RU2833537C2 - Novel versions of interleukin-2 for treating cancer - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to human interleukin-2 (IL-2) muteins, and can be used in medicine in treating cancer. Disclosed is modified IL-2, which contains only two amino acid substitutions in positions P65 and S125 SEQ ID NO: 3; or only three amino acid substitutions in positions of amino acid residues L19, P65 and S125 of SEQ ID NO: 3, or only four amino acid substitutions in positions of amino acid residues L19, P65, S125 and Q126 SEQ ID NO: 3.

EFFECT: invention provides obtaining mutant IL-2, which demonstrates reduced binding to IL-2Rα (interleukin-2 alpha receptor) with reduced activity on Treg (regulatory T-cell) compared to the polypeptide presented in SEQ ID NO: 3, but retaining the ability to bind to IL 2Rβγ complex (interleukin-2 beta gamma receptor) and activate it.

14 cl, 34 dwg, 15 tbl, 16 ex

Description

RELATED PATENT APPLICATIONS

This application claims priority to U.S. Provisional Patent Application No. 62/947,806, filed December 13, 2019, and U.S. Provisional Patent Application No. 62/861,651, filed June 14, 2019, each of which is incorporated herein by reference in its entirety.

PRIOR ART

Interleukin-2 (IL-2) was the first growth factor described for T cells. Since its discovery, it has been shown to stimulate T cell proliferation and survival in vitro (Smith, KA (1988) Science. 240, 1169-76) and their ability to stimulate immune responses in the context of T virus infections (Blattman, J N, et al. (2003) Nat Med 9, 540-7) or vaccines (Fishman, M, et al. (2008) J Immunother. 31, 72-80; Kudo-Saito, C, et al. (2007) Cancer Immunol Immunother. 56, 1897-910; Lin, C T., et al. (2007) Immunol Lett. 114, 86-93).

IL-2 has applications in cancer therapy. Recombinant human IL-2 is an effective immunotherapy for metastatic melanoma and renal cell carcinoma, with durable responses in approximately 10% of patients. However, optimal dosing of IL-2 is limited by its short half-life and severe toxicity. In addition, IL-2 also binds to its heterotrimeric receptor IL-2Rαβγ with higher affinity, resulting in preferential expansion of immunosuppressive regulatory T cells (Treg) with high levels of constitutive IL-2Rα expression. From a cancer immunotherapy perspective, Treg expansion is an undesirable effect of IL-2. Therefore, successful cancer immunotherapy using IL-2 requires addressing two fundamental issues: (1) how to limit side effects while maintaining desired activity; and (2) how to ensure preferential activation of effector T cells while limiting Treg stimulation.

Recently, it has been discovered that IL-2 can be modified to selectively stimulate cytotoxic effector T cells. Various techniques have produced IL-2 variants with improved and selective immunostimulatory abilities. Some of these IL-2 variants were designed to enhance the ability of the molecule to signal primarily through the high-affinity receptor (alpha, beta, and gamma chains) rather than the intermediate-affinity receptor (beta and gamma chains). The basic idea was to enhance signaling in T cells but not in NK cells, which are thought to be responsible for the observed toxic effects. The following inventions relate to this line of work: U.S. Patent No. 7,186,804, U.S. Patent No. 7,105,653, U.S. Patent No. 6,955,807, U.S. Patent No. 5,229,109, U.S. Patent Application 20050142106. It is important to note that none of these inventions relate to IL-2 variants that have greater therapeutic efficacy in vivo than native IL-2.

In conclusion, IL-2 is a highly pleiotropic cytokine that is important for the biological activity of various cell populations. This property makes IL-2 an important node in the regulation of the immune response and an attractive target for various types of therapy and complex immunomodulation. In addition, IL-2 variants more prone to binding to certain receptor subunits can be generated to provide selective IL-2-mediated immunomodulation with preferential proliferation and activation of Teff cells that attack cancer cells, while reducing the proliferation and activation of Treg cells.

DESCRIPTION OF THE INVENTION

In one aspect, the present invention relates to the production of mutant variants of IL-2 characterized in that they are selective agonists of IL-2 activity with reduced or eliminated binding ability to IL-2Rα. Specifically, these variants will provide a method for overcoming the limitations observed in native IL-2 therapy due to its proven ability to increase the number of natural regulatory T cells in vivo. The present invention relates to polypeptides having the same primary sequence as human IL-2, except for several amino acids that have been changed by mutations. The introduced mutations significantly reduce the ability of these polypeptides to stimulate Treg cells and increase the efficacy of IL-2. In addition, the introduced mutations are expected to reduce CD25-mediated VLS (vascular leak syndrome) and CD25-mediated clearance effect. The present invention relates to polypeptides having the same primary sequence as human IL-2, except for one or more amino acids that have been altered by mutations. The present invention also includes therapeutic uses of these mutant variants, alone or in combination with vaccines or immune checkpoint inhibitors or biological agents targeting tumor-associated antigens (TAAs), or as part of a bifunctional fusion construct for the treatment of diseases such as cancer or infections where regulatory T cell (Treg) activity is undesirable.

In one aspect, the present invention relates to the production of mutant variants of IL-2 characterized in that they are selective agonists of IL-2 activity with an optimally modulated overall activity by reducing the interaction with IL-2Rβγ, in addition to a reduced or eliminated binding ability to IL-2Rα. The introduced mutations prevent excessive activation of the IL-2 pathway, reduce unwanted toxicity due to target binding outside the target tissue, reduce potential clearance, reduce activation exhaustion of cells associated with excessive stimulation of lymphocytes, inhibit receptor-mediated internalization of IL-2 and, thus, increase the half-life in vivo and lead to slow and prolonged pharmacodynamics, improving biodistribution, bioavailability, function and antitumor efficacy. The present invention also contemplates that the use of IL-2 variants with reduced/abstracted binding to IL-2Rα and weakened activity against IL-2Rβγ should help establish a stoichiometric balance between the cytokine and antibody portions, which exhibit significantly different activities and molecular weights, allowing for optimal doses to be determined and for maintaining the function of each portion. The present invention relates to polypeptides having the same primary sequence as human IL-2, except for one or more amino acids that have been altered by mutations. The present invention also includes therapeutic uses of these mutant variants, alone or in combination with vaccines or immune checkpoint inhibitors or biological agents targeting tumor-associated antigens (TAAs), or as part of a bifunctional fusion construct for the treatment of diseases such as cancer or infections.

In one aspect, the present invention relates to obtaining mutant variants of IL-2 characterized in that they are selective agonists of IL-2 activity with reduced interaction with IL-2Rβγ, in addition to reduced or eliminated binding ability to IL-2Rα. The introduced mutations provide long-lasting and prolonged pharmacodynamics and, possibly, pharmacokinetics. In addition, the introduced mutations reduce cell exhaustion and cell death caused by their activation and enhance prolonged reactivity of lymphocytes. As a result, the introduced mutations allow for a less frequent administration regimen and provide ease of administration in clinical use. In addition, according to assumptions, this will lead to a decrease in cost. The present invention relates to polypeptides having the same primary sequence as human IL-2, except for one or more amino acids that have been changed by mutations. The present invention also includes therapeutic uses of these mutant variants, alone or in combination with vaccines or immune checkpoint inhibitors or biological agents targeting tumor-associated antigens (TAAs), or as part of a bifunctional fusion construct for the treatment of diseases such as cancer or infections.

In one aspect, the present invention relates to the production of mutant variants of IL-2 characterized in that they are selective agonists of IL-2 activity with abolished binding to IL-2Rα and enhanced effector T and NK cell responses of unexpectedly high intensity, not corresponding to their wild-type equivalent. Mutations that abolish binding to CD25 are expected to reduce clearance due to binding to CD25 or CD25+ cells and, therefore, increase accessibility to IL-2Rβγ. Increased receptor occupancy leads to a strong cytotoxic cellular response and high efficiency of tumor cell killing. The present invention relates to polypeptides having the same primary sequence as human IL-2, except for one or more amino acids that have been altered by mutations. The present invention also includes therapeutic uses of these mutant variants, alone or in combination with vaccines or immune checkpoint inhibitors or biological agents targeting tumor-associated antigens (TAAs), or as part of a bifunctional fusion construct for the treatment of diseases such as cancer or infections.

In one aspect of the present invention, the introduced mutations reduced the ability to bind to IL-2Rα (CD25), but maintained low levels of the Treg response. Residual immunoregulatory Tregs provide an immune counterbalance, increasing systemic tolerance and preventing an excessive shift in the immune balance towards cytotoxic effector cells. The finely tuned Treg response does not reduce the efficiency of tumor cell killing, but is strong enough to maintain peripheral tolerance. The present invention relates to polypeptides having the same primary sequence as human IL-2, except for one or more amino acids that have been altered by mutations. The present invention also includes therapeutic uses of these mutant variants, alone or in combination with vaccines or immune checkpoint inhibitors or biological agents targeting tumor-associated antigens (TAAs), or as part of a bifunctional fusion construct for the treatment of diseases such as cancer or infections.

In one aspect, the present invention relates to the production of mutant variants of IL-2 having reduced aggregation, increased expression, improved manufacturing and development capabilities with a combination of properties including, for example, significantly reduced ability to stimulate Treg cells, less receptor overactivation, less unwanted toxicity due to target binding outside the target tissue, and prolonged pharmacodynamics for improved biodistribution, bioavailability, function and antitumor efficacy. The present invention relates to polypeptides having the same primary sequence as human IL-2, except for one or more amino acids that have been altered by mutations. The present invention also includes therapeutic uses of these mutant variants, alone or in combination with vaccines or immune checkpoint inhibitors or biological agents targeting tumor-associated antigens (TAAs), or as part of a bifunctional fusion construct for the treatment of diseases such as cancer or infections.

In one aspect, the present invention relates to the production of mutant variants of IL-2 characterized by a decrease in severe toxicity, such as vascular leakage syndrome (VLS), associated with high doses of IL-2 used in clinical settings for the treatment of renal cell carcinoma and melanoma. Specifically, the introduced mutations significantly reduce the ability to bind to IL-2Rα (CD25), thereby reducing binding to CD25+ lung endothelial cells and are expected to prevent endothelial cell injury and significantly reduce VLS. The present invention relates to polypeptides having the same primary sequence as human IL-2, except for one or more amino acids that have been altered by mutations. The present invention also includes therapeutic uses of these mutant variants, alone or in combination with vaccines or immune checkpoint modulators or biological agents targeting tumor-associated antigens (TAAs), or as part of a bifunctional fusion construct for the treatment of diseases such as cancer or infections, to improve the safety profile.

The present invention provides significant improvements to existing IL-2-based immunomodulatory techniques for the treatment of cancer. Specifically, replacement of native IL-2 with the mutant variants described herein will result in a lack of preferential stimulation of Treg cells over cytotoxic effector cells, a reduction in unwanted toxicity due to target binding outside of the target tissue, a minimization of cellular exhaustion associated with excessive stimulation, and improved pharmacodynamics and possibly pharmacokinetics. The mutations are expected to impair binding to CD25+ lung endothelial cells and, as a result, will reduce VLS. In various embodiments, the IL-2 variant (or mutant) comprises an IL-2 variant (or mutant) sequence derived from the mature human IL-2 polypeptide sequence set forth in SEQ ID NO: 3. In various embodiments, the IL-2 variant functions as an IL-2 agonist. In various embodiments, the IL-2 variant functions as an IL-2 antagonist. In various embodiments, the IL-2 variants comprise SEQ ID NO: 31-66, or SEQ ID NO: 111-120, or amino acids 9-133, 10-133, and 11-113 of SEQ ID NO: 47.

In another aspect, the IL-2 variants of the present invention are fused to at least one heterologous protein. In various embodiments, the IL-2 variants are fused to at least one polypeptide that imparts an increased half-life to the fusion molecule. Such polypeptides include IgG Fc or other polypeptides that bind to a neonatal Fc receptor, human serum albumin, or polypeptides that bind to a protein that has an increased serum half-life. In various embodiments, the IL-2 variant is fused to an IgG Fc molecule. In various embodiments, the Fc domain is a human IgG Fc domain. In various embodiments, the Fc domain is derived from a human IgG1 heavy chain constant domain sequence set forth in SEQ ID NO: 6. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO: 7. In various embodiments, the Fc domain is an Fc domain having the amino acid sequence set forth in SEQ ID NO: 8. In various embodiments, the Fc domain is derived from a human IgG2 heavy chain constant domain sequence. In various embodiments, the Fc domain is derived from a human IgG4 heavy chain constant domain sequence.

In various embodiments, IL-2 variants may be linked to the N-terminus or C-terminus of the Fc region of IgG.

The term "Fc" refers to a molecule or sequence comprising the sequence of a fragment of a whole antibody other than an antigen-binding fragment, in monomeric or multimeric form. The immunoglobulin that is the source of native Fc is preferably of human origin and may be any of the immunoglobulins disclosed in the art. Native Fc is composed of monomeric polypeptides that may be linked to form dimeric or multimeric forms by covalent (i.e., disulfide) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules varies from 1 to 4 depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is the disulfide-linked dimer produced by papain cleavage of IgG (see Ellison et al. (1982) Nucleic Acids Res. 10: 4071-9). As used herein, the term "native Fc" is a generic term for monomeric, dimeric, and multimeric forms. Fc domains contain binding sites for protein A, protein G, various Fc receptors, and complement proteins.

In various embodiments, the term "Fc variant" refers to a molecule or sequence that is modified relative to a native Fc but still contains a binding site for the salvage receptor FcRn. Exemplary Fc variants, as well as interactions with the salvage receptor, are described in International Applications WO 97/34631 (published September 25, 1997) and WO 96/32458, incorporated herein by reference. In addition, a native Fc contains sites that may be removed because they impart structural characteristics or biological activity that are not essential to the fusion molecules of the present invention. Thus, in various embodiments, the term "Fc variant" includes a molecule or sequence that lacks one or more sites or residues of a native Fc that affect or are involved in (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity when expressed in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether produced by cleavage of a whole antibody, recombinant gene expression, or otherwise. In various embodiments, an "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO: 6), which typically contains all or a portion of a hinge region. In various embodiments, mutations of the Fc domain are possible to eliminate effector functions. In various embodiments, each of the Fc domain monomers in an Fc domain comprises amino acid substitutions in the CH2 constant domain of the antibody to reduce interaction or binding between the Fc domain and an Fcγ receptor. In various embodiments, each Fc domain subunit comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function, wherein said amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO: 7). In various embodiments, each Fc domain subunit comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function, wherein said amino acid substitutions are L234A, L235A, and P329G.

In various embodiments, the Fc domain may be mutated to further increase the in vivo half-life. In various embodiments, each Fc domain subunit comprises the three amino acid substitutions M252Y, S254T, and T256E disclosed in U.S. Patent Publication No. 7,658,921 that enhance binding to human FcRn. In various embodiments, each Fc domain subunit comprises the amino acid substitution N434A (SEQ ID NO: 8) disclosed in U.S. Patent Publication No. 7,371,826. In various embodiments, each Fc domain subunit comprises the amino acid substitution M428L or N434S disclosed in U.S. Patent Publication No. 8,546,543 that enhance binding to human FcRn. In various embodiments, combinations of mutations that increase half-life with amino acid substitutions that reduce binding to the activating Fc receptor and/or effector function are possible.

In various embodiments, the IL-2 variant Fc fusion protein will be monomeric, i.e., it will contain only one IL-2 mutein molecule. In such embodiments, the fusion protein is expressed together with a heterodimeric Fc (e.g., Knob-Fc having the sequence set forth in SEQ ID NO: 9) linked to an IL-2 variant and a corresponding heterodimeric Fc (e.g., Hole-Fc having the sequence set forth in SEQ ID NO: 10). The protein obtained by forming a heterodimer of two Fc-containing polypeptides contains only a monovalent IL-2 variant. In various embodiments, the heterodimeric Fc domain used to generate the monovalent IL-2 Fc fusion proteins is a Knob Fc domain with reduced/eliminated effector function and increased half-life (SEQ ID NO: 134) and a Hole Fc domain with reduced/eliminated effector function and increased half-life (SEQ ID NO: 135).

In various embodiments, the IL-2 variants of the present invention may be linked to an antibody that imparts an increased half-life to the fusion molecule, such as an antibody against keyhole limpet hemocyanin (KLH). Such an antibody recognizes a foreign antigen, imparts an increased half-life, but does not have a biological function or harmfulness to humans. The IgG class may be IgG, IgA, IgE, or a subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgA2).

In various embodiments, the IL-2 variant constructs of the present invention comprise a targeting moiety in the form of an antibody, antibody fragment, protein or peptide that binds to a molecule that is elevated in cancer tissue, such as a tumor-associated antigen (TAA).

The TAA may be any molecule, macromolecule, combination of molecules, etc., against which an immune response is desired. The TAA may be a protein comprising more than one polypeptide subunit. For example, the protein may be a dimer, trimer, or higher order multimer. In various embodiments, two or more protein subunits may be joined by a covalent bond, such as, for example, a disulfide bond. In various embodiments, the protein subunits may be held together by non-covalent interactions. Thus, the TAA may be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, against which one skilled in the art desires to induce an immune response. In various embodiments, the TAA is a peptide comprising about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 amino acids. In various embodiments, the peptide, polypeptide, or protein is a molecule typically administered to subjects by injection.

In various embodiments, the tumor-specific antibody or binding protein acts as a targeting moiety, directing the IL-2 variant to a disease site, such as a tumor lesion, where it can stimulate more optimal anti-tumor immune responses while avoiding the systemic toxicity that occurs with free cytokine therapy. In the case of a full IL-2 agonist, the localization of the immunocytokine may be determined by IL-2 interactions with the IL-2R rather than by antibody binding to antigen, directing it, at typical antibody doses, to cells expressing the IL-2 receptor rather than to tumor cells. In various embodiments, the use of IL-2 variants with reduced/abstracted IL-2Rα binding and attenuated activity in an antibody fusion protein helps establish a stoichiometric balance between IL-2 and the targeting antibody to determine optimal doses at which the antibody achieves sufficient target occupancy while the IL-2 moiety does not cause excessive activation of its pathway. The use of IL-2 variants with reduced/abstracted IL-2Rα binding and attenuated activity in IL-2 antibody fusion proteins further enhances tumor targeting via the antibody, minimizes peripheral activation and AICD, reduces clearance due to antigen binding, and stimulates tumor targeting via the antibody-containing moiety.

In various embodiments, the IL-2 variants of the present invention may be linked to a targeting/bifunctional moiety, i.e., an antibody, antibody fragment, protein, or peptide targeting immune checkpoint modulators.

A number of immune checkpoint protein antigens have been reported to be expressed on various immune cells, including, for example, SIRP (expressed on macrophages, monocytes, dendritic cells), CD47 (highly expressed on tumor cells and other cell types), VISTA (expressed on monocytes, dendritic cells, B cells, T cells), CD 152 (expressed by activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed on tumor-infiltrating lymphocytes, expressed by activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, anergic T cells, monocytes, dendritic cells), CD274 (expressed on T cells, B cells, dendritic cells, vascular endothelium, pancreatic islet cells) and CD223 (expressed on activated T cells, regulatory T cells, anergic T cells, NK cells, NKT cells, and plasmacytoid dendritic cells) (see, e.g., Pardoll, D., Nature Reviews Cancer, 12: 252-264, 2012). Antibodies that bind to an antigen identified as an immune checkpoint protein are known to those skilled in the art. For example, various antibodies against CD276 have been described in the art (see, e.g., US Patent Application Publication No. 20120294796 (Johnson et al.) and references cited therein); Various anti-CD272 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20140017255 (Mataraza et al.) and references cited therein); Various anti-CD152/CTLA-4 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20130136749 (Korman et al.) and references cited therein); Various anti-LAG-3/CD223 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20110150892 (Thudium et al.) and references cited therein); Various anti-CD279 (PD-1 (programmed cell death protein 1)) antibodies have been described in the art (see, e.g., U.S. Patent No. 7,488,802 (Collins et al.) and references cited therein); Various anti-CD274 (PD-L1 (PD-1 ligand)) antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20130122014 (Korman et al.) and references cited therein); Various anti-TIM-3 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20140044728 (Takayanagi et al.) and references cited therein); and Various anti-B7-H4 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20110085970 (Terrett et al.) and references cited therein); and various antibodies against TIGIT (T-cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine inhibitory motif) domains) have been described in the art (see, e.g., U.S. Patent Application Publication No. 20180169239 A1 (Grogan) and references cited therein). Each of these references is herein incorporated by reference in its entirety with respect to the particular antibodies and sequences set forth therein.

In various embodiments, the IL-2 variant may be fused to an antibody, antibody fragment, or protein or peptide that exhibits binding to an immune checkpoint protein antigen present on the surface of an immune cell. In various embodiments, the immune checkpoint protein antigen is selected from the group consisting of, but not limited to, CD279 (PD-1), CD274 (PDL-1), CD276, CD272, CD 152, CD223 (LAG-3), CD40, SIRPα, CD47, OX-40, GITR, ICOS, CD27, 4-1BB, TIM-3, B7-H3, B7-H4, TIGIT, and VISTA.

In various embodiments, the antibody is an antagonist antibody or antagonist antibody fragment to FAP (fibroblast activation protein). In various embodiments, the antibody is a humanized antagonist antibody to FAP comprising the variable domain sequences set forth in SEQ ID NOs: 136 and 137. In various embodiments, the heterologous protein is an antibody or antibody fragment to an immune checkpoint modulator. In various embodiments, the antibody is an antagonist antibody or antagonist antibody fragment to PD-1. In various embodiments, the antibody is an anti-PD-1 antagonist antibody comprising the variable domain sequences set forth in SEQ ID NOs: 138 and 139, SEQ ID NOs: 140 and 141, SEQ ID NOs: 142 and 143, SEQ ID NOs: 144 and 145, or SEQ ID NOs: 146 and 147. In various embodiments, the antibody is an anti-human PD-L1 antagonist antibody comprising the variable domain sequences set forth in SEQ ID NOs: 148 and 149. In various embodiments, the antibody is an anti-CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) antagonist antibody comprising the variable domain sequences set forth in SEQ ID NOs: 150 and 151. In various embodiments, the heterologous protein is linked to the IL-2 variant via a linker and/or hinge linker peptide. The linker or hinge linker may be an artificial sequence of 5, 10, 15, 20, 30, 40 or more amino acids that is relatively free of secondary structure.

In various embodiments, the heterologous protein is linked to the IL-2 variant via a rigid linker peptide of 10, 15, 20, 30, 40 or more amino acids that exhibit an α-helical conformation and can act as rigid spacers between protein domains.

In another aspect, the IL-2 variant can be linked to various non-proteinaceous polymers, including, but not limited to, various polyols such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes as described in U.S. Patent Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. In various embodiments, amino acid substitutions at various positions of the IL-2 variants are possible to facilitate the attachment of polymers such as PEG. In various embodiments, such PEGylated proteins can have an increased half-life and/or reduced immunogenicity compared to non-PEGylated proteins.

In various embodiments, IL-2 variants may be non-covalently or covalently linked to IgG Fc or other neonatal Fcγ receptor binding polypeptides, human serum albumin or extended serum half-life protein binding polypeptides, or various non-proteinaceous polymers at the N-terminus or C-terminus.

In another aspect, the present invention provides a pharmaceutical composition comprising isolated IL-2 variants in admixture with a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a method for treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention to a subject in need thereof. In one embodiment, the subject is a human subject. In various embodiments, the cancer is selected from, but not limited to, pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, myelodysplastic syndrome, lung cancer, prostate cancer, brain cancer, bladder cancer, head and neck cancer, or rhabdomyosarcoma.

In another aspect, the present invention provides a method of treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention in combination with a second therapy selected from the group consisting of cytotoxic chemotherapy, immunotherapy, targeted therapy with small molecule kinase inhibitors, surgery, radiation therapy, and stem cell transplantation. In various embodiments, the combination therapy may comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but not limited to: treatment using depleting antibodies to certain tumor antigens; treatment using antibody-drug conjugates; treatment with agonist, antagonist or blocking antibodies to costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, PD-L1, OX-40, CD137, TIGIT, GITR, LAG3, TIM-3, CD47, SIRPa, ICOS and VISTA; treatment with bispecific T-cell engaging antibodies (BiTE®) such as blinatumomab; treatment involving the administration of biological response modifiers such as the TNF (tumor necrosis factor) family, IL-1, IL-4, IL-7, IL-12, IL-15, IL-17, IL-21, IL-22, GM-CSF (granulocyte macrophage colony-stimulating factor), IFN-α (interferon-α), IFN-β and IFN-γ; treatment using therapeutic vaccines such as sipuleucel-T; treatment using dendritic cell vaccines or tumor antigen peptide vaccines; treatment using chimeric antigen receptor T cells (CAR-T cells); treatment using CAR-NK cells; treatment using tumor-infiltrating lymphocytes (TILs); treatment with adoptively transferred anti-tumor T cells (ex vivo cultured and/or TCR (T cell receptor) transgenic); treatment with TALL-104 cells; and treatment with immunostimulatory agents such as Toll-like receptor (TLR: TLR7, TLR8 and TLR9) CpG agonists and imiquimod; wherein the combination therapy provides enhanced tumor cell killing by effector cells, i.e., synergy occurs when IL-2 variants and immunotherapy are administered together.

In another aspect, the invention provides the use of IL-2 variants for the manufacture of a medicament for the treatment of cancer.

