr Leu Thr His Arg 210 215 220 Gln Thr Asn Thr Ile Ile Asp Val Gln Ile Ser Thr Pro Arg Pro Val 225 230 235 240 Lys Leu Leu Arg Gly His Thr Leu Val Leu Asn Cys Thr Ala Thr Thr 245 250 255 Pro Leu Asn Thr Arg Val Gln Met Thr Trp Ser Tyr Pro Asp Glu Lys 260 265 270 Asn Lys Arg Ala Ser Val Arg Arg Arg Ile Asp Gln Ser Asn Ser His 275 280 285 Ala Asn Ile Phe Tyr Ser Val Leu Thr Ile Asp Lys Met Gln Asn Lys 290 295 300 Asp Lys Gly Leu Tyr Thr Cys Arg Val Arg Ser Gly Pro Ser Phe Lys 305 310 315 320 Ser Val Asn Thr Ser Val His Ile Tyr Asp Lys Ala Phe Ile Thr Val 325 330 335 Lys His Arg Lys Gln Gln Val Leu Glu Thr Val Ala Gly Lys Arg Ser 340 345 350 Tyr Arg Leu Ser Met Lys Val Lys Ala Phe Pro Ser Pro Glu Val Val 355 360 365 Trp Leu Lys Asp Gly Leu Pro Ala Thr Glu Lys Ser Ala Arg Tyr Leu 370 375 380 Thr Arg Gly Tyr Ser Leu Ile Ile Lys Asp Val Thr Glu Glu Asp Ala 385 390 395 400 Gly Asn Tyr Thr Ile Leu Leu Ser Ile Lys Gln Ser Asn Val Phe Lys 405 410 415 Asn Leu Thr Ala Thr Leu Ile Val Asn Val Lys Pro Gln Ile Tyr Glu 420 425 430 Lys Ala Val Ser Ser Phe Pro Asp Pro Ala Leu Tyr Pro Leu Gly Ser 435 440 445 Arg Gln Ile Leu Thr Cys Thr Ala Tyr Gly Ile Pro Gln Pro Thr Ile 450 455 460<00030s38>Lys Trp Phe Trp His Pro Cys Asn His Asn His Ser Glu Ala Arg Cys 465 470 475 480 Asp Phe Cys Ser Asn Asn Glu Glu Ser Phe Ile Leu Asp Ala Asp Ser 485 490 495 Asn Met Gly Asn Arg Ile Glu Ser Ile Thr Gln Arg Met Ala Ile Ile 500 505 510 Glu Gly Lys Asn Lys Leu Pro Pro Ala Asn Ser Ser Phe Met Leu Pro 515 520 525 Pro Thr Ser Phe Ser Ser Asn Tyr Phe His Phe Leu Pro 530 535 540 <210> 95 <211> 13 <212> PRT <213> Homo sapiens <400> 95 Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu 1 5 10 It should be noted that there may be an error in the original text where "<00030s38>" is likely a miswriting. It should probably be " ". This has been translated as accurately as possible based on the given text. <210> 96 <211> 14 <212> PRT <213> Homo sapiens <400> 96 Ile Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr 1 5 10 <210> 97 <211> 9 <212> PRT <213> Homo sapiens <400> 97 Asp Thr Gly Arg Pro Phe Val Glu Met 1 5 <210> 98 <211> 8 <212> PRT <213> Homo sapiens <400> 98 Glu Ile Pro Glu Ile Ile His Met 1 5 <210> 99 <211> 10 <212> PRT <213> Homo sapiens <400> 99 Tyr Ser Glu Ile Pro Glu Ile Ile His Met 1 5 10 <210> 100 <211> 15 <212> PRT <213> Homo sapiens <400> 100 Tyr Ser Glu Ile Pro Glu Ile Ile His Met Thr Glu Gly Arg Glu 1 5 10 15 <210> 101 <211> 29 <212> PRT <213> Homo sapiens <400> 101 Ile Pro Asp Gly Lys Arg Ile Ile Trp Asp Ser Arg Lys Gly Phe Ile 1 5 10 15 Ile Ser Asn Ala Thr Tyr Lys Glu Ile Gly Leu Leu Thr 20 25 <210> 102 <211> 6 <212> PRT <213> Homo sapiens <400> 102 Tyr Lys Glu Ile Gly Leu 1 5 <210> 103 <211> 11 <212> PRT <213> Homo sapiens <400> 103 Ile Ser Asp Thr Gly Arg Pro Phe Val Glu Met 1 5 10 <210> 104 <211> 238 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 104 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 1 5 10 15 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 20 25 30 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 35 40 45 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 50 55 60 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 65 70 75 80 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 85 90 95 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 100 105 110 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 115 120 125 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 130 135 140 Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 145 150 155 160 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 165 170 175 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 180 185 190 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 195 200 205 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 210 215 220 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 225 230 235 <210> 105 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Oligopeptide <400> 105 Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly 1 5 10 15 Gly Gly Gly Ala Pro 20 <210> 106 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Oligopeptide <400> 106<00031​​​​​​​​​​​​​​​​​​​​​​<223> Synthetic oligopeptide <400> 107 Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly Gly Gly 1 5 10 15 Gly Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala Ala Gly 20 25 30 Gly Gly Gly Gly Gly Ala Pro Gly Gly Gly Gly Gly Ala Ala Ala Ala 35 40 45 Ala Gly Gly Gly Gly Gly Gly Ala Pro 50 55 <210> 108 <211> 475 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <220> <221> misc_feature <222> (475)..(475) <223> Xaa can be any naturally occurring amino acid <400> 108 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asp Tyr Ser Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ser Ala Ile Ser Trp Ser Gly Asp Ser Thr Tyr Tyr Ala 65 70 75 80 Glu Ser Val Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr 115 120 125 Tyr Gly Ser Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 130 135 140 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 145 150 155 160 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 165 170 175 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 180 185 190 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 195 200 205 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 210 215 220 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 225 230 235 240 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 245 250 255 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 260 265 270 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 275 280 285 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 290 295 300 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 305 310 315 320 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 325 330 335 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 340 345 350 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 355 360 365 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 370 375 380 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 385 390 395 400 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 405 410 415 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 420 425 430 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 435 440 445 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 450 455 460 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Xaa 465 470 475 <210> 109 <211> 455 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 109 Glu Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ser Ala Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Trp Ser Gly Asp Ser Thr Tyr Tyr Ala Glu Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Trp Ala Thr Pro Ile Glu Ser Leu Tyr Tyr Tyr Gly Ser 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser 130 135 140 Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His 165 170 175 Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser 180 185 190 Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys 195 200 205 Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu 210 215 220 Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 225 230 235 240 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 245 250 255 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 260 265 270 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 275 280 285 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 290 295 300 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 305 310 315 320 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 325 330 335 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 340 345 350 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 355 360 365 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 370 375 380 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 385 390 395 400 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 405 410 415 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 420 425 430 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 435 440 445 Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 110 <211> 234 <​​​​​​​​​​​​​<223> Xaa can be any naturally occurring amino acid <400> 110 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Ser Tyr Glu Leu Thr Gln Pro Leu Ser Val Ser Val Ala 20 25 30 Leu Arg Gln Ala Ala Lys Ile Thr Cys Gly Gly Asn Asn Ile Gly Ser 35 40 45 Gln Thr Ala Gln Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu 50 55 60 Val Ile Tyr Ala Asn Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe 65 70 75 80 Ser Gly Ser Lys Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Arg Ala 85 90 95 Gln Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ala Ser 100 105 110 Thr Gln Ala Ile Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly 115 120 125 Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu 130 135 140 Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe 145 150 155 160 Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val 165 170 175 Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys 180 185 190 Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser 195 200 205 His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu 210 215 220 Lys Thr Val Ala Pro Thr Glu Cys Ser Xaa 225 230 <210> 111 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic oligopeptides <400> 111 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser Brief description of the attached diagram

[0029] The doctrine described herein will be more fully understood from the following descriptions of the various illustrative embodiments when read in conjunction with the accompanying drawings. It should be understood that the drawings described below are for illustrative purposes only and are not intended to limit the scope of the doctrine in any way.

[0030] Figure 1AExemplary results depicting the clearance of the antibody after intravenous administration of anti-Flt-1 antibody 13B4 to mice at a dose of 10 mg / kg are shown.

[0031] Figure 1B Exemplary results depicting the clearance of the antibody after intravenous administration of anti-Flt-1 antibody 10G12 to mice at a dose of 10 mg / kg are shown.

[0032] Figure 2A Exemplary results depicting peak serum anti-Flt-1 antibody levels are shown. Figure 2B Exemplary results depicting trough serum anti-Flt-1 antibody levels are shown.

[0033] Figure 3 Exemplary results depicting a decrease in serum levels of free soluble Flt-1 (sFlt-1) following administration of an anti-Flt-1 antibody (13B4 or 10G12), an isotype control antibody, a commercial antibody (Angio), or a single mediator to mdx mice are shown.

[0034] Figure 4 Exemplary results depicting increased serum VEGF levels after administration of anti-Flt-1 antibody (13B4 or 10G12), isotype control antibody, commercial antibody (Angio), or a single mediator to mdx mice are shown.

[0035] Figures 5A-5D Exemplary results of CD31 staining are shown for tissue sections obtained from the diaphragm of mdx mice administered anti-Flt-1 antibody (13B4 or 10G12), isotype control antibody, or commercial antibody (Angio).

[0036] Figure 6A Exemplary results show the quantification of CD31-positive area as a percentage of the total stained area in a tissue section obtained from the diaphragm. Figure 6B Exemplary results show the quantification of CD31-positive area as a percentage of the total stained area in a tissue section obtained from the tibialis anterior (TA) muscle.

[0037] Figure 6B Exemplary results are shown as a quantification of the CD31-positive area as a percentage of the total stained area in tissue sections obtained from the tibialis anterior (TA) muscle of mdx mice administered anti-Flt-1 antibody.

[0038] Figure 7 Exemplary results depicting the binding of anti-Flt-1 antibody to recombinant sFlt-1 as determined by ELISA are shown.

[0039] Figure 8Exemplary results are shown depicting the inhibition of sFlt-1 binding to VEGF by anti-Flt-1 antibody in a competitive ELISA assay.

[0040] Figure 9 Exemplary results depicting the rescue of VEGF R2 phosphorylation by anti-Flt-1 antibody are shown. Human primary venous endothelial cells (HUVECs) were treated with VEGF in the presence of sFlt-1 and anti-Flt-1 antibody, and VEGF R2 phosphorylation levels were measured. The rescue percentage indicates the VEGF R2 phosphorylation level relative to the phosphorylation level of VEGF R2 when HUVECs were treated with VEGF and sFlt-1 alone (i.e., without anti-Flt-1 antibody).

[0041] Figure 10 Exemplary results are shown depicting the inhibition of soluble Flt-1 binding to VEGF by anti-Flt-1 antibody in a competitive ELISA assay.

[0042] Figure 11 Exemplary results show the clearance of anti-Flt-1 antibodies from serum within 672 hours following intravenous administration of anti-Flt-1 antibodies to mice at a dose of 10 mg / kg.

[0043] Figures 12A-12C Exemplary results of CD31 staining from tissue sections from the diaphragm are shown. Figure 12D-12F Exemplary results of CD31 staining of tissue sections obtained from the tibialis anterior muscle are shown.

[0044] Figures 13A-13C Exemplary biodistribution of the antibodies in the diaphragm, tibialis muscle, and gastrocnemius muscle of mice over a period of 256 hours following administration of anti-Flt-1 antibodies 27H9, 13B4, and 21B3 is shown.

[0045] Figure 14A Exemplary results depicting peak anti-Flt-1 antibody 21B3 levels at peak exposure are shown. Figure 14B Exemplary results depicting the trough level of anti-Flt-1 antibody 21B3 are shown.

[0046] Figure 15 Exemplary results depicting free sFlt-1 after administration of anti-Flt-1 antibody 21B3 to mdx mice are shown.

[0047] Figure 16 Exemplary results depicting VEGF levels after administration of anti-Flt-1 antibody 21B3 to mdx mice are shown.

[0048] Figures 17A-17EExemplary results of CD31 staining are shown for tissue sections obtained from the diaphragm of mdx mice administered anti-Flt-1 antibody 21B3 or an isotype control antibody.

[0049] Figure 18 Exemplary results show the quantification of the normalized percentage of CD31 positivity in tissue sections obtained from the diaphragm of mdx mice administered anti-Flt-1 antibody 21B3 or an isotype control antibody.

[0050] Figures 19A-19E Exemplary results of CD31 staining are shown for tissue sections obtained from the anterior tibialis muscle of mdx mice administered anti-Flt-1 antibody 21B3 or an isotype control antibody.

[0051] Figure 20 Exemplary results show the quantification of the normalized percentage of CD31 positivity in tissue sections obtained from the anterior tibialis muscle of mdx mice administered anti-Flt-1 antibody 21B3 or an isotype control antibody.

[0052] Figure 21A Exemplary results are shown for reversed-phase liquid chromatography / mass spectrometry (RP-LC / MS) analysis used to determine the molecular weight of deglycosylated 21B3 antibody. Figure 21B Exemplary results of the analysis of glycosylation patterns of heavy chains are shown.

[0053] Figure 22A and 22B Exemplary results depicting the rescue of VEGF R2 phosphorylation by anti-Flt-1 antibody are shown. Human primary venous endothelial cells (HUVECs) were treated with VEGF in the presence of sFlt-1 and anti-Flt-1 antibody, and VEGF R2 phosphorylation levels were measured. The rescue percentage indicates the VEGF R2 phosphorylation level relative to the phosphorylation level of VEGF R2 when HUVECs were treated with VEGF and sFlt-1 alone (i.e., without anti-Flt-1 antibody).

[0054] Figure 23 Exemplary results depicting the binding of anti-Flt-1 antibody to recombinant sFlt-1 as determined by ELISA are shown.

[0055] Figure 24 Exemplary results depicting serum levels of free anti-Flt-1 antibody 21B3 and isotype control antibody in mdx mice are shown.

[0056] Figure 25 Exemplary results depicting serum levels of free sFlt-1 in mdx mice treated with anti-Flt-1 antibody 21B3 or an isotype control antibody are shown.

[0057] Figure 26 Exemplary results depicting serum VEGF levels in mdx mice treated with anti-Flt-1 antibody 21B3 or an isotype control antibody are shown.

[0058] Figures 27A-27H Exemplary results of CD31 staining are shown for tissue sections obtained from the diaphragm of mdx mice administered anti-Flt-1 antibody 21B3 or a mediator control for 6 (27A-27D) or 12 (27E-27H) weeks.

[0059] Figures 28A-28H Exemplary results of CD31 staining are shown for tissue sections obtained from the gastrocnemius muscle of mdx mice administered anti-Flt-1 antibody 21B3 or the mediator control for 6 weeks (28A-28D) or 12 weeks (28E-28H).

[0060] Figures 29A-29H Exemplary results of CD31 staining of tissue sections obtained from the tibialis muscle of mdx mice administered anti-Flt-1 antibody 21B3 or the mediator control for 6 weeks (29A-29D) or 12 weeks (29E-29H) are shown.

[0061] Figures 30A-30C Exemplary results show the quantification of the percentage of positive CD31 staining in tissue sections obtained from the diaphragm, gastrocnemius, and tibialis muscles of mdx mice administered anti-Flt-1 antibody 21B3 or a mediator control antibody for 6 or 12 weeks.

[0062] Figures 31A-31H Exemplary results of immunohistochemical staining of type I collagen are shown for tissue sections obtained from the diaphragm of mdx mice administered anti-Flt-1 antibody 21B3 or a mediator control for 6 weeks (31A-31D) or 12 weeks (31E-31H).

[0063] Figures 32A-32H Exemplary results of immunohistochemical staining of type I collagen are shown in tissue sections obtained from the gastrocnemius muscle of mdx mice administered anti-Flt-1 antibody 21B3 or a mediator control for 6 weeks (32A-32D) or 12 weeks (32E-32H).

[0064] Figures 33A-33H Exemplary results of immunohistochemical staining of type I collagen are shown for tissue sections obtained from the tibialis muscle of mdx mice administered anti-Flt-1 antibody 21B3 or a mediator control for 6 weeks (33A-33D) or 12 weeks (33E-33H).

[0065] Figures 34A-34CExemplary results show the quantification of the positive percentage of type I collagen staining in tissue sections obtained from the diaphragm, gastrocnemius, and tibialis muscles of mdx mice administered anti-Flt-1 antibody 21B3 or a mediator control antibody for 6 or 12 weeks.

[0066] Figure 35A and 35B Exemplary results show the percentage of gastrocnemius muscle necrosis in mdx mice administered anti-Flt-1 antibody 21B3 or the mediator control antibody for 6 or 12 weeks.

[0067] Figure 36 Exemplary results are shown in the differential thermogram depicting the hydrogen / deuterium exchange of human sFlt-1 alone versus that of a mixture of human sFlt-1 and anti-Flt-1 antibody (21B3). Gray: No deuterium protection; Blue: Deuterium protection after Fab binding.

[0068] Figure 37 Exemplary results are shown in the differential thermogram depicting the hydrogen / deuterium exchange of human sFlt-1 alone versus that of a mixture of human sFlt-1 and anti-Flt-1 antibody (21C6). Gray: No deuterium protection; Blue: Deuterium protection after Fab binding.

