IL-2 fusion protein that preferentially binds to IL-2Rα

Abstract

The present disclosure provides novel isolated IL-2 fusion molecules that preferentially activate regulatory T cells (Tregs) in vitro and in vivo. Further included are methods of preparing and using the novel fusion molecules to treat inflammatory diseases and autoimmune diseases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 885,471, filed on August 12, 2019; U.S. Provisional Application No. 63 / 015,644, filed on April 26, 2020; U.S. Provisional Application No. 63 / 019,319, filed on May 2, 2020; and U.S. Provisional Application No. 63 / 044,294, filed on June 25, 2020. The contents of the priority applications are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on August 12, 2020 is named 025471_W0005_SL.txt and is 163,708 bytes in size. Background Art

[0005] Interleukin-2 (IL-2) plays a central role in lymphocyte production, survival, and homeostasis. The IL-2 has 133 amino acids and consists of four antiparallel, amphipathic α helices that form a quaternary structure that is essential for its function (Smith, Science (1988) 240: 1169-76; Bazan, Science (1992) 257: 410-13). IL-2 exerts its activity by binding to the IL-2 receptor (IL-2R), which consists of up to three separate subunits. α (CD25 or Tac antigen), β (CD122) and γ (γ c The association of the IL-2 receptor (K D ~0.01nM). The dimeric IL-2 receptor composed of β and γ subunits is called intermediate affinity IL-2R (K D ~1nM). The α subunit alone forms a monomeric low-affinity IL-2 receptor (K D~10nM). See, e.g., Kim et al., Cytokine Growth Factor Rev. (2006) 17:349-66). Although the dimeric intermediate affinity IL-2 receptor binds IL-2 with approximately 1 / 100 of the affinity of the trimeric high affinity receptor, both dimeric and trimeric IL-2 receptors can transmit signals upon IL-2 binding (Minami et al., Annu Rev Immunol (1993) 11:245-68). Therefore, while it helps to confer high affinity binding of the receptor to IL-2, the α subunit does not appear to be essential for IL-2 signal transduction. However, the β and γ subunits are essential for IL-2 signal transduction (Krieg et al., Proc Natl Acad Sci. (2010) 107:11906-11). The trimeric IL-2 receptor is composed of CD4 + FOXP3 + Regulatory T (Treg) cell expression. Treg cells continuously express the highest levels of IL-2Rα in vivo (Fontenot et al., Nature Immunol. (2005) 6: 1142-51). Trimeric IL-2 receptors are also transiently induced on conventional activated T cells, whereas in the resting state, these cells express only dimeric IL-2 receptors.

[0006] Based on the published crystal structure of the IL-2 / IL-2R complex (Wang et al., Science (2005) 310: 1159-63), researchers have made mutations in IL-2 to modulate its interaction with CD25, CD122 and / or CD132. In one example, it is reported that mutations at D20, N88 or Q126 of human IL-2 show different effects in activating T cells and NK cells (U.S. Patent 6955807). In another example, it is shown that IL-2 mutated at positions 69 and 74 binds tightly to CD25, while mutations at positions 88 or 91 block its interaction with CD122, and mutations at position 126 block its interaction with CD132 (PCT Publication WO 2009 / 061853).

[0007] Treg cells are crucial for suppressing autoimmunity and regulating inflammation. FOXP3 - CD25 + T effector cells (Teff) may be CD4 + or CD8 +cells, both of which play a role in inflammation, autoimmunity, organ transplant rejection or graft-versus-host disease (GVHD). IL-2-stimulated STAT5 signaling is critical for normal Treg cell growth and survival as well as high FOXP3 expression.

[0008] Although IL-2 plays a role in Treg activity, safe and effective IL-2-based therapies for modulating Treg activity have not been clinically demonstrated. Therefore, there is still a need to develop IL-2-based therapies that preferentially expand or stimulate Treg cells to treat inflammatory and autoimmune diseases. Summary of the invention

[0009] The present disclosure provides an isolated IL-2 fusion molecule comprising a carrier portion, a cytokine portion, and one or more masking portions, wherein the cytokine portion is fused to the carrier portion or the masking portion, the one or more masking portions are fused to the carrier portion or the cytokine portion, the cytokine portion comprises an IL-2 polypeptide, the IL-2 polypeptide comprises: (i) a C125A or C125S substitution; or (ii) an IL-2 amino acid sequence, the IL-2 amino acid sequence comprising one or more substitutions selected from T3A, C125S, V69A, and Q74P (numbered according to SEQ ID NO: 1), the one or more masking portions bind to the cytokine portion and inhibit the cytokine portion from binding to IL-2Rβ and / or IL-2Rγ on immune cells (e.g., T cells and NK cells), but does not inhibit its binding to IL-2Rα. In some embodiments, the IL-2 polypeptide binds to IL-2Rα with an affinity similar to or greater than that of wild-type IL-2.

[0010] The present disclosure also provides a method for treating an inflammatory condition or an autoimmune disease, the method comprising administering to a subject in need thereof a therapeutic amount of an isolated IL-2 fusion molecule, the isolated IL-2 fusion molecule comprising a carrier portion, a cytokine portion, and one or more masking portions, wherein the cytokine portion is fused to the carrier portion or the masking portion, the one or more masking portions are fused to the carrier portion or the cytokine portion, the cytokine portion comprises an IL-2 polypeptide, and the one or more masking portions bind to the cytokine portion and inhibit the cytokine portion from binding to IL-2Rβ and / or IL-2Rγ on immune cells (e.g., T cells and NK cells), but does not inhibit its binding to IL-2α. In some embodiments, the inflammatory condition or autoimmune disease is selected from the group consisting of: asthma, type I diabetes, rheumatoid arthritis, allergies, systemic lupus erythematosus, multiple sclerosis, organ transplant rejection, and graft-versus-host disease.

[0011] In some embodiments, the IL-2 polypeptide binds IL-2Rα with an affinity similar to or greater than that of wild-type IL-2.

[0012] In some embodiments, the IL-2Rβ ECD or a functional analog thereof has an amino acid sequence that is at least 95% (e.g., at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 3. In some embodiments, the IL-2Rγ ECD or a functional analog thereof has an amino acid sequence that is at least 95% (e.g., at least 97%, at least 98%, or at least 99%) identical to SEQ ID NO: 6. In some embodiments, the IL-2 polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1, optionally wherein the amino acid sequence is SEQ ID NO: 2.

[0013] In some embodiments, the IL-2 fusion molecule comprises a masking moiety comprising an extracellular domain (ECD) of IL-2Rβ or IL-2Rγ or a functional analog thereof, wherein the masking moiety is fused to the carrier moiety with or without a peptide linker. In other embodiments, the IL-2 fusion molecule comprises: a first masking moiety comprising an extracellular domain (ECD) of IL-2Rβ or IL-2Rγ or a functional analog thereof, wherein the first masking moiety is fused to the carrier moiety with or without a peptide linker; and a second masking moiety comprising an ECD of IL-2Rγ or IL-2Rβ or a functional analog thereof, wherein the second masking moiety is fused to the cytokine moiety or to the first masking moiety with or without a peptide linker. In some embodiments, the IL-2 fusion molecules of the present disclosure include at least two masking moieties, one of the two masking moieties is the ECD of IL-2Rα or a functional analog thereof, wherein the IL-2RαECD masking moiety is fused to the cytokine moiety, the carrier moiety or another masking moiety via a cleavable peptide linker. In a specific embodiment, the IL-2RαECD moiety includes an amino acid sequence that is at least 95% identical to SEQ ID NO:7.

[0014] In some embodiments, the cytokine portion is fused to the carrier portion or the masking portion via a non-cleavable peptide linker, and the masking portion is fused to the carrier portion or the cytokine portion via a non-cleavable peptide linker. In specific embodiments, the masking portion is fused to the carrier portion or the cytokine portion via a peptide linker comprising at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 22 amino acids, at least 25 amino acids, at least 30 amino acids, or at most 44 amino acids.

[0015] In some embodiments, the carrier portion is selected from a PEG molecule, an albumin, an albumin fragment, an antibody Fc domain, an antibody or an antigen-binding fragment thereof. In some embodiments, the carrier portion is an antibody Fc domain, and the fusion molecule is a heterodimer comprising: a first polypeptide chain, the first polypeptide chain comprising a molecular formula selected from F1-L1-E1, F1-L1-E1-L2-E2 and F1-L1-E2-L2-E1 from N-terminus to C-terminus; and a second polypeptide chain, the second polypeptide chain comprising a molecular formula F2-L3-C from N-terminus to C-terminus, wherein F1 and F2 are subunits of the Fc domain, L1, L2 and L3 are peptide linkers, E1 is IL-2RβECD or a functional analog thereof, and E2 is IL-2RγECD or a functional analog thereof, and C is the cytokine portion. In other embodiments, the carrier portion is an antibody Fc domain, and wherein the fusion molecule is a heterodimer comprising: a first polypeptide chain, the first polypeptide chain comprising a molecular formula selected from E1-L1-F1, E1-L1-E2-L2-F1 and E2-L1-E1-L2-F1 from N-terminus to C-terminus; and a second polypeptide chain, the second polypeptide chain comprising a molecular formula C-L3-F2 from N-terminus to C-terminus, wherein F1 and F2 are subunits of the Fc domain, L1, L2 and L3 are peptide linkers, E1 is IL-2RβECD or a functional analog thereof, and E2 is IL-2RγECD or a functional analog thereof, and C is the cytokine portion. In other embodiments, the carrier portion is an antibody Fc domain, and wherein the fusion molecule is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, the first polypeptide chain and the second polypeptide chain comprising a molecular formula selected from the following pairs from N-terminus to C-terminus:

[0016] F1-L1-E1 and F2-L2-C-L3-E2;

[0017] F1-L1-E1 and F2-L2-E2-L3-C;

[0018] F1-L1-E2 and F2-L2-C-L3-E1;

[0019] F1-L1-E2 and F2-L2-E1-L3-C;

[0020] E1-L1-F1 and E2-L2-C-L3-F2;

[0021] E1-L1-F1 and C-L2-E2-L3-F2;

[0022] E2-L1-F1 and E2-L2-C-L3-F2; and

[0023] E2-L1-F1 and C-L2-E1-L3-F2, where

[0024] F1 and F2 are subunits of the Fc domain, L1, L2 and L3 are peptide linkers, E1 is IL-2RβECD or a functional analog thereof, and E2 is IL-2RγECD or a functional analog thereof, and C is the cytokine portion. In some embodiments, the peptide linkers L1, L2 and L3 are non-cleavable. In specific embodiments, L1, L2 and L3 independently have an amino acid sequence selected from SEQ ID NO: 40-46, 55-57 and 59. In other specific embodiments, at least one of L1, L2 and L3 has an amino acid sequence comprising 20-44 amino acids.

[0025] In a specific embodiment, the IL-2 fusion molecule of the present disclosure comprises: a first polypeptide chain comprising an amino acid sequence at least 99% identical to SEQ ID NO: 50, 51 or 52; and a second polypeptide chain comprising an amino acid sequence at least 99% identical to SEQ ID NO: 53 or 54. In a specific embodiment, the IL-2 fusion molecule of the present disclosure comprises: a first polypeptide chain comprising an amino acid sequence at least 99% identical to SEQ ID NO: 50; and a second polypeptide chain comprising an amino acid sequence at least 99% identical to SEQ ID NO: 53.

[0026] In some embodiments, the IL-2 fusion molecules disclosed herein have one or more of the following properties:

[0027] (a) the IL-2 fusion molecule binds to a high affinity IL-2 receptor having α, β, and γ subunits (IL-2Rαβγ) with an affinity that is at least 100 times greater than the affinity that it binds to an intermediate affinity IL-2 receptor having β and γ subunits (IL-2Rβγ);

[0028] (b) the IL-2 fusion molecule binds to IL-2Rβγ with a KD of greater than about 5 nM or greater than 10 nM as measured in a surface plasmon resonance assay at 37°C;

[0029] (c) the IL-2 fusion molecule has an EC50 value of less than about 1 nM and greater than 0.01 nM, 0.25 nM or 0.05 nM in a CTLL-2 cell proliferation assay;

[0030] (d) the IL-2 fusion molecule has an EC50 value of greater than about 0.05 nM, 0.1 nM, 0.25 nM or 0.5 nM in a NK92 cell proliferation assay;

[0031] (e) in a NK92 cell proliferation assay, the Emax value of the IL-2 fusion molecule in the presence of a neutralizing CD25 antibody is at most 1 / 5 or at most 1 / 10 of the Emax value in the absence of the neutralizing CD25 antibody;

[0032] (f) the IL-2 fusion molecule preferentially stimulates FOXP3+ T regulatory cells relative to T effector cells or NK cells;

[0033] (g) the IL-2 fusion molecule promotes the growth or survival of FOXP3+ regulatory T cells; and

[0034] (h) The IL-2 fusion molecule induces STAT5 phosphorylation in FOXP3+ T cells, but has a reduced ability to induce STAT5 phosphorylation in FOXP3- T cells.

[0035] In other aspects, the present disclosure also provides: a pharmaceutical composition, the pharmaceutical composition comprising the IL-2 fusion molecule of the present disclosure and a pharmaceutically acceptable excipient; one or more polynucleotides, the one or more polynucleotides encoding the IL-2 fusion molecule; one or more expression vectors, the one or more expression vectors comprising the one or more polynucleotides; and a host cell comprising the vector, wherein the host cell can be a prokaryotic cell or a eukaryotic cell, such as a mammalian cell. In some embodiments, the gene encoding uPA, MMP-2 and / or MMP-9 in the mammalian host cell is knocked out (e.g., containing an invalid mutation of one or more of these genes). Therefore, the present disclosure also provides a method for preparing the IL-2 fusion molecule, the method comprising: culturing the host cell under conditions that allow the expression of the IL-2 fusion molecule, wherein the host cell is a mammalian cell; and isolating the IL-2 fusion molecule.

[0036] Other features, purposes and advantages of the present invention are apparent in the following detailed description. However, it should be understood that the detailed description, although indicating embodiments and aspects of the present invention, is provided only in an illustrative and non-restrictive manner. Based on the detailed description, various changes and modifications within the scope of the present invention will be apparent to those skilled in the art to which the present invention belongs. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A and 1B is a schematic representation of an IL-2 fusion molecule in which the IL-2Rβ extracellular domain (ECD) and the IL-2 polypeptide are fused to the C-terminus of an Fc domain. The IL-2 polypeptide is fused to the C-terminus of an Fc polypeptide via a non-cleavable linker. The IL-2Rβ ECD is fused to the C-terminus of an Fc polypeptide via a non-cleavable linker ( Figure 1A ) or a cleavable linker ( Figure 1B ) is fused to the C-terminus of another Fc polypeptide. "Knobs-into-holes" refers to a knobs-into-holes mutation in an Fc polypeptide.

[0038] Figure 2A and 2B is a schematic representation of an IL-2 fusion molecule in which the IL-2RβECD and the IL-2 polypeptide are fused to the C-terminus of the Fc domain, and the IL-2RγECD is fused to the C-terminus of the IL-2RβECD. The IL-2 polypeptide is fused to the C-terminus of one Fc polypeptide via a non-cleavable linker. The IL-2RβECD is fused to the C-terminus of another Fc polypeptide via a non-cleavable linker. The IL-2RγECD is fused to the C-terminus of the Fc polypeptide via a non-cleavable linker. Figure 2A ) or a cleavable linker ( Figure 2B ) was fused to the C-terminus of IL-2Rβ ECD.

