RU2836849C2 - Il-10 variant molecules and method of treating malignant tumour - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to biotechnology, in particular to interleukin 10 (IL-10) fusion proteins and a method of treating a malignant tumour. Disclosed is a fused protein of formula (I-VII): IL-10-L¹-X¹-L¹-X²-L¹-IL-10 (Formula I); (Z)_n-X¹-L²-Y²-L¹-IL-10 (Formula II); IL-10-L¹-Y¹-L²-X²-(Z)_n (Formula III); X1-L²-X²-L¹-IL-10 (Formula IV); IL-10-L¹-X¹-L²-X² (Formula V); X¹-L¹-IL-10 (Formula VI); and IL-10-L¹-X² (Formula VII). "IL-10" is a monomer sequence selected from SEQ ID NO: 1, 3, 14, 15, 16, 18, 19, 55, 57 or 59. "L¹" and "L²" are linkers. "X¹" and "X²" are the VH and VL regions obtained from the first antibody. "Y¹" and "Y²" are the VH and VL regions obtained from the second antibody. "Z" is a cytokine.

EFFECT: inventions provide creation of IL-10 receptor agonists with altered binding affinity to IL-10 receptor and/or altered interdomain angles in IL-10, which can be used for treatment of malignant tumour.

17 cl, 21 dwg, 7 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Patent Application No. 62/814,669, filed March 6, 2019, U.S. Provisional Patent Application No. 62/899,504, filed September 12, 2019, and U.S. Provisional Patent Application No. 62/962,332, filed January 17, 2020, the contents of which are incorporated herein by reference in their entireties.

INTRODUCTION

The invention relates to variant forms of Interleukin 10 (IL-10) that include modifications of the IL-10 receptor binding region and/or domains responsible for interdomain angles that exist in the IL-10 molecule. By altering one or both of these domains in IL-10, the inventors have unexpectedly discovered that the resulting biological function of the IL-10 receptor can be tuned or modulated to elicit a specific biological response. The invention also relates to IL-10 or a variant IL-10 molecule with an increased half-life that include non-protein moieties to increase serum half-life, as well as protein-based methods for increasing half-life. The invention also relates to fusion proteins comprising variant IL-10 molecules.

PRIOR ART

IL-10 has been described as a cytokine synthesis inhibitory factor due to its ability to inhibit both (i) the secretion of proinflammatory cytokines by monocytes/macrophages in response to lipopolysaccharide and (ii) the secretion of interleukin 2 (IL-2) and the proliferation of CD4 ⁺ T cells. When viral analogs of IL-10 were discovered and reported to have similar or identical functions to human IL-10, it was postulated that these viral IL-10 analogs enhance viral virulence by taking over the function of a suppressive cytokine found in the human genome.

Further investigation of the suppressive effects of IL-10 was made possible by the generation of IL-10 knockout mice that develop chronic enterocolitis. Data obtained with these mice clearly demonstrated that IL-10 knockout mice develop severe inflammation throughout the gastrointestinal tract, predominantly due to chronic secretion of inflammatory cytokines by monocytes/macrophages and CD4 ⁺ T cells, consistent with initial in vitro observations.

Taken together, these data suggest that IL-10 plays a dominant role in suppressing inflammation. Specifically, patients who lack functional IL-10 receptors or the ability to produce IL-10 are at increased risk for developing inflammation-related gastrointestinal diseases.

Numerous clinical trials have been conducted to evaluate the anti-inflammatory function of IL-10 in the context of psoriasis, rheumatoid arthritis, and Crohn's disease. In general, treatment with recombinant human IL-10 (rHuIL-10) has been shown to be safe but ineffective. In particular, treatment of patients with Crohn's disease with rHuIL-10 resulted in an inverse dose-response relationship, whereby low doses appeared to moderately suppress inflammation and the suppressive effect was lost at higher doses. A rigorous analysis of the latest study for Crohn's disease found that patients treated with 10 and 20 μg/kg rHuIL-10 demonstrated increased concentrations of interferon gamma (IFNγ) and neopterin. IFNγ is known to worsen the pathogenesis of inflammatory bowel disease and Crohn's disease. The data suggest that at high doses, IL-10 treatment induces IFNγ, which in turn worsens inflammatory disease.

Further analysis of the effects of IL-10 in healthy individuals suggests that administration of IL-10 prior to exposure to the pro-inflammatory factor lipopolysaccharide (LPS) inhibits the production of pro-inflammatory cytokines. However, administration of rHuIL-10 after LPS exposure enhances the secretion of pro-inflammatory cytokines. Since LPS is a product of both normal and foreign gut bacteria in patients with inflammatory bowel disease (IBD), these patients will never be “free” of LPS and thus will never be in a state where IL-10 treatment could be administered prior to LPS. Thus, these data suggest that patients with Crohn’s disease and other inflammatory diseases will never see the therapeutic benefits of IL-10 treatment.

Adding to the confusion about the role of IL-10 are accumulating data indicating that IL-10 activates the immune system to elicit antitumor responses. Initial data suggested that IL-10 activated NK cells, but further studies showed that IL-10 treatment inhibited tumor growth in a CD8+ T cell- and IFNγ-dependent manner. Further studies showed that IL-10 treatment inhibited the proliferation of regulatory FoxP3 ⁺ CD4 ⁺ T cells and enhanced scavenging by Kupffer cells, all of which suggest that IL-10 is a potent immunostimulant rather than a suppressor. Finally, these stimulatory actions were confirmed in clinical trials of cancer patients treated with pegylated IL-10.

Taken together, these data show that IL-10 treatment in patients suffering from autoimmune inflammation did not provide a therapeutic effect, suggesting that IL-10 is not a general immune suppressor. Consistent with this, treatment of patients with malignancies with (PEG) IL-10 results in potent and therapeutically beneficial immune activation, in particular induction of dose-dependent serum IFNγ, as seen in patients with Crohn's disease treated with IL-10, indicating that IL-10 is a potent immunostimulant.

It remains unclear why viruses acquire the IL-10 sequence. Further analysis of Epstein-Barr virus (EBV-IL10) and cytomegalovirus (CMV-IL10) homologs revealed that these viruses have altered the native IL-10 sequence in two predominant ways. EBV-IL10 appears to retain a similar tertiary angle of homodimer interaction, resulting in a distinct ligand-receptor interaction angle, although it essentially decreases its affinity for the IL-10 receptor. CMV-IL10 exhibits increased affinity for the IL-10 receptor, essentially altering the angle of interaction with IL-10 receptors. Although the sequences of EBV-IL10 and CMV-IL10, respectively, retain approximately 80% and 27% homology with native IL-10, each viral homolog has very different affinities for the IL-10 receptor, different angles of interaction with the receptor, and each viral homolog appears to exhibit anti-inflammatory functions very similar to native IL-10. Thus, it is unclear how IL-10 receptor affinity and/or angle of interaction with the receptor influence subsequent downstream IL-10 signaling.

NATURE OF VARIOUS PREFERRED IMPLEMENTATION OPTIONS

The present application relates generally to novel variant IL-10 molecules that modulate IL-10 receptor signaling. Thus, the application relates to variant IL-10 molecules that include modifications in the structure of the IL-10 molecule, as a result of which the novel variant IL-10 molecules have altered binding affinity for the IL-10 receptor and/or altered interdomain angles in IL-10. The inventor has unexpectedly discovered that modification of IL-10 in key domains affecting IL-10 receptor affinity and/or interdomain angles of IL-10 leads to the creation of IL-10 receptor agonists that can be used to treat immune diseases, inflammatory diseases or conditions, as well as in the treatment of cancer. In addition, variant IL-10 molecules may also have an increased serum half-life by incorporating variant IL-10 molecules as part of a fusion protein, such as different antibody domains (Fc or variable domains), without preventing IL-10 monomers or IL-10 variants from forming an IL-10 homodimer.

In certain embodiments, the variant IL-10 molecule is a modified human IL-10. In other embodiments, the variant IL-10 molecule is a modified murine IL-10. In preferred embodiments, the variant IL-10 molecule is a modified viral IL-10 homolog. In a more preferred embodiment, the viral IL-10 homolog is CMV-IL10. In a most preferred embodiment, the viral IL-10 homolog is EBV-IL10.

In further embodiments, the variant IL-10 molecule comprises at least one or more amino acid insertions, deletions or substitutions in the receptor binding region. In yet other embodiments, the variant IL-10 molecule comprises at least one or more amino acid insertions, deletions or substitutions in the region responsible for forming the interdomain angle of IL-10. In a preferred embodiment, the variant IL-10 molecule comprises one or both modifications of the receptor binding domain and/or the region responsible for the interdomain angle. In a most preferred embodiment, the variant IL-10 molecule comprises one or both modifications in the EBV-IL10 protein molecule.

In various embodiments, the variant IL-10 molecule has an increased affinity for the IL-10 receptor or IL-10 receptor complex compared to wild-type IL-10. In other embodiments, the variant IL-10 molecule has a decreased affinity for the IL-10 receptor or IL-10 receptor complex compared to wild-type IL-10. In other embodiments, the variant IL-10 molecule forms a narrower or restricted interdomain angle compared to wild-type IL-10, more preferably EBV IL10. In another embodiment, the variant IL-10 molecule forms a wider or weakened interdomain angle compared to wild-type IL-10, more preferably EBV IL-10.

In additional embodiments, the variant EBV IL-10 molecules comprise at least one or more amino acid insertions, deletions, or substitutions in the receptor binding region located in helix A, loop AB, and/or helix F ("site 1") of EBV IL-10. Modifications in the receptor binding domain region may, in certain embodiments, increase or enhance affinity for the IL-10 receptor or receptor complex. In some embodiments, modifications in the receptor binding region may decrease or reduce affinity for the IL-10 receptor or receptor complex.

In additional embodiments, the variant EBV IL-10 molecules include at least one or more amino acid insertions, deletions, or substitutions in regions of EBV IL-10 responsible for interdomain angle formation. In preferred embodiments, modifications may occur in the DE loop of EBV IL-10. Modifications in the DE loop result in variant EBV IL-10 molecules that have a limited interdomain angle or a weakened interdomain angle.

In other aspects, the variant IL-10 molecules are chimeric or fusion molecules. In one embodiment, the chimeric or fusion protein will contain one or more domains from different proteins or mutations within one protein that impart characteristics of another protein. In a preferred embodiment, the chimeric or fusion protein will comprise a first portion comprising an IL-10 molecule as provided herein fused to another molecule, including, but not limited to, albumin, enzymes, glycosyltransferases, galactosyltransferases, IgG hinge regions (e.g., the Fc region), one or more variable domains (such as, but not limited to, a heavy chain variable region or a light chain variable region) of one or more antibodies, cytokines (such as IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, GM-CSF, G-CSF, interferons-α, -β, -γ, TGF-β, and tumor necrosis factor-α, -β), labels, agents, chemotherapeutic agents, radioactive isotopes, half-life extending agents (such as hydroxyethyl starch (HEKylation), polysialic acid, heparosan polymers, elastin-like polypeptides, and hyaluronic acid). In another embodiment, a variant IL-10 molecule is fused to one or more variable regions of a heavy or light chain of an antibody. Fusion proteins may include one or more linkers that are covalently linked to different portions of the fusion protein. A fusion protein may form a non-covalently associated complex with another fusion protein of the same type. Such a fusion protein will allow IL-10 monomers or variant IL-10 molecule monomers to associate together into a functional IL-10 homodimer or variant IL-10.

In other embodiments, the variant IL-10 molecule is part of an engineered fusion protein. The fusion protein will comprise at least one IL-10 monomer or variant IL-10 molecule conjugated at a first end of the fusion protein to a linker or spacer, wherein the one or more spacers are used to link different portions of the fusion protein, which are then conjugated to at least one other molecule conjugated at the other end, wherein the molecule is selected from at least one cytokine or monomer thereof, a therapeutic agent, a label, a molecule that increases serum half-life, or a protein (such as, but not limited to, a receptor, a ligand, or different portions of an antibody). In a preferred embodiment, the fusion protein comprises an IL-10 monomer or a monomer of a variant IL-10 molecule conjugated via linkers or spacers to at least one variable region of a heavy and/or light chain. In a most preferred embodiment, the fusion protein comprises an EBV IL-10 monomer or a monomer of a variant EBV IL-10 molecule conjugated via linkers or spacers to at least one region of a heavy and/or light chain. In a most preferred embodiment, the EBV IL-10 monomer or the monomer of a variant EBV IL-10 molecule is conjugated to one variable region of a heavy chain and one variable region of a light chain, wherein the monomers together form a dimeric complex. An EBV IL-10 monomer or a monomer of a variant EBV IL-10 molecule may be conjugated to either the amino terminus or the carboxy terminus of the variable region of the heavy or light chain.

In some embodiments, a therapeutic amount of a variant IL-10 molecule, a fusion protein thereof, or a chimeric protein thereof is administered to a subject suffering from an inflammatory disease or condition, such as, but not limited to, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, nonalcoholic steatohepatitis (NASH), and nonalcoholic fatty liver disease (NAFLD). In another embodiment, a therapeutic amount of a therapeutic amount of a variant IL-10 molecule, a fusion protein thereof, or a chimeric protein thereof is administered to a subject suffering from a cancer. Treatment of subjects having more than one pathological condition is also contemplated. In a more preferred embodiment, the variant IL-10 molecule is an EBV-IL10 variant, a fusion protein thereof, or a chimeric protein thereof. In another embodiment, the variant IL-10 molecule, fusion protein or chimeric protein thereof is used in a combination therapy. For example, the variant IL-10 molecule, fusion protein or chimeric protein thereof can be administered individually in combination with other therapies. In yet other embodiments, a therapeutic amount of the variant IL-10 molecule, fusion protein or chimeric protein thereof is administered to an individual suffering from lipid-related diseases, such as, but not limited to, diseases with elevated cholesterol levels.

In various embodiments, the variant IL-10 molecule or fusion protein thereof of the present application is delivered as an isolated and purified protein. Delivery may occur as a subcutaneous bolus injection or as multiple microinjections into the dermis and subcutaneous tissue. In another embodiment, the variant IL-10 molecule may be delivered as a nucleic acid vector comprising a sequence encoding the IL-10 variant, fusion protein, or chimeric protein. The nucleic acid vector may be a plasmid or a viral particle carrying a viral vector. In one embodiment, the variant IL-10 molecules may be delivered to an individual by genetic medicine methods generally known to those skilled in the art.

In other embodiments, the variant IL-10 molecule may also be administered as part of a combination treatment regimen. In one embodiment, the variant IL-10 molecule may be administered in combination with Bispecific T-Cell Engagers (BITES), a currently available immunotherapy for the treatment of cancer, IBD, Crohn's disease, NAFLD, NASH, and autoimmune diseases, for example.

In another embodiment, the nucleic acid vector is an adeno-associated virus (AAV) vector comprising one or more AAV inverted terminal repeat (ITR) sequence elements and control elements for directing expression of a sequence encoding a variant IL-10 molecule in a target cell, into which the AAV vector can be introduced either as a plasmid (naked DNA) or packaged into an AAV particle. In another embodiment, the nucleic acid vector is a vaccinia virus vector. A number of vaccinia virus vectors derived from different strains can be used to introduce the variant IL-10 molecules of the present application, such as the WR strain (ATCC VR-119), the Wyeth strain (ATCC VR-325), the Lederle-Chorioallantoic strain (ATCC VR-325), the CL strain (ATCC VR-117), and others; All these strains are available from the American Type Culture Collection (Manassas, VA).

In another embodiment, any of the IL-10 variant molecules described herein, including but not limited to pegylated IL-10 variant molecules, can be delivered to an individual by any method described herein or by a method generally known in the art.

These and other embodiments of this application will be readily apparent to those skilled in the art in view of the description herein.

BRIEF DESCRIPTION OF DRAWINGS

Figure 1 shows a ribbon diagram (Josephson et al, Immunity, 15, p. 35-46) of the monomeric IL-10 molecule. The parts highlighted in red represent receptor contact interface site I, and those in green represent receptor contact interface site II.

Figures 2A-2C compare the ability of IL-10 and EBV IL-10 to activate monocytes/macrophages (Mθ) by assaying IL-1β (Figure 2A) and TNFα (Figure 2B) cytokine production and to stimulate T cells by assaying IFNγ production (Figure 2C) in Donor 1.

Figures 3A-3C compare the ability of IL-10 and EBV IL-10 to activate monocytes/macrophages (Mθ) by assaying IL-1β (Figure 3A) and TNFα (Figure 3B) cytokine production and T cell stimulation by assaying IFNγ production (Figure 3C) in Donor 1.

Figures 4A-B show the amount of IFNγ induced by T cells after stimulation with IL-10 or EBV IL-10.

Figures 5A-5C show the effect of N-terminal 5 kDa mono- and di-PEGylated EBV-IL-10 on MC/9 cell proliferation (Figure 5A), TNFα secretion by monocytes/macrophages (Mθ) in response to LPS (Figure 5B), and IFNγ secretion by T cells in response to T cell receptor stimulation (Figure 5C).

Figures 6A-6E show specific amino acid sequences for various variant IL-10 molecules and fusion proteins containing EBV-IL-10.

Figure 7 is a schematic representation of a fusion protein comprising IL-10 or a variant IL-10 molecule conjugated to a linker or spacer (in this case, an scFv). This typical example shows IL-10 or variant IL-10 molecules conjugated at both the N-terminus and the C-terminus.

Figures 8A-8C are schematic representations of various IL-10 variants, such as variants made in EBV IL-10. Figure 8A (DV05) is an IL-10 variant containing a single point mutation at amino acid position 31 (V31L - V31L) of SEQ ID NO: 3. Figure 8B (DV06) is an IL-10 variant containing a single point mutation at amino acid position 75 (A75I A75I) of SEQ ID NO: 3. Figure 8C (DV07) is an IL-10 variant containing two point mutations at amino acid positions 31 and 75 (V31L and A75I) of SEQ ID NO: 3.

Figure 8D shows assays of the monocyte/macrophage response to various forms of IL-10, including wild-type human IL-10, EBV-IL-10, DV05, DV06, and DV07. All forms, including the IL-10 variants DV05, DV06, and DV07, suppress TNFα secretion in response to LPS.

Figure 8E shows T cell response assays to different forms of IL-10 - human wild-type IL-10, EBV IL-10, DV05, DV06, and DV07 - and not all forms induce IFN-gamma.

Figures 9A-9F are schematic representations of various configurations of IL-10 fusion protein/immunoconjugate/diabody constructs. Figures 9(a)-(c) represent a fusion protein complex (i.e., a diabody), wherein each fusion protein comprises VH and VL regions derived from two different antibodies linked to an IL-10 monomer (which may also be replaced by a variant IL-10 molecule) via a carboxyl-terminal linker or an amino-terminal linker, with (a) a single mutation, such as at amino acid position 31, affecting binding to the IL-10 receptor; (b) a single mutation, such as at amino acid position 75, affecting binding to the IL-10 receptor; and (c) with two mutations, for example, at amino acid positions 31 and 75, affecting binding to the IL-10 receptor. Figures 9(d)-(f) represent a fusion protein complex (i.e., a miniantibody), wherein each fusion protein comprises one VH or VL region derived from a single antibody linked to an IL-10 monomer (which may also be replaced by a variant IL-10 molecule) via a carboxy-terminal linker or an amino-terminal linker, with (d) a single mutation, for example, at amino acid position 31, affecting binding to the IL-10 receptor; (e) a single mutation, for example, at amino acid position 75, affecting binding to the IL-10 receptor; and (f) with two mutations, for example at amino acid positions 31 and 75, affecting binding to the IL-10 receptor.

Figures 10(A)-10(F) are schematic representations of various configurations of IL-10 fusion protein/immunoconjugate/diabody constructs. Figures 10(a)-(c) represent a single fusion protein (i.e., a minibody) wherein each IL-10 monomer (which may also be replaced by a variant IL-10 molecule) is linked via a carboxyl-terminal linker or an amino-terminal linker to either a VH or VL from the same antibody, and the VH and VL are linked together. The IL-10 monomers comprise (a) a single mutation, such as at amino acid position 31, affecting binding to the IL-10 receptor; (b) a single mutation, such as at amino acid position 75, affecting binding to the IL-10 receptor; and (c) two mutations, for example, at amino acid positions 31 and 75, affecting binding to the IL-10 receptor. Figures 10(d)-(f) represent a single fusion protein wherein IL-10 monomers (which may also be replaced by a variant IL-10 molecule) are linked together and each IL-10 monomer is further linked via a carboxy-terminal linker or an amino-terminal linker to a single VH or VL region derived from a single antibody. The IL-10 monomers comprise (d) a single mutation (e.g., at amino acid position 31) affecting binding to the IL-10 receptor; (e) a single mutation, for example, at amino acid position 75, affecting binding to the IL-10 receptor; and (f) two mutations, for example at amino acid positions 31 and 75, affecting binding to the IL-10 receptor.

Figure 11 shows the in vivo tumor volume reduction using a diabody containing the DV07 mutation. A buffer formulation (1× phosphate-buffered saline; “control”) and DV07 diabody (a fusion protein complex containing the EBV IL-10 variant with V31 to V31L and A75I of the mature protein and the variable domains from anti-CD3α and anti-EGFR, neither of which binds to the murine target) were administered as a single dose on day 3. Dose concentrations of 1 mg/kg and 0.4 mg/kg were tested for the DV07 diabody.

Figure 12 shows the in vitro T cell response to a published IL-10 variant (diamond), wild-type IL-10 (circle), and an alternative form of the DV07 diabody called DHivDEbo:DV07 (diamond; a fusion protein complex containing the EBV IL-10 variant with the V31L and A75I mutations and the variable domains from anti-HIV and anti-Ebola (neither of these VH/VL pairs binds to mouse proteins), where the diabody has the structure schematically shown in Figure 9(c)).

Figure 13A compares the suppression of TNFα induced by LPS exposure of monocytes/macrophages using different forms of the EBV IL-10 variant molecule with the V31L and A75I mutations in diabody form. "Wt" is human IL-10; "EBV" is EBV IL-10; "DCd3DEgfr:DV05" is a diabody comprising the VH and VL regions of an anti-CD3 antibody and an anti-EGFR antibody associated with an EBV IL-10 variant containing the V31 mutation; "DCd3DEgfr:DV07" is a diabody comprising the VH and VL regions of an anti-CD3 antibody and an anti-EGFR antibody associated with an EBV IL-10 variant containing the V31 and A75I mutations; "DHivDEbo:DV07" is a diabody containing the VH and VL regions from an anti-HIV antibody and an anti-Ebola virus antibody linked to an EBV IL-10 variant containing the V31L and A75I mutations.

Figure 13B compares IFNγ secretion in T cells in a response assay using human IL-10 (“wt”), EBV IL-10 (“EBV”), and different diabody forms containing variant EBV IL-10 molecules with V31L and A75I mutations and VH and VL regions from different antibodies. The DHivDEgfr:DV07 form is an EBV IL-10 diabody with V31 and A75I substitutions containing variable regions from anti-HIV and anti-EGFR antibodies. The DHivDEbo:DV07 form is an EBV IL-10 diabody with V31 and A75I mutations containing variable regions from anti-HIV and anti-Ebola antibodies.

Figures 14A-14B compare two forms of the EBV IL-10 diabody, DH:DV07 and DHDE:DV07, on monocytes/macrophages (Figure 14A) and T cells (Figure 14B) isolated from two donors. The DHDV07 form is an EBV IL-10 variant diabody with V31 and A75I substitutions containing variable regions from antibodies to HIV and EGFR. The DHDE:DV07 form is an EBV IL-10 diabody with V31 and A75I substitutions containing variable regions from anti-HIV and anti-Ebola. Figure 14A compares the suppression of LPS-induced TNFα on isolated monocytes/macrophages using human IL-10 (wt), DH:DV07, and DHDE:DV07. Figure 14B compares IFNγ secretion by isolated T cells in response to IL-10 (wt), EBV IL-10, DH:DV07, and DHDE:DV07.

Figure 15 is a direct comparison of different EBV-10 variant diabody forms on MC/9 mast cells. This assay compares IL-10, EBV IL-10, D:DV05 (EBV IL-10 with the V31 mutation, containing variable regions from anti-CD3α and anti-EGFR), D:DV06 (EBV IL-10 with the A75I mutation, containing variable regions from anti-CD3α and anti-EGFR), D:DV07 with the V31L and A75I mutations, containing variable regions from anti-CD3α and anti-EGFR), and DhivDEbo:DV07 (EBV IL-10 variant diabody with the V31L and A75I substitutions, containing variable regions from anti-HIV and anti-Ebola).

Figures 16A-16C show the results of an in vivo tumor assay using D:DV07 (with V31 and A75I mutations, including variable regions from anti-CD3α and anti-EGFR). In vivo tumor volume was assessed following administration of dosing buffer (“control”), 0.4 mg/kg three times a week (q3w), 0.2 mg/kg three times a week (q3w), 0.2 mg/kg daily and two days off (qd), and 0.1 mg/kg daily and two days off (qd).

Figures 17A-17B show the results of in vivo studies of two IL-10 variant fusion protein formats, large molecular weight (L) and small molecular weight (S), which correspond to the schematic diagrams in Figures 9C and 9F, respectively. The IL-10 variant includes the V31L and A75I mutations. These results tested the effects of the non-targetable IL-10 fusion proteins on tumor size reduction. The VH and VL regions of the fusion proteins are non-targeting sequences from either anti-HIV and anti-Ebola (L) or anti-Ebola (S). Figure 17A shows a dosing study of the non-targeted small format with five days on and two days off dosing compared to pegylated IL-10 (0.75 mg/kg per day). Figure 17B presents a dosing study of the untargeted small (1 mg/kg, 0.5 mg/kg, 0.25 mg/kg) and large (0.2 mg/kg) formats with three times weekly dosing compared to pegylated recombinant human IL-10 (0.75 mg/kg daily).

Figures 18A-18C show the results of an in vivo study of two IL-10 variant fusion protein formats, large and small, which correspond to the schematic diagrams of Figures 9C and 9F, respectively. These results tested the effect of IL-10 fusion proteins with targeting capabilities on tumor size reduction. The IL-10 variant includes the V31L and A75I mutations. In the large format, one set of VH and VL regions from the fusion proteins represents the anti-EGFR antibody, while the other set of VH and VL regions represents the anti-Ebola antibody. The small format includes VH and VL from the anti-EGFR antibody only. Figure 18A shows the results of a daily dosing study of large (0.25 mg/kg), small (0.25 mg/kg), and small non-targeted IL-10 fusion proteins compared to pegylated recombinant human IL-10 (0.75 mg/kg). Figure 18B shows the results of a thrice-weekly dosing study of large (1 mg/kg, 0.25 mg/kg, and 0.25 mg/kg daily) targeted IL-10 fusion protein compared to large non-targeted IL-10 fusion protein (DhDe:DV07 at 0.2 mg/kg) and pegylated IL-10 (0.75 mg/kg daily). Figure 18C presents the results of a three-week dosing study of small format targeted IL-10 fusion protein (1 mg/kg, 0.25 mg/kg) compared to small format non-targeted IL-10 fusion protein (Debo:DV07 at 1 mg/kg) and pegylated IL-10 (0.75 mg/kg per day).

Figures 19A-19B show the results of an in vivo cholesterol assay using an EBV IL-10 variant diabody with the V31 mutation, including variable regions from anti-CD3α and anti-EGFR.

Figures 20A-20B show the results of a comparative study of two variant IL-10 fusion proteins in macrophages and T cells in vitro . The assays examine the efficacy of two forms of the DV06 fusion protein in vitro compared to human IL-10. Figure 20A is a monocyte/macrophage assay using DhivDebo:DV06 (SEQ ID NOS: 26 and 27) and DmadcamDEbo:DV06 (SEQ ID NOS: 41 and 42). Figure 20B is an assay of the T cell response measured by IFN-gamma using DhivDebo:DV06 (SEQ ID NOS: 26 and 27) and DmadcamDEbo:DV06 (SEQ ID NOS: 41 and 42).

Figures 21A-21D are amino acid sequences of EBV IL-10. Figure 21A is EBV IL-10. Figure 21B is DV05 including the V31 substitution. Figure 21C is DV06 including the A75I substitution. Figure 21D is DV07 including the V31L and A75I substitutions.

DETAILED DESCRIPTION OF VARIOUS PREFERRED IMPLEMENTATION OPTIONS

Before describing in detail the various embodiments of the application, it should be understood that this application is not limited to specific compositions or process parameters, as these, of course, may vary. It should also be understood that the terminology used herein is intended only to describe the various embodiments and is not intended to be limiting.

Although a number of methods and materials similar or equivalent to those described herein can be used in practicing the various embodiments described herein, the materials and methods described herein are preferred.

Unless otherwise indicated, embodiments are described herein using conventional methods and techniques of molecular biology, biochemistry, pharmacology, chemistry, and immunology well known to those skilled in the art. Many of the general methods for constructing and making IL-10 variants, including but not limited to human IL-10 and CMV and/or EBV forms of IL-10, as well as assays for testing IL-10 variants, are well known methods that are readily available and have been described in detail in the art. See, e.g., Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell eds., Blackwell Scientific Publications); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current supplement). The chemistry of N-terminal aldehyde-based PEGylation is also well known in the art.

The following terms will be used to describe the various embodiments discussed herein and are intended to be defined as follows.

As used herein in describing various embodiments, the singular forms include plural references unless the content clearly indicates otherwise.

The term "about" refers to a deviation of 0.0001-5% from the stated number or range of numbers. In one embodiment, the term "about" refers to a deviation of 1-10% from the stated number or range of numbers. In one embodiment, the term "about" refers to a deviation of up to 25% from the stated number or range of numbers. In a more specific embodiment, the term "refers to" a difference of 1-25% in nucleotide sequence homology or amino acid sequence homology compared to a wild-type sequence.

The term "interleukin-10" or "IL-10" refers to a protein comprising two subunits non-covalently associated to form a homodimer, wherein IL-10 is an intercalated dimer of two bundles of six helices (helix A-F). As used herein, unless otherwise indicated, "interleukin-10" and "IL-10" may refer to the protein (SEQ ID NO: 1) or nucleic acid (SEQ ID NO: 2) of human IL-10 ("hIL-10"; GeneBank Accession No. NP_000563; or U.S. Patent No. 6,217,857); to the protein (SEQ ID NO: 7) or nucleic acid (SEQ ID NO: 8) of murine IL-10 ("mIL-10"; Genbank Accession No. M37897; or U.S. Patent No. 6,217,857); or to viral IL-10, ("vIL-10"). Viral IL-10 homologs may be from EBV or CMV (GeneBank Accession Nos. NC_007605 and DQ367962, respectively). The term EBV-IL10 refers to a homolog of the protein (SEQ ID NO: 3) or nucleic acid (SEQ ID NO: 4) of IL-10 from EBV. The term CMV-IL10 refers to a homologue of the protein (SEQ ID NO: 5) or nucleic acid (SEQ ID NO: 6) of IL-10 from CMV. As used herein, the term "monomeric IL-10" refers to individual IL-10 subunits or an IL-10 variant that, when non-covalently associated, form an IL-10 homodimer or IL-10 variant. The terms "wild type", "wt" and "native" are used interchangeably herein to refer to a protein sequence (e.g., IL-10, CMV-IL10 or EBV-IL10) that is commonly found in nature in the species from which the particular IL-10 in question is derived. For example, the term "wild type" or "native" EBV-IL10 would thus correspond to the amino acid sequence that is most commonly found in nature.