[0069] Figures 38A-38E Exemplary results are shown depicting MS / MS spectra of identified peptides containing amino acid residues from epitope regions.

[0070] definition

[0071] To facilitate understanding of this disclosure, certain terms are defined below. Further definitions for the following and other terms are listed throughout the specification.

[0072] Affinity: As is known in the art, “affinity” is a measure of the tightness with which a particular ligand binds to its mate. In some embodiments, the ligand or mate is Flt-1. In some embodiments, the ligand or mate is soluble Flt-1. In some embodiments, the ligand or mate is recombinant Flt-1. In one specific embodiment, the ligand or mate is human sFlt-1. In one specific embodiment, the ligand or mate is recombinant sFlt-1. In other embodiments, the ligand or mate is an anti-Flt-1 antibody. Affinity can be measured in different ways. In some embodiments, affinity is measured by quantitative determination. In some such embodiments, the mate concentration can be fixed above the ligand concentration to simulate physiological conditions. Or, additionally, in some embodiments, the mate concentration and / or ligand concentration can be varied. In some such embodiments, affinity can be compared to a reference under comparable conditions (e.g., concentration).

[0073] Affinity maturation (or affinity maturation antibody): As used herein, the term "affinity maturation" or "affinity maturation antibody" refers to an antibody having one or more variations in one or more of its CDRs, said variations causing an increase in the affinity of said antibody for an antigen compared to a parent antibody without those variations. In some embodiments, the affinity maturation antibody will have nanomolar or even picomolar affinity for the target antigen. Affinity maturation antibodies can be produced by any of a variety of procedures known in the art. Mark et al., BioTechnology 10:779-783 (1992) describes this via V H and V L Domain shuffling leads to affinity maturation. Random mutagenesis of CDR and / or framework residues is described in the following literature: Barbas et al., Proc. Nat. Acad. Sci. USA91:3809-3813 (1994); Schier et al., Gene 169:147-155 (1995); Yelton et al., J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al., J. Mol. Biol. 226:889-896 (1992).

[0074] Improvement: As used herein, the term “improvement” means the prevention, reduction, or relief of the subject’s condition or an improvement in said condition. Improvement includes, but does not require, complete recovery or complete prevention of the disease symptoms.

[0075] Animal: As used herein, the term "animal" means any member of the animal kingdom. In some embodiments, "animal" means a human being at any developmental stage. In some embodiments, "animal" means a non-human animal at any developmental stage. In some embodiments, a non-human animal is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates, and / or pigs). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and / or worms. In some embodiments, an animal may be a transgenic animal, a genetically engineered animal, and / or a clone.

[0076] Antibody: As used herein, the term "antibody" refers to any immunoglobulin, whether naturally occurring or wholly or partially synthetically produced. All its derivatives that maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain that is homologous or substantially homologous to an immunoglobulin binding domain. Such proteins may be derived from natural sources or be wholly or partially synthetically produced. Antibodies may be monoclonal or polyclonal. Antibodies may be members of any immunoglobulin isotype, including any human isotype: IgG, IgM, IgA, IgD, and IgE. In some embodiments, antibodies may be members of the IgG immunoglobulin class (e.g., IgG1, IgG2, IgG3, etc.). In some embodiments, antibodies may be human antibodies. In some embodiments, antibodies may be humanized antibodies.

[0077] As is known to those skilled in the art, naturally occurring antibodies typically consist of four polypeptide chains: two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy and light chain is composed of variable regions (abbreviated herein as HCVR, VH, or V, respectively). H And LCVR, VL or V L It consists of a constant region and a C0 region. The constant region of the heavy chain contains C0. H 1. C H 2 and C H 3. Structural domains (and optionally, in the cases of IgM and IgE, C) H 4. Structural Domains). The constant region of a light chain contains a structural domain, C. L V H and V L The region also contains highly variable regions called complementary determinant regions (CDRs), interspersed with more conservative regions called frame regions (FRs). Each V H and V LIt consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The binding regions of the heavy and light chains contain binding domains that interact with the antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, including different cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system.

[0078] Antigen-binding moiety: As used herein, the term "antigen-binding moiety" or "antigen-binding fragment" refers to one or more fragments or portions of an antibody that retains its ability to specifically bind to an antigen (e.g., Flt-1). Examples of antigen-binding moieties include (i) the Fab fragment, a type of antibody produced by V... H V L C H 1 and C L (ii) a monovalent segment composed of structural domains; (iii) a bivalent segment consisting of two Fab segments connected by a disulfide bridge in the hinge region; and (iv) a segment composed of V H and C H (iv) The Fd fragment composed of a single-arm antibody; H and V L Fv fragments composed of domains; (v) dAb fragments containing a single variable domain (Ward et al., (1989) Nature 341:544-546); (vi) separated complementarity-determining regions (CDRs); (vii) Fab' fragments, which are essentially Fab fragments with a portion of a hinge region; (viii) nanobodies containing a heavy chain variable region with a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment (V... H and V L These genes are encoded by individual genes, but they can be joined by synthetic linkers using recombination methods, which enable them to be made into genes containing V. H and V L The antigen-binding fragment of an antibody may optionally comprise a single-chain antibody fragment. Alternatively, the antigen-binding fragment of an antibody may comprise, for example, multiple chains linked together by disulfide bonds. The antigen-binding fragment of an antibody may optionally comprise a single-chain antibody fragment. Or, additionally, the antigen-binding fragment of an antibody may comprise, for example, multiple chains linked together by disulfide bonds. The antigen-binding fragment of an antibody may optionally comprise a multi-molecular complex. Functional antibody fragments typically contain at least about 50 amino acids, and more typically contain at least about 200 amino acids.

[0079] In some embodiments, the antibody fragment contains a sufficient sequence of a parent antibody, wherein the antibody fragment is a fragment that binds to the same antigen as the parent antibody; in some embodiments, the fragment binds to the antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to the antigen.

[0080] Those skilled in the art will understand that the term "antibody fragment" does not imply, and is not limited to, any particular mode of production. Antibody fragments can be produced using any suitable method, including but not limited to the lysis, chemical synthesis, and recombinant production of whole antibodies. The utility of fragments can be screened in the same manner as whole antibodies.

[0081] Approximately or about: As used herein, when applied to one or more target values, the term “approximately” or “about” refers to a value similar to the stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values ​​(greater than or less than) 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in any direction of the stated reference value, unless otherwise stated or otherwise apparent from the context (except where such a value would exceed 100% of the possible value).

[0082] Associating with: If the presence, level, and / or form of one event or entity is related to the presence, level, and / or form of another event or entity, then the two events or entities are “associated” with each other, as the term is used herein. For example, if the presence, level, and / or form of a particular entity (e.g., a polypeptide) is related to the incidence and / or susceptibility to a particular disease, condition, or / or symptom (e.g., across relevant populations), then the particular entity is considered to be associated with the disease, condition, or symptom. In some embodiments, if two or more entities interact directly or indirectly, they are physically “associated” with each other such that they are and remain physically close to each other. In some embodiments, two or more entities that are physically associated with each other are covalently linked; in some embodiments, two or more entities that are physically associated with each other are not covalently linked, but are non-covalently associated, for example, through hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, and combinations thereof.

[0083] Carrier or diluent: As used herein, the terms “carrier” and “diluent” refer to pharmaceutically acceptable (e.g., safe and non-toxic for human administration) carriers or diluents useful in the preparation of pharmaceutical formulations. Exemplary diluents include sterile water, water for sterile injection (BWFI), pH buffer solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or glucose solution.

[0084] CDR: As used herein, refers to the complementarity-determining region within the antibody variable region. Three CDRs exist in each variable region of the heavy and light chains, designated as CDR1, CDR2, and CDR3 for each variable region. A “set of CDRs” or “group of CDRs” refers to a group of three or six CDRs present in a single variable region capable of binding the antigen, or CDRs in homologous heavy and light chain variable regions capable of binding the antigen. Certain systems for defining CDR boundaries have been established in the art (e.g., Kabat, Chothia, etc.); those skilled in the art understand the differences between these systems and are able to understand CDR boundaries to the extent necessary for understanding and practicing the claimed invention.

[0085] Chimeric antibodies: As used herein, refer to antibodies whose amino acid sequence contains V found in the first species. H and V L The chimeric antibody contains a region sequence and a constant region sequence found in a second species different from the first species. In many embodiments, the chimeric antibody has a mouse V antibody linked to the human constant region. H and V L Region. In some implementations, a human V region connected to a non-human constant region (e.g., a mouse constant region) is included. H and V L Antibodies in this region are called "reverse chimeric antibodies".

[0086] Dosage Form: As used herein, the terms “dosage form” and “unit dosage form” refer to a physically discrete unit of a therapeutic protein (e.g., an antibody) intended for use in a patient to be treated. Each unit contains a predetermined amount of active substance calculated to produce the desired therapeutic effect. However, it will be understood that the total dose of the composition will be determined by the attending physician within the bounds of reasonable medical judgment.

[0087] Dysfunction: As used herein, the term “dysfunction” refers to abnormal function. Dysfunction of a molecule (e.g., a protein) can be caused by an increase or decrease in the activity associated with that molecule. Dysfunction of a molecule can be caused by defects in the molecule itself or in relation to other molecules that directly or indirectly interact with or regulate the molecule.

[0088] Epitope: As used herein, includes any portion specifically recognized by an immunoglobulin-binding component (e.g., an antibody, its antibody fragment, or receptor). In some embodiments, an epitope consists of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts an associated three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, at least some of such chemical atoms or groups are physically separated from each other when the antigen adopts an alternative conformation (e.g., linearization).

[0089] Fc Region: As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides," each containing a constant region of the antibody excluding the first constant region immunoglobulin domain. In some embodiments, the "Fc region" comprises two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. An "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also contain part or all of the flexible hinge at the N-terminus of these domains. For IgG, the "Fc polypeptide" contains the immunoglobulin domains Cgamma2 (Cγ2) and Cgamma3 (Cγ3), and the lower portion of the hinge between Cgamma1 (Cγ1) and Cγ2. Although the boundaries of Fc peptides can vary, human IgG heavy chain Fc peptides are generally defined as containing residues originating from T223, C226, or P230 up to their carboxyl terminus, numbered according to the EU index in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). For IgA, the Fc peptide contains the immunoglobulin domains Calpha2 (Cα2) and Calpha3 (Cα3), and the lower part of the hinge between Calpha1 (Cα1) and Cα2. The Fc region can be synthetic, recombinant, or derived from natural sources such as IVIG.

[0090] Frame or frame region: As used herein, this refers to the sequence of the variable region minus the CDR. Because the CDR sequence can be determined by different systems, the frame sequence is also subject to corresponding different interpretations. The six CDRs also divide the frame regions on the heavy and light chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Unless a specific sub-region is designated as FR1, FR2, FR3, or FR4, as otherwise mentioned, a frame region refers to a combination of FRs within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four sub-regions, FR1, for example, represents the first frame region closest to the amino terminus of the variable region and 5' relative to CDR1, and FRs represent two or more sub-regions constituting a frame region.

[0091] Half-life: As used herein, the term “half-life” is the time required for an amount of protein, such as concentration or activity, to decrease to half of its value measured at the beginning of a period of time.

[0092] High affinity: As used herein, when referring to IgG type antibodies, the term "high affinity" refers to an affinity of 10 for the Flt-1 domain. -8 M or smaller, more preferably 10 -9 M or smaller and even more preferably 10 -10 M or smaller K D The antibody is called "high affinity". However, for other antibody isotypes, "high affinity" binding can vary. For example, for the IgM isotype, "high affinity" binding refers to binding with a 10-1... -7 M or smaller, more preferably 10 -8 M or smaller, or even more preferably 10 -9 M or smaller K D Antibodies.

[0093] Human antibody: As used herein, it is intended to include antibodies having variable and constant regions generated (or assembled) from human immunoglobulin sequences. In some embodiments, the antibody (or antibody component) may be considered “human” even though the amino acid sequence of the antibody (or antibody component) contains residues or elements not encoded by human germline immunoglobulin sequences (e.g., containing sequence variations that may have been introduced (initially) through in vitro random or site-specific mutagenesis or in vivo somatic mutations), such as in one or more CDRs and particularly CDR3.

[0094] Human monoclonal antibody: As used herein, it is intended to refer to an antibody having a variable region that exhibits single binding specificity, wherein both the framework region and the CDR region are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising B cells obtained from a transgenic nonhuman animal (e.g., a transgenic mouse) having a genome containing human heavy chain transgenes and light chain transgenes fused to immortalized cells.

[0095] Humanization: As is known in the art, the term "humanization" is generally used to refer to a substance whose amino acid sequence contains V from a reference antibody produced in a non-human species (e.g., mouse, llama). H and V L Humanized antibodies (or antibody components) are antibodies (or antibody components) that are immunoglobulin-specifically bound to a target antigen and include modifications in those sequences relative to a reference antibody, intended to make them more “human-like,” i.e., more similar to human germline variable sequences. In some embodiments, “humanized” antibodies (or antibody components) are antibodies (or antibody components) that are immunospecifically bound to a target antigen and have a framework (FR) region having an amino acid sequence substantially similar to that of human antibodies and a complementarity-determining region (CDR) having an amino acid sequence substantially similar to that of non-human antibodies (e.g., mouse, llama). Humanized antibodies comprise substantially all of at least one and typically two variable domains (Fab, Fab', F(ab')2, FabC, Fv), wherein all or substantially all of the CDR regions correspond to those CDR regions of non-human immunoglobulins (i.e., donor immunoglobulins) and all or substantially all of the framework regions are those framework regions of human immunoglobulin common sequences. In some embodiments, humanized antibodies also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of the human immunoglobulin constant region. In some implementations, the humanized antibody contains a light chain and at least a variable domain of the heavy chain. The antibody may also include a C0 constant region of the heavy chain. H 1. Hinges, C H 2. C H 3 and optional location C H Zone 4. In some implementation schemes, the humanized antibody contains only humanized V. L In some implementation schemes, the humanized antibody contains only humanized V. H In some specific implementations, the humanized antibody contains humanized V. H and V L district.

[0096] Hypertrophy: As used in this article, the term “hypertrophy” refers to an increase in the volume of an organ or tissue due to the enlargement of its constituent cells.

[0097] Improvement, increase, or decrease: As used herein, the terms “improvement,” “increase,” or “decrease,” or their grammatical equivalents, refer to values ​​relative to baseline measurements, such as measurements in the same individual prior to the commencement of the treatment described herein, or measurements in control individuals (or multiple control individuals) where the treatment described herein is not present. A “control individual” is an individual suffering from the same form of disease as the individual being treated, and who is approximately the same age as the individual being treated (to ensure that the stage of disease in the treated individual is comparable to that in the control individual).

[0098] Inhibition: As used herein, the terms “inhibition,” “inhibit,” and “inhibiting” refer to a process or method of reducing or decreasing the activity and / or expression of a target protein or gene. Generally, inhibiting a protein or gene means reducing the expression or associated activity of a protein or gene by at least 10% or more, such as 20%, 30%, 40%, or 50%, 60%, 70%, 80%, 90%, or more, or by more than 1, 2, 3, 4, 5, 10, 50, 100, or more, as measured by one or more methods described herein or recognized in the art.

[0099] In vitro: As used herein, the term “in vitro” refers to events that occur in an artificial environment, such as in test tubes or reaction vessels, cell cultures, etc., rather than in multicellular organisms.

[0100] In vivo: As used herein, the term "in vivo" refers to events occurring in multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events occurring within living cells (as opposed to, for example, in vitro systems).

[0101] Isolated antibody: As used herein, the term "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to Flt-1). Furthermore, isolated antibodies may be substantially free of other cellular material and / or chemicals.

[0102] K a As used herein, "K" refers to the specific rate of association of antibody-antigen interactions; while the term "K" as used herein refers to... d "Intended to refer to the dissociation rate of a specific antibody-antigen interaction. As used herein, the term "K" refers to the rate of dissociation of the antibody-antigen interaction. D "The intention refers to the dissociation constant, which is derived from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). The Kd of the antibody..." D The value can be determined using methods well-established in the art. The K value used to determine the antibody... DThe preferred method is to use surface plasmon resonance, preferably using a biosensor system, such as... system.

[0103] Light chain shuffling: As used herein, the term "light chain shuffling" is intended to refer to the affinity maturation step in which the heavy chain sequence remains constant and a library of light chain sequences is generated. Light chain libraries are screened against the heavy chain to identify antibodies with enhanced binding affinity. Enhanced binding affinity can be in the nanomolar or picomolar range.

[0104] Monoclonal antibody: As used herein, the term "monoclonal antibody" is intended to refer to a formulation of an antibody molecule consisting of a single molecule. Monoclonal antibody compositions exhibit single binding specificity and affinity for a specific epitope.

[0105] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” means a substance that, to the extent of reasonable medical judgment, is suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications, and is commensurate with a reasonable benefit / risk ratio.

[0106] Polypeptide: As used herein, the term "polypeptide" refers to a continuous chain of amino acids linked together by peptide bonds. The term is used to refer to an amino acid chain of any length, but those skilled in the art will understand that the term is not limited to lengthy chains and can refer to the smallest chain containing two amino acids linked together by peptide bonds. As is known to those skilled in the art, polypeptides can be processed and / or modified.