[0039] Figure 3A and 3B is a schematic representation of an IL-2 fusion molecule in which the IL-2RγECD and the IL-2 polypeptide are fused to the C-terminus of the Fc domain, and the IL-2RβECD is fused to the C-terminus of the IL-2RγECD. The IL-2 polypeptide is fused to the C-terminus of one Fc polypeptide via a non-cleavable linker. The IL-2RγECD is fused to the C-terminus of another Fc polypeptide via a non-cleavable linker. The IL-2RβECD is fused to the C-terminus of the other Fc polypeptide via a non-cleavable linker. Figure 3A ) or a cleavable linker ( Figure 3B ) was fused to the C-terminus of IL-2RγECD.

[0040] Figure 4A and 4B is a schematic representation of an IL-2 fusion molecule in which the IL-2RβECD and the IL-2 polypeptide are fused to the C-terminus of the Fc domain, and the IL-2RγECD is fused to the C-terminus of the IL-2 polypeptide. The IL-2 polypeptide is fused to the C-terminus of one Fc polypeptide via a non-cleavable linker. The IL-2RγECD is fused to the C-terminus of the IL-2 polypeptide via a cleavable linker. The IL-2RβECD is fused to the C-terminus of the IL-2 polypeptide via a non-cleavable linker ( Figure 4A ) or a cleavable linker ( Figure 4B ) is fused to the C-terminus of another Fc polypeptide.

[0041] Figure 5A and 5Bis a schematic representation of an IL-2 fusion molecule in which the IL-2RγECD and the IL-2 polypeptide are fused to the C-terminus of the Fc domain, and the IL-2RβECD is fused to the C-terminus of the IL-2 polypeptide. The IL-2 polypeptide is fused to the C-terminus of one Fc polypeptide via a non-cleavable linker. The IL-2Rβ is fused to the C-terminus of the IL-2 polypeptide via a cleavable linker. The IL-2RγECD is fused to the C-terminus of the IL-2 polypeptide via a non-cleavable linker ( Figure 5A ) or a cleavable linker ( Figure 5B ) is fused to the C-terminus of another Fc polypeptide.

[0042] Fig. 6A and 6B is a schematic representation of an IL-2 fusion molecule, in which IL-2RβECD and IL-2RγECD are fused to the C-terminus of the Fc domain, and the IL-2 polypeptide is fused to the C-terminus of IL-2RβECD or IL-2RγECD. Fig. 6A In the present invention, the IL-2RγECD is fused to the C-terminus of one Fc polypeptide via a cleavable linker, the IL-2RβECD is fused to the C-terminus of another Fc polypeptide via a non-cleavable linker, and the IL-2 polypeptide is fused to the C-terminus of the IL-2RβECD via a non-cleavable linker. Figure 6B In the method, the IL-2RβECD is fused to the C-terminus of one Fc polypeptide via a cleavable linker, the IL-2RγECD is fused to the C-terminus of another Fc polypeptide via a non-cleavable linker, and the IL-2 polypeptide is fused to the C-terminus of the IL-2RγECD via a non-cleavable linker.

[0043] Fig. 7A and 7B is a schematic representation of an IL-2 fusion molecule in which the IL-2RβECD and the IL-2 polypeptide are fused to the N-terminus of an Fc domain. The IL-2 polypeptide is fused to the N-terminus of an Fc polypeptide. The IL-2RβECD is linked to the Fc domain via a non-cleavable linker ( Fig. 7A ) or a cleavable linker ( Figure 7B ) is fused to the N-terminus of another Fc polypeptide.

[0044] Figure 8 It shows that Figure 1B SDS-PAGE analysis of the IL-2 fusion molecule JR3.116.5 with the schematic structure shown in , which fusion molecule includes two polypeptide chains having amino acid sequences as shown in SEQ ID NO: 12 and 23, respectively.

[0045] Fig. 9 Shown are the results of a CTLL2-based bioactivity assay of the IL-2 fusion molecule JR3.116.5 before and after activation by protease treatment.

[0046] Fig. 10A and 10B Schematic representation of IL-2 fusion molecules 982C1, 982C2, 982D1 and 982D2. 982C1 and 982C2 have two masking moieties, IL-2Rβ ECD and IL-2Rγ ECD, and the IL-2 mutein is fused to the C-terminus of the IgG4 Fc domain. Fig. 10A The IL-2RγECD is shown fused to the C-terminus of one IgG4 Fc polypeptide via a (G4S)2AA(G4S)2 (SEQ ID NO:59) non-cleavable linker. The IL-2RβECD is fused to the C-terminus of the IL-2RγECD via a 43 amino acid long non-cleavable linker, as shown in SEQ ID NO:46. The IL-2 mutein is fused to the C-terminus of another IgG4 polypeptide via a non-cleavable linker. The IL-2 mutein has a C125A substitution (982C1) or substitutions T3A / C125S / V69A / Q74P (982C2). Fig. 10B The IL-2Rβ ECD is shown fused to the C-terminus of one IgG4 Fc polypeptide via a (G4S)2AA(G4S)2 (SEQ ID NO:59) non-cleavable linker. The IL-2 mutein is fused to the C-terminus of another IgG4 polypeptide via a non-cleavable linker. The IL-2 mutein has a C125A substitution (982D1) or substitutions T3A / C125S / V69A / Q74P (982D2).

[0047] Fig.11 NK92 cell proliferation assays of 982D1, 982D2, IL-2 and reference molecules in the presence or absence of neutralizing antibodies against CD25 are shown. Reference molecule (982Ref) is an Fc-IL-2 fusion molecule in which IL-2 has mutations V91K and C125A. 982-Ref is a homodimeric Fc-fusion-IL-2 mutant protein molecule in which each chain includes the amino acid sequence of SEQ ID NO: 58.

[0048] FIG. 12 shows the interaction of IL-2 fusion molecules 982D1 and 982D2 with rat CD4 + Binding of T cells. NC stands for negative control.

[0049] Fig.13A and 13B The IL-2 fusion molecules 982D1, 982C1 and 982D2 and 982Ref are shown with CD4 + CD25 + T cells and CD4 + CD25 - Binding of T cells. NC stands for negative control.

[0050] Fig.14 CD4 T cells induced by 982D1, 982C1, 982D2 and 982Ref IL-2 fusion molecules are shown. + CD25 + T cells and CD4+CD25 - Concentration-dependent proliferation of T cells IL-2 alone was also tested.

[0051] Fig.15 Shown is the change in serum plasma concentrations over time of 982C1, 982D1 and 982Ref IL-2 fusion molecules in a rat PK study. The molecules were injected subcutaneously into rats.

[0052] Fig.16 Shown are the changes in serum plasma concentrations over time of 982C1, 982D1, and 982Ref IL-2 fusion molecules from the second rat PK study.

[0053] Fig.17A and 17B Figure 2 shows the rat CD4 T cells induced by 982C1, 982D1 and 982Ref IL-2 fusion molecules. + CD4 in T cells + FOXP3 + and CD4 + FOXP3 - Changes in the percentage of cells over time.

[0054] Fig.18A and 18B CD4 Tgp1 induced by 982C1, 982D1 and 982Ref in rats from the first PK study is shown. + CD25 + and CD4 + CD25 - Changes in the proliferation status of cells (as indicated by the proliferation marker Ki67) over time.

[0055] Fig.19A and 19B The rat CD4 + CD4 T cells induced by 982IL-2 fusion molecules + FOXP3 + and CD4 + FOXP3 - Changes in the percentage of cells over time.

[0056] Fig. 20A and 20B Shown is the CD4 induction by 982IL-2 fusion molecule in rats +CD25 + and CD4 + CD25 - Changes in the proliferation state of cells over time.

[0057] Fig.21 Shown are body weights for each treatment group following a single subcutaneous administration of 982D1, 982D2, and 982Ref. DETAILED DESCRIPTION

[0058] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. References herein to "about" a value or parameter include (and describe) variations on the value or parameter itself. For example, a description referring to "about X" includes a description of "X." In addition, the use of "about" before any series of numbers includes "about" each number listed in the series. For example, a description referring to "about X, Y, or Z" is intended to describe "about X, about Y, or about Z."

[0059] The term "antigen binding portion" refers to a polypeptide or a group of interacting polypeptides that specifically bind to an antigen, and includes but is not limited to antibodies (e.g., monoclonal antibodies, polyclonal antibodies, multispecific antibodies, dual-specific or bispecific antibodies, anti-idiotypic antibodies, or bifunctional hybrid antibodies) or antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies (dAb) or bifunctional antibodies), single-chain antibodies, and Fc-containing polypeptides, such as immunoadhesins. In some embodiments, the antibody may be of any heavy chain isotype (e.g., IgG, IgA, IgM, IgE, or IgD) or subtype (e.g., IgG1, IgG2, IgG3, or IgG4). In some embodiments, the antibody may be of any light chain isotype (e.g., κ or λ). The antibody can be human, non-human (e.g., from mouse, rat, rabbit, goat or another non-human animal), chimeric (e.g., having non-human variable regions and human constant regions), or humanized (e.g., having non-human CDRs and human framework and constant regions). In some embodiments, the antibody is a derivatized antibody.

[0060] The term "cytokine agonist polypeptide" refers to a wild-type cytokine or an analog thereof. The analog of a wild-type cytokine has the same biological specificity as the wild-type cytokine (e.g., binds to the same receptor and activates the same target cell), even if the activity level of the analog may be different from the activity level of the wild-type cytokine. The analog can be, for example, a mutant protein (i.e., a mutant polypeptide) of a wild-type cytokine, and can include at least one mutation, at least two mutations, at least three mutations, at least four mutations, at least five mutations, at least six mutations, at least seven mutations, at least eight mutations, at least nine mutations, or at least ten mutations relative to the wild-type cytokine.

[0061] The term "cytokine antagonist" or "cytokine masking agent (mask)" refers to a portion (e.g., polypeptide) that binds to a cytokine, thereby inhibiting the cytokine from binding to its receptor on the surface of a target cell and / or exerting its biological function when bound by the antagonist or masking agent. Examples of cytokine antagonists or masking agents include, but are not limited to, polypeptides derived from the extracellular domain of the natural receptor of the cytokine that contacts the cytokine.

[0062] The term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition or disease, such as to ameliorate, alleviate, lessen and / or delay one or more of its symptoms.

[0063] The term "functional analog" refers to a molecule that has the same biological specificity (eg, binds to the same ligand) and / or activity (eg, activates or inhibits a target cell) as a reference molecule.

[0064] The term "fused" or "fusion" with respect to two polypeptide sequences refers to the joining of two polypeptide sequences by a backbone peptide bond. The two polypeptides can be fused directly or by a peptide linker of one or more amino acids in length. Fusion polypeptides can be prepared by recombinant technology from coding sequences containing the corresponding coding sequences of the two fusion partners, with or without the coding sequence of a peptide linker in between. In some embodiments, fusion encompasses chemical conjugation.

[0065] The term "pharmaceutically acceptable excipient" when used to refer to an ingredient in a composition means that the excipient is suitable for administration to a therapeutic subject, including a human subject, without producing undue deleterious side effects to the subject and without affecting the biological activity of the active pharmaceutical ingredient (API).

[0066] The term "subject" refers to a mammal, and includes, but is not limited to, a human, a pet (eg, a canine or a feline), a farm animal (eg, a bovine or a horse), a rodent, or a primate.

[0067] As used herein, "treatment" or "treating" is a method for obtaining a beneficial or desired clinical outcome. A beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, improving the disease state, stabilizing the disease (e.g., preventing or delaying the deterioration or progression of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, providing partial or complete relief of the disease, reducing the dosage of one or more other drugs required for treating the disease, improving the patient's quality of life and / or prolonging survival. The method disclosed herein encompasses any one or more of these treatment aspects.

[0068] It should be understood that one, some or all of the features in the various embodiments described herein can be combined to form other embodiments of the present invention.The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described below.

[0069] Separate IL-2 Fusion molecules

[0070] The present disclosure provides IL-2 fusion molecules that can be used to treat inflammatory diseases and autoimmune diseases. To the surprise of the inventors, the desired in vivo activity can be achieved without the need to cut or remove the masking portion. Compared with the cleavable masked IL-2 fusion molecules, the masked IL-2 fusion molecules with non-cleavable peptide linkers have many significant advantages. For example, the cleavable masked IL-2 molecules require protease to cut the linker and remove the masking portion to be activated. Due to the uneven distribution of proteases at the disease site, its cytokine activation level will change, which may increase the variability of therapeutic efficacy. In addition, nonspecific activation may also occur during circulation and / or production, thereby increasing the safety issues and production complexity of the cleavable masked molecules.

[0071] In some embodiments, the IL-2 fusion molecules of the present disclosure have a reduced affinity (e.g., K D greater than 1 nM, greater than 5 nM, greater than 10 nM, greater than 100 nM, or greater than 1 μM), while retaining wild-type affinity for IL-2Rα (CD25) (e.g., K D about 10 nM), or have an affinity similar to the wild-type affinity of IL-2Rα (e.g., K D about 1-20 nM), or even higher than the wild-type affinity (e.g., K DThe isolated IL-2 fusion molecule may include an IL-2 polypeptide (cytokine portion), a carrier (carrier portion), and an IL-2 antagonist (masking portion or cytokine antagonist), wherein the IL-2 polypeptide is fused to the carrier directly or through a cleavable or non-cleavable peptide linker, and the IL-2 antagonist is linked to the IL-2 polypeptide or the carrier through a non-cleavable or cleavable peptide linker. In some embodiments, the cytokine portion may be fused to the masking portion, and the masking portion may be fused to the carrier portion directly or through a cleavable or non-cleavable linker.

[0072] In a preferred embodiment, the IL-2 polypeptide is fused to the carrier via a non-cleavable peptide linker, and the IL-2 antagonist is linked to the carrier or the IL-2 polypeptide via a non-cleavable peptide linker. For example, the IL-2 antagonist can be fused to the carrier via a non-cleavable peptide linker of SEQ ID NO: 59. In some embodiments, the IL-2 polypeptide is a wild-type IL-2 polypeptide or does not include a mutation that reduces the binding affinity of the polypeptide to CD25.

[0073] The IL-2 fusion molecule of the present invention may include an IL-2 polypeptide (cytokine portion) connected to a carrier portion and masked (bound) by a cytokine antagonist (masking portion). The cytokine antagonist is selected from the following: an extracellular domain (ECD) of IL-2Rβ (CD122), a functional analog of IL-2RβECD, IL-2RγECD (CD132), a functional analog of IL-2RγECD, and a combination of IL-2RβECD and IL-2RγECD. In some embodiments, the cytokine antagonist inhibits the binding of the cytokine portion to IL-2Rγ and / or IL-2Rβ on T cells of patients in need, while the cytokine portion bound to IL-2Rα (CD25) remains intact. Because IL-2Rα (CD25) is preferentially expressed on Treg cells, the IL-2 fusion molecule of the present invention can preferentially stimulate Treg cell proliferation while minimizing the effect on non-Treg cells.

[0074] In some embodiments, the carrier portion is an Fc domain. In some embodiments, the IL-2 fusion molecule of the present invention is a heterodimer, comprising: a first polypeptide chain, the first polypeptide chain comprising a molecular formula selected from F1-L1-E1, F1-L1-E1-L2-E2 and F1-L1-E2-L2-E1 from N-terminus to C-terminus; and a second polypeptide chain, the second polypeptide chain comprising a molecular formula F2-L3-C from N-terminus to C-terminus, wherein F1 and F2 are subunits of the heterodimeric Fc domain, L1, L2 and L3 are peptide linkers, E1 is IL-2RβECD or a functional analog thereof, E2 is IL-2RγECD or a functional analog thereof, and C is a cytokine portion comprising an IL-2 polypeptide (e.g., wild-type human IL-2 or a mutant protein thereof).