In another aspect, the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding an IL-2 variant of the present invention. In another aspect, the present invention provides vectors comprising the nucleic acids described herein. In various embodiments, the vector is an expression vector. In another aspect, the present invention provides isolated cells comprising the nucleic acids of the invention. In various embodiments, the cell is a host cell comprising an expression vector of the invention. In another aspect, methods for producing IL-2 variants are provided, comprising culturing the host cells under conditions conducive to expression of the proteins or polypeptides.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

FIG. 1 shows the purity determined by SDS-PAGE (under non-reducing (lane 1) and reducing conditions (lane 2)) and the percentage of monomers estimated by SEC-HPLC (size exclusion high performance liquid chromatography) of representative Fc-fusion proteins of IL-2 variants P-0635 (1A) and P-0704 (1B). P-0635 and P-0704 have the same P65R amino acid substitution in wild-type IL-2. P-0635 contains a bivalent IL-2 variant fused to an Fc homodimer, while P-0704 contains a monovalent IL-2 variant fused to a heterodimeric Fc with a knob-into-hole modification.

FIG. 2 shows the size exclusion chromatogram of representative Fc-fusion proteins IL-2P-0250 (2A), P-0318 (2B), P-0317 (2C), and P-0531 (2D) after purification using protein A.

FIG. 3 shows the effect of IL-2 valency on the binding strength of IL-2 Fc fusion proteins to IL-2Rα in an ELISA. P-0531 and P-0689 share the same design-enhancing amino acid substitution S1251 in wild-type IL-2. P-0531 contains a bivalent IL-2 variant fused to an Fc homodimer, while P-0689 contains monovalent IL-2 fused to a heterodimeric Fc with a knob-into-hole modification.

FIG. 4 shows the effect of different mutations on the binding strength of IL-2 variant Fc fusion proteins to IL-2Rα in ELISA. (4A) IL-2 variant Fc fusion proteins contain T41 amino acid substitutions; (4B) IL-2 variant Fc fusion proteins contain Y107 amino acid substitutions; (4C and 4D) IL-2 variant Fc fusion proteins contain R38 amino acid substitutions.

FIG. 5 shows the effect of IL-2 E68 substitutions on the binding strength of IL-2 variant Fc fusion proteins to IL-2Rα in ELISA.

FIG. 6 shows the effect of IL-2 E62 substitutions on the binding strength of IL-2 variant Fc fusion proteins to IL-2Rα in ELISA.

FIG. 7 shows the effect of different IL-2 P65 substitutions on the binding strength of IL-2 variant Fc fusion proteins to IL-2Rα in ELISA. (7A-7B) P65 substitutions resulted in enhanced binding to IL-2Rα; (7C) IL-2 P65 substitutions resulted in decreased binding to IL-2Rα; (7D) IL-2 P65 substitutions resulted in complete loss of binding to IL-2Rα.

FIG. 8 shows the effect of combinations of IL-2 amino acid substitutions on the strength of binding to IL-2Rα in ELISA. (8A) Effect of IL-2 substitution F42A on the strength of binding to IL-2Rα; (8B) The combination of F42A and the substitution E62F, which disrupts interaction with CD25, resulted in complete loss of binding to IL-2Rα; (8C) The combination of F42A and the substitution P65H, which disrupts interaction with CD25, resulted in complete loss of binding to IL-2Rα.

FIG. 9 shows the differential effects of IL-2 variant Fc fusion proteins on dose-dependent induction of STAT5 phosphorylation in CD4+ Treg cells compared to the wild-type fusion protein (P-0531) and the reference protein (P-0551) in a human PBMC (peripheral blood mononuclear cell) assay. The IL-2 variant panel includes mutations affecting interaction with CD25, which result in increased, decreased, or abolished binding to IL-2Rα.

FIG. 10 shows complete conservation of binding of a panel of IL-2 variant Fc fusion proteins to IL-2Rβγ in ELISA compared to the wild-type IL-2 fusion protein P-0531 and the reference protein P-0551. The panel of IL-2 variants includes mutations affecting interaction with CD25 that result in increased, decreased, or abolished binding to IL-2Rα.

FIG. 11 shows that a panel of IL-2 variant Fc fusion proteins demonstrated comparable activity in inducing Ki67 expression in human PBMC CD8+ T cells (11A) and NK cells (11B). The IL-2 variant panel includes mutations affecting interaction with CD25 that result in increased, decreased, or abolished binding to IL-2Rα. Wild-type IL-2 fusion protein P-0531 and the reference protein P-0511 are included for comparison.

FIG. 12 shows the effect of IL-2 valency on activity in inducing Ki67 expression in human PBMC CD8+ T cells. P-0531 and P-0689 are analogous wild-type IL-2 bivalent and monovalent Fc fusion proteins. P-0635 and P-0704 are equivalent P65R IL-2 bivalent and monovalent Fc fusion proteins.

FIG. 13 shows the effect of various amino acid substitutions modulating interaction with IL-2Rβ/γc or N-terminal deletions on the activity of inducing pSTAT5 expression in CD4+ T cells compared to their wild-type counterpart. (13A) IL-2 mutants with amino acid substitutions at position D20; (13B-13C) IL-2 mutants with amino acid substitutions at position L19; (13D) IL-2 Q126E mutation; and (13E) IL-2 mutants with N-terminal amino acid deletions.

FIG. 14 shows the effect of amino acid substitutions that disrupt interaction with IL-2Rβ or γc on the strength of binding to IL-2Rβγ in ELISA (14A) and on the activity in inducing Ki67 expression in human PBMC CD8+ T cells (14B). P-0689 is a monovalent Fc fusion protein of wild-type IL-2, and P-0704 is a monovalent Fc fusion protein of IL-2 P65R that no longer binds to IL-2Rα but fully retains affinity and functional activity for the dimeric IL-2Rβγ receptor.

FIG. 15 shows the effect of various amino acid changes that disrupt interaction with IL-2Rβ on the activity of Fc-fusion proteins of IL-2 variants in inducing Ki67 expression in CD8+ T cells (15A), NK cells (15B), and CD4+ T cells (15C) of human PBMC. P-0704 and a reference molecule (the monomeric form of P-0551) were included for comparison.

FIG. 16 shows the time-dependent effects of P-0704 on the expansion of Treg (16A), CD8+ T (16B), and NK cells (16C) in peripheral blood after a single injection in Ba1b/C mice. P-0704 is a monovalent Fc fusion protein of IL-2 P65R; P-0689, a monovalent Fc fusion protein of wild-type IL-2, was included for comparison. Blood was collected on days 3 and 5 for lymphocyte phenotyping by fluorescence-activated cell sorting (FACS) analysis.

FIG. 17 shows the effect of the fusion format on the dose-dependent induction of STAT5 phosphorylation in CD4+ Tregs (17A), CD8+ T cells (17B), and NK cells (17C) in a human PBMC assay. P-0704 is a monovalent IL-2 P65R Fc fusion protein, and P-0803 is an antibody fusion protein containing the same IL-2 moiety.

FIG. 18 shows the differential effects of IL-2 variant antibody fusion proteins on dose-dependent induction of STAT5 phosphorylation in CD4+ Tregs (18A), CD8+ T cells (18B), and NK cells (18C) compared to the wild-type fusion protein (P-0837) in a human PBMC assay. P-0838 contains the P65Q IL-2 mutation, which significantly reduced binding to IL-2Rα, and P-0782 has the IL-2 P65R moiety with abolished binding to IL-2Rα.

FIG. 19 shows the effect of amino acid changes that modulate interaction with IL-2Rβ on the activity of IL-2 variant antibody fusion proteins in stimulating STAT5 phosphorylation in CD8+ T (19A) and NK cells (19B) and inducing Ki67 expression in CD8+ T (19C) and NK cells (19D) of human PBMCs. All three compounds contain the P65R mutation in the IL-2 moiety, and P-0786 and P-0783 contain additional mutations that disrupt interaction with IL-2Rβ, L19Q and L19H, respectively.

FIG. 20 shows the effect of amino acid changes that modulate interaction with IL-2Rβ on the activity of IL-2 variant antibody fusion proteins in stimulating STAT5 phosphorylation in CD4+ Tregs (20A), CD8+ T cells (20B), and NK cells (20C) and inducing Ki67 expression in CD8+ T cells (20D) and NK cells (20E) in a human PBMC assay. All three compounds, P-0838, P-0790, and P-0787, contain the P65Q mutation in the IL-2 moiety, and P-0790 and P-0787 contain additional IL-2Rβ interaction-modulating mutations, L19Q and L19H, respectively. P-0837 is an analogous fusion protein of wild-type IL-2.

FIG. 21 shows the effect of amino acid changes that modulate interaction with IL-2Rβ on the activity of IL-2 variant antibody fusion proteins in CTLL-2 cell proliferation. P-0782, P-0783, and P-0786 contain the P65R mutation in the IL-2 moiety; P-0786 and P-0783 contain additional IL-2Rβ interaction-modulating mutations L19Q and L19H, respectively. P-0837 is a similar wild-type IL-2 fusion protein.

FIG. 22 shows the minimal effect of the IL-2 variant fusion on direct binding (22A) and competitive inhibition of ligand binding (22B) for the antibody moiety in ELISA, and similarly, in FACS analysis, the IL-2 variant with anti-human PD-1 antibody demonstrated binding to cell-surface expressed PD-1 similar to the parent antibody (FIG. 22C). P-0795 is an anti-human PD-1 antibody, P-0803, P-0880, and P-0885 have the monomeric P65R variant of IL-2 covalently linked to the C-terminus of the heavy chain of P-0795. P-0803 and P-0885 have the same P65R/S125I substitutions in IL-2 but different linkers ((Gr3S)2 and (G4S)3, respectively). P-0885 contains one additional L19Q mutation compared to P-0880. P-0704 and P-0859 are Fc-fusion analogs of P-0880 and P-0885, respectively.

FIG. 23 shows the differential effects of IL-2 variant antibody fusion proteins on the dose-dependent induction of STAT5 phosphorylation in human PBMCs CD4+ Tregs (23A and 23B), CD8+ T cells (23C and 23D), and NK cells (23E and 23F). P-0803 and P-0804 are IL-2 variant fusion proteins with an anti-human PD-1 antibody containing the P65R and L19H/P65R mutations, respectively. P-0782 is a fusion protein of IL-2 P65R with a surrogate anti-mouse PD-1 antibody, and P-0783 contains an additional L19H mutation compared to P-0782.

FIG. 24 shows size exclusion chromatograms of fusion proteins of IL-2 variants with anti-human PD-1 antibody P-0840 (24A), P-0841 (24B), P-0803 (24C), and P-0880 (24D) after purification using protein A.

FIG. 25 shows the effect of linker length in IL-2 variant antibody fusion proteins on the dose-dependent induction of STAT5 phosphorylation in CD8+ T (25A and 25B) and NK cells (25C and 25D) in a human PBMC assay. P-0840 and P-0841 are fusion proteins of the L19Q/P65Q variant of IL-2 with an antibody to human PD-1; P-0840 contains a (G3S)2 linker while P-0841 has a (G4S)3 linker. Similarly, P-0803 and P-0880 are fusion proteins of the P65R variant of IL-2 with an antibody to human PD-1; P-0803 contains a (G3S)2 linker, while P-0880 has a (G4S)3 linker.

FIG. 26 shows the effect of amino acid changes that modulate interaction with IL-2Rβ on the activity of IL-2 variant fusion proteins with anti-human PD-1 antibody in stimulating STAT5 phosphorylation in CD8+ T (26A) and NK cells (26B) and inducing Ki67 expression in human PBMC CD8+ T (26C) and NK cells (26D). All three compounds, P-0880, P-0885, and P-0882, contain the P65R mutation in the IL-2 moiety, while P-0885 and P-0882 contain additional L19Q and L19H mutations that modulate interaction with IL-2Rβ, respectively. P-0849 is an equivalent wild-type IL-2 fusion protein. All compounds have a (G4S)3 linker connecting the heavy chain of the PD-1 and IL-2 antibody.

FIG. 27 shows the time-dependent effects of IL-2 variant fusion proteins with the anti-mouse PD-1 surrogate antibody P-0782, P-0838, P-0781 (reference), and P-0837 on Ki67 expression in CD8+ T (27A) and NK cells (27B) and the effects on CD8 (27C) and NK cell (27D) expansion following a single injection in C57BL6 mice. Cell expansion was expressed as fold change from baseline. P-0782 contains the P65R mutation in the IL-2 moiety, P-0838 contains the P65Q mutation, P-0781 contains the reference IL-2 variant with ablated binding to IL-2Rα, and P-0837 is the analogous wild-type IL-2 fusion protein.

FIG. 28 shows the time- and dose-dependent effects of the IL-2 variant fusion protein with the anti-mouse PD-1 surrogate antibody P-0786 on Ki67 expression in CD8+ T (28A) and NK cells (28B) and the effects on the expansion of CD8+ T (28C) and NK cells (28D) after a single injection in C57BL6 mice. The expansion was expressed as the fold change in cell number from baseline. P-0786 contains the L19Q/P65R mutation, which abolishes binding to IL-2Rα and reduces overall activity. P-0837, a similar wild-type IL-2 fusion protein, was included for comparison.

FIG. 29 shows the time- and dose-dependent effects of the IL-2 variant fusion protein with the anti-mouse PD-1 surrogate antibody P-0783 on Ki67 expression in CD8+ T (29A) and NK cells (29B) and the effects on the expansion of CD8 (29C) and NK cells (29D) after a single injection in C57BL6 mice. The expansion was expressed as the fold change in cell numbers from baseline. P-0783 contains the L19H/P65R mutation, which abolishes binding to IL-2Rα and reduces overall activity. P-0837, a similar wild-type IL-2 fusion protein, was included for comparison.

FIG. 30 shows the change in body weight in C57BL/6 mice administered with IL-2 variant fusion proteins with the surrogate anti-mouse PD-1 antibody P-0782, P-0786, and P-0783. All compounds contain the P65R mutation in the IL-2 moiety, P-0781 contains the reference IL-2 variant with ablated binding to IL-2Rα, and P-0786 and P-0783 contain additional mutations L19Q and L19H, respectively, that disrupt interaction with IL-2Rβ. Data are presented as mean plus/minus SEM.

FIG. 31 shows the antitumor efficacy (31A) and body weight change (31B) following repeated administration of IL-2 variant fusion proteins with the surrogate mouse PD-1 antibody Q7D (once every 7 days) in a B16F10 subcutaneous murine melanoma model. All three antibody fusion proteins contain the L65Q mutation in IL-2 to reduce binding to IL-2Rα; P-0790 and P-0787 contain additional L19Q and L19H mutations, respectively, to further modulate overall activity. Data are presented as mean plus/minus SEM.

FIG. 32 shows the antitumor efficacy (32A) and body weight change (32B) following repeated administration of P-0787 Q7D at two different doses in a B16F10 murine subcutaneous melanoma tumor model. P-0787 is a fusion protein of an IL-2 variant with a surrogate antibody to murine PD-1 containing the L19H/P65Q mutations. Data are presented as mean plus/minus SEM.

FIG. 33 shows the antitumor efficacy of IL-2 variant fusion proteins with the surrogate murine PD-1 antibody P-0782 and P-0786 in a B16F10 murine subcutaneous melanoma tumor model following repeated Q7D administration. P-0722, a surrogate murine PD-1 antibody, was included for comparison. Both P-0782 and P-0786 contain the P65R mutation, which abolishes binding to IL-2Rα, while P-0786 contains an additional L19Q mutation to modulate overall activity. Data are presented as mean plus/minus SEM.

FIG. 34 shows dose-dependent inhibition of lung metastasis by P-0790 in the B16F10 mouse lung metastasis model. (34A) Average lung nodule counts; (34B) lung images from a representative animal from each group. P-0790 is an IL-2 L19Q/P65Q fusion protein with an anti-mouse PD-1 surrogate antibody with significantly reduced IL-2Rα binding and modulated total activity. Data are presented as mean plus/minus SEM. Statistical analysis was performed by one-way ANOVA followed by Tukey's post hoc test. *p less than 0.05.

EMBODIMENT(S) OF THE INVENTION

The present invention relates to polypeptides having the same primary sequence as human IL-2, except for one or more amino acids that have been altered by mutations. The IL-2 variants contain mutations that significantly reduce the ability of these polypeptides to stimulate Treg cells and increase their effectiveness in the treatment of tumors. It also includes therapeutic uses of these mutant variants used alone or in combination with vaccines or biological agents targeting TAA or an immune checkpoint blocker, or as part of a bifunctional molecule construct for the treatment of diseases such as cancer or infections where regulatory T cell (Treg) activity is undesirable. In another aspect, the present invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the present invention relates to therapeutic use of the disclosed polypeptides and pharmaceutical compositions due to their selective modulating effect on the immune system in cancer and various infectious diseases.

Definitions

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. In various embodiments, "peptides", "polypeptides" and "proteins" are chains of amino acids whose alpha carbons are linked by peptide bonds. Thus, the terminal amino acid at one end of the chain (the N-terminal) has a free amino group, while the terminal amino acid at the other end of the chain (the C-terminal) has a free carboxyl group. As used herein, the term "N-terminal" refers to the free α-amino group of the amino acid at the N-terminal of the peptide or to the α-amino group (the amino group when involved in a peptide bond) of the amino acid at any other location within the peptide in question. Similarly, the term "C-terminal" refers to the free carboxyl group at the C-terminal of the peptide or the carboxyl group of the amino acid at any other location within the peptide in question. Peptides also include virtually any polyamino acid, including, without limitation, peptide mimetics such as amino acids linked by ester rather than amide bonds.

The polypeptides of the invention include polypeptides that have been modified in any manner and for any reason, such as to: (1) reduce susceptibility to proteolysis; (2) reduce susceptibility to oxidation; (3) alter binding affinity for forming protein complexes; (4) alter binding affinity; and (5) impart or modify other physicochemical or functional properties.

As used herein, "amino acid substitution" refers to the replacement of one amino acid of a polypeptide at a particular position in the parent polypeptide sequence with another amino acid. Amino acid substitutions may be made using genetic or chemical methods well known in the art. For example, a single amino acid substitution or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in a naturally occurring sequence (e.g., in a portion of a polypeptide outside the domain(s) that form(s) intermolecular contacts). A "conservative amino acid substitution" refers to the replacement of an amino acid of a polypeptide with a functionally similar amino acid. Each of the following six groups includes amino acids that are conservative substitutions for each other:

1) alanine (A), series (S) and threonine (T);

2) aspartic acid (D) and glutamic acid (E);

3) asparagine (N) and glutamine (Q);

4) arginine (R) and lysine (K);

5) isoleucine (I), leucine (L), methionine (M) and valine (V);

6) phenylalanine (F), tyrosine (Y) and tryptophan (W).

"Non-conservative amino acid substitution" refers to the replacement of a member of one of these classes with a member of another class. In making such changes, according to various embodiments, the hydrophobicity index of the amino acids may be taken into account. Each amino acid has been assigned a hydrophobicity index based on its hydrophobicity and charge characteristics. It is: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the hydrophobicity index of amino acids in imparting biological interaction function to a protein is known in the art (see, e.g., Kyte et al., 1982, J. Mol. Biol. 157:105-131). It is known that certain amino acids can be substituted with other amino acids having a similar hydrophobicity index or value while maintaining similar biological activity. In various embodiments, changes based on the hydrophobicity index include substitutions of amino acids whose hydrophobicity indices differ by no more than 2. In various embodiments, substitutions of amino acids whose hydrophobicity indices differ by no more than 1 are included, and, in various embodiments, substitutions of amino acids whose hydrophobicity indices differ by no more than 0.5 are included.

It is also known in the art that substitution of similar amino acids can be effectively carried out based on hydrophilicity, especially where the biologically functional protein or peptide thus obtained is intended for use in immunological embodiments as disclosed herein. In various embodiments, the highest local average hydrophilicity of a protein, determined by the hydrophilicity of its adjacent amino acids, correlates with its immunogenicity and antigenicity, i.e., with the biological properties of the protein.

The following hydrophilicity values were assigned to the listed amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 plus or minus 0.1); glutamate (+3.0 plus or minus 0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 plus or minus 0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In various embodiments, changes based on similar hydrophilicity values include substitutions of amino acids whose hydrophilicity values differ by no more than 2, in various embodiments, substitutions of amino acids whose hydrophilicity values differ by no more than 1, and, in various embodiments, substitutions of amino acids whose hydrophilicity values differ by no more than 0.5.

Examples of amino acid substitutions are given in Table 1.

One skilled in the art will be able to determine suitable variants of the polypeptides as described herein using well-known techniques. In various embodiments, one skilled in the art will be able to determine suitable regions of the molecule that can be altered without disrupting activity by targeting regions that are not expected to be important for activity. In other embodiments, one skilled in the art will be able to determine residues and portions of the molecule that are conserved among similar polypeptides. In other embodiments, even regions that may be important for biological activity or for structure can be subject to conservative amino acid substitutions without disrupting biological activity or without adversely affecting the structure of the polypeptide.

In addition, a person skilled in the art will be able to review structure-function studies that identify residues of similar polypeptides that are important for their activity or structure. Based on such a comparison, a person skilled in the art will be able to predict the importance of amino acid residues of a polypeptide that correspond to amino acid residues that are important for the activity or structure of similar polypeptides. A person skilled in the art may prefer to replace such predicted important amino acid residues with chemically similar amino acid residues.

A person skilled in the art can also analyze the three-dimensional structure and amino acid sequence in comparison with such a structure of similar polypeptides. Based on such information, a person skilled in the art can predict the alignment of amino acid residues of the polypeptide in relation to its three-dimensional structure. In various embodiments, a person skilled in the art can avoid radical changes in amino acid residues presumably located on the surface of the polypeptide, since such residues can be involved in important interactions with other molecules. Moreover, a person skilled in the art can obtain test variants containing a single amino acid substitution of each desired amino acid residue. These variants can then be screened using activity assays known to those skilled in the art. Such variants can be used to obtain information about suitable variants. For example, if it is discovered that a change in a certain amino acid residue led to a disruption, an undesirable decrease in activity, or an inappropriate activity, variants with such a change can be avoided. In other words, based on the information obtained in such routine experiments, one skilled in the art can easily identify amino acids where further substitutions, either alone or in combination with other mutations, should be avoided.

As used herein, the terms "polypeptide fragment" and "truncated polypeptide" refer to a polypeptide having an N-terminal and/or C-terminal deletion compared to the corresponding full-length protein. In various embodiments, the fragments can be, for example, at least 5, at least 10, at least 25, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900, or at least 1000 amino acids long. In various embodiments, the length of the fragments can also be, for example, no more than 1000, no more than 900, no more than 800, no more than 700, no more than 600, no more than 500, no more than 450, no more than 400, no more than 350, no more than 300, no more than 250, no more than 200, no more than 150, no more than 100, no more than 50, no more than 25, no more than 10, or no more than 5 amino acids. The fragment can further comprise, at one or both ends thereof, one or more additional amino acids, such as an amino acid sequence from another naturally occurring protein (e.g., an Fc domain or a leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

As used herein, the terms "polypeptide variant", "hybrid polypeptide" and "polypeptide mutant" refer to a polypeptide comprising an amino acid sequence with an insertion, deletion and/or substitution of one or more amino acid residues of the amino acid sequence relative to the sequence of another polypeptide. In various embodiments, the number of amino acid residues to be inserted, deleted or substituted can be, for example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, at least 300, at least 350, at least 400, at least 450 or at least 500 amino acids in length. Hybrids of the present invention include fusion proteins.

A "derivative" of a polypeptide is a polypeptide that has been chemically modified, such as by conjugation with another chemical moiety such as, for example, polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation.

The term "% sequence identity" is used herein interchangeably with the term "% identity" and refers to the level of amino acid sequence identity between two or more peptide sequences or the level of nucleotide sequence identity between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% identity means the same as 80% sequence identity as determined by a given algorithm and means that a given sequence is at least 80% identical to another region of another sequence. In various embodiments, the % identity is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, or greater sequence identity to a given sequence. In various embodiments, the % identity ranges from, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

The term "% sequence homology" is used herein interchangeably with the term "% homology" and refers to the level of amino acid sequence homology between two or more peptide sequences or the level of nucleotide sequence homology between two or more nucleotide sequences when aligned using a sequence alignment program. For example, as used herein, 80% homology means the same as 80% sequence homology as determined by a given algorithm, and accordingly, a homolog of a given sequence has more than 80% sequence homology over a region of the given sequence. In various embodiments, the % homology is selected from, for example, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%, or more sequence homology to a given sequence. In various embodiments, the % homology ranges from, for example, about 60% to about 70%, about 70% to about 80%, about 80% to about 85%, about 85% to about 90%, about 90% to about 95%, or about 95% to about 99%.

Typical computer programs that can be used to determine the identity of two sequences include, but are not limited to, the BLAST suite of programs, such as BLASTN, BLASTX, TBLASTX, BLASTP, and TBLASTN, which are publicly available on the Internet at the NCBI website. See also Altschul et al, J. Mol. Biol. 215:403-10, 1990 (with special reference to the published default settings, i.e., w of 4 and t of 17) and Altschul et al, Nucleic Acids Res., 25:3389-3402, 1997. When evaluating a given amino acid sequence against amino acid sequences in GenBank Protein Sequences and other publicly available databases, sequence searches are typically performed using the BLASTP program. BLASTX is the program of choice for searching nucleic acid sequences translated in all reading frames against amino acid sequences from GenBank Protein Sequences and other publicly available databases. Both BLASTP and BLASTX are run using a gap-opening penalty of 11.0 and a gap-extension penalty of 1.0, which are the default parameters, and the BLOSUM-62 matrix.