The term "derived," "derived," "derived from," or "derived from" is used herein to identify the original source of a molecule, such as a viral form of the IL-10 molecule, but is not intended to limit the method by which the molecule is prepared, produced, manufactured, or made. The method could include, but is not limited to, methods such as chemical or recombinant methods.

The term "derivative" is intended to include any suitable modification of the reference molecule of interest or an analogue thereof, such as sulfation, acetylation, glycosylation, phosphorylation, conjugation (e.g. with polyethylene glycol), hesylation or other addition of foreign groups, so long as the desired biological activity (e.g. anti-inflammatory effect and/or lack of T-cell stimulation) of the reference molecule or variant is maintained.

The terms "variant", "analog" and "mutein" refer to biologically active derivatives of a reference molecule that retain a desired activity, such as anti-inflammatory activity. In general, the terms "variant", "variants", "analog" and "mutein", as they relate to a polypeptide, refer to a compound or compounds having a native polypeptide sequence and structure with one or more amino acid insertions, substitutions (which are conservative in nature) and/or deletions relative to the native molecule. Thus, the terms "IL-10 variant", "IL-10 variant molecule" and grammatical variations and plural forms thereof are intended to be equivalent terms that refer to an IL-10 amino acid sequence (or nucleic acid sequence) that differs from wild-type IL-10 in sequence identity or homology by 1-25%. Thus, for example, a variant EBV IL-10 molecule differs from wild-type EBV IL-10 by having one or more insertions, substitutions, and/or deletions in the amino acid sequence (or the nucleotide sequence encoding the amino acid). Thus, in one form, a variant EBV IL-10 is a variant that differs from the wild-type sequence of SEQ ID NO::3 by having about 1% to 25% difference in sequence homology, which is about 1-42 amino acids.

The term "fusion protein" refers to a combination or conjugation of two or more proteins or polypeptides that results in a novel protein arrangement that does not normally exist in nature. A fusion protein is the result of covalent linkages between two or more proteins or polypeptides. The two or more proteins that comprise a fusion protein may have any configuration from the amino terminus to the carboxy terminus. Thus, for example, the carboxy terminus of one protein may be covalently linked to the carboxy terminus or amino terminus of another protein. Typical fusion proteins may include a combination of monomeric IL-10 or a monomeric IL-10 variant molecule with one or more antibody variable domains. Fusion proteins may also form dimers or link with other fusion proteins of the same type, resulting in the formation of a fusion protein complex. In some cases, the formation of a fusion protein complex may activate or enhance the functionality of the fusion protein compared to a fusion protein that is not part of the complex. For example, a monomeric IL-10 or a monomeric variant IL-10 molecule with one or more antibody variable domains may have limited or reduced ability to bind to the IL-10 receptor; however, when the fusion protein forms a complex, the monomeric forms of IL-10 or variant IL-10 molecule become a homodimer and the variable domains associate into a functional diabody.

A "functional variant" is a variant IL-10 molecule that includes modifications (e.g., insertions, substitutions, and/or deletions) that do not destroy the biological activity of the reference molecule. These variants may be "homologous" to the reference molecule, as defined below. Generally, the amino acid sequences of such analogues will have a high degree of sequence homology to the reference sequence, such as an amino acid sequence homology of greater than 50%, generally greater than 60%-70%, even more particularly 80%-85% or more, such as at least 90%-95% or more when the two sequences are aligned. Often, analogues include the same number of amino acids, but include substitutions. A functional variant retains biological activity that is increased, decreased, or substantially the same as that of the native molecule. Specifically, the term "variant" IL-10 molecule, which is interchangeable with the terms "engineered" IL-10 molecule or variant IL-10 molecule or "engineered" variant IL-10, refers to an IL-10 molecule or protein that includes one or both modifications in the receptor binding domain(s) of IL-10 and/or in the regions responsible for forming the interdomain angle or interhomodimer angle in the IL-10 molecule or protein. A "fusion protein" or "diabody" or "fusion" of variant IL-10 generally refers to the formation of a fusion protein (or fusion protein complex) comprising a variant IL-10 (in any monomeric or homodimeric form) and at least one other protein. As used herein, the term "variant IL-10 or fusion protein thereof" may be used throughout this disclosure to describe such a variant IL-10 fusion protein.

An "analog" or "analogs" may contain substitutions that are conservative in nature. For example, conservative substitutions may include, but are not limited to, substitutions of a similar type, such as (1) an acidic substitution between aspartate and glutamate; (2) a basic substitution between lysine, arginine, or histidine; (3) a non-polar substitution between alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan; and (4) an uncharged polar substitution between any of glycine, asparagine, glutamine, cysteine, serine, threonine, or tyrosine. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. It is also possible that, provided that the desired and specific biological activity remains intact, an isolated substitution of leucine with isoleucine or valine, aspartate with glutamate, threonine with serine, or similar conservative substitution of an amino acid with a structurally related amino acid may be made. For example, a polypeptide of interest may include up to about 1-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer from 1 to 50, provided that the desired function of the molecule remains intact. One skilled in the art can readily determine regions of a molecule of interest that can tolerate changes well known in the art.

"Mutein" further includes polypeptides comprising one or more amino acid-like molecules, including but not limited to compounds comprising only amino and/or imino molecules, polypeptides comprising one or more amino acid analogs (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both natural and unnatural (e.g., synthetic), cyclized, branched molecules, etc. Preferably, the analog or mutein will retain some biological activity that is enhanced, diminished, or substantially the same as that of the native molecule. Methods for creating polypeptide analogs and muteins are well known in the art.

The term "homolog", "homology", "homologous" or "substantially homologous" refers to the percent identity between at least two polynucleotide sequences or at least two polypeptide sequences. Sequences are homologous to each other when they exhibit at least about 50%, preferably at least about 80%-85%, more preferably at least about 90%, and most at least about 80%-85%, at least about 90%, and most preferably at least about 95%-98% sequence identity over a given length of molecules.

"Sequence identity" refers to an exact nucleotide-to-nucleotide or amino acid-to-amino acid match. Sequence identity can range from 100% sequence identity to 50% sequence identity. Percent sequence identity can be determined using a number of methods, including, but not limited to, direct comparison of sequence information between two molecules (a reference sequence and a sequence with an unknown % identity to the reference sequence) by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the reference sequence, and multiplying the result by 100. Readily available computer programs can be used to assist in determining percent identity.

The term "fragment" is intended to include a portion of a molecule of a full-length amino acid or polynucleotide sequence and/or structure. A fragment of a polypeptide may include, for example, a C-terminal deletion, an N-terminal deletion, and/or an internal deletion of a native polypeptide. An active or functional fragment of a given protein generally includes at least about 5-10 contiguous amino acid residues of the full-length molecule, preferably at least about 15-25 contiguous amino acid residues of the full-length molecule, and most preferably at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 5 amino acids and the full-length sequence, provided that the fragment in question retains biological activity, such as anti-inflammatory activity. With respect to an antibody, an antibody fragment refers to a portion of an intact antibody comprising the antigen-binding site or the variable region (heavy chain and/or light chain regions) of the intact antibody. They may include, for example, Fab, Fab', Fab'-SH, (Fab') ₂ , Fv fragments, diabodies, single-chain Fv (ScFv), single-chain polypeptides with one light chain variable domain, a fragment having three CDRs of a light chain variable domain or a heavy chain variable domain.

The term "substantially purified" generally refers to the isolation of a substance such that the substance constitutes a major percentage of the sample in which it is found. The substantially purified component constitutes 50%, preferably 80-85%, more preferably 90-95% of the sample. Similarly, the term "isolated" means, when referring to a polypeptide or polynucleotide, that said molecule is separate and discrete from the entire organism in which the molecule naturally occurs, or is present in the substantial absence of other biological macromolecules of the same type.

The terms "subject," "individual," or "patient" are used interchangeably herein and refer to a vertebrate, preferably a mammal. Mammals include, but are not limited to, mice, rodents, monkeys, humans, farm animals, sport animals, and certain domestic animals.

The term "administration" includes routes of administration that allow the active ingredient of the invention to perform its intended functions.

"A therapeutically effective amount," when referring to, for example, the administration of the variant EBV-IL-10 or fusion proteins thereof described herein, refers to a sufficient amount of the variant EBV-IL-10 or fusion proteins thereof to induce certain biological activities. Such activities may include, for example, suppression of myeloid cell function, enhancement of Kupffer cell activity, and/or no effect on CD8 ⁺ T cells or enhancement of CD8 ⁺ T cell activity, as well as blockade of Fc receptor upregulation by mast cells or prevention of degranulation. Thus, an "effective amount" will improve or prevent a symptom or sign of a disease. An effective amount also means an amount sufficient to make or facilitate a diagnosis.

The term "treat" or "treatment" refers to a method of reducing the effects of a disease or condition. Treatment may also refer to a method of reducing the underlying cause of the disease or condition itself, rather than just the symptoms. Treatment may be any reduction from the natural level and may be, as non-limiting examples, the complete elimination of the disease, condition, or symptoms of the disease or condition.

A "chemotherapeutic" agent is a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN™); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichine, phenesterine, prednimustine, trofosfamide, uromustine; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycins, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, calicheamicin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, kelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zoristatin; Anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); Folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; Purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; Pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; Androgens such as calusterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; Adrenal suppressants such as aminoglutethimide, mitotane, trilostane; Folic acid such as frolinic acid; Aceglatone; Aldophosphamide Glycoside; Aminolevulinic Acid; Amsacrine; Bestrabucil; Bisantrene; Edatraxate; Defofamine; Demecolcine; Diaziquone; Elphornithine; Elliptinium Acetate; Etoglucide; Gallium Nitrate; Hydroxyurea; Lentinan; Lonidamine; Mitoguazone; Mitoxantrone; Mopidamol; Nitracrine; Pentostatin; Phenamet; Pirarubicin; Podophyllinic Acid; 2-Ethylhydrazide; Procarbazine; PSK®; Razoxane; Sizofiran; Spirogermanium; Tenuazonic Acid; Triaziquone; 2,2',2''-Trichlorotriethylamine; Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; mitolactol; pipobroman; gacytosin; arabinoside (Ara-C); cyclophosphamide; thiotepa; taxoids such as paclitaxel (TAXOL® Bristol-Myers Squibb Oncology, Princeton, N.J) and doxetaxel (Taxotere™, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; Xeloda® Roche, Switzerland; ibandronate; CPT11; the topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing. Also included within this definition are antihormonal agents that regulate or inhibit the action of a hormone on tumors, such as anti-estrogens, including, for example, tamoxifen, raloxifene, aromatase-inhibiting 4(5) imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids, or derivatives of any of the foregoing.

The term "conjugate", "conjugated", "conjugation" or "conjugation" as used herein refers to the linking of two or more molecular parts together. The linking occurs through a covalent bond (e.g., a peptide bond).

IL-10 variant proteins

The variant IL-10 molecule of the present application includes modifications to any form of IL-10. These modifications in the IL-10 molecule include insertions, deletions and/or substitutions of one or more amino acids in regions and/or domains involved in IL-10 receptor binding and/or in those involved in the formation of an interdomain or interhomodimer angle in the IL-10 molecule. Examples of IL-10 sequences that can be used in constructing the IL-10 variant molecules of the present application include, but are not limited to, homologs from Epstein-Barr virus ("EBV"; see, e.g., Moore et al., Science (1990) 248: 1230-1234; Hsu et al., Science (1990) 250: 830-832; Suzuki et al., J. Exp. Med. (1995) 182: 477-486), cytomegalovirus ("CMV"; see, e.g., Lockridge et al., Virol. (2000) 268: 272-280; Kotenko et al., Proc. Natl. Acad. Sci. USA (2000) 97: 1695-1700), and equine herpesvirus (see, e.g., Rode et al., Virus Genes (1993) 7: 111–116), OrF virus (see, e.g., Imlach et al., J. Gen. Virol. (2002) 83: 1049–1058 and Fleming et al., Virus Genes (2000) 21: 85–95). Other representative IL-10 sequences include those reported in NCBI with accession numbers NM010548, AF307012, M37897, M84340 (mouse sequences); U38200 (equine); U39569, AF060520 (feline sequences); U00799 (bovine); U11421, Z29362 (ovine sequences); L26031, L26029 (macaque sequences); AF294758 (monkey); U33843 (canine); AF088887, AF068058 (rabbit sequences); AF012909, AF120030 (marmot sequences); AF026277 (opossum); AF097510 (guinea pig); U11767 (deer); L37781 (gerbil); AB107649 (llama and camel).

In one embodiment, the variant IL-10 molecules described herein are obtained by modifying the sequences of the human wild-type (SEQ ID NO::1), CMV (SEQ ID NO:5), EBV (SEQ ID NO:3), or mouse (SEQ ID NO:7) IL-10 protein. Representative examples of various variant IL-10 molecules are provided in SEQ ID NOs:9-23.

Modifications relative to wild-type IL-10 include insertions, deletions, and/or substitutions of one or more amino acids in regions responsible for (i) IL-10 receptor binding and/or (ii) formation of the interdomain or interhomodimer angle of the IL-10 molecule.

Variant IL-10 molecules: IL-10 receptor binding regions

The regions responsible for receptor binding include any amino acid moiety located in regions that are directly involved in or responsible for the binding of IL-10 to IL-10 receptor 1 (IL10R1) and/or IL-10 receptor 2 (IL10R2). These regions may include, for example, discontinuous portions of the IL-10 molecule previously discussed and mapped in this area (see, for example, Yoon 2005; Josephson 2001). For example, modifications of any region, such as, but not limited to, helix A, helix F, and loop AB, responsible for forming contact points and clefts associated with the binding of IL-10 to the IL-10 receptor are contemplated by this application. In a preferred embodiment, the modification (e.g., insertion, deletion, and/or substitution) of the receptor binding domain comprises amino acid 31 and/or 75 of SEQ ID NO: 3. In a particularly preferred embodiment, the modification comprises substitution of valine at position 31 with leucine (V31L, herein referred to as "DV05") in SEQ ID NO: 3, substitution of alanine at position 75 with isoleucine (A75I, herein referred to as "DV06") in SEQ ID NO: 3, or both V31L and A75I (herein referred to as "DV07") in SEQ ID NO: 3. In one embodiment, DV05 is SEQ ID NO: 55, DV06 is SEQ ID NO: 57, and DV07 is SEQ ID NO: 59.

In one embodiment, the receptor binding domain comprises any one or more of the site Ia and/or site Ib interface contact points discussed in Josephson et al. (Immunity, 2001, 15, pp. 35-46, Figure 1). In one embodiment, the site Ia interface contact points comprise one or more amino acids located in the bend of helix F and in the AB loop. In another embodiment, the site Ib interface contact points comprise one or more amino acids located at the N-terminus of helix A and at the C-terminus of helix F. In another embodiment, any one or more amino acids located on IL-10 responsible for binding to the receptor will comprise any one or more of amino acids 1-10 within helix A and amino acids 1-7 within the AB loop. In another embodiment, the receptor binding region may include one or more modifications of the following amino acids or 1-10 amino acids centered thereon, wherein the amino acids are Glu-142, Lys-138, Asp-144, Gln-38, Ser-141, Asp-44, Gln-42, Gln-38, Arg-27, Glu-151, Arg-24, Pro-20, Ile-158, or any combination thereof.

In one aspect, the modifications of the receptor binding domain include any one or more of the site IIa and/or site IIb interface contact points discussed in Josephson et al. (Immunity, 2001, 15, p. 35-46, Figure 1). In one embodiment, the site IIa interface contact points include one or more amino acids located in the DE loop. In another embodiment, the receptor binding region can include one or more modifications of the following amino acids or 1-10 amino acids centered thereon, wherein the amino acids are Ser-11, Thr-13, Asn-18, Arg-104, Arg-107, or any combination thereof. In one embodiment, the variant IL-10 molecule will comprise 1-100 (or any integer within the range) amino acid insertions, deletions and/or substitutions that affect the receptor binding domain, wherein such insertions, deletions and/or substitutions either increase or decrease the binding affinity of the variant IL-10 molecule for the IL-10 receptor.

Variant IL-10 molecules: Modifications of the interdomain angle

The regions responsible for forming the interdomain (or interhomodimer, which is used interchangeably) angle of the IL-10 molecule include any amino acid moiety located in regions that are directly involved in or responsible for forming certain interdomain angles of IL-10 homodimers. Wild-type IL-10 forms an "L-shaped" dimer when two IL-10 monomer units intertwine antiparallel. The resulting interdomain angle for human IL-10 and EBV-IL10 is approximately 89 and 97 degrees, respectively. In order to tune IL-10 receptor signaling, in one embodiment, the present invention is directed to modifying the amino acids responsible for forming the interdomain angle in the DE loop, the portions of helix D or helix E responsible for forming the L-shaped dimer in each monomer. In another embodiment, the regions responsible for the interdomain angle include a linker region of approximately 12 amino acids located between helix D and helix E of the IL-10 protein. The modifications by insertion, deletion or substitution result in a restricted or weakened interdomain angle of IL-10 compared to human IL-10 or EBV-IL10. When a monomeric IL-10 molecule is modified, resulting in a restricted/tight/closed or relaxed/loose/open interdomain angle of IL-10 upon homodimerization, the modified IL-10 molecule will produce a variant IL-10 molecule that has an altered interdomain angle that engages and modulates its receptor (IL-10 receptor). In another embodiment, the substitution comprises the introduction of a proline into the amino acid segment located between helix D and helix E of EBV-IL10 and/or between helix C and helix D.

Thus, in an embodiment, the variant IL-10 molecule will comprise one or more insertions, deletions, and/or substitutions that exhibit an altered intermolecular angle or an altered interdomain angle compared to wild-type IL-10. The altered intermolecular angle or the altered interdomain angle can dimerize with the same or a different variant IL-10 molecule, resulting in a variant IL-10 molecule that interacts with the IL-10 receptor with a different interaction angle compared to the wild-type IL-10 molecule. The different interaction angle of the variant IL-10 molecule results in the ability to modulate or "tune" IL-10 receptor signaling to activate or suppress inflammation and/or the immune response. In a preferred embodiment, the variant IL-10 molecule is EBV-IL10. In another preferred embodiment, the variant IL-10 molecule uses an EBV-IL10 molecule as a basis for modification. In yet another embodiment, the variant IL-10 molecule is a hybrid molecule with portions and domains from other IL-10 molecules (such as, but not limited to, human IL-10, murine IL-10, and/or CMV-IL10).

In another preferred embodiment, the variant IL-10 molecule produces a weakened interdomain angle that will suppress the response of inflammatory cells (cells of the myeloid lineage) and will not drive the activation of lymphocytes such as T cells. In combination with modifications of the receptor binding domain, preferably with modifications that result in reduced or unchanged affinity for the receptor, the variant IL-10 molecules with a weakened interdomain angle will be effective in suppressing the secretion of cytokines by myeloid cells (monocytes, macrophages, neutrophils, granulocytes, mast cells, Kupffer cells) in response to proinflammatory stimuli. This configuration of the variant IL-10 molecule is suitable, for example, for the treatment of inflammatory diseases, such as, but not limited to, IBD, Crohn's disease, psoriasis, rheumatoid arthritis, NAFLD and NASH.

In another preferred embodiment, the variant IL-10 molecule produces a restricted interdomain angle that will enhance the activation of immune cells, such as T cells. In combination with modifications of the receptor binding domain, preferably with modifications that lead to higher affinity for the receptor, variant IL-10 molecules with a restricted interdomain angle will be effective in enhancing, for example, CD8 ⁺ T cells, NK cells and the utilization of Kupffer cells. This configuration of the variant IL-10 molecule is suitable, for example, for the treatment of a number of solid and hematological malignancies, including metastatic malignancies.

The regions responsible for forming the interdomain angle may be continuous or discontinuous portions located within the IL-10 molecule. In one embodiment, the interdomain angle for a variant IL-10 molecule will have a degree change of 1-25 degrees, in another preferred embodiment, the degree change is 1-10 degrees, in a more preferred embodiment, the degree change is 1-5 degrees, in the most preferred embodiment, the degree change is less than 5 degrees. In one embodiment, the variant IL-10 molecule will contain 1-100 (or any integer in the range) amino acid insertions, deletions and/or substitutions that affect the interdomain angle.

In one embodiment of the invention, IL-10 molecules will be designed and created using computer modeling to predict the region or regions most responsible for IL-10 receptor binding and/or interdomain angles. Computer modeling will help provide a faster and more efficient means of predicting regions that will benefit most from modification of the receptor binding domain and/or interdomain angles.

In another embodiment, the molecule of the present application includes derivatives of variant IL-10 molecules. These may include modifications of the variant molecules to include moieties that increase the size, half-life, and bioavailability of the variant molecules.

Variant IL-10 molecules: Modifications with PEG

In one embodiment, the variant IL-10 molecules may include the addition of polyethyleneglycol (PEG). PEGylated IL-10 variants will include the attachment of at least one PEG molecule. Without being bound by any particular theory, the attachment of PEG to the variant IL-10 may protect against proteolysis, reduce immunogenicity, promote destabilization of the variant IL-10 on the receptor to maintain its suppressive effect on myeloid cells, and prevent T cell activation.

In its most common form, PEG is a linear or branched polyether terminated with hydroxyl groups and has the general structure:

HO-(CH ₂ CH ₂ O) _n -CH ₂ CH ₂ -OH

The methods for coupling PEG to the IL-10 variant molecules in the present application will follow those methods/protocols already known in the art. For example, conjugation or coupling of PEG requires activation of PEG by preparing a PEG derivative having a functional group at one or both ends. The most common method for conjugating proteins to PEG has been the activation of PEG with functional groups suitable for reaction with lysine and N-terminal amino acid groups. In particular, the most common reactive groups involved in PEG coupling to polypeptides are the alpha or epsilon amino group of lysine.

The PEGylation reaction of the linker with the variant IL-10 molecule results in the addition of a PEG group predominantly at the following sites: the alpha-amino group at the N-terminus of the protein, the epsilon-amino group on the side chain of lysine residues, and the imidazole group on the side chain of histidine residues. In some embodiments, since the variant IL-10 molecules are recombinant proteins that have one alpha and a number of epsilon-amino and imidazole groups, multiple positional isomers can be obtained depending on the chemical composition of the linker.

Two widely used first-generation activated monomethoxy-PEGs (mPEGs) were succinimidyl carbonate PEG (SC-PEG; see, e.g., Zalipsky, et al. (1992) Biotechnol. Appl. Biochem 15: 100–114; and Miron and Wilcheck (1993) Bioconjug. Chem. 4: 568–569) and benzotriazole carbonate PEG (BTC-PEG; see, e.g., Dolence, et al., U.S. Patent No. 5,650,234), which react preferentially with lysine residues to form a carbamate linkage, but are also known to react with histidine and tyrosine residues. The linkage to histidine residues on IFNα has been shown to be a hydrolytically unstable imidazolecarbamate linkage (see, e.g., Lee and McNemar, U.S. Patent No. 5,985,263, which is incorporated by reference in its entirety).

Second-generation PEGylation technology was developed to avoid these unstable linkages as well as the lack of selectivity with respect to residual reactivity. Using a PEG-aldehyde linker, it targets a single site at the N-terminus of a polypeptide and/or protein subunit via reductive amination. IL-10 can be PEGylated using different types of linkers and pH to produce different forms of the PEGylated molecule (see, e.g., U.S. Patent No. 5,252,714, U.S. Patent No. 5,643,575, U.S. Patent No. 5,919,455, U.S. Patent No. 5,932,462, U.S. Patent No. 5,932,462, U.S. Patent No. 5,252,714, U.S. Patent No. 5,643,575, U.S. Patent No. 5,643,575, U.S. Patent No. 5,932,462, U.S. Patent No. 5,985,263, U.S. Patent No. 7,052,686, which are incorporated by reference in their entireties).

IL-10 Mimetic Molecules

In another embodiment, the present application includes mimetic molecules that mimic the biological function of variant IL-10 molecules. These mimetics include, but are not limited to, peptides, small molecules, modified hormones, and antibodies that have structures and functions that are the same or substantially the same as those derived from variant IL-10 molecules. IL-10 mimetic molecules that can form the basis for modification to reproduce or replicate variant IL-10 molecules include those described in US20080139478, US20120238505, and/or US20150218222, all of which are incorporated by reference in their entireties.

IL-10 hybrid molecules and IL-10 fusion proteins

In another embodiment, the present application includes variant IL-10 molecules that are hybrid molecules comprised of portions derived from IL-10, EBV-IL10, and/or CMV-IL10. For example, different domains within each of IL-10, EBV-IL10, and/or CMV-IL10 can be combined together to create a hybrid molecule such that all or portions of the receptor binding domain and/or the domains responsible for the interdomain angle in IL-10 are in the combination.

In yet another embodiment, the variant IL-10 molecule is part of an engineered fusion protein. The linker or spacer may be a random amino acid sequence (e.g., SSGGGGS (SEQ ID NO: 30, GGGGGSGGGGSGGGGS (SEQ ID NO: 31), or SSGGGGSGGGGSGGGGS (SEQ ID NO: 54)) a constant region of an antibody, scFv, or diabody. The constant region may be, but is not limited to, derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE. The linker or spacer may be advantageously a heavy chain constant (CH) region 1, CH2, or CH3. In a more preferred embodiment, the linker or spacer is a random amino acid sequence of SEQ ID NO: 30 and/or 31. In another aspect, the linker or spacer may further comprise at least two interchain disulfide bonds.

The fusion protein may also include at least one IL-10 or variant IL-10 molecule monomer conjugated to the N-terminus of the fusion protein, the C-terminus, or both. In another embodiment, a fusion protein comprising IL-10 or variant IL-10 may also include at least one cytokine conjugated to the opposite end of IL-10 or variant IL-10 and includes IL-2, IL-7, IL-15, IL-26, IL-27, IL-28, IL-29, IL-10, variant IL-10 molecule, IFN-alpha, TGF-beta, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or any combination thereof. In some preferred embodiments, the fusion protein comprises two monomeric forms of IL-10 or variant IL-10 molecules conjugated to the N-terminus of the fusion protein and two IL-10 or two variant IL-10 molecules conjugated to the C-terminus of the fusion protein; the fusion protein comprises two monomeric forms of IL-10 or variant IL-10 molecules conjugated to the N-terminus of the fusion protein and at least one IL-2 molecule conjugated to the C-terminus of the fusion protein; the fusion protein comprises two IL-10 or two variant IL-10 molecules conjugated to the N-terminus of the fusion protein and at least one IL-15 molecule conjugated to the C-terminus of the fusion protein. In another embodiment, the C-terminus of the fusion protein may have at least two different cytokines selected from IL-2, IL-7, IL-15, IL-26, IL-27, IL-28, IL-29, IL-10, a variant IL-10 molecule, IFN-alpha, TGF-beta, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13.

In another embodiment, the fusion protein is made using a single chain variable fragment (scFv), diabody, Fab, or any antibody fragment as a backbone onto which one or two monomers of IL-10, one or two monomers of a variant IL-10 molecule, IL-2, IL-7, IL-15, IL-26, IL-27, IL-28, IL-29, IFN-alpha, TGF-beta, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or a combination thereof, are conjugated.

In one particularly preferred embodiment of the invention, the fusion protein comprises at least one variable region having a heavy chain variable region (VH) and/or a light chain variable region (VL) linked to IL-10 or a variant IL-10 molecule. In this configuration, the fusion protein comprises an IL-10 monomer or a variant IL-10 molecule monomer linked to at least one variable region of an antibody. In one aspect, the fusion protein is a linear contiguous sequence comprising an IL-10 monomer or a variant IL-10 molecule monomer linked to a VH linked to a VL linked to an IL-10 monomer. The variable region of the antibody can be a heavy chain variable region (VH), a light chain variable region (VL), or both. The first fusion protein comprises a protein sequence having a linear configuration such that the IL-10 monomer or the variant IL-10 monomer is conjugated to the carboxy terminus of the variable region (VH or VL or both). The second fusion protein may comprise a protein sequence having a linear configuration such that the IL-10 monomer or the variant IL-10 monomer is linked to the amino terminus of the variable region (VH or VL or both). A typical example of the first fusion protein described above may include the following configuration:

a) _NH2 (Ab1VL) _COOH -(linker)- _NH2 (monoIL10) _COOH

A typical example of the second fusion protein described above might include the following configuration:

b) _NH2 (monoIL10) _COOH -(linker)- _NH2 (Ab ₁ VH) _COOH

Together, the first (a) and second (b) fusion proteins form a functional protein complex in an antiparallel manner, whereby the end-linked IL-10 or variant IL-10 monomers form a functional homodimer and the variable regions are together capable of forming a functional antigen-binding site (“ABS”) (see, for example, Figures 9(a)-(f)).

In an alternative embodiment, the IL-10 monomer or the variant IL-10 monomer may be conjugated to at least two variable regions from the same antibody or from two different antibodies. In this configuration, the at least two variable regions are VH and VL. An example of such a configuration might include a first fusion protein with a linear contiguous protein sequence of a VH region from a first antibody linked at its carboxy terminus to the amino terminus of a VL region from a second antibody linked to the amino terminus of an IL-10 monomer or a variant IL-10 monomer. An alternative configuration might include a second fusion protein with a linear contiguous protein sequence of an IL-10 monomer or a variant IL-10 monomer linked at the carboxy terminus to the amino terminus of a VH region from a second antibody linked to the amino terminus of a VL region from the first antibody. A typical example of the first fusion protein described above might include the following configuration:

a) _NH2 (Ab _1- VH) _COOH- -(linker)- _NH2 (Ab ₂ VL) _COOH -(linker)- _NH2 (monoIL10) _COOH

A typical example of the second fusion protein described above might include the following configuration:

b) _NH2 (monoIL10) _COOH -(linker)- _NH2 (Ab ₂ VH) _COOH -(linker)- _NH2 (Ab _1- VL) _COOH

Together, the first (a) and second (b) fusion proteins form a functional protein complex in an antiparallel manner, whereby the end-linked IL-10 or variant IL-10 monomers form a functional homodimer and the variable regions are together capable of forming an ABS (see, for example, Figures 9(a)-(c)).

In another embodiment, the fusion protein comprises two IL-10 monomers or two variant IL-10 monomers that are fused together and one or more VH and VL regions. Each monomer is individually linked to one or more VH regions and/or VL regions of an antibody. When more than one VH and/or VL region is involved in this fusion protein configuration, the VH and VL regions may be from the same antibody or from at least two different antibodies. In one particular configuration of this fusion protein, the VH or VL region is linked to the amino terminus of a first monomer, which is then linked at its carboxy terminus to the amino terminus of a second monomer, which is then linked to the amino terminus of VL or VH. Optionally, additional VH or VL regions can be linked to the amino or carboxy termini, wherein the VH or VL regions can be from the same antibody or from different antibodies. Typical examples of the fusion protein described above may include the following configurations (see, for example, Figures 10(d)-(f)):

_NH2 (Ab _1- VH) _COOH- -(linker)- _NH2 (monoIL10) _COOH -(linker)- _NH2 (monoIL10) _COOH- (linker) _-NH2 (Ab _1- VL) _COOH

The fusion protein described above would be able to fold such that IL-10 monomers form a homodimer and the variable domains (VH and VL) of the antibody form a functional ABS.