[0107] Prevention: As used herein, the term “prevent” or “prevention” when used in connection with the occurrence of a disease, condition, and / or symptom means reducing the risk of developing said disease, condition, and / or symptom. See the definition of “risk”.

[0108] Protein: As used herein, the term "protein" refers to one or more polypeptides that function as discrete units. The terms "polypeptide" and "protein" may be used interchangeably if a single polypeptide is a discrete functional unit and does not require permanent or temporary physical association with other polypeptides to form a discrete functional unit. If a discrete functional unit comprises more than one polypeptide physically associated with each other, the term "protein" refers to multiple polypeptides that are physically linked and together function as discrete units.

[0109] Risk: As will be understood from the context, “risk” of a disease, condition, and / or symptom includes the likelihood that a particular individual will develop a disease, condition, and / or symptom (e.g., DMD). In some embodiments, risk is expressed as a percentage. In some embodiments, risk is from 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% up to 100%. In some embodiments, risk is expressed as a risk relative to the risk associated with a reference sample or a set of reference samples. In some embodiments, the reference sample or set of reference samples has a known risk of disease, condition, symptom, and / or event (e.g., DMD). In some embodiments, the reference sample or set of reference samples is from individuals comparable to the specific individual. In some embodiments, relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0110] Selective binding: As used herein, “selective binding,” “selectively binding,” “specifically binding,” or “specifically binding” refers to the preferential binding of the binding moiety to the target rather than to an entity that is not a target, relating to the binding moiety and the target. A degree of non-specific binding may occur between the binding moiety and the non-target. In some embodiments, the binding moiety selectively binds to the target if the binding between the binding moiety and the target is greater than 2, 5, 10, or 100 times greater than the binding between the binding moiety and the non-target. In some embodiments, if the binding affinity is less than about 10... -5 M, less than approximately 10 -6 M, less than approximately 10 -7 M, less than approximately 10 -8 M or less than approximately 10 -9 M, on the other hand, selectively binds to the target.

[0111] Striated muscle: As used herein, the term "striated muscle" refers to multinucleated muscle tissue with a regular arrangement of its intracellular contractile units, sarcomeres, resulting in striations visible under a microscope and under voluntary control. Typically, striated muscle can be cardiac muscle, skeletal muscle, or branchial muscle.

[0112] Smooth muscle: As used in this article, the term "smooth muscle" refers to non-striated muscle that is not under voluntary control, including single and multi-unit muscles.

[0113] Subject: As used herein, the term "subject" refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate). Human includes both prenatal and postnatal forms. In many embodiments, the subject is a human being. A subject can be a patient, which refers to a person referred to a healthcare provider for the diagnosis or treatment of a disease. The term "subject" is used interchangeably herein with "individual" or "patient." A subject may have or be susceptible to a disease or condition but may or may not exhibit symptoms of said disease or condition.

[0114] Essentially: As used herein, the term “essentially” refers to a qualitative situation that exhibits a target characteristic or property of general or near-general extent or degree. Those skilled in the art of biology will understand that biological and chemical phenomena rarely (if they have ever occurred) reach completeness and / or proceed to completeness or achieve or avoid absolute results. Therefore, this article uses the term “essentially” to capture the inherent lack of completeness in many biological and chemical phenomena.

[0115] Fundamental homology: The phrase “fundamental homology” is used herein to refer to a comparison between amino acid sequences or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered “fundamentally homologous” if they contain homologous residues at corresponding positions. Homologous residues can be identical residues. Alternatively, homologous residues can be dissimilar residues that would have appropriately similar structural and / or functional characteristics. For example, as is well known to those skilled in the art, certain amino acids are generally classified as “hydrophobic” or “hydrophilic” amino acids, and / or as having “polar” or “nonpolar” side chains. Substituting one amino acid for another of the same type is generally considered a “homologous” substitution.

[0116] As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, vacancy BLAST, and PSI-BLAST for amino acid sequences. Examples of such procedures are described in Altschul et al., basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., "GappedBLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, these procedures typically provide an indication of the degree of homology. In some embodiments, two sequences are considered substantially homologous if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of corresponding residues in the relevant segment are homologous. In some embodiments, the relevant segment is a complete sequence. In some implementations, the relevant segment is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0117] Substantial Identity: The phrase “substantial identity” is used herein to refer to comparisons between amino acid sequences or nucleic acid sequences. As will be understood by those skilled in the art, two sequences are generally considered “substantially identical” if they contain the same residues at corresponding positions. As is well known in the art, amino acid sequences or nucleic acid sequences can be compared using any of a variety of algorithms, including those available in commercial computer programs, such as BLASTN for nucleotide sequences and BLASTP, vacancy BLAST, and PSI-BLAST for amino acid sequences. Examples of such procedures are described in Altschul et al., Basic local alignment search tool, J. Mol. Biol., 215(3):403-410, 1990; Altschul et al., Methods in Enzymology; Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener et al. (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, these procedures typically provide an indication of the degree of identity. In some embodiments, two sequences are considered substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of corresponding residues in the relevant segment are identical. In some embodiments, the relevant segment is a complete sequence. In some implementations, the relevant segment is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0118] Surface plasmon resonance: As used herein, it refers to an optical phenomenon that allows for the real-time analysis of specific binding interactions, for example, by using a Biacore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, NJ) to detect changes in protein concentration within a biosensor matrix. For further description, see Jonsson, U. et al., (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U. et al., (1991) Biotechniques 11:620-627; Johnsson, B. et al., (1995) J. Mol. Recognit. 8:125-131; and Johnsson, B. et al., (1991) Anal. Biochem. 198:268-277.

[0119] "Having": An individual who has been diagnosed with or exhibits one or more symptoms of the disease, condition and / or symptom (such as DMD).

[0120] Susceptible: An individual who is "susceptible" to a disease, condition, and / or symptom has not been diagnosed with the disease, condition, and / or symptom. In some embodiments, an individual susceptible to a disease, condition, and / or symptom may not exhibit symptoms of the disease, condition, and / or symptom. In some embodiments, an individual susceptible to a disease, condition, symptom, or event (e.g., DMD) may have one or more of the following characteristics: (1) a gene mutation associated with the development of the disease, condition, and / or symptom; (2) a genetic polymorphism associated with the development of the disease, condition, and / or symptom; (3) increased and / or decreased expression and / or activity of a protein associated with the disease, condition, and / or symptom; (4) habits and / or lifestyles associated with the development of the disease, condition, symptom, and / or event; (5) has experienced, plans to experience, or needs a transplant. In some embodiments, an individual susceptible to a disease, condition, and / or symptom will develop the disease, condition, and / or symptom. In some implementations, individuals susceptible to diseases, conditions, and / or symptoms will not develop the diseases, conditions, and / or symptoms.

[0121] Target tissue: As used herein, the term "target tissue" refers to any tissue affected by a disease to be treated, such as DMD. In some embodiments, target tissue includes tissue exhibiting disease-related pathology, symptoms, or characteristics, including but not limited to muscle wasting, skeletal deformities, cardiomyopathy, muscle ischemia, cognitive impairment, and impaired respiratory function. In some embodiments, target tissue is smooth muscle, striated muscle, or cardiac muscle.

[0122] Therapeutic effective amount: As used herein, the term "therapeutic effective amount" of a therapeutic agent means an amount sufficient to treat, diagnose, prevent symptoms of, and / or delay the onset of a disease, condition, and / or symptom when administered to a subject who has or is susceptible to such a disease, condition, and / or symptom. Those skilled in the art will understand that a therapeutic effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[0123] Treatment: As used herein, the term "treatment" means any method used to partially or completely alleviate, improve, mitigate, suppress, prevent, or delay the onset of one or more symptoms or features of a particular disease, condition, and / or symptom (such as DMD), reduce its severity, and / or decrease its incidence. Treatment may be administered to subjects who do not exhibit signs of the disease and / or only exhibit early signs of the disease for the purpose of reducing the risk of developing disease-related pathology.

[0124] Detailed Explanation

[0125] The present invention particularly provides methods and compositions for treating muscular dystrophy, including Duchenne muscular dystrophy (DMD) and / or Becker muscular dystrophy, based on the use of an anti-Flt-1 antibody or its antigen-binding fragment as a therapeutic agent for treating muscular dystrophy. In some embodiments, the present invention provides a method for treating DMD, the method comprising administering a therapeutically effective amount of an anti-Flt-1 antibody or its antigen-binding fragment to an individual suffering from or susceptible to DMD, such that at least one symptom or feature of DMD is reduced or delayed in terms of intensity, severity, or frequency.

[0126] Various aspects of the present invention are described in detail in the following sections. The use of sections is not intended to limit the invention. Each section may be applied to any aspect of the invention. In this application, unless otherwise indicated, the use of "or" means "and / or".

[0127] Duchenne muscular dystrophy (DMD)

[0128] DMD is a disease characterized by progressive deterioration of muscles and loss of muscle-related function throughout the body. This invention is intended to provide methods and compositions for slowing, delaying, or preventing muscle degeneration, regenerating muscle, and reversing, eliminating, delaying, preventing, or minimizing fibrosis, inflammation, and other symptoms or features associated with DMD and other muscular dystrophys in various muscle tissues.

[0129] muscle tissue

[0130] There are two main types of muscle tissue in animals: striated muscle and smooth muscle. As used herein, the term "striated muscle" refers to muscle tissue containing repetitive sarcomeres. Striated muscles tend to be voluntary and attach to bones. Striated muscles allow for voluntary movement of the body and include major muscle groups such as the quadriceps, gastrocnemius, biceps, triceps, trapezius, deltoid, and others. Striated muscles tend to be long, and many can function independently. However, some striated muscles do not attach to bones, including those in the mouth, anus, heart, and upper esophagus.

[0131] On the other hand, smooth muscle has a very different structure. Smooth muscle tends to be arranged in continuous sheets, with mechanical connections between smooth muscle cells, rather than a series of long muscles with individual skeletal appendages. Smooth muscle is usually located in the walls of hollow organs and is generally not under voluntary control. Smooth muscle lining a particular organ must simultaneously bear the same load and contract. Smooth muscle functions at least partially to handle changes in load on hollow organs caused by movement and / or changes in posture or pressure. This dual function means that smooth muscle must not only be able to contract like striated muscle, but also must be able to contract tensely to maintain organ size against sustained loads. Examples of smooth muscle are those lining blood vessels, bronchioles, the bladder, and the gastrointestinal tract such as the rectum.

[0132] Muscle strength depends on the number and size of muscle cells and their anatomical structure. Increasing the diameter of muscle fibers by synthesizing new myofibrils (hypertrophy) and / or forming more muscle cells (hyperplasia) will increase the muscle's force-generating capacity.

[0133] Muscles can also be grouped according to location or function. In some embodiments, the Flt-1 antibody or its antigen-binding fragment is targeted to one or more muscles of the face, one or more muscles for chewing, one or more muscles of the tongue and neck, one or more muscles of the chest, one or more muscles of the shoulder girdle and arm, one or more muscles of the arm and shoulder, one or more ventral and dorsal forearm muscles, one or more muscles of the hand, one or more muscles of the erector spinae, one or more muscles of the pelvic girdle and leg, and / or one or more muscles of the foreleg and foot.

[0134] In some implementations, facial muscles include, but are not limited to, intraocular muscles such as the ciliary muscle, iris dilator, and iris sphincter; ear muscles such as the auricular muscles, temporoparietal muscles, stapedius muscles, and tensor tympanic membrane muscles; nasal muscles such as the depressor supercilii, nasal muscles, nasal dilator, depressor nasal septum, and levator labii superioris alaeque nasi; and oral muscles such as the levator anguli oris, depressor anguli oris, orbicularis oris, buccinator, zygomaticus major and minor, platysma, levator labii superioris, depressor labii inferioris, risorius, mentalis, and / or corrugator supercilii.

[0135] In some embodiments, the masticatory muscles include, but are not limited to, the masseter, temporalis, medial pterygoid, and lateral pterygoid. In some embodiments, the muscles of the tongue and neck include, but are not limited to, the genioglossus, stylohyoidus, palatoglossus, hyoidoglossus, digastric, stylohyoid, mandibular, geniohyoid, omohyoid, sternohyoid, sternothyroid, thyrohyoid, sternocleidomastoid, anterior scalene, middle scalene, and / or posterior scalene.

[0136] In some implementations, the muscles of the chest, shoulder girdle, and arm include, but are not limited to, the subclavian, pectoralis major, pectoralis minor, rectus abdominis, external oblique, internal oblique, transversus abdominis, diaphragm, external intercostal muscles, internal intercostal muscles, serratus anterior, trapezius, levator scapulae, rhomboid major, rhomboid minor, latissimus dorsi, deltoid, subscapularis, supraspinatus, infraspinatus, teres major, teres minor, and / or coracobrachialis.

[0137] In some implementations, the muscles of the arm and shoulder include, but are not limited to, the long head of the biceps brachii, the short head of the biceps brachii, the long head of the triceps brachii, the lateral head of the triceps brachii, the medial head of the triceps brachii, the anconeus, the pronator teres, the supinator, and / or the brachialis.

[0138] In some implementations, the muscles of the ventral and dorsal forearms include, but are not limited to, the brachioradialis, flexor carpi radialis, flexor carpi ulnaris, palmaris longus, extensor carpi ulnaris, extensor carpi radialis longus, extensor carpi radialis brevis, extensor digitorum, and extensor digiti minimi.

[0139] In some implementations, the muscles of the hand include, but are not limited to, the intrinsic muscles of the hand, such as the thenar muscles, abductor pollicis brevis, flexor pollicis brevis, opponens pollicis, hypothenar muscles, abductor digiti minimi, flexor digiti minimi brevis, opponens digiti minimi, palmar interosseous muscles, dorsal interosseous muscles, and / or lumbrical muscles.

[0140] In some implementations, the erector spinae muscles include, but are not limited to, the cervical muscles, spinae, longissimus, and / or iliocostalis.

[0141] In some implementations, the muscles of the pelvic girdle and legs include, but are not limited to, the psoas major, iliacus, quadratus femoris, adductor longus, adductor brevis, adductor magnus, gracilis, sartorius, quadriceps femoris such as rectus femoris, vastus lateralis, vastus medialis, vastus intermedius, gastrocnemius, peroneus (peroneus) longus, soleus, gluteus maximus, gluteus medius, gluteus minimus, hamstring tendons: biceps femoris: long head, hamstring tendons: biceps femoris: short head, hamstring tendons: semitendinosus, hamstring tendons: semimembranosus, tensor fasciae latae, pectineus and / or tibialis anterior.

[0142] In some implementations, the muscles of the foreleg and foot include, but are not limited to, extensor digitorum longus, extensor peroneus brevis, plantar muscles, tibialis posterior, flexor digitorum longus, extensor digitorum brevis, extensor hallucis brevis, abductor hallucis, flexor hallucis brevis, abductor digiti minimi, flexor digiti minimi brevis, antiplantarus of digiti minimi, extensor digiti minimi brevis, lumbrical muscles of the foot, quadratus plantaris or accessory flexor digiti minimi, flexor digiti minimi brevis, dorsal interosseous muscles, and / or interosseous plantar muscles.

[0143] Exemplary muscle targets are summarized in Table 1.

[0144] Table 1.

[0145]

[0146]

[0147] Muscular dystrophy

[0148] Muscular dystrophy is a group of inherited disorders that cause muscle degeneration, leading to weakness and impaired movement. A central characteristic of all muscular dystrophys is that they are progressive in nature. Muscular dystrophys include, but are not limited to: Duchenne muscular dystrophy (DMD), Becker muscular dystrophy, Emery-Dreifuss muscular dystrophy, facioscapulohumeral muscular dystrophy, limb-girdle muscular dystrophy, and myotonic dystrophy type 1 and 2, including the congenital form of myotonic dystrophy type 1. Symptoms can vary depending on the type of muscular dystrophy, with some or all of the muscles affected. Exemplary symptoms of muscular dystrophy include delayed development of motor skills, difficulty using one or more muscle groups, difficulty swallowing, speaking, or eating, drooling, ptosis (drooping eyelids), frequent falls, loss of strength in adult muscles or muscle groups, loss of muscle size, gait impairment due to weakness or altered biomechanics, and / or cognitive or behavioral impairments / intellectual disability.

[0149] While there is no known cure for muscular dystrophy, several supportive treatments are used, including symptom and disease-modifying therapies. Corticosteroids, ACE inhibitors, angiotensin receptor blockers, physical therapy, orthotics, wheelchairs, or other assistive medical devices for ADL and lung function are commonly used in muscular dystrophy. Cardiac pacemakers are used to prevent sudden death due to arrhythmias in myotonic dystrophy. Antimyotonic agents that improve myotonia (inability to relax) include mexilitine, and in some cases phenytoin, procainamide, and quinine.

[0150] Duchenne muscular dystrophy

[0151] Duchenne muscular dystrophy (DMD) is a latent X-linked form of muscular dystrophy that leads to muscle degeneration and eventual death. DMD is characterized by proximal muscle weakness, gait abnormalities, hypertrophy of the gastrocnemius (calf) muscles, and elevated creatine kinase levels. Many DMD patients are diagnosed around age 5, at which point symptoms / signs usually become more pronounced. Affected individuals typically cease walking around age 10–13 and die in their mid-to-late 20s or earlier due to respiratory complications and cardiomyopathy.