[0075] In some embodiments, the IL-2 fusion molecule of the present invention is a heterodimer, which comprises: a first polypeptide chain, which comprises a molecular formula selected from E1-L1-F1, E1-L1-E2-L2-F1 and E2-L1-E1-L2-F1 from N-terminus to C-terminus; and a second polypeptide chain, which comprises a molecular formula C-L3-F2 from N-terminus to C-terminus, wherein F1 and F2 are subunits of the heterodimeric Fc domain, L1, L2 and L3 are peptide linkers, E1 is IL-2RβECD or a functional analogue thereof, E2 is IL-2RγECD or a functional analogue thereof, and C is a cytokine portion comprising an IL-2 polypeptide (e.g., wild-type human IL-2 or a mutant protein thereof).

[0076] In some embodiments, the IL-2 fusion molecule of the present invention is a heterodimer comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain comprise a molecular formula selected from the following pairs from N-terminus to C-terminus:

[0077] a.F1-L1-E1 and F2-L2-C-L3-E2;

[0078] b. F1-L1-E1 and F2-L2-E2-L3-C;

[0079] c.F1-L1-E2 and F2-L2-C-L3-E1;

[0080] d. F1-L1-E2 and F2-L2-E1-L3-C;

[0081] e.E1-L1-F1 and E2-L2-C-L3-F2;

[0082] f.E1-L1-F1 and C-L2-E2-L3-F2;

[0083] g. E2-L1-F1 and E2-L2-C-L3-F2; and

[0084] h.E2-L1-F1 and C-L2-E1-L3-F2;

[0085] wherein F1 and F2 are subunits of a heterodimeric Fc domain, L1, L2 and L3 are peptide linkers, E1 is IL-2Rβ ECD or a functional analogue thereof, E2 is IL-2Rγ ECD or a functional analogue thereof, and C is a cytokine portion.

[0086] In some embodiments, peptide linkers L1, L2, and L3 independently have an amino acid sequence selected from SEQ ID NOs: 40-49 and 55-57.

[0087] In some embodiments, at least one of the peptide linkers L1, L2, and L3 has an amino acid sequence comprising at least 20-44 amino acids (e.g., at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, at least 45 amino acids, or at least 55 amino acids). In some embodiments, at least one of the peptide linkers has at least 16 amino acids, 18 amino acids, 20 amino acids, 22 amino acids, 24 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 31 amino acids, 32 amino acids, 33 amino acids, 34 amino acids, 36 amino acids, 37 amino acids, 38 amino acids, 39 amino acids, 41 amino acids, or 42 amino acids.

[0088] In some embodiments, the IL-2 fusion molecules of the present invention have Figure 1A , Figure 1B , Figure 2A , Figure 2B , Figure 3A , Figure 3B , Figure 4A , Figure 4B , Figure 5A , Figure 5B , Fig. 6A , Figure 6B , Fig. 7A or Figure 7B In a specific embodiment, the IL-2 fusion molecule has Fig. 10A Or the structure shown in 10B.

[0089] In some embodiments, the isolated fusion molecule comprises: a first polypeptide chain comprising an amino acid sequence that is at least 99% identical to any amino acid sequence selected from SEQ ID NOs: 50, 51, and 52; and a second polypeptide chain, the amino acid sequence of the second polypeptide chain being at least 99% identical to any amino acid sequence selected from SEQ ID NOs: 53 and 54.

[0090] Isolated IL-2 fusion molecules 982C1, C2, D1, D2 and 982Ref include two polypeptide chains having the amino acid sequences shown in Table 1. The 982C1 and 982C2 molecules each include two masking moieties, which are IL-2RγECD and IL-2RβECD. 982D1 and 982D2 each include one masking moiety, which is IL-2RβECD. The IL-2 portions of 982C2 and 982D2 each include mutations T3A, V69A, P74Q and C125S (numbered according to SEQ ID NO: 1).

[0091] Table 1. Sequences of 982C1, C2, D1, D2 and 982Ref

[0092] Molecule name Polypeptide chain 1 Polypeptide chain 2 982Cl SEQ ID NO:50 SEQ ID NO:54 982C2 SEQ ID NO:52 SEQ ID NO:54 982D1 SEQ ID NO:50 SEQ ID NO:53 982D2 SEQ ID NO:52 SEQ ID NO:53 982Ref SEQ ID NO:58 SEQ ID NO:58

[0093] A.IL-2 Peptide or mutant protein

[0094] In the IL-2 fusion molecules of the present invention, the IL-2 polypeptide can be a wild-type IL-2 polypeptide, such as a wild-type human IL-2 polypeptide (SEQ ID NO: 1), or an IL-2 mutant protein, such as an IL-2 mutant protein derived from human IL-2. The IL-2 mutant protein is an IL-2 derivative that retains at least one or more aspects of the biological activity of IL-2. In some embodiments, the IL-2 mutant protein includes an amino acid sequence that is at least 95% identical to SEQ ID NO: 1. In certain embodiments, the IL-2 mutant protein has the same length as SEQ ID NO: 1, but differs therefrom in that it has no more than 7 amino acid residues (e.g., no more than 6 amino acid residues, no more than 5 amino acid residues, no more than 4 amino acid residues, no more than 3 amino acid residues, or no more than 2 amino acid residues). The IL-2 mutant protein may have a binding affinity for CD4 T cells. The invention further comprises one or more mutations selected from the group consisting of L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T, D20W, M23R, R81A, R81G, R81E 26I, Q126L, Q126M, Q126N, Q126R, Q126V, and Q126Y. Unless otherwise indicated, all residue numbering in IL-2 is according to that of SEQ ID NO: 1. In some embodiments, the IL-2 mutant protein may have a mutation that results in an enhanced affinity for CD25. Such mutations may be selected from mutations at positions 69 and 74. In some embodiments, the IL-2 mutant protein may include one or more mutations selected from the following: T3A, C125A, C125S, and C125G.

[0095] B. Masking Moieties of Isolated IL-2 Fusion Molecules

[0096] The cytokine antagonist in the isolated IL-2 fusion molecule of the present invention, i.e., the masking part, is an IL-2Rβ or IL-2Rγ extracellular domain or a functional analog thereof, such as IL-2Rβ or IL-2Rγ derived from humans (e.g., one of SEQ ID NOs: 3-6). In some embodiments, the IL-2 fusion molecule includes at least one masking part. For example, the fusion molecule may include both IL-2RβECD and IL-2RγECD or only one of these ECDs. ECD may include the entire extracellular domain of human IL-2Rβ or IL-2Rγ, or only a portion thereof, as long as the portion is still able to bind to the IL-2 portion or otherwise inhibit the IL-2 portion from binding to IL-2Rβ or IL-2Rγ on T cells.

[0097] In some embodiments, the IL-2 fusion molecule includes an additional masking moiety, which is an ECD of IL-2Rα (e.g., SEQ ID NO: 7) or a functional analog thereof, wherein the IL-2Rα ECD masking moiety is fused to the cytokine moiety, the carrier moiety, or another masking moiety in the fusion molecule via a cleavable peptide linker. The presence of the IL-2Rα masking moiety connected to the fusion molecule via a cleavable linker allows the fusion molecule to home to the targeted site without binding to cells in non-targeted sites; once at the targeted site, the cleavable linker is cleaved by a protease present at a high concentration at the targeted site, thereby allowing the activated fusion molecule to bind to IL-2Rα on cells (e.g., Treg cells) at the targeted site and stimulate the bound cells.

[0098] A functional analog of the ECD of an IL-2R subunit (α, β, or γ) refers to a polypeptide that has a similar affinity for IL-2 as the wild-type ECD. For example, a functional analog contains the IL-2 binding core region of the wild-type ECD and can have a sequence that is at least 95% (e.g., at least 96%, 97%, 98, or 99%) identical to the wild-type ECD (e.g., SEQ ID NOs: 3-7, below) over the entire length of the analog.

[0099] C. The carrier portion of the isolated IL-2 fusion molecule

[0100] The carrier portion of the IL-2 fusion molecule of the present invention can be an antigen binding portion or a portion that is not an antigen binding portion. The carrier portion can improve the PK properties of the cytokine agonist polypeptide, such as serum half-life, and can also target the cytokine agonist polypeptide to a target site in the body, such as a tumor site.

[0101] 1. Antigen binding carrier part

[0102] The carrier portion can be an antibody or an antigen binding fragment thereof, or an immunoadhesin. In some embodiments, the antigen binding portion is a full-length antibody having two heavy chains and two light chains, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, a disulfide-bonded Fv fragment, a single domain antibody, a nanobody, or a single chain variable fragment (scFv). In some embodiments, the antigen binding portion is a bispecific antigen binding portion and can bind to two different antigens or two different epitopes on the same antigen. The antigen binding portion can provide additional and potentially synergistic therapeutic efficacy for the cytokine agonist polypeptide.

[0103] The IL-2 polypeptide and its masking agent can be fused to the N-terminus or C-terminus of the light chain and / or heavy chain of the antigen binding portion. For example, the IL-2 polypeptide and its masking agent can be fused to the antibody heavy chain or its antigen binding fragment or to the antibody light chain or its antigen binding fragment. In some embodiments, the IL-2 polypeptide is fused to the C-terminus of one or both heavy chains of the antibody, and the cytokine masking agent is fused to the other end of the cytokine portion through a non-cleavable or cleavable peptide linker. In some embodiments, the IL-2 polypeptide is fused to the C-terminus of one of the heavy chains of the antibody, and the cytokine masking agent is fused to the C-terminus of the other heavy chain of the antibody through a non-cleavable or cleavable peptide linker, wherein the two heavy chains contain mutations that allow specific pairing of two different heavy chains.

[0104] Strategies for forming heterodimers are well known (see, e.g., Spies et al., Mol Immunol. (2015) 67(2)(A):95-106). For example, the two heavy chain polypeptides in an isolated IL-2 fusion molecule can form a stable heterodimer by a "knob-hole" mutation. The "knob-hole" mutation is performed to promote the formation of heterodimers of antibody heavy chains, and the mutation is often used to prepare bispecific antibodies (see, e.g., U.S. Patent 8,642,745). For example, the Fc domain of an antibody can include a T366W mutation in the CH3 domain of the "knob chain" and a T366S, L368A and / or Y407V mutation in the CH3 domain of the "hole chain". Additional interchain disulfide bonds between CH3 domains can also be used, for example by introducing a Y349C mutation into the CH3 domain of the "knob chain" and an E356C or S354C mutation into the CH3 domain of the "hole chain" (see, e.g., Merchant et al., Nature Biotech (1998) 16:677-81). In other embodiments, the antibody portion can include a Y349C and / or T366W mutation in one of the two CH3 domains and an E356C, T366S, L368A and / or Y407V mutation in the other CH3 domain. In certain embodiments, the antibody portion may include a Y349C and / or T366W mutation in one of the two CH3 domains and a S354C (or E356C), T366S, L368A and / or Y407V mutation in the other CH3 domain, wherein the additional Y349C mutation is located in one CH3 domain and the additional E356C or S354C mutation is located in the other CH3 domain, thereby forming an interchain disulfide bond (always numbered according to the EU index of Kabat; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Other knob-in-mold techniques, such as the knob-in-mold technique described in EP1870459A1, may be used alternatively or additionally. Thus, another example of a knob-hole mutation in an antibody portion is one having R409D / K370E mutations in the CH3 domain of the "knob chain" and D399K / E357K mutations in the CH3 domain of the "hole chain" (EU numbering).

[0105] In some embodiments, the antibody portion of the isolated IL-2 fusion molecule includes L234A and L235A ("LALA") mutations in its Fc domain. The LALA mutation abolishes complement binding and fixation and Fcγ-dependent ADCC (see, e.g., Hezareh et al., J. Virol. (2001) 75(24): 12161-8). In other embodiments, the LALA mutation is present in addition to the knob-hole mutation in the antibody portion.

[0106] In some embodiments, the antibody portion includes M252Y / S254T / T256E ("YTE") mutations in the Fc domain. The YTE mutation allows for simultaneous modulation of serum half-life, tissue distribution, and activity of IgG1 (see Dall'Acqua et al., J Biol Chem. (2006) 281(33):23514-24; and Robbie et al., Antimicrob Agents Chemother. (2013) 57(12):6147-53). In further embodiments, the YTE mutation is present in the antibody portion in addition to the knob-in-hole mutation. In specific embodiments, the antibody portion has YTE, LALA, and knob-in-hole mutations, or any combination thereof.

[0107] In some embodiments, the antigen binding portion binds to IL-1β, IL-1β receptor, IL-4, IL-4 receptor, IL-6, IL-6 receptor, IL-13, IL-13 receptor, IL-17, IL-17 receptor, IL-23, IL-23 receptor, TNFα, or TNFα receptor.

[0108] 2. Other carrier parts

[0109] Other non-antigen binding carrier moieties can be used for the isolated IL-2 fusion molecules of the present invention. For example, an antibody Fc domain (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc), a polymer (e.g., PEG), an albumin (e.g., human albumin) or a fragment thereof or a nanoparticle can be used.

[0110] For example, IL-2 polypeptides and antagonists thereof can be fused to antibody Fc domains to form Fc fusion proteins. In some embodiments, the IL-2 polypeptide is fused (directly or through a peptide linker) to the C-terminus or N-terminus of one of the Fc domain polypeptide chains, and the cytokine masking agent is fused to the C-terminus or N-terminus of another Fc domain polypeptide chain through a non-cleavable or cleavable peptide linker, wherein the two Fc domain polypeptide chains contain mutations that allow specific pairing of two different Fc chains. In some embodiments, the Fc domain includes the above-mentioned hole-to-hole mutation. In further embodiments, the Fc domain may also include the above-mentioned YTE and / or LALA mutations. In some embodiments, the Fc domain includes a mutation at N297 (EU numbering).

[0111] The carrier portion of the isolated IL-2 fusion molecule may include albumin (e.g., human serum albumin) or a fragment thereof. In some embodiments, the albumin or albumin fragment is about 85% or more, about 90% or more, about 91% or more, about 92% or more, about 93% or more, about 94% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, about 99.5% or more, or about 99.8% or more identical to human serum albumin or a fragment thereof.

[0112] In some embodiments, the carrier portion comprises an albumin fragment (e.g., a human serum albumin fragment) of about 10 amino acids or more, 20 amino acids or more, 30 amino acids or more, 40 amino acids or more, 50 amino acids or more, 60 amino acids or more, 70 amino acids or more, 80 amino acids or more, 90 amino acids or more, 100 amino acids or more, 120 amino acids or more, 140 amino acids or more, 160 amino acids or more, 180 amino acids or more, 200 amino acids or more, 250 amino acids or more, 300 amino acids or more, 350 amino acids or more, 400 amino acids or more, 450 amino acids or more, 500 amino acids or more, or 550 amino acids or more. In some embodiments, the length of the albumin fragment is between about 10 amino acids and about 584 amino acids (e.g., between about 10 amino acids and about 20 amino acids, between about 20 amino acids and about 40 amino acids, between about 40 amino acids and about 80 amino acids, between about 80 amino acids and about 160 amino acids, between about 160 amino acids and about 250 amino acids, between about 250 amino acids and about 350 amino acids, between about 350 amino acids and about 450 amino acids, or between about 450 amino acids and about 550 amino acids in length). In some embodiments, the albumin fragment comprises a Sudlow I domain or a fragment thereof, or a Sudlow II domain or a fragment thereof.