In another embodiment, the fusion protein comprises two IL-10 monomers or two variant IL-10 monomers located at opposite ends of the fusion protein and at least one VH and VL region, wherein the VH and VL regions are linked together. In this configuration, the VH and VL regions are fused together and each monomer is individually linked to either a VL region or a VH region from a first antibody. In this configuration, each of the IL-10 monomers or variant IL-10 monomers is individually linked to either a VH or a VL from the first antibody. A typical example of a fusion protein as described above may include the following configurations (see, for example, Figures 10(a)-(c)):

a) _NH2 (monoIL10) _COOH - (linker) _-NH2 (Ab _1- VH) _COOH- (linker) - _NH2 (Ab _1- VL) _COOH- (linker) _-NH2 (monoIL10) _COOH

b) _NH2 (monoIL10) _COOH - (linker) _-NH2 (Ab _1- VL) _COOH- (linker) - _NH2 (Ab _1- VH) _COOH- (linker) _-NH2 (monoIL10) _COOH

The IL-10 monomer or the variant IL-10 monomer may be linked to the VH or VL sequence via a linker sequence. The linker may be a carboxy-terminal linker linking the carboxy-terminus of the variable chain region (VH or VL) to the amino-terminus of the IL-10 monomer or the monomer of the variant IL-10 molecule. Alternatively, the linker may be an amino-terminal linker whereby the carboxy-terminus of the monomeric IL-10 or the monomeric variant IL-10 molecule is linked to the amino-terminus of the variable chain region (VH or VL).

Thus, in one form, the fusion protein comprises a monomer of an IL-10 molecule or a variant IL-10 molecule linked to two variable regions from at least two different antibodies, wherein the two variable regions are configured as a VH region from a first antibody linked to a VL region from a second antibody, or a VL from the first antibody linked to a VH from a second antibody. A fusion protein according to this form may include a monomeric IL-10 molecule or a variant thereof, including at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor. The amino acid substitution affecting binding to the IL-10 receptor may be in IL-10 from human, CMV or EBV. The amino acid substitution may preferably be in EBV IL-10 and include an amino acid substitution at position 31, 75 or both. The amino acids may include any one or more substitutions of V31 or A75I, or both. In addition to amino acid substitutions that affect IL-10 receptor binding affinity, the variant IL-10 may also include modifications that affect the interdomain angle. In another embodiment, the fusion protein comprises a configuration selected from: (a) a VH region from a first antibody linked at its carboxy terminus to the amino terminus of a VL region from a second antibody linked to the carboxy terminus of an IL-10 monomer or variant thereof; or (b) an IL-10 molecule or variant thereof linked at its carboxy terminus to the amino terminus of a VH region from a second antibody then linked to the amino terminus of a VL region from the first antibody. These fusion protein configurations include, in one preferred embodiment, the sequences of SEQ ID NOs: 24-28, 29, and 33-53. These fusion protein sequences are capable of forming a complex where IL-10 monomers or variant IL-10 monomers are capable of forming a homodimer. Such a complex may comprise and/or be configured as a diabody complex.

In another form, the fusion protein can be formed as an immunoconjugate comprising a first fusion protein comprising at its amino terminus a heavy chain variable region (VH) from a first antibody linked to a light chain variable region (VL) from a second antibody, further linked to an IL-10 monomer; and a second fusion protein comprising at its amino terminus an IL-10 monomer linked to a VH from a second antibody, further linked to a VL from the first antibody, wherein the VH and VL from the first and second antibodies associate into a diabody and the IL-10 monomers form a functional dimeric IL-10 molecule. In another preferred embodiment, the immunoconjugate complex comprises a first fusion protein comprising at its amino terminus a VH region from a first antibody and a monomeric IL-10 molecule linked via its amino terminus; and a second fusion protein comprising at its amino terminus an IL-10 monomer linked to a VL from a first antibody, wherein the VH region from the first antibody associates with the VL region from the first antibody, thereby allowing the monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer. The IL-10 monomer(s) or IL-10 variant(s) may comprise, as described above, amino acid modifications that affect binding to the IL-10 receptor and/or the interdomain angle. In another preferred embodiment, the immunoconjugate complex comprises a first fusion protein comprising at its amino terminus a VH region from a first antibody linked to a monomeric IL-10 molecule; and a second fusion protein comprising at its amino terminus an IL-10 monomer linked to a VL from a first antibody, wherein the VH region from the first antibody associates with the VL region from the first antibody, thereby allowing the monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer. In another embodiment, the immunoconjugate comprises at its amino terminus a first IL-10 monomer (or variant IL-10 molecule) linked to a VH region from the first antibody linked to a VL region from the first antibody linked to a second IL-10 monomer (or variant IL-10 molecule), wherein the two IL-10 monomers are capable of associating together to form a functional IL-10 dimer. The described VH and VL regions are capable of forming an antigen-binding site that specifically targets an antigen (e.g., a receptor, a protein, a nucleic acid, etc.). Thus, there are two chains, chain 1 and chain 2, which together form a fusion protein complex that creates a functional IL-10 homodimer (or IL-10 variant molecule). Typical fusion protein chains (i.e., chain 1 and chain 2) include the following:

Chain 1 Chain 2 SEQ ID NO:24 SEQ ID NO:25 SEQ ID NO:26 SEQ ID NO:27 SEQ ID NO:28 SEQ ID NO:29 SEQ ID NO:35 SEQ ID NO:36 SEQ ID NO:38 SEQ ID NO:39 SEQ ID NO:41 SEQ ID NO:42 SEQ ID NO:46 SEQ ID NO:47 SEQ ID NO:48 SEQ ID NO:49 SEQ ID NO:50 SEQ ID NO:51

Fusion proteins include a VH and VL pair from at least one antibody. The VH and VL pair acts as a scaffold to which monomers of IL-10 or its variants can be attached so that they can homodimerize into a functional IL-10 molecule. One skilled in the art will thus appreciate that the VH and VL scaffold used in the fusion protein can be selected based on the desired physical characteristics necessary for proper dimerization of the IL-10 protein or IL-10 variant and/or the desire to maintain the targeting properties of the VH and VL. Similarly, one skilled in the art will appreciate that the CDR regions of the VH and VL pair can also be replaced with other CDR regions to produce a specifically targeted fusion protein. It is also contemplated that if the fusion protein is not intended to target any specific antigen, the VH and VL pair may be selected as a backbone that does not target any specific antigen (or is an antigen at low levels in vivo ), such as a VH and VL pair from anti-HIV and/or anti-Ebola. The fusion protein may include 1-4 variable regions. The variable regions may be derived from the same antibody or from at least two different antibodies. The variable chains of the antibody may be derived from or may be derived from multiple antibodies (e.g., those that target proteins, cell receptors, and/or tumor-associated antigens, etc.). In another embodiment, the variable regions are derived from antibodies that target antigens associated with various diseases (e.g., cancer), or that are typically not detected or are rarely detected in the serum of a healthy individual, such as variable regions from antibodies directed to EGFR, PDGFR, VEGFR, Her2Neu, FGFR, GPC3 or other tumor-associated antigens, MadCam, ICAM, VCAM or other inflammation-associated cell surface proteins, HIV and/or Ebola virus. Thus, in one embodiment, the variable regions are derived from or are derived from anti-EGFR, anti-MadCam, anti-HIV (Chan et al, J. Virol, 2018, 92 (18): e006411-19), anti-ICAM, anti-VCAM, or anti-Ebola (US Published Application 2018/0180614, incorporated by reference in its entirety, especially the mAbs described in Tables 2, 3, and 4), for example. In another embodiment, the variable regions are derived from or are derived from antibodies that are capable of increasing the concentration of cytokines, such as IL-10, in a particular target region to allow IL-10 to exert its biological effect. Such antibodies may include antibodies that target overexpressed or activated receptors or antigens in certain disease regions, or that are specifically expressed in certain disease regions. For example, variable regions can be derived from an antibody specific for the epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as, but not limited to, PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ trap; MadCam, integrin β7 subunit; integrin α4β7; integrin α4 SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; and SR-J1 and many others. An IL-10 monomer (e.g., human, CMV, or EBV) or a variant IL-10 molecule (as described herein) is conjugated to either the amino terminus or the carboxy terminus of the variable region (VH or VL) such that the IL-10 or variant IL-10 molecule is capable of dimerizing with one another.

The fusion protein or fusion protein complex may also have an antigen-targeting function. The fusion protein or fusion protein complex will include VH and VL regions that are capable of associating together to form an antigen-binding site or ABS. In some configurations, IL-10 or a variant IL-10 molecule or monomers thereof will be covalently linked to the end containing the antigen-binding site. These targeting fusion proteins may include at least one functional variable region or paired VH and VL at one end of the fusion protein such that the fusion protein retains the ability to target an antigen and also has a functional homodimer of IL-10 or a variant IL-10 molecule (see, Figs. 9(a)-(e) and 10(a)-(e)). The variable regions may be further altered (e.g., by insertion, deletion, or substitution) by changing one or more amino acids that reduce antigenicity in an individual. The VH and VL pair forms a scaffold onto which CDR regions derived from a variety of antibodies can be grafted. Such antibody CDR regions include those antibodies known and described above. For example, CDR regions from any antibody can be grafted to a VH and VL pair such as those described in SEQ ID NO: 37, 44, or 45 or those fusion proteins capable of forming a fusion protein complex such as those described in SEQ ID NO: 46 and 47; 48 and 49; or 50 and 51. The CDR regions in the VH and VL scaffolds described above will include the following number of amino acid positions available for CDR grafting/insertion:

Heavy Chain CDR1 3-7 amino acids Heavy Chain CDR2 7-11 amino acids Heavy Chain CDR3 7-11 amino acids CDR1 Light Chain 9-14 amino acids Light chain CDR2 5-9 amino acids CDR3 light chain 7-11 amino acids

In another aspect, the above-described fusion protein may be represented by one of the following general formulas:

1) IL10-L ¹ -X ¹ -L ¹ -X ² -L ¹ -IL10 (Formula I);

2) (Z) _n -X ¹ -L ² -Y ² -L ¹ -IL10 (Formula II);

3) IL10-L ¹ -Y ¹ -L ² -X ² -(Z) _n (Formula III);

4) X ¹ -L ² -X ² -L ¹ -IL10 (Formula IV);

5) IL10-L ¹ -X ¹ -L ² -X ² (Formula V);

6) X ¹ -L ¹ -IL10 (Formula VI); And

7) IL10-L ¹ -X ² (Formula VII)

Where

"IL-10" is human IL-10 (SEQ ID NO: 1); EBV IL-10 (SEQ ID NO: 3), DV05 (SEQ ID NO: 14, 18, or 55), DV06 (SEQ ID NO: 15, 19, or 57), or DV07 (SEQ ID NO: 16, 20, or 59), in a preferred embodiment "IL-10" consists of DV05, DV06, or DV07, more preferably "IL-10" consists of SEQ ID NO: 55, 57, or 59;

"L ¹ " is a linker of SEQ ID NO: 31 or 54;

"L ² " is the linker of SEQ ID NO: 30;

"X ¹ " is the VH region derived from the first antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as but not limited to PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ trap; MadCam, integrin β7 subunit; integrin α4β7; integrin α4 SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; and SR-J1; HIV, or Ebola;

"X2" represents the VL region derived from the same antibody as X1;

"Y1" is a VH region derived from a second antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as but not limited to PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ trap; MadCam, integrin β7 subunit; integrin α4β7; integrin α4 SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; and SR-J1; HIV, or Ebola;

"Y2" represents the VL region derived from the same antibody as Y1;

where X and Y are obtained from the same or different antibodies;

"Z" is a cytokine selected from IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL_15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons -α, -β, -γ, TGF-β, or tumor necrosis factor -α, -β, basic FGF, EGF, PDGF, IL-4, IL-11 or IL-13;

"n" is an integer chosen from 0-2.

In an embodiment, the substituents of the above Formula I-VII are preferably selected from the following: IL-10 is preferably DV05, DV06 or DV07, more preferably IL-10 comprises DV05, DV06 or DV07 or most preferably IL-10 consists of SEQ ID NO: 55, 57 or 59; X1 and X2 are preferably anti-EGFR, anti-PDGFR, anti-FGFR, anti-VEGF, anti-Her2Neu, anti-GPC3, anti-MAdCAM, anti-ICAM-1, -2, -3, -4, anti-VCAM, anti-HIV or anti-Ebola; Y1 and Y2 are preferably anti-EGFR, anti-MAdCAM, anti-ICAM-1, -2, -3, -4, anti-VCAM, anti-HIV or anti-Ebola; Z is selected from IL-2, IL-7 or IL-15; and n is 1. In a most preferred embodiment, the fusion proteins are any of SEQ ID NOs: 33-53, 61, 63, 65 or 67. Those skilled in the art will appreciate that the histidine tag is used during the purification of the fusion protein and may remain intact or be removed from the final product. Those skilled in the art will also appreciate that the VH and VL framework regions of any of the antibodies described above may be replaced with other complementarity determining regions (CDRs). For example, if the VH and VL regions are derived from an antibody to the Ebola virus, the six CDR regions (i.e., CDRs 1-3 of both VH and VL) may be replaced with the six CDR regions of an antibody to EGFR (e.g., cetuximab). Thus, in one preferred embodiment, the fusion protein is SEQ ID NO: 33-34, 52, or 53. In another preferred embodiment, the fusion protein is a protein having a framework represented by SEQ ID NO: 37; 44; 45; 46-47; 48-49; or 50-51, wherein any six CDR regions from any antibody may be grafted. In other preferred embodiments, the CDR regions from the VH and VL regions of an Ebola virus antibody may be grafted with the CDR regions from anti-MAdCAM, anti-VCAM, or anti-ICAM-1, -2, -3, -4 antibodies, wherein in a preferred embodiment, the CDR regions may be grafted into a fusion protein of SEQ ID NO: 37. The fusion proteins as described in the above formulas II and III, formulas IV and V, and formulas VI and VII are intended to associate together to form a biologically active IL-10 homodimer (or variant thereof). The above-described fusion proteins are intended to be either non-targeting or targeting depending on the pair of VH and VL regions selected and/or the CDR regions inserted into the VH and VL. The term "non-targetable" is intended to describe a region of VH and VL that is unable to target a specific antigen located in vivo because the antigen is absent or the antigen-binding site (ABS) has been disabled or altered to eliminate ABS functionality.

The above-described fusion proteins can be further conjugated with additional proteins/molecules. An additional protein as used herein describes a protein that is conjugated to a fusion protein or fusion protein complex such that it is linked to the side opposite to the side of the IL-10 monomer or the IL-10 variant molecule monomer. The addition of an additional protein effectively creates a multifunctional molecule that includes both the functionality of IL-10 or the IL-10 variant molecule and the functionality of the additional protein. The addition of additional proteins (e.g., IL-2, IL-7, IL-12, IL-15 cytokines, etc.) can occur, for example, to a fusion protein containing a VH and VL framework at the N-terminus of the VH region. For example, with respect to fusion protein complexes or a fusion protein for treating oncology, the accessory protein includes, but is not limited to, IL-10, a variant IL-10 molecule, IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons α, β, γ, TGF-β, or tumor necrosis factor α, β, basic FGF, EGF, PDGF, IL-4, IL-11 or IL-13, or any combination thereof. With respect to fusion protein complexes or fusion proteins for treating inflammatory diseases, the accessory protein includes, but is not limited to, TGF-β. With respect to fusion protein complexes or fusion proteins for the treatment of autoimmune diseases (such as, but not limited to, fatty liver disease), the additional molecule includes, but is not limited to, obticholic acid, aramchol, elafibranor, liraglutide, selonsertib or simtuzumab. The additional proteins defined above, when described using formulas I-V above, can be attached to a substituent X1 or Y1 on the N-terminal side of the VH portion of the framework.

The fusion proteins described above may also include additional amino acid sequences that aid in the recovery or purification of the fusion proteins during manufacturing. These sequences may include various sequence modifications or affinity tags such as, but not limited to, protein A, albumin binding protein, alkaline phosphatase, FLAG epitope, galactose binding protein, histidine tags, and any other tags that are well known in the art. See, for example, Kimple et al. (Curr. Protoc. Protein Sci., 2013, 73: Unit 9.9, Table 9.91, incorporated by reference in its entirety). In one aspect, the affinity tag is a histidine tag having the amino acid sequence HHHHHH (SEQ ID NO::32). The histidine tag may be removed from the final product or left intact. In another embodiment, the affinity tag is a modification of Protein A that is included in a fusion protein (e.g., in the VH region of the fusion proteins described herein) as described in SEQ ID NO: 34 or 44-53. One skilled in the art will appreciate that any fusion protein sequence described herein can be altered to include a modification of Protein A by inserting point amino acid substitutions into the framework regions of the antibody as described in the art.

In another embodiment, the various fusion proteins described above can be used in a method of treating a cancer, treating or preventing IBD or Crohn's disease, an autoimmune disease, NAFLD or NASH.

IL-10 variant polynucleotides

Also included within the scope of the present application are polynucleotide sequences that encode the variant IL-10 molecules and the various fusion proteins and/or immunocytokines described above. Once the key IL-10 receptor binding regions and/or interdomain angle regions in the variant IL-10 molecules of the present invention have been localized, the DNA modifications necessary to effect the desired modifications in the amino acid sequences are within the skill of the skilled artisan. Such modifications will utilize conventional and recombinant DNA techniques. For example, the addition or substitution of specific amino acid sequences can be introduced into the IL-10 sequence at the nucleic acid (DNA) level using site-directed mutagenesis techniques using synthetic oligonucleotides, which are also well known in the art.

In another embodiment, polynucleotides encoding a variant IL-10 sequence can be obtained using standard molecular biology techniques. For example, polynucleotide sequences encoding the above-described variant molecules can be obtained by recombinant methods, such as by screening cDNA and genomic libraries from cells expressing the gene, or by obtaining the gene from a vector known to include the gene. The gene of interest can also be produced synthetically, rather than cloned, based on known sequences. Molecules can be designed with the appropriate codons for a particular sequence. The complete sequence is then assembled from overlapping oligonucleotides prepared by standard techniques and assembled into the complete coding sequence. See, e.g., Edge, Nature (1981) 292: 756; Nambair et al., Science (1984) 223: 1299; and Jay et al., J. Biol. Chem. (1984) 259: 6311.

In one embodiment, the variant IL-10 molecule or proteins thereof is a nucleic acid molecule encoding any of SEQ ID NOs: 9-29, 33-53, 55, 57, or 59. In another embodiment, the variant IL-10 molecule is DV05, DV06, or DV07 of SEQ ID NOs: 56, 58, or 60, respectively. The nucleic acid molecule encoding DV05, DV06, or DV07 may include insertions, deletions, or substitutions (e.g., degenerate code) that do not alter the functionality of the variant IL-10 molecule. The nucleotide sequences encoding the variant IL-10 and fusion proteins described herein may differ from the sequences of SEQ ID NO: 1, 3, 5, 7, 9-29, 33-53, 55, 57, 59, 61, 63, 65, or 67 due to the degeneracy of the genetic code and may be 70-99%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homologous to the above sequences. The nucleotide sequence encoding the variant IL-10 and the fusion proteins of SEQ ID NO: 1, 3, 5, 7, 9-29, 33-53, 55, 57, 59, 61, 63, 65 or 67 may also be 70%-99%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homologous due to insertions, deletions or substitutions of at least one nucleotide. Also described herein are nucleotide sequences that have 70-99%, preferably 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence homology with SEQ ID NO: 2, 4, 6, 8, 56, 58, 60, 62, 64, 66 and 68 due to the insertion, addition, deletion or substitution of at least one nucleotide. In addition, the nucleotide sequences encoding the IL-10 variant and fusion proteins described herein may further comprise well-known sequences that assist, for example, in the expression, production or secretion of the proteins. Such sequences may include, for example, a leader sequence, a signal peptide, and/or translation initiation sites/sequence (e.g., a Kozak consensus sequence). The nucleotide sequences described herein may also include one of several restriction enzyme recognition sites that allow insertion into various expression systems/vectors.

In another embodiment, the polynucleotides are contained in vectors containing the desired sequences of the desired IL-10, or are artificially synthesized using oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR). See, e.g., Sambrook, supra. In another embodiment, nucleotide sequences encoding variant IL-10 molecules are prepared by an annealing process of complementary overlapping synthetic oligonucleotides produced in an automated polynucleotide synthesizer, followed by ligation and amplification of the ligated nucleotide sequences by PCR. See, e.g., Jayaraman et al., Proc. Natl. Acad. Sci. USA (1991) 88: 4084-4088. In addition, oligonucleotide-directed synthesis (Jones et al., Nature (1986) 54: 75-82), oligonucleotide-directed mutagenesis of pre-existing nucleotide regions (Riechmann et al., Nature (1988) 332: 323-327 and Verhoeyen et al., Science (1988) 239: 1534-1536), and enzymatic filling of missing oligonucleotides using T4 DNA polymerase (Queen et al., Proc. Natl. Acad. Sci. USA (1989) 86: 10029-10033) can be used to generate molecules for use in the methods.

A number of suitable expression vectors that can be used to express and deliver variant IL-10 molecules and fusion proteins are well known to those skilled in the art. These vectors include, for example, pUC type vectors, pBR type vectors, pBI type vectors, pGA type vectors, pBinl9, pBI121, pGreen series, pCAMBRIA series, pPZP series, pPCV001, pGA482, pCLD04541, pBIBAC series, pYLTAC series, pSB11, pSB1, pGPTV series, and viral vectors and the like.

Vectors carrying variant IL-10 molecules may also include other vector components necessary for the operation of the vectors. For example, the vector may include signal sequences, tag sequences, protease recognition sequences, selectable markers, and other regulatory sequences such as promoters necessary for the proper replication and expression of the variant IL-10 molecules. The particular promoters used in the vector are not particularly limited, provided that they can drive the expression of the variant IL-10 molecule in a variety of host cell types. Likewise, the type of tag sequence is not limited, provided that the tag sequence simplifies or facilitates purification of the expressed variant IL-10 molecule. These may include, for example, 6-histidine, GST, MBP, HAT, HN, S, TF, Trx, Nus, biotin, FLAG, myc, RCFP, GFP, and the like. The recognition sequences of the protease are not particularly limited, for example, recognition sequences such as Factor Xa, thrombin, HRV, protease 3C can be used. The selected markers are not particularly limited as long as they can detect transformed rice plant cells, for example, neomycin resistance genes, kanamycin resistance genes, hygromycin resistance genes, etc. can be used.

The resulting DNA constructs carrying the desired IL-10 variants or fusion protein can then be used directly in gene therapy or can be used to produce recombinant IL-10 variants or fusion proteins. In one embodiment, the variant IL-10 molecules or fusion protein of the present invention can be delivered by any method known in the art, including direct administration of the mutant IL-10 protein and gene therapy with a vector encoding the mutant IL-10 protein. Gene therapy can be performed using plasmid DNA or a viral vector, such as an adeno-associated viral vector, an adenoviral vector, a retroviral vector, etc. In some embodiments, the viral vectors of the invention are administered as viral particles, while in others they are administered as plasmids (e.g., as naked DNA).

Other methods of delivering nucleotide sequences include those already known in the art. These methods may include delivering nucleotide sequences such as, but not limited to, DNA, RNA, siRNA, mRNA, oligonucleotides or variants thereof encoding IL-10 or variant IL-10 molecules via a cell penetrating peptide, a hydrophobic group, an electrostatic complex, a liposome, a ligand, liposomal nanoparticles, a lipoprotein (preferably HDL or LDL), a folate-targeted liposome, an antibody (e.g., to a folate receptor, a transferrin receptor), a targeting peptide or an aptamer. The nucleotide sequences encoding variant IL-10 molecules can be delivered to an individual by direct injection, infusion, patches, bandages, aerosol or mist, or by delivery via a thin film. The nucleotide (or protein) can be directed to any area that requires targeted delivery of the cytokine stimulus. Areas may include, for example, the lung, gastrointestinal tract, skin, liver, brain via intracranial injection, deep metastatic tumor lesions via ultrasound-guided injection.

Testing IL-10 variants

In one embodiment, the variant IL-10 molecules or fusion proteins thereof will be screened for new functions that have not previously been generated using IL-10 homodimer sequences that suppress the secretion of inflammatory cytokines by macrophages but do not activate T cells. In one preferred embodiment, the variant IL-10 molecule or fusion proteins thereof will be based on the EBV-IL10 backbone, which has an anti-inflammatory response but does not have the ability to stimulate T cells. These variant EBV-IL10 molecules or fusion proteins will include modifications of the receptor binding domains and previously uncharacterized binding regions that alter the primary, secondary and tertiary structure to limit or expand the angle between IL-10 homodimers. In another embodiment, the variant IL-10 molecules containing modifications in the binding regions or in the regions responsible for interdomain angle formation will have a CD8 ⁺ T cell enhancing function. In another embodiment, the variant IL-10 molecules or proteins thereof containing modifications in the binding region or in the regions responsible for interdomain angle formation will have a myeloid cell suppressive function, but not a Kupffer cell enhancing function.

Once the variant IL-10 molecules or fusion proteins thereof are constructed and expressed, one skilled in the art can analyze the variant IL-10 molecules or fusion proteins thereof to determine whether the molecules possess the desired biological functions conveyed by modifications to the IL-10 receptor binding regions and/or interdomain angle. Many screening assays are known and available to test for the desired biological function. In one embodiment, the desired biological function includes, but is not limited to, reducing the anti-inflammatory response, reducing T cell stimulation, enhancing T cell function, enhancing Kupffer cell functionality, and reducing mast cell degranulation.

For example, it is known that exposure to IL-10 causes T cells to generate and secrete more IFNγ upon stimulation of the T cell receptor. Concomitantly, exposure to IL-10 prevents the secretion of TNFα, IL-6, and other proinflammatory cytokines secreted by monocytes/macrophages in response to LPS. IL-10 also suppresses the proliferation of FoxP3 ⁺ CD4 ⁺ Tregs. In one embodiment, those variant IL-10 molecules or fusion proteins thereof that maximize monocyte/macrophage suppression but do not affect T cells, including both stimulatory and suppressive responses, will be positively selected. In one embodiment, a screen for variant IL-10 molecules or fusion proteins thereof that have enhanced anti-inflammatory effects will be positively evaluated for the treatment of an autoimmune disease, an anti-inflammatory disease, or both. In other embodiments, variant IL-10 molecules or fusion proteins thereof that enhance the salvage functions of Kupffer cells and do not suppress Tregs will also be selected for development for the treatment of non-alcoholic steatotic hepatitis (NASH) and/or non-alcoholic fatty liver disease (NAFLD). In yet another embodiment, IL-10 variants that maximize T cell biology, including both stimulatory and suppressive responses, and have enhanced salvage in Kupffer cells will be selected for development for the treatment of cancer.

The literature is replete with descriptions of assays for the effect of cytokines on immune system cells such as T cells, monocytes/macrophages, Kupffer cells, Treg cells, and mast cells, for example. The present invention will employ these assay systems to test for a biological response using similar assays by contacting the IL-10 variant molecules or fusion proteins of the present invention.

Various methods are described in the art for assaying the efficiency of inducing a T cell response. Any of these methods are applicable to testing the variant IL-10 molecules described herein. For example, Chan et al. (2015) describe one such method applicable to variant IL-10 molecules. CD8 ⁺ T cells are isolated from peripheral blood mononuclear cells (PBMCs) using anti-CD8 microbeads. The isolated CD8 ⁺ T cells are activated with anti-CD3 and anti-CD28 antibody. For example, activation can occur using plates coated with at least about 5 to 20 μg/ml anti-CD3 antibody and at least about 1 to 5 μg/ml anti-CD28 antibody for a period of about 3 days. Following activation, T cells were harvested, plated, and treated with EBV-IL10 variants or their fusion proteins for approximately 3-5 days. Commercially available recombinant pegylated human IL-10 or EBV-IL10 could be used as a control. Following treatment with EBV-IL10 variants, T cells were treated with soluble anti-CD3. Following anti-CD3 treatment, cell culture medium was collected and analyzed by ELISA for interferon gamma (IFNγ) secretion.

Various methods have been described in the art for assaying monocyte/macrophage stimulation by cytokines. Any of these methods are applicable to testing the IL-10 variant molecules described herein. For example, Conway et al (2017) describe one such method that is applicable to IL-10 variant molecules. Human monocytes are isolated from the buffy coat of fresh donor blood using a Ficoll gradient followed by hypertonic density centrifugation in Percoll. After 30 min of culture in RPMI supplemented with 5% human serum and 1% L-glutamine, monocytes become attached to the substrate and are washed with SMEM Spinner medium to remove contaminating lymphocytes. Solutions and materials are tested for the absence of LPS. After four days of culture, monocytes/macrophages are contacted or incubated with 10 ng/ml LPS and various concentrations of variant IL-10 molecules for at least 24 hours. Culture supernatants are collected and TNF-α and IL-1β concentrations are determined by ELISA.

Various methods have been described in the art for analyzing the response of Kupffer cells to cytokines. Any of these methods are applicable to testing the variant IL-10 molecules described herein. For example, Chan et al (2016) describe one such method that is applicable to variant IL-10 molecules. Kupffer cells are seeded in 24-well or 96-well plates and incubated overnight in hepatocyte incubation medium (RPMI without phenol red, penicillin/streptavidin, cell maintenance supplement B (Invitrogen)). The cells are washed and exposed to variant IL-10 molecules for 24 hours. Cells were washed once and exposed to 15-20 μl DiI-LDL, DiI-VLDL, DiI-OxLDL or DiI-AcLDL, 2 μl DMSO, 15 μl Cytochalasin D, and uptake was measured after 4 h. All cells were washed once in 1× PBS and lysed in 110 μl cell lysis buffer. 45 μl of cell lysate were transferred to clear bottom black-walled plates, where fluorescence was read at 575 nm.

Various methods have been described in the art for analyzing the efficiency of cytokine stimulation of T regulatory cell responses. Any of these methods are applicable to testing the variant IL-10 molecules described herein. For example, Chan et al (2016) describe one such method applicable to variant IL-10 molecules. CD4 ⁺ T cells are isolated using CD4 ⁺ microbeads and cultured for 5-6 days in AIMV media containing varying concentrations of variant IL-10 molecule and 2 μg/ml immobilized anti-CD3 and 1 mg/ml anti-CD28. The cells are analyzed for FoxP3 expression by flow cytometry to determine whether TGF-β or IL-2 is induced in FOXP3 ⁺ CD4 ⁺ regulatory T cells.

Various methods have been described in the art for assaying the efficiency of mast cell proliferation in response to cytokine stimulation. The murine mast cell line MC/9 is a common cell line used for production testing of IL-10 molecules. In particular, IL-10 and variant IL-10 molecules induce dose-dependent proliferation of mast cells. Conversely, IL-10 suppresses Fc expression by mast cells, suggesting that IL-10 has both stimulatory and suppressive effects on these cells. Any of these methods are applicable to testing the variant IL-10 molecules described herein. For example, Thompson-Snipes et al. (1991) describe one such method that is applicable to variant IL-10 molecules. MC/9 mast cells were seeded in flat-bottomed 24-well plates containing 1 ml RPMI 1640, 10% FBS, 50 mM 2-ME and different concentrations of variant cytokines. After 3 days of culture, cells were counted using a cell counter to determine the effect of variant IL-10 molecules on mast cell proliferation.

IL-10 is known to play a role in inhibiting mast cell expression of the IgE receptor, FcεRI, and IgE-mediated cytokine production. Accordingly, methods for testing the effects of IL-10 on mast cells have been described in the prior art. These methods are applicable to testing the IL-10 variant molecules described herein. For example, Kennedy Norton et al (2008) describe one such method. Human mast cells are isolated from donor skin samples and cultured in stem cell factor (SCF)-containing medium in the presence or absence of IL-10. FcεRI expression is determined by flow cytometry using anti-FcεRI specific antibodies followed by FITC-labeled anti-mouse F(ab') ₂ antibodies.