[0152] In individuals with DMD, serum creatine kinase levels can be increased more than 10-fold compared to unaffected individuals. In some embodiments, application of the provided composition to the affected individual produces a reduction in serum creatine kinase levels compared to baseline levels prior to treatment. Typically, baseline levels are measured immediately prior to treatment. In some embodiments, application of the provided composition results in a reduction of serum creatine kinase levels of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% compared to baseline serum creatine kinase levels immediately prior to treatment. In some embodiments, administration of the provided composition results in a reduction of serum creatine kinase levels to less than about 3500 IU / L, 3000 IU / L, 2500 IU / L, 2000 IU / L, 1500 IU / L, 1000 IU / L, 750 IU / L, 500 IU / L, 250 IU / L, 100 IU / L, 90 IU / L, 80 IU / L, 70 IU / L, or 60 IU / L. In some embodiments, administration of the provided composition produces a reduced serum creatine kinase level compared to the serum creatine kinase level of an untreated subject.

[0153] The disease DMD is caused by mutations in the dystrophin gene located on the human X chromosome. This gene encodes the protein dystrophin, an important structural component in muscle tissue that provides structural stability to the dystrophin-associated proteoglycan complex (DGC) of the cell membrane. Dystrophin connects the internal cytoplasmic actin filament network to the extracellular matrix, thus providing physical strength to muscle fibers. Therefore, alterations or absence of dystrophin lead to abnormal muscle membrane tearing and necrosis of muscle fibers. Although both sexes can carry the mutation, women rarely exhibit severe disease symptoms.

[0154] The main symptom of DMD is muscle weakness associated with muscle wasting, which usually affects voluntary muscles first, particularly those in the hip, pelvic region, thigh, shoulder, and calf. Muscle weakness can also occur in the arms, neck, and other areas. The calves are often enlarged. Signs and symptoms usually appear before age 6, but can appear as early as infancy. Cardiomyopathy usually occurs in DMD patients after age 18. Other physical symptoms include, but are not limited to, delayed ability to walk independently, progressive difficulty walking, stepping, or running, and eventual loss of walking ability (usually by age 12); frequent falls; fatigue; difficulty with motor skills (running, jumping, hopping); increased lumbar lordosis leading to shortening of the hip flexors; impaired function of the Achilles and hamstrings, connective tissue fibrosis; myofibril deformities; pseudohypertrophy (enlargement) of the tongue and calf muscles due to the replacement of muscle tissue with fat and connective tissue; high risk of non-progressive weakening in neurobehavioral disorders (e.g., ADHD), learning disabilities (dyslexia), and specific cognitive skills (especially short-term nonverbal memory); and skeletal deformities (including scoliosis in some cases).

[0155] Muscle changes observed in DMD are accompanied by an increase in connective tissue (i.e., the development of fibrosis) and are caused by responses or repair processes involving mechanical, humoral, and / or cytokine factors. A lack of functional dystrophin leads to instability of the muscle fiber membrane, and as a result, cells are less resistant to mechanical shearing and tend to experience excessive electrolyte influx, leading to tissue damage. Because muscle tissue in DMD is damaged, recovery is limited by the ability of satellite cells to proliferate. This results in necrosis, inflammation, fibrosis, and adipocyte replacement. The increase in connective tissue occurs early in the disease process, as the conjunctival tissue that encloses each muscle cell in a sheath and covers the sarcolemma (i.e., the endomysium) increases before observable muscle damage. Increased collagenous connective tissue is a factor in the muscle pathology of DMD, adversely affecting the nutrient supply to the affected muscle cells and, secondarily, the age at which muscle strength and mobility are lost.

[0156] In some embodiments, administration of an anti-Flt-1 antibody or its antigen-binding fragment in vivo results in a reduction of fibrosis in muscle tissue. In some embodiments, the muscle is skeletal muscle. In specific embodiments, the muscle is cardiac muscle, diaphragm, gastrocnemius muscle, and / or tibialis anterior (TA) muscle. In some embodiments, reduced fibrosis is demonstrated by reduced collagen staining. In some embodiments, the collagen is type I collagen. In some embodiments, reduced fibrosis can be measured, for example, by measuring the percentage of collagen-positive area in the muscle of a mouse administered the anti-Flt-1 antibody or its antigen-binding fragment. For example, the percentage of collagen-positive area in the diaphragm of a mouse administered the anti-Flt-1 antibody or its antigen-binding fragment could be at least about 3.0%, about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, or about 9.0% of the total tissue area. In one specific implementation, the percentage of collagen-positive area in the diaphragm of mice administered anti-Flt-1 antibody was significantly lower than that of mice administered isotype control antibody.

[0157] In some embodiments, administration of an anti-Flt-1 antibody or its antigen-binding fragment in vivo results in a reduction of muscle tissue necrosis. In some embodiments, the muscle is skeletal muscle. In specific embodiments, the muscle is cardiac muscle, diaphragm, gastrocnemius muscle, and / or tibialis (TA) muscle. In some embodiments, the reduction in necrosis can be measured, for example, by measuring the percentage of necrotic positive area in the muscle of a mouse administered the anti-Flt-1 antibody or its antigen-binding fragment. For example, the percentage of necrotic positive area in the gastrocnemius muscle of a mouse administered the anti-Flt-1 antibody or its antigen-binding fragment may be at least about 0.5%, about 0.45%, about 0.4%, about 0.35%, about 0.3%, about 0.25%, about 0.2%, about 0.15%, about 0.1%, about 0.05%, or about 0.025% of the total tissue area. In one specific embodiment, the percentage of necrotic positive area in the gastrocnemius muscle of a mouse administered the anti-Flt-1 antibody may be significantly lower than the percentage of necrotic positive area in the gastrocnemius muscle of a mouse administered an isotype control antibody.

[0158] In some implementations, administration of anti-Flt-1 antibody or its antigen-binding fragment in vivo produces increased muscle strength and / or anti-fatigue properties.

[0159] Flt-1 receptor

[0160] The Flt-1 receptor (also known as vascular endothelial growth factor receptor 1 (VEGFR-1) or Flt-1) is a receptor encoded by the FLT1 gene and expressed on the cell membranes of endothelial cells and monocytes. The vascular endothelial growth factor (VEGF) family of signaling glycoproteins acts as potential promoters of angiogenesis during embryogenesis and postnatal growth. Specifically, the binding of VEGF-A ligands to VEGF receptors has been shown to promote vascular permeability and also induce endothelial cell migration, proliferation, and survival, with newly formed endothelial cells providing the basic structure of the new vascular system. VEGF-A, the major VEGF signaling molecule for angiogenesis, mediates its signaling through VEGF receptor-1 (VEGFR-1, also known as Flt-1) and VEGF receptor-2 (VEGFR-2, also known as Flk-1). A soluble form of Flt-1 (sFlt-1) also exists, but lacks an intracellular signal transduction domain and is therefore thought to function solely through chelation of VEGF-A or other ligands bound to it in terms of regulatory capacity. sFlt-1 and other molecules containing Flt-1 binding sites that are not involved in intracellular signal transduction pathways are collectively referred to as "decoy receptors." Flt-1 and Flk-1 receptors contain an extracellular VEGF-A binding domain and an intracellular tyrosine kinase domain, and are expressed during developmental stages and tissue regeneration in angiogenic and endothelial cell lineages. Compared to Flk-1, Flt-1 has approximately 10-fold binding affinity for VEGF-A (Kk). d Approximately 2 -10 While the VEGF-A concentration was pM, the weaker tyrosine kinase activity indicated that the angiogenic signaling pathway following VEGF-A binding to Flt-1 was weaker than that resulting from VEGF-A binding to Flk-1. Therefore, homozygous Flt-1 knockout mice died during embryonic development due to excessive endothelial cell production and vascular tissue destruction. Conversely, homozygous Flk-1 knockout mice died due to defects in organized vascular development caused by the lack of yolk sac blood island formation during embryogenesis. Both Flt-1 and Flk-1 receptors are required for normal development, but selectively increased VEGF-A concentration allows for greater binding to the Flk-1 receptor and induces increased capillary density, promoting muscle regeneration, reducing fibrosis and inflammation, and alleviating symptoms and features associated with DMD and other muscular dystrophy in various muscle tissues.

[0161] As used herein, the term "Flt-1 receptor" refers to both soluble and membrane-associated Flt-1 receptors or a functional fragment thereof.

[0162] Anti-Flt-1 antibody

[0163] As used herein, the term "anti-Flt-1 antibody" refers to any antibody or antigen-binding fragment thereof that binds to a Flt-1 receptor (e.g., a soluble or membrane-associated Flt-1 receptor). In some embodiments, anti-Flt-1 antibodies are generated that bind to the Flt-1 receptor with high affinity. It is not intended to be theoretically binding, but it is believed that anti-Flt-1 antibodies binding to the Flt-1 receptor inhibit the binding of one or more endogenous ligands to Flt-1, thereby allowing a greater quantity of available ligands to associate with other VEGF receptors such as the Flk-1 receptor. Increased VEGF availability promotes angiogenesis, accompanied by increased blood flow to muscle to counteract functional ischemia and improve the structural and functional characteristics of DMD. In some embodiments, antibodies binding to the Flt-1 receptor increase the amount of VEGF available for binding to other VEGF receptors.

[0164] In some implementations, the anti-Flt-1 antibody or its antigen-binding fragment comprises the sequences provided in Table 2.

[0165] Table 2.

[0166]

[0167]

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment comprises one or more complementarity-determining regions (CDRs), said CDRs being selected from the group consisting of: VL CDR1, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:19 to SEQ ID NO:21; VLCDR2, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:22 to SEQ ID NO:24; VL CDR3, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:25 to SEQ ID NO:34; VH chain CDR1, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:19 to SEQ ID NO:21; and VH chain CDR1, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:19 to SEQ ID NO:21. The amino acid sequence defined by any one of SEQ ID NO:4 having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:5 to SEQ ID NO:14; VH CDR2, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:15 to SEQ ID NO:18; and VHCDR3, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:15 to SEQ ID NO:18. In some embodiments, VL CDR3 is not SEQ ID NO:25. In some embodiments, VH CDR3 is not SEQ ID NO:15.

[0178] In some embodiments, the one or more CDRs include VL CDR3, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:25 to SEQ ID NO:34; and VH CDR3, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:15 to SEQ ID NO:18. In some embodiments, VL CDR3 is not SEQ ID NO:25. In some embodiments, VH CDR3 is not SEQ ID NO:15.

[0179] In some embodiments, the one or more CDRs include VL CDR1, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any of SEQ ID NO:19 to SEQ ID NO:21; VL CDR2, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any of SEQ ID NO:22 to SEQ ID NO:24; and VL CDR3, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any of SEQ ID NO:25 to SEQ ID NO:34. In some embodiments, VL CDR3 is not SEQ ID NO:25.

[0180] In some embodiments, the one or more CDRs include VH CDR1, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any of SEQ ID NO:1 to SEQ ID NO:4; VH CDR2, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any of SEQ ID NO:5 to SEQ ID NO:14; and VH CDR3, defined by an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any of SEQ ID NO:15 to SEQ ID NO:18. In some embodiments, VH CDR3 is not SEQ ID NO:15.

[0181] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VL chain, the VL chain containing VL CDR1, VLCDR2, and VL CDR3 as defined by the amino acid sequences of SEQ ID NO:19, SEQ ID NO:22, and SEQ ID NO:25, respectively. In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VL chain, the VL chain containing VLCDR1, VL CDR2, and VL CDR3 as defined by the amino acid sequences of SEQ ID NO:20, SEQ ID NO:23, and SEQ ID NO:25, respectively. In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VL chain, the VL chain comprising VL CDR1 and VL CDR2 defined by the amino acid sequences of SEQ ID NO:21 and SEQ ID NO:24, respectively, and VLCDR3 defined by the amino acid sequences of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, or SEQ ID NO:34. In one specific embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VL chain, the VL chain comprising VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, VLCDR2 defined by the amino acid sequence of SEQ ID NO:24, and VL CDR3 defined by the amino acid sequence of SEQ ID NO:32. In some embodiments, VL CDR3 is not SEQ ID NO:25.

[0182] In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VH chain, the VH chain containing VH CDR1, VHCDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO:1, SEQ ID NO:5, and SEQ ID NO:15, respectively. In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VH chain, the VH chain containing VHCDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:6, and SEQ ID NO:16, respectively. In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VH chain, the VH chain containing VH CDR1, VH CDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:18, respectively. In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VH chain, the VH chain containing VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:17, respectively, and VH CDR2 defined by the amino acid sequences of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:13, or SEQ ID NO:14. In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VH chain, the VH chain containing VH CDR1 and VH CDR3 defined by the amino acid sequences of SEQ ID NO:3 and SEQ ID NO:17, respectively, and VHCDR2 defined by the amino acid sequences of SEQ ID NO:9, SEQ ID NO:11, or SEQ ID NO:12. In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VH chain, the VH chain containing VH CDR1, VHCDR2, and VH CDR3 defined by the amino acid sequences of SEQ ID NO:4, SEQ ID NO:9, and SEQ ID NO:17, respectively. In one embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a VH chain, the VH chain containing VH CDR1 defined by the amino acid sequence of SEQ ID NO:3, VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and VH CDR3 defined by the amino acid sequence of SEQ ID NO:17. In some embodiments, VH CDR3 is not SEQ ID NO:15.

[0183] In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a light chain VL region containing an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:49 to SEQ ID NO:61; and / or a heavy chain VH region containing an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:35 to SEQ ID NO:48. In one specific embodiment, the VL region contains the amino acid sequence of SEQ ID NO:60, and the VH region contains the amino acid sequence of SEQ ID NO:45. In another embodiment, the antibody further comprises a heavy chain constant region containing an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:87 to SEQ ID NO:89. In some embodiments, the VL region is not SEQ ID NO:49 or SEQ ID NO:50. In some embodiments, the VH region is not SEQ ID NO:35 or SEQ ID NO:36.

[0184] In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment comprises a light chain having an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:75 to SEQ ID NO:86; and / or a heavy chain having an amino acid sequence having at least 60%, 70%, 80%, 90%, 95%, or 98% identity with any one of SEQ ID NO:62 to SEQ ID NO:74. In one specific embodiment, the light chain comprises the amino acid sequence of SEQ ID NO:76, and the heavy chain region comprises the amino acid sequence of SEQ ID NO:71.

[0185] In some embodiments, the heavy chain of the anti-Flt-1 antibody or its antigen-binding fragment contains an amino acid sequence.

[0186] MGWSCIILFLVATATGVHSELQLVESGGGLVQPGGSLRLSCAASGF

[0187] TFSDYSASWVRQAPGKGLEWVSAISWSGDSTYYAESVKGRFTIFR

[0188] DNSKNTLYLQMNSLRAEDTAVYYCAKSWATPIESLYYYGSDYWG

[0189] QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV

[0190] TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYIC

[0191] NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPP

[0192] KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT

[0193] KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE

[0194] KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV

[0195] EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKX (SEQ ID NO: 108). In some embodiments, the heavy chain of the anti-Flt-1 antibody or its antigen-binding fragment contains the amino acid sequence ELQLVESGGGLVQPGGSLRLSCAASGFTFSDYSASWVRQAPGKGLEWVSAISWSGDSTYYAESVKGRFTIFRDNSKNTLYLQMNSLRAED TAVYYCAKSWATPIESLYYYGSDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPS RDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 109).

[0196] In some embodiments, the light chain of the anti-Flt-1 antibody or its antigen-binding fragment contains an amino acid sequence.

[0197] MGWSCIILFLVATATGVHSSYELTQPLSVSVALRQAAKITCGGNNIG

[0198] SQTAQWYQQKPGQAPVLVIYANNRRPSGIPERFSGSKSGNTATLTIS

[0199] RAQAGDEADYYCQVWDASTQAIVFGGGTKLTVLGQPKAAPSVTL

[0200] FPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTT

[0201] PSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAP TECSX (SEQ ID NO: 110).

[0202] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment comprises the heavy chain of SEQ ID NO:108 or SEQ ID NO:109 and the light chain of SEQ ID NO:110 or SEQ ID NO:76.

[0203] In some implementations, the anti-Flt-1 antibody or its antigen-binding fragment has an affinity for human Flt-1 greater than about 10. -7 M, greater than approximately 0.5 x 10 -7 Greater than approximately 10 -8 Greater than approximately 0.5 x 10 -8 Greater than approximately 10 -9 M, greater than approximately 0.5 x 10 -9 Greater than approximately 10 -10 M, greater than approximately 0.5 x 10 -10 M, greater than approximately 10 -11 M, greater than approximately 0.5 x 10 -11 M, greater than approximately 10 -12 M or greater than approximately 0.5 x 10 -12 M. In other embodiments, the anti-Flt-1 antibody or its antigen-binding fragment binds to mouse Flt-1 with an affinity greater than about 10. -7 M, greater than approximately 0.5 x 10 -7 Greater than approximately 10 -8 Greater than approximately 0.5 x 10 -8 Greater than approximately 10 -9 M, greater than approximately 0.5 x 10 -9 Greater than approximately 10 -10 M, greater than approximately 0.5 x 10 -10 M, greater than approximately 10 -11 M, greater than approximately 0.5 x 10 -11 M, greater than approximately 10 -12 M or greater than approximately 0.5 x 10 -12 The affinity of the M. Flt-1 antibody can be measured, for example, in a surface plasmon resonance assay, such as the BIACORE assay.