In addition to calculating percent identity, the BLAST algorithm also allows for statistical analysis of the similarity of two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA, 90:5873-5787, 1993). One measure of similarity when using the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability of a coincidence between two nucleotide or amino acid sequences occurring by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability when comparing the analyzed nucleic acid to the reference nucleic acid is, e.g., less than about 0.1, less than about 0.01, or less than about 0.001.

As used herein, the term “modification” refers to any manipulation of the peptide backbone (e.g., amino acid sequence) or post-translational modifications (e.g., glycosylation) of the polypeptide.

As used herein, the term "knob-into-hole modification" refers to a modification of the facing surfaces of two immunoglobulin heavy chains in the CH3 domain. In one embodiment, the "knob-into-hole modification" comprises an amino acid substitution of T366W and optionally an amino acid substitution of S354C in one heavy chain of an antibody and amino acid substitutions of T366S, L368A, Y407V, and optionally Y349C in the other heavy chain of the antibody. Knob-into-hole technology is described, for example, in U.S. Patent No. 5,731,168; U.S. Patent No. 7,695,936; Ridgway et al, Prot Eng 9, 617-621 (1996); and Carter, J Immunol Meth 248, 7-15 (2001).

As used herein, the term "fusion protein" refers to a fusion polypeptide molecule comprising two or more genes originally encoding separate proteins, wherein the components of the fusion protein are joined to each other by peptide bonds, either directly or through peptide linkers. As used herein, the term "fusion" refers to components joined by peptide bonds, either directly or through one or more peptide linkers.

"Linker" refers to a molecule that joins two other molecules, either covalently or through ionic, van der Waals, or hydrogen bonds, such as a nucleic acid molecule that hybridizes to one complementary sequence at the 5' end and to another complementary sequence at the 3' end, thereby joining two non-complementary sequences. "Cleavable linker" refers to a linker that can be cleaved or otherwise separated to separate the two components joined by the cleavable linker. Cleavable linkers are generally cleaved by enzymes, typically peptidases, proteases, nucleases, lipases, and the like. It is also possible for cleavable linkers to be cleaved by environmental factors, such as, for example, changes in temperature, pH, salt concentration, and the like.

As used herein, the term "peptide linker" refers to a peptide comprising one or more amino acids, typically about 2-20 amino acids. Peptide linkers are known in the art or described herein. Suitable non-immunogenic linker peptides include, for example, (G ₄ S) _n , (SG ₄ ) _n , or G4(SG ₄ ) _n peptide linkers. In most cases, "n" is a number from 1 to 10, typically from 2 to 4.

"Pharmaceutical composition" refers to a composition suitable for pharmaceutical use in an animal. The pharmaceutical composition comprises a pharmacologically effective amount of an active agent and a pharmaceutically acceptable carrier. "Pharmacologically effective amount" refers to that amount of the agent effective to produce the intended pharmacological result. "Pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, vehicles, buffers and excipients such as phosphate buffered saline, 5% aqueous dextrose, emulsions such as oil/water or water/oil emulsion, and various types of wetting agents and/or adjuvants. Suitable pharmaceutical carriers and compositions are described in Remington's Pharmaceutical Sciences, 21st Ed. 2005, Mack Publishing Co, Easton. A "pharmaceutically acceptable salt" is a salt that can be incorporated into a compound for pharmaceutical use, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

As used herein, "treatment" (and its grammatical variations such as "treat") refers to a clinical intervention in an attempt to alter the natural course of a disease in the individual being treated, and may be performed prophylactically or during the progression of a clinical pathology. Desired effects of treatment include, but are not limited to, prevention of development or recurrence of a disease, relief of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or palliation of a disease state, and remission or improvement of prognosis. As used herein, "alleviation" of a disease, disorder, or condition means a decrease in the severity and/or frequency of symptoms of the disease, disorder, or condition. Additionally, reference to "treatment" herein includes reference to curative, palliative, and prophylactic treatment.

As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat the specified disorder, condition, or disease, such as ameliorating, alleviating, reducing, and/or delaying one or more of its symptoms. With respect to cancer or other unwanted cell proliferation, an effective amount includes an amount sufficient to: (1) reduce the number of cancer cells; (2) reduce the size of a tumor; (3) inhibit, slow down, somewhat slow down, and preferably stop the infiltration of peripheral organs by cancer cells; (4) inhibit (i.e., somewhat slow down, and preferably stop) tumor metastasis; (5) inhibit tumor growth; (6) prevent or delay the development and/or recurrence of a tumor; and/or (7) somewhat alleviate one or more of the symptoms associated with the cancer. An effective amount may be administered in one or more administrations.

The phrase "administration" or "acts leading to administration" refers to the actions taken by a healthcare professional (e.g., a physician) or a person in control of the healthcare of a patient who supervises and/or authorizes the administration of the agent(s)/compound(s) to the patient. Acts leading to administration may include diagnosis, and/or determination of an appropriate therapeutic regimen, and/or prescription of the agent(s)/compound(s) to the patient. Such prescription may include, for example, filling out a prescription form, recording in a patient's medical record, and the like. When describing administration, "acts leading to administration" is also meant herein.

The terms "patient," "individual," and "subject" may be used interchangeably and refer to a mammal, preferably a human or non-human primate, but also to domesticated mammals (e.g., cats or dogs), laboratory mammals (e.g., mice, rats, rabbits, hamsters, guinea pigs), and farm mammals (e.g., horses, bovines, pigs, sheep). In various embodiments, a patient may be a human (e.g., an adult male, an adult female, an adolescent male, an adolescent female, a child male, a child female) being cared for by a physician or other health care professional in an inpatient, mental health, outpatient, or other clinical setting. In various embodiments, the patient may be an immunocompromised patient or a patient with a weakened immune system, including, but not limited to, patients with primary immunodeficiency, AIDS, cancer patients, and transplant patients receiving certain immunosuppressive drugs, and patients with inherited diseases that affect the immune system (e.g., congenital agammaglobulinemia, congenital IgA deficiency). In various embodiments, the patient is a patient with an immunogenic cancer, including, but not limited to, bladder cancer, lung cancer, melanoma, and other cancers that have been reported to have an increased mutation rate (Lawrence et al., Nature, 499(7457):214-218, 2013).

The term "immunotherapy" refers to the treatment of cancer including, but not limited to: treatment using depleting antibodies to certain tumor antigens; treatment using antibody-drug conjugates; treatment using agonist, antagonist, or blocking antibodies to costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, OX-40, CD 137, GITR, LAG3, TIM-3, SIRP, CD47, and VISTA; treatment using bispecific T-cell engager (BiTE®) antibodies such as blinatumomab; treatment involving the administration of biological response modifiers such as IL-2, IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatment with therapeutic vaccines such as sipuleucel-T; treatment with dendritic cell vaccines or tumor antigen peptide vaccines; treatment with chimeric antigen receptor T cells (CAR T cells); treatment with CAR-NK cells; treatment with tumor-infiltrating lymphocytes (TILs); treatment with adoptively transferred anti-tumor T cells (ex vivo cultured and/or TCR transgenic); treatment with TALL-104 cells; and treatment with immunostimulatory agents such as Toll-like receptor (TLR) CpG agonists and imiquimod.

"Resistant or refractory cancer" refers to tumor cells or cancer that do not respond to previous anti-cancer therapy, including, for example, chemotherapy, surgery, radiation therapy, stem cell transplantation, and immunotherapy. Tumor cells may be resistant or refractory at the start of treatment, or they may become resistant or refractory during treatment. Refractory tumor cells include tumors that do not respond at the start of treatment or that initially respond for a short period but ultimately do not respond to treatment. Refractory tumor cells also include tumors that respond to anti-cancer therapy but do not respond to subsequent rounds of therapy. For purposes of the present invention, refractory tumor cells also include tumors that are suppressed by anti-cancer therapy but relapse within five years, sometimes within ten years or more, after cessation of therapy. Cancer therapy may involve chemotherapeutic agents alone, radiation alone, targeted therapy alone, surgery alone, or combinations thereof. For ease of description and not limitation, it should be understood that "refractory tumor cells" are interchangeable with "resistant tumor."

As used herein, the term "Fc domain" or "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. The Fc region of IgG comprises a CH2 domain of IgG and a CH3 domain of IgG. The CH3 region may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain that has a "bump" introduced into one chain and a corresponding "cavity" introduced into the other chain; see U.S. Patent No. 5,821,333, expressly incorporated herein by reference). Such variant CH3 domains may be used to promote heterodimerization of two non-identical immunoglobulin heavy chains, as described herein. Unless otherwise indicated herein, the numbering of amino acid residues in the Fc region or constant region follows the EU numbering system.

As used herein, the term "effector functions" refers to biological activities inherent in the Fc region of an immunoglobulin, which vary among immunoglobulin isotypes. Examples of immunoglobulin effector functions include: C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. Effector functions may also refer to similar immune responses elicited by effector immune cells such as CD8 and NK cells.

As used herein, the term "regulatory T cell" or "Treg cell" refers to a specialized type of CD4+ T cell that can suppress the responses of other T cells (effector T cells). Tregs are characterized by the expression of CD4, the IL-2 receptor alpha subunit (CD25), and the transcription factor forkhead box P3 (FOXP3) (Sakaguchi, Annu Rev Immunol 22, 531-62 (2004)) and play a critical role in the development and maintenance of peripheral self-tolerance to antigens, including antigens expressed by tumors.

When used herein, the term "conventional CD4+ T cells" refers to CD4+ T cells other than regulatory T cells. Conventional CD4+ T cells express CD3 and CD4. In the absence of antigen stimulation, they do not express the IL-2 receptor α subunit (CD25), but do express the IL-2 receptor βγ subunit.

The term "CD8 T cells" represents a type of cytotoxic T lymphocyte characterized by the expression of CD3 and CD8. CD8 T cells express primarily the βγ subunit of the IL-2 receptor and play a critical role in the killing of cancer cells, cells infected with viruses, or cells that are otherwise damaged.

The term "NK cells" represents a type of cytotoxic lymphocyte that is critical to the innate immune system. NK cells express primarily the βγ subunit of the IL-2 receptor and mediate rapid responses to virus-infected cells and tumor formation.

As used herein, "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an immunoglobulin to bind to a particular antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR).

As used herein, the terms "affinity" or "binding affinity" refer to the strength of the sum of the noncovalent interactions between one binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). The affinity of a molecule X for its partner Y can typically be represented as a dissociation constant (K _D ), which is the ratio of the dissociation and association rate constants (k _off and k _on , respectively). A special method for measuring affinity is surface plasmon resonance (SPR).

As used herein, the term "reduced binding" refers to a decrease in the affinity of the relevant interaction, as measured, for example, by SPR. Conversely, "increased binding" refers to an increase in the binding affinity of the relevant interaction.

As used herein, the term "polymer" generally includes, without limitation: homopolymers; copolymers, such as, for example, block copolymers, graft, random and alternating copolymers; and terpolymers; and blends and modifications thereof. Furthermore, unless expressly limited, the term "polymer" includes all possible geometric configurations of the substance. These configurations include, without limitation, isotactic, syndiotactic and random symmetry.

"Polyethylene glycol" or "PEG" means a polyalkylene glycol compound or derivative thereof, with or without coupling agents or derivatization using coupling or activating moieties (e.g., using an aldehyde, hydroxysuccinimidyl, hydrazide, thiol, triflate, tresylate, azirdine, oxirane, orthopyridyl, disulfide, vinyl sulfone, iodoacetamide, or maleimide moiety). In various embodiments, PEG includes substantially linear unbranched PEG, branched PEG, or dendritic PEG. PEG is a well-known water-soluble polymer that can be purchased from commercial sources or prepared by ring-opening polymerization of ethylene glycol by methods well known in the art (Sandler and Kara, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161).

"Polynucleotide" refers to a polymer composed of nucleotide units. Polynucleotides include naturally occurring nucleic acids such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs include analogs containing bases that are not naturally occurring, nucleotides that form linkages with other nucleotides other than a naturally occurring phosphodiester bond, or that contain bases attached through linkages other than phosphodiester linkages. Thus, nucleotide analogs include, for example and without limitation, phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, organophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNA), and the like. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid" generally refers to large polynucleotides. The term "oligonucleotide" generally refers to short polynucleotides, usually of no more than about 50 nucleotides. It should be understood that the representation of a nucleotide sequence as a DNA sequence (i.e., A, T, G, C) also includes an RNA sequence (i.e., A, U, G, C) in which "T" is replaced by "U".

The conventional notation used to describe polynucleotide sequences is as follows: the left end of a single-stranded polynucleotide sequence is called the 5' end; the left direction of a double-stranded polynucleotide sequence is called the 5' direction. The 5'-3' direction of addition of nucleotides to the synthesized RNA transcripts is called the direction of transcription. The DNA strand having the same sequence as the mRNA is called the "coding strand"; the sequences of the DNA strand having the same sequence as the mRNA transcribed from it and located 5' to the 5' end of the RNA transcript are called the "upstream sequences"; the sequences of the DNA strand having the same sequence as the RNA and located 3' to the 3' end of the coding RNA transcript are called the "downstream sequences".

"Complementarity" refers to the topological compatibility or mutual comparability of the interacting surfaces of two polynucleotides. Thus, two molecules can be described as complementary and, in addition, the characteristics of their contact surfaces are complementary to each other. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions.

"Specifically hybridizes to", "specific hybridization", or "selective hybridization" refers to the preferential binding, duplexing, or hybridization of a nucleic acid molecule to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) of DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will hybridize preferentially to its target subsequence and to a lesser extent to other sequences, or not at all to other sequences. "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridization are sequence dependent and vary under different environmental conditions. Comprehensive guidelines for nucleic acid hybridization are provided by Tijssen, 1993, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes, part I, chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, N.Y.; Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd sup.rd ed., NY; and Ausubel et al., eds., Current Edition, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY.

Typically, highly stringent hybridization and washing conditions are selected to be approximately 5°C below the melting temperature (Tm) of a particular sequence at a given ionic strength and pH. Tm is the temperature (at a given ionic strength and pH) at which the target sequence hybridizes to 50% of its ideally matched probe. Very stringent conditions are selected to be equal to the Tm of the corresponding probe. An example of stringent hybridization conditions for hybridization of complementary nucleic acids having more than approximately 100 complementary residues on a Southern or Northern blot filter is 50% formalin with 1 mg heparin at 42°C, with hybridization performed overnight. An example of high stringency wash conditions is 0.15 M NaCl at 72°C for approximately 15 minutes. An example of stringent wash conditions is a wash using 0.2×SSC (saline-sodium citrate buffer) at 65°C for 15 minutes. SSC buffer is described in Sambrook et al. A wash under high stringency conditions may be preceded by a wash under low stringency conditions to remove background probe signal. A typical medium stringency wash for a duplex of, for example, more than about 100 nucleotides is 1×SSC at 45°C for 15 minutes. A typical low stringency wash for a duplex of, for example, more than about 100 nucleotides is 4-6×SSC at 40°C for 15 minutes. Typically, a signal-to-noise ratio 2 (or more) times that observed for an unrelated probe in a given hybridization assay indicates detection of specific hybridization.

"Primer" refers to a polynucleotide capable of hybridizing specifically to a designated polynucleotide template and providing a starting point for the synthesis of a complementary polynucleotide. Such synthesis occurs when the polynucleotide primer is placed under conditions that initiate synthesis, i.e., in the presence of nucleotides, a complementary polynucleotide template, and a polymerizing agent such as DNA polymerase. The primer is usually single-stranded, but may be double-stranded. Primers are usually deoxyribonucleic acids, but in many cases a wide variety of synthetic and naturally occurring primers are useful. A primer is complementary to the template to which it is designed to hybridize, providing a starting point for synthesis, but it does not necessarily reflect the exact sequence of the template. Specific hybridization of the primer to the template then depends on the stringency of the hybridization conditions. Primers can be labeled with, for example, chromogenic, radioactive or fluorescent moieties and used as detectable moieties.

When used in connection with a polynucleotide, "probe" refers to a polynucleotide capable of specifically hybridizing to a designated sequence of another polynucleotide. The probe specifically hybridizes to a target complementary polynucleotide, but does not necessarily reflect the exact complementary sequence of the template. In such a case, specific hybridization of the probe to the target polynucleotide depends on the stringency of the hybridization conditions. Probes may be labeled with, for example, chromogenic, radioactive, or fluorescent moieties and used as detectable moieties. In cases where the probe provides a starting point for the synthesis of a complementary polynucleotide, the probe may also be a primer.

A "vector" is a polynucleotide that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional segments of nucleic acid can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), where additional DNA segments can be introduced into the viral genome. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (e.g., bacterial vectors containing the bacterial replicator and episomal vectors for mammalian cells). Other vectors (e.g., non-episomal vectors for mammalian cells) integrate into the host cell's genome upon introduction into the host cell and are thereby replicated along with the host genome. An "expression vector" is a type of vector that is capable of directing the expression of a selected polynucleotide.

A "regulatory sequence" is a nucleic acid that influences the expression (e.g., level, timing, or location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence may, for example, exert its effects on the nucleic acid being regulated directly or through the action of one or more other molecules (e.g., polypeptides linked to the regulatory sequence and/or the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Other examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California, and Baron et al., 1995, Nucleic Acids Res. 23:3605-06. A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, or location of expression) of the nucleotide sequence.

"A host cell" is a cell that can be used to express a polynucleotide of the invention. The host cell can be a prokaryote, such as E. coli, or it can be a eukaryote, such as a unicellular eukaryote (e.g., yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Typically, the host cell is a cultured cell that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. The phrase "recombinant host cell" can be used to refer to a host cell transformed or transfected with a nucleic acid to be expressed. A host cell may also be a cell that contains a nucleic acid but does not express it at a desired level, unless a regulatory sequence has been introduced into the host cell such that it becomes operably linked to the nucleic acid. It should be understood that the term "host cell" refers not only to the particular cell in question, but also to the progeny or potential progeny of such a cell. Since certain modifications are possible in subsequent generations, due to, for example, mutations or environmental influences, such progeny may not actually be identical to the original cell, but the scope of the term, as used herein, still includes such progeny.

The term "isolated molecule" (where the molecule is, for example, a polypeptide or a polynucleotide) is a molecule that, by virtue of its source: (1) is not associated with the components with which it is naturally associated in its native state; (2) is substantially free of other molecules of the same species; (3) is expressed by a cell of a different species; or (4) is not found in nature. Thus, a chemically synthesized molecule or a molecule expressed in a cellular system other than the cell of its natural source will be "isolated" from the components with which it is naturally associated. The molecule may also be made substantially free of the components with which it is naturally associated by isolation using purification techniques well known in the art. The purity or homogeneity of the molecule can be analyzed using a variety of techniques well known in the art. For example, the purity of a polypeptide sample can be analyzed using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide using techniques well known in the art. For certain applications, higher resolution can be achieved using HPLC (high performance liquid chromatography) or other techniques well known in the art.

A protein or polypeptide is "substantially pure," "substantially uniform," or "substantially purified" when at least about 60% to 75% of the sample is a single species of polypeptide. The polypeptide or protein may be monomeric or multimeric. A substantially pure polypeptide or protein will typically comprise about 50%, 60%, 70%, 80%, or 90%, w/w, of the protein sample, more typically about 95%, and will preferably be greater than 99% pure. The purity or uniformity of a protein can be demonstrated by a variety of techniques well known in the art, such as polyacrylamide gel electrophoresis of a protein sample followed by visualization of a single band of the polypeptide upon staining the gel with a dye well known in the art. For certain purposes, higher resolution can be achieved using HPLC or other techniques well known in the art.

As used herein, the terms "label" or "labeled"/"labeled" refer to the incorporation of another molecule into an antibody. In one embodiment, the label is a detectable marker, such as by incorporating a radiolabeled amino acid or by attaching biotinyl moieties to a polypeptide that can be detected by avidin containing a marker (e.g., streptavidin containing a fluorescent marker or having enzymatic activity that can be detected by optical or calorimetric methods). In another embodiment, the label or marker can be therapeutic, such as a drug conjugate or a toxin. Various methods for labeling polypeptides and glycoproteins known in the art can be used. Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., ³ H, ¹⁴ C, ¹⁵ N, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope labels), magnetic agents such as gadolinium chelates, toxins such as pertussis toxin, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoside, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinatedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol and puromycin, and analogs and homologues thereof. In various embodiments, the labels are attached via spacers of varying lengths to reduce potential steric hindrance.

As used herein, the term "heterologous" refers to a composition or structure that is not native or present in nature, such as a composition or structure that can be obtained by replacing an existing natural composition or structure with a composition or structure derived from another source. Similarly, expression of a protein in an organism other than the organism that naturally expresses the protein constitutes a heterologous expression system and a heterologous protein.

It should be understood that aspects and embodiments of the invention described herein include aspects and embodiments “consisting of” and/or “consisting essentially of” the described aspects and embodiments.

A reference to "approximately" in a description of a value or parameter herein includes (and describes) variations that directly address that value or parameter. For example, a description that states "approximately X" includes the description "X".

Unless the context clearly dictates otherwise, as used herein and in the appended claims, the singular forms include the plural. It is to be understood that aspects and variants of the invention described herein include aspects and variants "consisting of" and/or "consisting essentially of" the described aspects and variants.

IL-2

Interleukin-2 (IL-2), a classical Th1 cytokine, is produced by T cells following activation via the T cell antigen receptor and the costimulatory molecule CD28. IL-2 is regulated through activation of signaling pathways and transcription factors acting on the IL-2 promoter, leading to new gene transcription, but also includes modulation of IL-2 mRNA stability. IL-2 binds to a receptor containing several chains, including the highly regulated α chain and the β and γ chains that mediate signaling through the Jak-STAT pathway. IL-2 transmits activation, growth, and differentiation signals to T cells, B cells, and NK cells. IL-2 is also important in mediating T cell activation-induced death, a function that is a critical mechanism for terminating immune responses. A commercially available preparation of nonglycosylated human recombinant IL-2, aldesleukin (available under the trade name Proleukin® as desalanil-1-series-125 human interleukin-2 from Prometheus Laboratories Inc., San Diego, CA), is approved for administration to patients with metastatic renal cell carcinoma and metastatic melanoma. IL-2 has also been proposed for administration to patients infected with hepatitis C virus (HCV), human immunodeficiency virus (HIV), patients with acute myeloid leukemia, non-Hodgkin lymphoma, cutaneous T-cell lymphoma, juvenile rheumatoid arthritis, atopic dermatitis, breast cancer, and bladder cancer. Unfortunately, optimal dosing of IL-2 is limited by its short half-life and significant toxicity.

When used herein in the context of proteins or polypeptides, the terms "native IL-2" and "native interleukin-2" refer to any naturally occurring amino acid sequence of mammalian interleukin-2, including both immature or precursor forms and mature forms. Non-limiting examples of amino acid sequence accession numbers of various native mammalian interleukin-2 species in GenBank include NP_032392.1 (Mus musculus, immature form), NP_001040595.1 (Macaca mulatta, immature form), NP_000577.2 (human, precursor form), CAA01199.1 (human, immature form), AAD48509.1 (human, immature form), and AAB20900.1 (human). In various embodiments of the present invention, native IL-2 is an immature or precursor form of naturally occurring mammalian IL-2. In other embodiments, native IL-2 is a mature form of naturally occurring mammalian IL-2. In various embodiments, native IL-2 is a precursor form of naturally occurring human IL-2. In various embodiments, native IL-2 is a mature form of naturally occurring human IL-2. In various embodiments, the IL-2-based D2 domain is derived from the amino acid sequence of the precursor human IL-2 set forth in SEQ ID NO: 1:

In various embodiments, the IL-2 based D2 domain comprises the amino acid sequence of the mature form of wild-type human IL-2 as set forth in SEQ ID NO: 3, comprising a cysteine substitution at position 125 with serine, but does not alter binding to the IL-2 receptor compared to naturally occurring IL-2:

IL-2 variants

The present invention relates to polypeptides having the same primary sequence as human IL-2, except for one or more amino acids that have been altered by mutations. One panel of IL-2 variants includes mutations that substantially reduce the ability of these polypeptides to stimulate Treg cells and increase their efficacy in the treatment of tumors. It also includes the therapeutic use of these mutant variants, alone or in combination with vaccines or biological agents targeting TAA or an immune checkpoint blocker, or as part of a bifunctional molecule construct for the treatment of diseases such as cancer or infections where regulatory T cell (Treg) activity is undesirable. In another aspect, the present invention relates to pharmaceutical compositions comprising the disclosed polypeptides. Finally, the present invention relates to the therapeutic use of the disclosed polypeptides and pharmaceutical compositions due to their selective modulating effect on the immune system in diseases such as autoimmune and inflammatory disorders or cancer, and in various infectious diseases.

The present invention relates to polypeptides of 100 to 500 amino acids in length, preferably 140 residues in size, with an average molecular weight of at least 15 kDa. These polypeptides retain a high, more than 90%, sequence identity to native IL-2. In one region of their sequence, these polypeptides contain mutations with the introduction of amino acid residues that differ from the amino acid residues in the same positions of native IL-2.

The polypeptides of the present invention may be called, inter alia, immunomodulatory polypeptides, IL-2 analogs or IL-2 variants. These polypeptides are designed based on the three-dimensional structure of the IL-2 receptor complex (presented in the publicly available PDB database), with mutations introduced primarily at those positions of IL-2 that correspond to amino acids interacting with the IL-2 receptor α-subunit.