Compositions and formulations containing variant IL-10 molecules

The variant IL-10 molecules or fusion proteins thereof of the present invention can also be formulated as a pharmaceutical composition comprising a therapeutically effective amount of the variant IL-10 molecule and pharmaceutical carriers and/or pharmaceutically acceptable excipients. The pharmaceutical composition can be formulated with commonly used buffers, excipients, preservatives, stabilizers. The pharmaceutical composition is formulated for administration to a patient in a therapeutically effective amount sufficient to provide the desired therapeutic result. Preferably, such an amount has minimal negative side effects. In one embodiment, the amount of the variant IL-10 molecule or fusion protein thereof administered will be sufficient to treat inflammatory diseases or conditions. In another embodiment, the amount of the variant IL-10 molecule or fusion proteins thereof administered will be sufficient to treat a malignant tumor. The amount administered may vary from patient to patient and will need to be determined taking into account the disease or condition of the individual or patient, the general health of the patient, the route of administration, the severity of side effects, etc. In a preferred embodiment, the pharmaceutical composition will comprise a variant IL-10 molecule or a fusion protein thereof that includes one or both of the modifications of the receptor binding domain and/or the interdomain angle of IL-10. In another embodiment, the variant IL-10 molecule is a pegylated form of the variant IL-10 molecule. In an even more preferred embodiment, the pharmaceutical composition comprises a variant IL-10 molecule included as a fusion protein or immunocytokine and pharmaceutical excipients.

The effective amount for a particular patient may vary depending on factors such as the condition being treated, the general health of the patient, the route and dose of administration, and the severity of side effects. The appropriate dose to administer to a patient is typically determined by the physician using parameters or factors that are known or suspected to affect treatment in the field, or are expected to affect treatment. Generally, the dose is started at an amount slightly less than the optimal dose and then increased in small increments until the desired or optimal effect is achieved relative to any negative side effects. Important diagnostic measurements include measurements of symptoms, such as inflammation or levels of inflammatory cytokines produced.

The dose administered to a patient can also be optimized based on the addition of modifications to the IL-10 or variant IL-10 molecule to increase a specific serum half-life (see, for example, pegylated IL-10, as described in detail in, for example, U.S. Patents 9943568, 10010588, and 10143726, is known to improve the circulating half-life of IL-10). The fusion protein, immunoconjugates, minibodies, and diabodies comprising IL-10 or variant IL-10 described herein are also capable of prolonging the circulating half-life while maintaining high binding affinity for the IL-10 receptor. Thus, in one embodiment, various diseases, disorders or conditions associated with IL-10 or that can be ameliorated by administering a fusion protein, a complex of fusion proteins, an immunoconjugate or a diabody comprising IL-10 or a variant IL-10 can be administered to a patient in need thereof. In one preferred embodiment, diseases, disorders or conditions associated with IL-10 can be treated or prevented by administering to a patient in need thereof a therapeutically effective amount of an immunoconjugate complex, a fusion protein or a diabody with EBV IL-10 or a variant thereof (including a variant that affects affinity for the IL-10 receptor), wherein the immunoconjugate complex, fusion protein or diabody has a molecular weight of about 60 to 155 kDa, wherein the therapeutically effective amount is in the range of about 0.5 μg/kg to 100 micrograms/kilogram. The immunoconjugate, fusion protein, diabody described herein may be administered daily, three times per week, twice per week, once per week, once every two months, or once per month. The EBV IL-10 portion and the variable regions of the immunoconjugate, fusion protein, or diabody may have any configuration or combination of the structures discussed herein. The molecules with increased half-life described herein will be effective in the treatment of a number of diseases, including, but not limited to, cancer, inflammatory diseases, autoimmune diseases (e.g., non-alcoholic steatotic hepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD)) and high cholesterol.

Methods of co-administering or treating with a second therapeutic agent, such as a cytokine, steroid, chemotherapeutic agent, antibiotic, anti-inflammatory agents, or radiation, are well known in the art. This may include combination therapy with other therapeutic agents such as, but not limited to, one or more of the following: interferon-β, such as IFNβ-1α and IFN-β-1β; myelin basic protein mimicking protein; corticosteroids; IL-1 inhibitors; TNF inhibitors; anti-TNFα antibodies, anti-IL-6 antibodies, IL-1br-Ig fusion, anti-IL-23 antibodies, anti-CD40, ligand, and CD80 antibodies; IL-12 and IL-23 antagonists, such as IL-12 and IL-23 p40 subunit antagonists (e.g., inhibitory antibodies against the p40 subunit); IL-22 antagonists; small molecule inhibitors, such as methotrexate, leflunomide, sirolimus (rapamycin) and its analogues, such as CCI-779; Cox-2 and cPLA2 inhibitors; NSAIDs; p38 inhibitors; TPL-2; Mk-2; NFkβ inhibitors; RAGE or soluble RAGE; P-selectin or PSGL-1 inhibitors (e.g. small molecule inhibitors, antibodies to them, such as anti-P-selectin antibodies); estrogen receptor beta (ERB) agonists or ERB-NFkβ antagonists.

Additionally, combination therapy useful for administration with variant IL-10 molecules or fusion proteins thereof may include TNF inhibitors, including, for example, chimeric, humanized, effectively human, human, or in vitro generated antibodies or antigen-binding fragments thereof that bind to TNF; soluble fragments of the TNF receptor, for example, p55 or p75 human TNF or derivatives thereof, for example, 75 kDa TNFR-IgG receptor (75 kDa TNF-IgG receptor fusion protein, ENBREL™), p55 kDa TNF-IgG receptor fusion protein; and TNF enzyme antagonists, for example, TNFα-converting enzyme (TACE) inhibitors. Other combination treatments with anti-inflammatory agents/medicines include, but are not limited to, standard non-steroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase-2 inhibitors. NSAIDs may include aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, oxaprozin, piroxicam, salsalate, sulindac and/or tolmetin. The cyclooxygenase-2 inhibitor used in the composition according to the application may, for example, be celecoxib or rofecoxib.

Additional therapeutic agents that may be co-administered and/or co-formulated with variant IL-10 molecules or proteins thereof include one or more of: interferon-β, such as IFN β-1α and IFN β-1β; COPAXONE®; corticosteroids; IL-1 inhibitors; TNF antagonists (e.g., a soluble fragment of the TNF receptor, e.g., p55 or p75 human TNF receptor or derivatives thereof, e.g., 75 kdTNFR-IgG; antibodies to CD40 and CD80 ligand; IL-12 and/or IL-23 antagonists, e.g., IL-12 and IL-23 p40 subunit antagonists (e.g., inhibitory antibodies that bind to the p40 subunit of IL-12 and IL-23); methotrexate, leflunomide, and sirolimus (rapamycin) or an analogue thereof, e.g., CCI-779. Other therapeutic agents may include Imphimzi or Atezolizumab.

For the treatment of NASH, for example, variant IL-10 molecules or fusion proteins thereof can be combined with cholesterol-lowering agents such as statins and non-statin drugs. These agents include, but are not limited to, simvastatin, atorvastatin, rosuvastatin, lovastatin, pravastatin, gemfibrozil, fluvastatin, cholestyramine, fenofibrate, cholesterol absorption inhibitors, bile acid binding resins or sequestrants, and/or microsomal triglyceride transfer protein (MTP) inhibitors.

An effective amount of therapeutic agents will affect the level of inflammation or disease or condition by alleviating symptoms. For example, the effect may include an effect level that is at least 10%, at least 20%, at least about 30%, at least 40%, at least 50%; or more, such that the disease or condition is alleviated or completely cured.

Pharmaceutical compositions comprising a variant IL-10 molecule or fusion proteins thereof are mixed with a pharmaceutically acceptable carrier or excipient. Various pharmaceutical carriers are known in the art and can be used in the pharmaceutical composition. For example, the carrier can be any compatible, non-toxic substance suitable for delivering the variant IL-10 molecule compositions of the invention to a patient. Examples of suitable carriers include normal saline, Ringer's solution, dextrose solution, and Hank's solution. The carriers may also include any poloxamers generally known to those skilled in the art, including, but not limited to, those having molecular weights of 2900 (L64), 3400 (P65), 4200 (P84), 4600 (P85), 11400 (F88), 4950 (P103), 5900 (P104), 6500 (P105), 14600 (F108), 5750 (P123), and 12600 (F127). The carriers may also contain emulsifiers, including, but not limited to, polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, and many others. Non-aqueous carriers such as fixed oils and ethyl oleate may also be used. The carrier may also contain additives such as substances that enhance isotonicity and chemical stability, e.g., buffers and preservatives, see, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984). Therapeutic and diagnostic formulations can be prepared by mixing with physiologically acceptable carriers, excipients, or stabilizers in the form of lyophilized powders, suspensions, aqueous solutions, or suspensions, e.g.

The compositions of the invention can be administered orally or injected into the body. Formulations for oral use can also include compounds for additional protection of the variant IL-10 molecules from protease in the gastrointestinal tract. Injections are typically intramuscular, subcutaneous, intradermal or intravenous. Alternatively, intra-articular injection or other routes can be used in appropriate circumstances. Parenterally administered variant IL-10 molecules are advantageously formulated in the form of an injection dosage unit (solution, suspension, emulsion) in combination with pharmaceutical carriers and / or pharmaceutically acceptable excipients. In other embodiments, the composition can be administered to a patient using an implantable or injection drug delivery system.

Therapeutic applications of IL-10 variants

In one embodiment, the present application relates to methods of treating, alleviating or reducing symptoms associated with inflammation, an inflammatory disease or an autoimmune disease. The present application also relates to IL-10, variant IL-10 molecules, fusion proteins thereof or chimeric molecules thereof for use as a medicament for inflammation, an inflammatory disease, an autoimmune disease, a cancer or oncology. The present application also relates to the use of IL-10, variant IL-10 molecules, fusion proteins thereof or chimeric molecules for the treatment of inflammation or an inflammatory disease, or an autoimmune disease, a cancer or oncology. These include, for example, IBD, Crohn's disease, ulcerative colitis, NASH, NAFLD, hypercholesterolemia, cancer and many others. The method involves administering a therapeutically effective amount of one or more variant IL-10 molecules or fusion proteins thereof described herein. In one embodiment, the invention includes a method of treating inflammatory diseases or autoimmune diseases, comprising administering a therapeutically effective amount of a variant IL-10 molecule comprising one or more modifications associated with the receptor binding domain and/or the region responsible for forming an interdomain angle. In one preferred embodiment, the method comprises administering a variant EBV-IL10 molecule or fusion proteins thereof. In one preferred embodiment, variant IL-10 molecules or fusion proteins thereof suitable for treating an inflammatory disease include variant molecules that have limited interdomain angles compared to wild-type IL-10 molecules and/or also exhibit lower affinity for the receptor. In other embodiments, variant IL-10 molecules or fusion proteins thereof suitable for treating an inflammatory disease include variant molecules that have weakened interdomain angles compared to wild-type IL-10 molecules and/or also exhibit lower receptor affinity. PEGylated forms of variant IL-10 molecules are also contemplated as part of the present invention for an inflammatory disease or inflammation.

Inflammatory diseases or autoimmune diseases according to the present invention include any disease or condition associated with unwanted or unnecessary inflammation and immune response. These diseases include, but are not limited to, inflammatory bowel disease (IBD), Crohn's disease, rheumatoid arthritis, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH). In other embodiments, the diseases or conditions include neurodegenerative disorders such as Parkinson's disease, amelotrophic lateral sclerosis (ALS), fatal familial insomnia, Rasmussen encephalitis, Down syndrome, Huntington's disease, Gerstmann-Straussler-Scheinker disease, tuberous sclerosis, neuronal ceroid lipofuscidosis, subacute sclerosing panencephalitis, Lyme disease; tsetse disease (African sleeping sickness), HIV dementia, bovine spongiform encephalopathy (mad cow disease); Creutzfeldt-Jakob disease; Herpes simplex encephalitis, herpes zoster cerebellitis, general paresis (syphilis), tuberculous meningitis, tuberculous encephalitis, optic neuritis, granulomatous angiitis, temporal arthritis, cerebral vasculitis, Spatz-Lindenberg disease, methamphetamine-associated vasculitis, cocaine-associated vasculitis, traumatic brain injury, stroke, Lance-Adams syndrome, post-anoxic encephalopathy, radiation necrosis, limbic encephalitis, Alzheimer's disease, progressive supranuclear palsy, striatonigral degeneration, corticobasal ganglionic degeneration, primary progressive spinal aphasia, frontotemporal dementia associated with chromosome 17, spinal muscular atrophy, HIV-associated myelopathy, HTLV-1-associated myelopathy (tropical spastic paraparesis), tabes dorsalis (syphilis), transverse myelitis, post-polio syndrome, spinal cord injury, radiation myelopathy, Charcot-Marie-Tooth syndrome, HIV-associated polyneuropathies, campylobacter-associated motor axonopathies, chronic inflammatory demyelinating polyneuropathy, diabetic amyotrophy, phantom limb syndrome, complex regional pain syndrome, diabetic neuropathies, paraneoplastic neuropathies, myotonic dystrophy, HTLV-1-associated myopathy, trichinosis, inflammatory myopathies, dermonymic myositis (polymyositis, myositis) sickle cell disease, alpha-1 antitrypsin deficiency, tuberculosis, subacute bacterial endocarditis, chronic viral hepatitis, viral cardiomyopathy, Chagas disease, malaria, coxsackie B infection, macular degeneration, retinitis pigmentosa, vasculitis, inflammatory bowel disease, rheumatoid arthritis, pemphigus, Churg-Strauss syndrome, myocardial infarction, toxic epidermal necrolysis, shock (eg, anaphylactic shock), type 1 diabetes, autoimmune thyroiditis, lymphoma, ovarian cancer, lupus (systemic lupus erythematosus), asthma, progeria, sarcoidosis, type 2 diabetes, and metabolic syndrome. Other diseases or conditions associated with inflammation that are embodiments of the invention include inflammatory disorders of the lung, such as bronchitis, oxidant-induced lung injury and chronic obstructive pulmonary disease; inflammatory disorders of the eye, including corneal dystrophy, ocular hypertension, trachoma, onchocerciasis, retinitis, uveitis, sympathetic ophthalmitis and endophthalmitis; chronic inflammatory disorders of the gums, including periodontitis; chronic inflammatory disorders of the joints, including arthritis, septic arthritis and osteoarthritis, tuberculous arthritis, leprous arthritis, sarcoid arthritis; skin disorders, including scleroderma, sunburn, psoriasis and eczema; encephalomyelitis and viral or autoimmune encephalitis; autoimmune diseases, including immune complex disease, vasculitis, heart disease, including coronary artery disease, heart failure and cardiomyopathy. Other non-limiting examples of diseases for which IL-10 variant molecules or fusion proteins thereof may be useful include adrenal insufficiency, hypercholesterolemia, atherosclerosis, bone disease associated with increased bone resorption such as osteoporosis, preeclampsia, eclampsia, uremic complications, chronic liver failure and other inflammation-related disorders such as cystic fibrosis, tuberculosis, cachexia, ischemia/reperfusion, hemodialysis-related conditions, glomerulonephritis, restenosis, the inflammatory consequences of viral infection, hypoxia, hyperbaric seizures and toxicity, dementia, Sydenham's disease, Huntington's disease, epilepsy, Korsakoff's disease, dementia associated with cerebrovascular accident, nitric oxide-mediated cerebral injury and related sequelae, ischemic cerebral edema (stroke), migraine, vomiting, immune disease, allograft rejection, infections caused by invasive microorganisms; and aging.

The most effective IL-10 variant or fusion protein thereof for the treatment of anti-inflammatory diseases or conditions include those that have a reduced ability to stimulate T cells. Thus, modification of the receptor binding domain by an amino acid substitution at position 75 has been shown by the inventor to induce the least amount of T cell stimulation. In particular, EBV IL-10 bearing the A75I substitution in SEQ ID NO: 3 (or SEQ ID NO: 57) has been shown to reduce T cell stimulation (see, for example, Figure 8E, designated as DV06). Thus, one particularly preferred embodiment relates to the use of a diabody and a monobody with variant IL-10 molecules bearing a mutation based on DV06 (amino acid substitution at position 75 in SEQ ID NO: 3 or SEQ ID NO: 57). In a more preferred embodiment, the methods for an inflammatory disease will use a fusion protein complex or a fusion protein comprising SEQ ID NO: 26-27; 37; 40; 41-42, 43, 48-49, or a combination thereof.

In another embodiment of the invention, a method of treatment comprises administering IL-10 or a variant IL-10 molecule or fusion proteins thereof to treat or reduce symptoms associated with a cancer. The method involves administering a therapeutically effective amount of one or more of the IL-10 or variant IL-10 molecule or fusion proteins thereof described herein. In one embodiment, the invention includes a method of treating or reducing symptoms associated with a cancer, comprising administering a therapeutically effective amount of a variant IL-10 molecule comprising one or more modifications associated with a receptor-binding domain and/or a region responsible for forming an interdomain angle. In one preferred embodiment, the method comprises administering a variant EBV-IL10 molecule or fusion proteins thereof. Variant IL-10 molecules or fusion proteins thereof suitable for treating a malignant tumor include variant molecules that have a limited interdomain angle compared to wild-type IL-10 molecules and/or also exhibit a higher affinity for the receptor. Variant IL-10 molecules or fusion proteins thereof useful for treating or reducing symptoms associated with a malignant tumor include variant molecules that have weakened interdomain angles compared to wild-type IL-10 molecules and/or also exhibit a higher affinity for the receptor. PEGylated forms of variant IL-10 molecules are also contemplated as part of the present invention for treating a malignant tumor. One specific example of a fusion protein capable of reducing tumor volume in vivo comprises a variant IL-10 bearing two substitutions at amino acid positions 31 and 75 of SEQ ID NO: 3, which include the specific substitutions V31 and A75I, designated DV07 (e.g., SEQ ID NO::59). Figures 16A-C demonstrate that, at various doses, a fusion protein comprising a DV07 EBV IL-10 molecule conjugated to a structural diabody designated D:DV07 reduced tumor volume over a period of seven and ten days. Thus, one particularly preferred embodiment involves the use of a diabody and a monobody with variant IL-10 molecules bearing a mutation based on DV07 (substitutions at amino acid positions 31 and 75 of SEQ ID NO: 3; or SEQ ID NO: 59). In a more preferred embodiment, the methods of treating a cancer or oncology or tumors will use a fusion protein complex or fusion protein comprising SEQ ID NO: 28-29; 33; 34; 35-36; 38-39; 46-47, 61, 63, 65, or 67, or a combination thereof.

A malignant tumor or proliferative disorder treatable with the IL-10 variant molecules or fusion proteins thereof described herein include various forms of malignancy, including, but not limited to, malignancy of the uterus, cervix, breast, prostate, testicles, penis, gastrointestinal tract, such as the esophagus, oropharynx, stomach, small or large intestine, colon or rectum, kidney, renal cell, bladder, bone, bone marrow, skin, head or neck, skin, liver, gallbladder, heart, lung, pancreas, salivary gland, adrenal gland, thyroid, brain, such as glioma, ganglia, central nervous system (CNS) and peripheral nervous system (PNS), and the immune system, such as the spleen or thymus. The present application relates to methods of treating, for example, immunogenic tumors, non-immunogenic tumors, dormant tumors, virus-induced malignancies, for example, epithelial cell cancer, endothelial cell cancer, squamous cell carcinoma, papillomavirus, adenocarcinomas, lymphomas, carcinomas, melanomas, leukemias, myelomas, sarcomas, teratocarcinomas, chemically induced malignancies, metastasis and angiogenesis. The application also contemplates reducing tolerance to a tumor cell or a malignant cell antigen, for example, by modulating the activity of a regulatory T cell (Treg) or CD8 ⁺ T cell. In a preferred embodiment, the variant IL-10 molecules are particularly suitable for treating patients or individuals with metastatic liver disease.

In other embodiments, methods for treating or reducing symptoms associated with an inflammatory disease or cancer comprise administering variant IL-10 molecules or fusion proteins thereof or derivative forms thereof (e.g., PEGylation) in combination with other therapeutic agents. These therapeutic agents include, but are not limited to, a cytokine or cytokine antagonist such as IL-12, IL-2, IL-15, interferon-alpha or an anti-epidermal growth factor receptor agent, doxorubicin, epirubicin, an antifolate such as methotrexate or fluorouracil, irinotecan, cyclophosphamide, radiation therapy, hormonal or anti-hormonal therapy such as an androgen, estrogen, an antiestrogen, flutamide or diethylstilbestrol, surgery, tamoxifen, ifosfamide, mitolactol, an alkylating agent such as melfolactol, an alkylating agent such as melphalan or cis-platin, etoposide, vinorelbine, vinblastine, vindesine, a glucocorticoid, a histamine receptor antagonist, an angiogenesis inhibitor, radiation, radiation sensitizer, anthracycline, vinca alkaloid, taxane such as paclitelax and doaxcetel, cell cycle inhibitor such as cyclin-dependent kinase inhibitor, monoclonal antibody against another tumor antigen, monoclonal antibody-toxin complex, T-cell adjuvant, bone marrow transplant, or antigen-presenting cells such as dendritic cell therapy.

In other embodiments, the present application also includes methods for treating lipid-related disorders such as hypercholesterolemia and hypertriglyceridemia and/or improving lipid parameters such as total cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, very-low-density lipoprotein (VLDL) cholesterol, triglycerides, and non-HDL cholesterol, comprising administering variant IL-10 molecules or derivative forms thereof (e.g., PEGylation).

The most effective IL-10 variant or fusion protein thereof for the treatment of lipid-related diseases or disorders include those that have the least inhibitory effect on macrophages. Thus, alteration of the receptor binding domain by amino acid substitution at position 31 (in combination with an increased interdomain angle in the homodimer) has been shown by the inventor to induce the least macrophage response. In particular, EBV IL-10 bearing the V31L substitution in SEQ ID NO: 3 has been shown to reduce the macrophage response (see, for example, Figure 8A, designated as DV05, or SEQ ID NO: 55). Thus, one particularly preferred embodiment involves the use of a diabody and a monobody with variant IL-10 molecules bearing a mutation based on DV05 (substitution at amino acid positions 31 of SEQ ID NO: 3 or SEQ ID NO: 55). In a more preferred embodiment, methods of treating lipid-based diseases or disorders will utilize a fusion protein complex or fusion protein comprising SEQ ID NO: 24-25, 50-51, or 45.

In another embodiment, variant IL-10 molecules or fusion proteins thereof, viral IL-10 (including EBV or CMV IL-10), or wild-type IL-10, either of which may optionally include pegylation or hesylation, are used in a method of targeting mast cells to reduce mast cell degranulation. In a preferred embodiment, the method of targeting mast cells comprises contacting viral IL-10 or variant IL-10 molecules to treat seasonal allergies or an acute anaphylactic response. In another aspect, variant IL-10 molecules, viral IL-10 (including EBV or CMV IL-10), or wild-type IL-10 are used to reduce IgE responsiveness.

In another embodiment, the variant IL-10 molecules or fusion proteins thereof of the present invention are preferably useful in the disclosed methods (e.g., anti-inflammatory and/or anti-cancer) in screening a patient population. In one embodiment, those patients who exhibit a profile where there is an increased or high IFNγ response are most responsive or ideal for the use of variant IL-10 molecules for the treatment of cancer. In another embodiment, those patients who exhibit a profile where there is a decreased or low IFNγ response are most responsive or ideal for the use of variant IL-10 molecules for the treatment of inflammation.

The broad scope of this application is best understood by reference to the following examples, which are not intended to limit the invention to any particular embodiments. All citations herein are hereby incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Many modifications and variations of this invention can be made without departing from the spirit and scope thereof, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example, and the invention should be limited by the terms of the appended claims together with the full scope of equivalents to which such claims are entitled; and the invention should not be limited to the specific embodiments that have been presented herein by way of example. Additionally, all references, patents and patent applications cited in the above description are incorporated herein by reference.

EXAMPLES

The following examples are merely illustrative of various embodiments of the application and should not be construed in any way as limiting the scope of the application.

Example 1

EBV-IL-10 variants or proteins thereof are constructed by altering the primary sequence using standard molecular biology cloning techniques. The alterations in the primary sequence are designed to alter the affinity of the receptor-binding domains and to open or close interdomain angles. Receptor affinity can be altered by substituting amino acids at and near positions 31 and/or 75 in the mature secreted sequence. Interdomain angles can be altered by, for example, but not limited to, the introduction of prolines into the non-alpha helical sequences between helices C and D, and D and E. Prolines cause the primary amino acid sequence to bend in a linear direction, potentially altering subsequent interdomain angles governed by the secondary and tertiary structures of helices D and E. Similarly, the introduction of amino acids with bulky side chains, such as tryptophan, can introduce less significant changes in the linear structure of the amino acid backbones, resulting in less profound changes in the secondary and tertiary structures.

Example 2

The following examples provide a description of how IL-10 variant molecules or its fusion proteins are assessed in macrophages.

Human blood was collected from healthy patient populations or patients with inflammatory disease (e.g., Crohn's disease), and freshly drawn buffy coats were processed to obtain PBMC using standard Ficoll density gradient centrifugation procedures. PBMC were then enriched for CD14 ⁺ monocytic cells using the EasySep™ Human Monocyte Enrichment kit (Catalog #19059, Stem Cell Technologies) according to the manufacturer's instructions. Enrichment efficiency was assessed by standard flow cytometry.

Enriched monocytes are seeded in 24-well plates at 2 x 10 ⁶ cells/ml/well in RPMI medium supplemented with 5% human serum and PSG. Cells are treated with serial dilutions (0, 0.1, 1, 10, 100, 1000 ng/ml) of the variant IL-10 molecule for 1 h at 37°C/5% CO ₂ in a humidity-controlled incubator and then exposed to 10 ng/ml LPS (Catalog # L4391, Sigma-Aldrich) for 12-16 h. After overnight incubation, supernatants are collected and inflammatory cytokines (IL-6, TNFα, IL-1β) are measured either by standard ELISA or by the iQue Screener (Intellicyt).

The study utilizes the above-described method to compare the effects of non-PEGylated EBV-IL10 and non-PEGylated human IL-10 on immunosuppressive abilities toward macrophages. Figures 2A, 2B and 3A, 3B show that EBV-IL10 retained the ability to suppress the inflammatory cytokines IL-1β and TNFα, indicating that despite the differences in interdomain angle, EBV-IL10 is able to maintain its suppressive anti-inflammatory functions in a manner similar to human IL-10.

Example 3

The following examples provide a description of how IL-10 variant molecules or its fusion proteins are assessed on human CD8 ⁺ T cells.

Human blood was collected from a population of healthy patients or patients with an inflammatory disease (e.g., Crohn's disease), and freshly collected buffy coats were processed to obtain PBMCs using standard Ficoll density gradient centrifugation methods. PBMCs were then enriched for CD8 ⁺ T cells using the EasySep™ Human CD8 ⁺ T Cell Enrichment Kit (Catalog #19053, Stem Cell Technologies) according to the manufacturer's instructions. Enrichment efficiency was assessed by standard flow cytometry methods. Enriched cells were suspended in AIMV culture medium (Thermo Fisher Scientific, catalog #12055083). 24-well plates were coated with 10 μg/ml anti-CD3 (catalog #16-0039-85, Thermo Fisher Scientific) and 2 μg/ml anti-CD28 (catalog #16-0289-85, Thermo Fisher Scientific) for 2 h by incubation at 37°C/5% _CO2 in a humidity-controlled cell culture incubator followed by 1–2 washes with 1× PBS.

Enriched CD8 ⁺ T cells (3× ¹⁰⁶ /ml/well) were added to anti-CD3/anti-CD28 coated plates and incubated for 72 h at 37°C/5% CO2 in a humidity-controlled cell culture incubator.

After 72 h, cells were harvested, counted and 100 μl were transferred to a 96-well round-bottom plate (2 x 10 ⁵ cells/well) in the presence/absence of serial dilutions (0, 0.1, 1, 10, 100, 1000 ng/ml with 100 μl/well added) of IL-10 variant molecules or control sample. Testing was performed in triplicate. Cells with IL-10 variant molecules or its fusion proteins were incubated for 72 h at 37°C/5%CO ₂ in a humidity-controlled incubator. After 72 hours, cells were harvested, washed, and placed in a fresh 96-well round-bottom plate containing soluble anti-CD3 antibody (catalog #16-0039-85, Thermo Fisher Scientific) for 4 hours at 37°C/5% _CO2 in a humidity-controlled incubator.

The study uses the above method to compare the effects of non-PEGylated EBV-IL10 and non-PEGylated human IL-10 on CD8 ⁺ T cell stimulation. Figures 2C and 3C show that EBV-IL10 exhibits reduced levels of IFNγ (an indicator of T cell stimulation) compared to human IL-10, indicating that by altering the interdomain angle, there is an ability to modulate T cell stimulation. Figures 4A and 4B show that half of the treated donors exhibit the desired full anti-inflammatory effect and half do not. The variants selected for development would mimic the response of Donor 1, complete suppression of inflammatory cytokine secretion by macrophage cells in response to LPS and no induction of IFNγ by activated CD8 ⁺ T cells. Donor 2 showed the same suppression of inflammatory cytokine secretion by monocytes/macrophages as donor 1, but there was a rightward shift in the curve and maximal activation of IFNγ secretion by T cells. A variant of the IL-10 molecule that alters the receptor affinity and interdomain angle should further reduce T cell activation in patients similar to donor 2.

Example 4

Human monocytes/macrophages, T cells, and murine MC/9 cells purchased from ATCC were cultured as previously and the response to mono- or di-PEGylated EBV-IL10 at the N-terminus of 5 kDa was assessed. PEGylation of EBV-IL10 resulted in a slight reduction in the macrophage response to LPS (Figure 5B) but almost complete suppression of IFNγ induction in stimulated T cells (Figure 5C). Similarly, PEGylation of EBV-IL10 nearly abolished its stimulatory effect on MC/9 cells (Figure 5A).

Different forms of the EBV-IL-10 variant diabody with VH and VL regions from anti-CD3α and anti-EGFR were tested using the MC/9 cell proliferation assay. The EBV-10 variant portions included D:DV05 (EBV IL-10 with the V31 mutation), D:DV06 (EBV IL-10 with the A75I mutation), and D:DV07 with the V31L and A75I mutations). In addition, the DV07 diabody, which includes VH and VL regions from anti-HIV and anti-Ebola, was tested. The different forms of the variant diabody were compared with human IL-10 and EBV IL-10. The results are shown in Fig. 15.

Other forms of EBV-IL-10 fusion proteins were also tested in vitro . Specifically, DhivDebo:DV06 (SEQ ID NOS: 26 and 27) and DmadcamDebo:DV06 (SEQ ID NOS: 41 and 42) were compared to human IL-10 in the macrophage and T cell response assays described herein. The results are shown in Figures 20A and 20B.

Example 5

The following example presents a typical protocol for testing IL-10 and a variant IL-10 molecule and its fusion proteins in an in vivo tumor model. All in vivo studies are performed in accordance with standard operating procedures and established guidelines approved by the Institutional Animal Care and Use Committee (“IACUC”).

Eight-week-old female Balb/C mice were purchased from a qualified retailer, quarantined for one week, and maintained on normal food and water with bedding changed once per week under a standard 24-hour light/dark cycle.