[0204] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is characterized by an IC50 value that, in a competitive assay with human Flt-1, 50Below approximately 500 pM, below approximately 400 pM, below approximately 300 pM, below approximately 200 pM, below approximately 100 pM, below approximately 50 pM, below approximately 25 pM, below approximately 10 pM, below approximately 5 pM, or below approximately 1 pM. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is characterized by an IC50 value lower than that of mouse Flt-1 in a competitive assay. 50 Below approximately 500 pM, below approximately 400 pM, below approximately 300 pM, below approximately 200 pM, below approximately 100 pM, below approximately 50 pM, below approximately 25 pM, below approximately 10 pM, below approximately 5 pM, or below approximately 1 pM.

[0205] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment inhibits the binding and / or activity of VEGF at the Flt-1 receptor. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is characterized by an IC50 value for inhibition of VEGF binding to human Flt-1 in a competitive assay. 50 Below approximately 500 pM, below approximately 400 pM, below approximately 300 pM, below approximately 200 pM, below approximately 100 pM, below approximately 50 pM, below approximately 25 pM, below approximately 10 pM, below approximately 5 pM, or below approximately 1 pM.

[0206] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment competitively binds to soluble Flt-1 and / or inhibits the binding of VEGF to soluble Flt-1. In other embodiments, the competition and / or inhibition are in a dose-dependent manner. In a specific embodiment, inhibition of VEGF binding to Flt-1 leads to increased phosphorylation of VEGF R2. Not wishing to be bound by theory, the binding of the anti-Flt-1 antibody or its antigen-binding fragment to Flt-1 inhibits the binding of VEGF to Flt-1. Unbound VEGF binds to VEGF R2, which can be demonstrated by measuring VEGF R2 phosphorylation. In a specific embodiment, the anti-Flt-1 antibody or antigen-binding fragment rescues VEGF R2 phosphorylation in a dose-dependent manner. For example, VEGF R2 phosphorylation can be rescued by at least about 100%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%.

[0207] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment provides rescue of greater than about 95%, greater than about 90%, greater than about 85%, greater than about 80%, greater than about 75%, greater than about 70%, greater than about 65%, greater than about 60%, greater than about 55%, greater than about 50%, greater than about 45%, greater than about 40%, greater than about 35%, greater than about 30%, greater than about 25%, greater than about 20%, greater than about 15%, or greater than about 10% in the bioassay. In one specific embodiment, the bioassay comprises human primary venous endothelial cells (HUVECs) stimulated with VEGF in the presence of sFlt-1 and the anti-Flt-1 antibody or its antigen-binding fragment. VEGF-induced cell activation can be determined by determining the phosphorylation status of the VEGF R2 receptor. In the presence of sFlt-1 alone (e.g., without the anti-Flt-1 antibody), the data can be expressed as the percentage of VEGF R2 receptor phosphorylation rescue relative to the phosphorylation of the VEGF R2 receptor.

[0208] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment has a half-life of greater than about 200 hours, greater than about 150 hours, greater than about 100 hours, greater than about 95 hours, greater than about 90 hours, greater than about 85 hours, greater than about 80 hours, greater than about 75 hours, greater than about 70 hours, greater than about 65 hours, greater than about 60 hours, greater than about 55 hours, greater than about 50 hours, or greater than about 45 hours, and ranges thereof. In some embodiments, the half-life is measured in mice.

[0209] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment has a maximum serum concentration greater than about 400 ug / mL, greater than 375 ug / mL, greater than about 350 ug / mL, greater than about 325 ug / mL, greater than about 300 ug / mL, greater than about 275 ug / mL, greater than about 250 ug / mL, greater than about 225 ug / mL, greater than about 200 ug / mL, greater than about 175 ug / mL, greater than about 150 ug / mL, greater than about 125 ug / mL, greater than about 100 ug / mL, greater than about 75 ug / mL, or greater than about 50 ug / mL, and ranges thereof. In some embodiments, said maximum serum concentration is measured in mice.

[0210] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment selectively binds to Flt-1 and has minimal or no significant binding to other VEGF receptors. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment selectively binds to Flt-1 and has minimal or no significant binding to VEGF R2 (Flk-1) and / or VEGF R3 (Flt-4).

[0211] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a ka greater than about 1 x 10 -3 M -1 sec -1 , greater than about 1 x 10 -4 M -1 sec -1 , greater than about 1 x 10 -5 M -1 sec -1 , greater than about 1 x 10 -6 M -1 sec -1 or greater than about 1 x 10 -7 M -1 sec -1 when binding to human Flt-1.

[0212] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a kd greater than about 1 x 10 -3 sec -1 , greater than about 1 x 10 -4 sec -1 , greater than about 1 x 10 -5 sec -1 or greater than about 1 x 10 -6 sec -1 when binding to human Flt-1.

[0213] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a K -8 greater than about 1 x 10 -9 M, greater than about 1 x {{10}} -10 M, greater than about 1 x {{10}} -11 M, greater than about 1 x {{10}} -12 M or greater than about 1 x {{10}} D .

[0214] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof binds to soluble Flt-1. In certain embodiments, the binding is dose-dependent, where higher concentrations of the antibody or antigen-binding fragment bind greater amounts of soluble Flt-1.

[0215] In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment thereof has a percent human identity greater than about 99%, greater than about 98%, greater than about 97%, greater than about 96%, greater than about 95%, greater than about 94%, greater than about 93%, greater than about 92%, greater than about 91%, greater than about 90% or greater than about 80%.

[0216] In some implementations, the anti-Flt-1 antibody or its antigen-binding fragment has a human homology percentage greater than about 99%, greater than about 98%, greater than about 97%, greater than about 96%, greater than about 95%, greater than about 94%, greater than about 93%, greater than about 92%, greater than about 91%, greater than about 90%, or greater than about 80%.

[0217] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment binds to the Flt-1 protein. In some embodiments, the Flt-1 protein is a recombinant protein, such as recombinant sFlt-1. In one specific embodiment, the anti-Flt-1 antibody or its antigen-binding fragment binds to human Flt-1 isotype 1 (NP_002010.2GI:156104876; SEQ ID NO:90) (Table 13).

[0218] In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment binds to human Flt-1 isotype X1 (XP_011533316.1GI:767977511; SEQ ID NO:91). In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment binds to human Flt-1 isotype 2 precursor (NP_001153392.1GI:229892220; SEQ ID NO:92). In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment binds to human Flt-1 isotype 3 precursor (NP_001153502.1GI:229892300; SEQ ID NO:93). In another embodiment, the anti-Flt-1 antibody or its antigen-binding fragment binds to human Flt-1 isotype 4 precursor (NP_001153503.1GI:229892302; SEQ ID NO:94).

[0219] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment binds to a specific epitope of the Flt-1 protein. For example, the anti-Flt-1 antibody or its antigen-binding fragment binds to an amino acid sequence as provided in Table 3.

[0220] Table 3.

[0221]

[0222] In some embodiments, administration of the anti-Flt-1 antibody or its antigen-binding fragment in vivo produces peak serum antibody levels of at least about 700 ug / mL, at least about 650 ug / mL, at least about 600 ug / mL, at least about 550 ug / mL, at least about 500 ug / mL, at least about 450 ug / mL, at least about 400 ug / mL, at least about 350 ug / mL, at least about 300 ug / mL, at least about 250 ug / mL, at least about 200 ug / mL, at least about 150 ug / mL, at least about 100 ug / mL, at least about 50 ug / mL, at least about 40 ug / mL, at least about 30 ug / mL, at least about 20 ug / mL, at least about 10 ug / mL, or at least about 5 ug / mL, and ranges thereof. In some embodiments, the peak serum antibody levels are dose-dependent.

[0223] In some embodiments, administration of anti-Flt-1 antibody or its antigen-binding fragment in vivo produces trough serum antibody levels of at least about 450 ug / mL, at least about 400 ug / mL, at least about 350 ug / mL, at least about 300 ug / mL, at least about 250 ug / mL, at least about 200 ug / mL, at least about 150 ug / mL, at least about 100 ug / mL, at least about 50 ug / mL, or at least about 25 ug / mL, and ranges thereof. In some embodiments, the trough serum antibody level is dose-dependent.

[0224] In some embodiments, administration of an anti-Flt-1 antibody or its antigen-binding fragment to the body produces a reduced serum level of soluble Flt-1 compared to baseline levels or compared to levels in a subject administered the agent alone. Typically, baseline levels are measured immediately prior to administration. In some embodiments, administration of the anti-Flt-1 antibody or its antigen-binding fragment results in a reduction of serum levels of soluble Flt-1 by at least about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% compared to baseline serum levels of soluble Flt-1 immediately prior to administration. In some implementations, administration of anti-Flt-1 antibody or its antigen-binding fragment reduces serum levels of soluble Flt-1 to less than approximately 4000 pg / mL, approximately 3500 pg / mL, approximately 3000 pg / mL, approximately 2500 pg / mL, approximately 2000 pg / mL, approximately 1750 pg / mL, approximately 1500 pg / mL, approximately 1250 pg / mL, approximately 1000 pg / mL, approximately 900 pg / mL, approximately 800 pg / mL, approximately 700 pg / mL, approximately 600 pg / mL, approximately 500 pg / mL, approximately 450 pg / mL, approximately 400 pg / mL, approximately 350 pg / mL, approximately 300 pg / mL, approximately 250 pg / mL. pg / mL, about 200 pg / mL, about 150 pg / mL, about 100 pg / mL, about 50 pg / mL, or about 10 pg / mL, and ranges thereof. In some embodiments, administration of said antibody or its antigen-binding fragment produces a reduced serum level of soluble Flt-1 compared to the serum level of soluble Flt-1 in subjects who have not been administered the anti-Flt-1 antibody or its antigen-binding fragment. In some embodiments, the reduced serum level of soluble Flt-1 is dose-dependent.

[0225] In some embodiments, administration of an anti-Flt-1 antibody or its antigen-binding fragment to the body results in an increase in serum VEGF levels compared to baseline levels or compared to levels in subjects treated with the mediator alone. Typically, baseline levels are measured immediately prior to treatment. In some embodiments, administration of the anti-Flt-1 antibody or its antigen-binding fragment to the body results in an increase in serum VEGF levels of at least about 95%, about 90%, about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, or about 10% compared to baseline serum VEGF levels immediately prior to administration. In some embodiments, administration of the anti-Flt-1 antibody or its antigen-binding fragment results in an increase in serum VEGF levels of up to and including approximately 500 pg / mL, approximately 450 pg / mL, approximately 400 pg / mL, approximately 350 pg / mL, approximately 300 pg / mL, approximately 250 pg / mL, approximately 200 pg / mL, approximately 150 pg / mL, approximately 100 pg / mL, approximately 50 pg / mL, or approximately 25 pg / mL, and ranges thereof. In some embodiments, administration of the anti-Flt-1 antibody or its antigen-binding fragment produces an increased serum VEGF level compared to the serum VEGF level in untreated subjects. In some embodiments, the increased serum VEGF level is dose-dependent.

[0226] In some embodiments, administration of an anti-Flt-1 antibody or its antigen-binding fragment in vivo results in increased angiogenesis in muscle tissue. In some embodiments, the muscle is skeletal muscle. In specific embodiments, the muscle is the diaphragm, gastrocnemius, and / or tibialis anterior (TA) muscle. In some embodiments, the increased angiogenesis is demonstrated by increased CD31 staining of endothelial cell markers such as CD31. In some embodiments, the increased staining can be measured, for example, by measuring the percentage of CD31-positive area in the muscle of mice administered the anti-Flt-1 antibody or its antigen-binding fragment. For example, the percentage of CD31-positive area in the diaphragm of mice administered anti-Flt-1 antibody or its antigen-binding fragment may be at least about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, or about 2.5% of the total tissue area. In another example, the percentage of CD31-positive area in the TA muscle of mice administered anti-Flt-1 antibody or its antigen-binding fragment may be at least about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, or about 1.0% of the total tissue area. In one specific implementation, the percentage of CD31-positive area in the diaphragm or TA muscle of mice administered anti-Flt-1 antibody was significantly higher than that in mice administered isotype control antibody.

[0227] In some embodiments, the increased staining of the endothelial cell marker can be measured, for example, by measuring the normalized percentage of CD31 positivity in the muscle of mice administered anti-Flt-1 antibody or its antigen-binding fragment. In specific embodiments, the increase in CD31 staining in the muscle of mice administered anti-Flt-1 antibody or its antigen-binding fragment is relative to CD31 staining measured in the muscle of mice administered an isotype control antibody. For example, the normalized percentage of CD31 positivity in the diaphragm of mice administered anti-Flt-1 antibody or its antigen-binding fragment can be at least about 200%, about 190%, about 180%, about 170%, about 160%, about 150%, about 140%, about 130%, about 120%, or about 110%. In another instance, the standardized CD31 positivity percentage in the TA muscle of mice administered anti-Flt-1 antibody or its antigen-binding fragment can be at least about 300%, about 290%, about 280%, about 270%, about 260%, about 250%, about 240%, about 230%, about 220%, about 210%, about 200%, about 190%, about 180%, about 170%, about 160%, about 150%, about 140%, about 130%, about 120%, or about 110%, and ranges thereof.

[0228] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment selectively binds to human Flt-1 and has minimal or no significant binding to other mammalian Flt-1 receptors (e.g., binding affinity less than 10). -7 M or 10 -6 M). In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment selectively binds to human Flt-1 and not to monkey Flt-1. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment selectively binds to human Flt-1 and not to mouse Flt-1.

[0229] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment binds to human Flt-1 and monkey Flt-1. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment binds to cynomolgus monkey Flt-1. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment binds to human Flt-1 and mouse Flt-1.

[0230] In some implementations, the anti-Flt-1 antibody or its antigen-binding fragment is selected from the group consisting of: IgG, F(ab')2, F(ab)2, Fab', Fab, ScFv, biantibody, triantibody, and tetraantibody.

[0231] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is IgG. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is IgG1.

[0232] Constant zone of engineering renovation

[0233] In some embodiments, a suitable anti-Flt-1 antibody contains an Fc domain or a portion thereof that binds to an FcRn receptor. As a non-limiting example, a suitable Fc domain may be derived from an immunoglobulin subclass, such as IgG. In some embodiments, a suitable Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. Particularly suitable Fc domains include those derived from human or humanized antibodies.

[0234] The improved binding between the Fc domain and the FcRn receptor is expected to result in an extended serum half-life. Therefore, in some embodiments, a suitable Fc domain (SEQ ID NO: 104) comprises one or more amino acid mutations that lead to improved binding with FcRn. Various mutations within the Fc domain that affect improved binding with FcRn are known in the art and are suitable for practicing the present invention. In some embodiments, a suitable Fc domain comprises one or more mutations at one or more positions corresponding to Leu234, Leu235, Gly237, Thr250, Met 252, Ser254, Thr256, Thr 307, Glu 380, Met428, His 433, and / or Asn434 of human IgG1.

[0235] Some mutations in the Fc domain lead to reduced binding of IgG to the FcRn receptor, thereby inhibiting effector function. In some embodiments, a suitable Fc domain (SEQ ID NO: 104) comprises one or more mutations at one or more sites corresponding to Leu 234, Leu 235, and Gly 237 of human IgG1. In one specific embodiment, Leu 234 is mutated to Ala. In another embodiment, Leu 235 is mutated to Ala. In yet another embodiment, Gly 237 is mutated to Ala.

[0236] In some embodiments, the anti-FLT-1 antibody or antigen-binding fragment contains a spacer region and / or is linked to another entity. In some embodiments, the linker or spacer region contains a linker to... GAP GGGGGAAAAAGGGGG GAP(SEQ ID NO:105) (GAG connector) has a sequence with at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity. In some embodiments, the connector or spacer region contains a sequence with... GAP GGGGGAAAAAGGGGG GAP GGGGGAAAAAGGGGG GAP (SEQ ID NO:106) (GAG2 connector) has a sequence with at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity. In some embodiments, the connector or spacer region contains a sequence with... GAP GGGGGAAAAAGGGGG GAP GGGGGAAAAAGGGGG GAP GG GGGAAAAAGGGGG GAP (SEQ ID NO:107) (GAG3 connector) has a sequence with at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identity.

[0237] Production of anti-Flt-1 antibody and antigen-binding fragments

[0238] The recombinant anti-Flt-1 antibody or its antigen-binding fragment suitable for this invention can be generated by any available means. For example, the recombinant anti-Flt-1 antibody or antigen-binding fragment can be recombinantly generated using a host cell system engineered to express nucleic acids encoding the recombinant anti-Flt-1 antibody or antigen-binding fragment.