[0113] D. Linker portion of isolated fusion molecules

[0114] The IL-2 polypeptide can be fused to the carrier portion with or without a peptide linker. The peptide linker can be cleavable or non-cleavable. In some embodiments, the cytokine portion is fused to the carrier via a peptide linker, wherein the peptide linker is selected from SEQ ID NOs: 40-46 and 55-57. In particular embodiments, the peptide linker comprises an amino acid sequence of SEQ ID NOs: 42, 44, 45, 46, 55, 56 or 57. The masking portion can be fused to the cytokine portion or the carrier via a non-cleavable or cleavable linker or without a peptide linker. The cleavable linker can contain one or more (e.g., two or three) cleavable moieties (CM). Each CM can be a substrate for an enzyme or protease selected from the group consisting of legumin, plasmin, TMPRSS-3 / 4, MMP-2, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase, β-secretase, uPA and PSA. In some embodiments, the masking moiety is fused to the carrier via a peptide linker, wherein the peptide linker is selected from SEQ ID NOs: 40-46, 55, 56, and 57. In specific embodiments, the peptide linker comprises an amino acid sequence of SEQ ID NOs: 42, 44, 45, 46, 55, 56, or 67. In some embodiments, the peptide linker comprises at least 10 amino acids, 12 amino acids, 14 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 25 amino acids, 27 amino acids, or 30 amino acids.

[0115] Specific non-limiting examples of IL-2 polypeptides, cytokine masking agents, carriers, peptide linkers, and isolated IL-2 fusion molecules are shown in the sequence section below. In addition, the isolated fusion molecules of the present disclosure can be prepared by well-known recombinant techniques. For example, one or more expression vectors comprising the coding sequence of the polypeptide chain of the isolated fusion molecule can be transfected into a mammalian host cell (e.g., CHO cells), and the cells are cultured under conditions that allow expression of the coding sequence and assembly of the expressed polypeptide into the isolated IL-2 fusion molecule complex.

[0116] Pharmaceutical composition

[0117] Pharmaceutical compositions comprising the isolated IL-2 fusion molecules of the present disclosure (i.e., active pharmaceutical ingredients or APIs) can be prepared by mixing the API having the desired purity with one or more optional pharmaceutically acceptable excipients in the form of a lyophilized formulation or an aqueous solution (see, e.g., Remington's Pharmaceutical Sciences, 16th edition, Osol, A., ed. (1980)). Pharmaceutically acceptable excipients (or carriers) are generally nontoxic to recipients at the dosages and concentrations employed and include, but are not limited to: a buffer containing, for example, phosphate, citrate, succinate, histidine, acetate, or another inorganic or organic acid or salt thereof; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenolic, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol) ; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including sucrose, glucose, mannose or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0118] Buffer is used for pH control in the scope of optimizing therapeutic effect, especially when stability depends on pH.Buffer preferably exists in the concentration of about 50mM to about 250mM with the scope.Suitable buffer used together with the present invention comprises organic acid and inorganic acid and salt thereof, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate and acetate.In addition, buffer can comprise histidine and trimethylamine salt, such as Tris.

[0119] Preservatives are added to slow down microbial growth and are typically present in the range of 0.2%-1.0% (w / v). Suitable preservatives for use with the present invention include: octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl alcohol or benzyl alcohol; alkyl parabens, such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol, 3-pentanol and m-cresol.

[0120] Tension agent, sometimes referred to as " stabilizer ", exists in order to regulate or maintain the tension force of liquid in the composition.When used together with large charged biomolecules such as protein and antibody, tension agent is commonly referred to as " stabilizer ", because it can interact with the charged group of amino acid side chain, thus reducing the possibility of intermolecular and intramolecular interaction.Considering the relative amount of other components, tension agent can exist with any amount between 0.1 % by weight to 25 % by weight or more preferably between 1 % by weight to 5 % by weight.Preferred tension agent comprises polyol, preferably trivalent or higher sugar alcohol, such as glycerol, erythritol, arabitol, xylitol, sorbitol and mannitol.

[0121] A nonionic surfactant or detergent (also referred to as a "wetting agent") is present to help dissolve the therapeutic agent and protect the therapeutic protein from agitation-induced aggregation, which also allows the formulation to be exposed to shear surface stresses without causing denaturation of the active therapeutic protein or antibody. The nonionic surfactant is present in the range of about 0.05 mg / ml to about 1.0 mg / ml, preferably about 0.07 mg / ml to about 0.2 mg / ml.

[0122] Suitable nonionic surfactants include polysorbate (20, 40, 60, 65, 80, etc.), poloxamer (184, 188, etc.), Polyols, Polyoxyethylene sorbitan monoether ( -20, -80, etc.), lauromacrogol 400, polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glyceryl monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, sodium hexadecyl sulfosuccinate and sodium dioctyl sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0123] The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.The pharmaceutical composition may additionally include any suitable binders, lubricants, suspending agents, coating agents or solubilizing agents.

[0124] Depending on the delivery system, there may be different composition / formulation requirements. For example, the pharmaceutical compositions useful in the present invention can be formulated for administration using a micropump or by a mucosal route, such as a nasal spray or aerosol for inhalation or an ingestible solution, or by formulating the composition in an injectable form for parenteral delivery, such as by an intravenous route, an intramuscular route, or a subcutaneous route.

[0125] In some embodiments, the pharmaceutical composition of the present disclosure is a lyophilized protein formulation. In other embodiments, the pharmaceutical composition can be an aqueous liquid formulation.

[0126] Treatment

[0127] IL-2 fusion molecules can be used to treat inflammatory diseases or autoimmune diseases. In some embodiments, a method of treating a disease (such as an autoimmune disease) in a subject comprises administering to the subject an effective amount of an isolated IL-2 fusion molecule disclosed herein. In some embodiments, the inflammatory disease or autoimmune disease is selected from the group consisting of asthma, diabetes (e.g., type I diabetes or latent autoimmune diabetes), lupus (e.g., systemic lupus erythematosus), arthritis (e.g., rheumatoid arthritis), allergies, organ transplant rejection, GVHD, Addison's disease, ankylosing spondylitis, anti-glomerular basement membrane disease, autoimmune hepatitis, dermatitis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barre syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schonlein purpura (HSP), juvenile myositis, Kawasaki disease, inflammatory bowel disease (e.g., Crohn's disease), disease and ulcerative colitis), multiple sclerosis, myasthenia gravis, neuromyelitis optica, PANDAS, psoriasis, psoriatic arthritis, Sjogren's syndrome, systemic scleroderma, systemic sclerosis, thrombocytopenic purpura, uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada Disease.

[0128] Typically, the dosage and route of the pharmaceutical composition of the present invention are determined according to the size and condition of the subject according to standard pharmaceutical practice. In certain embodiments, the pharmaceutical composition is administered to the subject by any route, including oral administration, transdermal administration, administration by inhalation, intravenous administration, intraarterial administration, intramuscular administration, direct application to the wound site, application to the surgical site, intraperitoneal administration, administration by suppository, subcutaneous administration, intradermal administration, transdermal administration, administration by atomization, intrapleural administration, intraventricular administration, intraarticular administration, intraocular administration, intracranial administration, or intraspinal administration. In certain embodiments, the composition is administered intravenously to the subject.

[0129] In some embodiments, the dosage of the pharmaceutical composition is a single dose or repeated dose. In some embodiments, the dosage is administered to the subject once a day, twice a day, three times a day, or four times a day or more. In some embodiments, about 1 or more (e.g., about 2, 3, 4, 5, 6 or 7 or more) doses are administered in a week. In some embodiments, the pharmaceutical composition is administered once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every two weeks in 3 weeks, or once every 3 weeks in 4 weeks. In some embodiments, multiple doses are administered over the course of several days, weeks, months, or years. In some embodiments, a course of treatment is about 1 dose or more doses (e.g., about 2 doses, 3 doses, 4 doses, 5 doses, 7 doses, 10 doses, 15 doses, or 20 or more doses).

[0130] Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present disclosure should have the meanings commonly understood by those of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In the event of a conflict, this specification (including definitions) shall prevail. Typically, the term list and technology used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization as described herein are well-known and commonly used term lists and technologies in the art. Enzymatic reactions and purification techniques are performed according to the instructions of manufacturers as generally implemented in the art or as described herein. In addition, unless the context requires otherwise, singular terms should include plural meanings and plural terms should include singular meanings. Throughout the specification and examples, the words "have", "comprise" or variations such as "has", "having", "comprises" or "comprising" should be understood to imply the inclusion of a stated integer or groups of integers but not the exclusion of any other integer or groups of integers. It should be understood that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" the aspects and variations. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0131] Exemplary Embodiments

[0132] Additional specific embodiments of the present disclosure are described below. These embodiments are intended to illustrate the compositions and methods described in the present disclosure and are not intended to limit the scope of the present disclosure.

[0133] 1. An IL-2Rβ-ECD mutant comprising one or more point mutations, wherein the IL-2Rβ-ECD mutant has enhanced thermal stability compared to the wild type.

[0134] 2. The IL-2Rβ-ECD mutant according to Example 1, comprising one or more mutations at positions selected from the group consisting of: Δ1-5 (deletion of the first five amino acids) F11, V21, L28, W38, L51, P52, V53,163, P67,177, V88, V92, M93,195, M107,1110, VI15, P156, L157, Q162, Q164, W166, P174, L187, F191, P196, P200, P207, W90, H150, W152, W166, W194 and W197 (numbered according to SEQ ID NO: 3).

[0135] 3. The IL-2Rβ-ECD mutant according to Example 1, comprising a mutation at a site selected from the following group (numbered according to SEQ ID NO: 3):

[0136] a. F11 and F191;

[0137] b.L51, P52 and V53;

[0138] c.V92, M93, 195;

[0139] d.M107, P196, 1110; and

[0140] e.P156 and L157.

[0141] 4. The IL-2Rβ-ECD mutant according to embodiment 2 or 3, wherein the hydrophobic amino acid is mutated to a hydrophilic amino acid selected from S, G, N, T and Q.

[0142] 5. The IL-2Rβ-ECD mutant according to Example 3, comprising a mutation selected from the following group (numbered according to SEQ ID NO: 3):

[0143] a.F11S and F191G;

[0144] b.L51S, P52G and V53S;

[0145] c.V92S, M93G, I95G;

[0146] d.M107G, P196S, I110G; and

[0147] e.P156S and L157G;

[0148] f.W166N;

[0149] g.Q164E;

[0150] h.W166N, V115S;

[0151] i.W152N;

[0152] j.W152N, W166N;

[0153] k.V92S;

[0154] l.W166N, V92S;

[0155] m.L157S;

[0156] n.W165N, W157S.

[0157] 6. The IL-2Rβ-ECD mutant according to Example 1, comprising an amino acid sequence selected from SEQ ID NO: 47, 48 and 49.

[0158] 7. An isolated IL-2 fusion molecule that can be used to treat inflammatory diseases and autoimmune diseases, the isolated IL-2 fusion molecule comprising a cytokine portion and a masking portion, wherein the cytokine portion comprises an IL-2 polypeptide or an IL-2 mutant protein, and the masking portion comprises the extracellular domain (ECD) of IL-2Rβ or a functional analog or mutant thereof; and wherein the fusion molecule preferentially stimulates T regulatory cells relative to other T cells or NK cells in an in vitro assay.

[0159] 8. The isolated IL-2 fusion molecule of embodiment 7, wherein the fusion molecule has an EC50 value of less than about 1 nM in a CTLL-2 cell proliferation assay.

[0160] 9. The isolated IL-2 fusion molecule of embodiment 7, wherein the fusion molecule has an EC50 value of less than about 0.1 nM in a CTLL-2 cell proliferation assay.

[0161] 10. The isolated fusion molecule of any one of embodiments 7 to 9, wherein the masking moiety comprises the IL-2Rβ-ECD mutant of any one of embodiments 1 to 6.

[0162] 11. The isolated fusion molecule according to any one of embodiments 7 to 10, further comprising the extracellular domain (ECD) of IL-2Rγ or a functional analog thereof.

[0163] 12. The isolated fusion molecule of any one of embodiments 7 to 11, further comprising a vector.

[0164] 13. The isolated fusion molecule of embodiment 12, wherein the masking moiety is linked to the carrier moiety via a cleavable or non-cleavable peptide linker.

[0165] 14. An isolated fusion molecule according to any one of embodiments 7 to 13, wherein the IL-2 polypeptide or IL-2 mutant protein comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1; and wherein the IL-2RβECD or a functional analog or mutant thereof has an amino acid sequence that is at least 95% identical to SEQ ID NO: 3.

[0166] 15. The isolated fusion molecule of any one of embodiments 7 to 13, wherein the IL-2 mutant protein has the amino acid sequence as shown in SEQ ID NO: 2.

[0167] 16. The isolated fusion molecule of any one of embodiments 7 to 13, wherein the IL-2 mutant protein has at least one mutation selected from the group consisting of L12G, L12K, L12Q, L12S, Q.13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F , D20G, D20T, D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q 26I, Q126L, Q126M, Q126N, Q126R, Q126V and Q126Y (numbered according to SEQ ID NO: 1).

[0168] 17. The isolated fusion molecule according to any one of embodiments 7 to 16, wherein the carrier moiety is selected from: a PEG molecule, albumin, an albumin fragment, an antibody Fc domain, or an antibody or an antigen-binding fragment thereof.

[0169] 18. The isolated fusion molecule of embodiment 17, wherein the carrier portion comprises an antibody Fc domain (EU numbering) having a mutation at N297 and / or mutations L234A and L235A ("LALA").

[0170] 19. An isolated fusion molecule according to embodiment 17 or 18, wherein the carrier portion comprises an antibody Fc domain, the antibody Fc domain comprises a knob-to-hole mutation, and wherein the cytokine portion and the masking portion are fused to different polypeptide chains of the antibody Fc domain.

[0171] 20. The isolated fusion molecule of embodiment 19, wherein the cytokine portion and the masking portion are fused to the C-terminus of the two different polypeptide chains of the Fc domain or to the C-terminus of the two different heavy chains of the antibody.

[0172] 21. The isolated fusion molecule of embodiment 19, wherein the carrier is an antibody Fc domain, and wherein the cytokine portion and the masking portion are fused to the N-termini of the two different polypeptide chains of the Fc domain.

[0173] 22. An isolated fusion molecule according to embodiment 12, wherein the carrier portion is an antibody Fc domain, and wherein the isolated fusion molecule comprises: a first polypeptide chain, the first polypeptide chain comprising a molecular formula selected from F1-L1-E1, F1-L1-E1-L2-E2 and F1-L1-E2-L2-E1; and a second polypeptide chain, the second polypeptide chain comprising a molecular formula F2-L3-C, wherein F1 and F2 are subunits of the Fc domain that form a heterodimer, L1, L2 and L3 are peptide linkers, E1 is IL-2RβECD or a functional analog thereof, and E2 is IL-2RγECD or a functional analog thereof, and C is the cytokine portion.

[0174] 23. An isolated fusion molecule according to embodiment 12, wherein the carrier portion is an antibody Fc domain, and wherein the isolated fusion molecule comprises: a first polypeptide chain, the first polypeptide chain comprising a molecular formula selected from E1-L1-F1, E1-L1-E2-L2-F1 and E2-L1-E1-L2-F1; and a second polypeptide chain, the second polypeptide chain comprising the molecular formula C-L3-F2, wherein F1 and F2 are subunits of the Fc domain forming a heterodimer, L1, L2 and L3 are peptide linkers, E1 is IL-2RβECD or a functional analog thereof, and E2 is IL-2RγECD or a functional analog thereof, and C is the cytokine portion.