In various embodiments, the IL-2 variant (or mutant) comprises a sequence derived from the mature human IL-2 polypeptide sequence set forth in SEQ ID NO: 3. In various embodiments, the IL-2 variant comprises an amino acid sequence different from the native IL-2 protein (or wild-type IL-2 protein). In various embodiments, the IL-2 variant interacts with an IL-2 receptor polypeptide and functions as an IL-2 agonist or antagonist. In various embodiments, IL-2 variants with agonist activity have superagonist activity. In various embodiments, the IL-2 variant can function as an IL-2 agonist or antagonist independent of its binding to IL-2Rα. IL-2 agonists are characterized by comparable or increased biological activity compared to wild-type IL-2. IL-2 antagonists are characterized by reduced biological activity compared to wild-type IL-2 or the ability to inhibit IL-2-mediated responses. In various embodiments, the IL-2 variant sequence has at least one amino acid change, such as a substitution or deletion, compared to the sequence of native IL-2, wherein such changes result in IL-2 agonist or antagonist activity. In various embodiments, IL-2 variants have amino acid sequences as set forth in SEQ ID NOs: 31-66, with reduced/aborted binding to IL-2Rα for selective activation and proliferation of effector T cells (Teff). In various embodiments, IL-2 variants have amino acid sequences set forth in SEQ ID NOs: 111-120, containing mutations that modulate interaction with IL-2Rβ or γc, in addition to mutations that result in decreased/aborted binding to IL-2Rα, for selective activation and proliferation of effector T cells, with attenuated activity for reduced IL-2Rβ or γc-associated toxicity, decreased cellular exhaustion, and improved long-term pharmacodynamics. In various embodiments, IL-2 variants have the amino acid sequences shown in SEQ ID NOs: 189 (amino acids 462-586), 190 (amino acids 462-585) and 191 (amino acids 462-584) containing N-terminal deletions, in addition to mutations resulting in decreased/aborted binding to IL-2Rα, for selective activation and proliferation of effector T cells with impaired activity. In various embodiments, IL-2 variants with the amino acid sequences shown in SEQ ID NOs: 31-66, 111-120, and amino acids 9-133, 10-133 and 11-133 of SEQ ID NO: 47 also contain the amino acid substitution S125I to expand the possibilities for the development of IL-2 and related fusion proteins.

Representative IL-2 variants with amino acid substitutions introduced into the regions interacting with IL-2Rα are presented in Table 2.

A key aspect of the present invention is to improve the selectivity of IL-2 over wild-type IL-2 for IL-2Rβγ (but not IL-2Rα)-expressing cells over IL-2Rαβγ-expressing cells for the treatment of cancer. One approach used by the present inventors is to generate highly selective IL-2 Fc-fusion proteins by introducing CD25-impairing mutations into the cytokine component. The selection of CD25-impairing mutations was based on analysis of the IL-2/IL-2R co-crystal structure (PDB accession no. 2B51). Multiple mutations of one or two involved amino acid residues in the regions interacting with the IL-2 receptor α subunit were introduced, including R38, T41, F42, F44, E62, P65, E68, and Y107, aimed at reducing or eliminating binding to IL-2Rα. These constructs also contained the S125I mutation to significantly expand design options. Furthermore, reducing binding of IL-2 variants to IL-2Rα+ lung endothelial cells is expected to prevent endothelial cell injury and significantly reduce VLS. Furthermore, reducing binding to CD25 is predicted to also reduce clearance mediated by CD25 antigen binding, increase cytokine receptor occupancy on IL-2Rβγ-expressing cells, and therefore enhance the in v/vo response and tumor cell killing efficacy.

Since all of the IL-2 target residues, R38, T41, F42, F44, E62, P65, E68, and Y107, are located in regions that interact with IL-2Rα and form hydrogen bonds/salt bonds or hydrophobic interactions with multiple IL-2Rα residues (Mathias Rickert, et al. (2005) Science 308, 1477-80), it was hypothesized that the IL-2 variants listed in Table 2 and similar variants would disrupt the interaction with IL-2Rα and result in IL-2 variants with reduced or abolished binding to IL-2Rα. However, it was found that mutations at specific sites and different substitutions at the same site can lead to significant differences in their effects on IL-2Rα binding that could not be predicted by structure-based mutagenesis, and some of these differences are particularly unexpected (see Examples 4 and 5).

In addition, it has been proposed that additional substitutions that modulate the interaction with IL-2Rβγ may be necessary to attenuate overall activity and achieve optimal activity. Agonists with modulated IL-2Rβγ activity may prevent excessive cytotoxic lymphocyte activation and minimize toxicity due to target binding outside the target tissue. In addition, they may minimize cellular exhaustion and apoptosis due to excessive stimulation. Furthermore, decreased binding affinity for the cytokine signaling molecule may reduce receptor-mediated internalization, decrease unwanted clearance due to target binding, and result in persistent receptor activation and prolonged pharmacodynamics and pharmacokinetics. Therefore, substitutions that modulate interactions with IL-2Rβγ have the potential to reduce toxicity and improve pharmacokinetics and pharmacodynamics, as well as the therapeutic index.

Representative IL-2 variants with amino acid substitutions including mutations that disrupt interaction with IL-2Rβ or γc, in IL-2 variants with reduced/abstracted binding to IL-2Rα, are presented in Table 3.

The present invention also includes further modifications of the class of IL-2 variants indicated above and in particular described in Table 2 and Table 3, including deletions of 8 or 9 or 10 N-terminal residues of the IL-2 variants indicated above for selective activation and proliferation of effector T cells with different levels of attenuated activity. The nature and scope of the present invention include mutants with any further combination of mutations to change their affinity for certain components of the IL-2 receptor or to improve their pharmacodynamics in vivo: increasing the half-life or reducing their internalization by T cells. These further mutations can be obtained by rational design using bioinformatic tools or using combinatorial molecular libraries of various natures (phage libraries, yeast or bacterial gene expression libraries). In another aspect, the present invention relates to a fusion protein comprising any of the immunomodulatory polypeptides described above in combination with a carrier protein. The carrier protein may be albumin or the Fc region of human immunoglobulins.

In various embodiments, IL-2RαSushi having the amino acid sequence set forth in SEQ ID NO: 170 was linked between the IL-2 and Fc domains using linkers of varying length and composition. The Fc domain can be at the N-terminus or C-terminus. It is expected that the IL-2-IL-2RαSushi-Fc fusion protein having the amino acid sequence set forth in SEQ ID NO: 171-172 will have reduced binding to IL-2Rα, providing selective activation and proliferation of effector T cells.

In various embodiments, IL-2 and IL-2RαSushi form a non-covalent complex. IL-2 has been fused to the N- or C-terminus of the Hole-Fc chain (SEQ ID NO: 10), and IL-2RαSushi has been fused to the N- or C-terminus of the Knob-Fc chain (SEQ ID NO: 9). The non-covalent C-terminal IL-2-IL-2RαSushi-Fc fusion protein has the amino acid sequence set forth in SEQ ID NOs: 173-174.

Fc domains

Immunoglobulins of the IgG class are among the most abundant proteins in human blood. Their half-life in circulation can be as long as 21 days. Fusion proteins containing combinations of the Fc regions of IgG with domains of other proteins, such as various cytokines and receptors, have been reported (see, e.g., Capon et al., Nature, 337:525–531, 1989; Chamow et al., Trends Biotechnol, 14:52–60, 1996; U.S. Patents 5,116,964 and 5,541,087). The prototype fusion protein is a homodimeric protein linked through cysteine residues in the hinge Fc region of IgG to produce a molecule similar to an IgG molecule, lacking the variable domain and the CH1 domain of the heavy chain and light chains. The dimeric nature of Fc-domain containing fusion proteins may be advantageous in terms of enabling higher order interactions (i.e., bivalent or bispecific binding) with other molecules. Due to structural homology, the in vivo pharmacokinetic profile of Fc-fusion proteins is comparable to that of isotype-matched human IgG.

The term "Fc" refers to a molecule or sequence comprising the sequence of a fragment of a whole antibody other than an antigen-binding fragment, in monomeric or multimeric form. The immunoglobulin that is the original source of native Fc is preferably of human origin and may be any of the immunoglobulins, but IgG1 and IgG2 are preferred. Native Fc are composed of monomeric polypeptides that may be linked to form dimeric or multimeric forms by covalent (i.e., disulfide) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of native Fc molecules varies from 1 to 4, depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2). One example of a native Fc is the disulfide-linked dimer produced by papain cleavage of IgG (see Ellison et al. (1982) Nucleic Acids Res. 10: 4071-9). As used herein, the term "native Fc" is a generic term for monomeric, dimeric, and multimeric forms. Fc domains contain binding sites for protein A, protein G, various Fc receptors, and complement proteins.

In various embodiments, the term "Fc variant" refers to a molecule or sequence that is modified relative to a native Fc but still contains a binding site for the salvage receptor FcRn. Exemplary Fc variants, as well as interactions with the salvage receptor, are described in International Applications WO 97/34631 (published September 25, 1997) and WO 96/32478, incorporated herein by reference. In addition, a native Fc contains sites that may be removed because they impart structural characteristics or biological activity that are not essential to the fusion molecules of the present invention. Thus, in various embodiments, the term "Fc variant" includes a molecule or sequence that lacks one or more sites or residues of a native Fc that affect or are involved in (1) disulfide bond formation, (2) incompatibility with the selected host cell, (3) N-terminal heterogeneity when expressed in the selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to an Fc receptor other than a salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC).

The term "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined above. As with Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether produced by cleavage of a whole antibody, recombinant gene expression, or otherwise. In various embodiments, an "Fc domain" refers to a dimer of two Fc domain monomers (SEQ ID NO: 6), which typically contains all or a portion of a hinge region. In various embodiments, mutations of the Fc domain are possible to eliminate effector functions. In various embodiments, each of the Fc domain monomers in an Fc domain comprises amino acid substitutions in the CH2 constant domain of the antibody to reduce interaction or binding between the Fc domain and an Fcγ receptor. In various embodiments, each Fc domain subunit comprises three amino acid substitutions that reduce binding to an activating Fc receptor and/or effector function, wherein said amino acid substitutions are L234A, L235A, and G237A (SEQ ID NO: 7).

In various embodiments, each of the two Fc domain monomers in the Fc domain comprises amino acid substitutions that facilitate heterodimerization of the two monomers. In various other embodiments, heterodimerization of the Fc domain monomers can be facilitated by introducing different but compatible substitutions into the two Fc domain monomers, such as knob-into-hole residue pairs. Knob-into-hole technology is also disclosed in U.S. Patent Publication No. 8,216,805. In yet another embodiment, one Fc domain monomer comprises the knob mutation T366W and the other Fc domain monomer comprises the hole mutations T366S, L358A, and Y407V. In various embodiments, two Cys residues (S354C on the knob side and Y349C on the hole side) were introduced to form a stabilizing disulfide bond (SEQ ID NOs: 9 and 10). The use of a heterodimeric Fc can result in a monovalent variant of IL-2.

In various embodiments, the Fc domain sequence used to generate the dimeric Fc fusion proteins of IL-2 variants is the human IgG1 Fc domain sequence shown in SEQ ID NO: 7:

where SEQ ID NO: 7 contains amino acid substitutions (underlined) that abolish binding to FcγR and C1q.

In various embodiments, the Fc domain sequence used to generate the dimeric IL-2 Fc fusion proteins is the IgG1 Fc domain sequence shown in SEQ ID NO: 8:

wherein SEQ ID NO: 8 contains amino acid substitutions (underlined) that abolish binding to FcγR and C1q and an amino acid substitution (in bold) to increase half-life.

In various embodiments, the heterodimeric Fc domain sequence used to produce the monomeric IL-2 variant fusion proteins is the Knob Fc domain sequence shown in SEQ ID NO: 9:

where SEQ ID NO: 9 contains amino acid substitutions (underlined) that abolish binding to FcγR and C1q.

In various embodiments, the heterodimeric Fc domain sequence used to generate IL-2 variants is the Hole Fc domain sequence shown in SEQ ID NO: 10:

where SEQ ID NO: 10 contains amino acid substitutions (underlined) that abolish binding to FcγR and C1q.

In various embodiments, the heterodimeric Fc domain used to generate monomeric IL-2 Fc fusion proteins is a Knob Fc domain with reduced/aborted effector function and increased half-life, having the amino acid sequence set forth in SEQ ID NO: 134:

wherein SEQ ID NO: 134 contains amino acid substitutions (underlined) that eliminate binding to FcγR and C1q, and an amino acid substitution (in bold) to increase half-life.

In various embodiments, the heterodimeric Fc domain used to generate monomeric IL-2 Fc fusion proteins is a Hole Fc domain with reduced/aborted effector function and increased half-life, having the amino acid sequence set forth in SEQ ID NO: 135:

wherein SEQ ID NO: 135 contains amino acid substitutions (underlined) that abolish binding to FcγR and C1q and an amino acid substitution (in bold) to increase half-life.

Antibodies as targeting moieties

In various embodiments, the IL-2 variant constructs of the present invention comprise a targeting moiety in the form of an antibody, antibody fragment, protein or peptide that binds to a molecule that is elevated in cancer tissue, such as a tumor-associated antigen (TAA).

The TAA may be any molecule, macromolecule, combination of molecules, etc., against which an immune response is desired. The TAA may be a protein comprising more than one polypeptide subunit. For example, the protein may be a dimer, trimer, or higher order multimer. In various embodiments, two or more protein subunits may be joined by a covalent bond, such as, for example, a disulfide bond. In various embodiments, the protein subunits may be held together by non-covalent interactions. Thus, the TAA may be any peptide, polypeptide, protein, nucleic acid, lipid, carbohydrate, or small organic molecule, or any combination thereof, against which one skilled in the art desires to induce an immune response. In various embodiments, the TAA is a peptide comprising about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 150, about 200, about 250, about 300, about 400, about 500, about 600, about 700, about 800, about 900, or about 1000 amino acids. In various embodiments, the peptide, polypeptide or protein is a molecule typically administered to subjects by injection. In various embodiments, upon administration, the tumor-specific antibody or binding protein acts as a targeting moiety, directing the IL-2 variant to a disease site, such as a cancer site, where the active domain can be released and interact with its cognate receptors on diseased cells.

Any of the above markers can be used as a TAA target for the IL-2 variants of the present invention. In various embodiments, one or more TAA, TAA variant, or TAA mutant contemplated for use in the IL-2 variant constructs and methods of the present invention are selected from the list presented in Table 5 or are derived from antigens presented in this list.

In various embodiments, the IL-2 variants of the present invention may be linked to a targeting/bifunctional moiety, i.e., an antibody, antibody fragment, protein, or peptide targeting immune checkpoint modulators.

A variety of immune checkpoint protein antigens have been reported to be expressed by various immune cells, including, for example, SIRP (expressed on macrophages, monocytes, dendritic cells), CD47 (highly expressed on tumor cells and other cell types), VISTA (expressed on monocytes, dendritic cells, B cells, T cells), CD152 (expressed on activated CD8+ T cells, CD4+ T cells, and regulatory T cells), CD279 (expressed on tumor-infiltrating lymphocytes, expressed by activated T cells (both CD4 and CD8), regulatory T cells, activated B cells, activated NK cells, anergic T cells, monocytes, dendritic cells), CD274 (expressed on T cells, B cells, dendritic cells, vascular endothelial cells, pancreatic islet cells) and CD223 (expressed on activated T cells, regulatory T cells, anergic T cells, NK cells, NKT cells, and plasmacytoid dendritic cells) (see, e.g., Pardoll, D., Nature Reviews Cancer, 12:252-264, 2012). Antibodies that bind to an antigen identified as an immune checkpoint protein are known to those skilled in the art. For example, various antibodies against CD276 have been described in the art (see, e.g., US Patent Application Publication No. 20120294796 (Johnson et al.) and references cited therein); Various anti-CD272 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20140017255 (Mataraza et al.) and references cited therein); Various anti-CD152/CTLA-4 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20130136749 (Korman et al.) and references cited therein); Various anti-LAG-3/CD223 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20110150892 (Thudium et al.) and references cited therein); Various anti-CD279 (PD-1) antibodies have been described in the art (see, e.g., U.S. Patent No. 7,488,802 (Collins et al.) and references cited therein); Various anti-CD274 (PD-L1) antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20130122014 (Korman et al.) and references cited therein); various anti-TIM-3 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20140044728 (Takayanagi et al.) and references cited therein); and various anti-B7-H4 antibodies have been described in the art (see, e.g., U.S. Patent Application Publication No. 20110085970 (Terrett et al.) and references cited therein). Each of these references is hereby incorporated by reference in its entirety with respect to the specific antibodies and sequences disclosed therein.

In various embodiments, the fusion partner of IL-2 can be an antibody, an antibody fragment, or a protein or peptide that exhibits binding to an immune checkpoint protein antigen present on the surface of an immune cell. In various embodiments, the immune checkpoint protein antigen is selected from the group consisting of, but not limited to, PD1 (CD279), PDL-1 (CD274), CD276, CD272, CD152 (CTLA-4), CD223, CD279, CD274, CD40, SIRPα, CD47, OX-40, GITR, ICOS, CD27, 4-1BB, TIM-3, B7-H3, B7-H4, TIGIT, and VISTA.

In various embodiments, the antibody is an antagonist antibody or antagonist antibody fragment to FAP. In various embodiments, the antibody is a humanized antagonist antibody to FAP comprising the variable domain sequences set forth in SEQ ID NOs: 136 and 137. In various embodiments, the heterologous protein is an antibody or antibody fragment to an immune checkpoint modulator. In various embodiments, the antibody is an antagonist antibody to human TIGIT. In various embodiments, the antibody is an antagonist antibody or antagonist antibody fragment to PD-1. In various embodiments, the antibody is an anti-PD-1 antagonist antibody comprising the variable domain sequences set forth in SEQ ID NOs: 138 and 139, SEQ ID NOs: 140 and 141, SEQ ID NOs: 142 and 143, SEQ ID NOs: 144 and 145, or SEQ ID NOs: 146 and 147. In various embodiments, the antibody is an anti-human PD-L1 antagonist antibody comprising the variable domain sequences set forth in SEQ ID NOs: 148 and 149. In various embodiments, the antibody is an anti-human CTLA-4 antagonist antibody comprising the variable domain sequences set forth in SEQ ID NOs: 150 and 151. In various embodiments, exemplary bifunctional IL-2 fusion proteins with antibody to PD-1 are presented in Table 12.

Bifunctional fusion proteins of IL-2 variants with PD-1 antibodies

In various embodiments, immune checkpoint blocking antibodies that bypass immunosuppressive effects in the tumor microenvironment or immunostimulatory antibodies to enhance existing responses are used to construct IL-2 antibody fusion proteins. Expression levels of negative immune checkpoints are particularly high in exhausted T cells that have been exposed to tumor antigens and infiltrate the tumor microenvironment. In various embodiments, binding of IL-2 variants to immune checkpoint targeting antibodies is expected to direct IL-2 to exhausted T cells and immunologically activate the tumor microenvironment. In various embodiments, bifunctional fusion proteins of IL-2 variants with anti-checkpoint inhibitor antibodies enable delivery of IL-2 preferentially to cells expressing checkpoint inhibitors, such as exhausted T cells exposed to tumor antigens and infiltrating the tumor microenvironment, to promote selective signaling and enhancement of activity at a desired tumor site. In various embodiments, bifunctional fusion proteins of IL-2 variants with anti-checkpoint inhibitor antibodies provide synergy by eliminating downregulation, reactivating T cell function, and increasing Teff cells to further enhance the anti-tumor activity of the immune system.

In various embodiments, bifunctional fusion proteins of IL-2 variants with antibodies to checkpoint inhibitors reduce systemic IL-2 exposure and non-target toxicity. In various embodiments, the use of IL-2 variants with reduced/aborted binding to IL-2Rα and attenuated/modulated activity against IL-2Rβγ helps establish a stoichiometric balance between IL-2 cytokine activity and antibody activity. Variants with attenuated IL-2 activity with adequately targeted antibody or cis-activation of depleted Teff cells will allow for optimal dosing and preserve the function of each moiety. In addition, IL-2-deficient variants fused to antibodies are expected to minimize peripheral activation, reduce T cell AICD, decrease antigen-binding-mediated clearance, and stimulate tumor cell killing via tumor lesion-targeted antibody moiety and/or immune cells.

In various embodiments, the IL-2 variants of the present invention can be coupled to a checkpoint inhibitor that is an antibody, antibody fragment, protein, or peptide directed to immune checkpoint modulators. In various embodiments, the checkpoint inhibitor is an antagonist PD-1 antibody. In various embodiments, the anti-PD-1 antibody comprises the variable domain sequences set forth in SEQ ID NOs: 138 and 139, SEQ ID NOs: 140 and 141, SEQ ID NOs: 142 and 143, SEQ ID NOs: 144 and 145, or SEQ ID NOs: 146 and 147. In various embodiments, exemplary bifunctional IL-2 anti-PD-1 antibody fusion proteins are set forth in Table 12.

Linkers

In various embodiments, the heterologous protein is linked to the IL-2 variant via a linker and/or hinge linker peptide. The linker or hinge linker may be an artificial sequence of 5, 10, 15, 20, 30, 40 or more amino acids that are relatively free of secondary structure or exhibit an α-helical conformation.

The peptide linker provides covalent attachment and additional structural and/or spatial flexibility between protein domains. As is known in the art, peptide linkers comprise flexible amino acid residues such as glycine and serine. In various embodiments, the peptide linker may comprise 1-100 amino acids. In various embodiments, the spacer may comprise a GGGSGGGS motif (SEQ ID NO: 18). In other embodiments, the linker may comprise a GGGGS motif (SEQ ID NO: 21)n, where n is an integer from 1 to 10. In other embodiments, the linker may also comprise amino acids other than glycine and serine. In another embodiment, the linker may comprise other protein motifs, including, but not limited to, sequences with an α-helical conformation, such as AEAAAKEAAAKEAAAKA (SEQ ID NO: 16). In various embodiments, the length and composition of the linker can be selected to optimize activity or development capabilities, including, but not limited to, expression level and aggregation propensity. In another embodiment, the peptide linker can be a simple chemical bond, such as an amide bond (e.g., in PEG chemical conjugation).

Typical peptide linkers are shown in Table 6.

Polynucleotides

In another aspect, the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding IL-2, an IL-2 variant, an IL-2 fusion protein, or an IL-2 variant fusion protein of the present invention. The nucleic acids in question may be single-stranded or double-stranded. Such nucleic acids may be DNA or RNA molecules. DNA includes, for example, cDNA (complementary DNA), genomic DNA, synthetic DNA, PCR-amplified DNA (polymerase chain reaction), and combinations thereof. Genomic DNA encoding IL-2 polypeptides is obtained from genomic libraries available from a number of species. Synthetic DNA can be obtained by chemical synthesis of overlapping oligonucleotide fragments, followed by assembly of the fragments to restore part or all of the coding regions and flanking sequences. RNA can be obtained using prokaryotic expression vectors that result in high levels of mRNA synthesis, such as vectors with promoters and T7 RNA polymerase. cDNA is obtained from libraries prepared from mRNA isolated from various tissues expressing IL-2. The DNA molecules of the invention include full-length genes, as well as polynucleotides and fragments thereof. A full-length gene may also contain sequences encoding an N-terminal signal sequence. Such nucleic acids can be used, for example, in methods for producing new IL-2 variants.

In various embodiments, the isolated nucleic acid molecules comprise the polynucleotides described herein and further comprise a polynucleotide encoding at least one heterologous protein described herein. In various embodiments, the nucleic acid molecules further comprise polynucleotides encoding linkers or hinge linkers described herein.

In various embodiments, the recombinant nucleic acids of the present invention may be operably linked to one or more regulatory nucleotide sequences in an expression construct. Regulatory sequences are known in the art and are selected to direct expression of the IL-2 variant. Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosome binding sites, transcription start and termination sequences, translation start and termination sequences, and enhancer or activator sequences. The present invention contemplates constitutive or inducible promoters known in the art. The promoters may be naturally occurring promoters or hybrid promoters that combine elements of more than one promoter. The expression construct may be present in the cell on an episome such as a plasmid, or the expression construct may be introduced into the chromosome. In various embodiments, the expression vector contains a selectable marker gene that allows selection of transformed host cells. Selectable marker genes are well known in the art and will vary depending on the host cell used.

In another aspect of the present invention, the nucleic acid in question is presented in an expression vector comprising a nucleotide sequence encoding an IL-2 variant and operably linked to at least one regulatory sequence. The term "expression vector" refers to a plasmid, phage, virus or vector for expressing a polypeptide with a polynucleotide sequence. Vectors suitable for expression in host cells are readily available and nucleic acid molecules are introduced into vectors using standard recombinant DNA techniques. Such vectors may contain a wide range of expression control sequences which, when operably linked to a DNA sequence, control its expression and can be used in these vectors to express DNA sequences encoding an IL-2 variant. Such useful expression control sequences include, for example, the SV40 early and late promoters, the tet promoter, the adenovirus or cytomegalovirus immediate early promoter, the RSV promoters, the lac system, the trp system, the TAC or TRC system, the T7 promoter expressed by T7 RNA polymerase, the basic operator and promoter regions of the lambda phage, the control regions of the coat protein fd, the promoter of 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatase promoters such as PhoS, yeast mating factor a-promoters, the polyhedrin promoter of the baculovirus system, and other known sequences that control the expression of genes in prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It will be appreciated that the structure of the expression vector may depend on factors such as the choice of host cell to be transformed and/or the type of protein desired to be expressed. Moreover, the copy number of the vector, the ability to control this copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, should be taken into account. Exemplary expression vectors suitable for the expression of IL-2 include pDSRa (described in WO 90/14363, incorporated herein by reference) and derivatives thereof containing IL-2 polynucleotides, as well as any additional suitable vectors known in the art or described below.