[0239] Therefore, the present invention further provides polynucleotide sequences encoding the various amino acid sequences described herein. In some embodiments, the present invention provides a polynucleotide sequence encoding the heavy or light chain amino acid sequence of the anti-Flt-1 antibody described herein (e.g., any one of SEQ ID NO: 62-86 or SEQ ID NO: 108-110). In some embodiments, the present invention provides a polynucleotide sequence encoding a variable region (e.g., any one of SEQ NO: 35-61) of the heavy or light chain amino acid sequence of the anti-Flt-1 antibody described herein. In some embodiments, the present invention provides a polynucleotide sequence encoding a CDR region (e.g., any one of SEQ ID NO: 1-34) of the heavy or light chain amino acid sequence of the anti-Flt-1 antibody described herein. In some embodiments, the present invention provides a polynucleotide sequence encoding the constant region amino acid sequence of the anti-Flt-1 antibody described herein (e.g., any one of SEQ ID NO: 87-89). In some embodiments, the present invention provides a polynucleotide sequence encoding the Fc region amino acid sequence of the anti-Flt-1 antibody described herein (e.g., SEQ ID NO: 104). In some embodiments, the present invention provides a polynucleotide sequence encoding the amino acid sequence of the anti-Flt-1 antibody linker described herein (e.g., SEQ ID NO: 105-107).

[0240] In some embodiments, the multinucleotide sequence encoding the amino acid sequence of the heavy chain, light chain, variable region, CDR region, Fc region, or linker region of the anti-Flt-1 antibody also includes a sequence encoding a signal peptide. As a non-limiting example, a suitable signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO:111).

[0241] The various polynucleotide sequences described in this article can be embodied in various vector systems used to express recombinant anti-Flt-1 antibodies or their antigen-binding fragments.

[0242] When recombinant antibodies are generated, any expression system can be used. To give some examples, known expression systems include, for instance, eggs, baculoviruses, plants, yeast, or mammalian cells.

[0243] In some embodiments, the recombinant anti-Flt-1 antibody or its antigen-binding fragment suitable for use in this invention is generated in mammalian cells. Non-limiting examples of mammalian cells that can be used according to this invention include the BALB / c mouse myeloma line (NSO / l, ECACC number: 85110503); human retinal cells (PER.C6, CruCell, Leiden, The Netherlands); and the monkey kidney CV1 line transformed from SV40 (COS-7, ATCC CRL 1651).

[0244] In some embodiments, the present invention provides a recombinant anti-Flt-1 antibody or an antigen-binding fragment thereof produced by human cells. In some embodiments, the present invention provides an anti-Flt-1 antibody or an antigen-binding fragment thereof produced by CHO cells.

[0245] Pharmaceutical compositions containing the antibodies of the present invention

[0246] The present invention further provides pharmaceutical compositions comprising a therapeutically active ingredient according to the invention (e.g., an anti-Flt-1 antibody or an antigen-binding fragment thereof) together with one or more pharmaceutically acceptable carriers or excipients. Such pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances.

[0247] While the description of the pharmaceutical compositions provided herein primarily pertains to those suitable for administration to humans by a physician's prescription, those skilled in the art will understand that such compositions are generally suitable for administration to all species of animals. It is well known that pharmaceutical compositions suitable for human administration can be modified to make them suitable for administration to a wide variety of animals, and such modifications can be designed and / or performed by a veterinary pharmacologist simply through routine experiments (if available).

[0248] The formulations of the pharmaceutical compositions described herein can be prepared by any method known in or subsequently developed in the field of pharmacology. Typically, such preparation methods include the steps of associating the active ingredient with a diluent or another excipient or carrier and / or one or more other auxiliary ingredients, and then, if necessary and / or desired, shaping and / or packaging the product into the desired single-dose or multi-dose units.

[0249] The pharmaceutical compositions according to the invention can be prepared, packaged, and / or marketed in single unit doses and / or in multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or an appropriate fraction of such a dose, such as half or one-third of such a dose.

[0250] The relative amounts of the active ingredient, pharmaceutically acceptable excipients or carriers, and / or any other components in the pharmaceutical compositions according to the invention will vary depending on the identity, size, and / or condition of the subject being treated and, more importantly, on the route of administration of the composition. For example, the composition may contain between 0.1% and 100% (w / w) of the active ingredient.

[0251] Pharmaceutical formulations may additionally contain pharmaceutically acceptable excipients or carriers, such as those used herein, including any and all solvents, dispersion media, diluents or other liquid media, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, etc., suitable for the desired specific dosage form. Remington's *The Science and Practice of Pharmacy*, 21st edition, ARGennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; which is incorporated herein by reference) discloses various excipients for formulating pharmaceutical compositions and known techniques for their preparation. The use of such excipients or carriers is covered within the scope of this invention, except for any conventional excipient media or carriers that are incompatible with the substance or its derivatives by producing any undesirable biological effects or otherwise harmfully interacting with any other component of the pharmaceutical composition.

[0252] In some embodiments, the pharmaceutically acceptable excipient or carrier is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient or carrier is approved for human and veterinary use. In some embodiments, the excipient or carrier is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient or carrier is pharmaceutical grade. In some embodiments, the excipient or carrier meets the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0253] Pharmaceutically acceptable excipients or carriers used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants and / or oils. Such excipients or carriers may optionally be included in the pharmaceutical composition. Excipients or carriers such as cocoa butter and suppository waxes, colorants, coatings, sweeteners, flavorings and / or aromatizers may be present in the composition at the formulator's discretion.

[0254] Suitable pharmaceutically acceptable excipients or carriers include, but are not limited to, water, salt solutions (e.g., NaCl), saline, buffered saline, alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (such as lactose, amylose, or starch), sugars (such as mannitol, sucrose, or others), dextrose, magnesium stearate, talc, silica, viscous paraffin, aromatic oils, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, and combinations thereof. If desired, the pharmaceutical formulation may be mixed with adjuvants (e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts affecting osmotic pressure, buffers, colorants, flavoring agents, and / or aromatic substances, etc.) that do not adversely react with or interfere with the activity of the active compound. In a preferred embodiment, a water-soluble carrier suitable for intravenous administration is used.

[0255] If desired, suitable pharmaceutical compositions or formulations may also contain small amounts of wetting agents, emulsifiers, or pH buffers. Compositions may be liquid solutions, suspensions, emulsions, tablets, pills, capsules, sustained-release formulations, or powders. Compositions may also be formulated as suppositories using conventional binders and carriers (such as triglycerides). Oral formulations may contain standard carriers such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, polyvinylpyrrolidone, sodium saccharin, cellulose, magnesium carbonate, etc.

[0256] Pharmaceutical compositions or agents can be formulated according to conventional procedures to be suitable for administration to humans. For example, in some embodiments, compositions for intravenous administration are typically solutions in sterile isotonic buffer solutions. Where necessary, the composition may also contain solubilizers and local anesthetics to reduce pain at the injection site. Typically, the components are supplied separately or mixed together in unit dosage forms, such as in the form of dried lyophilized powders or anhydrous concentrates in airtight containers such as ampoules or capsules indicating the amount of active agent. When the composition is intended for administration by infusion, it can be dispensed using infusion bottles containing sterile pharmaceutical-grade water, saline, or dextran / water. When the composition is administered by injection, ampoules of sterile water for injection or saline can be provided so that the components can be mixed prior to administration.

[0257] General considerations for the formulation and / or manufacture of pharmaceutical preparations can be found, for example, in Remington: The Science and Practice of Pharmacy, 21st edition, Lippincott Williams & Wilkins, 2005 (incorporated hereby by reference).

[0258] Application route

[0259] The anti-Flt-1 antibody or its antigen-binding fragment (or a composition or pharmaceutical agent containing the anti-Flt-1 antibody or its antigen-binding fragment described herein) described herein may be administered via any suitable route. In some embodiments, the anti-Flt-1 antibody or antigen-binding fragment protein or a pharmaceutical composition containing said anti-Flt-1 antibody or antigen-binding fragment protein may be administered parenterally. Parenteral administration may be intravenous, intradermal, intrathecal, inhalation, percutaneous (topical), intraocular, intramuscular, subcutaneous, intramuscular, and / or transmucosal administration. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment or a pharmaceutical composition containing said anti-Flt-1 antibody or its antigen-binding fragment may be administered subcutaneously. As used herein, the term "subcutaneous tissue" is defined as the layer of loose, irregular connective tissue directly beneath the skin. For example, subcutaneous administration may be performed by injecting the composition into areas including, but not limited to, the thigh region, abdominal region, buttock region, or scapular region. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment or a pharmaceutical composition containing said anti-Flt-1 antibody or its antigen-binding fragment may be administered intravenously. In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment, or a pharmaceutical composition containing said anti-Flt-1 antibody or its antigen-binding fragment, is administered orally. If desired, more than one route may be used simultaneously.

[0260] In some implementations, administration produces a local effect only in an individual, while in other implementations, administration produces an effect in multiple parts of the individual, such as a systemic effect. Typically, administration results in the delivery of an anti-Flt-1 antibody or antigen-binding fragment to one or more target tissues, including but not limited to the kidney, liver, brain, spinal cord, intestine, eye, lung, spleen, and heart (including myocardial, striated, and smooth muscle).

[0261] In some implementations, the striated muscles are selected from the group consisting of: triceps, tibialis anterior, soleus, gastrocnemius, quadriceps, and diaphragm.

[0262] In some implementations, smooth muscle is the muscle that lines blood vessels, bronchioles, the bladder, and the gastrointestinal tract, such as the rectum.

[0263] Dosage form and dosing regimen

[0264] In some embodiments, the composition is administered at a therapeutically effective amount and / or according to a dosing regimen associated with a specific desired outcome (e.g., treating muscular dystrophy such as Duchenne muscular dystrophy or reducing its risk).

[0265] The specific dose or amount to be administered according to the present invention may vary, for example, depending on the nature and / or extent of the desired outcome, the details of the route and / or timing of administration, and / or one or more characteristics (e.g., weight, age, personal history, genetic characteristics, lifestyle parameters, severity of cardiac defects and / or risk level of cardiac defects, or combinations thereof). Such doses or amounts can be determined by those skilled in the art. In some embodiments, the appropriate dose or amount is determined according to standard clinical techniques. Alternatively, in some embodiments, the appropriate dose or amount is determined by using one or more in vitro or in vivo assays to help identify the desired or optimal range or amount of dose to be administered.

[0266] In various implementations, the anti-Flt-1 antibody or its antigen-binding fragment is administered in a therapeutically effective amount. Typically, a therapeutically effective amount is sufficient to achieve a meaningful benefit to the subject (e.g., treatment, modulation, cure, prevention, and / or improvement of an underlying disease or symptom). In some specific implementations, the appropriate dose or amount to be administered can be deduced from dose-response curves derived from in vitro or animal model testing systems.

[0267] In some embodiments, the provided composition is provided as a pharmaceutical preparation. In some embodiments, the pharmaceutical preparation is or comprises a unit dose for administration according to a dosing regimen associated with achieving a reduction in the incidence or risk of muscular dystrophy, such as Duchenne muscular dystrophy.

[0268] In some embodiments, the formulation comprising the anti-Flt-1 antibody or antigen-binding fragment described herein is administered as a single dose. In some embodiments, the formulation comprising the anti-Flt-1 antibody or antigen-binding fragment described herein is administered at regular time intervals. As used herein, administration at a certain “time interval” indicates the periodic (as opposed to a single dose) administration of a therapeutically effective amount. The time interval can be determined using standard clinical techniques. In some embodiments, the formulation comprising the anti-Flt-1 antibody or antigen-binding fragment described herein is administered once every two months, once a month, twice a month, once every three weeks, once every two weeks, once a week, twice a week, three times a week, once daily, twice daily, or every six hours. The administration interval for an individual is not necessarily a fixed time interval but may vary over time according to the individual’s needs. In one specific embodiment, the anti-Flt-1 antibody or its antigen-binding fragment is administered twice a week.

[0269] As used herein, the term “once every two months” means once every two months (i.e., once every two months); the term “once a month” means once a month; the term “once every three weeks” means once every three weeks (i.e., once every three weeks); the term “once every two weeks” means once every two weeks (i.e., once every two weeks); the term “once a week” means once a week; and the term “once a day” means once a day.

[0270] In some embodiments, the formulation comprising the anti-Flt-1 antibody or antigen-binding fragment described herein is administered at indeterminate, regular time intervals. In some embodiments, the formulation comprising the anti-Flt-1 antibody or antigen-binding fragment described herein is administered at regular time intervals for a defined period of time.

[0271] As described herein, the term "therapeutic effective amount" is determined largely based on the total amount of therapeutic agent contained in the pharmaceutical compositions of the present invention. Therapeutic effective amounts are typically administered in a dosing regimen that may include multiple unit doses. For any particular composition, the therapeutic effective amount (and / or the appropriate unit dose within an effective dosing regimen) may vary, for example, depending on the route of administration or combination with other pharmaceutical agents.

[0272] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is administered at a dose ranging from about 0.1 mg / kg to about 50 mg / kg. In other embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is administered at doses ranging from about 0.1 mg / kg to about 40 mg / kg, about 0.1 mg / kg to about 30 mg / kg, about 0.1 mg / kg to about 20 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, about 1.0 mg / kg to about 30 mg / kg, about 1.0 mg / kg to about 20 mg / kg, about 1.0 mg / kg to about 10 mg / kg, about 1.0 mg / kg to about 5 mg / kg, or about 1.0 mg / kg to about 3 mg / kg. In specific implementation schemes, anti-Flt-1 antibody or its antigen-binding fragment is administered at doses of approximately 1.0 mg / kg, approximately 3.0 mg / kg, approximately 10 mg / kg, or approximately 20 mg / kg.

[0273] In some embodiments, administration of an anti-Flt-1 antibody or its antigen-binding fragment reduces the intensity, severity, or frequency of at least one sign or symptom of DMD or delays its onset. The DMD sign or symptom is selected from the group consisting of: muscle wasting, skeletal deformities, cardiomyopathy, muscle ischemia, cognitive impairment, and impaired respiratory function.

[0274] In some implementations, such as those measured by the 6-minute walk test, quantitative muscle strength test, and timed motor performance test, administration of anti-Flt-1 antibody or its antigen-binding fragment can improve clinical outcomes. Other methods include the Brooke and Vignos Limb Function Scale, pulmonary function tests (forced expiratory volume in one second, peak expiratory flow rate, maximum inspiratory and expiratory pressure), health-related quality of life, knee and elbow flexor strength, elbow extensor strength, shoulder abduction, grip strength, time to rise from supine position, North Start Ambulatory Assessment, timed 10-meter walk / run, Egen-Klassification Scale, Gowers score, Hammersmith motor ability, hand-held myometry, range of motion, goniometry, hypercapnia, the Nayley Scales of Infant and Toddler Development, and / or the caregiver burden scale.

[0275] Combination therapy

[0276] In some embodiments, the anti-Flt-1 antibody or its antigen-binding fragment is administered in combination with one or more additional therapeutic agents. In one embodiment, the additional therapeutic agent is a corticosteroid, such as prednisone. In another embodiment, the additional therapeutic agent is a glucocorticoid, such as defcodone. In another embodiment, the additional therapeutic agent is follistatin or a recombinant protein thereof. In yet another embodiment, the additional therapeutic agent is an RNA-regulating therapeutic agent. The RNA-regulating therapeutic agent may be an exon-skipping therapeutic agent or a gene therapy. RNA-regulating therapeutic agents may be, for example, Drispersen, PRO044, PRO045, Eteplirsen (AVI-4658), SRP-4053, SRP-4045, SRP-4050, SRP-4044, SRP-4052, SRP-4055, or SRP-4008. In some embodiments, the additional therapeutic agent is currently used to treat muscular dystrophy. In other embodiments, the additional therapeutic agent may also be used to treat other diseases or conditions. In some embodiments, the known therapeutic agent is administered according to its standard or approved dosing regimen and / or schedule. In some embodiments, the known therapeutic agent is administered according to a modified regimen compared to its standard or approved dosing regimen and / or schedule. In some embodiments, this modified regimen differs from the standard or approved method of administration in that the amount of one or more unit doses is changed (e.g., reduced or increased), and / or in that the dosing frequency is changed (e.g., the time interval between one or more unit doses is increased, resulting in a lower frequency, or the time interval between one or more unit doses is decreased, resulting in a higher frequency). Example

[0277] Example 1. Generation and characterization of high-affinity anti-Flt-1 antibody

[0278] Antibody production

[0279] Monoclonal antibodies against soluble Flt-1 were generated using a llama monoclonal antibody method. In short, llamas were immunized with recombinant human soluble Flt-1 (purchased from ABCAM), and serum was collected.

[0280] Antibody characterization

[0281] Antibodies bound to human and mouse Flt-1 were further characterized for 1) the VH family; 2) affinity for Flt-1; 3) IC50; 4) dissociation rate screening by Biacore assay; 5) cross-reactivity with cynomolgus monkey Flt-1; and 6) binding to VEGF R2 and VEGF R3. Candidate antibodies against human Flt-1 (hFl-1) and mouse Flt-1 (mFlt-1) were characterized as shown in Table 4.

[0282] Table 4.

[0283]

[0284] The pharmacokinetic properties of antibodies 13B4 and 10G12 were investigated in mice by intravenous administration of 10 mg / kg of each antibody (Table 5). Data showed that antibody 10G12 was undetectable after 288 hours, while antibody 13B4 was detectable after 672 hours. Figure 1A-1B ).

[0285] Table 5.