[0175] 24. The isolated fusion molecule of embodiment 12, wherein the carrier moiety is an antibody Fc domain, and wherein the isolated fusion molecule comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain comprise a molecular formula selected from the following pairs:

[0176] a.F1-L1-E1 and F2-L2-C-L3-E2;

[0177] b. F1-L1-E1 and F2-L2-E2-L3-C;

[0178] c.F1-L1-E2 and F2-L2-C-L3-E1;

[0179] d. F1-L1-E2 and F2-L2-E1-L3-C;

[0180] e.E1-L1-F1 and E2-L2-C-L3-F2;

[0181] f.E1-L1-F1 and C-L2-E2-L3-F2;

[0182] g. E2-L1-F1 and E2-L2-C-L3-F2; and

[0183] h.E2-L1-F1 and C-L2-E1-L3-F2;

[0184] Wherein F1 and F2 are subunits of the Fc domain forming a heterodimer, L1, L2 and L3 are peptide linkers, E1 is IL-2RβECD or a functional analogue thereof, and E2 is IL-2RγECD or a functional analogue thereof, and C is the cytokine portion.

[0185] 25. An isolated fusion molecule according to any one of embodiments 22 to 24, wherein the IL-2RβECD has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:3, the IL-2RγECD has an amino acid sequence as shown in SEQ ID NO:6, and the cytokine portion includes an IL-2 mutant protein having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:2.

[0186] 26. The isolated fusion molecule of any one of embodiments 21 to 25, wherein the Fc domain comprises a knob-to-hole mutation.

[0187] 27. An isolated fusion molecule according to any one of embodiments 18 to 26, wherein the knob-to-hole mutation comprises a T366Y "knob" mutation on a polypeptide chain of the Fc domain and a Y407T "hole" mutation in another polypeptide of the Fc domain (EU numbering).

[0188] 28. An isolated fusion molecule according to any one of embodiments 18 to 21 and 26, wherein the knob-to-hole mutations include Y349C and / or T366W mutations in the CH3 domain of the "knob chain" and E356C, T366S, L368A and / or Y407V mutations in the CH3 domain of the "hole chain" (EU numbering).

[0189] 29. An isolated fusion molecule according to embodiment 12, wherein the carrier portion is an antibody Fc domain, and wherein the fusion molecule comprises: a first polypeptide chain, the first polypeptide chain comprising an amino acid sequence that is at least 99% identical to any amino acid sequence selected from SEQ ID NOs: 8-11, 28, 29 and 30; and a second polypeptide chain, the second polypeptide chain having an amino acid sequence that is at least 99% identical to any amino acid sequence selected from SEQ ID NOs: 16-21.

[0190] 30. An isolated fusion molecule according to embodiment 12, wherein the carrier portion is an antibody Fc domain, and wherein the fusion molecule comprises: a first polypeptide chain, the first polypeptide chain comprising an amino acid sequence that is at least 99% identical to any amino acid sequence selected from SEQ ID NOs: 12-15, 31, 32 and 33; and a second polypeptide chain, the second polypeptide chain having an amino acid sequence that is at least 99% identical to any amino acid sequence selected from SEQ ID NOs: 22-27.

[0191] 31. An isolated fusion molecule according to embodiment 29 or 30, wherein the Fc domain further comprises a mutation of N297A or N297G (EU numbering).

[0192] 32. The isolated fusion molecule of embodiment 12, wherein the carrier is an IgG4 Fc further comprising a knob-to-hole mutation.

[0193] 33. An isolated fusion molecule according to embodiment 32, comprising: a first polypeptide chain, which comprises an amino acid sequence that is at least 99% identical or 100% identical to any amino acid sequence selected from SEQ ID NO:50, 51 and 52; and a second polypeptide chain, which has an amino acid sequence that is at least 99% identical or 100% identical to any amino acid sequence selected from SEQ ID NO:53 and 54.

[0194] 34. The isolated fusion molecule of any one of embodiments 22 to 24, wherein the peptide linkers L1, L2 and L3 independently have an amino acid sequence selected from SEQ ID NO: 40-46, 55-57, 59 and 60.

[0195] 35. The isolated fusion molecule according to any one of embodiments 22 to 24, wherein at least one of said peptide linkers L1, L2 and L3 has an amino acid sequence comprising 20-44 amino acids.

[0196] 36. An isolated fusion molecule according to any one of embodiments 7 to 11, wherein the affinity of the fusion molecule binding to the high affinity IL-2 receptor having α, β and γ subunits (IL-2Rαβγ) is at least 100 times that of the medium affinity IL-2 receptor (IL-2Rβγ) formed with β and γ subunits.

[0197] 37. The isolated fusion molecule of any one of embodiments 7 to 11, which binds to IL-2Rβγ with a binding dissociation equilibrium constant (KD) greater than about 5 nM as measured in a surface plasmon resonance assay at 37°C.

[0198] 38. The isolated fusion molecule of any one of embodiments 7 to 37, which promotes the growth or survival of FOXP3-positive regulatory T cells in vitro.

[0199] 39. The isolated fusion molecule of any one of embodiments 7 to 37, which induces STAT5 phosphorylation in FOXP3-positive T cells ex vivo, said FOXP3-positive T cells comprising a functional IL-2 receptor complex, but has reduced ability to induce STAT5 phosphorylation in FOXP3-negative T cells.

[0200] 40. The fusion molecule according to any one of embodiments 7 to 11, further comprising an extracellular domain (ECD) of IL-2Rα or a functional analogue thereof; wherein the IL-2Rα ECD or a functional analogue thereof is linked to the fusion molecule via a cleavable peptide linker.

[0201] 41. According to the fusion molecule of embodiment 40, the IL-2Rα ECD or a functional analog thereof comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:7.

[0202] 42. A polynucleotide encoding the fusion molecule according to any one of embodiments 7 to 41 or the IL-2Rβ-ECD mutant according to any one of embodiments 1 to 6.

[0203] 43. An expression vector comprising the polynucleotide according to embodiment 42.

[0204] 44. A host cell comprising the vector of embodiment 43.

[0205] 45. A method of making the isolated fusion molecule of any one of embodiments 7 to 41, the method comprising culturing the host cell of claim 44 under conditions that allow expression of the fusion molecule and isolating the fusion molecule.

[0206] 46. ​​A pharmaceutical composition comprising the isolated fusion molecule of any one of embodiments 7 to 41 and a pharmaceutically acceptable excipient.

[0207] 47. A method of treating an inflammatory disease or an autoimmune disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of the isolated fusion molecule of any one of embodiments 7 to 41.

[0208] 48. A method of treating an inflammatory disease or an autoimmune disease in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition according to Examples to 46.

[0209] 49. The method of embodiment 48, wherein the inflammatory disease or autoimmune disease is selected from the group consisting of asthma, diabetes, arthritis, allergies, organ transplant rejection, and graft-versus-host disease.

[0210] Examples

[0211] Transient transfection

[0212] For transient transfection using HEK293 cells, the expression plasmid was co-transfected into 3 × 10 cells using PEI (polyethyleneimine) at 2.5-3 μg / mL. 6 cells / mL in freestyle HEK293 cells. For IL-2 fusion molecules for Fc-based IL-2 isolation, the ratio of Fc-IL-2 mutein fusion polypeptide to Fc masking moiety fusion polypeptide was 1:2. For IL-2 fusion molecules for antibody-based IL-2 isolation, the molar ratio of knob heavy chain (containing IL-2 agonist polypeptide) to hole heavy chain (containing masking moiety) to light chain DNA was 2:1:2. Six days after transfection, cell cultures were harvested by centrifugation at 9,000 rpm for 45 minutes followed by 0.22 μM filtration.

[0213] For transient transfection using ExpiCHO cells, co-transfect the expression plasmid into 6 × 10 cells using Expifectamine CHO reagent at 1-2 μg / mL. 6cells / mL ExpiCHO-S cells. For 982D1, the ratio of knob heavy chain IL-2 mutant protein fusion polypeptide to hole heavy chain (containing a β-shielding portion polypeptide) was 1:4. Similarly, for 982D2, the ratio of IL-2E mutant protein polypeptide knob heavy chain to hole heavy chain (containing a β-shielding portion polypeptide) was 1:4. Approximately 7 days after transfection, cell cultures were harvested by centrifugation at 12,000 rpm for 40 minutes followed by 0.2 or 0.45 μM filtration.

[0214] Protein purification

[0215] Using Protein A affinity chromatography Protein IL-2 fusion molecule (protein) was purified using 1% 1% 1% 1% 2 ... Q FF resin or Further purification was performed using QHP resin followed by Capto TM MMC ImpRes resin was used for the third column step. For 982D1 and 982D2 samples, anion exchange chromatography was used in flow-through mode with Further purification was performed using Q HP resin followed by Capto TM SP ImpRes resin was used for the third column step. and Capto TM Resins were ordered from GE Healthcare Life Sciences (now Cytiva, Marlborough, MA). Prior to in vivo studies, samples were purified to at least 98% purity by SEC-HPLC analysis. Samples were formulated in 20 mM histidine, 7% sucrose, 0.03% polysorbate-20. Samples were stored in a -80°C freezer until use.

[0216] Proteolytic treatment

[0217] 0.1 μg / μL human MMP2 (Sino Biological No. 10082-HNAH) was activated with 1 mM p-aminophenylmercuric acetate (APMA, Sigma No. A-9563). Two hundred (200) μg of IL-2 fusion molecules were incubated with 0.5 μg of human MMP2 in HBS buffer (20 mM HEPES, 150 mM NaCl2, pH 7.4) containing 2 mM CaCl2 and 10 μM ZnCl2 at 37°C for 16 hours (overnight).

[0218] SDS-PAGE analysis

[0219] Ten (10) μL of culture supernatant or 20 μg of purified protein sample were mixed with Bolt TM LDS sample buffer (Novex) was mixed. The samples were heated at 70°C for 3 minutes and then loaded onto NuPAGE TM 4-12% BisTris gel (Invitrogen). The gel was run on NuPAGE TM The samples were run at 200 volts for 40 min in MOPS SDS running buffer (Invitrogen) and then stained with Coomassie blue.

[0220] Figure 8 The results of SDS-PAGE analysis of the isolated IL-2 fusion molecule JR3.116.5 before (unreduced and reduced) and after activation by the above-mentioned protease treatment are shown. JR3.116.5 includes two polypeptide chains whose amino acid sequences are shown in SEQ ID NO: 12 and 23, respectively, and has Figure 1B The structure shown. The data indicate that the majority of the Protein A column pool is the expected heterodimeric molecule of JR3.116.5. There appears to be a small band of mortar chain homodimers (SEQ ID NO:23). Surprisingly, there is no obvious band of unpaired chains or any homodimers of the knob chain. The interaction between the cytokine portion and the masking agent portion may promote the correct heterodimerization between the knob chain (SEQ ID NO:12) and the mortar chain (SEQ ID NO:23).

[0221] CTLL-2 assay

[0222] CTLL-2 cells were grown in RPMI 1640 medium supplemented with L-glutamine, 10% fetal bovine serum, 10% non-essential amino acids, 10% sodium pyruvate, and 55 μM β-mercaptoethanol. CTLL-2 cells are non-adherent cells and were maintained at 5×10 cells / mL in medium with 100 ng / mL IL-2. 4 -1×10 6 Cells were divided twice a week. For bioassays, cells were preferably used no less than 48 hours after passaging.

[0223] The samples were diluted in 50 μL / well at 2× concentration in a 96-well plate. The IL-2 standard was titrated from 20 ng / mL (2× concentration) to 3× serial dilutions in 12 wells. When appropriate, the samples were titered. The CTLL-2 cells were washed 5 times to remove IL-2, 5000 cells / well were distributed in 50 μL and cultured with the samples overnight or for at least 18 hours. Subsequently, 100 μL / well Cell Titer Glo reagent (Promega) was added and luminescence was measured. Fig. 9 The results of the CTLL-2 analysis are shown. The data show that the masking moiety reduces the activity of JR3.116.5 by approximately 20-fold. In addition, this masking effect is reversible, as activation of the masking moiety by protease cleavage restores the activity of the fusion molecule.

[0224] NK92 cell proliferation assay

[0225] NK92 cell line is a factor-dependent cell line that requires IL-2 to grow and survive. Before the assay, NK92 cells were washed to remove IL-2 and cultured overnight in the absence of growth factors. Cells were collected and washed again to remove residual growth factors. The cells were resuspended to 4000,000 cells / mL. Then cells (20,000 / well) were added to 96-well plates. Anti-CD25 antibody, i.e. basiliximab, was added to half of the plates (48 wells) at 10 μg / mL. Cell incubation was continued for 15 minutes. Continuous titration of IL-2 fusion molecules was added to each well at 50 μL / well. The plate was incubated overnight, and cell titer Glo (Promega) was added before measuring luminescence. This provides a measure of ATP levels as a cell viability indicator. Fig.11NK92 proliferation assays of 982D1, 982D1 and 982Ref in the presence and absence of anti-CD25 neutralizing antibodies are shown. The reference molecule (982Ref) is an IL-2 fusion molecule with substitution mutations V91K and C125A in IL-2 (IL-2 portion numbered according to SEQ ID NO: 1). For assays with anti-CD25 antibodies, anti-CD25 antibodies were added to cells at 10 μg / mL.

[0226] The data showed that 982Ref was more active than 982D1 and 982D2 in stimulating NK92 cell proliferation. All tested fusion molecules showed minimal activity when neutralizing anti-CD25 antibody was added.

[0227] Binding assay: rat CD4+ T cells

[0228] Blood samples collected from male Sprague-Dawley rats with jugular cannulae were lysed to remove red blood cells. The remaining cells were incubated on ice with various concentrations of test article 982D1 or 982D2 for approximately 60 minutes. Detection antibody, goat anti-human IgG Fcγ-APC (Jackson ImmunoResearch Lab. catalog number 109-135-170) was added to each well. After incubation and subsequent washing, anti-rat CD4 antibody (BD Bioscience, catalog number 554866) was added to stain rat CD4 T cells. The stained samples were washed again, and flow cytometry analysis was then performed to detect the fusion molecules of IL-2 and rat CD4 + Binding of T cells.

[0229] Figure 12 shows the relationship between 982D1 and 982D2 and rat CD4 + Binding activity of T cells. NC represents irrelevant Ab control. Surprisingly, 982D1 bound to rat CD4+ T cells to a minimal degree stronger than 82D2, but the difference was not significant.

[0230] Binding Assay: Human CD4+CD25+ T Cells

[0231] Human peripheral blood mononuclear cells (hPBMC) were isolated from buffy coat blood (BioIVT and RBC) and cultured overnight in complete medium RPMI1640 (Life Technologies, catalog number 12633-020) containing 10% FBS (Life Technologies, catalog number 10099141). The next day, human PBMC were treated with anti-human CD3 antibody (Biolegend, catalog number 317302) for 2 days, washed 3 times with complete medium RPMI 1640, and then left to stand for 3 days. The cells were adjusted to 4-5×10 6 The concentration of cells / mL was then added to 50 μL of cells (200-250K cells / well), followed by 50 μL of IL-2 fusion molecules 982C1, 982D1, 982D2 and 982Ref loaded into the corresponding wells of 96-well plates at various concentrations. Unrelated Ab was added within the same concentration range as the negative control. After incubation on ice for about 60 minutes, the cells were washed and then detection antibodies, goat anti-human IgG Fcγ-APC (Jackson ImmunoResearch Laboratories, catalog number 109-135-170) were added. After sufficient washing to remove free IgG Fcγ-APC, FITC-conjugated mouse anti-human CD4Ab (BD Biosciences, catalog number 555346) and PE-conjugated mouse anti-human CD25 Ab (BD Biosciences, catalog number 555432) were added to the wells for cell staining. Finally, the stained samples were subjected to flow cytometry analysis to detect the expression of IL-2 fusion molecules with Treg (CD4 + CD25+) and T eff (CD4 + CD25 - ) cell binding.