The recombinant nucleic acid of the present invention can be produced by ligating the cloned gene or a portion thereof into a vector suitable for expression in prokaryotic cells and/or eukaryotic cells (yeast, bird, insect or mammalian cells). Expression vehicles for producing a recombinant IL-2 polypeptide include plasmids and other vectors. For example, suitable vectors include the following types of plasmids: pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derived plasmids, and pUC-derived plasmids for expression in prokaryotic cells such as E. coli.

Some mammalian cell expression vectors contain both prokaryotic sequences that facilitate vector propagation in bacteria and one or more eukaryotic transcription units that are expressed in eukaryotic cells. Examples of mammalian cell expression vectors suitable for transfection of eukaryotic cells include those derived from pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo, and pHyg. Some of these vectors are modified with sequences from bacterial plasmids such as pBR322 to facilitate replication and selection for drug resistance in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as bovine papillomavirus (BPV-1) or Epstein-Barr virus (pHEBo, derived from pREP and p205) can be used for transient expression of proteins in eukaryotic cells. Examples of viral (including retroviral) expression systems are given below under the heading of delivery systems for gene therapy. The various techniques used in preparing plasmids and transforming host organisms are well known in the art. Other suitable expression systems for both prokaryotic and eukaryotic cells, as well as general recombinant techniques, are given in Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989) Chapters 16 and 17. In some cases, it may be desirable to express recombinant polypeptides using a baculovirus expression system. Examples of such baculovirus expression systems include pVL-derived vectors (such as pVL1392, pVL1393, and pVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derived vectors (such as pBlueBac III, which contains B-gal).

In various embodiments, a vector will be developed for producing the subject IL-2 variants in CHO cells, such as the Pcmv-Script vector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen, Carlsbad, Calif.), and pCI-neo vectors (Promega, Madison, Wis.). As will be appreciated, the subject gene constructs can be used to express the subject IL-2 variants in cultured cells, for example, to produce proteins, including fusion proteins or protein variants, for purification.

The present invention also relates to a host cell transfected with a recombinant gene comprising a nucleotide sequence encoding an amino acid sequence of one or more of the subject IL-2 variants. The host cell may be any prokaryotic or eukaryotic cell. For example, an IL-2 variant of the present invention may be expressed in bacterial cells such as E. coli, insect cells (e.g., using a baculovirus expression system), yeast cells, or mammalian cells. Other suitable host cells are known to those skilled in the art.

Accordingly, the present invention also relates to methods for producing the subject IL-2 variants. For example, a host cell transfected with an expression vector encoding an IL-2 variant can be cultured under suitable conditions allowing it to express the IL-2 variant. The IL-2 variant can be secreted and isolated from a mixture of cells and medium containing the IL-2 variant. Alternatively, the IL-2 variant can remain in the cytoplasmic or membrane fraction, and the cells can be harvested and lysed, followed by isolation of the protein. The cell culture comprises host cells, media, and other by-products. Suitable cell culture media are well known in the art.

The polypeptides and proteins of the present invention can be purified according to protein purification techniques well known to those skilled in the art. These techniques include, at one level, the separation of crude protein and non-protein fractions. Once the peptides and polypeptides have been separated from other proteins, the peptide or polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). As used herein, the term "isolated polypeptide" or "purified polypeptide" is intended to refer to a composition that can be separated from other components, wherein the polypeptide is purified to any extent compared to its state in which it is naturally obtained. Thus, a purified polypeptide also refers to a polypeptide that is free from the environment in which it may be naturally found. Typically, "purified" will refer to a polypeptide composition that has been fractionated to remove various other components and substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a peptide or polypeptide composition wherein the polypeptide or peptide is a major component of the composition, such as comprising about 50%, about 60%, about 70%, about 80%, about 85%, or about 90% or more of the proteins of the composition.

Various techniques suitable for use in purification will be well known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies (immunoprecipitation), and the like, or by thermal denaturation followed by centrifugation; chromatography such as affinity chromatography (protein A columns), ion exchange chromatography, gel filtration, reverse phase chromatography, hydroxyapatite chromatography, hydrophobic interaction chromatography; isoelectric focusing; gel electrophoresis; and combinations of these techniques. As is well known in the art, it is contemplated that the order of the various purification steps can be varied, or that certain steps can be omitted, and the method will still be suitable for obtaining a substantially purified polypeptide.

Pharmaceutical compositions

In another aspect, the present invention provides a pharmaceutical composition comprising IL-2 variants or IL-2 variant fusion proteins in admixture with a pharmaceutically acceptable carrier. Such pharmaceutically acceptable carriers are well known and understood by those skilled in the art and are described in many references (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990). Pharmaceutically acceptable carriers can be included to modify, maintain, or preserve, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, absorption, or penetration of the composition. Such pharmaceutical compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the polypeptide. Suitable pharmaceutically acceptable carriers include, but are not limited to: amino acids (such as glycine, glutamine, asparagine, arginine, or lysine); antimicrobial agents; antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins); proteins (such as serum albumin, gelatin, or immunoglobulins); colors; flavors and diluents; emulsifiers; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, cyclodextrin, sorbic acid, or hydrogen peroxide); solvents (such as glycerol, propylene glycol, or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surface-active or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, Triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides (preferably sodium or potassium chloride), mannitol, sorbitol); delivery vehicles; diluents; excipients; and/or pharmaceutical adjuvants.

The main vehicle or carrier in the pharmaceutical composition may be of aqueous or non-aqueous nature. For example, a suitable vehicle or carrier may be water for injection, physiological saline or artificial cerebrospinal fluid, optionally supplemented with other substances frequently used in parenteral compositions. Other typical vehicles are neutral buffered physiological saline or physiological saline mixed with serum albumin. Other typical pharmaceutical compositions contain a Tris buffer of about pH 7.0-8.5 or an acetate buffer of about pH 4.0-5.5, which may additionally contain sorbitol or a suitable substitute. In one embodiment of the present invention, the compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with additional agents of the composition (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized mass or an aqueous solution. In addition, the therapeutic composition can be prepared in the form of a lyophilisate using suitable excipients such as sucrose. The optimal pharmaceutical composition will be determined by a person skilled in the art depending on, for example, the intended route of administration, the delivery format and the desired dose.

When parenteral administration is contemplated, the therapeutic pharmaceutical compositions can be in the form of a pyrogen-free aqueous solution suitable for parenteral administration containing the desired IL-2 polypeptide or IL-2 polypeptide fusion protein in a pharmaceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile distilled water, wherein the polypeptide is included in the composition in the form of a sterile isotonic solution with the desired preservative(s). In various embodiments, pharmaceutical compositions suitable for injection can be prepared in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution or buffered saline. Aqueous injection suspensions can contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In addition, suspensions of the active compounds can be prepared in the form of suitable oily injection suspensions. Optionally, the suspension may also contain suitable stabilizers or agents to enhance the solubility of the compounds, allowing highly concentrated solutions to be obtained.

In various embodiments, therapeutic pharmaceutical compositions can be prepared for targeted delivery using a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, granules, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Examples of lipids useful in preparing liposomes include phosphatidyl compounds such as phosphatidylglycerol, phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine, sphingolipids, cerebrosides, and gangliosides. Exemplary phospholipids include egg phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphosphatidylcholine. Targeting of liposomes based on, for example, their specificity for organs, cells, and organelles is also possible, as is known in the art.

In various embodiments, oral administration of the pharmaceutical compositions is contemplated. The pharmaceutical compositions administered in this manner may be prepared with or without those carriers that are commonly used in the preparation of solid dosage forms such as tablets and capsules. In solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like), one or more therapeutic compounds of the present invention may be mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or calcium hydrogen phosphate, and/or any of the following: (1) fillers or excipients, such as starches, lactose, sucrose, glucose, mannitol and/or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates and sodium carbonate; (5) agents retarding dissolution, such as paraffin; (6) agents accelerating absorption, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricating agents, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions can also contain buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethyleneglycols, and the like. Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as aqueous or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (especially cottonseed, peanut, corn, germ, olive, castor and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycols, sorbitan esters of fatty acids and mixtures thereof. In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents, coloring agents, perfumes and preservatives.

In various embodiments, topical administration of the pharmaceutical compositions to the skin or mucous membranes is envisaged. The compositions for topical administration may further comprise one or more of a wide range of known agents that effectively enhance penetration through the skin or stratum corneum. Examples thereof are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide, dimethylformamide, propylene glycol, methyl or isopropyl alcohol, dimethyl sulfoxide and azone. In addition, additional agents may be included to ensure cosmetic acceptability of the composition. Examples thereof are fats, waxes, oils, dyes, fragrances, preservatives, stabilizers and surface-active agents. It is also possible to include keratolytic agents, such as keratolytic agents known in the art. Examples are salicylic acid and sulfur. Dosage forms for topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and dosage forms for inhalation administration. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier and with any preservatives, buffers or propellants that may be necessary. Ointments, pastes, creams and gels can contain, in addition to the subject compound of the invention (e.g., an IL-2 variant), excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Additional pharmaceutical compositions contemplated for use herein include compositions comprising polypeptides in sustained or controlled delivery compositions. In various embodiments, the pharmaceutical compositions can be formulated as nanoparticles as a slow-release hydrogel or incorporated into oncolytic viruses. Such nanoparticle-based methods include, for example, encapsulation in nanoparticles composed of polymers with a hydrophobic backbone and hydrophilic side chains as drug carriers, encapsulation in microparticles, incorporation into liposomes in emulsions, and conjugation with other molecules. Examples of nanoparticles include chitosan and carbopol coated mucoadhesive nanoparticles (Takeuchi et al., Adv. Drag Deliv. Rev. 47(1):39-54, 2001) and nanoparticles containing charged combination polyesters, poly(2-sulfobutylvinyl alcohol) and poly(D,L-lactic and glycolic acid) (Jung et al., Eur. J. Pharm. Biopharm. 50(1):147-160, 2000). Albumin-based nanoparticle compositions have been developed as a drug delivery system for the delivery of hydrophobic drugs such as taxane. See, for example, U.S. Patents Nos. 5,916,596, 6,506,405, 6,749,868, 6,537,579, 7,820,788, and 7,923,536. Abraxane®, a nanoparticle formulation of paclitaxel stabilized with albumin, was approved in the United States in 2005 and subsequently in various other countries for the treatment of metastatic breast cancer.

Techniques for preparing a variety of other means for sustained or controlled delivery, such as liposomal carriers, biodegradable microparticles or porous beads, and depot injections are also known to those skilled in the art.

An effective amount of a pharmaceutical composition for therapeutic use will depend, for example, on the therapeutic context and objectives. One skilled in the art will appreciate that appropriate dosage levels for treatment will thus vary depending, in part, on the molecule being delivered, the indication for use of the polypeptide, the route of administration, and the biometric data (body weight, body surface area, or organ size) and condition (age and general health) of the patient. Accordingly, a clinician can adjust the dosage and modify the route of administration to obtain an optimal therapeutic effect. A typical dosage can range from about 0.001 mg/kg to about 100 mg/kg or more, depending on the factors noted above. Preferably, the polypeptide compositions are injected or administered intravenously. Long-acting pharmaceutical compositions can be administered once every three or four days, once a week, or once every two weeks, depending on the half-life and clearance rate of the particular composition. The frequency of dosing will depend on the pharmacokinetic parameters of the polypeptide in the composition used. Typically, the composition is administered until a dose is reached that provides the desired effect. Thus, the composition can be administered as a single dose or in multiple doses (at the same or different concentrations/doses) over time or in the form of a continuous infusion. In addition, titration of the appropriate dosage is usually performed. Appropriate dosages can be determined using available dose-response data.

The route of administration of the pharmaceutical composition corresponds to known methods, for example oral administration, administration by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional injection, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, or intraperitoneal, or intratumoral administration, as well as administration by intranasal, enteral, local, sublingual, urethral, vaginal or rectal route, using systems for prolonged delivery or implantable devices. Where desired, the compositions can be administered by bolus injection or continuously by infusion or using an implantable device. Alternatively or additionally, the composition can be administered locally by implantation of a membrane, sponge or other suitable material on which the desired molecule is absorbed or encapsulated. When using an implantable device, it can be implanted into any suitable tissue or organ and delivery of the desired molecule can occur via diffusion, time-dependent release bolus administration, or continuous administration.

Therapeutic use

In one aspect, the present invention provides a method of treating cancer cells in a subject, comprising administering to said subject a therapeutically effective amount (as a monotherapy or in a combination therapy regimen) of an IL-2 variant or IL-2 variant fusion proteins of the present invention in a pharmaceutically acceptable carrier, wherein such administration inhibits the growth and/or proliferation of the cancer cell. Specifically, the IL-2 variant or IL-2 variant fusion protein of the present invention is useful in the treatment of disorders characterized as cancer. Such disorders include, but are not limited to, solid tumors such as cancer of the breast, respiratory system, brain, reproductive organs, digestive system, urinary system, eye, liver, skin, head and neck, thyroid, parathyroid glands and distant metastases thereof, lymphomas, sarcomas, multiple myeloma and leukemia. Examples of breast cancer include, but are not limited to, invasive ductal carcinoma, invasive lobular carcinoma, ductal carcinoma in situ, and lobular carcinoma in situ. Examples of respiratory cancer include, but are not limited to, small cell and non-small cell lung carcinoma, as well as bronchial adenoma and pleuropulmonary blastoma. Examples of brain cancer include, but are not limited to, brainstem and hypothalamic glioma, cerebellar and cerebral astrocytoma, medulloblastoma, ependymoma, and neuroectodermal and pineal tumors. Tumors of the male reproductive organs include, but are not limited to, prostate and testicular cancer. Tumors of the female reproductive organs include, but are not limited to, endometrial, cervical, ovarian, vaginal, and vulvar cancer, and uterine sarcoma. Tumors of the digestive system include, but are not limited to, anal cancer, colon cancer, colorectal cancer, esophageal cancer, gallbladder cancer, stomach cancer, pancreas cancer, rectum cancer, small intestine cancer, and salivary gland cancer. Tumors of the urinary system include, but are not limited to, bladder cancer, penis cancer, kidney cancer, renal pelvis cancer, ureter cancer, and urethra cancer. Eye cancer includes, but is not limited to, intraocular melanoma and retinoblastoma. Examples of liver cancer include, but are not limited to, hepatocellular carcinoma (hepatocellular carcinoma with or without the fibrolamellar variant), cholangiocarcinoma (cancer of the intrahepatic bile ducts), and mixed hepatocellular cholangiocarcinoma. Skin cancer includes, but is not limited to, squamous cell carcinoma, Kaposi's sarcoma, malignant melanoma, Merkel cell skin cancer, and non-melanoma skin cancer. Head and neck cancers include, but are not limited to, nasopharyngeal cancer and cancer of the lip and oral cavity. Lymphomas include, but are not limited to, AIDS-associated lymphoma, non-Hodgkin's lymphoma, cutaneous T-cell lymphoma, Hodgkin's disease, and central nervous system lymphoma. Sarcomas include, but are not limited to, soft tissue sarcoma, osteosarcoma, malignant fibrous histiocytoma, lymphosarcoma, and rhabdomyosarcoma. Leukemias include, but are not limited to, acute myelogenous leukemia, acute lymphoblastic leukemia, various lymphocytic leukemias, various myelogenous leukemias, and hairy cell leukemia. In various embodiments, the cancer will be a cancer with high expression of a member of the TGF-β family, such as activin A, myostatin, TGF-β and GDF15, such as pancreatic cancer, gastric cancer, ovarian cancer, colorectal cancer, melanoma, leukemia, lung cancer, prostate cancer, brain cancer, bladder cancer and head and neck cancer.

"Therapeutically effective amount" or "therapeutically effective dose" refers to that amount of a therapeutic agent administered that will alleviate to some extent one or more of the symptoms of the disorder being treated.

An initial estimate of the therapeutically effective dose can be made in cell culture assays by determining the _EC50 . A dose can then be selected in animal models to achieve a circulating plasma concentration range that includes the _EC50 determined in cell culture. Such information can be used to more accurately determine useful doses for humans. Plasma levels can be measured, for example, by HPLC. The exact composition, route of administration, and dose can be selected by the individual physician based on the condition of the subject.

The administration regimens can be adjusted to provide the optimal desired response (e.g., a therapeutic or prophylactic response). For example, a single bolus administration can be given, several divided doses (multiple, repeat, or maintenance) can be given over a period of time, and the dose can be proportionally decreased or increased depending on the particular therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the mammalian subjects to be treated, each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The characteristics of the dosage unit forms of the present invention will be dictated primarily by the unique properties of the antibody and the particular therapeutic or prophylactic effect to be achieved.

Thus, based on the description provided herein, it will be clear to one skilled in the art that the dose and administration schedule are adjusted in accordance with methods well known in the therapeutic field. That is, the maximum tolerated dose can be readily determined, and the effective amount that provides a detectable therapeutic benefit in a subject can also be determined, as can the time requirements for administering each agent to provide a detectable therapeutic benefit in a subject. Accordingly, although specific doses and administration schedules are exemplified herein, these examples in no way limit the dose and administration schedule that can be used in a subject in practicing the present invention.

It should be noted that dosage values may vary depending on the type and severity of the condition being treated, and may include single or multiple doses. It should also be understood that for any particular subject, the said administration regimens should be adjusted over time according to the individual needs and professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges provided herein are merely examples and are not intended to limit the scope or practical application of the claimed composition. In addition, the administration regimen of the compositions of the present invention can be based on a variety of factors, including the type of disease, age, body weight, sex and health of the subject, the severity of the condition, the route of administration and the particular antibody used. Thus, the administration regimen can vary widely, but can be routinely determined using standard techniques. For example, doses can be adjusted based on pharmacokinetic or pharmacodynamic parameters, which can include clinical effects, such as toxic effects, and/or laboratory parameters. Thus, the present invention encompasses increasing the dose administered to a subject at the discretion of the skilled artisan. Determination of appropriate doses and administration schedules is well known in the art and will be apparent to the skilled artisan having the information disclosed herein.

Non-limiting examples of daily dosage ranges for a therapeutically or prophylactically effective amount of an IL-2 variant or IL-2 variant fusion protein of the invention may be ranges of from 0.001 to 100 mg/kg, from 0.001 to 90 mg/kg, from 0.001 to 80 mg/kg, from 0.001 to 70 mg/kg, from 0.001 to 60 mg/kg, from 0.001 to 50 mg/kg, from 0.001 to 40 mg/kg, from 0.001 to 30 mg/kg, from 0.001 to 20 mg/kg, from 0.001 to 10 mg/kg, from 0.001 to 5 mg/kg, from 0.001 to 4 mg/kg, from 0.001 to 3 mg/kg, from 0.001 to 2 mg/kg, from 0.001 to 1 mg/kg, from 0.010 to 50 mg/kg, from 0.010 to 40 mg/kg, from 0.010 to 30 mg/kg, from 0.010 to 20 mg/kg, from 0.010 to 10 mg/kg, from 0.010 to 5 mg/kg, from 0.010 to 4 mg/kg, from 0.010 to 3 mg/kg, from 0.010 to 2 mg/kg, from 0.010 to 1 mg/kg, from 0.1 to 50 mg/kg, from 0.1 to 40 mg/kg, from 0.1 to 30 mg/kg, from 0.1 to 20 mg/kg, from 0.1 to 10 mg/kg, from 0.1 to 5 mg/kg, from 0.1 to 4 mg/kg, 0.1 to 3 mg/kg, 0.1 to 2 mg/kg, 0.1 to 1 mg/kg, 1 to 50 mg/kg, 1 to 40 mg/kg, 1 to 30 mg/kg, 1 to 20 mg/kg, 1 to 10 mg/kg, 1 to 5 mg/kg, 1 to 4 mg/kg, 1 to 3 mg/kg, 1 to 2 mg/kg, or 1 to 1 mg/kg of body weight. It should be noted that dosage values may vary depending on the type and severity of the conditions to be relieved. It should also be understood that for any particular subject, specific administration regimens should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges given herein are merely examples and are not intended to limit the scope or practical application of the claimed composition.

The toxicity and therapeutic index of the pharmaceutical compositions of the invention can be determined by standard pharmaceutical techniques in cell cultures or in experimental animals, for example, by techniques for determining LD ₅₀ (the dose lethal to 50% of the population) and ED ₅₀ (the dose therapeutically effective for 50% of the population). The ratio of the toxic and therapeutically effective dose is the therapeutic index, and it can be expressed as the ratio LD ₅₀ /ED ₅₀ . Compositions exhibiting large therapeutic indices are generally preferred.

The frequency of administration of the IL-2 variant pharmaceutical composition or IL-2 variant fusion protein depends on the nature of the therapy and the specific disorder being treated. The composition can be administered to the subject at regular intervals, such as twice a week, once a week, or once a month, until the desired therapeutic result is achieved. Examples of dosing frequencies include, but are not limited to: once a week without a break; once every 2 weeks; once every 3 weeks; once a week without a break for 2 weeks, then once a month; once a week without a break for 3 weeks, then once a month; once a month; once every two months; once every three months; once every four months; once every five months; or once every six months; or once a year.

Combination therapy

As used herein, the terms "co-administration", "co-administered" and "in combination with" referring to an IL-2 variant or IL-2 variant fusion protein of the invention and one or more other therapeutic agents are intended to mean and include the following: the simultaneous administration of such a combination of an IL-2 variant or IL-2 variant fusion protein of the invention and therapeutic agent(s) to a subject in need of treatment, wherein such components are formulated together in a single dosage form that releases said components in said subject at substantially the same time; the substantially simultaneous administration of such a combination of an IL-2 variant or IL-2 variant fusion protein of the invention and therapeutic agent(s) to a subject in need of treatment, wherein such components are formulated separately from one another in separate dosage forms administered to said subject at substantially the same time, whereby said components are released in said subject at substantially the same time; sequentially administering such a combination of an IL-2 variant or an IL-2 variant fusion protein of the invention and a therapeutic agent(s) to a subject in need of treatment, wherein such components are separately formulated in separate dosage forms administered to said subject sequentially with a substantial time interval between each administration, whereby said components are released in said subject at substantially different times; and sequentially administering such a combination of an IL-2 variant or an IL-2 variant fusion protein of the invention and a therapeutic agent(s) to a subject in need of treatment, wherein such components are formulated together in a single dosage form releasing said components in a controlled manner, whereby said subject releases them simultaneously, sequentially and/or overlappingly, at the same and/or at different times, wherein all parts may be administered by the same route of administration or by different routes of administration.

In another aspect, the present invention provides a method of treating cancer or cancer metastasis in a subject, comprising administering a therapeutically effective amount of the pharmaceutical compositions of the invention in combination with a second therapy, including, but not limited to, immunotherapy, cytotoxic chemotherapy, targeted therapy with small molecule kinase inhibitors, surgery, radiation therapy and stem cell transplantation. For example, such methods can be used in prophylactic prevention of cancer, prevention of recurrence and metastasis of cancer after surgery and as an adjunct to other standard cancer therapy. According to the present invention, the use of the combination methods described herein can improve the effectiveness of standard cancer therapy (e.g., chemotherapy, radiation therapy, phototherapy, immunotherapy and surgery).

A wide range of standard compounds have been shown to have antitumor activity. These compounds are used as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent metastasis and further growth, or reduce the number of malignant T cells in leukemia or bone marrow malignancies. Despite the effectiveness of chemotherapy in the treatment of various types of malignancies, many antitumor compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, they can act synergistically, allowing the dose of each to be reduced, thereby reducing the deleterious side effects produced by each compound at higher doses. In other cases, malignancies that are refractory to treatment may respond to combination therapy of two or more different treatments.

In various embodiments, the patient will be administered a second anti-cancer agent, such as a chemotherapeutic agent. Typical chemotherapeutic agents include, but are not limited to, daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin, nitrogen mustard, chlorambucil, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, bendamustine, cytarabine (CA), 5-fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine, vincristine, vinblastine, etoposide, teniposide, cisplatin, carboplatin, oxaliplatin, pentostatin, cladribine, cytarabine, gemcitabine, pralatrexate, mitoxantrone, diethylstilbestrol (DES), fludarabine, ifosfamide, hydroxyureatoxanes (such as paclitaxel and docetaxel) and/or anthracycline antibiotics, as well as combinations of agents such as, but not limited to, DA-EPOCH, CHOP, CVP or FOLFOX. In various embodiments, dosages of such chemotherapeutic agents include, without limitation, any of about 10 mg/ ^m2 , 20 mg/ ^m2 , 30 mg/ ^m2 , 40 mg/ ^m2 , 50 mg/ ^m2 , 60 mg/ ^m2 , 75 mg/ ^m2 , 80 mg/ ^m2 , 90 mg/ ^m2 , 100 mg/ ^m2 , 120 mg/ ^m2 , 150 mg/ ^m2 , 175 mg/ ^m2 , 200 mg/ ^m2 , 210 mg/ ^m2 , 220 mg/ ^m2 , 230 mg/ ^m2 , 240 mg/ ^m2 , 250 mg/ ^m2 , 260 mg/ ^m2 , and 300 mg/ ^m2 .