CT26 tumor cells (2×10 ⁵ ) were suspended in Hanks' buffered saline and implanted subcutaneously into eight-week-old mice and allowed to grow. Balb/C Envigo wild-type or B-cell knockout (Jackson) mice bearing CT26 tumors (average 50-150 ^mm3 ) were treated with 0.4 and 0.2 mg/kg three times a week (q3w), 0.2 and 0.1 mg/kg daily (qd) for 5 days every other day with IL-10 or variant IL-10 molecules or its fusion proteins (e.g., the EBV IL-10 variant molecule bearing two substitutions in the receptor-binding domain, DV07 (Figure 8C), covalently linked to VH and VL from two different antibodies or diabody) subcutaneously (scruff of the neck) for 10 days. Tumor length and width were measured every three days with electronic calipers and tumor volume was calculated ((L× ^W2 )/2)). B cells were depleted from wild-type mice by intravenous (i.v.) administration of 200 μg/mouse anti-mouse CD20 antibody. The results of one such study are shown in Figure 16, which used a variant IL-10 molecule designated D:DV07, which is a variant IL-10 carrying the V31 and A75I mutations and containing variable regions from anti-CD3α and anti-EGFR.

Figures 17A and 17B compare two formats of variant IL-10 fusion proteins (i.e., variant IL-10 including both the V31L and A75I mutations, DV07) shown in Figures 9C (large format) and 9f (small format) in an in vivo tumor model. The fusion proteins are non-targeted proteins and contain the VH and VL regions from an anti-HIV antibody and an anti-Ebola antibody (large format) and the VH and VL regions from an anti-Ebola antibody. A dosing study examined the effects of the small format non-targeted IL-10 fusion protein administered five days on and two days off (Figure 17A) compared to pegylated recombinant human IL-10 (0.75 mg/kg per day). The dosing study also examined the effects of large and small formats of non-targeted IL-10 fusion protein administered three times weekly (Figure 17B) compared to pegylated IL-10 (0.75 mg/kg per day).

Studies using small and large format IL-10 fusion proteins (i.e., variant IL-10 containing both the V31l and A75I mutations, DV07) with tumor targeting capabilities were also conducted in vivo . Figure 18A presents the results of daily administration of various targeted variant IL-10 fusion proteins, where the large format (DegfDebo:DV07) and small format (Degf:DV07) were compared with the small format non-targeting IL-10 fusion protein (Debo:DV07) and PEGylated IL-10. Figure 18B shows the results of thrice weekly administration of various large format targeted variant IL-10 fusion proteins, where the large format (DegfDebo:DV07) at different doses (1 mg/kg and 0.25 mg/kg) was compared to the small format non-targeting IL-10 fusion protein (DhDe:DV07) and PEGylated IL-10. Figure 18C shows the results of thrice weekly administration of various small format targeted variant IL-10 fusion proteins, where the small format (Degf:DV07) at different doses (1 mg/kg and 0.25 mg/kg) was compared to the small format non-targeting IL-10 fusion protein (Debo:DV07) and PEGylated IL-10.

Example 6

The following example presents a typical protocol for testing IL-10 and variant IL-10 molecules and their fusion proteins in an in vivo cholesterol model. All in vivo studies are performed in accordance with standard operating procedures and established guidelines approved by the IACUC.

Eight-week-old female C57BL/6J mice were purchased from a qualified retailer, quarantined for one week, and maintained on normal food and water with bedding changed once per week under a standard 24-hour light/dark cycle.

Eight-week-old female C57BL/6J mice from Jackson Laboratories were maintained on a high-fat diet (Envigo) for three weeks. Plasma samples were obtained by retro-orbital bleeding from each mouse prior to treatment with variant IL-10 or IL-10 or fusion protein (e.g., variant EBV IL-10 containing a single substitution at amino acid position 31 (V31L) of SEQ ID NO:3 and linked to a diabody (variant D:DV05 EBV IL-10)). Mice were treated subcutaneously for two weeks with 0.4 and 0.2 mg/kg three times weekly (q3w) and 0.2 and 0.1 mg/kg weekly (qd) for 5 treatment days with a 2-day therapeutic rest. Animals were treated for two weeks and blood was collected, after which pre- and post-dose cholesterol was quantified. One day before treatment, B cells were depleted by intravenous (i.v.) administration of 200 μg/mouse anti-mouse CD20 antibody. Results from one such study are shown in Figures 19A and 19B.

Example 7

The following example presents a typical protocol for testing IL-10 and its fusion proteins in the dextran sodium sulfate (“DSS”) in vivo inflammation model. All in vivo studies are performed in accordance with standard operating procedures and established guidelines approved by the IACUC.

Eight-week-old female Balb/C mice were purchased from a qualified retailer, quarantined for one week, and maintained on normal food and water with weekly bedding changes under a standard 24-hour light/dark cycle. B-cell knockout mice (Jackson) were fed 4% DSS in water ad libitum for six days, after which they were provided normal water. On day 5, mice are treated with 0.4 and 0.2 mg/kg three times a week (q3w), 0.2 and 0.1 mg/kg daily (qd) for five days every other day with IL-10 or a variant IL-10 or its fusion protein (e.g., a variant EBV IL-10 containing a single substitution at amino acid position 75 (A75I) of SEQ ID NO: 3, associated with a diabody (variant D:DV06 EBV IL-10)) subcutaneously (scruff of the neck) for 10 days. Mice are scored daily for:

1) Masse

2) Blood in the stool

3) Extensive bleeding

4) Stool consistency.

The disease activity index is determined by adding up the points:

1. Loss of mass

2. Stool consistency

3. Bleeding (divided by 3)

Each score determines the following: change in weight (0: <1%, 1: 1-5%, 2: 5-10%, 3: 10-15%, 4>15%), blood in the stool (0: negative, 2: positive) or severe bleeding (4), and stool consistency (0: normal, 2: loose stools, 4: diarrhea).

LIST OF PREFERRED IMPLEMENTATION OPTIONS

1. A variant Epstein-Barr virus IL-10 protein (EBV-IL10) containing one or more amino acid insertions, deletions and/or substitutions that exhibits an altered interdomain angle and/or altered affinity for its receptor compared to wild-type EBV-IL10, wherein the altered interdomain angle, upon dimerization, modulates the interaction angle with its receptor.

2. The EBV-IL10 protein of the preceding embodiment, wherein one or more amino acid insertions, deletions and/or substitutions are located in the receptor binding domain of IL-10.

3. The EBV-IL10 protein of any of the preceding embodiments, wherein one or more amino acid insertions, deletions and/or substitutions are located in alpha helix A and/or helix D.

4. The EBV-IL10 protein of any of the preceding embodiments, wherein one or more amino acid insertions, deletions and/or substitutions are located in the binding domain of EBV-IL10.

5. The EBV-IL10 protein of any of the preceding embodiments, wherein one or more amino acid insertions, deletions and/or substitutions are located in the DE loop of EBV-IL10.

6. The EBV-IL10 protein of any of the preceding embodiments, wherein one or more amino acid insertions, deletions and/or substitutions are located in the twelve amino acid linker region found between alpha helix D and alpha helix E, or alpha helix C and alpha helix D, preferably the addition or substitution of proline in the twelve amino acid linker region.

7. The EBV-IL10 protein of any of the preceding embodiments, wherein the altered affinity for its receptor comprises one or more amino acid insertions, deletions, and/or substitutions in the receptor binding domain of IL-10.

8. The EBV-IL10 protein of any of the preceding embodiments, further comprising one or more amino acid insertions, deletions and/or substitutions located within alpha helix A and/or alpha helix D.

9. The EBV-IL10 protein of any of the preceding embodiments, further comprising one or more amino acid insertions, deletions and/or substitutions in the receptor binding domain of IL-10.

10. The EBV-IL10 protein of any of the preceding embodiments, further comprising one or more amino acid insertions, deletions and/or substitutions located within alpha helix A and/or alpha helix D.

11. The EBV-IL10 protein of any of the preceding embodiments, wherein one or more amino acid insertions, deletions and/or substitutions are at amino acid position 31 and/or 75 of SEQ ID NO: 3.

12. A monomeric recombinant protein comprising six alpha helices numbered A-F, capable of forming a homodimer with an identical monomeric protein, wherein alpha helices D and E are connected by an interchain amino acid linker, wherein the linker is modified by the insertion, deletion or substitution of at least one amino acid that alters the intermolecular angle of the protein upon homodimerization.

13. The recombinant protein of the preceding embodiment, wherein the protein is a protein from a virus.

14. The recombinant protein of any of the preceding embodiments, wherein the virus is Epstein-Barr virus (EBV).

15. The recombinant protein of any of the preceding embodiments, wherein the homodimer formed by two identical monomeric proteins forms a specific angle of interaction with its receptor.

16. The recombinant protein of any of the preceding embodiments, wherein the interaction angle is greater than that of the natural wild-type protein.

17. The recombinant protein of any of the preceding embodiments, wherein the interaction angle formed by homodimerization results in the protein having a higher affinity for its receptor.

18. The recombinant protein of any of the preceding embodiments, wherein the interaction angle formed by homodimerization results in the protein having a lower affinity for its receptor.

19. The recombinant protein of any of the preceding embodiments, wherein the interaction angle is smaller than that of the natural wild-type protein.

20. The recombinant protein of any of the preceding embodiments, wherein the interaction angle results in the protein having a higher affinity for its receptor.

21. The recombinant protein of any of the preceding embodiments, wherein the interaction angle results in the protein having a lower affinity for its receptor.

22. The recombinant protein of any of the preceding embodiments, wherein the monomeric protein is interleukin 10.

23. The recombinant protein of any of the preceding embodiments, wherein the monomeric protein is EBV-IL10.

24. The recombinant protein of any of the preceding embodiments, wherein the angle of the protein is provided by modifications in the linker, resulting in an angle of interaction with its receptor.

25. A recombinant variant of the Epstein-Barr virus IL-10 protein (EBV-IL10) comprising at least one amino acid insertion, deletion or substitution in the linker region between the alpha helix D and E of EBV-IL10 and/or in the receptor-binding region of EBV-IL10.

26. The recombinant protein of the preceding embodiment, wherein the variant EBV-IL10 protein interacts with an identical protein resulting in a homodimer with an altered angle of interaction with its receptor and/or an altered interhomodimer angle.

27. The recombinant protein of any of the preceding embodiments, wherein the variant EBV-IL10 protein forms an interaction angle and/or an altered interhomodimer angle that is larger than that of the wild-type EBV-IL10 protein.

28. The recombinant protein of any of the preceding embodiments, wherein the variant EBV-IL10 protein forms an interaction angle and/or an altered interhomodimer angle that is smaller than that of the wild-type EBV-IL10 protein.

29. The recombinant protein of any of the preceding embodiments, wherein the interaction angle upon formation of the homodimer results in the variant EBV-IL10 protein having increased affinity for its receptor.

30. The recombinant protein of any of the preceding embodiments, wherein the interaction angle during homodimer formation results in the variant EBV-IL10 protein having reduced affinity for its receptor.

31. The recombinant protein of any of the preceding embodiments, wherein the interaction angle confers an increase in affinity for its receptor.

32. The recombinant protein of any of the preceding embodiments, wherein the interaction angle imparts a decrease in affinity for its receptor.

33. An isolated recombinant polynucleotide encoding a protein according to any of the preceding embodiments.

34. An isolated recombinant polynucleotide encoding a protein according to any of the preceding embodiments.

35. A vector comprising a nucleic acid encoding a protein according to any of the preceding embodiments.

36. A host cell comprising a polynucleotide according to any of the preceding embodiments.

37. A method of treating or preventing inflammation in an individual, comprising administering to the individual a therapeutically effective amount of a variant protein of any of the preceding embodiments.

38. The method of the preceding embodiment, wherein the altered angle of the variant protein is less than that of wild-type EBV-IL10.

39. The method of any one of the preceding embodiments, wherein the variant protein binds with moderate affinity to the IL10 receptor compared to wild-type EBV-IL10.

40. The method of any of the preceding embodiments, wherein the inflammation is inflammatory bowel disease (IBD), Crohn's disease, non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), psoriasis, rheumatoid arthritis, acute anaphylactic shock, and/or seasonal allergies.

41. A method of treating or preventing an autoimmune disease in an individual, comprising administering to the individual a therapeutically effective amount of a variant protein of any of the preceding embodiments.

42. The method of the preceding embodiment, wherein the altered angle of the variant protein is smaller than that of wild-type EBV-IL10.

43. The method of any one of the preceding embodiments, wherein the variant protein binds with moderate affinity to the IL10 receptor compared to wild-type EBV-IL10.

44. A method of treating or preventing IBD or Crohn's disease in an individual, comprising administering to the individual a therapeutically effective amount of a variant protein of any of the preceding embodiments.

45. The method according to the preceding embodiment, wherein the altered angle of the variant protein is smaller than that of wild-type EBV-IL10.

46. The method of any one of the preceding embodiments, wherein the variant protein binds with moderate affinity to the IL10 receptor compared to wild-type EBV-IL10.

47. A method for treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in an individual, comprising administering to the individual a therapeutically effective amount of the variant protein of claim 1.

48. The method of the preceding embodiment, wherein the altered angle of the variant protein is smaller than that of wild-type EBV-IL10.

49. The method of any one of the preceding embodiments, wherein the variant protein binds with moderate affinity to the IL10 receptor compared to wild-type EBV-IL10.

50. A method of treating or preventing a malignant tumor in an individual, comprising administering to the individual a therapeutically effective amount of a variant protein of any of the preceding embodiments.

51. The method of the preceding embodiment, wherein the altered angle of the variant protein is greater than that of wild-type EBV-IL10.

52. The method of any one of the preceding embodiments, wherein the variant protein binds with increased affinity to the IL10 receptor compared to wild-type EBV-IL10.

53. An engineered fusion protein comprising at least one IL-10 or IL-10 variant molecule monomer conjugated to a first end of the fusion protein, at least one cytokine or its monomer conjugated to a second end of the fusion protein, and a linker or spacer,

where a linker or spacer connects the first and second ends.

54. The fusion protein of the preceding embodiment, wherein the linker or spacer is a constant region of an antibody.

55. The fusion protein of any of the preceding embodiments, wherein the constant region is derived from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgD, or IgE.

56. The fusion protein of any of the preceding embodiments, wherein the linker or spacer further comprises at least two interchain disulfide bonds.

57. The fusion protein of any of the preceding embodiments, wherein the linker or spacer is an scFv, diabody, or fragments thereof.

58. The fusion protein of any of the preceding embodiments, wherein the constant region is a heavy chain constant region (CH)1, CH2, CH3, or any combination thereof.

59. The fusion protein of any of the preceding embodiments, wherein at least one IL-10 or variant IL-10 molecule is conjugated to the N-terminus, C-terminus, or both ends of the fusion protein.

60. The fusion protein of any one of the preceding embodiments, wherein the at least one cytokine conjugated at the other end comprises IL-10, a variant IL-10 molecule, IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons-α, -β, -γ, TGF-β, or tumor necrosis factors-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13, or any combination thereof.

61. The fusion protein of any one of the preceding embodiments, wherein the fusion protein comprises two IL-10 or two variant IL-10 molecules conjugated to the N-terminus of the fusion protein and two IL-10 or two variant IL-10 molecules conjugated to the C-terminus of the fusion protein.

62. The fusion protein of any of the preceding embodiments, wherein the fusion protein comprises two IL-10 or two variant IL-10 molecules conjugated to the N-terminus of the fusion protein and at least one IL-2 molecule conjugated to the C-terminus of the fusion protein.

63. The fusion protein of any of the preceding embodiments, wherein the C-terminus further comprises IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons-α, -β, -γ, TGF-β, or tumor necrosis factors-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13.

64. The fusion protein of any of the preceding embodiments, wherein the fusion protein comprises two IL-10 or two variant IL-10 molecules conjugated to the N-terminus of the fusion protein and at least one IL-15 molecule conjugated to the C-terminus of the fusion protein.

65. A fusion protein wherein the C-terminus further comprises IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons -α, -β, -γ, TGF-β, or tumor necrosis factors -α, -β, basic FGF, EGF, PDGF, IL-4, IL-11 or IL-13.

66. The fusion protein of any of the preceding embodiments, wherein the fusion protein comprises two IL-10 or two variant IL-10 molecules conjugated to the N-terminus of the fusion protein and at least one IL-2 molecule conjugated to the C-terminus of the fusion protein.

67. The fusion protein of any one of the preceding embodiments, wherein the C-terminus further comprises IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons-α, -β, -γ, TGF-β, or tumor necrosis factors-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13.

68. The fusion protein of any of the preceding embodiments, wherein the fusion protein is produced on a single chain variable fragment (scFv) framework.

69. The fusion protein of any of the preceding embodiments, wherein the fusion protein is produced on a diabody scaffold.

70. The fusion protein of any of the preceding embodiments, wherein the fusion protein is produced on a Fab backbone.

71. The fusion protein of any one of the preceding embodiments, wherein the fusion protein forms a complex with another fusion protein having at least one IL-10 monomer, or variant IL-10 molecule, conjugated to a first end of the fusion protein, at least one cytokine or monomer thereof, conjugated to a second end of the fusion protein, and a linker or spacer,

where a linker or spacer connects the first and second ends.

72. A method of treating a cancer in an individual in need thereof, comprising administering to the individual an engineered fusion protein of any of the preceding embodiments.

73. A method for treating or preventing IBD or Crohn's disease comprising administering to an individual an engineered fusion protein of any of the preceding embodiments.

74. A method for treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in an individual, comprising administering to the individual a therapeutically effective amount of the engineered fusion protein of any of the preceding embodiments.

75. A method of activating a CD8 positive T cell comprising administering an engineered fusion protein of any of the preceding embodiments.

76. The method according to any of the preceding embodiments, wherein the administration is in vitro administration.

77. The method of any of the preceding embodiments, wherein the administration is in vivo administration to an individual in need thereof, wherein the individual has been diagnosed with a cancer, IBD or Crohn's disease, or NAFLD or NASH.

78. The method according to any of the preceding embodiments, wherein the fusion protein comprises IL-10 or a variant IL-10 molecule at a first end of the fusion protein and IL-2 and/or IL-15 at a second end of the fusion protein.

79. A method of treating a malignant tumor in an individual in need thereof, comprising administering to the individual a bispecific T-cell activator (BITE) and IL-10, a variant IL-10 molecule, or an engineered fusion protein comprising IL-10 or a variant IL-10 molecule.

80. The method of the preceding embodiment, wherein the engineered fusion protein comprises at least one IL-10 or one variant IL-10 molecule conjugated to a first end of the fusion protein, at least one cytokine conjugated to a second end of the fusion protein, and a linker or spacer, wherein the linker or spacer connects the first and second ends.

81. The method of any one of the preceding embodiments, wherein IL-10, a variant IL-10 molecule, or an engineered fusion protein comprising IL-10 or a variant IL-10 molecule enhances and maintains T cell receptor complex (CD3) signaling.

82. A method for treating or preventing inflammation in an individual comprising administering to the individual a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

83. The method according to the previous embodiment, wherein the nucleotide sequence is DNA, RNA, or modified versions thereof.

84. The method according to any of the preceding embodiments, wherein the nucleotide sequence is mRNA or modified mRNA linked to a nucleoside.

85. The method of any of the preceding embodiments, wherein the nucleotide sequence is capable of in vivo expression of a variant IL-10 molecule in a cell, tissue, or organism.

86. The method of any of the preceding embodiments, wherein the nucleotide sequence is delivered to a cell, tissue, or organism using a cell-penetrating peptide, a hydrophobic group, an electrostatic complex, a liposome, a ligand, a liposomal nanoparticle, a lipoprotein (preferably HDL or LDL), a folate-targeted liposome, an antibody (e.g., to a folate receptor, a transferrin receptor), a targeted peptide, or an aptamer.

87. A method for treating or preventing an autoimmune disease in an individual, comprising administering to the individual a therapeutically effective amount of a nucleotide sequence encoding variant IL-10 molecules.

88. The method according to the previous embodiment, wherein the nucleotide sequence is DNA, RNA, or modified versions thereof.

89. The method according to any of the preceding embodiments, wherein the nucleotide sequence is mRNA or modified mRNA linked to a nucleoside.

90. The method of any of the preceding embodiments, wherein the nucleotide sequence is capable of in vivo expression of a variant IL-10 molecule in a cell, tissue, or organism.

91. The method of any of the preceding embodiments, wherein the nucleotide sequence is delivered to a cell, tissue, or organism using a cell penetrating peptide, a hydrophobic group, an electrostatic complex, a liposome, a ligand, liposomal nanoparticles, a lipoprotein (preferably HDL or LDL), a folate-targeted liposome, an antibody (e.g., to a folate receptor, a transferrin receptor), a targeted peptide, or an aptamer.

92. A method of treating or preventing IBD or Crohn's disease in an individual, comprising administering to the individual a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

93. The method according to the previous embodiment, wherein the nucleotide sequence is DNA, RNA, or modified versions thereof.

94. The method according to any of the preceding embodiments, wherein the nucleotide sequence is mRNA or modified mRNA linked to a nucleoside.

95. The method of any of the preceding embodiments, wherein the nucleotide sequence is capable of in vivo expression of a variant IL-10 molecule in a cell, tissue, or organism.

96. The method of any of the preceding embodiments, wherein the nucleotide sequence is delivered to a cell, tissue, or organism using a cell penetrating peptide, a hydrophobic group, an electrostatic complex, a liposome, a ligand, liposomal nanoparticles, a lipoprotein (preferably HDL or LDL), a folate-targeted liposome, an antibody (e.g., to a folate receptor, a transferrin receptor), a targeted peptide, or an aptamer.

97. A method for treating or preventing non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH) in an individual, comprising administering to the individual a therapeutically effective amount of a nucleotide sequence encoding a variant IL-10 molecule.

98. The method according to any of the preceding embodiments, wherein the nucleotide sequence is DNA, RNA, or modified versions thereof.

99. The method according to any of the preceding embodiments, wherein the nucleotide sequence is mRNA or modified mRNA linked to a nucleoside.

100. The method of any of the preceding embodiments, wherein the nucleotide sequence is capable of in vivo expression of a variant IL-10 molecule in a cell, tissue, or organism.

101. The method according to any of the preceding embodiments, wherein the nucleotide sequence is delivered to a cell, tissue, or organism using a cell penetrating peptide, a hydrophobic group, an electrostatic complex, a liposome, a ligand, liposomal nanoparticles, a lipoprotein (preferably HDL or LDL), a folate-targeted liposome, an antibody (e.g., to a folate receptor, a transferrin receptor), a targeted peptide, or an aptamer.

102. A fusion protein comprising a monomeric IL-10 molecule or variant thereof linked to two variable regions from at least two different antibodies, wherein the two variable regions are configured as a heavy chain variable region (VH) from a first antibody linked to a light chain variable region (VL) from a second antibody, or a VL from the first antibody linked to a VH from the second antibody

103. The fusion protein of any of the preceding embodiments, wherein the monomeric IL-10 molecule or variant thereof comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

104. The fusion protein of any of the preceding embodiments, wherein the monomeric IL-10 molecule or variant thereof comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

105. The fusion protein of any of the preceding embodiments, wherein the monomeric IL-10 molecule or variant thereof is an IL-10 homologue from the Epstein-Barr virus (EBV) of SEQ ID NO: 3.

106. The fusion protein of any of the preceding embodiments, wherein the EBV IL-10 homologue comprises an amino acid substitution at position 31, 75, or both.

107. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 31.

108. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 75.

109. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at positions 31 and 75.

110. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises a V31L amino acid substitution.

111. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an A75I amino acid substitution.

112. The fusion protein of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution of V31L and A75I.

113. The fusion protein of any one of the preceding embodiments, wherein the VH region of the first antibody is derived from a monoclonal antibody to HIV and the VL region of the second antibody is derived from a monoclonal antibody to Ebola.

114. The fusion protein of any one of the preceding embodiments, wherein the VH region of the first antibody is derived from a monoclonal antibody to Ebola and the VL region of the second antibody is derived from a monoclonal antibody to HIV.

115. The fusion protein of any of the preceding embodiments, wherein the fusion protein is an amino acid sequence selected from SEQ ID NO: 24-28, 29, 33-51, 61, 63, 65, or 67.

116. The fusion protein of any one of the preceding embodiments, wherein the fusion protein is a diabody.

117. The fusion protein of any one of the preceding embodiments, wherein the fusion protein comprises a configuration selected from: (a) a VH region from a first antibody linked at the carboxy terminus to the amino terminus of a VL region from a second antibody, then linked to the amino terminus of an IL-10 monomer or variant thereof; or

(b) an IL-10 molecule or variant thereof linked at the carboxy terminus to the amino terminus of the VH region of a second antibody, then linked to the amino terminus of the VL region of a first antibody.

118. The fusion protein of any one of the preceding embodiments, wherein configurations (a) and (b) together form a diabody complex.

119. The fusion protein of any of the preceding embodiments further comprising a linker between the VH region and the VL region.

120. The fusion protein of any of the preceding embodiments, wherein the amino acid sequence is selected from SEQ ID NO: 24-28, 29, 33-53, 61, 63, 65, 67.

121. The fusion protein of any of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in the individual.

122. An immunoconjugate complex comprising i) a first fusion protein comprising at the amino terminus a heavy chain variable region (VH) from a first antibody linked to a light chain variable region (VL) of a second antibody, further linked to an IL-10 monomer or variant thereof; and ii) a second fusion protein comprising at the amino terminus an IL-10 monomer or variant thereof linked to a VH from a second antibody, further linked to a VL of the first antibody, wherein the VH and VL from the first and second antibodies associate into a diabody, and the IL-10 monomers form a functional IL-10 dimeric molecule.

123. The immunoconjugate complex of any of the preceding embodiments, wherein the IL-10 monomer comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

124. The immunoconjugate complex of any one of the preceding embodiments, wherein the IL-10 molecule comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

125. The immunoconjugate complex of any one of the preceding embodiments, wherein the IL-10 monomer is an IL-10 homolog from the Epstein-Barr virus (EBV) of SEQ ID NO: 3.

126. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV IL-10 homolog comprises an amino acid substitution at position 31, 75, or both.

127. The immunoconjugate complex of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 31.

128. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 75.

129. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at positions 31 and 75.

130. The immunoconjugate complex of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises a V31L amino acid substitution.

131. The immunoconjugate complex of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an A75I amino acid substitution.

132. The immunoconjugate complex of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution of V31L and A75I.

133. The immunoconjugate complex of any of the preceding embodiments, wherein the first antibody is derived from a monoclonal antibody to HIV and the second antibody is derived from a monoclonal antibody to Ebola.

134. The immunoconjugate complex of any one of the preceding embodiments, wherein the first fusion protein is an amino acid sequence selected from SEQ ID NO: 24, 26, 28, 35, 38, 41, 46, 48, or 50.

135. The immunoconjugate complex of any one of the preceding embodiments, wherein the second fusion protein is an amino acid sequence selected from SEQ ID NO: 25, 27, 29, 36, 39, 42, 47, 49, or 51.

136. The immunoconjugate complex of any of the preceding embodiments, further comprising a linker between the VH region and the VL region.

137. The immunoconjugate complex of any one of the preceding embodiments, wherein the IL-10 monomer in the first fusion protein is linked to the VL region via the amino terminus.

138. The immunoconjugate complex of any one of the preceding embodiments, wherein the IL-10 monomer in the second fusion protein is linked to the VH region via the carboxy terminus.

139. The immunoconjugate complex of any of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in the individual.

140. A diabody comprising a first peptide chain comprising a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and monomeric IL-10; and a second peptide chain comprising VH and VL from the second antibody and a monomeric IL-10 molecule, wherein the VH region from the first antibody associates with the VL region from the first antibody and the VH region from the second antibody associates with the VL from the second antibody, thereby allowing monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer.

141. The diabody of any of the preceding embodiments, wherein the monomeric IL-10 comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

142. The diabody of any of the preceding embodiments, wherein the monomeric IL-10 molecule comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

143. The diabody of any of the preceding embodiments, wherein the monomeric IL-10 molecule is an IL-10 homologue from the Epstein-Barr virus (EBV) of SEQ ID NO: 3.

144. The diabody of any of the preceding embodiments, wherein the EBV IL-10 homologue comprises an amino acid substitution at position 31, 75, or both.

145. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 31.

146. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 75.

147. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at positions 31 and 75.

148. The diabody of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises a V31L amino acid substitution.

149. The diabody of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution of A75I.

150. The diabody of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution of V31L and A75I.

151. The diabody of any of the preceding embodiments, wherein the first antibody is derived from a monoclonal antibody to HIV and the second antibody is derived from a monoclonal antibody to Ebola.

152. The diabody of any of the preceding embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NO: 24, 26, 28, 35, 38, 41, 46, 48, or 50.

153. The diabody of any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NO: 25, 27, 29, 36, 39, 42, 47, 49 or 51.

154. The diabody of any of the preceding embodiments further comprising a linker between the VH and VL regions.

155. The diabody of any of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in the individual.

156. The diabody of any of the preceding embodiments, wherein the IL-10 monomer is linked to the first and second peptide chains via its carboxy terminus.

157. The diabody of any of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in the individual.

158. An immunoconjugate complex comprising i) a first fusion protein comprising at the amino terminus a heavy chain variable region (VH) from a first antibody and a monomeric IL-10 molecule linked via the amino terminus; and ii) a second fusion protein comprising at the amino terminus a monomer of IL-10 linked to a light chain variable region (VL) of the first antibody, wherein the VH region from the first antibody associates with the VL region from the first antibody, thereby allowing the monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer.

159. The immunoconjugate complex of any of the preceding embodiments, wherein the IL-10 monomer comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

160. An immunoconjugate complex according to any of the preceding embodiments, wherein the IL-10 molecule comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

161. The immunoconjugate complex of any one of the preceding embodiments, wherein the IL-10 monomer is an IL-10 homolog from the Epstein-Barr virus (EBV) of SEQ ID NO: 3.

162. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV IL-10 homologue comprises an amino acid substitution at position 31, 75, or both.

163. The immunoconjugate complex of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 31.

164. The immunoconjugate complex of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 75.

165. The immunoconjugate complex of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at positions 31 and 75.

166. The immunoconjugate complex of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises a V31L amino acid substitution.

167. The immunoconjugate complex of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an A75I amino acid substitution.

168. The immunoconjugate complex of any one of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution of V31L and A75I.

169. The immunoconjugate complex of any one of the preceding embodiments, wherein the VH and VL regions of the first antibody are derived from a monoclonal antibody to the epidermal growth factor receptor (EGFR).

170. The immunoconjugate complex of any of the preceding embodiments, wherein the first fusion protein further comprises a VL region from a second antibody linking a VH region from the first antibody to monomeric IL-10 and wherein the second fusion protein further comprises a VH region from the second antibody linking monomeric IL-10 to the VL region.

171. The immunoconjugate complex of any one of the preceding embodiments, wherein the VH and VL regions of the second antibody are derived from a monoclonal antibody to Ebola.

172. The immunoconjugate complex of any of the preceding embodiments, wherein the variable regions are linked to IL-10 monomers via linkers.

173. The immunoconjugate complex of any one of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in the individual.

174. The immunoconjugate complex of any one of the preceding embodiments, wherein the first and second antibodies comprise one or more amino acid substitutions that reduce antigenicity in the individual.

175. A fusion protein comprising the variable region of the light chain (VL) and the variable region of the heavy chain (VH) from the first antibody fused to IL-10 monomers, wherein the IL-10 monomers are directly linked to each other.

176. The fusion protein of any one of the preceding embodiments, wherein the IL-10 monomers are linked at the carboxy terminus of the first IL-10 monomer to the amino terminus of the second IL-10 monomer.