[0232] Fig.13A and 13B The binding of 982C1, 982D1 and 982Ref to human CD4 + / CD25 + T cells and CD4 + / CD25 - The results show that 982Ref binds to CD4 + / CD25 +The binding affinity of T cells is stronger than that of 982D2, 982D1 and 982C1. In this assay, PBMCs were treated with anti-CD3 antibodies for 2 days, left to stand for 3 days, and then incubated with various concentrations of IL-2 fusion molecules 982C1, 982D1, 982Ref and buffer control (NC) at room temperature for approximately 40 minutes. Anti-hFc secondary antibodies were added, followed by staining with anti-CD4 and anti-CD25 antibodies. Flow cytometry analysis was performed on the stained samples to detect the effects of IL-2 fusion molecules on Treg (CD4 + CD25 + ) and T eff (CD4 + CD25 - ) cells. Subsequent potency comparisons between 982C1, 982D1, and 982D2 showed a rank order of binding potency of 982D2>982D1>982C1. The IL-2 portion of 982D2 includes two point mutations that enhance its binding to CD25. These results suggest that masking with IL-2Rβ-ECD reduces the binding of 982D1, while double masking with IL-2Rβ-ECD and IL-2Rγ-ECD results in a further reduction in the binding of the masked IL-2 fusion molecule 982C1. Despite the + CD25 - A similar rank order of binding activity was observed in T cells, but the corresponding MFIs of 982Ref, 982D1, 982D2, and 982Cl binding were similar to those in CD4 + CD25 + The IL-2 fusion molecules preferentially bind to CD4 + CD25 + T cells.

[0233] T cell proliferation assay

[0234] Human PBMCs isolated from buffy coat blood (BioIVT and RBC) were treated with anti-CD3 antibody (Bio-Biotech, catalog number 317302) for 2 days and then left to stand for 3 days. The cells were incubated with various concentrations of IL-2 fusion molecules 982C1, 982D1, 982D2 or 982Ref or IL-2 as indicated in a 37°C, 5% CO2 incubator for 3 days. The cells were then lysed / fixed / permeabilized and subsequently stained with mouse anti-human CD4-FITC (BD Biosciences, catalog number 555346), mouse anti-human CD25-PE (BD Biosciences, catalog number 555432) and mouse anti-human Ki67Alex-647 (BD Biosciences, catalog number 558615). After washing, the stained cells were respectively stained for Treg (CD4 + CD25 + ) and T eff (CD4 + CD25 - ) cells were analyzed by flow cytometry.

[0235] Fig.14 CD4 T cells induced by 982D1, 982C1, 982D2 and 982Ref IL-2 fusion molecules are shown. + CD25 + T cells and CD4+CD25 - Concentration-dependent proliferation of T cells. In this assay, PBMCs were treated with anti-CD3 antibodies for 2 days and left to stand for 3 days. PBMCs were then incubated with various concentrations of IL-2 fusion molecules 982C1, 982D1, 982D2 or 982Ref or IL-2 as indicated in a 37°C, 5% CO2 incubator for 3 days. The cells were then lysed / fixed / permeabilized and stained with anti-CD4, CD25 and Ki67 antibodies. After washing, the stained cells were stained for Treg (CD4 + CD25 + ) and Teff (CD4 + CD25 - )Ki67 on cells + (proliferation markers) cells were analyzed by flow cytometry.

[0236] The in vitro activity of IL-2 fusion molecules is in the following order from strong to weak: 982Ref, 982D2, 982D1 and 982C1. + CD25 + The overall proliferation ratio observed in T cells was + CD25 -The overall proliferation observed in T cells is much greater. In summary, the results show that 982Ref has the strongest activity in all three in vitro assays, followed by 982D2, 982D1 and 982C1. These results are consistent with the binding activity results shown in Figure 13.

[0237] Rat PK and PD studies

[0238] Male Sprague-Dawley rats with jugular vein cannulae were administered 1 mg / kg or 3 mg / kg of IL-2 fusion molecule subcutaneously. Blood was sampled at various time points from 0 to 144 hours.

[0239] For PK analysis, serum samples of test articles were measured by ELISA. In short, ELISA plates were coated with 100 μL / well F(ab')2 goat anti-human IgG Fcγ (Jackson ImmunoResearch Laboratories, catalog number 109-006-170) at 2 μg / mL in PBS. The plates were incubated overnight at 4°C. The plates were sealed with 100 μL / well PBS / 10% goat serum. After incubation for 1 hour and subsequent washing (washing four times with DI water), 100 μL of serum samples diluted in PBS / 10% goat serum or standards were added to each well. After incubation (1 hour) and washing (6 times with DI water), 100 μL of 0.5 μg / mL secondary antibody (anti-IL2-biotin (R&D Systems BAF202) in PBS / 10% goat serum was added to each well. After incubation (1 hour) and washing (6 times with DI water), 100 μL of streptavidin-HRP (Jackson ImmunoResearch Laboratories, catalog number 016-30-84, 1:1000) in PBS / 10% goat serum was added to each well. After incubation (1 hour) and washing (8 times with DI water), the color reaction was started by adding 100 μL of TMB substrate to each well. The reaction was stopped by adding 100 μL / well of 1N H2SO4 solution. The OD450 was then measured.

[0240] Fig.15Serum plasma concentrations of 982C1, 982D1 and 982Ref IL-2 fusion molecules from rat PK studies are shown over time. In this assay, 1 mg / kg of IL-2 fusion molecules 982C1, 982D1 and 982Ref were administered subcutaneously to male Sprague-Dawley rats with jugular vein cannulas. Blood was sampled at 0, 1, 3, 6, 10, 24, 48, 72, 96, 120 and 144 hours. IL-2 fusion molecules in serum samples were determined by ELISA using goat anti-human IgG Fcγ capture and anti-human IL-2 biotin as detection reagents. The AUC of 982C1 (0-t) (area under the concentration-time curve to the last measurable concentration) is greater than 982D1, while the AUC (0-t) Both are significantly greater than 982Ref.

[0241] Fig.16 The serum plasma concentration of 982D1, 982Ref and 982D2 IL-2 fusion molecules from the second rat study is shown as a function of time. In this assay, 1 mg / kg of IL-2 fusion molecules 982D1, 982D2 and 982Ref and 3 mg / kg of 982D1 were injected subcutaneously to male Sprague-Dawley rats with jugular vein cannulae, as indicated. Blood was sampled at 0 hour, 1 hour, 3 hours, 6 hours, 10 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours and 144 hours. Goat anti-human IgG Fc γ was used to capture and anti-human IL-2 biotin as a detection agent to measure the test article in the serum sample by ELISA. The AUC of 982D2 (0-t) is greater than 982D1, and the AUC of both (0-t) All were significantly greater than 982Ref. The serum plasma concentration results over time showed that the masked IL-2 fusion molecule had better PK properties than 982Ref, which had a V91K mutation in its IL-2 portion.

[0242] For PD analysis, blood was sampled at various time points between 0 and 144 h after subcutaneous injection of 982 molecules. One volume of each blood sample was mixed with a freshly prepared and pre-warmed BD Phosflow TMLysis / fixation buffer (1×, BD Biosciences, catalog number 558049) was mixed to lyse and fix blood samples collected from rats treated with 982IL-2 fusion molecules in K2 EDTA blood collection tubes. Blood samples were then washed twice with PBS containing 2% FBS according to the manufacturer's instructions and then permeabilized on ice for 30 minutes with cold permeabilization buffer II (BD Biosciences, catalog number 558052, -20°C). Cells were then washed extensively 4 times with PBS containing 2% FBS, and cell pellets were stored at 4°C or resuspended in staining buffer.

[0243] For FOXP3 and Ki67 measurements, aliquots of the above-mentioned fixed / permeabilized rat blood cells (300k-400K cells / well) from 50 μL / well of each sampling were added to a 96-well working plate. Then a mixture of 50 μL of Ab containing mouse anti-rat CD4-FITC (Baijin Biotech, catalog number 201505), mouse anti-rat CD25-PE (BD Biosciences, catalog number 554866) and mouse anti-rat FOXP3-APC (Baijin Biotech, catalog number 320014); or mouse anti-Ki67-APC (Baijin Biotech, catalog number 320514) was added to each well, and the cells in the plate were incubated at room temperature for 1 hour. The plate was washed twice with FACS buffer, and then Treg (CD4 + FOXP3 + ) and Teff (CD4 + FOXP3 - ) cells were analyzed by flow cytometry to obtain the % change over time. + CD25 + ) and T eff (CD4 + CD25 - )Ki67 in cells + (Proliferation markers) cells were analyzed by flow cytometry to obtain % change over time.

[0244] Fig.17A and 17B The results show that 982C1, 982D1 and 982Ref IL-2 fusion molecules are expressed in CD4 + / FOXP3 + and CD4 + / FOXP3 - Changes induced in cells (in rats). Fig.18A and 18BCD4 T cells induced by 982C1, 982D1 and 982Ref IL-2 fusion molecules in rats from the first study are shown. + CD25 + and CD4 + CD25 - Cell proliferation. Male Sprague-Dawley rats with jugular vein cannulae were subcutaneously administered 1 mg / kg of IL-2 fusion molecules 982C1, 982D1, and 982Ref. Blood was sampled at 0, 24, 48, 96, and 144 hours, and Ab staining was performed after blood sample lysis / fixation / permeabilization. Treg (CD4 + FOXP3 + ) and Teff (CD4 + FOXP3 - ) cells were analyzed by flow cytometry to obtain the % change over time ( Fig.17A and 17B ) or gated Treg (CD4 + CD25 + ) and T eff (CD4XD25 - ) cells were analyzed by flow cytometry to obtain the % change over time ( Fig.18A and 18B ). Fig.19A and 19B Shown is the CD4 induction by 982IL-2 fusion molecule in rats + / FOXP3 + and CD4 + / FOXP3 - The result of cellular changes. Fig. 20A and 20B CD4 in rats is shown + / CD25 + and CD4 + / CD25 - Results of the proliferation induced by the 982-IL-2 fusion molecule of cells. Male Sprague-Dawley rats with jugular vein cannula were subcutaneously administered with 1 mg / kg and 3 mg / kg of IL-2 fusion molecule 982D1, 1 mg / kg of 982D2 and 1 mg / kg of 982Ref. Blood was sampled at 0 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours and 144 hours, and Ab staining was performed after blood sample lysis / fixation / permeabilization. Treg (CD4 + FOXP3 + ) and Teff (CD4 + FOXP3- ) cells were analyzed by flow cytometry to obtain the % change over time ( Fig.19A and 19B ) or gated Treg (CD4 + CD25 + ) and T eff (CD4 + CD25 - ) cells were analyzed by flow cytometry to obtain the % change over time ( Fig. 20A and 20B ).

[0245] The results showed that 982D1 had a significantly higher effect on CD4 + FOXP3 + T cells and CD4 + CD25 + T cells have a stronger effect and longer duration of action. Surprisingly, the observation was made considering the significantly higher in vitro activity of 982Ref. Similar observations were made in a second in vivo rat study ( Fig.19A , 19B , 20A and 20B). Surprisingly, 982D1 stimulated CD4 + / FOXP3 + T cells and CD4 + / CD25 + and CD4 + / CD25 - The cell proliferation also showed that it was higher than 982D2 ( Fig.19A , 19B , 20A and 20B). This is consistent with the finding that 982D1 showed slightly higher binding activity to rat CD4 T cells than 982D2 in the binding assay (Figure 12). However, the observed difference in activity between 982D1 and 982D2 was more pronounced in vivo than in vitro (Figure 13). Fig.19A and 20A ).

[0246] Although both 982D1 and 982Ref preferentially stimulate Treg cells rather than T eff 982D1 showed selectivity in stimulating T cells, but it is clear that 982D1 has better selectivity than 982Ref, as shown by the fact that 982D1 has a higher selectivity than 982Ref in stimulating T cells. eff Almost no cell activity ( Fig.18B and 20B ).

[0247] weight

[0248] To evaluate the safety of the IL-2 fusion molecules, the body weight of the animals was also measured during the 6-day study. The animals received a single subcutaneous administration of 1 mg / kg and 3 mg / kg of the IL-2 fusion molecules 982D1, 1 mg / kg of 982D2, and 1 mg / kg of 982Ref. The body weight (BW) of each animal was measured daily between day 0 (dosing) and day 6. Fig.21 The results are shown in . The data show that rats receiving a single injection of 1 mg / kg and 3 mg / kg of 982D1 gained more weight than rats receiving 1 mg / kg of 982Ref.

[0249] In summary, the above in vitro and in vivo studies show that the masked IL-2 fusion molecule 982D1 has more surprising PK properties than 982Ref, which is a homodimeric IL-2 fusion molecule including mutations V91K and C125A in its IL-2 portion. Surprisingly, 982D1 has stronger in vivo activity than 982Ref in stimulating the proliferation of CD4+CD25+T cells and CD4+FOXP3+T cells in rats ( Fig.17A , 17B , 18A, 18B, 19A, 19B, 20A and 20B). In addition, the PK of all three masked IL-2 fusion molecules (982C1, 982D1 and 982D2) was longer than that of 982Ref ( Fig.15 and 16 ). Also surprisingly, 982D1 had greater in vivo activity in rats than 982D2. Although 982D1 had relatively modest in vitro activity compared to 982D2 and 982Ref, 982D1 also had better in vivo activity compared to the other molecules tested in the same rat study. In addition, the body weight data ( Fig.21 ) suggests that 982D1 may be safer than 982Ref. Also surprising is that the effective and selective in vivo activity of the masked IL-2 fusion molecule 982D1 can be achieved without the need for protease-dependent cleavage and removal of the masking moiety, because 982D1 does not include any cleavable peptide linker. This new mode of action is desirable because the distribution of proteases at the disease site may be uneven, and nonspecific cleavage and removal (or "leakage") of the masking moiety may occur in the circulation or outside other normal tissues and disease sites.

[0250] Without wishing to be bound by theory, differences in PK properties may partially explain the superior in vivo activity of 982D1 compared to 982Ref. Species cross-reactivity may partially explain the differences in in vivo activity observed between 982D1 and D2. Without wishing to be bound by theory, when both endogenous IL-2Rα and IL-2Rγ are present, the long linker between the masking portion and the carrier in 982D1 may also promote competition between the endogenous IL-2RβECD and the masking portion after the fusion molecule binds to CD25. The binding of the cytokine portion to both endogenous IL-2Rβ and IL-2Rγ is necessary for 982D1 to stimulate the expansion of Treg cells. The long linker between the masking portion IL-2Rβ-ECD and the carrier can provide the flexibility required for the cytokine portion to form a tetrameric complex with endogenous IL-2Rα, IL-2Rβ and IL-2Rγ. If the linker between the masking moiety IL-2Rβ-ECD and the carrier is short, and especially if the linker between the cytokine moiety and the carrier is also short, the masking moiety will likely become a particular constraint in the formation of the tetrameric complex. Fig.11 982D2 and rat CD4 + T cells were slightly weaker than D1, although 982D2 had stronger in vitro activity with human T cells than 982D1. + The differences in cell binding are relatively modest and may not explain the differences in D1 vs. D2 ( Fig.19A , 19B , 20A and 20B).