In various embodiments, the combination therapy methods of the present invention may further comprise administering to the subject a therapeutically effective amount of immunotherapy, including, but not limited to: treatment using depleting antibodies to certain tumor antigens; treatment using antibody-drug conjugates; treatment using agonist, antagonist, or blocking antibodies to costimulatory or coinhibitory molecules (immune checkpoints) such as CTLA-4, PD-1, OX-40, CD137, GITR, LAG3, TIM-3, SIRP, CD47, CD40, TIGIT, and VISTA; treatment using bispecific T cell engager (BiTE®) antibodies such as blinatumomab; treatment involving administration of biological response modifiers such as IL-12, IL-15, IL-21, GM-CSF, IFN-α, IFN-β, and IFN-γ; treatment using therapeutic vaccines such as sipuleucel-T; treatment using dendritic cell vaccines or tumor antigen peptide vaccines; treatment using chimeric antigen receptor T cells (CAR-T cells); treatment using CAR-NK cells; treatment using tumor-infiltrating lymphocytes (TILs); treatment using adoptively transferred anti-tumor T cells (ex vivo cultured and/or TCR transgenic); treatment using TALL-104 cells; and treatment using immunostimulatory agents such as Toll-like receptor (TLR) CpG agonists and imiquimod; where combination therapy provides enhanced destruction of tumor cells by effector cells, i.e., synergy occurs when IL-2 variants and immunotherapy are used together.

In various embodiments, the combination therapy comprises administering the IL-2 variant and the second agent composition simultaneously, in the same pharmaceutical composition or in separate pharmaceutical compositions. In various embodiments, the IL-2 variant composition and the second agent composition are administered sequentially, i.e., the IL-2 variant composition is administered before or after the second agent composition. In various embodiments, the IL-2 variant composition and the second agent composition are administered simultaneously, i.e., the periods of administration of the IL-2 variant composition and the second agent composition overlap with each other. In various embodiments, the IL-2 variant composition and the second agent composition are not administered simultaneously. For example, in various embodiments, administration of the IL-2 variant composition is stopped before administration of the second agent composition. In various embodiments, administration of the second agent composition is stopped before administration of the IL-2 variant composition.

The following examples are provided to more fully explain and describe the invention, but should not be construed as limiting the scope of the invention.

Example 1

Construction and production of IL-2 Fc-fusion constructs

All genes synthesized and subcloned into the recipient mammalian cell expression vector (GenScript) were codon optimized for expression in mammalian cells. Protein expression is driven by the CMV promoter, and a synthetic SV40 poly-A signal sequence is present at the 3' end of the CDS. A leader sequence was designed at the N-terminus of the constructs to provide the necessary signaling and processing for secretion.

Constructs were generated by cotransfection of HEK293-F cells grown in suspension with mammalian expression vectors using polyethyleneimine (PEI, linear, molecular weight 25,000, Polysciences). When two or more expression vectors were available, they were used for transfection at a 1:1 ratio. For transfection, HEK293 cells were cultured in FreeStyle™293 serum-free expression medium (ThermoFisher). For production in 1000 ml shake flasks (working volume 330 ml), HEK293 cells were seeded at a density of 0.8 × 10 ⁶ cells/ml 24 h before transfection. Expression vectors, to a total DNA amount of 330 μg, were mixed with 16.7 ml Opti-MEM medium (ThermoFisher). After adding 0.33 mg PEI diluted in 16.7 ml Opti-MEM medium, the mixture was vortexed for 15 sec and then incubated for 10 min at room temperature. Then, the DNA/PEI solution was added to the cells and incubated at 37°C in an 8% CO ₂ incubator. On day 4, sodium butyrate (Millipore Sigma) was added to the cells at a final concentration of 2 mM to promote stable protein expression. After 6 days of culturing, the supernatant was collected for purification by centrifugation for 20 min at 2200 rpm. The resulting solution was sterilized by filtration (0.22 μm filter, Corning). Secreted protein was purified from cell culture supernatants using protein A affinity chromatography.

Alternatively, constructs were generated in ExpiCHO cells (ThermoFisher) following the manufacturer's instructions.

For affinity chromatography, the supernatant was loaded onto a HiTrap MabSelect SuRe column (CV = 5 mL, GE Healthcare) equilibrated with 25 mL of phosphate buffered saline, pH 7.2 (ThermoFisher). Unbound protein was removed by washing with 5 column volumes of PBS (phosphate buffered saline), pH 7.2, and the target protein was eluted with 25 mM sodium citrate, 25 mM sodium chloride, pH 3.2. The protein solution was neutralized by adding 3% 1 M Tris, pH 10.2. Ion exchange chromatography or mixed chromatography, including but not limited to CaptoMMC (GE Healthcare), ceramic hydroxyapatite, or ceramic fluoroapatite (Bio-Rad), were also used for further purification of protein A purified materials as needed. The target protein was concentrated using an Amicon® Ultra-15 concentrator with a molecular weight cutoff (NMWC) of 10 kDa (Merck Millipore Ltd.).

Purity and molecular weight of purified constructs were analyzed by SDS-PAGE in the presence and absence of reducing agent and Coomassie staining (Imperial ^R Stain). The NuPAGE® Pre-Cast gel system (4-12% or 8-16% Bis-Tris, ThermoFisher) was used following the manufacturer's instructions. Protein concentration of the purified protein sample was determined by measuring the UV absorbance at 280 nm (Nanodrop spectrophotometer, ThermoFisher) divided by the molar extinction coefficient calculated from the amino acid sequence. The aggregate content of the constructs was analyzed on an Agilent 1200 high-performance liquid chromatography (HPLC) system. Samples were loaded onto an AdvanceBio size-exclusion column (300 , 4.6×150 mm, 2.7 μm, LC column, Agilent) using 150 μM sodium phosphate, pH 7.0, as mobile phase at 25°C.

SDS-PAGE and size exclusion chromatogram analysis of representative IL-2 Fc fusion constructs P-0635 and P-0704 purified on Protein A are shown in FIG. 1. P-0635 (SEQ ID NO:85; FIG. 1A) and P-0704 (SEQ ID NOS:96 and 10; FIG. 1B) have the same P65R amino acid substitution in IL-2. P-0635 contains a bivalent variant of IL-2 fused to an Fc homodimer, while P-0704 contains a monovalent variant of IL-2 fused to a heterodimeric Fc with a knob-into-hole modification. SDS-PAGE analysis showed that both molecules exhibited a high degree of protein purity, and samples analyzed under reducing conditions (lane 2) showed the expected MW for both the homodimeric Fc chain of P-0635 and the heterodimeric Fc chains of P-0704. Analysis of size exclusion chromatograms showed that both molecules exhibited a low propensity to aggregate, with the degree of aggregation being less than 5% after the initial capture step on Protein A.

Example 2

A single amino acid change in IL-2 leads to a versatile expansion of fusion design options

The use of design techniques to find a combination of mutations that would produce a protein variant with the desired biological properties has proven to be very difficult in the case of IL-2. It is known in the art that the naturally occurring IL-2 protein tends to have very low stability and is prone to aggregation. In our experiments, we demonstrated that a wild-type IL-2 Fc fusion protein (P-0250) expressed at low levels (approximately 3 mg/L, transiently, in HEK-293F cells) has a high propensity to aggregate, as shown in the SEC chromatogram shown in FIG. 2A. Design was greatly hampered because amino acid substitutions in IL-2 aimed at achieving the desired biological activity typically resulted in even less stable mutant proteins. A significant proportion of the IL-2 variants obtained in the early phase of these studies showed extremely low levels of expression, and some variants were much more prone to aggregation, as exemplified by the SEC chromatogram of P-0318 (IL-2 D20I/N88I Fc fusion protein) shown in FIG. 2B. This presents a challenge in terms of manufacturing and storage of the therapeutic agent.

It was also noted that the expression profiles and aggregation propensities of the IL-2 variant fusion proteins varied significantly between constructs with different mutation sites or between mutants that had the same mutation site but different substituted residues. Examples of this observation are P-0317 (IL-2 D20I/N88R Fc fusion protein) and P-0318 (IL-2 D20I/N88I Fc fusion protein). The fusion proteins of both of these variants have the same mutation sites at residues 20 and 88, differ in only one amino acid, and have similar low expression levels. As seen in FIG. 2B, P-0318 is highly aggregation prone and contains 65% of the high molecular weight species, forming the expected minor species peak in the chromatogram, indicated by the arrow. In contrast, P-0317 is relatively pure and contains 7.5% aggregates (FIG. 2C). It could be concluded that the N88R mutation may reduce the propensity of the resulting fusion proteins to aggregate. However, IL-2 with a single N88R mutation or with a double D20T/N88R mutation and the resulting fusion proteins, P-0254 and P-0324, respectively, were prone to aggregation and contained 30-40% aggregates. Thus, the impact of individual amino acid substitutions on protein stability appears to be context dependent.

The fact that amino acid substitutions of IL-2 generally result in a less stable protein was compounded by the unpredictable impact of substitutions of different residues on protein stability. Thus, it is highly desirable to find residue substitution(s) that could universally enhance protein design options, including increased stability, higher expression levels, and reduced aggregation propensity.

The amino acid substitutions at position 125 were originally aimed at increasing the selectivity of IL-2, since this residue is located in close proximity to Q126, which is required for interaction with γc. Naturally occurring IL-2 contains an unpaired cysteine at position 125, which is replaced by Ser in Proleukin®. IL-2 containing an alanine substitution at position 125 is also widely used. Substitution of Ser or Alan from cysteine 125 retained full biological activity; bulky charged or hydrophobic residues including Glu, Lys, Try, His, and Iso were introduced at position 125 to interfere with the interaction of Q126 with γc and to obtain altered biological activity. All of the resulting fusion molecules, except the fusion molecule containing Iso125 (P-0531), were expressed at levels too low to be characterized. Compared to its S125 equivalent (P-0250), P-0531 was expressed at a significantly higher level (titer 29.5 mg/L vs. 3.1 mg/L) with a significantly lower tendency to aggregate (aggregation 0.7% vs. 25.7%). This impressive expansion of development capabilities, especially with respect to product purity, prompted the inventors to explore the possibility of reproducing this effect of substitution with isoleucine at position 125 in a different mutational context.

Thus, the S125I substitution was introduced into several Fc fusion molecules of IL-2 variants. Constructs containing a substitution of Ile at amino acid position 125 (125I) of IL-2 were expressed using the same vector and under the same culture conditions as their Ser-125 counterparts and purified using MabSelect SuRe. A summary of the expression level in mg/L and the purity of the material, assessed by SEC chromatography, as % aggregation for representative molecules is presented in Table 7. Two molecules in the same row of Table 7 have the same other amino acid substitution(s) and differ only at residue 125, which is serine or isoleucine. In addition, in FIG. 2D shows the SEC profile of P-0531 (SEQ ID NO:68), an Fc-fusion protein of the S125I-equivalent of wild-type IL-2. It is clear from Table 7 that the substitution with isoleucine at position 125 resulted in a 4- to 11-fold increase in expression and a similarly low propensity to aggregate.

It is evident from the present invention that isoleucine at position 125 resulted in a versatile expansion of the design possibilities for IL-2 fusion constructs. This finding is particularly valuable since engineering IL-2 to achieve desired biological properties has been hampered by the fact that alteration of wild-type IL-2, which is borderline stable, typically results in even less stable mutant proteins. These problems inherent in IL-2 engineering can be partially overcome by a single amino acid substitution of isoleucine at position 125.

Example 3

Development of IL-2 constructs to improve selectivity for effector T cells and NK cells

A key aspect of the present invention is to improve the selectivity of IL-2 over wild-type IL-2 for IL-2Rβγ (but not IL-2Rα)-expressing cells over IL-2Rαβγ-expressing cells for the treatment of cancer. One approach used by the present inventors is to generate highly selective IL-2 Fc-fusion proteins by introducing CD25-impairing mutations into the cytokine component. The selection of CD25-impairing mutations was based on analysis of the IL-2/IL-2R co-crystal structure (PDB code 2B51). Multiple substitutions of one or two relevant amino acid residues in the regions interacting with the IL-2 receptor α subunit were introduced, including R38, T41, F42, F44, E62, P65, E68, and Y107, aimed at reducing or eliminating binding to IL-2Rα. These constructs also contained the S125I mutation to significantly expand design options. Furthermore, reducing binding of IL-2 variants to IL-2Rα+ lung endothelial cells is expected to prevent endothelial cell injury and significantly reduce VLS. Furthermore, reducing binding to CD25 is predicted to also reduce clearance mediated by CD25 antigen binding, increase cytokine receptor occupancy on IL-2Rβγ-expressing cells, and therefore enhance the in vivo response and tumor cell killing efficacy.

A summary of the panel of IL-2 muteins expressed as C-terminal fusion proteins with an Fc homodimer or Fc heterodimer is presented in Table 3. A panel of IL-2 variants (SEQ ID NOs:31-66) containing one or two amino acid substitutions of residues in the IL-2 receptor α-subunit interacting regions were fused via the “GGGSGGGS” linker (SEQ ID NO:18) to the C-terminus of the Fc homodimer in the form of bivalent IL-2 fusion proteins (SEQ ID NOs:69-95) or the Fc heterodimer in the form of monovalent IL-2 fusion proteins (SEQ ID NOs:96-106).

Example 4

Effect of IL-2 mutations introduced into IL-2Rα-interacting regions on binding to the α-subunit of the receptor

A panel of IL-2 muteins as C-terminal fusion proteins with Fc homodimer or Fc heterodimer were expressed and screened for binding to IL-2Rα in an enzyme-linked immunosorbent assay (ELISA). Briefly, IL-2Rα ECD (SEQ ID NO:5) was coated onto wells of Nunc Maxisorp 96-well microplates at 0.1 μg/well. After overnight incubation at 4°C and blocking with superblock (ThermoFisher), 100 μl of 3-fold serial dilutions of IL-2 Fc fusion proteins starting at 100 nM were added to each well. After incubation for one hour at room temperature, 100 μl of goat anti-human IgG Fc conjugated to HRP (horseradish peroxidase) (1:5000 dilution in diluent) were added to each well and incubated at room temperature for 1 hour. After each step, the wells were carefully aspirated and washed three times with PBS/0.05% Tween-20. Finally, 100 μl of TMB substrate were added to each well, the plate was developed at room temperature in the dark for 10 minutes, and stop solution (2 H sulfuric acid, Ricca Chemical) was added at 100 μl per well. Optical density was determined at 450 nm and curves were plotted using Prism software (GraphPad).

First, the binding of Fc fusion proteins of the S125I equivalent of wild-type IL-2, P-0531 and P-0689, to CD25 was analyzed. P-0531 contains a bivalent IL-2 moiety fused to an Fc homodimer (SEQ ID NO:68), and P-0689 (SEQ ID NO:107+10) is a monovalent analog of P-0531. As shown in FIG. 3, the 2-fold difference in _EC50 binding between P-0531 and P-0689 (0.21 nM and 0.51 nM, respectively) was consistent with the difference in IL-2 valency.

Since all of the IL-2 target residues, R38, T41, F42, F44, E62, P65, E68, and Y107, are located in IL-2Rα-interacting regions and form hydrogen bonds/salt bonds or hydrophobic interactions with multiple IL-2Rα residues (Mathias Rickert, et al. (2005) Science 308, 1477-80), it was hypothesized that amino acid substitutions at these sites would disrupt interactions with IL-2Rα and result in IL-2 variants with reduced or abolished binding to IL-2Rα. However, our binding data revealed that the effects of different IL-2 mutations on IL-2Rα binding vary considerably.

As shown in FIG. 4, IL-2 Fc homodimer fusion proteins containing various substitutions at position T41 (examples of which are P-0603, P-0604, and P-0605 in FIG. 4A) or Y107 (examples of which are P-0610, P-0611, and P-0612 in FIG. 4B) fully retained binding potency to IL-2Rα. These data indicate that residues T41 and Y107 are likely not of critical functional importance, despite their location in regions interacting with various residues of IL-2Rα.

The R38 residue was found at an energetically active point of the IL-2/IL-2Rα interaction where it is involved in the formation of critical hydrogen bonds; multiple redesign attempts, such as Keith M. Heaton, et al., (1993) Cancer Res. 53. 2597–2602, and Peisheng Hu, et. al, (2003) Blood 101: 4853–4861, showed that many R38 substitutions resulted in impaired interaction with IL-2Rα. It was therefore somewhat surprising to observe that various mutations, exemplified by P-0602 (R38A), P-0614 (R38F), and P-0615 (R38G), resulted in no or only minimal (up to 3-fold) reduction in IL-2Rα binding strength. The binding data are shown in FIGS. 4C-4D.

Similarly, residue E68 is involved in multiple hydrogen bonding with interacting residues of IL-2Rα, but none of the substitutions of E68 with amino acids with different properties, exemplified by E68A (P-0628), E68F (P-0629), E68H (P-0630), and E68L (P-0631), resulted in decreased binding to IL-2Rα. Interestingly, P-0629 and P-0630 actually showed a 3-fold and 14-fold increase in binding to IL-2Rα, respectively (FIG. 5).

In conclusion, substitution of IL-2 residues T41, R38, E68, and Y107 generally did not disrupt interaction with IL-2Rα, and the resulting IL-2 Fc homodimer fusion proteins retained all or almost all binding to IL-2Rα. A summary of the _EC50 binding of the various IL-2 muteins in ELISA, normalized to P-0531, is presented in Table 8.

In contrast, all amino acid substitutions of residue E62, exemplified by P-0624 (E62A), P-0625 (E62F), P-0626 (E62H), and P-0627 (E62L), resulted in decreased binding to IL-2Rα, indicating that E62 is indeed located at an energetically active point in the IL-2/IL-2Rα interaction. As shown in FIG. 6, while the E62H and E62L substitutions resulted in only a slight 2- to 3-fold decrease in IL-2Rα binding, the E62A and E62F mutations appeared to significantly disrupt the interaction with this IL-2R subunit, resulting in a 60- and 150-fold decrease in IL-2Rα binding, respectively. Furthermore, it is well documented in the literature that the F42A mutation in IL-2 (P-0613) disrupts interaction with the α receptor, as shown in FIG. 8A, reducing binding to IL-2Rα by 15-fold.

In conclusion, F42 and E62 are IL-2 residues whose substitution usually disrupted the interaction with IL-2Rα, and the resulting IL-2 variants showed decreased binding to IL-2Rα. The EC ₅₀ binding of the various IL-2 muteins in ELISA were normalized to P-0531 and are shown in Table 9.

Example 5

Amino acid substitutions at residue P65 had unexpectedly diverse effects on receptor α-subunit binding

The IL-2 P65 residue forms van der Waals interactions with a number of critical interacting residues of IL-2Rα, including R36 and L42, but does not form salt or hydrogen bonds with IL-2Rα. It was therefore expected that P65 substitutions would only slightly disrupt the interaction with this IL-2R subunit and would likely have little effect on IL-2Rα binding. However, the effects of P65 substitutions on IL-2Rα interaction were in stark contrast to this prediction and were unexpectedly diverse, including complete retention/enhancement, reduction, or complete abolition of IL-2Rα binding.

Numerous P65 substitutions were introduced, examples of which are P65G, P65E, P65A, P65H, P65N, P65Q, P65R, P65K, and the resulting IL-2 muteins were expressed as C-terminal fusion proteins with an Fc homodimer or an Fc heterodimer. This panel of IL-2 muteins was then screened in ELISA for their binding to CD25. The resulting binding data are shown in FIG. 7, and a summary of the ELISA binding _EC50 data for the IL-2 muteins, normalized to P-0531 or P-0689 based on the valency of each construct, is presented in Table 10.

As shown in FIGS. 7A and 7B, the mutations P65G (P-0608), P65E (P-0633), P65A (P-0706) did not appear to result in any impairment of interaction with the IL-2Rα subunit; instead, the binding strength to IL-2Rα was increased by 18-, 10-, and 10-fold, respectively, compared to their wild-type counterparts.

Another panel of IL-2 Fc fusion mutein proteins, P-0634, P-0708, and P-0709, contained mutations in P65 that significantly disrupted the interaction of IL-2 with the IL-2Rα subunit. As shown in FIG. 7C and Table 9, P65N (P-0708) resulted in a modest 8.6-fold decrease in IL-2Rα binding, while the P65H (P-0634) and P65H (P-0709) substitutions resulted in a more pronounced effect, as indicated by a 23-fold and 43-fold decrease in IL-2Rα binding, respectively.

Another category of IL-2 P65 substitutions, P65R and P65K, appeared to significantly disrupt the interaction of IL-2 and IL-R2Rα and abolish the binding of P-0635, P-0704, and P-0707 to IL-2Rα (FIG. 7D). P-0635 and P-0704 are equivalent bivalent and monovalent IL-2 Fc fusion proteins containing the P65R substitution, and P-0707 contains the P65K amino acid substitution. In FIG. Figure 7D shows that all three IL-2 Fc-fusion mutein proteins showed minimal signal at 100 nM IL-2Rα, comparable to a reference molecule containing three F42A/Y45A/L72G mutations that disrupt interaction with CD25, which demonstrated abolition of binding (Christian Klein, et. al, Oncomimunology (2017), 6:3, e1277306).

As shown in FIGS. 7A-7C and Tables 9 and 10, substitutions of the P65 residue resulted in unexpectedly diverse effects on IL-2Rα binding. Importantly, its substitution could result in complete retention/enhancement, reduction, or complete abolition of the resulting IL-2 variants' binding to IL-2Rα. As will be appreciated by those skilled in the art, this level of change in activity resulting from single amino acid changes is not predictable by structure-based mutagenesis. Complete abolition of IL-2Rα binding was also not expected or described in the prior art, since the P65 mutations altered only a limited portion of the surface involved in van der Waals interactions.

Example 6

Amino acid substitution combinations to modulate IL-2 binding to the receptor α subunit

As will be apparent to those skilled in the art, the mutations disclosed in the present invention can be alternatively and independently combined in any manner to optimally modulate IL-2 binding to the receptor α subunit. Here, the inventors demonstrate the development of IL-2 compounds that are incapable of binding to IL-2Rα by combinations of two amino acid substitutions that disrupt the interaction with IL-2Rα.

P-0613 contains the F42A mutation, which resulted in a 15-fold decrease in IL-2Rα binding (FIG. 8A); P-0625 and P-0634 contain the E62F and P65H substitutions, which decreased IL-2Rα binding by 150-fold and 23-fold, respectively. Combinations of two F42A and E62F mutations in P-0702 and two F42A and P65H mutations in P-0703 abolished IL-2Rα binding (FIGS. 8B and 8C). As expected, P-0766, which contained two F42/E62A amino acid changes, and P-0767, which had two F42A/E62H substitutions, were unable to bind IL-2Rα (data not shown).

In addition to their use in efficiently designing IL-2 muteins with ablated IL-2Rα binding, combinations of amino acids can also be used to modulate the level of binding activity. One example shown here is P-0765, which contains one mutation that disrupts interaction with CD25, F42A, and one mutation that enhances interaction with CD25, P65A, and showed a modest 6.8-fold decrease in IL-2Rα binding strength compared to its wild-type counterpart P-0689 (data not shown), consistent with the combination of individual mutations. Summary ELISA _EC50 data for IL-2 muteins normalized to P-0689 are presented in Table 11.

In conclusion, combinations of amino acid substitutions are a versatile way to modulate IL-2 binding to the receptor α subunit. It allows complete abolition of IL-2Rα binding by combining two residues that disrupt interaction with CD25, or it can be used to modulate IL-2Rα binding with varying degrees of attenuation.

Example 7

Modulation of IL-2Rα Binding Strength Correlates with IL-2 Treg Cell Stimulating Activity in an Ex Vivo Functional Assay

A panel of IL-2 variant Fc fusion proteins were then analyzed for their ability to differentially stimulate STAT5 phosphorylation in CD4+ Treg cells compared to the wild-type fusion protein P-0531 and the reference molecule P-0551 (SEQ ID NO:95). STAT5 is known to be involved in the signaling cascade downstream of IL-2 binding to IL-2 transmembrane receptors. STAT5 phosphorylation in lymphocyte subsets was measured using fresh human peripheral blood mononuclear cells (PBMCs), and the Forkhead transcription factor FOXP3 was used to define the Treg population in FACS analysis.

Purified PBMCs were maintained in serum-free MACS buffer at 4°C for 1 h. Then, 2 x ¹⁰⁵ PBMCs were treated with serial dilutions of test compounds for 30 min at 37°C. Cells were fixed and permeabilized using the Foxp3/Transcription Factor Staining Buffer Set (EBIO) by incubation with 1x Foxp3 fixation/permeabilization working solution for 30 min and washing with 1x permeabilization buffer. Cells were further fixed with Cytofix, permeabilized with Perm Buffer III (BD Biosciences), and then washed. After blocking Fc receptors with Human TruStain FcX (diluted 1:50), cells were stained with anti-CD25 with PE, anti-FOXP3 with APC, anti-pSTAT5 with FITC, and anti-CD4 with PerCP-Cy5.5 at the concentrations recommended by the manufacturer for 45 min at room temperature. Cells were collected by centrifugation, washed, resuspended in FACS buffer, and analyzed by flow cytometry. Flow cytometric data of Treg cell subsets were analyzed by the CD4+/Foxp3+/CD25 _high group. Data are expressed as the percentage of pStat5-positive cells in the analyzed population.

This panel of IL-2 variant Fc fusion proteins includes amino acid substitutions that enhance binding (P-0608), decrease binding (P-0626, P-0634, and P-0624), or abolish binding (P-0635) to IL-2Rα. In addition, P-0626, P-0634, and P-0624 exhibited varying levels of attenuation of IL-2Rα binding strength; P-0626, P-0634, and P-0624 exhibited 2.6-, 23-, and 60-fold reductions in binding, respectively. Reflecting the trend and level of IL-2Rα binding modulation, the differential potency of the various IL-2 variant Fc fusion proteins in stimulating STAT5 phosphorylation in CD4+ Treg cells (FIG. 9). Accordingly, P-0608 with enhanced IL-2Rα binding showed a trend toward higher potency than P-0531 in stimulating STAT5 phosphorylation in Tregs. P-0626, P-0624, and P-0634 showed reduced pSTAT5 activity consistent with their reduced IL-2Rα binding. Their preserved, albeit reduced, IL-2Rα binding still resulted in higher Treg activity than P-0635 and the IL-2Rα-ablative reference P-0551. P-0635 and P-0551 have comparable 5-fold log right shifts in pSTAT5 induction activity in Tregs, and this low level of Treg signaling is likely due to the activation of IL-Rβγ expressed on Tregs. Therefore, these mutants are expected to achieve the desired Treg activation at a concentration that also activated CD8+ T and NK cells. Surprisingly, complete abolition of IL-2Rα binding resulted in a greater than 5-log reduction in Treg activity (FIG. 9).