[0286]

[0287] In vivo efficacy of antibodies

[0288] Mdx mice (a mouse model of Duchenne muscular dystrophy) were treated with either antibody 13B4 or antibody 10G12 via intravenous administration twice weekly for one month, starting at 4 weeks of age. Control mice were treated with a mediator-only, isotype control antibody that does not bind to Flt-1, or a commercial anti-Flt-1 antibody (AngioProteomie, catalog number AP-MAB0702) known as the Flt-1:VEGF antagonist. Blood was collected 4 days after the fifth intravenous dose to assess serum antibody concentrations at the trough exposure point. Blood was collected 24 hours after the last dose to assess serum antibody concentrations at the peak exposure point. Peak and trough concentrations of antibodies 13B4 and 10G12 were [data missing]. Figure 2A and 2B As shown in the figure, the concentrations of free antibody 13B4 and free antibody 10G12 in the blood were higher than those of the isotype control antibody and the commercial control antibody at the peak and trough exposure time points.

[0289] To assess serum free sFlt-1 and VEGF levels, blood was collected 24 hours after the fifth intravenous dose and before sacrifice. Administration of antibodies 13B4 and 10G12, as well as the commercial control antibody, significantly reduced serum sFlt-1 concentrations compared to the isotype control antibody (p<0.0001). Figure 3 Compared with the isotype control antibody, administration of antibody 13B4 and antibody 10G12 significantly increased serum VEGF levels (p<0.001). Figure 4 Compared with the isotype control antibody, administration of the commercial control antibody also produced a significant increase in blood VEGF levels (p<0.05). Figure 4 ).

[0290] Histopathology

[0291] Mice were sacrificed at the end of the 30-day treatment period, and diaphragmatic and tibialis anterior (TA) muscles were collected and sectioned to determine whether anti-Flt-1 antibody induced angiogenesis in skeletal muscle. Muscle sections were stained with the endothelial cell marker CD31. A significant increase in capillary density was observed in the diaphragm of mice treated with antibodies 13B4, 10G12, or a commercial control antibody compared to mice treated with an isotype control antibody. Figures 5A-5D Data were quantified using automated quantitative imaging software as shown in Figures 6A-6B. The CD31-positive area in the diaphragm of mice treated with commercial control antibodies (p<0.05), antibody 13B4 (p<0.01), and antibody 10G12 (p<0.0001) was significantly increased compared to the diaphragm of mice treated with allotype control antibodies. A significant increase in the CD31-positive area of ​​the tibialis anterior (TA) muscle was also demonstrated in mice treated with antibody 10G12 compared to the tibialis anterior muscle treated with allotype control antibodies (p<0.01).

[0292] This study demonstrates that administration of Flt-1 antibodies (e.g., 10G12 and 13B4) to mdx mice resulted in a significant increase in endothelial cell proliferation, a decrease in serum soluble Flt-1, and an increase in serum VEGF concentration. These antibodies demonstrated binding affinity for the Flt-1 target in the pM range (see Table 4), an IC50 for Flt-1 binding less than 100 pM (see Table 4), and greater than 50% rescue of VEGF signaling in bioassays.

[0293] Example 2. Generation and characterization of high-affinity anti-Flt-1 antibody

[0294] Additional anti-Flt-1 monoclonal antibodies were generated as described above. The binding affinity of these antibodies to the sFlt-1 antigen was further characterized (by ELISA and Biacore); their performance in competing with VEGF in an sFlt-1:VEGF competitive ELISA; and their performance in cell-based assays.

[0295] Antibody characterization – binding to the target

[0296] The binding of monoclonal anti-Flt-1 antibody to recombinant sFlt-1 antigen was measured in an ELISA assay. Figure 7 All antibodies showed a dose-dependent increase in binding. The binding affinity of anti-Flt-1 antibodies to mouse and human Flt-1 antigens was measured by a surface plasmon resonance method (i.e., Biacore) (Table 6). Antibodies binding to human Flt-1 in the nanomolar range with antibody 11A11 showed the highest binding affinity to human Flt-1. Biacore analysis also demonstrated that the antibodies did not cross-react with VEGF R2 or VEGF R3 (Table 6); however, all antibodies did cross-react with cynomolgus monkey Flt-1.

[0297] Table 6.

[0298]

[0299] Antibody characterization – competitive / antagonistic effects

[0300] To estimate antibody potency, human sFlt-1 and VEGF were measured in a competitive ELISA. The antibody concentrations tested ranged from 0.1 mg / mL to 10,000 ng / mL. A commercial anti-Flt-1 antibody served as a control. All antibodies, except 11A11, were able to block the binding of VEGF to sFlt-1. Figure 8 ).

[0301] Antibody characterization – cell-based assay

[0302] Human primary venous endothelial cells (HUVECs) were stimulated with VEGF in the presence of sFlt-1 and monoclonal antibodies 02G07, 11A11, and 13B4. VEGF-induced cell activation was determined by assessing the phosphorylation status of the VEGF R2 receptor. In the presence of sFlt-1 alone (e.g., without anti-Flt-1 antibody), data are expressed as the percentage of VEGFR2 receptor phosphorylation rescued relative to VEGF R2 receptor phosphorylation. Monoclonal antibodies rescue cell activation (i.e., phosphorylation) by antagonizing soluble Flt-1. Figure 9 ).

[0303] Example 3. Characterization of high-affinity anti-Flt-1 antibody generated by light chain shuffling

[0304] The light chains of antibodies 18B6, 11A11, and 13B4 described in Example 2 were shuffled to improve the affinity and potency of the candidate antibodies.

[0305] Antibody characterization – binding to the target

[0306] The resulting antibodies exhibited increased affinity for the Flt-1 antigen. For example, the K of antibody 21C6... D K compared to parental antibody 11A11 D Increased approximately 10-fold. Similarly, the K of antibody 21B3... D K compared to parental antibody 13B4 D An increase of approximately 5 times (Table 7).

[0307] Table 7.

[0308]

[0309] Antibody characterization – competitive / antagonistic effects

[0310] To estimate antibody potency, antibodies were measured in a competitive ELISA using human sFlt-1 and VEGF after light chain shuffling. The antibody concentrations tested ranged from 0.2 mg / mL to 200 ng / mL. The ability of parental antibody 13B4 to competitively bind to sFlt-1 was compared with that of antibodies generated via light chain shuffling. All antibodies demonstrated dose-dependent inhibition of VEGF binding to human sFlt-1, with clone 21B3 being the most potent competitor. Figure 10 ).

[0311] Internal effects

[0312] To determine the serum half-life and pharmacokinetic characteristics of the light chain shuffled antibodies, mice were administered a single 10 mg / kg dose of the light chain shuffled antibodies 27H9 and 21B3, as well as the parental antibody 13B4 (each administered as IgA). 125 (Label). Serum was collected at 0.083, 0.25, 0.5, 1, 4, 8, and 24 hours, and at 3, 5, 7, 14, 21, and 28 days, and serum antibody concentrations were determined. Compared with parental antibodies, serum half-life was decreased in light chain shuffled antibodies ( Figure 11 However, the light chain shuffled antibodies showed improved pharmacodynamic characteristics compared to the parental antibody. For example, antibody 27H9 reached a maximum concentration of 222.4 μg / mL in 0.083 hours, while the parental antibody 13B4 reached a maximum concentration of 217 μg / mL in 0.5 hours (Table 8).

[0313] Table 8.

[0314]

[0315] Histopathology

[0316] To determine whether light chain shuffle antibodies and parental antibodies could induce endothelial cell proliferation, MDX mice were treated with 20 mg / kg of antibodies intravenously every two weeks for 4 weeks. At the end of the treatment period, mice were sacrificed, and diaphragmatic and anterior tibialis anterior muscle tissues were collected and sectioned to determine whether the antibodies induced angiogenesis in skeletal muscle. The muscle sections were stained with the endothelial cell marker CD31. A significant increase in capillary density was observed in the diaphragm of mice treated with antibodies 13B4 and 21B3 compared to mice treated with the isotype control antibody. Figures 12A-12C Furthermore, compared with tibialis anterior muscle from mice treated with the isotype control antibody, capillary density was significantly increased in the tibialis anterior muscle from mice treated with the light chain shuffle antibody 21B3. Figure 12D-12F ).

[0317] use 125 I-labeled antibodies were used to determine the biodistribution of antibodies 27H9, 13B4, and 21B3 in the diaphragm, tibialis muscle, and gastrocnemius muscle. The diaphragm showed the highest exposure to all antibodies during the stated time period. Figures 13A-13C ).

[0318] These studies demonstrate that administration of Flt-1 antibodies (i.e., 13B4 and 21B3) to mdx mice resulted in a significant increase in endothelial cell proliferation.

[0319] Example 4. In vivo efficacy of high-affinity anti-Flt-1 antibody 21B3

[0320] Mdx mice were treated with 1, 3, 10, or 20 mg / kg antibody 21B3 or 20 mg / kg isotype control antibody twice weekly for one month, starting at 4 weeks of age. Blood was collected 4 days after the fifth intravenous dose to assess serum antibody concentrations at the trough exposure point. Blood was collected 24 hours after the last dose to assess blood antibody concentrations at the peak exposure point. Peak and trough serum concentrations of antibody 21B3 and isotype control antibody were compared. Figure 14A and 14B As shown in the figure, the peak and trough levels of antibody 21B3 were dose-dependent and higher than those of the isotype control antibody.

[0321] To assess serum levels of free sFlt-1, blood was collected on days 0, 14, and 28. Administration of antibody 21B3 induced a dose-dependent decrease in serum-free sFlt-1 levels. As observed on days 14 and 28, the response was more durable at doses of 10 and 20 mg / kg. However, statistically significant reductions in free sFlt-1 levels were observed at doses of 3, 10, and 20 mg / kg compared to levels in mice treated with the medium alone. Figure 15 ).

[0322] To assess serum VEGF levels, blood was collected on days 0, 14, and 28. Administration of antibody 21B3 induced a dose-dependent increase in serum VEGF levels. As observed with free sFlt-1 levels, the response was more durable at doses of 10 and 20 mg / kg and on days 14 and 28. Indeed, a statistically significant increase in serum VEGF was observed at antibody 21B3 doses of 10 and 20 mg / kg compared to serum VEGF levels in mice treated with the medium alone. Figure 16 ).

[0323] Histopathology

[0324] Mice were sacrificed at the end of the 30-day treatment period, and diaphragmatic and anterior tibialis anterior muscles were collected and sectioned to determine whether anti-Flt-1 antibody induced angiogenesis in skeletal muscle. Muscle sections were stained against the endothelial cell marker CD31. A significant increase in capillary density in the diaphragm of mice treated with antibody 21B3 was observed compared to that in mice treated with an isotype control antibody. Figures 17A-17E ). Use as Figure 18 The data were quantified by automated quantitative imaging software. A significant increase in the CD31-positive area in the diaphragm of mice treated with 10 mg / kg or 20 mg / kg was observed compared to that in mice treated with an allotype control antibody (p < 0.0001).

[0325] A significant increase in capillary density in the tibialis anterior muscle was also observed in mice treated with antibody 21B3 compared to mice treated with an isotype control antibody. Figures 19A-19E ). Use as Figure 20 The data were quantified by automated quantitative imaging software. A significant increase in the CD31-positive area in the tibialis anterior muscle of mice treated with 10 mg / kg or 20 mg / kg was observed compared to that in mice treated with an allotype control antibody (p < 0.0001).

[0326] RP-LC / MS characterization

[0327] The molecular weight of the deglycosylated 21B3 antibody was determined by reversed-phase liquid chromatography / mass spectrometry (RP-LC / MS). Figure 21A Following the reduction reaction, the molecular weights of the light and heavy chains were determined. The glycosylation pattern of the heavy chain was also determined. Figure 21B ).

[0328] These results demonstrate that administration of Flt-1 antibody (i.e., 21B3) to mdx mice resulted in a significant increase in endothelial cell proliferation, a decrease in serum soluble Flt-1, and an increase in serum VEGF concentration.

[0329] Example 5. Characterization of humanized high-affinity anti-Flt-1 antibody

[0330] The light chain shuffling antibodies described in Example 3 were further modified to introduce sequence variations in the CDR region and / or Fc effector region. These antibodies were evaluated using surface plasmon resonance methods (e.g., Biacore) to determine their binding characteristics (Table 9). Antibody 27H9 NG / NAAAA exhibited approximately a 2-fold reduced binding affinity to Flt-1.

[0331] Table 9.

[0332] ka(1 / Ms) kd(1 / s) Rmax(RU) KD(M) <![CDATA[Chi 2 ]]> experiment 21B3WT 5.4E+05 1.7E-04 292 3.2E-10 290 2 21B3AAA 5.2E+05 1.1E-04 305 2.1E-10 371 2 27H9old07 / 05 3.5E+05 7.9E-05 306 2.3E-10 235 1 27H9WT23 / 07 3.3E+05 8.4E-05 323 2.6E-10 236 1 27H9NG / QG 3.7E+05 1.3E-04 315 3.6E-10 280 1 27H9NG / NA 2.9E+05 1.1E-04 298 3.9E-10 158 1 27H9NG / NA 2.9E+05 1.5E-04 240 5.1E-10 96.4 2 27H9NG / NAAAA 2.9E+05 1.5E-04 236 5.1E-10 72.5 2

[0333] The ability of the antibody to rescue cell activation by antagonizing sFlt-1 was also evaluated in cell-based assays. Human primary venous endothelial cells (HUVECs) were stimulated with VEGF in the presence of sFlt-1 and the monoclonal antibody. VEGF-induced cell activation was determined by assessing the phosphorylation status of the VEGF R2 receptor. The monoclonal antibody rescued cell activation (i.e., phosphorylation) by antagonizing soluble Flt-1. Figure 22A Furthermore, antibody 27H9 NG / NAAAA (NA+AAA) has similar potency to the unmutated parental antibody (wt).

[0334] Example 6. Antibody Optimization

[0335] Candidate antibodies were analyzed to identify the closest human VH and VL germline sequences, and to identify distinct residues within the framework region and oxidation / isomerization sites of the CDR. Fab libraries containing human and wild-type residues were constructed and fused to human constant domains. Phage display was applied to identify Fabs with the same or better dissociation rates (i.e., no loss of affinity) as the parent antibody 21B3. Fabs with the desired dissociation rates were sequenced and compared with human germlines, and those with the highest identity (e.g., VH+VL identity >95%) and homology (e.g., >96% homology) were selected for conversion into human monoclonal antibodies. Fabs were also analyzed for unwanted amino acids. Table 10 provides Fabs sorted by percentage of human identity, with some clones exhibiting up to 97.6% human identity and 98.8% homology.

[0336] Table 10.

[0337]

[0338] Example 7. Characterization of anti-Flt-1 monoclonal antibody

[0339] The thermostability of monoclonal antibodies was analyzed using the Biacore method. Each monoclonal antibody was incubated for 1 hour at different temperatures in phosphate-buffered saline at a concentration of 100 μg / mL. After 1 hour of incubation, the antibodies were slowly cooled to 25°C over two hours and then incubated overnight at 4°C. The percentage of functional antibodies was then measured by Biocore assay of binding to human Flt-1 (see Table 11). The thermostability of wild-type antibodies was consistent with previous experiments. However, mutations in the VH or VL regions, except for the 27H6DG / DA clone, lowered the melting temperature by approximately 2°C. The AAA mutation in the Fc region had no effect on the thermostability of the antibodies.

[0340] Table 11.

[0341]

[0342] The binding affinity of humanized clones was analyzed using Biacore (Table 12). The ability of antibodies 27H4, 27H6, and 27H9 to rescue VEGF signaling was determined in a cell-based assay for VEGF:sFlt-1. Figure 22BIn short, human primary venous endothelial cells (HUVECs) were stimulated with VEGF in the presence of sFlt-1 and monoclonal antibodies 27H4, 27H6, and 27H9. VEGF-induced cell activation was determined by assessing the phosphorylation status of the VEGF R2 receptor. In the presence of sFlt-1 alone (e.g., without anti-Flt-1 antibody), data are expressed as the rescue percentage of VEGF R2 receptor phosphorylation relative to the phosphorylation of the VEGF R2 receptor. The ability of antibodies 27H4, 27H6, and 27H9 to antagonize the binding of VEGF and sFlt-1 was also determined by ELISA. Figure 23 ).

[0343] Table 12.

[0344] ka(1 / Ms) kd(1 / s) Rmax(RU) KD(M) 21B3 5.4E+05 1.7E-04 292 3.2E-10 27H4 3.4E+05 8.2E-05 305 2.5E-10 27H9 3.3E+05 8.4E-05 323 2.6E-10 27H6 9.1E+05 1.4E-04 436 1.6E-10

[0345] Example 8. In vitro study of anti-Flt-1 antibody on muscle pathology

[0346] Mdx mice were treated with 1, 3, or 10 mg / kg of anti-Flt-1 antibody 21B3 or 10 mg / kg of IgG1 isotype control antibody by intravenous administration twice a week for 6 or 12 weeks starting at 3 weeks of age.