[0251] sequence

[0252] In the sequences below, boxed residues indicate mutations. Underlined residues in cleavable linkers indicate protease substrate sequences.

[0253] SEQ ID NO: 1—Human IL-2

[0254]

[0255] SEQ ID NO:2—Human IL-2 mutant protein

[0256]

[0257] Where X aa3 Selected from T and A, and X aa125 Select from A, S and G.

[0258] SEQ ID NO:3—Human IL-2 receptor β subunit extracellular domain (https: / / www.uniprot.org / uniprot / P147 84)

[0259]

[0260] SEQ ID NO:4—Human IL-2 receptor β subunit extracellular domain mutant D68E (https: / / www.uniprot.org / uniprot / P14784)

[0261]

[0262] SEQ ID NO:5—Human IL-2 receptor β subunit extracellular domain mutant E136Q / H138R (https: / / www.uniprot.org / uniprot / P147 84)

[0263]

[0264] SEQ ID NO:6—Human IL-2 receptor gamma subunit extracellular domain (http: / / www.uniprot.org / uniprot / P317 85)

[0265]

[0266] SEQ ID NO:7 IL-2Rα extracellular domain

[0267]

[0268] SEQ ID NO:8-IgG1FC (with LALA and knob)-IL-2-T3A / C125S

[0269]

[0270] SEQ ID NO:9-IgG1FC (with LALA and knob)-IL-2-T3A / C125S / N88R

[0271]

[0272] SEQ ID NO: 10—IgG1FC (with LALA and knob)-IL-2-T3A / C125S / V91K

[0273]

[0274] SEQ ID NO: 11 - IgG1FC (with LALA and knob) -IL-2-T3A / C125S / Q126N

[0275]

[0276] SEQ ID NO: 12-IgG1FC (with LALA / YTE and knob)-IL-2-T3A / C125S

[0277]

[0278] SEQ ID NO: 13—IgG1FC (with LALA / YTE and knob)-IL-2-T3A / C125S / N88R

[0279]

[0280] SEQ ID NO: 14 - IgG1FC (with LALA / YTE and knob)-IL-2-T3A / C125S / V91K

[0281]

[0282] SEQ ID NO: 15—IgG1FC (with LALA / YTE and knob)-IL-2-T3A / C125S / Q126N

[0283]

[0284] SEQ ID NO: 16—IgG1 Fc with LALA / Hodgkin / IL-2Rβ

[0285]

[0286] SEQ ID NO: 17—IgG1 Fc with LALA / hole / IL-2Rβ / cleavable linker

[0287]

[0288] SEQ ID NO: 18—IgG1 Fc with LALA / IL2Rβ / IL2Rγ

[0289]

[0290] SEQ ID NO: 19—IgG1 Fc with LALA / IL2Rγ / IL2Rβ

[0291]

[0292] SEQ ID NO:20—IgG1 Fc with LALA / hole / IL2Rβ / cleavable linker / IL2Rγ

[0293]

[0294] SEQ ID NO:21—IgG1 Fc with LALA / hole / IL2Rγ / cleavable linker / IL2Rβ

[0295]

[0296]

[0297] SEQ ID NO:22—IgG1 Fc with YTE / LALA / hole / IL-2Rβ

[0298]

[0299] SEQ ID NO:23—IgG1 Fc with YTE / LALA / hole / IL-2Rβ / cleavable linker

[0300]

[0301] SEQ ID NO:24—IgG1 Fc with YTE / LALA / tert-butyl / IL2Rβ / IL2Rγ

[0302]

[0303] SEQ ID NO:25—IgG1 Fc with YTE / LALA / tert-butyl / IL2Rγ / IL2Rβ

[0304]

[0305] SEQ ID NO:26—IgG1 Fc with YTE / LALA / hole / IL2Rβ / cleavable linker / IL2Rγ

[0306]

[0307] SEQ ID NO:27—IgG1 Fc with YTE / LALA / hole / IL2Rγ / cleavable linker / IL2Rβ

[0308]

[0309] SEQ ID NO:28-IgG1FC (with LALA and knob)-IL-2-T3A / C125S / Q126G

[0310]

[0311] SEQ ID NO:29-IgG1FC (with LALA and knob)-IL-2-T3A / C125S / Q126E

[0312]

[0313] SEQ ID NO:30—IgG1FC (with LALA and knob)-IL-2-T3A / C125S / I92T

[0314]

[0315] SEQ ID NO:31 - IgG1FC (with LALA / YTE and knob)-IL-2-T3A / C125S / Q126G

[0316]

[0317] SEQ ID NO:32—IgG1FC (with LALA / YTE and knob)-IL-2-T3A / C125S / Q126E

[0318]

[0319] SEQ ID NO:33—IgG1FC (with LALA / YTE and knob)-IL-2-T3A / C125S / I92T

[0320]

[0321] SEQ ID NO:34-IL-2-T3A / C125S / N88R

[0322]

[0323] SEQ ID NO:35-IL-2-T3A / C125S / V91K

[0324]

[0325] SEQ ID NO:36-IL-2-T3A / C125S / Q126N

[0326]

[0327] SEQ ID NO:37-IL-2-T3A / C125S / Q126G

[0328]

[0329] SEQ ID NO:38-IL-2-T3A / C125S / Q126E

[0330]

[0331] SEQ ID NO:39 - IL - 2 - T3A / C125S / I92T

[0332]

[0333] SEQ ID NO:40 - 46 Non - cleavable peptide linker

[0334]

[0335] SEQ ID NO:47 - 49 Cleavable peptide linker

[0336] GPLGVR (SEQ ID NO:47)

[0337] GPANVR (SEQ ID NO:48)_

[0338] GPASGE (SEQ ID NO:49)

[0339] SEQ ID NO:50 - 982_CX7_56_5, IgG4 Fc - IL2(C125A), pestle chain

[0340]

[0341] SEQ ID NO:51 - 982_CX7_56_5, IgG4 Fc - IL2(C125A), pestle chain

[0342]

[0343] SEQ ID NO:52 - 982CX7 72 2, Fc - IGG4 - pestle - 2xG4SAA2xG4S - IL2(C125S, V69A / Q74P)

[0344]

[0345] SEQ ID NO:53 - 982_CX7_56_6, IgG4 Fc - IL2Rβ - ECD with long linker, mortar chain

[0346] SEQ ID NO:54 - 982_CX7_56_4, mortar chain with longer peptide linker between γ and βECD

[0347]

[0348] GGGS GPASGEGGGGS (SEQ ID NO:55)

[0349] GGGGSGGGS G PASGE GGGGS (SEQ ID NO:56)

[0350] GGGGSGGGS G PASGE GGGGS GGGGS (SEQ ID NO:57)

[0351] SEQ ID NO:58 - 982-Ref with IL-2 mutant protein including mutations V91K and C125A

[0352]