Example 8

Effect of IL-2 mutations introduced into IL-2Rα-interacting regions on interaction with IL-2Rβγ

To investigate whether IL-2 mutations introduced into IL-2Rα interacting regions affect the interaction of IL-2 with IL-2Rβγ, the same panel of IL-2 variant Fc fusion proteins as in Example 7 were assayed in an ELISA for binding to IL-2Rβγ.

Briefly, recombinant IL-2Rβγ heterodimer containing IL-2Rβ ECD (SEQ ID NO:109) fused to the N-terminus of the Fc Hole chain (SEQ ID NO:10) and γc ECD (SEQ ID NO:110) fused to the N-terminus of the Fc Knob chain (SEQ ID NO:9) was coated onto wells of 96-well Nunc Maxisorp microplates at 2 μg per well. After overnight incubation at 4°C and blocking with 1% BSA, 100 μl of 3-fold serial dilutions of IL-2 Fc fusion proteins, starting at 10 nM, were added to each well. After incubation for one hour at room temperature, 100 μl of mouse anti-human IL-2 antibody clone B33-2 (BD Biosiences), 0.5 μg/ml, were added to each well and incubated at room temperature for 1 hour. Then, 100 μl of streptavidin-HRP (1:5000 dilution in diluent) were added to each well and incubated at room temperature for 40 min. After each step, the wells were carefully aspirated and washed three times with PBS/0.05% Tween-20. Finally, 100 μl of TMB substrate were added to each well, the plate was developed at room temperature in the dark for 10 min, and stop solution (2 H sulfuric acid, Ricca Chemical) was added, 100 μl per well. Optical density was determined at 450 nm and curves were plotted using Prism software (GraphPad).

As shown in FIG. 10, compared with the wild-type IL-2 fusion protein P-0531, all representative Fc fusion proteins of IL-2 variants containing mutations that enhance, reduce, or eliminate binding to IL-2Rα showed unchanged binding to IL-2Rβγ. The data obtained confirmed that the analyzed IL-2 mutations introduced into the regions interacting with IL-2Rα indeed interfere only with binding to CD25 and do not affect the interaction with IL-2Rβγ.

A panel of representative IL-2 variant Fc fusion proteins were also characterized for induction of Ki67 expression in human CD8+ T cells and NK cells by flow cytometry. Freshly isolated NK cells and CD8+ T cells express no or very low levels of CD25, and IL-2R signaling is mediated primarily by the βγ subunits of the intermediate-affinity receptor. Ki67 is a nuclear protein used as a marker of cell proliferation.

Briefly, human PBMCs were isolated by Ficoll-Hypaque centrifugation from healthy donor buffy coat. Purified human PBMCs were treated with serial dilutions of IL-2 variant Fc fusion proteins and incubated at 37°C for 5 days. On day 5, cells were washed once with FACS buffer (1% FBS/PBS) and first stained with Fc blocking solution and antibodies to surface markers, anti-human CD56 with FITC, anti-human CD8 with APC. After 30 min of incubation and washing, cell pellets were completely resuspended by adding 1x Foxp3 fixative and permeabilizing working solution, 200 μl/well, and incubated for 30 min at room temperature in the dark. After centrifugation, 200 μl of 1x permeabilization buffer was added to each well for another wash. Cell pellets were resuspended in permeabilization buffer with anti-human Ki67 antibody with PE (1:25 dilution). After 30 min of incubation at room temperature, cells were collected and washed, resuspended in FACS buffer and analyzed by flow cytometry. Data are expressed as % Ki67-positive cells in the analyzed population.

Dose-dependent increases in Ki67 expression in CD8+ T cells and NK cells in response to Fc-fusion proteins of IL-2 variants, compared to P-0531 and P-0551, are shown in FIGS. 11A and 11B. Introduction of mutations that interfere with interaction with CD25 resulted in Fc-fusion constructs with activity comparable to P-0531, a bivalent wild-type IL-2 fusion protein.

In addition, P-0689 and P-0704, monovalent analogs of P-0531 and P-0635, respectively, were characterized for their induction of Ki67 expression in human CD8+ T cells. As shown in FIG. 11C, P-0689 (wild-type IL-2) and P-0704, which contains the P65R mutation that abolishes binding to IL-2Rα, demonstrated similar dose-dependent increases in Ki67 expression in CD8+ T cells. Taken together, the ex vivo functional data further confirmed that IL-2 mutations introduced into IL-2Rα-interacting regions have minimal or no effect on interaction with IL-2Rβγ. Moreover, the differences in activity between P-0531 and P-0689 and between P-0635 and P-0704 were consistent with their differences in IL-2 valence.

Example 9

Introduction of substitutions that disrupt interaction with IL-2Rβ or γc into IL-2 variants with reduced binding to IL-2Rα to attenuate overall activity

A full IL-2 agonist may result in excessive activation of the IL-2 pathway and unwanted toxicity due to target binding outside the target tissue. This may be particularly pronounced in the case of a selective full IL-2Rβγ agonist; due to its increased selectivity and decreased clearance due to CD25 binding, a selective full IL-2Rβγ agonist can stimulate robust CD4+, CD8+ effector T and NK cell responses in vivo. Acute toxicity with severe weight loss may result. Furthermore, cellular exhaustion or cell death due to excessive stimulation may result in loss of response in vivo after repeated administration. It has been hypothesized that reducing overall activity may prevent excessive activation of the IL-2 pathway and reduce unwanted clearance due to target binding; therefore, it could potentially reduce toxicity and improve pharmacokinetics and pharmacodynamics. Therefore, for optimal activity, substitutions modulating the interaction with IL-2Rβγ to attenuate overall activity were introduced into IL-2 variants with reduced/abstracted binding to IL-2Rα. Reduced binding affinity to IL-2Rβγ would also reduce receptor-mediated internalization of IL-2, resulting in slow but persistent receptor activation and prolonged pharmacodynamics compared to wild-type IL-2.

The selection of mutations disrupting interaction with IL-2Rβ or γc was based on the analysis of the IL-2/IL-2R co-crystal structure (PDB code 2B51). Substitution of residues located in or near the interacting regions that directly contact the IL-2Rβ or γc subunits can result in decreased binding to IL-2Rβγ and thus modulate overall activity in pathway activation. For example, D20 is involved in an extensive hydrogen bonding network with the receptor subunit side chains in the IL-2Rβ-interacting regions. Similarly, N88 is an energetically active IL-2/IL-2Rβ interaction site, participating in critical hydrogen bonds with the receptor chain. Q126 is required for interaction with γc. However, amino acid substitutions at the energetic hotspot can result in a significant reduction in activity to a suboptimal level, as exemplified by the various mutations at position D20 (D20E, D20T, D20N, D20Q, D20S) in FIG. 13A. All mutations were introduced into IL-2 P-0250 (SEQ ID:67) and expressed as IL-2 variant Fc fusion proteins. As shown in FIG. 13A, most of the D20 mutations resulted in a significant reduction or abolition of activity upon stimulation of pSTAT5 expression in CD4+ Tconv cells expressing only IL-2Rβγ subunits. Similarly, most of the mutations at position N88 also resulted in abolition of activity upon activation of CD4+ Tconv cells (data not shown).

Therefore, amino acid substitutions were made at the residue at position L19, which only undergoes van der Waals interactions with IL-2Rβ, and the resulting mutants only modulate but do not abolish the functional activity of IL-2. FIGS. 13B and 13C show that IL-2 variants with different mutations at position 19 exhibited a spectrum of activity levels in inducing STAT5 phosphorylation in CD4+ Tconv cells. Compared with the wild type, mutations L19Y, L19R, L19Q resulted in a slight decrease in activity, while L19N and L19H moderately decreased activity. In the case of L19D, the decrease in activity was significant. The different levels of decrease in activity resulting from mutations at position L19 facilitate fine-tuning of activity to obtain optimal activity to reduce toxicity and improve pharmacokinetics and in vivo pharmacokinetics.

In addition, IL-2 variants with amino acid changes at residue Q126, which is required for interaction with γc, were similarly generated. The functional activity of the IL-2 Fc fusion protein Q126E in inducing STAT5 phosphorylation in CD4+ Tconv cells is shown in FIG. 13D. Compared with the wild-type counterpart, Q126E resulted in a slight decrease in activity.

Furthermore, since the interaction with IL-2Rβγ involves mainly the N-terminal amino acids of IL-2, deletion of the N-terminal amino acids was considered as another way to modulate the overall activity. Therefore, N-terminal deletion mutants (deletions of 5, 7, 9 or 11 N-terminal amino acids) were constructed from the L19H/S125I/Q126E variant of IL-2 and analyzed in a human PBMC assay. Since the parent molecule, the L19H/S125I/Q126E variant of IL-2, fully retains binding to IL-2Rα but has reduced binding to IL-2Rβγ, it can be reliably analyzed only using Treg cells, which still allows the effect of the mutations on overall activity to be dissected. In the case of the IL-2 Fc variant with an 11 amino acid deletion, the material obtained was insufficient to characterize it. As shown in FIG. 13E, while deletions of 5 and 7 amino acids fully retained activity, deletion of 9 amino acids resulted in a 25-fold decrease in activity (18 pM versus 0.74 pM). Therefore, it is expected that various IL-2 variants with different activities can be further modified to obtain a desired activity profile by deleting 7, 8, 9, or 10 N-terminal amino acids.

P-0704 was modified with L19H, L19Q, L19Y mutations that disrupt interaction with IL-2Rβ and Q126E mutation that disrupts interaction with γc to generate P-0731, P-0759, P-0761 and P-0732, respectively. P-0704 contains P65R amino acid substitution that results in complete loss of binding to IL-2Rα. P-0731, P-0759, P-0761 and P-0732 were analyzed for binding to IL-2Rβγ in ELISA and for induction of Ki67 expression in human CD8+ T cells, CD4+ T cells and NK cells by flow cytometry in comparison with P-0704.

As shown in FIG. 14A, compared with P-0689 and P-0704, all representative IL-2 variant Fc fusion proteins showed varying levels of reduction in binding to IL-2Rβγ. Since IL-2 binding to either the β- or γ-subunit of the receptor was weak and characterized by a high off-rate, the binding activity for each individual subunit could not be reliably assessed by ELISA (data not shown). However, it was suggested that the reduction in binding to the IL-2Rβγ heterodimer was due to amino acid changes that disrupted the interaction with the corresponding β- or γ-subunit of the receptor.

The reduced activity caused by the IL-2Rβ-impairing L19H substitution in P-0731 and the γc-impairing Q126E mutation in P-0732 was assessed by determining the activity in inducing Ki67 expression in human CD8+ T cells in human PBMC. P-0689, the S125I equivalent of the wild-type monomeric IL-2 Fc fusion protein, and P-0704, which lacked IL-2Rα binding but fully retained affinity and functional activity for the dimeric IL-2Rβγ receptor, were included for comparison. As shown in FIG. 14B, all monomeric IL-2 Fc fusion proteins resulted in an increased percentage of Ki67-positive CD8+ T cells in a dose-dependent manner; P-0731 showed approximately a 30-fold decrease in activity compared to P-0704. P-0732 showed the lowest activity with an EC ₅₀ increase of more than 100-fold compared to P-0704.

Dose-dependent enhancement of human CD8+ T cell, NK cell, and CD4+ T cell proliferation by P-0731, P-759, and P-0761 is shown in FIGS. 15A, 15B, and 15C, respectively. The IL-2 Fc fusion proteins of the P-0731, P-0759, and P-0761 variants contain mutations at position L19 that disrupt interaction with IL-2Rβ, in addition to a P65R substitution that abolishes binding to IL-2Rα in P-0704. Compared to P-0704, all variants demonstrated the expected reduction in activity in human CD8+ T cell, NK cell, and CD4+ T cell proliferation. P-0759 (L19Q) and P-0761 (L19Y) showed a slight decrease in activity by 3-5-fold, while the L19H mutation in P-0731 resulted in a more significant 30-fold decrease in activity. The degree of attenuation of activity by L19Q and L19H substitutions showed the same trend in all cell subsets analyzed and was consistent with the degree of decrease in activity in inducing pSTAT5 expression in CD4+ Tconv cells (FIGS. 13B and 13C) and the degree of attenuation of binding to recombinant IL-2Rβγ protein (FIG. 14A). The reference molecule showed comparable, but slightly reduced, activity in inducing cell proliferation compared to P-0704.

In conclusion, in addition to introducing a CD25-impairing substitution in IL-2 to curb unwanted expansion of immunosuppressive Tregs, it may be possible to additionally introduce substitutions or N-terminal deletions that disrupt interaction with IL-2Rβγ to attenuate overall activity to ensure optimal activity. Reducing activity may prevent excessive activation of the IL-2 pathway and reduce unwanted clearance due to target binding; therefore, it may potentially reduce toxicity and improve pharmacokinetics and pharmacodynamics.

Example 10

Pharmacodynamic effects of Fc-fusion proteins of IL-2 variants in mice after a single injection

The dynamics of cell number growth in different lymphocyte subsets after administration of P-0704 (SEQ ID NOs:96 and 10), a C-terminal monovalent Fc fusion protein of an IL-2 variant with ablated binding to IL-2Rα, was assessed in Balb/C mice after a single injection. The effect on the growth of peripheral blood lymphocytes was monitored over time. In addition, the immunopharmacodynamic profiles of P-0704 and P-0689 (SEQ ID NOs:107 and 10), an analogue of wild-type IL-2, were compared.

Seven-week-old female Balb/c mice were obtained from Charles River Laboratory and acclimated at our own research facility for at least 7 days prior to testing. Vehicle, P-0704, and P-0689 at 0.6 mg/kg were injected intraperitoneally once on day 0. Blood samples were collected on days 3 and 5 post-injection. Each group included 4 mice.

For immune phenotyping, heparinized whole blood was used. After lysis of red blood cells using BD Pharm lysis buffer, the total number of viable blood mononuclear cells was calculated by exclusion of nonviable cells using trypan blue, followed by intracellular Ki67 staining. Cell pellets were completely resuspended by adding 1x Foxp3 fixative/permeabilization working solution, 200 μl per well, and incubated for 30 min at room temperature in the dark. After centrifugation, 200 μl of 1x permeabilization buffer was added to each well for another wash. After Fc receptor blocking with purified anti-mouse CD16/CD32 antibody (1:50 dilution), cells were stained with APC-cy7 CD3, BV510 CD4, FITC Foxp3, PE Ki67, APC CD335, and Percpcy5.5 CD8 (1:50 dilution). After 30 min of incubation, cells were collected and washed, resuspended in FACS buffer, and analyzed by flow cytometry.

As shown in FIG. 16A, wild-type IL-2 in P-0689 resulted in a marked increase in Treg cell numbers (6-fold increase in cell counts) peaking at day 3, which is undesirable for cancer treatment, while P-0704 did not result in an increase in Treg cell numbers at day 3 and resulted in only a minimal increase in Treg cell numbers at day 5. In contrast, P-0704 increased the percentage of CD8+ T cells in the total CD3+ lymphocyte population at day 3 and continued to increase the CD8 population at day 5 from 19% (baseline) to 67% (FIG. 16B). In contrast, P-0689 resulted in a minimal increase in CD8+ T cell numbers (FIG. 16B). Regarding NK cells, a 5.4-fold increase in cell count was observed at day 3 after P-0704 administration, which continued to result in a 64-fold increase in cell count at day 5. P-0689 increased NK cell count by 7.8-fold at day 3, but this effect quickly disappeared, returning to baseline values at day 5 (FIG. 16C).

In conclusion, P-0704 demonstrated almost complete abolition of Treg expansion and significantly enhanced CD8 and NK cell expansion, a cell expansion profile that is significantly different from P-0689. This observation is consistent with the significant difference in their IL-2Rα subunit binding affinity and, therefore, Treg cell responsiveness. Furthermore, as a full IL-2Rβγ-selective agonist, P-0704 can stimulate robust CD8+ effector T and NK cell responses in vivo due to its enhanced selectivity and reduced clearance mediated by CD25 binding.

Example 11

Construction, expression and purification of IL-2 antibody fusion proteins

In this example, various IL-2 antibody fusion proteins were generated and analyzed. Linking IL-2 variants to immune checkpoint-targeting antibodies is expected to direct IL-2 to exhausted T cells and immunologically reactivate the tumor microenvironment. This strategy also reduces systemic IL-2 exposure and off-target toxicity. Bifunctional fusion proteins of immune checkpoint inhibitors with IL-2 variants are also expected to provide synergy by eliminating downregulation, reactivating T cell function, and expanding T cell numbers. Cytokine fusion proteins with immune checkpoint blocking antibodies are expected to further enhance the immune system's antitumor activity. The present inventors propose that the use of IL-2 variants with reduced/abstracted binding to IL-2Rα and weakened activity against IL-2Rβγ should help establish a stoichiometric balance between the cytokine and antibody portions, which exhibit significantly different activities and molecular weights, allowing for optimal dose determination and maintenance of the function of each portion. In addition, attenuation of cytokine activity is expected to minimize peripheral activation, reduce clearance due to antigen binding, and promote tumor targeting via the antibody portion.

Because checkpoint inhibitor targets such as PD-1 are expressed on cytotoxic T cells or other lymphocyte subsets that also express IL-2Rβγ, IL-2 fusion proteins with an anti-PD-1 antibody would be expected to deliver the IL-2 variant preferentially to PD-1+ cells such as activated and exhausted CD8+ T cells in the tumor microenvironment, facilitating selective signaling.

Following this concept, various IL-2 antibody fusion proteins were constructed.

To obtain IL-2 fusion proteins with an antibody, the CH1-CH2-CH3 domain (residues 118-447 of the antibody according to EU numbering) of the heavy chains of the above antibodies was replaced with the IgG1 sequence presented in SEQ ID NO: 162, containing the mutations L234A, L235A, G237A to eliminate binding to FcγR and C1q, but retain binding to FcRn or PK (pharmakinetics). The peptide of the IL-2 variant was fused via a peptide linker, the sequence of which is given in Table 6, to the C-terminus of the Fc domain. Alternatively, to express a monovalent IL-2 variant, the CH1-CH2-CH3 domain of the heavy chains of the above antibodies was replaced with the heterodimeric chains presented in SEQ ID NO: 163-164. The IL-2 variant peptide is fused via a peptide linker, the sequence of which is shown in Table 6, to the C-terminus of a heterodimeric heavy chain containing a knob-type modification constructed using knob-into-hole technology. Mutations that increase half-life, such as N434α, may also be included in the homodimeric or heterodimeric Fc chains. Exemplary IL-2 fusion proteins with a PD-1 antagonist antibody are shown in Table 12. In addition, P-0844 is a reference IL-2 variant fusion protein with a PD-1 antagonist antibody comprising SEQ ID NOs:182-184.

Gene synthesis, construction of expression vectors, and production, purification, and characterization of proteins were performed following the same procedures as described in detail in Example 1.

Fusion proteins of IL-2 variants with a surrogate anti-mouse PD-1 antibody were similarly prepared for use in in vivo tumor models in immunocompetent mice. The surrogate anti-mouse PD-1 antibody contains SEQ ID NOs:185-187 with Fc mutations to abolish effector function and to allow heterodimerization; the IL-2 variant was fused to the C-terminus of HC chain 2 of the anti-mouse PD-1 antibody (SEQ ID NO:186) via a (G ₄ S) ₃ linker (SEQ ID NO:15). Table 13 lists the IL-2 variants in each representative fusion protein of IL-2 variants with a surrogate anti-mouse PD-1 antibody.

Example 12

Fusion proteins of IL-2 variants with antibody fully retain IL-2 activity and activity profiles in ex vivo functional assays

The murine PD-1 antagonist surrogate antibody (SEQ ID NOs:185-187) used in this study does not cross-react with human antigen; therefore, it was used as a non-functional antibody in human cells to assess the impact of the antibody fusion protein format on the activity and activity profile of IL-2 variants in stimulating and proliferating lymphocyte subsets.

The effect of the antibody fusion protein format was examined using P-0782 in comparison to its Fc fusion counterpart P-0704. Both P-0782 and P-0704 contain the monomeric P65R variant of IL-2 linked to the C-terminus of the heterodimeric Fc domain via a flexible linker ( _G3S ) ₂ (SEQ ID NO:18). Substitution of P65R in IL-2 abolished binding to IL-2Rα (FIG. 7D). As shown in FIGS. 17A-17C, P-0782 and P-0704 were equivalent in inducing dose-dependent STAT5 phosphorylation in CD4+ Treg cells (FIG. 17A), CD8+ T cells (FIG. 17B), and NK cells (FIG. 17C). The obtained data confirmed that the IL-2 moiety fused to the antibody fully retains its activity as in its corresponding Fc-fusion protein.

In addition, three IL-2 variant fusion proteins with anti-mouse PD-1 antibody, P-0837, P-0838, and P-0782, were compared for their activity in stimulating pSTAT5 in human PBMCs. The IL-2 mutations in P-0838 and P-0782 are P65Q and P65R, respectively. P-0837 contains the wild-type IL-2 moiety (SEQ ID NO:4). Compared to the wild type, P65Q reduced the binding strength to IL-2Rα by 43-fold (Table 10), and P65R abolished binding to IL-2Rα. Confirming the data obtained using IL-2 Fc fusion molecules in the previous examples, FIG. 18A-18C show that IL-2 mutations introduced into the regions interacting with IL-2Rα indeed only interfere with binding to CD25 and do not affect interaction with IL-2Rβγ. Since antigen-naive human PBMC CD8+ T and NK cells express CD25 at very low levels, all three molecules exhibit identical activity in dose-dependently stimulating pSTAT5 expression in these two lymphocyte subsets (FIGS. 18B and 18C). In contrast, Treg cells constitutively express CD25 at high levels, and therefore P-0838 and P-0782 showed a significantly reduced response to stimulating pSTAT5 expression in Treg cells compared to their wild-type counterpart P-0837 (FIG. 18A); P-0837, P-0838, and P-0782 had _EC50 values of 0.45 pM, 0.36 nM (800-fold weaker than P-0837), and 4.5 nM (10,000-fold weaker than P-0837), respectively. Both the CD25-depleted mutant P-0838 and the CD25-deficient mutant P-0782 retained similar activity in stimulating CD8 and NK cells as their wild-type counterpart. Furthermore, ablation of IL-2Rα binding in P-0782 resulted in an _EC50 Treg/CD8 ratio of approximately 1, indicating no preferential stimulation of Tregs over cytotoxic effector cells ( _EC50 4.5 nM for Tregs vs. 4.6 nM for CD8+ T cells). The presence of IL-2Rα binding, albeit significantly attenuated, in P-0838 resulted in a 13-fold enhancement of pSTAT5 responsiveness of Tregs compared to CD8+ T cells (0.36 nM for Tregs vs. 4.6 nM for CD8+ T cells).

In functional assays of the ex vivo IL-2 antibody fusion protein format, IL-2 variants with reduced activity and L19Q and L19H mutations that disrupt interaction with IL-2Rβ, in addition to reduced binding to IL-2Rα, were also studied. Compared to P-0782, P-0786 contains one additional L19Q substitution and P-0783 contains L19H. The Fc analogs of P-0782, P-0786, and P-0783 are P-0704, P-0759, and P-0731, respectively.

The dose-dependent induction of STAT5 phosphorylation by P-0782, P-0786, and P-0783 in human CD8+ T cells and NK cells is shown in FIGS. 19A and 19B, respectively; the dose-dependent enhancement of proliferation of the same lymphocyte subsets is shown in FIGS. 19C and 19D, respectively. Compared with P-0782, P-0786 showed a slight 2- to 3-fold decrease in activity in inducing STAT5 phosphorylation in CD8+ T cells (FIG. 19A) and NK cells (FIG. 19B), while the L19H mutation in P-0783 resulted in a more pronounced 20- to 30-fold decrease in activity (FIGS. 19A and 19B). A similar level of attenuation was observed in the dose-dependent increase in Ki67 in CD8+ T cells (FIG. 19C) and NK cells (FIG. 19D). The level of attenuation for the antibody fusion proteins P-0782, P-0786, and P-0783 showed the same trend as their corresponding Fc fusion proteins P-0704, P-0759, and P-0731, respectively (FIG. 15A and 15B).

Attenuation of activity by mutations that disrupt interaction with IL-2Rβ was also assessed in the context of the P65Q mutation in the IL-2 antibody fusion protein format. L19Q and L19H were introduced into P-0838 to generate P-0790 and P-0787, respectively. FIGS. 20A, 20B, and 20C show their activity in stimulating STAT5 phosphorylation in Tregs, CD8+ T, and NK cells. FIGS. 20D and 20E show a dose-dependent increase in the proliferation marker Ki67 in CD8+ T cells and NK cells. The attenuation levels showed the same trend as the antibody fusion proteins based on the P65R mutation that abolishes interaction with IL-2Rα.