177. The fusion protein of any one of the preceding embodiments, wherein the fusion protein comprises the following configuration in the amino-terminal to carboxy-terminal direction: the VL region of the first antibody is linked to a first IL-10 monomer linked to a second IL-10 monomer linked to the VH region of the first antibody.

178. The fusion protein of any of the preceding embodiments, further comprising a VH region of the second antibody linked to the amino terminus of the VL region of the first antibody, and a VL region of the second antibody linked to the carboxy terminus of the VH region of the first antibody.

179. The fusion protein of any of the preceding embodiments, wherein each of the IL-10 monomers comprises at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

180. The fusion protein of any one of the preceding embodiments, wherein each of the IL-10 monomers comprises at least one amino acid substitution that increases affinity for the IL-10 receptor.

181. The fusion protein of any one of the preceding embodiments, wherein the IL-10 monomer is an IL-10 homolog from the Epstein-Barr virus (EBV) of SEQ ID NO: 3.

182. The fusion protein of claim 181, wherein the EBV IL-10 homologue comprises an amino acid substitution at position 31, 75, or both.

183. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 31.

184. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an amino acid substitution at position 75.

185. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises a V31L amino acid substitution.

186. The fusion protein of any of the preceding embodiments, wherein the EBV-IL-10 homologue comprises an A75I amino acid substitution.

187. The fusion protein of any one of the preceding embodiments, wherein the first antibody is a monoclonal antibody to Ebola.

188. The fusion protein of any of the preceding embodiments, wherein the first antibody is a monoclonal antibody to the epidermal growth factor receptor (EGFR).

189. The fusion protein of any of the preceding embodiments, wherein the first antibody is a monoclonal antibody to Ebola and the second antibody is a monoclonal antibody to EGFR.

190. A method of treating a disease, disorder, or condition in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of an immunoconjugate complex with IL-10 from Epstein-Barr virus (EBV), wherein the immunoconjugate complex has a molecular weight of about 60 to 155 kDa, wherein the therapeutically effective amount is in the range of about 0.5 micrograms/kilogram to 100 micrograms/kilogram, and wherein the EBV IL-10 portion of the immunoconjugate is derived from SEQ ID NO: 3.

191. The method of any of the preceding embodiments, wherein the immunoconjugate complex is administered once a month, twice a month, once a week, twice a week, three times a week, or daily.

192. The method of any one of the preceding embodiments, wherein the variant EBV IL-10 is a variant comprising at least one amino acid substitution that increases or decreases affinity for the IL-10 receptor.

193. The method of any one of the preceding embodiments, wherein the variant EBV IL-10 comprises an amino acid substitution at positions 31, 75, or both of SEQ ID NO: 3.

194. The method of any one of the preceding embodiments, wherein the variant EBV IL-10 comprises a V31L amino acid substitution.

195. The method according to any of the preceding embodiments, wherein the EBV IL-10 comprises an A75I amino acid substitution.

196. The method of any one of the preceding embodiments, wherein the EBV IL-10 comprises the amino acid substitutions V31L and A75I.

197. The method according to any of the preceding embodiments, wherein the immunoconjugate complex is a complex of two fusion proteins.

198. The method of any one of the preceding embodiments, wherein the immunoconjugate complex comprises i. a first fusion protein comprising at the amino terminus a heavy chain variable region (VH) of a first antibody linked to a light chain variable region (VL) of a second antibody, further linked to the carboxy terminus of an EBV IL-10 monomer; and

ii. a second fusion protein comprising at the amino terminus an EBV IL-10 monomer linked to the VH of a second antibody, further linked to the VL of a first antibody, wherein the VH and VL of the first and second antibodies associate into a diabody and the EBV IL-10 monomers form a functional dimeric EBV IL-10 molecule.

199. The method of any of the preceding embodiments, wherein the first antibody and the second antibody are different antibodies.

200. The method of any of the preceding embodiments, wherein the first antibody is a monoclonal antibody to HIV and the second antibody is a monoclonal antibody to Ebola.

201. The method according to claim 202, wherein the fusion protein is an amino acid sequence selected from SEQ ID NO: 24-51.

202. The method of claim 202, wherein the first fusion protein is an amino acid sequence selected from SEQ ID NO: 24, 26, 28, 35, 38, 41, 46, 48, or 50.

203. The method of claim 202, wherein the second fusion protein is an amino acid sequence selected from SEQ ID NO: 25, 27, 29, 36, 39, 42, 47, 49, or 51.

204. The method according to any of the preceding embodiments, wherein the immunoconjugate complex is a diabody comprising EBV IL-10 monomers fused at both ends thereof, wherein the EBV IL-10 monomers are capable of associating into a functional EBV IL-10 dimer.

205. The method of any of the preceding embodiments, wherein the disease, disorder, or condition is selected from a cancer, an inflammatory disease, an autoimmune disease, or cholesterol.

206. The method of any of the preceding embodiments, wherein the EBV IL-10 immunoconjugate complex is administered in an amount sufficient to maintain a steady-state concentration of IL-10 in the serum, based on administration at least every 2-3 days.

207. The method of any of the preceding embodiments, wherein the immunoconjugate is capable of suppressing TNFα secretion and inducing IFNγ production at concentrations similar to those of wild-type IL-10.

208. The method according to any of the preceding embodiments, wherein the immunoconjugate complex with EBV IL-10 has an activity similar to wild-type IL-10.

209. The method according to any of the preceding embodiments, wherein the immunoconjugate complex comprises:

(a) a VH region of a first antibody linked at the N-terminus to a VL region of a second antibody linked to the carboxy-terminus of an IL-10 molecule; and

(b) an IL-10 molecule linked to the VH region of a second antibody linked to the VL region of a first antibody.

210. A method for treating a malignant tumor in a patient in need thereof, comprising administering to the patient a diabody comprising a first peptide chain having a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and a monomeric IL-10 molecule; and a second peptide chain having a VH and VL from the second antibody and a monomeric IL-10 molecule, wherein the VH region of the first antibody associates with the VL region of the first antibody and the VH region of the second antibody associates with the VL region of the second antibody, thereby allowing the monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer.

211. The method of any one of the preceding embodiments, wherein the monomeric IL-10 is an IL-10 homologue from the Epstein-Barr virus (EBV) of SEQ ID NO: 3.

212. The method according to any of the preceding embodiments, wherein the EBV IL-10 comprises an amino acid substitution at position 31 of SEQ ID NO: 3.

213. The method of any one of the preceding embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

214. The method of any of the preceding embodiments, wherein the VH region of the first antibody is derived from a monoclonal antibody to HIV and the VL region of the second antibody is derived from a monoclonal antibody to Ebola.

215. The method according to any of the preceding embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NO: 28, 35, 38, 46.

216. The method according to any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NO: 29, 36, 39, 47.

217. The method of any one of the preceding embodiments, further comprising a linker between the VH regions and the VL regions.

218. The method of any one of the preceding embodiments, wherein each of the VH and VL comprises one or more amino acid substitutions that reduce antigenicity in the individual.

219. The method of any of the preceding embodiments, wherein the first antibody is derived from a monoclonal antibody to HIV and the second antibody is derived from a monoclonal antibody to Ebola.

220. The method of any of the preceding embodiments, wherein the diabody is formed by two peptide chains with the following configurations from amino-terminus to carboxy-terminus: (a) the first peptide comprises a VH region of a first antibody linked to a VL region of a second antibody, which is then linked to the amino-terminus of an IL-10 monomer or variant thereof; and

(b) the second peptide comprises an IL-10 monomer linked to the VH region of the second antibody, which is then linked to the VL region of the first antibody.

221. A method for treating cholesterol in a patient in need thereof, comprising administering to the patient a cholesterol-lowering amount of a diabody comprising a first peptide chain with a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and monomeric IL-10; and a second peptide chain with VH and VL from the second antibody and a monomeric IL-10 molecule, wherein the VH region of the first antibody associates with the VL region of the first antibody and the VH region of the second antibody associates with the VL region of the second antibody, thereby allowing the monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer.

222. The method of any one of the preceding embodiments, wherein the monomeric IL-10 is an IL-10 homologue from the Epstein-Barr virus (EBV) of SEQ ID NO:3.

223. The method according to any of the preceding embodiments, wherein the EBV IL-10 comprises an amino acid substitution at position 31 of SEQ ID NO: 3.

224. The method of any one of the preceding embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

225. The method of any of the preceding embodiments, wherein the VH region of the first antibody is derived from a monoclonal antibody to HIV and the VL region of the second antibody is derived from a monoclonal antibody to Ebola.

226. The method according to any of the preceding embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NO: 24 or 50.

227. The method according to any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NO: 25 or 51.

228. The method according to any of the preceding embodiments, further comprising a linker between the VH regions and the VL regions.

229. The method of any one of the preceding embodiments, wherein each of the VH and VL comprises one or more amino acid substitutions that reduce antigenicity in the individual.

230. The method of any of the preceding embodiments, wherein the first antibody is derived from a monoclonal antibody to HIV and the second antibody is derived from a monoclonal antibody to Ebola.

231. The method according to any of the preceding embodiments, wherein the diabody is formed by two peptide chains with the following configurations from amino-terminus to carboxy-terminus:

(a) the first peptide comprises a VH region of a first antibody linked to a VL region of a second antibody, which is then linked to the amino terminus of an IL-10 monomer or variant thereof; and

(b) the second peptide comprises an IL-10 monomer or variant thereof linked to the VH region of the second antibody, which is then linked to the VL region of the first antibody

232. A method for treating non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD) in a patient in need thereof, comprising administering to a patient with NASH or NAFLD an amount of a diabody comprising a first peptide chain with a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and monomeric IL-10; and a second peptide chain with VH and VL from the second antibody and a monomeric IL-10 molecule, wherein the VH region of the first antibody associates with the VL region of the first antibody and the VH region of the second antibody associates with the VL region of the second antibody, thereby allowing the monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer.

233. The method of any one of the preceding embodiments, wherein the monomeric IL-10 is an IL-10 homologue from the Epstein-Barr virus (EBV) of SEQ ID NO:3.

234. The method according to any of the preceding embodiments, wherein the EBV IL-10 comprises an amino acid substitution at position 31 of SEQ ID NO: 3.

235. The method according to any of the preceding embodiments, the EBV IL-10 comprises a V31L amino acid substitution.

236. The method of any of the preceding embodiments, wherein the VH region of the first antibody is derived from a monoclonal antibody to HIV and the VL region of the second antibody is derived from a monoclonal antibody to Ebola.

237. The method according to any of the preceding embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NO: 24 or 50.

238. The method according to any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NO: 25 or 51.

239. The method according to any of the preceding embodiments, further comprising a linker between the VH regions and the VL regions.

240. The method of any one of the preceding embodiments, wherein each of the VH and VL comprises one or more amino acid substitutions that reduce antigenicity in the patient.

241. The method according to any of the preceding embodiments, wherein the first antibody is derived from a monoclonal antibody to HIV and the second antibody is derived from a monoclonal antibody to Ebola.

242. A method for treating inflammation in a patient in need thereof, comprising administering to the patient an anti-inflammatory amount of a diabody comprising a first peptide chain with a heavy chain variable region (VH) from a first antibody, a light chain variable region (VL) from a second antibody, and monomeric IL-10; and a second peptide chain with VH and VL from the second antibody and a monomeric IL-10 molecule, wherein the VH region of the first antibody associates with the VL region of the first antibody and the VH region of the second antibody associates with the VL region of the second antibody, thereby allowing the monomeric IL-10 molecules on each peptide chain to form a functional IL-10 dimer.

243. The method of any one of the preceding embodiments, wherein the monomeric IL-10 is an IL-10 homologue from the Epstein-Barr virus (EBV) of SEQ ID NO:3.

244. The method according to any of the preceding embodiments, wherein the EBV IL-10 comprises an amino acid substitution at position 75 of SEQ ID NO: 3.

245. The method of any one of the preceding embodiments, wherein the EBV IL-10 comprises a V31L amino acid substitution.

246. The method according to any of the preceding embodiments, wherein the VH region of the first antibody is derived from a monoclonal antibody to HIV and the VL region of the second antibody is derived from a monoclonal antibody to Ebola.

247. The method according to any of the preceding embodiments, wherein the first peptide chain is an amino acid sequence selected from SEQ ID NO: 26, 41, or 48.

248. The method according to any of the preceding embodiments, wherein the second peptide chain is an amino acid sequence selected from SEQ ID NO: 27, 42, 49.

249. The method according to any of the preceding embodiments, further comprising a linker between the VH regions and the VL regions.

250. The method of any one of the preceding embodiments, wherein each of the VH and VL comprises one or more amino acid substitutions that reduce antigenicity in the patient.

251. The method of any of the preceding embodiments, wherein the first antibody is derived from a monoclonal antibody to HIV and the second antibody is derived from a monoclonal antibody to Ebola.

252. Fusion protein of Formula (I-VII)

IL10-L ¹ -X ¹ -L ¹ -X ² -L ¹ -IL10 (Formula I); (Z) _n -X ¹ -L ² -Y ² -L ¹ -IL10 (Formula II); IL10-L ¹ -Y ¹ -L ² -X ² -(Z) _n (Formula III); X ¹ -L ² -X ² -L ¹ -IL10 (Formula IV); IL10-L ¹ -X ¹ -L ² -X ² (Formula V); X ¹ -L ¹ -IL10 (Formula VI); and IL10-L ¹ -X ² (Formula VII), or any combination thereof;

Where

"IL-10" is a monomeric sequence selected from SEQ ID NO: 1, 3, 14, 15, 16, 18, 19, 55, 57, or 59; more preferably, "IL-10" consists of SEQ ID NO: 55, 57, or 59;

"L ¹ " is a linker of SEQ ID NO: 31 or 54;

"L ² " is the linker of SEQ ID NO: 30;

"X ¹ " is a VH region derived from the first antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as but not limited to PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ trap; MadCam, integrin β7 subunit; integrin α4β7; integrin α4 SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; and SR-J1; HIV, or Ebola;

"X ² " represents the VL region derived from the same antibody as X1;

"Y ¹ " is a VH region derived from a second antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as but not limited to PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ trap; MadCam, integrin β7 subunit; integrin α4β7; integrin α4 SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; and SR-J1; HIV, or Ebola;

"Y ² " represents the VL region derived from the same antibody as Y1;

where X and Y are obtained from the same or different antibodies;

"Z" is a cytokine selected from IL-10, a variant IL-10 molecule, IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons -α, -β, -γ, TGF-β, or tumor necrosis factors -α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13;

"n" is an integer chosen from 0-2.

253. The fusion protein of the preceding embodiment, wherein Formula II and III are capable of forming a fusion protein complex, wherein the IL-10 monomer from each of Formula II and III is capable of forming a functional homodimeric IL-10 or variant thereof.

254. The fusion protein of any of the preceding embodiments, wherein Formula II is SEQ ID NO: 24, 26, 28, 41, 48, or 50.

255. The fusion protein of any of the preceding embodiments, wherein Formula III is SEQ ID NO: 25, 27, 29, 42, 49, or 51.

256. The fusion protein of any of the preceding embodiments, wherein the fusion protein complex is formed between SEQ ID NOs: 24 and 25; 26 and 27, 28 and 29; 41 and 42; 48 and 49; or 50 and 51.

257. The fusion protein of any of the preceding embodiments, wherein Formula IV and V are capable of forming a fusion protein complex, wherein the IL-10 monomer from each of Formula IV and V is capable of forming a functional homodimeric IL-10 or variant thereof.

258. The fusion protein of any of the preceding embodiments, wherein Formula IV is SEQ ID NO: 35, 38, 46, 48, or 50.

259. The fusion protein of any of the preceding embodiments, wherein Formula V is SEQ ID NO: 36, 39, 47, 49, or 51.

260. The fusion protein of any of the preceding embodiments, wherein the fusion protein complex is formed between SEQ ID NOs: 35 and 36; 38 and 39, 46 and 47; 48 and 49; or 50 and 51.

261. The fusion protein of any of the preceding embodiments, wherein Formula VI and VII are capable of forming a fusion protein complex, wherein the IL-10 monomer from each of Formula VI is capable of forming a functional homodimeric IL-10 or variant thereof.

262. The fusion protein of any of the preceding embodiments, wherein Formula I is SEQ ID NO: 33-34, 40, 43-44, 45, 52, 53, 61, 63, 65, or 67.

263. The fusion protein of any of the preceding embodiments, wherein "n" 1 and Z represents IL-2, IL-7, IL-12, IL-15, or any combination thereof.

264. The fusion protein of any of the preceding embodiments, wherein Z is conjugated at the N-terminus of ^X1 , ^Y1 , or both.

265. A method for treating a malignant tumor, comprising administering to a patient in need thereof a composition comprising a fusion protein according to any of the preceding embodiments.

266. The method of any of the preceding embodiments, wherein the fusion protein is SEQ ID NO: 28-29, 35-36, 38-39, 46-47, 52-53, 61, 63, 65, or 67.

267. The method of any of the preceding embodiments, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID Nos: 28 and 29; 35 and 36; 38 and 39; or 46 and 47.

268. The method according to any of the preceding embodiments, wherein the composition comprises a fusion protein of SEQ ID NO: 33, 34, 44, 52 or 53, 61, 63, 65 or 67.

269. The method according to any of the preceding embodiments, wherein the fusion protein comprises IL-10 consisting of DV07 of SEQ ID NO: 59.

270. A method for treating an inflammatory disease, comprising administering to a patient in need thereof a composition comprising a fusion protein according to any of the preceding embodiments.

271. The method of any of the preceding embodiments, wherein the fusion protein is SEQ ID NO: 26-27, 41-42, 48, or 49.

272. The method according to any of the preceding embodiments, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID NOs: 26 and 27; 41 and 42; 48 and 49.

273. The method according to any of the preceding embodiments, wherein the composition comprises a fusion protein of SEQ ID NO: 37, 40, or 43.

274. The method of claim 18, wherein the fusion protein comprises IL-10 consisting of DV06 of SEQ ID NO: 57.

275. A method of treating a lipid-related disease comprising administering to a patient in need thereof a composition comprising a fusion protein according to any of the preceding embodiments.

276. The method of any of the preceding embodiments, wherein the fusion protein is SEQ ID NO: 24-25, 50, or 51.

277. The method according to any of the preceding embodiments, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID No: 24 and 25; and 50 and 51.

278. The method according to any of the preceding embodiments, wherein the composition comprises the fusion protein of SEQ ID NO: 45.

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LIST OF SEQUENCES

<110> DEKA BIOSCIENCES, INC.

<120> VARIANT IL-10 MOLECULES AND METHODS FOR THE TREATMENT OF INFLAMMATORY DISEASES

AND ONCOLOGY

<130> 039451.00022

<140>

<141>

<150> 62/962,332

<151> 2020-01-17

<150> 62/899,504

<151> 2019-09-12

<150> 62/814,669

<151> 2019-03-06

<160> 68

<170> PatentIn version 3.5

<210> 1

<211> 178

<212> Protein

<213> Homo sapiens

<400> 1

Met His Ser Ser Ala Leu Leu Cys Cys Leu Val Leu Leu Thr Gly Val

1 5 10 15

Arg Ala Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His

20 25 30

Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe

35 40 45

Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu

50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

65 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

85 90 95

Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu

100 105 110

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

115 120 125

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn

130 135 140

Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu

145 150 155 160

Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile

165 170 175

Arg Asn

<210> 2

<211> 1629

<212> DNA

<213> Homo sapiens

<400> 2

acacatcagg ggcttgctct tgcaaaacca aaccacaaga cagacttgca aaagaaggca 60

tgcacagctc agcactgctc tgttgcctgg tcctcctgac tggggtgagg gccagcccag 120

gccagggcac ccagtctgag aacagctgca cccacttccc aggcaacctg cctaacatgc 180

ttcgagatct ccgagatgcc ttcagcagag tgaagacttt ctttcaaatg aaggatcagc 240

tggacaactt gttgttaaag gagtccttgc tggaggactt taagggttac ctgggttgcc 300

aagccttgtc tgagatgatc cagttttacc tggaggaggt gatgccccaa gctgagaacc 360

aagacccaga catcaaggcg catgtgaact ccctggggga gaacctgaag accctcaggc 420

tgaggctacg gcgctgtcat cgatttcttc cctgtgaaaa caagagcaag gccgtggagc 480

aggtgaagaa tgcctttaat aagctccaag agaaaggcat ctacaaagcc atgagtgagt 540

ttgacatctt catcaactac atagaagcct acatgacaat gaagatacga aactgagaca 600

tcagggtggc gactctatag actctaggac ataaattaga ggtctccaaa atcggatctg 660

gggctctggg atagctgacc cagccccttg agaaacctta ttgtacctct cttatagaat 720

atttattacc tctgatacct caacccccat ttctatttat ttactgagct tctctgtgaa 780

cgatttagaa agaagcccaa tattataatt tttttcaata tttattattt tcacctgttt 840

ttaagctgtt tccatagggt gacacactat ggtatttgag tgttttaaga taaattataa 900

gttacataag ggaggaaaaa aaatgttctt tggggagcca acagaagctt ccattccaag 960

cctgaccacg ctttctagct gttgagctgt tttccctgac ctccctctaa tttatcttgt 1020

ctctgggctt ggggcttcct aactgctaca aatactctta ggaagagaaa ccagggagcc 1080

cctttgatga ttaattcacc ttccagtgtc tcggagggat tcccctaacc tcattcccca 1140

accacttcat tcttgaaagc tgtggccagc ttgttattta taacaaccta aatttggttc 1200

taggccgggc gcggtggctc acgcctgtaa tcccagcact ttgggaggct gaggcgggtg 1260

gatcacttga ggtcaggagt tcctaaccag cctggtcaac atggtgaaac cccgtctcta 1320

ctaaaaatac aaaaattagc cgggcatggt ggcgcgcacc tgtaatccca gctacttggg 1380

aggctgaggc aagagaattg cttgaaccca ggagatggaa gttgcagtga gctgatatca 1440

tgcccctgta ctccagcctg ggtgacagag caagactctg tctcaaaaaa taaaaataaa 1500

aataaatttg gttctaatag aactcagttt taactagaat ttattcaatt cctctgggaa 1560

tgttacattg tttgtctgtc ttcatagcag attttaattt tgaataaata aatgtatctt 1620

attcacatc 1629

<210> 3

<211> 147

<212> Protein

<213> Gammaherpesvirus 4 humans

<400> 3

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg

145

<210> 4

<211> 632

<212> DNA

<213> Gammaherpesvirus 4 humans

<400> 4

tataaatcac ttccctatct caggtaggcc tgcacacctt aggtatggag cgaaggttag 60

tggtcactct gcagtgcctg gtgctgcttt acctggcacc tgagtgtgga ggtacagacc 120

aatgtgacaa ttttccccaa atgttgaggg acctaagaga tgccttcagt cgtgttaaaa 180

cctttttcca gacaaaggac gaggtagata accttttgct caaggagtct ctgctagagg 240

actttaaggg ctaccttgga tgccaggccc tgtcagaaat gatccaattc tacctggagg 300

aagtcatgcc acaggctgaa aaccaggacc ctgaagccaa agaccatgtc aattctttgg 360

gtgaaaatct aaagacccta cggctccgcc tgcgcaggtg ccacaggttc ctgccgtgtg 420

agaacaagag taaagctgtg gaacagataa aaaatgcctt taacaagctg caggaaaaag 480

gaatttacaa agccatgagt gaatttgaca tttttattaa ctacatagaa gcatacatga 540

caattaaagc caggtgataa ttccataccc tggaagcagg agatgggtgc atttcacccc 600

aaccccccct ttcgactgtc atttacaata aa 632

<210> 5

<211> 177

<212> Protein

<213> Human betaherpesvirus 5

<400> 5

Met Leu Ser Val Met Val Ser Ser Ser Leu Val Leu Ile Val Phe Phe

1 5 10 15

Leu Gly Ala Ser Glu Glu Ala Lys Pro Ala Ala Thr Thr Thr Thr Ile

20 25 30

Lys Asn Thr Lys Pro Gln Cys Arg Pro Glu Asp Tyr Ala Ser Arg Leu

35 40 45

Gln Asp Leu Arg Val Thr Phe His Arg Val Lys Pro Thr Leu Gln Arg

50 55 60

Glu Asp Asp Tyr Ser Val Trp Leu Asp Gly Thr Val Val Lys Gly Cys

65 70 75 80

Trp Gly Cys Ser Val Met Asp Trp Leu Leu Arg Arg Tyr Leu Glu Ile

85 90 95

Val Phe Pro Ala Gly Asp His Val Tyr Pro Gly Leu Lys Thr Glu Leu

100 105 110

His Ser Met Arg Ser Thr Leu Glu Ser Ile Tyr Lys Asp Met Arg Gln

115 120 125

Cys Pro Leu Leu Gly Cys Gly Asp Lys Ser Val Ile Ser Arg Leu Ser

130 135 140

Gln Glu Ala Glu Arg Lys Ser Asp Asn Gly Thr Arg Lys Gly Leu Ser

145 150 155 160

Glu Leu Asp Thr Leu Phe Ser Arg Leu Glu Glu Tyr Leu His Ser Arg

165 170 175

Lys

<210> 6

<211> 693

<212> DNA

<213> Human betaherpesvirus 5

<400> 6

atgctgtcgg tgatggtctc ttcctctctg gtcctgatcg tcttttttct aggcgcttcc 60

gaggaggcga agccggcggc gacgacgacg acgataaaga atacaaagcc gcagtgtcgt 120

ccggaggatt acgcgagcag attgcaagat ctccgcgtca cctttcatcg agtaaaacct 180

acgttggtag gtcatgtagg tacggtttat tgcgacggtc tttcttttcc gcgtgtcggg 240

tgacgtagtt ttcctcttgt agcaacgtga ggacgactac tccgtgtggc tcgacggtac 300

ggtggtcaaa ggctgttggg gatgcagcgt catggactgg ttgttgaggc ggtatctgga 360

gatcgtgttc cccgcaggcg accacgtcta tcctggactt aagacggaat tgcatagtat 420

gcgctcgacg ctagaatcca tctacaaaga catgcggcaa tgcgtaagtg tctctgtggc 480

ggcgctgtcc gcgcagaggt aacaacgtgt tcatagcacg ctgttttact tttgtcgggc 540

tcccagcctc tgttaggttg cggagataag tccgtgatta gtcggctgtc tcaggaggcg 600

gaaaggaaat cggataacgg cacgcggaaa ggtctcagcg agttggacac gttgtttagc 660

cgtctcgaag agtatctgca ctcgagaaag tag 693

<210> 7

<211> 178

<212> Protein

<213> Mus musculus

<400> 7

Met Pro Gly Ser Ala Leu Leu Cys Cys Leu Leu Leu Leu Thr Gly Met

1 5 10 15

Arg Ile Ser Arg Gly Gln Tyr Ser Arg Glu Asp Asn Asn Cys Thr His

20 25 30

Phe Pro Val Gly Gln Ser His Met Leu Leu Glu Leu Arg Thr Ala Phe

35 40 45

Ser Gln Val Lys Thr Phe Phe Gln Thr Lys Asp Gln Leu Asp Asn Ile

50 55 60

Leu Leu Thr Asp Ser Leu Met Gln Asp Phe Lys Gly Tyr Leu Gly Cys

65 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Val Glu Val Met Pro

85 90 95

Gln Ala Glu Lys His Gly Pro Glu Ile Lys Glu His Leu Asn Ser Leu

100 105 110

Gly Glu Lys Leu Lys Thr Leu Arg Met Arg Leu Arg Arg Cys His Arg

115 120 125

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Ser

130 135 140

Asp Phe Asn Lys Leu Gln Asp Gln Gly Val Tyr Lys Ala Met Asn Glu

145 150 155 160

Phe Asp Ile Phe Ile Asn Cys Ile Glu Ala Tyr Met Met Ile Lys Met

165 170 175

Lys Ser

<210> 8

<211> 1314

<212> DNA

<213> Mus musculus

<400> 8

gggggggggg atttagagac ttgctcttgc actaccaaag ccacaaagca gccttgcaga 60

aaagagagct ccatcatgcc tggctcagca ctgctatgct gcctgctctt actgactggc 120

atgaggatca gcaggggcca gtacagccgg gaagacaata actgcaccca cttcccagtc 180

ggccagagcc acatgctcct agagctgcgg actgccttca gccaggtgaa gactttcttt 240

caaacaaagg accagctgga caacatactg ctaaccgact ccttaatgca ggactttaag 300

ggttacttgg gttgccaagc cttatcggaa atgatccagt tttacctggt agaagtgatg 360

ccccaggcag agaagcatgg cccagaaatc aaggagcatt tgaattccct gggtgagaag 420

ctgaagaccc tcaggatgcg gctgaggcgc tgtcatcgat ttctcccctg tgaaaataag 480

agcaaggcag tggagcaggt gaagagtgat tttaataagc tccaagacca aggtgtctac 540

aaggccatga atgaatttga catcttcatc aactgcatag aagcatacat gatgatcaaa 600

atgaaaagct aaaacacctg cagtgtgtat tgagtctgct ggactccagg acctagacag 660

agctctctaa atctgatcca gggatcttag ctaacggaaa caactccttg gaaaacctcg 720

tttgtacctc tctccgaaat atttattacc tctgatacct cagttcccat tctatttatt 780

cactgagctt ctctgtgaac tatttagaaa gaagcccaat attataattt tacagtattt 840

attattttta acctgtgttt aagctgtttc cattggggac actttatagt atttaaaggg 900

agattatatt atatgatggg aggggttctt ccttgggaag caattgaagc ttctattcta 960

aggctggcca cacttgagag ctgcagggcc ctttgctatg gtgtcctttc aattgctctc 1020

atccctgagt tcagagctcc taagagagtt gtgaagaaac tcatgggtct tgggaagaga 1080

aaccagggag atcctttgat gatcattcct gcagcagctc agagggttcc cctactgtca 1140

tcccccagcc gcttcatccc tgaaaactgt ggccagtttg ttatttataa ccacctaaaa 1200

ttagttctaa tagaactcat ttttaactag aagtaatgca attcctctgg gaatggtgta 1260

ttgtttgtct gcctttgtag cagcatctaa ttttgaataa atggatctta ttcg 1314

<210> 9

<211> 168

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 9

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Asp Glu Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu

50 55 60

Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln

65 70 75 80

Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu

85 90 95

Ala Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Leu Arg Leu

100 105 110

Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys

115 120 125

Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly

130 135 140

Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu

145 150 155 160

Ala Tyr Met Thr Ile Lys Ala Arg

165

<210> 10

<211> 167

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 10

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ala Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Gln Cys Pro Leu Leu Gly Cys Gly Asp Lys Ala

115 120 125

Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile

130 135 140

Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala

145 150 155 160

Tyr Met Thr Ile Lys Ala Arg

165

<210> 11

<211> 167

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 11

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ala Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Gln Cys Pro Leu Leu Gly Cys Gly Asp Lys Ala

115 120 125

Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile

130 135 140

Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala

145 150 155 160

Tyr Met Thr Ile Lys Ala Arg

165

<210> 12

<211> 167

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 12

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Gln Cys Pro Leu Leu Gly Cys Gly Asp Lys Ala

115 120 125

Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile

130 135 140

Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala

145 150 155 160

Tyr Met Thr Ile Lys Ala Arg

165

<210> 13

<211> 167

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 13

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Gln Cys Pro Leu Leu Gly Cys Gly Asp Lys Ala