[0353] SEQ ID NO:59-(G4S)2AA(G4S)2 linker

[0354] GGGGSGGGGS AAGGGGSGGGG S Sequence Listing <110> Ogilvy Pharmaceuticals Ltd. <120> IL-2 fusion protein that preferentially binds to IL-2Rα <130> 025471.WO005 <140> <141> <150> 63 / 044,294 <151> 2020-06-25 <150> 63 / 019,319 <151> 2020-05-02 <150> 63 / 015,644 <151> 2020-04-26 <150> 62 / 885,471 <151> 2019-08-12 <160> 59 <170> PatentIn version 3.5 <210> 1 <211> 133 <212> PRT <213> Homo sapiens <400> 1 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 2 <211> 133 <212> PRT <213> Homo sapiens <220> <221> Mutant <222> (3)..(3) <223> Substituted with "Ala" <220> <221> Mutant <222> (125)..(125) <223> Replace with "Ser" or "Gly" <220> <221> Location <222> (1)..(133) <223> At variable sites, there is no preference for variable residues in the sequence listing relative to the amino acids provided in the annotations <400> 2 Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 3 <211> 214 <212> PRT <213> Homo sapiens <400> 3 Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30 Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45 Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60 Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu 65 70 75 80 Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95 Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110 Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125 Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140 Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu 145 150 155 160 Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175 Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190 Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205 Ala Leu Gly Lys Asp Thr 210 <210> 4 <211> 214 <212> PRT <213> Homo sapiens <400> 4 Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30 Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45 Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60 Gly Ala Pro Glu Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu 65 70 75 80 Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95 Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110 Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125 Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg 130 135 140 Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu 145 150 155 160 Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175 Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190 Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205 Ala Leu Gly Lys Asp Thr 210 <210> 5 <211> 214 <212> PRT <213> Homo sapiens <400> 5 Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala 1 5 10 15 Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser 20 25 30 Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys 35 40 45 Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu 50 55 60 Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu 65 70 75 80 Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln 85 90 95 Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu 100 105 110 Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile 115 120 125 Ser Gln Ala Ser His Tyr Phe Gln Arg Arg Leu Glu Phe Glu Ala Arg 130 135 140 Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu 145 150 155 160 Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr 165 170 175 Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr 180 185 190 Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala 195 200 205 Ala Leu Gly Lys Asp Thr 210 <210> 6 <211> 240 <212> PRT <213> Homo sapiens <400> 6 Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala 1 5 10 15 Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr 20 25 30 Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met 35 40 45 Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr 50 55 60 Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys 65 70 75 80 Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln 85 90 95 Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp 100 105 110 Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn 115 120 125 Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser 130 135 140 Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys 145 150 155 160 Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr 165 170 175 Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp 180 185 190 Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu 195 200 205 Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp 210 215 220 Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala 225 230 235 240 <210> 7 <211> 219 <212> PRT <213> unknown <220> <221> source <223> Unknown Description: IL-2Rα extracellular domain" <400> 7 Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys 1 5 10 15 Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg 20 25 30 Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly 35 40 45 Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser 50 55 60 Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln 65 70 75 80 Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp 85 90 95 Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn 100 105 110 Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr 115 120 125 Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu 130 135 140 Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln 145 150 155 160 Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu 165 170 175 Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys 180 185 190 Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr Glu Met Ala Ala Thr 195 200 205 Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln 210 215 <210> 8 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: Synthetic peptide <400> 8 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 9 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: Synthetic peptide <400> 9 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 10 <211> 375 <212> PRT <213> artificial sequence <220> <221> Source <223> Artificial Sequence Description: Synthetic Polypeptide <400> 10 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Lys Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 11 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 11 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Asn 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 12 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 12 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 13 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 13 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 14 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 14 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Lys Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 15 <211> 375 <212> PRT <213> artificial sequence <220> <221> source <223> Artificial Sequence Description: Synthetic polypeptide <400> 15 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Asn 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 16 <211> 456 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 16 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser 245 250 255 Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp 260 265 270 Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln 275 280 285 Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu 290 295 300 Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val 305 310 315 320 Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala 325 330 335 Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile 340 345 350 Ser Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp 355 360 365 Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu 370 375 380 Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu 385 390 395 400 Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro 405 410 415 Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu 420 425 430 Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys 435 440 445 Pro Ala Ala Leu Gly Lys Asp Thr 450 455 <210> 17 <211> 462 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 17 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Pro Leu 225 230 235 240 Gly Val Arg Gly Gly Gly Gly Ser Ala Val Asn Gly Thr Ser Gln Phe 245 250 255 Thr Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp Ser Gln 260 265 270 Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp Pro Asp 275 280 285 Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser Gln Ala 290 295 300 Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu 305 310 315 320 Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu Gly Val 325 330 335 Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu 340 345 350 Arg Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu Thr His 355 360 365 Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu 370 375 380 Arg His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His Thr Trp 385 390 395 400 Glu Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys 405 410 415 Leu Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val Arg Val 420 425 430 Lys Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro 435 440 445 Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr 450 455 460 <210> 18 <211> 711 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 18 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser 245 250 255 Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp 260 265 270 Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln 275 280 285 Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu 290 295 300 Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val 305 310 315 320 Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala 325 330 335 Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile 340 345 350 Ser Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp 355 360 365 Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu 370 375 380 Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu 385 390 395 400 Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro 405 410 415 Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu 420 425 430 Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys 435 440 445 Pro Ala Ala Leu Gly Lys Asp Thr Gly Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Gly Gly Gly Ser Leu Asn Thr Thr Ile Leu Thr Pro Asn 465 470 475 480 Gly Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr 485 490 495 Asp Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe 500 505 510 Val Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu 515 520 525 Pro Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp 530 535 540 Asn Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile 545 550 555 560 Thr Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr 565 570 575 Phe Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr 580 585 590 Gln Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn 595 600 605 Leu Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn 610 615 620 Asn Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr 625 630 635 640 Asp Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys 645 650 655 Phe Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val 660 665 670 Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu 675 680 685 Trp Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro 690 695 700 Phe Leu Phe Ala Leu Glu Ala 705 710 <210> 19 <211> 711 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 19 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr 245 250 255 Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val 260 265 270 Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu 275 280 285 Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn 290 295 300 Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln 305 310 315 320 Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln 325 330 335 Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu 340 345 350 Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu 355 360 365 Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys 370 375 380 Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn 385 390 395 400 His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser 405 410 415 Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser 420 425 430 Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn 435 440 445 Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile 450 455 460 His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu 465 470 475 480 Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 485 490 495 Ser Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg 500 505 510 Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr 515 520 525 Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr 530 535 540 Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile 545 550 555 560 Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr 565 570 575 Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile 580 585 590 Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser 595 600 605 Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu 610 615 620 Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala 625 630 635 640 Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr 645 650 655 Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp 660 665 670 Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe 675 680 685 Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro 690 695 700 Ala Ala Leu Gly Lys Asp Thr 705 710 <210> 20 <211> 717 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 20 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser 245 250 255 Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp 260 265 270 Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln 275 280 285 Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu 290 295 300 Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val 305 310 315 320 Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala 325 330 335 Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile 340 345 350 Ser Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp 355 360 365 Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu 370 375 380 Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu 385 390 395 400 Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro 405 410 415 Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu 420 425 430 Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys 435 440 445 Pro Ala Ala Leu Gly Lys Asp Thr Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Gly Pro Leu Gly Val Arg Gly Gly Gly Gly Ser Leu Asn Thr 465 470 475 480 Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala Asp Phe Phe 485 490 495 Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr Leu Pro Leu 500 505 510 Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met Asn Cys Thr 515 520 525 Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr Leu His Tyr 530 535 540 Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys Ser His Tyr 545 550 555 560 Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln Lys Lys Glu 565 570 575 Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp Pro Arg Glu 580 585 590 Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn Leu Val Ile 595 600 605 Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser Glu Ser Gln 610 615 620 Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys Leu Glu His 625 630 635 640 Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr Glu Gln Ser 645 650 655 Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp Gly Gln Lys 660 665 670 Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser 675 680 685 Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp Gly Ser Asn 690 695 700 Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala 705 710 715 <210> twenty one <211> 717 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> twenty one Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr 245 250 255 Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val 260 265 270 Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu 275 280 285 Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn 290 295 300 Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln 305 310 315 320 Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln 325 330 335 Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu 340 345 350 Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu 355 360 365 Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys 370 375 380 Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn 385 390 395 400 His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser 405 410 415 Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser 420 425 430 Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn 435 440 445 Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile 450 455 460 His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu 465 470 475 480 Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Pro Leu Gly 485 490 495 Val Arg Gly Gly Gly Gly Ser Ala Val Asn Gly Thr Ser Gln Phe Thr 500 505 510 Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp 515 520 525 Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg 530 535 540 Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser 545 550 555 560 Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr 565 570 575 Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg 580 585 590 Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg 595 600 605 Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu Thr His Arg 610 615 620 Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg 625 630 635 640 His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu 645 650 655 Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu 660 665 670 Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys 675 680 685 Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu 690 695 700 Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr 705 710 715 <210> twenty two <211> 456 <212> PRT <213> Artificial sequence <220> <221> source <223> Human process description: Synthetic peptide <400> twenty two Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser 245 250 255 Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp 260 265 270 Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln 275 280 285 Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu 290 295 300 Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val 305 310 315 320 Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala 325 330 335 Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile 340 345 350 Ser Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp 355 360 365 Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu 370 375 380 Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu 385 390 395 400 Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro 405 410 415 Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu 420 425 430 Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys 435 440 445 Pro Ala Ala Leu Gly Lys Asp Thr 450 455 <210> twenty three <211> 462 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> twenty three Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Pro Leu 225 230 235 240 Gly Val Arg Gly Gly Gly Gly Ser Ala Val Asn Gly Thr Ser Gln Phe 245 250 255 Thr Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp Ser Gln 260 265 270 Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp Pro Asp 275 280 285 Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser Gln Ala 290 295 300 Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu 305 310 315 320 Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu Gly Val 325 330 335 Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu 340 345 350 Arg Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu Thr His 355 360 365 Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu 370 375 380 Arg His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His Thr Trp 385 390 395 400 Glu Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys 405 410 415 Leu Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val Arg Val 420 425 430 Lys Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro 435 440 445 Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr 450 455 460 <210> twenty four <211> 711 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> twenty four Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser 245 250 255 Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp 260 265 270 Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln 275 280 285 Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu 290 295 300 Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val 305 310 315 320 Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala 325 330 335 Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile 340 345 350 Ser Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp 355 360 365 Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu 370 375 380 Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu 385 390 395 400 Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro 405 410 415 Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu 420 425 430 Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys 435 440 445 Pro Ala Ala Leu Gly Lys Asp Thr Gly Gly Gly Gly Gly Ser Gly Gly 450 455 460 Gly Gly Ser Gly Gly Gly Ser Leu Asn Thr Thr Ile Leu Thr Pro Asn 465 470 475 480 Gly Asn Glu Asp Thr Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr 485 490 495 Asp Ser Leu Ser Val Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe 500 505 510 Val Phe Asn Val Glu Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu 515 520 525 Pro Gln Pro Thr Asn Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp 530 535 540 Asn Asp Lys Val Gln Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile 545 550 555 560 Thr Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr 565 570 575 Phe Val Val Gln Leu Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr 580 585 590 Gln Met Leu Lys Leu Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn 595 600 605 Leu Thr Leu His Lys Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn 610 615 620 Asn Arg Phe Leu Asn His Cys Leu Glu His Leu Val Gln Tyr Arg Thr 625 630 635 640 Asp Trp Asp His Ser Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys 645 650 655 Phe Ser Leu Pro Ser Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val 660 665 670 Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu 675 680 685 Trp Ser His Pro Ile His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro 690 695 700 Phe Leu Phe Ala Leu Glu Ala 705 710 <210> 25 <211> 711 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 25 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr 245 250 255 Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val 260 265 270 Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu 275 280 285 Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn 290 295 300 Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln 305 310 315 320 Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln 325 330 335 Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu 340 345 350 Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu 355 360 365 Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys 370 375 380 Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn 385 390 395 400 His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser 405 410 415 Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser 420 425 430 Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn 435 440 445 Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile 450 455 460 His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu 465 470 475 480 Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 485 490 495 Ser Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg 500 505 510 Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr 515 520 525 Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr 530 535 540 Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile 545 550 555 560 Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr 565 570 575 Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile 580 585 590 Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser 595 600 605 Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu 610 615 620 Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala 625 630 635 640 Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr 645 650 655 Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp 660 665 670 Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe 675 680 685 Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro 690 695 700 Ala Ala Leu Gly Lys Asp Thr 705 710 <210> 26 <211> 717 <212> PRT <213> Artificial sequence <220> <221> source <223> Human process description: Synthetic peptide <400> 26 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser 245 250 255 Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp 260 265 270 Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln 275 280 285 Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu 290 295 300 Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val 305 310 315 320 Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala 325 330 335 Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile 340 345 350 Ser Leu Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp 355 360 365 Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu 370 375 380 Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu 385 390 395 400 Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro 405 410 415 Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu 420 425 430 Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys 435 440 445 Pro Ala Ala Leu Gly Lys Asp Thr Gly Gly Gly Gly Ser Gly Gly Gly 450 455 460 Gly Ser Gly Pro Leu Gly Val Arg Gly Gly Gly Gly Ser Leu Asn Thr 465 470 475 480 Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala Asp Phe Phe 485 490 495 Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr Leu Pro Leu 500 505 510 Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met Asn Cys Thr 515 520 525 Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr Leu His Tyr 530 535 540 Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys Ser His Tyr 545 550 555 560 Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln Lys Lys Glu 565 570 575 Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp Pro Arg Glu 580 585 590 Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn Leu Val Ile 595 600 605 Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser Glu Ser Gln 610 615 620 Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys Leu Glu His 625 630 635 640 Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr Glu Gln Ser 645 650 655 Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp Gly Gln Lys 660 665 670 Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser 675 680 685 Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp Gly Ser Asn 690 695 700 Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu Glu Ala 705 710 715 <210> 27 <211> 717 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 27 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Thr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr 245 250 255 Thr Ala Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val 260 265 270 Ser Thr Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu 275 280 285 Tyr Met Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn 290 295 300 Leu Thr Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln 305 310 315 320 Lys Cys Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln 325 330 335 Leu Gln Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu 340 345 350 Gln Asp Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu 355 360 365 Gln Asn Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys 370 375 380 Leu Ser Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn 385 390 395 400 His Cys Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser 405 410 415 Trp Thr Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser 420 425 430 Val Asp Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn 435 440 445 Pro Leu Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile 450 455 460 His Trp Gly Ser Asn Thr Ser Lys Glu Asn Pro Phe Leu Phe Ala Leu 465 470 475 480 Glu Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Pro Leu Gly 485 490 495 Val Arg Gly Gly Gly Gly Ser Ala Val Asn Gly Thr Ser Gln Phe Thr 500 505 510 Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp Ser Gln Asp 515 520 525 Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp Pro Asp Arg 530 535 540 Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser Gln Ala Ser 545 550 555 560 Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu Thr 565 570 575 Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu Gly Val Arg 580 585 590 Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu Arg 595 600 605 Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu Thr His Arg 610 615 620 Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu Arg 625 630 635 640 His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His Thr Trp Glu 645 650 655 Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys Leu 660 665 670 Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val Arg Val Lys 675 680 685 Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro Leu 690 695 700 Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr 705 710 715 <210> 28 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 28 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gly 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 29 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 29 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Glu 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 30 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 30 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Thr Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 31 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 31 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gly 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 32 <211> 375 <212> PRT <213> artificial sequence <220> <221> source <223> Artificial Sequence Description: Synthetic polypeptide <400> 32 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Glu 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 33 <211> 375 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 33 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30 Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 225 230 235 240 Gly Ser Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu 245 250 255 Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn 260 265 270 Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met 275 280 285 Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu 290 295 300 Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe 305 310 315 320 His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Thr Val Leu 325 330 335 Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu 340 345 350 Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln 355 360 365 Ser Ile Ile Ser Thr Leu Thr 370 375 <210> 34 <211> 133 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 34 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Arg Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 35 <211> 133 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 35 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Lys Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 36 <211> 133 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 36 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Asn Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 37 <211> 133 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 37 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gly Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 38 <211> 133 <212> PRT <213> artificial sequence <220> <221> source <223> Artificial sequence description: Synthesized multiple peptides <400> 38 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Glu Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 39 <211> 133 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 39 Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His 1 5 10 15 Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys 20 25 30 Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys 35 40 45 Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys 50 55 60 Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu 65 70 75 80 Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Thr Val Leu Glu Leu 85 90 95 Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala 100 105 110 Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile 115 120 125 Ile Ser Thr Leu Thr 130 <210> 40 <211> 5 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 40 Gly Gly Gly Gly Ser 1 5 <210> 41 <211> 10 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 41 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 42 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 42 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 43 <211> 20 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <220> <221> Mutants <222> (10)..(10) <223> Replace with "Asn" <220> <221> Location <222> (1)..(20) <223> At variable sites, there is no preference for variable residues in the sequence listing relative to the amino acids provided in the annotations <400> 43 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 44 <211> 20 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <220> <221> Mutants <222> (10)..(10) <223> Replace with "Ala" or "Asn" <220> <221> Mutants <222> (15) <223> Replace with "Asn" <220> <221> Location <222> (1)..(20) <223> At variable sites, there is no preference for variable residues in the sequence listing relative to the amino acids provided in the annotations <400> 44 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ala Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 45 <211> twenty two <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequences: synthetic peptides <400> 45 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Gly Gly Gly Gly 1 5 10 15 Ser Gly Gly Gly Gly Ser 20 <210> 46 <211> 44 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 46 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala 1 5 10 15 Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 30 Ser Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 35 40 <210> 47 <211> 6 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 47 Gly Pro Leu Gly Val Arg 1 5 <210> 48 <211> 6 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 48 Gly Pro Ala Asn Val Arg 1 5 <210> 49 <211> 6 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 49 Gly Pro Ala Ser Gly Glu 1 5 <210> 50 <211> 395 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 50 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 20 25 30 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 65 70 75 80 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 85 90 95 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 115 120 125 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140 Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys 145 150 155 160 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 210 215 220 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 225 230 235 240 Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr 260 265 270 Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn 275 280 285 Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe 290 295 300 Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys 305 310 315 320 Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala Gln 325 330 335 Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn 340 345 350 Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu 355 360 365 Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile 370 375 380 Thr Phe Ala Gln Ser Ile Ile Ser Thr Leu Thr 385 390 395 <210> 51 <211> 402 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 51 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 20 25 30 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 65 70 75 80 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 85 90 95 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 115 120 125 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140 Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys 145 150 155 160 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 210 215 220 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 225 230 235 240 Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr 260 265 270 Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu 275 280 285 Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys 290 295 300 Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr 305 310 315 320 Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu 325 330 335 Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg 340 345 350 Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser 355 360 365 Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val 370 375 380 Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile Ile Ser Thr 385 390 395 400 Leu Thr <210> 52 <211> 402 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 52 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 20 25 30 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 65 70 75 80 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 85 90 95 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 115 120 125 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140 Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys 145 150 155 160 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 210 215 220 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 225 230 235 240 Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr 260 265 270 Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu 275 280 285 Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys 290 295 300 Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr 305 310 315 320 Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu 325 330 335 Glu Ala Leu Asn Leu Ala Pro Ser Lys Asn Phe His Leu Arg Pro Arg 340 345 350 Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu Lys Gly Ser 355 360 365 Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val 370 375 380 Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr 385 390 395 400 Leu Thr <210> 53 <211> 483 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 53 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 20 25 30 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 65 70 75 80 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 85 90 95 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 115 120 125 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 145 150 155 160 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 195 200 205 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 210 215 220 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 225 230 235 240 Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Val Asn 260 265 270 Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser 275 280 285 Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln Val 290 295 300 His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu 305 310 315 320 Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro 325 330 335 Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu 340 345 350 Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys 355 360 365 Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu Gln Val Val 370 375 380 His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala 385 390 395 400 Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg Thr Leu Ser 405 410 415 Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys 420 425 430 Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu 435 440 445 Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser 450 455 460 Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly 465 470 475 480 Lys Asp Thr <210> 54 <211> 718 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 54 Met Gly Val Lys Val Leu Phe Ala Leu Ile Cys Ile Ala Val Ala Glu 1 5 10 15 Ala Ala Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 20 25 30 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 65 70 75 80 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 85 90 95 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 115 120 125 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 145 150 155 160 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 195 200 205 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 210 215 220 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 225 230 235 240 Leu Ser Leu Ser Leu Gly Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly 245 250 255 Ser Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Leu Pro 260 265 270 Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met Asn Cys Thr Trp 275 280 285 Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr Leu His Tyr Trp 290 295 300 Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys Ser His Tyr Leu 305 310 315 320 Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln Lys Lys Glu Ile 325 330 335 His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp Pro Arg Glu Pro 340 345 350 Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn Leu Val Ile Pro 355 360 365 Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser Glu Ser Gln Leu 370 375 380 Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys Leu Glu His Leu 385 390 395 400 Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr Glu Gln Ser Val 405 410 415 Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp Gly Gln Lys Arg 420 425 430 Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu Cys Gly Ser Ala 435 440 445 Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Ala Gly Gly Gly 465 470 475 480 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Arg Gly Gly 485 490 495 Gly Gly Ser Gly Gly Gly Gly Ser Ala Val Asn Gly Thr Ser Gln Phe 500 505 510 Thr Cys Phe Tyr Asn Ser Arg Ala Asn Ile Ser Cys Val Trp Ser Gln 515 520 525 Asp Gly Ala Leu Gln Asp Thr Ser Cys Gln Val His Ala Trp Pro Asp 530 535 540 Arg Arg Arg Trp Asn Gln Thr Cys Glu Leu Leu Pro Val Ser Gln Ala 545 550 555 560 Ser Trp Ala Cys Asn Leu Ile Leu Gly Ala Pro Asp Ser Gln Lys Leu 565 570 575 Thr Thr Val Asp Ile Val Thr Leu Arg Val Leu Cys Arg Glu Gly Val 580 585 590 Arg Trp Arg Val Met Ala Ile Gln Asp Phe Lys Pro Phe Glu Asn Leu 595 600 605 Arg Leu Met Ala Pro Ile Ser Leu Gln Val Val His Val Glu Thr His 610 615 620 Arg Cys Asn Ile Ser Trp Glu Ile Ser Gln Ala Ser His Tyr Phe Glu 625 630 635 640 Arg His Leu Glu Phe Glu Ala Arg Thr Leu Ser Pro Gly His Thr Trp 645 650 655 Glu Glu Ala Pro Leu Leu Thr Leu Lys Gln Lys Gln Glu Trp Ile Cys 660 665 670 Leu Glu Thr Leu Thr Pro Asp Thr Gln Tyr Glu Phe Gln Val Arg Val 675 680 685 Lys Pro Leu Gln Gly Glu Phe Thr Thr Trp Ser Pro Trp Ser Gln Pro 690 695 700 Leu Ala Phe Arg Thr Lys Pro Ala Ala Leu Gly Lys Asp Thr 705 710 715 <210> 55 <211> 15 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 55 Gly Gly Gly Ser Gly Pro Ala Ser Gly Glu Gly Gly Gly Gly Ser 1 5 10 15 <210> 56 <211> 20 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 56 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Pro Ala Ser Gly Glu Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 57 <211> 25 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 57 Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Pro Ala Ser Gly Glu Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser 20 25 <210> 58 <211> 364 <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 58 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Gly Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys 225 230 235 240 Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu 245 250 255 Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr 260 265 270 Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln 275 280 285 Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu Ala 290 295 300 Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile 305 310 315 320 Asn Lys Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys 325 330 335 Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp 340 345 350 Ile Thr Phe Ala Gln Ser Ile Ile Ser Thr Leu Thr 355 360 <210> 59 <211> twenty one <212> PRT <213> Artificial sequence <220> <221> source <223> Artificial sequence description: synthetic peptide <400> 59 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Gly Gly Gly Gly Ser 1 5 10 15 Gly Gly Gly Gly Ser 20

Claims

1. An IL-2 fusion molecule, comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain is as shown in SEQ ID NO:50, and the second polypeptide chain is as shown in SEQ ID NO:

53.

2. A polynucleotide encoding the IL-2 fusion molecule according to claim 1. An expression vector comprising the polynucleotide according to claim 2. A host cell comprising the expression vector according to claim 3. 5 . A pharmaceutical composition comprising the IL-2 fusion molecule according to claim 1 and a pharmaceutically acceptable excipient.

Images