[0347] To assess serum levels of free antibody concentration, blood was collected from mice 4 days after intravenous doses administered at weeks 2, 4, 7, and 10. Samples were collected from euthanized mice 24 hours after the last dose. Figure 24 At a dose of 10 mg / kg, serum levels of free antibodies were statistically significant at all time points compared to mice receiving the isotype control antibody. At a dose of 3 mg / kg, serum levels of free antibodies were statistically significant at weeks 4, 7, and 10, and at sacrifice, compared to mice receiving the isotype control antibody. At a dose of 1 mg / kg, serum levels of free antibodies were statistically significant at sacrifice, compared to mice receiving the isotype control antibody.

[0348] To assess serum levels of free sFlt-1, blood was collected from mice 4 days after intravenous doses administered at weeks 2, 4, 7, and 10. Samples were collected from euthanized mice 24 hours after the last dose. Figure 25At a dose of 10 mg / kg, serum levels of free sFlt-1 were statistically significant at all time points compared to mice receiving the allotype control antibody. At a dose of 3 mg / kg, serum levels of free sFlt-1 were statistically significant at weeks 4, 7, and 10, and at sacrifice, compared to mice receiving the allotype control antibody. At a dose of 1 mg / kg, serum levels of free sFlt-1 were statistically significant at sacrifice, compared to mice receiving the allotype control antibody.

[0349] To assess serum VEGF levels, blood was collected from mice 4 days after intravenous doses administered at weeks 2, 4, 7, and 10. Samples were collected from euthanized mice 24 hours after the last dose. Figure 26 Antibody 21B3 administration induced a dose-dependent increase in serum VEGF levels. At a dose of 10 mg / kg, serum VEGF levels were statistically significant at all time points compared to mice receiving the isotype control antibody. At a dose of 3 mg / kg, serum VEGF levels were statistically significant at week 7 and at sacrifice compared to mice receiving the isotype control antibody. At a dose of 1 mg / kg, serum VEGF levels were statistically significant at sacrifice compared to mice receiving the isotype control antibody.

[0350] Histopathology

[0351] Mice were sacrificed at weeks 6 and 12 of the treatment period, and the diaphragm, gastrocnemius, and tibialis anterior muscles were collected and sectioned to determine whether treatment with anti-Flt-1 antibody induced angiogenesis and prevented fibrosis and necrosis in skeletal muscle.

[0352] Angiogenesis

[0353] Staining muscle sections with the endothelial cell marker CD31 ( Figures 27A-27H(28A-28H and 29A-29H). A significant increase in capillary density was observed in all muscle groups studied in mice treated with antibody 21B3 compared to the muscles of mice treated with the allotype control antibody. Data were quantified using automated quantitative imaging software. Mice treated with antibody 21B3 at 3 mg / kg (p<0.01) and 10 mg / kg (p<0.0001) showed a statistically significant increase in CD31-positive area in the diaphragm at weeks 6 and 12 compared to mice treated with the allotype control antibody. Mice treated with antibody 21B3 at 10 mg / kg showed a statistically significant increase in CD31-positive area in the gastrocnemius muscle at weeks 6 and 12 compared to mice treated with the allotype control antibody (p<0.05). Compared with mice treated with the same type control antibody, mice treated with 3 mg / kg antibody 21B3 showed a statistically significant increase in the area of ​​CD31-positive tibial anterior muscle at week 6 (p<0.05) and week 12 (p<0.0001), and mice treated with 10 mg / kg antibody 21B3 (p<0.0001) showed a statistically significant increase in the area of ​​CD31-positive tibial anterior muscle at week 6 and week 12. Figures 30A-30C ).

[0354] fibrosis

[0355] Muscle sections were also stained for type I collagen using immunohistochemistry (Figs. 31A-31H, 32A-32H, and 33A-33H). A significant reduction in type I collagen staining was observed in the diaphragm and gastrocnemius muscles of mice treated with antibody 21B3 compared to mice treated with an isotype control antibody. Statistically significant reductions in type I collagen staining in the diaphragm of mice treated with antibody 21B3 at 1 mg / kg (p<0.0001), 3 mg / kg (p<0.001), and 10 mg / kg (p<0.0001) were observed at 12 weeks compared to mice treated with an isotype control antibody. Compared with mice treated with the same type control antibody, mice treated with antibody 21B3 at doses of 1 mg / kg (p<0.01), 3 mg / kg (p<0.05), and 10 mg / kg (p<0.001) showed a statistically significant reduction in type I collagen staining in the gastrocnemius muscle at 12 weeks. Figures 34A-34C ).

[0356] Necrosis

[0357] The percentage of necrosis in the gastrocnemius muscle of mice treated with the 21B3 antibody was determined relative to the percentage of necrosis in the gastrocnemius muscle of mice treated with an isotype control antibody. A trend of improvement in necrosis was observed. Figures 35A-35B ).

[0358] Example 9. Localization of epitopes on human sFlt-1 targeted by anti-Flt-1 antibodies 21B3 and 21C6

[0359] Peptide-level epitopes on human sFlt-1 targeted by anti-human sFlt-1 monoclonal antibodies (mAbs) 21B3 and 21C6 were established by hydrogen-deuterium exchange (HDX) mass spectrometry.

[0360] Pepsin digestion and LC-MS

[0361] For pepsin digestion, 10 μg of sFlt-1 or a mixture of sFlt-1 and antibody (21B3) (10 μg:20 μg) or a mixture of sFlt-1 and antibody (21C6) (10 μg:20 μg) was denatured in 0.365 M TCEP and 1.7 M guanidine hydrochloride (pH 2.5). The mixtures were subjected to online pepsin digestion, and the resulting peptides were analyzed using a UPLC-MS system consisting of a WatersAcquity UPLC connected to a MicroTOF-Q2 mass spectrometer (Bruker). The peptides were separated on a 50 mm x 1 mm C8 column using a 19-minute gradient of 5%–28.5% solvent B (0.1% formic acid in acetonitrile). Solvent A was 0.1% formic acid in water. The solvent mixing valve, injection valve, C8 column, and all connecting stainless steel tubing were immersed in a refrigerated circulating water bath maintained at 0 °C. Peptide identification was performed using MS / MS data searched for the sFlt-1 sequence with Mascot. The mass tolerances for the precursor and product ions were 0.1 Da and 0.2 Da, respectively.

[0362] Deglycosylation

[0363] 200 μg of human sFlt-1 recombinant protein was incubated with 10 μl of PNGase F at 37°C for 4 hours.

[0364] Fab preparation

[0365] Pierce Fab Preparation Kit was used to prepare Fab from two anti-sFlt-1 mAbs (21B3 and 21C6) using papain digestion and protein A capture.

[0366] Size exclusion chromatography (SEC)

[0367] To examine the binding between native or deglycosylated human sFlt-1 and two anti-human sFlt-1 mAbs (21B3 and 21C6) on a SEC column, 10 μg of sFlt-1 (native or deglycosylated) was mixed with 40 μg of anti-sFlt-1 mAb. Native or deglycosylated sFlt-1 alone, anti-sFlt-1 mAb alone, or the complexes therein were injected into an SEC column with PBS as the mobile phase at a flow rate of 0.35 ml / min, and protein was monitored at 280 nm. Fab generated by anti-sFlt-1 mAb (21B3 and 21C6) and the binding between anti-sFlt-1 Fab and sFlt-1 were also evaluated using SEC.

[0368] HDX

[0369] 10 μL of human sFlt-1 (10 μg), or a mixture of sFlt-1 and mAb (21B3) (10 μg:20 μg), or a mixture of sFlt-1 and mAb (21C6) (10 μg:20 μg), was incubated with 90 μL of deuterium oxide labeling buffer (50 mM phosphate, 100 mM sodium chloride, pH 7.4) for 0 s, 30 s, 2 min, 10 min, 1 h, or 4 h. Deuterium exchange was quenched by adding 100 μL of 3.4 M guanidine hydrochloride and 0.73 M TCEP buffer (final pH 2.5), followed by the pepsin digestion and LC-MS analysis described above. Mass spectra were recorded in MS-only mode. Raw MS data were processed using HDExaminer software (Sierra Analytics, CA). Deuterium levels were calculated using the average mass difference between the deuterated peptide and its native form (t0).

[0370] result

[0371] To verify that glycan removal does not alter the binding of human sFlt-1 to antibodies, native and deglycosylated sFlt-1 proteins were mixed with anti-human sFlt-1 IgG (21B3 and 21C6), and complex formation was monitored on size exclusion chromatography. Data showed that native human sFlt-1 bound completely to both anti-human sFlt-1 IgGs (21B3 and 21C6), while deglycosylated human sFlt-1 did not bind completely to anti-human sFlt-1 mAb (21B3) or not to anti-human sFlt-1 mAb (21C6), indicating that deglycosylation disrupts the interaction between human sFlt-1 and the antibodies. Therefore, native human sFlt-1 was chosen for HD exchange experiments. Due to the non-uniform glycosylation and the high complexity of the 12 N-linked glycosylation sites, native human sFlt-1 initially showed poor sequence coverage. To improve sequence coverage, the quality of glycans at each glycosylation site was identified, and a high sequence coverage (85.2%) was achieved for natural human sFlt-1.

[0372] Natural human sFlt-1, alone or in complexes with anti-human sFlt-1 mAb (21B3) or anti-human sFlt-1 mAb (21C6), was incubated in deuterium oxide. Deuterium exchange was performed at room temperature for 0 s, 30 s, 2 min, 10 min, 60 min, and 240 min. The exchange reaction was quenched by low pH, and the protein was digested with pepsin. Deuterium levels at identified peptides were monitored by mass shift on LC-MS. Deuterium accumulation curves were plotted for all peptides over the exchange time. While most sFlt-1 peptides exhibited the same or similar deuterium levels with or without anti-human sFlt-1 mAb (21B3 and 21C6), several peptide segments showed significantly reduced deuterium incorporation upon binding to mAb 21B3 or mAb 21C6. Residues 117-129 (corresponding to amino acids 141-153 of SEQ ID NO:90) and 169-182 (corresponding to amino acids 193-206 of SEQ ID NO:90) derive strong deuterium protection upon binding to anti-human sFlt-1 mAb 21B3, while residues 106-114 (corresponding to amino acids 130-138 of SEQ ID NO:90) and 117-124 (corresponding to amino acids 141-148 of SEQ ID NO:90) derive strong deuterium protection upon binding to human sFlt-1 mAb 21C6. These strongly protected areas are designated as epitope peptides of anti-human sFlt-1 mAb (21B3 and 21C6) and in Figure 36 and Figure 37 The differential thermal plot shown is highlighted in blue. The MS / MS spectra of the identified peptides containing amino acid residues from the epitope region are shown in... Figures 38A-38EAs shown in the diagram, peptides 115-124 correspond to amino acids 139-148 of SEQ ID NO:90; peptides 115-129 correspond to amino acids 139-153 of SEQ ID NO:90; peptides 154-182 correspond to amino acids 178-206 of SEQ ID NO:90; peptides 175-180 correspond to amino acids 119-204 of SEQ ID NO:90; and peptides 104-114 correspond to amino acids 128-138 of SEQ ID NO:90.

[0373] in conclusion

[0374] For human sFlt-1, 85.2% sequence coverage was achieved. Residues 117-129 and 169-180 underwent strong deuterium protection upon binding to anti-human sFlt-1 mAb 21B3, while residues 106-114 and 117-124 underwent strong deuterium protection upon binding to anti-human sFlt-1 mAb 21C6. These strongly protected regions were designated as epitope peptides of the corresponding anti-human sFlt-1 mAb.

[0375] Equivalent Cases and Scope

[0376] Those skilled in the art will recognize or be able to determine many equivalent embodiments of the invention described herein using only conventional experiments. The scope of the invention is not intended to be limited to the foregoing description, but rather as set forth in the following claims.

[0377] Table 13.

[0378]

[0379]

[0380]

[0381] Table 14.

[0382]

Claims

1. An isolated antibody or antigen-binding fragment thereof, which competes with an anti-Flt-1 antibody for binding to human Flt-1, wherein the anti-Flt-1 antibody comprises a light chain and a heavy chain, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 83; and the heavy chain comprises the amino acid sequence of SEQ ID NO: 70, wherein the isolated antibody or antigen-binding fragment thereof comprises: (a) a light chain and a heavy chain, the light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO:76, the heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO:71, wherein the isolated antibody comprises a variable light (VL) chain CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VL CDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:32, a variable heavy (VH) chain CDR1 defined by the amino acid sequence of SEQ ID NO:3, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17, (b) a light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO:85, and a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO:72, wherein the isolated antibody comprises a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VLCDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:27, a VH CDR1 defined by the amino acid sequence of SEQ ID NO:2, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:7, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17, (c) a light chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:84, and a heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:71, wherein the isolated antibody comprises a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VLCDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:26, a VH CDR1 defined by the amino acid sequence of SEQ ID NO:3, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:12, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17, or (d) a light chain comprising an amino acid sequence at least 90% identical to SEQ ID NO:86, and a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO:73, wherein the isolated antibody comprises a VL CDR1 defined by the amino acid sequence of SEQ ID NO:21, a VLCDR2 defined by the amino acid sequence of SEQ ID NO:24, a VL CDR3 defined by the amino acid sequence of SEQ ID NO:28, a VH CDR1 defined by the amino acid sequence of SEQ ID NO:2, a VH CDR2 defined by the amino acid sequence of SEQ ID NO:8, and a VH CDR3 defined by the amino acid sequence of SEQ ID NO:17, and Wherein the isolated antibody or antigen-binding fragment thereof binds to human Flt-1.

2. An isolated antibody or antigen-binding fragment thereof as claimed in claim 1, wherein the isolated antibody or antigen-binding fragment recognizes a peptide or fragment thereof comprising the following amino acid sequence, the amino acid sequence corresponding to positions 139 to 148, positions 139 to 153, positions 178 to 206, positions 199 to 204 and positions 128 to 138 of SEQ ID NO:

90.

3. The isolated antibody or antigen-binding fragment thereof of claim 2, wherein the isolated antibody or antigen-binding fragment recognizes a peptide comprising the amino acid sequences of SEQ ID NO: 95 and SEQ ID NO:

96.

4. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 3, wherein the isolated antibody or antigen-binding fragment thereof is selected from the group consisting of IgG, F(ab')2, F(ab)2, Fab', Fab, ScFv, diabodies, triabodies and tetrabodies.

5. The isolated antibody or antigen-binding fragment thereof of claim 4, wherein the isolated antibody or antigen-binding fragment thereof is IgG.

6. The isolated antibody or antigen-binding fragment thereof of claim 5, wherein the isolated antibody or antigen-binding fragment thereof is IgG1.

7. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 6, wherein the isolated antibody or antigen-binding fragment thereof is a monoclonal antibody.

8. The isolated antibody or antigen-binding fragment thereof of claim 7, wherein the isolated antibody is a humanized monoclonal antibody.

9. The isolated antibody or antigen-binding fragment thereof of claim 8, wherein the humanized monoclonal antibody contains a human Fc region.

10. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 9, wherein the isolated antibody or antigen-binding fragment thereof comprises (a) a light chain and a heavy chain, the light chain comprising the amino acid sequence of SEQ ID NO: 76, and the heavy chain comprising the amino acid sequence of SEQ ID NO: 71, (b) a light chain and a heavy chain, the light chain comprising the amino acid sequence of SEQ ID NO: 85, and the heavy chain comprising the amino acid sequence of SEQ ID NO: 72, (c) a light chain comprising the amino acid sequence of SEQ ID NO:84 and a heavy chain comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:71, or (d) a light chain and a heavy chain, the light chain comprising the amino acid sequence of SEQ ID NO: 86, and the heavy chain comprising the amino acid sequence of SEQ ID NO:

73.

11. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-10, wherein the isolated antibody or antigen-binding fragment thereof does not bind to VEGF R2 and / or VEGF R3.

12. The isolated antibody or antigen-binding fragment thereof of any one of claims 1 to 11, wherein the isolated antibody or antigen-binding fragment thereof binds to human Flt-1 with an affinity greater than about 0.5 x 10 -9 M.

13. The isolated antibody or antigen-binding fragment thereof of claim 12, wherein the affinity is determined in a surface plasmon resonance analysis.

14. The isolated antibody or antigen-binding fragment thereof of claim 13, wherein the affinity is determined in a BIACORE assay.

15. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-14, wherein the isolated antibody or antigen-binding fragment thereof inhibits the binding and / or activity of VEGF at the Flt-1 receptor.

16. The isolated antibody or antigen-binding fragment thereof of any one of claims 15, wherein the isolated antibody or antigen-binding fragment thereof is characterized by an inhibition of VEGF binding to human Flt-1 in a competition assay, IC 50 Less than about 25pM.

17. The isolated antibody or antigen-binding fragment thereof of any one of claims 1-16, wherein the isolated antibody or antigen-binding fragment thereof provides greater than about 90% rescue of VEGF-R2 phosphorylation in a bioassay relative to the phosphorylation level of VEGF-R2 in the absence of the isolated antibody or antigen-binding fragment.

18. The isolated antibody or antigen-binding fragment thereof of claim 17, wherein the bioassay comprises human primary venous endothelial cells (HUVEC) stimulated with VEGF in the presence of sFlt-1 and the isolated antibody or antigen-binding fragment thereof.

19. A pharmaceutical composition comprising the isolated antibody or antigen-binding fragment thereof according to any one of claims 1 to 18 and a pharmaceutically acceptable carrier.

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