115 120 125

Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile

130 135 140

Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala

145 150 155 160

Tyr Met Thr Ile Lys Ala Arg

165

<210> 14

<211> 170

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 14

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ala Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys

115 120 125

Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu

130 135 140

Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr

145 150 155 160

Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

165 170

<210> 15

<211> 170

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 15

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys

115 120 125

Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu

130 135 140

Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr

145 150 155 160

Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

165 170

<210> 16

<211> 170

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 16

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys

115 120 125

Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu

130 135 140

Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr

145 150 155 160

Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

165 170

<210> 17

<211> 184

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 17

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ala Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly

115 120 125

Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala

130 135 140

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

145 150 155 160

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

165 170 175

Asp Tyr Lys Asp Asp Asp Asp Lys

180

<210> 18

<211> 184

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 18

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ala Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly

115 120 125

Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala

130 135 140

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

145 150 155 160

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

165 170 175

Asp Tyr Lys Asp Asp Asp Asp Lys

180

<210> 19

<211> 184

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 19

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly

115 120 125

Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala

130 135 140

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

145 150 155 160

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

165 170 175

Asp Tyr Lys Asp Asp Asp Asp Lys

180

<210> 20

<211> 184

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 20

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Gly

115 120 125

Gly Gly Ser Gly Gly Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala

130 135 140

Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe

145 150 155 160

Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

165 170 175

Asp Tyr Lys Asp Asp Asp Asp Lys

180

<210> 21

<211> 192

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 21

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Gln Ser Glu Asn Ser Cys Thr His

20 25 30

Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe

35 40 45

Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu

50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

65 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

85 90 95

Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu

100 105 110

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

115 120 125

Phe Leu Pro Cys Glu Asn Gly Gly Gly Ser Gly Gly Lys Ser Lys Ala

130 135 140

Val Glu Gln Val Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile

145 150 155 160

Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala

165 170 175

Tyr Met Thr Met Lys Ile Arg Asn Asp Tyr Lys Asp Asp Asp Asp Lys

180 185 190

<210> 22

<211> 186

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 22

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Gln Ser Glu Asn Ser Cys Thr His

20 25 30

Phe Pro Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe

35 40 45

Ser Arg Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu

50 55 60

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

65 70 75 80

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

85 90 95

Gln Ala Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu

100 105 110

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

115 120 125

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn

130 135 140

Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu

145 150 155 160

Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile

165 170 175

Arg Asn Asp Tyr Lys Asp Asp Asp Asp Lys

180 185

<210> 23

<211> 178

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 23

Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp

1 5 10 15

Leu Arg Gly Ala Arg Cys Gly Thr Asp Gln Cys Asp Asn Phe Pro Gln

20 25 30

Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe

35 40 45

Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu

50 55 60

Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile

65 70 75 80

Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro

85 90 95

Glu Ala Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu

100 105 110

Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys

115 120 125

Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu

130 135 140

Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr

145 150 155 160

Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg Asp Tyr Lys Asp Asp Asp

165 170 175

Asp Lys

<210> 24

<211> 398

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 24

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asn Phe Thr Thr Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Gly Gly Asn Gly Asp Thr Arg Tyr Ser Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu His Ser Leu Thr Ser Glu Asp Thr Ala Leu Phe Tyr Cys

85 90 95

Ala Arg Glu Ser Gly Asp Tyr Tyr Ser Glu Ile Ser Gly Ala Leu Asp

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

130 135 140

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Pro Arg Asn

145 150 155 160

Tyr Ile Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

165 170 175

Ile Tyr Gly Ala Ser Ser Arg Ala Ala Gly Phe Pro Asp Arg Phe Ser

180 185 190

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu

195 200 205

Pro Glu Asp Phe Ala Met Tyr Tyr Cys His Gln Tyr Asp Arg Leu Pro

210 215 220

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys Asp

245 250 255

Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val

260 265 270

Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys

275 280 285

Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu

290 295 300

Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu

305 310 315 320

Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn Ser Leu Gly Glu Asn

325 330 335

Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro

340 345 350

Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn

355 360 365

Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile

370 375 380

Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

385 390 395

<210> 25

<211> 397

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 25

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Thr Thr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Glu Val Asn Tyr Ser Gly Asn Ala Asn Tyr Asn Pro Ser

210 215 220

Leu Lys Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Arg Ile Arg Ser His Ile Ala Tyr Ser Trp Lys Gly Asp

260 265 270

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

275 280 285

Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly

290 295 300

Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Val Leu Pro Lys Lys Tyr

305 310 315 320

Ala Tyr Trp Tyr Gln Gln Lys Ser Gly Leu Ala Pro Val Leu Val Ile

325 330 335

Tyr Glu Asp Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly

340 345 350

Ser Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Ser Gly Ala Gln Val

355 360 365

Glu Asp Glu Gly Asp Tyr Tyr Cys Ser Ser Thr Asp Ser Ser Gly Asp

370 375 380

His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

385 390 395

<210> 26

<211> 398

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 26

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asn Phe Thr Thr Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Gly Gly Asn Gly Asp Thr Arg Tyr Ser Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu His Ser Leu Thr Ser Glu Asp Thr Ala Leu Phe Tyr Cys

85 90 95

Ala Arg Glu Ser Gly Asp Tyr Tyr Ser Glu Ile Ser Gly Ala Leu Asp

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

130 135 140

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Pro Arg Asn

145 150 155 160

Tyr Ile Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

165 170 175

Ile Tyr Gly Ala Ser Ser Arg Ala Ala Gly Phe Pro Asp Arg Phe Ser

180 185 190

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu

195 200 205

Pro Glu Asp Phe Ala Met Tyr Tyr Cys His Gln Tyr Asp Arg Leu Pro

210 215 220

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys Asp

245 250 255

Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val

260 265 270

Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu Lys

275 280 285

Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu

290 295 300

Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu

305 310 315 320

Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn

325 330 335

Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro

340 345 350

Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn

355 360 365

Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile

370 375 380

Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

385 390 395

<210> 27

<211> 397

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 27

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Thr Thr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Glu Val Asn Tyr Ser Gly Asn Ala Asn Tyr Asn Pro Ser

210 215 220

Leu Lys Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Arg Ile Arg Ser His Ile Ala Tyr Ser Trp Lys Gly Asp

260 265 270

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

275 280 285

Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly

290 295 300

Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Val Leu Pro Lys Lys Tyr

305 310 315 320

Ala Tyr Trp Tyr Gln Gln Lys Ser Gly Leu Ala Pro Val Leu Val Ile

325 330 335

Tyr Glu Asp Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly

340 345 350

Ser Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Ser Gly Ala Gln Val

355 360 365

Glu Asp Glu Gly Asp Tyr Tyr Cys Ser Ser Thr Asp Ser Ser Gly Asp

370 375 380

His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

385 390 395

<210> 28

<211> 398

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 28

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asn Phe Thr Thr Tyr

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Asn Gly Gly Asn Gly Asp Thr Arg Tyr Ser Gln Lys Phe

50 55 60

Arg Gly Arg Val Thr Ile Ser Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu His Ser Leu Thr Ser Glu Asp Thr Ala Leu Phe Tyr Cys

85 90 95

Ala Arg Glu Ser Gly Asp Tyr Tyr Ser Glu Ile Ser Gly Ala Leu Asp

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser

115 120 125

Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

130 135 140

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Pro Arg Asn

145 150 155 160

Tyr Ile Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

165 170 175

Ile Tyr Gly Ala Ser Ser Arg Ala Ala Gly Phe Pro Asp Arg Phe Ser

180 185 190

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu

195 200 205

Pro Glu Asp Phe Ala Met Tyr Tyr Cys His Gln Tyr Asp Arg Leu Pro

210 215 220

Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly

225 230 235 240

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys Asp

245 250 255

Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val

260 265 270

Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys

275 280 285

Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu

290 295 300

Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu

305 310 315 320

Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn

325 330 335

Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro

340 345 350

Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn

355 360 365

Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile

370 375 380

Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

385 390 395

<210> 29

<211> 397

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 29

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Thr Thr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Glu Val Asn Tyr Ser Gly Asn Ala Asn Tyr Asn Pro Ser

210 215 220

Leu Lys Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Arg Ile Arg Ser His Ile Ala Tyr Ser Trp Lys Gly Asp

260 265 270

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

275 280 285

Ser Ser Tyr Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly

290 295 300

Gln Thr Ala Arg Ile Thr Cys Ser Gly Asp Val Leu Pro Lys Lys Tyr

305 310 315 320

Ala Tyr Trp Tyr Gln Gln Lys Ser Gly Leu Ala Pro Val Leu Val Ile

325 330 335

Tyr Glu Asp Asn Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly

340 345 350

Ser Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Ser Gly Ala Gln Val

355 360 365

Glu Asp Glu Gly Asp Tyr Tyr Cys Ser Ser Thr Asp Ser Ser Gly Asp

370 375 380

His Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

385 390 395

<210> 30

<211> 7

<212> Protein

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: synthetic peptide

<400> 30

Ser Ser Gly Gly Gly Gly Ser

1 5

<210> 31

<211> 15

<212> Protein

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: synthetic peptide

<400> 31

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 32

<211> 6

<212> Protein

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: synthetic label

6xHis

<400> 32

His His His His His

1 5

<210> 33

<211> 567

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 33

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Thr Thr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Glu Val Asn Tyr Ser Gly Asn Ala Asn Tyr Asn Pro Ser

210 215 220

Leu Lys Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Arg Ile Arg Ser His Ile Ala Tyr Ser Trp Lys Gly Asp

260 265 270

Val Trp Gly Lys Gly Thr Thr Val Thr Val Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser

290 295 300

Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

305 310 315 320

Arg Ala Ser Gln Ser Val Pro Arg Asn Tyr Ile Gly Trp Phe Gln Gln

325 330 335

Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Ser Arg

340 345 350

Ala Ala Gly Phe Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp

355 360 365

Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr

370 375 380

Tyr Cys His Gln Tyr Asp Arg Leu Pro Tyr Thr Phe Gly Gln Gly Thr

385 390 395 400

Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

405 410 415

Gly Gly Gly Ser Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg

420 425 430

Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys

435 440 445

Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe

450 455 460

Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr

465 470 475 480

Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys

485 490 495

Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg

500 505 510

Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala

515 520 525

Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile

530 535 540

Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala

545 550 555 560

Tyr Met Thr Ile Lys Ala Arg

565

<210> 34

<211> 561

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 34

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Asn Tyr Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro

210 215 220

Phe Thr Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Gln Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Trp Gly Lys Gly

260 265 270

Thr Thr Val Thr Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser

290 295 300

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

305 310 315 320

Ile Gly Thr Asn Ile His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro

325 330 335

Arg Leu Leu Ile Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Phe Pro Asp

340 345 350

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr

355 360 365

Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln Asn Asn

370 375 380

Asn Trp Pro Thr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly

385 390 395 400

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp

405 410 415

Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe

420 425 430

Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp Asn Leu

435 440 445

Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys

450 455 460

Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro

465 470 475 480

Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn Ser Leu

485 490 495

Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg

500 505 510

Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn

515 520 525

Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu

530 535 540

Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala

545 550 555 560

Arg

<210> 35

<211> 396

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 35

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr Asn Tyr

20 25 30

Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

Ser Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Thr

85 90 95

Ser Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Trp Lys Gly Asp Val Trp

100 105 110

Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu

115 120 125

Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu

130 135 140

Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn Ile

145 150 155 160

His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr

165 170 175

Tyr Ala Ser Glu Ser Ile Ser Gly Phe Pro Asp Arg Phe Ser Gly Ser

180 185 190

Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro Glu

195 200 205

Asp Phe Ala Met Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr Thr

210 215 220

Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly

225 230 235 240

Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys Asp Asn Phe

245 250 255

Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr

260 265 270

Phe Phe Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu Ser

275 280 285

Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu

290 295 300

Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln

305 310 315 320

Asp Pro Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys

325 330 335

Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu

340 345 350

Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu

355 360 365

Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile

370 375 380

Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

385 390 395

<210> 36

<211> 403

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 36

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Asn

180 185 190

Tyr Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp

195 200 205

Ile Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe

210 215 220

Thr Ser Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

225 230 235 240

Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys

245 250 255

Thr Ser Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Trp Lys Gly Asp Val

260 265 270

Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser

275 280 285

Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

290 295 300

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

305 310 315 320

Ile His Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

325 330 335

Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Phe Pro Asp Arg Phe Ser Gly

340 345 350

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro

355 360 365

Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

370 375 380

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys Asp Asp

385 390 395 400

Asp Asp Lys

<210> 37

<211> 581

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<220>

<221> Modified residue

<222> (193)..(199)

<223> Any amino acid

<220>

<221> Other sign

<222> (193)..(199)

<223> This region may span 3-7 residues

<220>

<221> Modified residue

<222> (214)..(231)

<223> Any amino acid

<220>

<221> Other sign

<222> (214)..(231)

<223> This region may span 14-18 residues

<220>

<221> Modified residue

<222> (264)..(274)

<223> Any amino acid

<220>

<221> Other sign

<222> (264)..(274)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (329)..(342)

<223> Any amino acid

<220>

<221> Other sign

<222> (329)..(342)

<223> This region may span 9-14 residues

<220>

<221> Modified residue

<222> (358)..(366)

<223> Any amino acid

<220>

<221> Other sign

<222> (358)..(366)

<223> This region may span 5-9 residues

<220>

<221> Modified residue

<222> (399)..(409)

<223> Any amino acid

<220>

<221> Other sign

<222> (399)..(409)

<223> This region may span 7-11 residues

<220>

<223> See the attached description for a detailed description of the substitutions and preferred

embodiments

<400> 37

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly

195 200 205

Leu Glu Trp Ile Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

210 215 220

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Val Ala Ile Ser Val Asp Thr Ser

225 230 235 240

Lys Asn Gln Phe Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr

245 250 255

Ala Ile Tyr Tyr Cys Thr Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

260 265 270

Xaa Xaa Trp Lys Gly Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val

275 280 285

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

290 295 300

Ser Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro

305 310 315 320

Gly Glu Arg Ala Thr Leu Ser Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

325 330 335

Xaa Xaa Xaa Xaa Xaa Xaa Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro

340 345 350

Arg Leu Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe

355 360 365

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

370 375 380

Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Xaa Xaa

385 390 395 400

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly Thr Lys Leu

405 410 415

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

420 425 430

Gly Ser Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu

435 440 445

Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu

450 455 460

Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly

465 470 475 480

Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu

485 490 495

Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His

500 505 510

Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg

515 520 525

Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu

530 535 540

Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys

545 550 555 560

Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met

565 570 575

Th Ile Lys Ala Arg

580

<210> 38

<211> 397

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 38

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr Thr Thr

20 25 30

Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Val Asn Tyr Ser Gly Asn Ala Asn Tyr Asn Pro Ser Leu Lys

50 55 60

Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu

65 70 75 80

Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Thr

85 90 95

Ser Arg Ile Arg Ser His Ile Ala Tyr Ser Trp Lys Gly Asp Val Trp

100 105 110

Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu

115 120 125

Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu

130 135 140

Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Pro Arg Asn Tyr

145 150 155 160

Ile Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile

165 170 175

Tyr Gly Ala Ser Ser Arg Ala Ala Gly Phe Pro Asp Arg Phe Ser Gly

180 185 190

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro

195 200 205

Glu Asp Phe Ala Met Tyr Tyr Cys His Gln Tyr Asp Arg Leu Pro Tyr

210 215 220

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys Asp Asn

245 250 255

Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys

260 265 270

Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu

275 280 285

Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser

290 295 300

Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn

305 310 315 320

Gln Asp Pro Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu

325 330 335

Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys

340 345 350

Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys

355 360 365

Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe

370 375 380

Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

385 390 395

<210> 39

<211> 405

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 39

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Thr Thr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Glu Val Asn Tyr Ser Gly Asn Ala Asn Tyr Asn Pro Ser

210 215 220

Leu Lys Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Arg Ile Arg Ser His Ile Ala Tyr Ser Trp Lys Gly Asp

260 265 270

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

275 280 285

Ser Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro

290 295 300

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Pro Arg

305 310 315 320

Asn Tyr Ile Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

325 330 335

Leu Ile Tyr Gly Ala Ser Ser Arg Ala Ala Gly Phe Pro Asp Arg Phe

340 345 350

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu

355 360 365

Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys His Gln Tyr Asp Arg Leu

370 375 380

Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Asp Tyr Lys

385 390 395 400

Asp Asp Asp Asp Lys

405

<210> 40

<211> 569

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 40

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Thr Thr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Glu Val Asn Tyr Ser Gly Asn Ala Asn Tyr Asn Pro Ser

210 215 220

Leu Lys Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Arg Ile Arg Ser His Ile Ala Tyr Ser Trp Lys Gly Asp

260 265 270

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

275 280 285

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr

290 295 300

Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

305 310 315 320

Ser Cys Arg Ala Ser Gln Ser Val Pro Arg Asn Tyr Ile Gly Trp Phe

325 330 335

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser

340 345 350

Ser Arg Ala Ala Gly Phe Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

355 360 365

Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala

370 375 380

Met Tyr Tyr Cys His Gln Tyr Asp Arg Leu Pro Tyr Thr Phe Gly Gln

385 390 395 400

Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

405 410 415

Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys Asp Asn Phe Pro Gln Met

420 425 430

Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln

435 440 445

Thr Lys Asp Glu Val Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu

450 455 460

Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln

465 470 475 480

Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu

485 490 495

Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg

500 505 510

Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser

515 520 525

Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys

530 535 540

Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile

545 550 555 560

Glu Ala Tyr Met Thr Ile Lys Ala Arg

565

<210> 41

<211> 399

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 41

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Val Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys Val

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Asp Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Gly Ser Ser Ser Ser Gly Asp Tyr Tyr Tyr Gly Met Asp

100 105 110

Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

115 120 125

Ser Glu Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro

130 135 140

Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Pro Arg

145 150 155 160

Asn Tyr Ile Gly Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

165 170 175

Leu Ile Tyr Gly Ala Ser Ser Arg Ala Ala Gly Phe Pro Asp Arg Phe

180 185 190

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu

195 200 205

Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys His Gln Tyr Asp Arg Leu

210 215 220

Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly

225 230 235 240

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys

245 250 255

Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

260 265 270

Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu

275 280 285

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

290 295 300

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

305 310 315 320

Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu

325 330 335

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

340 345 350

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe

355 360 365

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

370 375 380

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

385 390 395

<210> 42

<211> 401

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 42

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Thr Thr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Glu Val Asn Tyr Ser Gly Asn Ala Asn Tyr Asn Pro Ser

210 215 220

Leu Lys Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Arg Ile Arg Ser His Ile Ala Tyr Ser Trp Lys Gly Asp

260 265 270

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

275 280 285

Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro

290 295 300

Gly Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His

305 310 315 320

Thr Asp Gly Thr Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln

325 330 335

Pro Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val

340 345 350

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys

355 360 365

Ile Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln

370 375 380

Asn Ile Gln Leu Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile

385 390 395 400

Lys

<210> 43

<211> 575

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 43

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro

165 170 175

Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr

180 185 190

Ser Tyr Gly Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu

195 200 205

Trp Met Gly Trp Ile Ser Val Tyr Ser Gly Asn Thr Asn Tyr Ala Gln

210 215 220

Lys Val Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr

225 230 235 240

Ala Tyr Met Asp Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr

245 250 255

Tyr Cys Ala Arg Glu Gly Ser Ser Ser Ser Gly Asp Tyr Tyr Tyr Gly

260 265 270

Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

290 295 300

Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly Gln Pro Ala

305 310 315 320

Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Thr Asp Gly Thr

325 330 335

Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro Gln Leu

340 345 350

Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe

355 360 365

Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val

370 375 380

Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Asn Ile Gln Leu

385 390 395 400

Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly

405 410 415

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys

420 425 430

Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

435 440 445

Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp Asn Leu Leu Leu

450 455 460

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

465 470 475 480

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

485 490 495

Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu

500 505 510

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

515 520 525

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe

530 535 540

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

545 550 555 560

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

565 570 575

<210> 44

<211> 573

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<220>

<221> Modified residue

<222> (193)..(199)

<223> Any amino acid

<220>

<221> Other sign

<222> (193)..(199)

<223> This region may span 3-7 residues

<220>

<221> Modified residue

<222> (213)..(230)

<223> Any amino acid

<220>

<221> Other sign

<222> (213)..(230)

<223> This region may span 14-18 residues

<220>

<221> Modified residue

<222> (263)..(273)

<223> Any amino acid

<220>

<221> Other sign

<222> (263)..(273)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (321)..(334)

<223> Any amino acid

<220>

<221> Other sign

<222> (321)..(334)

<223> This region may span 9-14 residues

<220>

<221> Modified residue

<222> (350)..(358)

<223> Any amino acid

<220>

<221> Other sign

<222> (350)..(358)

<223> This region may span 5-9 residues

<220>

<221> Modified residue

<222> (391)..(401)

<223> Any amino acid

<220>

<221> Other sign

<222> (391)..(401)

<223> This region may span 7-11 residues

<220>

<223> See the attached description for a detailed description of the substitutions and preferred

embodiments

<400> 44

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly

195 200 205

Leu Glu Trp Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

210 215 220

Xaa Xaa Xaa Xaa Xaa Xaa Arg Val Thr Ile Ser Val Asp Thr Ser Lys

225 230 235 240

Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Ala Ala Asp Thr Ala

245 250 255

Ile Tyr Tyr Cys Thr Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

260 265 270

Xaa Trp Gly Lys Gly Thr Thr Val Thr Val Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser

290 295 300

Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

305 310 315 320

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Phe

325 330 335

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Xaa Xaa Xaa

340 345 350

Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe Pro Asp Arg Phe Ser Gly Ser Gly

355 360 365

Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp

370 375 380

Phe Ala Met Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

385 390 395 400

Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

405 410 415

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys Asp Asn

420 425 430

Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys

435 440 445

Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu

450 455 460

Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser

465 470 475 480

Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn

485 490 495

Gln Asp Pro Glu Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu

500 505 510

Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys

515 520 525

Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys

530 535 540

Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe

545 550 555 560

Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

565 570

<210> 45

<211> 573

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<220>

<221> Modified residue

<222> (193)..(199)

<223> Any amino acid

<220>

<221> Other sign

<222> (193)..(199)

<223> This region may span 3-7 residues

<220>

<221> Modified residue

<222> (213)..(230)

<223> Any amino acid

<220>

<221> Other sign

<222> (213)..(230)

<223> This region may span 14-18 residues

<220>

<221> Modified residue

<222> (263)..(273)

<223> Any amino acid

<220>

<221> Other sign

<222> (263)..(273)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (321)..(334)

<223> Any amino acid

<220>

<221> Other sign

<222> (321)..(334)

<223> This region may span 9-14 residues

<220>

<221> Modified residue

<222> (350)..(358)

<223> Any amino acid

<220>

<221> Other sign

<222> (350)..(358)

<223> This region may span 5-9 residues

<220>

<221> Modified residue

<222> (391)..(401)

<223> Any amino acid

<220>

<221> Other sign

<222> (391)..(401)

<223> This region may span 7-11 residues

<220>

<223> See the attached description for a detailed description of the substitutions and preferred

embodiments

<400> 45

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly

195 200 205

Leu Glu Trp Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

210 215 220

Xaa Xaa Xaa Xaa Xaa Xaa Arg Val Thr Ile Ser Val Asp Thr Ser Lys

225 230 235 240

Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Ala Ala Asp Thr Ala

245 250 255

Ile Tyr Tyr Cys Thr Ser Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

260 265 270

Xaa Trp Gly Lys Gly Thr Thr Val Thr Val Gly Gly Gly Gly Ser Gly

275 280 285

Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser

290 295 300

Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys

305 310 315 320

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Phe

325 330 335

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Xaa Xaa Xaa

340 345 350

Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe Pro Asp Arg Phe Ser Gly Ser Gly

355 360 365

Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp

370 375 380

Phe Ala Met Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

385 390 395 400

Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser

405 410 415

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys Asp Asn

420 425 430

Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys

435 440 445

Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu

450 455 460

Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser

465 470 475 480

Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn

485 490 495

Gln Asp Pro Glu Ala Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu

500 505 510

Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys

515 520 525

Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys

530 535 540

Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe

545 550 555 560

Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys Ala Arg

565 570

<210> 46

<211> 402

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<220>

<221> Modified residue

<222> (31)..(37)

<223> Any amino acid

<220>

<221> Other sign

<222> (31)..(37)

<223> This region may span 3-7 residues

<220>

<221> Modified residue

<222> (52)..(62)

<223> Any amino acid

<220>

<221> Other sign

<222> (52)..(62)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (95)..(105)

<223> Any amino acid

<220>

<221> Other sign

<222> (95)..(105)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (150)..(163)

<223> Any amino acid

<220>

<221> Other sign

<222> (150)..(163)

<223> This region may span 9-14 residues

<220>

<221> Modified residue

<222> (179)..(187)

<223> Any amino acid

<220>

<221> Other sign

<222> (179)..(187)

<223> This region may span 5-9 residues

<220>

<221> Modified residue

<222> (220)..(230)

<223> Any amino acid

<220>

<221> Other sign

<222> (220)..(230)

<223> This region may span 7-11 residues

<220>

<223> See the attached description for a detailed description of the substitutions and preferred

embodiments

<400> 46

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Val

50 55 60

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn

65 70 75 80

Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Thr Ser Xaa Xaa

85 90 95

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Lys Gly Asp Val Trp Gly

100 105 110

Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile

115 120 125

Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg

130 135 140

Ala Thr Leu Ser Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

145 150 155 160

Xaa Xaa Xaa Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

165 170 175

Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe Pro Asp Arg

180 185 190

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg

195 200 205

Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa

210 215 220

Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr

245 250 255

Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala

260 265 270

Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp Asn

275 280 285

Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly

290 295 300

Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met

305 310 315 320

Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn Ser

325 330 335

Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His

340 345 350

Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys

355 360 365

Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser

370 375 380

Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys

385 390 395 400

Ala Arg

<210> 47

<211> 409

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<220>

<221> Modified residue

<222> (192)..(198)

<223> Any amino acid

<220>

<221> Other sign

<222> (192)..(198)

<223> This region may span 3-7 residues

<220>

<221> Modified residue

<222> (213)..(223)

<223> Any amino acid

<220>

<221> Other sign

<222> (213)..(223)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (256)..(266)

<223> Any amino acid

<220>

<221> Other sign

<222> (256)..(266)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (311)..(324)

<223> Any amino acid

<220>

<221> Other sign

<222> (311)..(324)

<223> This region may span 9-14 residues

<220>

<221> Modified residue

<222> (340)..(348)

<223> Any amino acid

<220>

<221> Other sign

<222> (340)..(348)

<223> This region may span 5-9 residues

<220>

<221> Modified residue

<222> (381)..(391)

<223> Any amino acid

<220>

<221> Other sign

<222> (381)..(391)

<223> This region may span 7-11 residues

<220>

<223> See the attached description for a detailed description of the substitutions and preferred

embodiments

<400> 47

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Xaa

180 185 190

Xaa Xaa Xaa Xaa Xaa Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu

195 200 205

Glu Trp Ile Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg

210 215 220

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu

225 230 235 240

Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Thr Ser Xaa

245 250 255

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Lys Gly Asp Val Trp

260 265 270

Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu

275 280 285

Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu

290 295 300

Arg Ala Thr Leu Ser Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

305 310 315 320

Xaa Xaa Xaa Xaa Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

325 330 335

Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe Pro Asp

340 345 350

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr

355 360 365

Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Xaa Xaa Xaa Xaa

370 375 380

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile

385 390 395 400

Lys Asp Tyr Lys Asp Asp Asp Asp Lys

405

<210> 48

<211> 402

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<220>

<221> Modified residue

<222> (31)..(37)

<223> Any amino acid

<220>

<221> Other sign

<222> (31)..(37)

<223> This region may span 3-7 residues

<220>

<221> Modified residue

<222> (52)..(62)

<223> Any amino acid

<220>

<221> Other sign

<222> (52)..(62)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (95)..(105)

<223> Any amino acid

<220>

<221> Other sign

<222> (95)..(105)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (150)..(163)

<223> Any amino acid

<220>

<221> Other sign

<222> (150)..(163)

<223> This region may span 9-14 residues

<220>

<221> Modified residue

<222> (179)..(187)

<223> Any amino acid

<220>

<221> Other sign

<222> (179)..(187)

<223> This region may span 5-9 residues

<220>

<221> Modified residue

<222> (220)..(230)

<223> Any amino acid

<220>

<221> Other sign

<222> (220)..(230)

<223> This region may span 7-11 residues

<220>

<223> See the attached description for a detailed description of the substitutions and preferred

embodiments

<400> 48

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Val

50 55 60

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn

65 70 75 80

Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Thr Ser Xaa Xaa

85 90 95

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Lys Gly Asp Val Trp Gly

100 105 110

Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile

115 120 125

Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg

130 135 140

Ala Thr Leu Ser Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

145 150 155 160

Xaa Xaa Xaa Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

165 170 175

Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe Pro Asp Arg

180 185 190

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg

195 200 205

Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa

210 215 220

Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr

245 250 255

Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala

260 265 270

Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp Asn

275 280 285

Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly

290 295 300

Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met

305 310 315 320

Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn Ser

325 330 335

Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His

340 345 350

Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys

355 360 365

Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser

370 375 380

Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys

385 390 395 400

Ala Arg

<210> 49

<211> 409

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<220>

<221> Modified residue

<222> (192)..(198)

<223> Any amino acid

<220>

<221> Other sign

<222> (192)..(198)

<223> This region may span 3-7 residues

<220>

<221> Modified residue

<222> (213)..(223)

<223> Any amino acid

<220>

<221> Other sign

<222> (213)..(223)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (256)..(266)

<223> Any amino acid

<220>

<221> Other sign

<222> (256)..(266)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (311)..(324)

<223> Any amino acid

<220>

<221> Other sign

<222> (311)..(324)

<223> This region may span 9-14 residues

<220>

<221> Modified residue

<222> (340)..(348)

<223> Any amino acid

<220>

<221> Other sign

<222> (340)..(348)

<223> This region may span 5-9 residues

<220>

<221> Modified residue

<222> (381)..(391)

<223> Any amino acid

<220>

<221> Other sign

<222> (381)..(391)

<223> This region may span 7-11 residues

<220>

<223> See the attached description for a detailed description of the substitutions and preferred

embodiments

<400> 49

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Xaa

180 185 190

Xaa Xaa Xaa Xaa Xaa Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu

195 200 205

Glu Trp Ile Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg

210 215 220

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu

225 230 235 240

Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Thr Ser Xaa

245 250 255

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Lys Gly Asp Val Trp

260 265 270

Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu

275 280 285

Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu

290 295 300

Arg Ala Thr Leu Ser Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

305 310 315 320

Xaa Xaa Xaa Xaa Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

325 330 335

Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe Pro Asp

340 345 350

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr

355 360 365

Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Xaa Xaa Xaa Xaa

370 375 380

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile

385 390 395 400

Lys Asp Tyr Lys Asp Asp Asp Asp Lys

405

<210> 50

<211> 402

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<220>

<221> Modified residue

<222> (31)..(37)

<223> Any amino acid

<220>

<221> Other sign

<222> (31)..(37)

<223> This region may span 3-7 residues

<220>

<221> Modified residue

<222> (52)..(62)

<223> Any amino acid

<220>

<221> Other sign

<222> (52)..(62)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (95)..(105)

<223> Any amino acid

<220>

<221> Other sign

<222> (95)..(105)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (150)..(163)

<223> Any amino acid

<220>

<221> Other sign

<222> (150)..(163)

<223> This region may span 9-14 residues

<220>

<221> Modified residue

<222> (179)..(187)

<223> Any amino acid

<220>

<221> Other sign

<222> (179)..(187)

<223> This region may span 5-9 residues

<220>

<221> Modified residue

<222> (220)..(230)

<223> Any amino acid

<220>

<221> Other sign

<222> (220)..(230)

<223> This region may span 7-11 residues

<220>

<223> See the attached description for a detailed description of the substitutions and preferred

embodiments

<400> 50

Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

1 5 10 15

Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr Xaa Xaa

20 25 30

Xaa Xaa Xaa Xaa Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

35 40 45

Trp Ile Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Val

50 55 60

Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn

65 70 75 80

Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Thr Ser Xaa Xaa

85 90 95

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Lys Gly Asp Val Trp Gly

100 105 110

Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu Ile

115 120 125

Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg

130 135 140

Ala Thr Leu Ser Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

145 150 155 160

Xaa Xaa Xaa Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu

165 170 175

Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe Pro Asp Arg

180 185 190

Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg

195 200 205

Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Xaa Xaa Xaa Xaa Xaa

210 215 220

Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

225 230 235 240

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr

245 250 255

Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp Ala

260 265 270

Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp Asn

275 280 285

Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly

290 295 300

Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met

305 310 315 320

Pro Gln Ala Glu Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn Ser

325 330 335

Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His

340 345 350

Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile Lys

355 360 365

Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser

370 375 380

Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile Lys

385 390 395 400

Ala Arg

<210> 51

<211> 409

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<220>

<221> Modified residue

<222> (192)..(198)

<223> Any amino acid

<220>

<221> Other sign

<222> (192)..(198)

<223> This region may span 3-7 residues

<220>

<221> Modified residue

<222> (213)..(223)

<223> Any amino acid

<220>

<221> Other sign

<222> (213)..(223)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (256)..(266)

<223> Any amino acid

<220>

<221> Other sign

<222> (256)..(266)

<223> This region may span 7-11 residues

<220>

<221> Modified residue

<222> (311)..(324)

<223> Any amino acid

<220>

<221> Other sign

<222> (311)..(324)

<223> This region may span 9-14 residues

<220>

<221> Modified residue

<222> (340)..(348)

<223> Any amino acid

<220>

<221> Other sign

<222> (340)..(348)

<223> This region may span 5-9 residues

<220>

<221> Modified residue

<222> (381)..(391)

<223> Any amino acid

<220>

<221> Other sign

<222> (381)..(391)

<223> This region may span 7-11 residues

<220>

<223> See the attached description for a detailed description of the substitutions and preferred

embodiments

<400> 51

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Xaa

180 185 190

Xaa Xaa Xaa Xaa Xaa Xaa Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu

195 200 205

Glu Trp Ile Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg

210 215 220

Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu

225 230 235 240

Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Thr Ser Xaa

245 250 255

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Trp Lys Gly Asp Val Trp

260 265 270

Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Glu

275 280 285

Ile Val Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu

290 295 300

Arg Ala Thr Leu Ser Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

305 310 315 320

Xaa Xaa Xaa Xaa Trp Phe Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu

325 330 335

Leu Ile Tyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Gly Phe Pro Asp

340 345 350

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr

355 360 365

Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Xaa Xaa Xaa Xaa

370 375 380

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile

385 390 395 400

Lys Asp Tyr Lys Asp Asp Asp Asp Lys

405

<210> 52

<211> 569

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 52

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Thr Thr Tyr Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Glu Val Asn Tyr Ser Gly Asn Ala Asn Tyr Asn Pro Ser

210 215 220

Leu Lys Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Arg Ile Arg Ser His Ile Ala Tyr Ser Trp Lys Gly Asp

260 265 270

Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly

275 280 285

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr

290 295 300

Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu

305 310 315 320

Ser Cys Arg Ala Ser Gln Ser Val Pro Arg Asn Tyr Ile Gly Trp Phe

325 330 335

Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser

340 345 350

Ser Arg Ala Ala Gly Phe Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly

355 360 365

Thr Asp Phe Thr Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala

370 375 380

Met Tyr Tyr Cys His Gln Tyr Asp Arg Leu Pro Tyr Thr Phe Gly Gln

385 390 395 400

Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly

405 410 415

Ser Gly Gly Gly Gly Ser Thr Asp Gln Cys Asp Asn Phe Pro Gln Met

420 425 430

Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln

435 440 445

Thr Lys Asp Glu Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu

450 455 460

Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln

465 470 475 480

Phe Tyr Leu Glu Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu

485 490 495

Ile Lys Asp His Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg

500 505 510

Leu Arg Leu Arg Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser

515 520 525

Lys Ala Val Glu Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys

530 535 540

Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile

545 550 555 560

Glu Ala Tyr Met Thr Ile Lys Ala Arg

565

<210> 53

<211> 563

<212> Protein

<213> Artificial sequence

<220>

<223> Description of artificial sequence: synthetic polypeptide

<400> 53

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Arg Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Thr

180 185 190

Asn Tyr Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro

210 215 220

Phe Thr Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Gln Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Trp Gly Lys Gly

260 265 270

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

275 280 285

Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro Gly Thr

290 295 300

Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

305 310 315 320

Gln Ser Ile Gly Thr Asn Ile His Trp Phe Gln Gln Lys Pro Gly Gln

325 330 335

Ala Pro Arg Leu Leu Ile Tyr Tyr Ala Ser Glu Ser Ile Ser Gly Phe

340 345 350

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

355 360 365

Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys Gln Gln

370 375 380

Asn Asn Asn Trp Pro Thr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

385 390 395 400

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

405 410 415

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

420 425 430

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

435 440 445

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

450 455 460

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

465 470 475 480

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

485 490 495

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

500 505 510

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

515 520 525

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

530 535 540

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

545 550 555 560

Lys Ala Arg

<210> 54

<211> 17

<212> Protein

<213> Artificial sequence

<220>

<223> Description of the artificial sequence: synthetic peptide

<400> 54

Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 55

<211> 147

<212> Protein

<213> Artificial sequence

<220>

<223> DV05 protein

<400> 55

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ala Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg

145

<210> 56

<211> 441

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid DV05

<220>

<221> Other sign

<222> (225)..(225)

<223> n is a, c, g, or t

<400> 56

accgaccagt gcgacaactt ccctcagatg ctgcgggacc tgagagatgc cttctccaga 60

gtgaaaacat tcttccagac caaggacgag ctggacaacc tgctgctgaa agagtccctg 120

ctggaagatt tcaagggcta cctgggctgt caggccctgt ccgagatgat ccagttctac 180

ctggaagaag tgatgcccca ggccgagaat caggaccccg aggcnaagga ccacgtgaac 240

tccctgggcg agaacctgaa aaccctgcgg ctgagactgc ggcggtgcca cagatttctg 300

ccctgcgaga acaagtccaa ggccgtggaa cagatcaaga acgccttcaa caagctgcaa 360

gagaagggca tctacaaggc catgagcgag ttcgacatct tcatcaacta catcgaggcc 420

tacatgacca tcaaggccag a 441

<210> 57

<211> 147

<212> Protein

<213> Artificial sequence

<220>

<223> DV06 protein

<400> 57

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Val Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg

145

<210> 58

<211> 441

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid DV06

<220>

<221> Other sign

<222> (93)..(93)

<223> n is a, c, g, or t

<400> 58

accgaccagt gcgacaactt ccctcagatg ctgcgggacc tgagagatgc cttctccaga 60

gtgaaaacat tcttccagac caaggacgag gtngacaacc tgctgctgaa agagtccctg 120

ctggaagatt tcaagggcta cctgggctgt caggccctgt ccgagatgat ccagttctac 180

ctggaagaag tgatgcccca ggccgagaat caggaccccg agatcaagga ccacgtgaac 240

tccctgggcg agaacctgaa aaccctgcgg ctgagactgc ggcggtgcca cagatttctg 300

ccctgcgaga acaagtccaa ggccgtggaa cagatcaaga acgccttcaa caagctgcaa 360

gagaagggca tctacaaggc catgagcgag ttcgacatct tcatcaacta catcgaggcc 420

tacatgacca tcaaggccag a 441

<210> 59

<211> 147

<212> Protein

<213> Artificial sequence

<220>

<223> DV07 protein

<400> 59

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg

145

<210> 60

<211> 441

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid DV07

<400> 60

accgaccagt gcgacaactt ccctcagatg ctgcgggacc tgagagatgc cttctccaga 60

gtgaaaacat tcttccagac caaggacgag ctggacaacc tgctgctgaa agagtccctg 120

ctggaagatt tcaagggcta cctgggctgt caggccctgt ccgagatgat ccagttctac 180

ctggaagaag tgatgcccca ggccgagaat caggaccccg agatcaagga ccacgtgaac 240

tccctgggcg agaacctgaa aaccctgcgg ctgagactgc ggcggtgcca cagatttctg 300

ccctgcgaga acaagtccaa ggccgtggaa cagatcaaga acgccttcaa caagctgcaa 360

gagaagggca tctacaaggc catgagcgag ttcgacatct tcatcaacta catcgaggcc 420

tacatgacca tcaaggccag a 441

<210> 61

<211> 565

<212> Protein

<213> Artificial sequence

<220>

<223> Option 1 DEboDV07

<400> 61

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Ser Gly Phe Ser Leu Thr

180 185 190

Asn Tyr Gly Val Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Pro Ser

210 215 220

Leu Lys Gly Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly

260 265 270

Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro

290 295 300

Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

305 310 315 320

Ala Ser Gln Ser Ile Gly Thr Asn Ile Gly Trp Phe Gln Gln Lys Pro

325 330 335

Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Arg Ala Ala

340 345 350

Gly Phe Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

355 360 365

Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys

370 375 380

Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Gln Gly Thr Lys Leu

385 390 395 400

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

405 410 415

Gly Ser Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu

420 425 430

Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu

435 440 445

Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly

450 455 460

Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu

465 470 475 480

Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His

485 490 495

Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg

500 505 510

Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu

515 520 525

Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys

530 535 540

Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met

545 550 555 560

Th Ile Lys Ala Arg

565

<210> 62

<211> 1698

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid variant 1 DEboDV07

<400> 62

accgaccagt gcgacaactt ccctcagatg ctgcgggacc tgagagatgc cttctccaga 60

gtgaaaacat tcttccagac caaggacgag ctggacaacc tgctgctgaa agagtccctg 120

ctggaagatt tcaagggcta cctgggctgt caggccctgt ccgagatgat ccagttctac 180

ctggaagaag tgatgcccca ggccgagaat caggaccccg agatcaagga ccacgtgaac 240

tccctgggcg agaacctgaa aaccctgcgg ctgagactgc ggcggtgcca cagatttctg 300

ccctgcgaga acaagtccaa ggccgtggaa cagatcaaga acgccttcaa caagctgcaa 360

gagaagggca tctacaaggc catgagcgag ttcgacatct tcatcaacta catcgaggcc 420

tacatgacca tcaaggccag aggcggcgga ggatctggcg gaggtggaag cggaggcggt 480

ggatctcagg ttcagttgca gcaatggggc gctggcctgc tgaagccttc tgagacactg 540

tctctgacct gcgccgtgtc cggcttctct ctgaccaatt acggcgtgaa ctggattcgg 600

cagcctcctg gcaaaggcct ggaatggatc ggagtgattt ggagcggcgg caacaccgac 660

tacaacccca gtctgaaggg cagagtggcc atctccgtgg acacctccaa gaaccagttc 720

tccctgagac tgaactccgt gaccgccgct gataccgcca tctactactg tgctagagcc 780

ctgacctact acgactacga gttcgcctat tggggcaagg gcaccaccgt gactgttagt 840

agtggtggtg gcggtagtgg cggaggcggc tcaggcggtg gtggatctga aatcgtgatg 900

acccagtctc ctggcactct gtctctgtct cccggcgaga gagctaccct gtcttgtaga 960

gcctctcagt ccatcggcac caacatcggc tggttccagc agaagcctgg acaggctccc 1020

cggctgctga ttaagtacgc ctctgagaga gccgctggct tccctgacag attctccggc 1080

tctggctctg gcaccgactt caccctgacc atcaccagac tggaacccga ggacttcgct 1140

atgtactact gccagcagaa caacaactgg cccaccacct ttggccaggg caccaagctg 1200

gaaatcaaag gtggcggtgg ttcaggtggc ggaggaagcg gcggaggcgg atctacagat 1260

cagtgtgaca attttcccca aatgctgagg gatctgcggg acgccttcag ccgggtcaag 1320

acattttttc agacaaagga tgaactcgat aacctcttgc tcaaagagag cctgctcgag 1380

gactttaagg gatatctggg atgccaggct ctgagcgaaa tgattcagtt ttatctcgag 1440

gaagtcatgc ctcaagcaga gaaccaggat ccagagatta aggatcatgt gaatagcctc 1500

ggggagaacc tcaagacact gagactccgg ctgagaagat gccaccggtt tctgccttgt 1560

gaaaacaaaa gcaaggctgt cgagcagatt aagaatgctt ttaacaaact ccaagaaaaa 1620

gggatctata aggctatgtc tgagtttgat atctttatca attatatcga agcttatatg 1680

actattaagg cccggtag 1698

<210> 63

<211> 565

<212> Protein

<213> Artificial sequence

<220>

<223> Option 2 DEboDV07

<400> 63

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Phe Ser Leu Thr

180 185 190

Asn Tyr Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Ile Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro

210 215 220

Phe Thr Ser Arg Val Ala Ile Ser Val Asp Thr Ser Lys Asn Gln Phe

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Thr Ser Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly

260 265 270

Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Met Thr Gln Ser Pro

290 295 300

Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

305 310 315 320

Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Phe Gln Gln Lys Pro

325 330 335

Gly Gln Ala Pro Arg Leu Leu Ile Tyr Tyr Ala Ser Glu Ser Ile Ser

340 345 350

Gly Phe Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

355 360 365

Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys

370 375 380

Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Gln Gly Thr Lys Leu

385 390 395 400

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

405 410 415

Gly Ser Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu

420 425 430

Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu

435 440 445

Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly

450 455 460

Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu

465 470 475 480

Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His

485 490 495

Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg

500 505 510

Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu

515 520 525

Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys

530 535 540

Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met

545 550 555 560

Th Ile Lys Ala Arg

565

<210> 64

<211> 1770

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid variant 2 DEboDV07

<400> 64

gctagcgccg cccacatggg atggtctttg atcctgctgt tcctggtggc cgtggctacc 60

agagtgcatt ctaccgacca gtgcgacaac ttccctcaga tgctgcggga cctgagagat 120

gccttctcca gagtgaaaac attcttccag accaaggacg agctggacaa cctgctgctg 180

aaagagtccc tgctggaaga tttcaagggc tacctgggct gtcaggccct gtccgagatg 240

atccagttct acctggaaga agtgatgccc caggccgaga atcaggaccc cgagatcaag 300

gaccacgtga actccctggg cgagaacctg aaaaccctgc ggctgagact gcggcggtgc 360

cacagatttc tgccctgcga gaacaagtcc aaggccgtgg aacagatcaa gaacgccttc 420

aacaagctgc aagagaaggg catctacaag gccatgagcg agttcgacat cttcatcaac 480

tacatcgagg cctacatgac catcaaggcc agaggcggcg gaggatctgg cggaggtgga 540

agcggaggcg gtggatctca ggttcagttg cagcaatggg gcgctggcct gctgaagcct 600

tctgagacac tgtctctgac ctgcgccgtg tacggcttct ccctgaccaa ttatggcgtg 660

cactggatca gacagcctcc aggcaaaggc ctggaatgga tcggagtgat ttggagcggc 720

ggcaacaccg actacaacac ccctttcacc tctagagtgg ccatctccgt ggacacctcc 780

aagaaccagt tcagcctgag actgaactcc gtgaccgccg ctgataccgc catctactac 840

tgcacctccg ctctgaccta ctacgactac gagttcgcct actggggcaa gggcaccaca 900

gtgactgtta gtagtggtgg cggaggtagc ggtggtggtg gtagtggcgg tggcggatct 960

gagatcgtga tgacccaatc tcctggcact ctgtctctgt ctcccggcga gagagctacc 1020

ctgtcttgta gagcctctca gtccatcggc accaacatcc actggttcca gcagaagcct 1080

ggacaggccc ctagactgct gatctactac gcctccgaga gcatcagcgg cttccctgac 1140

agattctccg gctctggctc tggcaccgac ttcaccctga caatcacccg gctggaacct 1200

gaggacttcg ctatgtacta ctgccagcag aacaacaact ggcccaccac ctttggccag 1260

ggcaccaagc tggaaatcaa aggcggaggc ggcagtggcg gcggtggctc cggcggaggc 1320

ggatctacag atcagtgtga caattttccc caaatgctga gggatctgcg ggacgccttc 1380

agccgggtca agacattttt tcagacaaag gatgaactcg ataacctctt gctcaaagag 1440

agcctgctcg aggacttcaa aggatatctg ggatgccagg ctctgagcga aatgattcag 1500

ttttatctcg aggaagtcat gccacaagca gagaaccagg atccagagat taaggatcat 1560

gtgaatagcc tcggggagaa cctcaagaca ctgagactcc ggctgagaag atgccaccgg 1620

tttctgcctt gtgaaaacaa aagcaaggct gtcgagcaga ttaagaatgc ttttaacaaa 1680

ctccaagaaa aagggatcta taaggctatg tctgagtttg atatctttat caattatatc 1740

gaagcttata tgactattaa ggcccggtag 1770

<210> 65

<211> 565

<212> Protein

<213> Artificial sequence

<220>

<223> Option 3 DEboDV07

<400> 65

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Phe Ser Leu Thr

180 185 190

Asn Tyr Gly Val His Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro

210 215 220

Phe Thr Ser Arg Val Ala Ile Ser Lys Asp Asn Ser Lys Asn Gln Val

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly

260 265 270

Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro

290 295 300

Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

305 310 315 320

Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Lys Pro

325 330 335

Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser

340 345 350

Gly Phe Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

355 360 365

Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala Met Tyr Tyr Cys

370 375 380

Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Gln Gly Thr Lys Leu

385 390 395 400

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

405 410 415

Gly Ser Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu

420 425 430

Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu

435 440 445

Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly

450 455 460

Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu

465 470 475 480

Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His

485 490 495

Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg

500 505 510

Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu

515 520 525

Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys

530 535 540

Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met

545 550 555 560

Th Ile Lys Ala Arg

565

<210> 66

<211> 1698

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid variant 3 DEboDV07

<400> 66

accgaccagt gcgacaactt ccctcagatg ctgcgggacc tgagagatgc cttctccaga 60

gtgaaaacat tcttccagac caaggacgag ctggacaacc tgctgctgaa agagtccctg 120

ctggaagatt tcaagggcta cctgggctgt caggccctgt ccgagatgat ccagttctac 180

ctggaagaag tgatgcccca ggccgagaat caggaccccg agatcaagga ccacgtgaac 240

tccctgggcg agaacctgaa aaccctgcgg ctgagactgc ggcggtgcca cagatttctg 300

ccctgcgaga acaagtccaa ggccgtggaa cagatcaaga acgccttcaa caagctgcaa 360

gagaagggca tctacaaggc catgagcgag ttcgacatct tcatcaacta catcgaggcc 420

tacatgacca tcaaggccag aggcggcgga ggatctggcg gaggtggaag cggaggcggt 480

ggatctcagg ttcagttgca gcaatggggc gctggcctgc tgaagccttc tgagacactg 540

tctctgacct gcgccgtgta cggcttctcc ctgaccaatt atggcgtgca ctggatcaga 600

cagcctccag gcaaaggcct ggaatggctg ggagtgattt ggagcggcgg caacaccgac 660

tacaacaccc ctttcacctc tagagtggcc atctccaagg acaactccaa gaaccaggtg 720

tccctgagac tgaactccgt gaccgctgcc gataccgcca tctactactg tgctagagcc 780

ctgacctact acgactacga gttcgcctat tggggcaagg gcaccaccgt gactgttagt 840

agtggtggtg gcggtagtgg cggaggcggc tcaggcggtg gtggatctga aattgtgctg 900

acccagtctc ctggcactct gtctttgagc cctggcgaga gagctaccct gtcctgtaga 960

gcctctcagt ccatcggcac caacatccac tggtatcagc agaagcctgg acaggcccct 1020

cggctgctga ttaagtacgc ctccgagtcc atcagcggct tccctgacag attctccggc 1080

tctggctctg gcaccgactt caccctgaca atcacccggc tggaacctga ggacttcgct 1140

atgtactact gccagcagaa caacaactgg cccaccacct ttggccaggg caccaagctg 1200

gaaatcaaag gtggcggtgg ttcaggtggc ggaggaagcg gcggaggcgg atctacagat 1260

cagtgtgaca attttcccca aatgctgagg gatctgcggg acgccttcag ccgggtcaag 1320

acattttttc agacaaagga tgaactcgat aacctcttgc tcaaagagag cctgctcgag 1380

gacttcaaag gatatctggg atgccaggct ctgagcgaaa tgattcagtt ttatctcgag 1440

gaagtcatgc ctcaagcaga gaaccaggat ccagagatta aggatcatgt gaatagcctc 1500

ggggagaacc tcaagacact gagactccgg ctgagaagat gccaccggtt tctgccttgt 1560

gaaaacaaaa gcaaggctgt cgagcagatt aagaatgctt ttaacaaatt gcaagaaaaa 1620

gggatctata aggctatgtc tgagtttgat atctttatca attatatcga agcttatatg 1680

actattaagg cccggtag 1698

<210> 67

<211> 565

<212> Protein

<213> Artificial sequence

<220>

<223> Option 4 DEboDV07

<400> 67

Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu Arg Asp

1 5 10 15

Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu Leu Asp

20 25 30

Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu

35 40 45

Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val

50 55 60

Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His Val Asn

65 70 75 80

Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys

85 90 95

His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Ile

100 105 110

Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met

115 120 125

Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Ile

130 135 140

Lys Ala Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

145 150 155 160

Gly Ser Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro

165 170 175

Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr

180 185 190

Asn Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu

195 200 205

Trp Leu Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro

210 215 220

Phe Thr Ser Arg Val Ala Ile Ser Lys Asp Asn Ser Lys Asn Gln Val

225 230 235 240

Ser Leu Arg Leu Asn Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr

245 250 255

Cys Ala Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly

260 265 270

Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

275 280 285

Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr Gln Ser Pro

290 295 300

Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

305 310 315 320

Ala Ser Gln Ser Ile Gly Thr Asn Ile His Trp Tyr Gln Gln Lys Pro

325 330 335

Gly Gln Ala Pro Arg Leu Leu Ile Lys Tyr Ala Ser Glu Ser Ile Ser

340 345 350

Gly Ile Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

355 360 365

Leu Thr Ile Thr Arg Leu Glu Pro Glu Asp Phe Ala Asp Tyr Tyr Cys

370 375 380

Gln Gln Asn Asn Asn Trp Pro Thr Thr Phe Gly Gln Gly Thr Lys Leu

385 390 395 400

Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly

405 410 415

Gly Ser Thr Asp Gln Cys Asp Asn Phe Pro Gln Met Leu Arg Asp Leu

420 425 430

Arg Asp Ala Phe Ser Arg Val Lys Thr Phe Phe Gln Thr Lys Asp Glu

435 440 445

Leu Asp Asn Leu Leu Leu Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly

450 455 460

Tyr Leu Gly Cys Gln Ala Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu

465 470 475 480

Glu Val Met Pro Gln Ala Glu Asn Gln Asp Pro Glu Ile Lys Asp His

485 490 495

Val Asn Ser Leu Gly Glu Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg

500 505 510

Arg Cys His Arg Phe Leu Pro Cys Glu Asn Lys Ser Lys Ala Val Glu

515 520 525

Gln Ile Lys Asn Ala Phe Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys

530 535 540

Ala Met Ser Glu Phe Asp Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met

545 550 555 560

Th Ile Lys Ala Arg

565

<210> 68

<211> 1695

<212> DNA

<213> Artificial sequence

<220>

<223> Nucleic acid variant 4 DEboDV07

<400> 68

accgaccagt gcgacaactt ccctcagatg ctgcgggacc tgagagatgc cttctccaga 60

gtgaaaacat tcttccagac caaggacgag ctggacaacc tgctgctgaa agagtccctg 120

ctggaagatt tcaagggcta cctgggctgt caggccctgt ccgagatgat ccagttctac 180

ctggaagaag tgatgcccca ggccgagaat caggaccccg agatcaagga ccacgtgaac 240

tccctgggcg agaacctgaa aaccctgcgg ctgagactgc ggcggtgcca cagatttctg 300

ccctgcgaga acaagtccaa ggccgtggaa cagatcaaga acgccttcaa caagctgcaa 360

gagaagggca tctacaaggc catgagcgag ttcgacatct tcatcaacta catcgaggcc 420

tacatgacca tcaaggccag aggcggcgga ggatctggcg gaggtggaag cggaggcggt 480

ggatctcagg ttcagttgca gcaatggggc gctggcctgc tgaagccttc tgagacactg 540

tctctgacct gcaccgtgtc cggcttctcc ctgaccaatt atggcgtgca ctgggtccga 600

cagcctccag gcaaaggatt ggaatggctg ggagtgattt ggagcggcgg caacaccgac 660

tacaacaccc ctttcacctc tagagtggcc atctccaagg acaactccaa gaaccaggtg 720

tccctgagac tgaactccgt gaccgctgcc gataccgcca tctactactg tgctagagcc 780

ctgacctact acgactacga gttcgcctat tggggcaagg gcaccaccgt gactgttagt 840

agtggtggtg gcggtagtgg cggaggcggc tcaggcggtg gtggatctga aattgtgctg 900

acccagtctc ctggcactct gtctttgagc cctggcgaga gagctaccct gtcctgtaga 960

gcctctcagt ccatcggcac caacatccac tggtatcagc agaagcctgg acaggcccct 1020

cggctgctga ttaagtacgc ctccgagtcc atcagcggca tccctgacag attctccggc 1080

tctggctctg gcaccgactt caccctgaca atcacccggc tggaacctga ggacttcgcc 1140

gactactact gccagcagaa caacaactgg cccaccacct ttggccaggg caccaagctg 1200

gaaatcaaag gtggcggtgg ttcaggtggc ggaggaagcg gcggaggcgg atctacagat 1260

cagtgtgaca attttcccca aatgctgagg gatctgcggg acgccttcag ccgggtcaag 1320

acattttttc agacaaagga tgaactcgat aacctcttgc tcaaagagag cctgctcgag 1380

gacttcaaag gatatctggg atgccaggct ctgagcgaaa tgattcagtt ttatctcgag 1440

gaagtcatgc ctcaagcaga gaaccaggat ccagagatta aggatcatgt gaatagcctc 1500

ggggagaacc tcaagacact gagactccgg ctgagaagat gccaccggtt tctgccttgt 1560

gaaaacaaaa gcaaggctgt cgagcagatt aagaatgctt ttaacaaatt gcaagaaaaa 1620

gggatctata aggctatgtc tgagtttgat atctttatca attatatcga agcttatatg 1680

actattaagg cccgg 1695

<---

Claims (35)

1. Fusion protein of Formula (I-VII), which is an IL-10 receptor agonist, IL-10-L ¹ -X ¹ -L ¹ -X ² -L ¹ -IL-10 (Formula I); (Z) _n -X ¹ -L ² -Y ² -L ¹ -IL-10 (Formula II); IL-10-L ¹ -Y ¹ -L ² -X ² -(Z) _n (Formula III); X ¹ -L ² -X ² -L ¹ -IL-10 (Formula IV); IL-10-L ¹ -X ¹ -L ² -X ² (Formula V); X ¹ -L ¹ -IL-10 (Formula VI); and IL-10-L ¹ -X ² (Formula VII); Where "IL-10" is a monomeric sequence selected from SEQ ID NO: 1, 3, 14, 15, 16, 18, 19, 55, 57, or 59; "L ¹ " is a linker of SEQ ID NO: 31 or 54; "L ² " is the linker of SEQ ID NO: 30; "X ¹ " is a VH region derived from the first antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as but not limited to PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ trap; MadCam, β7 integrin subunit; α4β7 integrin; α4 integrin SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV or Ebola; "X ² " represents the VL region derived from the same antibody as X1; "Y ¹ " is a VH region derived from a second antibody specific for epidermal growth factor receptor (EGFR); CD52; various immune checkpoint targets such as but not limited to PD-L1, PD-1, TIM3, BTLA, LAG3, or CTLA4; CD20; CD47; GD-2; HER2; EpCAM; ICAM (ICAM-1, -2, -3, -4, -5), VCAM, FAPα; 5T4; Trop2; EDB-FN; TGFβ trap; MadCam, integrin β7 subunit; integrin α4β7; integrin α4 SR-A1; SR-A3; SR-A4; SR-A5; SR-A6; SR-B; dSR-C1; SR-D1; SR-E1; SR-F1; SR-F2; SR-G; SR-H1; SR-H2; SR-I1; SR-J1; HIV or Ebola; "Y ² " represents the VL region derived from the same antibody as Y1; where X and Y are obtained from the same or different antibodies; "Z" is a cytokine selected from IL-6, IL-4, IL-1, IL-2, IL-3, IL-5, IL-7, IL-8, IL-9, IL-15, IL-26, IL-27, IL-28, IL-29, GM-CSF, G-CSF, interferons -α, -β, -γ, TGF-β, or tumor necrosis factor-α, -β, basic FGF, EGF, PDGF, IL-4, IL-11, or IL-13; "n" is an integer chosen from 0-2. 2. The fusion protein of claim 1, wherein Formulas II and III are capable of forming a fusion protein complex, wherein the IL-10 monomer from each of Formulas II and III is capable of forming a functional homodimeric IL-10 or a variant thereof. 3. The fusion protein of claim 2, wherein Formula II is SEQ ID NO: 24, 26, 28, 41, 48, or 50. 4. The fusion protein of claim 2, wherein Formula III is SEQ ID NO: 25, 27, 29, 42, 49, or 51. 5. The fusion protein of claim 2, wherein the fusion protein complex is formed between SEQ ID NO: 24 and 25; 26 and 27; 28 and 29; 41 and 42; 48 and 49; or 50 and 51. 6. The fusion protein of claim 1, wherein Formulas IV and V are capable of forming a fusion protein complex, wherein the IL-10 monomer from each of Formulas IV and V is capable of forming a functional homodimeric IL-10 or a variant thereof. 7. The fusion protein of claim 6, wherein Formula IV is SEQ ID NO: 35, 38, 46, 48 or 50. 8. The fusion protein of claim 6, wherein Formula V is SEQ ID No: 36, 39, 47, 49 or 51. 9. The fusion protein of claim 6, wherein the fusion protein complex is formed between SEQ ID NO: 35 and 36; 38 and 39; 46 and 47; 48 and 49; or 50 and 51. 10. The fusion protein of claim 1, wherein Formulas VI and VII are capable of forming a fusion protein complex, wherein the IL-10 monomer from each of Formulas VI and VII is capable of forming a functional homodimeric IL-10 or a variant thereof. 11. The fusion protein of claim 1, wherein Formula I is SEQ ID NO: 33, 34, 40, 43, 44, 45, 52 or 53. 12. The fusion protein of claim 1, wherein “n” ≥1 and Z is IL-2, IL-7, IL-12, IL-15, or any combination thereof. 13. A method for treating a malignant tumor, comprising administering to a patient in need thereof a composition containing the fusion protein of claim 1. 14. The method of claim 13, wherein the fusion protein is SEQ ID NO: 28, 29, 35, 36, 38, 39, 46, 47, 52, 53, 61, 63, 65, or 67. 15. The method according to claim 14, wherein the fusion protein forms a protein complex, and the protein complex is formed between SEQ ID NO: 28 and 29; 35 and 36; 38 and 39; or 46 and 47. 16. The method of claim 13, wherein the fusion protein comprises IL-10 consisting of DV07, which is SEQ ID NO: 59. 17. The method of claim 16, wherein the fusion protein is SEQ ID NO: 26, 27, 41, 42, 48 or 49.

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