P-0782, P-0786, and P-0783 were further analyzed for CTLL-2 proliferation activity along with P-0837 containing the S125I equivalent of wild-type IL-2. CTLL-2 cells are cytotoxic T cells derived from C57BL/6 mice and express the α-, β-, and γ-subunits of the receptor. Briefly, CTLL-2 cells were harvested, washed, resuspended in medium (RPMI1640, 10% FCS, 2 mM glutamine) without IL-2, and incubated for two hours (without IL-2). Subsequently, 50 μl of CTLL-2 cells resuspended at 50,000/ml in fresh IL-2-free medium were transferred to a 96-well U-bottom plate. Fifty μl of a serial dilution of the IL-2 antibody fusion protein was added to each well to give a final volume of 100 μl per well. Samples were incubated for 2 days and proliferation was assessed using CellTiter-Glo following the manufacturer's instructions and measuring luminescent signals. As shown in FIG. 21, the levels of attenuation mediated by L19Q and L19H were maintained when murine cells were used. P-0837, containing wild-type IL-2, showed significantly greater growth than P-0782 because CTLL-2 cells express the IL-2Rα subunit, similar to Treg cells.

In conclusion, in ex vivo functional assays, IL-2 variants in fusion protein format with a surrogate antibody to mouse PD-1 fully retained the activity and activity profiles observed for their Fc-fusion counterparts.

Example 13

In vitro characterization of IL-2 variant fusion proteins with anti-human PD-1 antibody

P-0795 is a human PD-1 antagonist antibody comprising SEQ ID NO: 140 as the heavy chain and SEQ ID NO: 141 as the light chain. P-0803 (SEQ ID NOS: 166, 169, and 141) is an immunoconjugate of P-0795 with a variant IL-2 fused to the C-terminus of a heterodimeric heavy chain containing a knob modification. The IL-2 variant in P-0803 contains a P65R mutation that abolishes binding to IL-2Rα and an S125I substitution that expands development options. The function of the antibody portion of the fusion protein with the antibody in P-0803 was analyzed in both a direct binding ELISA and a competitive inhibition of ligand binding ELISA.

For the direct binding assay, the same ELISA protocol was followed as in Example 4 using huPD-1-His as the adsorbed antigen. For the competitive inhibition of ligand binding (PD-L1) ELISA, a similar ELISA protocol was used with some modifications. Briefly, human PD-1 protein with Fc, 0.2 μg/well, was adsorbed onto the plate at 4°C overnight. After washing and blocking with 2% BSA, biotinylated human PD-L1 with Fc, 0.5 μg/mL, was mixed with serially diluted P-0795 or P-0803, 1:1 (v/v); 100 μL of the mixture was added to each well and incubated at 37°C for 1 hour. Streptavidin-HRP was added as a secondary antibody.

As shown in FIG. 22A, P-0803 and P-0795 had similar binding potency to PD-1 ( _EC50 of 0.6 nM). P-0803 and P-0795 also had similar potency in blocking human PD-1 binding to surface-immobilized PD-L1 ( _IC50 of 2.1 nM; FIG. 22B). Taken together, these data confirmed the full functionality of the antibody portion of the IL-2 antibody fusion protein.

Similarly, in FACS analysis, the IL-2 variant with anti-human PD-1 antibody demonstrated binding to cell-surface expressed PD-1 similar to the parent antibody (FIG. 22C). P-0795 is an anti-human PD-1 antibody, and both P-0880 and P-0885 contain monovalent IL-2 attached to the C-terminus of P-0795 via a ( _G4S ) ₃ linker. P-0880 contains P65R/S125I substitutions, while P-0885 contains L19Q/P65R/S125I mutations. P-0704 and P-0759 are Fc-fusion analogs of P-0880 and P-0885, respectively. As expected, due to the lack of a PD-1 targeting moiety, P-0704 and P-0759 did not bind to PD-1 expressing cells.

Because PD-1 binds to the PD-1 checkpoint inhibitor, the immunoconjugate is expected to deliver the IL-2 variant preferentially to PD-1+ cells, such as activated and exhausted CD8+ T cells in the tumor microenvironment, facilitating selective signaling. In healthy human PBMCs, CD8+ T cells and NK cells are typically PD-1 negative, while Tregs constitutively express PD-1 at low levels. Consequently, the IL-2 fusion proteins with the anti-huPD-1 antibody P-0803 and P-0804 were found to stimulate pSTAT5 in PD-1-positive T cells more than 15-fold more than their non-PD-1-targeted counterparts, P-0782 and P-0783, respectively (FIGS. 23A and 23B); However, in the case of PD-1-negative cells, differences in activity were minimal or insignificant (FIGS. 23C-23F). Fusion proteins with the huPD-1 antibody also showed a trend toward increased activity compared to their non-PD-1-targeted counterparts in non-activated CD8 and NK cells not exposed to antigen (FIGS. 23C-23F).

Higher levels of PD-1 expression on T cells are expected to increase the likelihood of targeting antibody-fusion proteins to achieve selective signaling. Therefore, in the tumor microenvironment, IL-2-PD-1 antibody-fusion proteins will preferentially bind to Teff rather than Tregs.

In addition, the effect of the length of the linker connecting the heavy chain of the antibody with a knob modification and the IL-2 variant on the expression profile and activity of the protein was studied. P-0840 (SEQ ID NOS: 168, 169, and 141) and P-0841 (SEQ ID NOS: 178, 175, and 141) are two IL-2 fusion proteins of P-0795 that differ only in the length of the linker. P-0840 contains a (G ₃ S) ₂ linker (SEQ ID NO: 18), while P-0841 has a (G ₄ S) ₃ linker (SEQ ID NO: 15). As shown in FIG. 24A and 24B, P-0841 purified on Protein A, produced by transient expression in ExpiCHO, showed significantly lower levels of low molecular weight impurities than P-0840 produced and purified by the same methods (16% vs. 3%). Similar differences in impurity levels were noted for P-0803 and P-0880 (SEQ ID NOS: 177, 175, and 141) (11% vs. 2.7%; FIGS. 24C and 24D) with ( _G3S ) ₂ and ( _G4S ) ₃ linkers, respectively, and otherwise identical sequences.

While the slightly longer linker in P-0841 and P-0880 resulted in increased purity compared to their respective shorter linker containing counterparts, the impact on the biological activity of the IL-2 moiety, as exemplified by pSTAT5 stimulating activity in cytotoxic lymphocytes, was either minimal or represented a borderline enhancement (FIG. 25). Given the positive impact on the design capability profile of the fusion proteins, a longer linker that does not otherwise have a negative impact is preferable to a short linker.

Several fusion proteins were constructed with P-0795 IL-2 variants fused to the C-terminus of a heterodimeric heavy chain containing a knob modification via a ( _G4S ) ₃ linker, P-0880, P-0882 (SEQ ID NOS: 176, 175, and 141), and P-0885 (SEQ ID NOS: 179, 175, and 141). As shown in FIG. 22C, the binding of the IL-2 variant fusion proteins to the anti-huPD-1 antibody and the longer linker to cell-surface expressed PD-1 was not altered compared to the anti-hPD-1 antibody alone. They were further analyzed in ex vivo functional assays to examine IL-2 activity in stimulating pSTAT5 and inducing Ki67 expression in CD8+ T cells and NK cells (FIG. 26). All three constructs contain the P65R mutation, abolishing interaction with IL-2Rα, while P-0882 and P-0885 contain additional L19H and L19Q mutations, respectively, to modulate overall activity. P-0849, a wild-type IL-2 analog, was included for comparison. A summary of the ex vivo functional activity data is presented in Table 14. The level of attenuation of activity for P-0885 and P-0882 compared to P-0880 showed the same trend across all cell subsets analyzed and was consistent with the level of attenuation observed for P-0759 and P-0731 compared to P-0704 (respective Fc fusion proteins; FIGS. 15A and 15B) and for P-0786 and P-0783 compared to P-0782 (respective anti-mouse PD-1 fusion proteins; FIG. 19). As expected, in CD8+ T and NK cells, the wild-type IL-2 fusion protein demonstrated activity comparable to P-0880.

Example 14

Pharmacodynamic effect of fusion proteins of IL-2 variants with a surrogate antibody to mouse PD-1 in C57BL6 mice

The pharmacodynamic effect of the IL-2 variant fusion proteins with anti-mouse PD-1 antibody was assessed in C57BL6 mice following a single injection. Seven-week-old female C57BL6 mice were obtained from Charles River Laboratory and acclimated at our own research facility for at least 7 days prior to testing. Vehicle and each IL-2 fusion protein with anti-mouse PD-1 antibody were injected intraperitoneally into mice once at time 0. Blood samples were collected at days 3, 5, 7, and 10 post-injection. Each group included 5 mice. Heparinized whole blood was used for immune phenotyping as described in Example 10.

P-0782 contains the IL-2 P65R moiety that abolished binding to IL-2Rα, P-0838 contains the IL-2 P65Q moiety that reduced binding to IL-2Rα, while P-0837 contains wild-type IL-2. P-0781, a similar fusion protein with an antibody to mouse PD-1 containing a reference variant of IL-2 (SEQ ID NO:188) that completely lacks binding to IL-2Rα, was included for comparison.

Following a single injection of 2 mg/kg, Ki67 stimulation in CD8 and NK cells was maximal for all compounds analyzed (FIGS. 27A–27B). In CD8+ T cells, Ki67 expression signals for each compound peaked at day 3. For P-0782, P-0838, and the reference P-0781, the signal persisted until day 7 and declined at day 10. In comparison, the Ki67 signal declined more rapidly for wild-type P-0837 (FIG. 27A). Similar Ki67 induction in NK cells was observed for all compounds tested (FIG. 27B).

The increases in CD8 and NK cells unexpectedly demonstrated significant differences between the compounds tested. P-0782, which has a mutation that abolishes binding to IL-2Rα, demonstrated robust increases in CD8+ T (FIG. 27C) and NK (FIG. 27D) cells. The increases in both lymphocyte subsets began at day 3 and continued to peak at day 7 with a 68-fold increase in CD8+ T cells and a 182-fold increase in NK cells. P-0838, which has a mutation that reduces binding to IL-2Rα, demonstrated increases in CD8 and NK cells similar to or slightly greater than the wild-type antibody fusion protein. The reference P-0781 demonstrated intermediate increases in both lymphocyte subsets compared to P-0780 and P-0838. In contrast, the increase in both lymphocyte subsets in response to wild-type P-0837 peaked at day 5 with significantly lower maximal signal (3.9-fold increase for CD8+ T cells and 6.8-fold increase for NK cells; FIGS. 27C and 27D).

It is possible that the mutation that abolishes CD25 binding may reduce the clearance effect of CD25 binding and thereby increase the availability of IL-2Rβγ. Increased receptor occupancy results in a robust expansion of cytotoxic cells. Mutants with residual CD25 binding activity may still be susceptible to the clearance effect of CD25 binding, resulting in wild-type-like activity on CD8 and NK cells. In conclusion, P-0782 had a strikingly different cell expansion profile than P-0838 and P-0837. P-0782 demonstrated significant proliferation and expansion of CD8+ T and NK cells compared to any compound tested, outperforming the reference compound P-0781. As full IL-2Rβγ-selective agonists, P-0782 and P-0781 can stimulate robust CD8+ effector T and NK cell responses in vivo due to enhanced selectivity and reduced clearance mediated by CD25 binding. Although P-0838 did not demonstrate robust expansion of CD8 and NK cells compared to wild type, the mutation introduced to reduce IL-2Rα (CD25) binding is expected to provide the benefit of reduced VLS. Additionally, residual immunoregulatory Treg responses may act as an immune counterbalance, enhancing systemic tolerability and preventing excessive immune bias towards cytotoxic effector cells. Fine-tuning of the Treg response may be possible without compromising tumor cell killing efficiency, yet maintaining sufficient potency to support peripheral tolerance.

The pharmacodynamics of bifunctional fusion proteins with an anti-PD-1 antibody and IL-2 variants containing mutations to reduce interaction with IL-2Rβγ, in addition to eliminating the ability to bind to IL-2Rα, were also analyzed. Both P-0786 and P-0783 are analogs of P-0782 where IL-2 activity is attenuated by the introduction of different mutations L19Q and L19H that modulate interaction with IL-2Rβ, respectively. FIGS. 19C and 19D show the differences in the activity of these three compounds in stimulating Ki67 expression in vitro. The effect of P-0786 and P-0783 on CD8+ and NK cell proliferation and expansion at two different dose levels is shown in FIGS. 28 and 29. As shown in FIG. 28A, the less potent compound, P-0786, resulted in a peak Ki67 signal in CD8+ T cells at day 5, but not at day 3 like wild-type P-0837. The greatest increase in Ki67 in NK cells was seen with both P-0783 and P-0837 (FIG. 28B), consistent with the finding that NK cells are more sensitive to IL-2 than CD8+ T cells.

The pharmacodynamic effect of the attenuated IL-2 fusion proteins with anti-PD-1 antibody was significantly superior to that of the wild-type fusion protein. FIGS. 28C and 28D show a significantly longer and enhanced dose-dependent effect of P-0786 on cell expansion compared to wild-type. The enhancement of CD8+ T and NK cell expansion was delayed but robust and long-lasting. The response in the 2 mg/kg group peaked at day 7 without returning to baseline at day 10, while the response in the 5 mg/kg group increased rapidly and continuously without peaking at day 10 post-dose. In contrast, the increase in CD8 and NK cells in the wild-type fusion protein group was marginal, peaking at day 5 and returning to baseline at day 7 (FIGS. 28C and 28D).

P-0783, which contains an even weaker IL-2 agonist, demonstrated a similar delayed but persistent and long-lasting effect as P-0786 in inducing Ki67 expression (FIGS. 29A and 29B) and dose-dependent increases in CD8+ and NK cell counts (FIGS. 29C and 29D). Summary data on days of peak cell counts and fold increases in cell counts for each compound are presented in Table 15.

Furthermore, as shown in FIG. 30, the level of activity and the corresponding increase in cytotoxic lymphocytes correlated with toxicity, as reflected by the decrease in body weight of the mice. As a full IL-2Rβγ-selective agonist, P-0782 resulted in a significant increase in CD8+ T and NK cells and the greatest decrease in body weight; the attenuated agonists P-0786 and P-0783 demonstrated better tolerability in vivo. P-0783 was slightly better tolerated than P-0786, consistent with the fact that P-0783 is a weaker agonist than P-0786.

In conclusion, P-0782 demonstrated potent pharmacodynamic effects on CD8+ T and NK cell proliferation and expansion. P-0786 and P-0783 showed attenuated but more robust signals. The ex vivo and in vivo potencies of the three compounds were generally consistent. Furthermore, the attenuated compounds P-0786 and P-0783 demonstrated improved pharmacodynamics and tolerability in vivo compared to the full agonist P-0782.

Example 15

In vivo efficacy of IL-2 variant fusion proteins with anti-PD-1 antibody in syngeneic mouse tumor models

The antitumor efficacy of fusion proteins of IL-2 variants with anti-mouse PD-1 antibody was analyzed in a subcutaneous murine melanoma model B16F10. Female C57BL/6 mice (7 weeks old) were randomized into treatment groups (n = 10 per group) by body weight after 4-7 days of acclimatization. B16F10 cells after passage 3 (5× ¹⁰⁵ cells per mouse) were injected subcutaneously (s.c.) into the right flank of mice on day -1. The compounds to be studied were injected intraperitoneally (i.p.) three times on days 0, 7, and 14 (once every 7 days, Q7D). All mice were carefully observed, measuring body weight three times a week. Tumors were measured three times a week using standard calipers, and tumor size was calculated using the standard formula: length × width (m ² ) × 0.5 in mm ³ . Mice were sacrificed when tumor size exceeded 1500 mm ³ .

Three antibody fusion proteins, P-0838, P-0790, and P-0787, were administered twice at a dose of 3 mg/kg once every 7 days. All three fusion proteins contain the L65Q mutation in IL-2 to reduce binding to IL-2Rα; P-0790 and P-0787 contain additional L19Q and L19H mutations, respectively, to further reduce activity against IL-2Rβγ and modulate overall activity. As shown in FIG. 31A, all compounds demonstrated potent antitumor efficacy as monotherapy with tumor growth inhibition of 78%, 64%, and 57% for P-0787, P-0790, and P-0838, respectively. The level of tumor inhibition efficacy correlated with the decrease in in vitro activity from P-0838 to P-0790 to P-0787.

Similar to FIG. 30, FIG. 31B shows that the full IL-2 agonist, P-0838, resulted in the earliest and most pronounced toxicity, as reflected by the greatest loss in body weight, and the attenuated agonists, P-0790 and P-0787, demonstrated better tolerability in vivo. P-0787 was slightly better tolerated than P-0790, consistent with the fact that P-0783 is a weaker agonist than P-0786. Overall, these data indicate that the IL-2Rβγ-selective and attenuated mutants exhibit good tumor cell killing efficacy and better tolerability.

In the case of P-0787, an additional dose-effect study on tumor inhibition and tolerability in vivo was conducted. As shown in FIG. 32A, P-0787 demonstrated similar antitumor effects when the dose was increased from 3 mg/kg to 5 mg/kg. Dose escalation did not result in significant reduction in body weight (FIG. 32B), indicating that the weak agonist facilitated high dose administration and improved tolerability.

Strong tumor growth inhibition was also observed with P-0782 and P-0786 administered twice daily at 1.5 mg/kg once every 7 days (FIG. 33). P-0722, a surrogate antibody to murine PD-1, failed to demonstrate antitumor activity in the syngeneic B16F10 model, whereas P-0782 and P-0786 demonstrated comparable strong tumor growth inhibition, despite P-0786 being an attenuated analogue of P-0782.

Finally, the IL-2 variant antibody fusion protein, P-0790, was studied in the B16F10 murine lung metastasis model. Briefly, 3× ¹⁰⁵ murine melanoma cells were injected intravenously into female B57BL6 mice (10–12 weeks old). On the following day (day 1), the test protein was administered intraperitoneally three times, once every 7 days. The treatment groups (n = 5 per group) included P-0790 at doses of 0.3, 1, and 3 mg/kg and the corresponding antibody P-0722 at a dose of 3 mg/kg. Vehicle (PBS) was included as a negative control. On day 24, all mice were sacrificed for tissue collection. Tumor nodules in the lungs were counted and the antimetastatic effect was presented as the different number of tumor nodules in different administration groups and in the control group, which was the vehicle.

P-0790 is an IL-2 L19Q/P65Q fusion protein with an anti-PD-1 antibody with significantly reduced binding to IL-2Rα and modulated overall activity. Similarly, P-0722, a surrogate antibody to murine PD-1, failed to effectively inhibit B16F10 tumor cell metastasis, while P-0790 resulted in dose-dependent inhibition of metastatic lung nodules. FIG. 34A shows the mean number of lung nodules, and FIG. 34B shows images of lungs from representative animals in each group. Data are presented as mean plus/minus SEM.

In conclusion, various fusion proteins of IL-2 variants with anti-mouse PD-1 antibody demonstrated potent antitumor effects as monotherapy. Attenuated IL-2 agonists demonstrated effective tumor growth inhibition and better tolerability, allowing higher doses to be used for increased efficacy.

Example 16

Evaluation of pharmacodynamics/pharmacokinetics and safety of selected fusion proteins of IL-2 variants with an anti-PD-1 antibody in cynomolgus monkeys

The PK/PD properties and safety of selected fusion proteins of IL-2 variants with an anti-PD-1 antibody will be assessed in cynomolgus monkeys. Drug-naive cynomolgus monkeys will be acclimated and trained for 2-3 weeks and randomized to one monkey per group with a baseline week before dosing. On day +1, one group will receive intravenous vehicle (PBS) and the other groups will receive intravenous dosing of the various test compounds.

Blood sampling is performed on days -3, 2, 4, 6, 8, 10, 12, 15. Peripheral blood mononuclear cells (PBMCs) are isolated from whole blood of monkeys and used for immunophenotyping of Tregs, non-regulatory CD4+ T cells, CD8+ T cells, CD8+ central memory T cells, CD8+ effector memory cells, CD8+ T cells not contacted with antigens, and peripheral blood NK cells by FACS to determine pharmacodynamics. Cell activation and proliferation will also be monitored by measuring CD25 and Ki67. Whole blood is also used for clinical blood count (CBC), determining 5 parameters: neutrophils, lymphocytes, monocytes, eosinophils, and basophils.

The PK properties of selected IL-2 variant fusion proteins with anti-PD-1 antibody will be assessed in cynomolgus monkey plasma samples by detecting full-length intact molecules using mouse anti-human IL-2 antibody (BD Pharmingen) for adsorption onto 96-well plates to capture the fusion proteins. Mouse anti-human IL-2 antibody with biotin (in-house) will be used for detection and then plasma concentrations of the test compounds will be quantified. In addition to plasma samples obtained on days -3, 2, 3, 4, 5, 6, 8, 10, 15, four more plasma samples were obtained on day 1 at 10 min, 1 h, 4 h and 8 h after administration of selected IL-2 variant fusion proteins with anti-PD-1 antibody.

Plasma samples obtained on days -7, 8, and 15 will also be used to assess the following biochemical parameters: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase, albumin, total bilirubin, creatinine, blood urea nitrogen, and C-reactive protein.

In addition, weekly monitoring of body weight of each animal will be conducted throughout the study period. Body temperature and blood pressure will be monitored on day -1 (before administration) and 6 hours, 24 hours, 96 hours and 168 hours after administration of the drugs.

In light of the present invention, all articles and methods disclosed and claimed herein can be made and carried out without undue experimentation. Although the articles and methods of the present invention have been described by way of preferred embodiments, it will be apparent to those skilled in the art that these articles and methods can be modified without departing from the spirit and scope of the invention. It is intended that all such modifications and equivalents apparent to those skilled in the art, whether existing at the time of filing of this application or later developed, be included within the spirit and scope of the invention as defined by the appended claims. All patents, patent applications, and publications mentioned in this specification are indicative of the level of those skilled in the art to which the invention pertains. All patents, patent applications, and publications are herein incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. The invention illustratively described herein may suitably be practiced in the absence of any element(s) not specifically disclosed herein. It should therefore be understood that, although the present invention has been particularly disclosed through preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may occur to those skilled in the art and that such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

List of sequences

The nucleic acid sequences and amino acid sequences provided in the appended sequence listing are shown using standard nucleotide base abbreviations and single-letter amino acid codes as defined in 37 C.F.R. 1.822.

SEQ ID NO: 1 is the amino acid sequence of human IL-2 precursor.

SEQ ID NO: 2 is the naturally occurring amino acid sequence of the mature form of human IL-2.

SEQ ID NO: 3 is the amino acid sequence of the mature form of wild-type human IL-2.

SEQ ID NO: 2 is the amino acid sequence of the mature form of human IL-2 containing the S125I substitution to enhance the design of fusion proteins.

SEQIDNO: 5 is the amino acid sequence of the extracellular domain of human IL-2Rα.

SEQ ID NO: 6 is the amino acid sequence of Fc of human IgG1.

SEQ ID NO: 7 is the Fc sequence of human IgG1 with reduced/abstracted effector function.

SEQ ID NO: 8 is the Fc sequence of human IgG1 with reduced/abstracted effector function and increased half-life.

SEQ ID NO: 9 is the amino acid sequence of Knob-Fc with reduced/abstracted effector function.

SEQ ID NO: 10 is the amino acid sequence of Hole-Fc with reduced/abstracted effector function.

SEQ ID NO: 11-30 are the amino acid sequences of various peptide linkers.

SEQ ID NO: 31-66 represent the amino acid sequences of different IL-2 variants with amino acid substitutions introduced in the region interacting with the IL-2 receptor alpha subunit.

SEQ ID NOs: 67-107 are the amino acid sequences of Fc fusion proteins of various IL-2 variants.

SEQ ID NO: 108 is the amino acid sequence of the Fc fusion protein of the reference variant IL-2.

SEQ ID NO: 109 is the amino acid sequence of the extracellular domain of human IL-2Rβ.

SEQ ID NO: 110 is the amino acid sequence of the extracellular domain of human cyn.

SEQ ID NO: 111-120 are the amino acid sequences of different IL-2 variants.

SEQ ID NOs: 121-133 are the amino acid sequences of Fc fusion proteins of various IL-2 variants.

SEQ ID NO: 134 is the amino acid sequence of Knob-Fc with reduced/abstracted effector function and increased half-life.

SEQ ID NO: 135 is the amino acid sequence of Hole-Fc with reduced/abstracted effector function and increased half-life.

SEQ ID NO: 136-137 are the amino acid sequences of the heavy chain and light chain of the humanized anti-FAP antibody.

SEQ ID NO: 138-139 are the amino acid sequences of the heavy chain and light chain of a human PD-1 antagonist antibody.

SEQ ID NO: 140-141 are the amino acid sequences of the heavy chain and light chain of the PD-1 antagonist antibody.

SEQ ID NO: 142-143 are the amino acid sequences of the heavy chain and light chain of the PD-1 antagonist antibody.

SEQ ID NO: 144-145 are the amino acid sequences of the heavy chain and light chain of the PD-1 antagonist antibody.

SEQ ID NO: 146-147 are the amino acid sequences of the heavy chain and light chain of the PD-1 antagonist antibody.

SEQ ID NO: 148-149 are the amino acid sequences of the heavy chain and light chain of the PD-L1 antagonist antibody.

SEQ ID NOs: 150-151 are the amino acid sequences of the heavy chain and light chain of a CTLA-4 antagonist antibody.

SEQ ID NO: 152-153 are the amino acid sequences of the heavy chain and light chain of a CD40 agonist antibody.

SEQ ID NO: 154-155 are the amino acid sequences of the heavy chain and light chain of a fibronectin antagonist antibody.

SEQ ID NO: 156-157 are the amino acid sequences of the heavy chain and light chain of a CD20 antagonist antibody.

SEQ ID NO: 158-159 are the amino acid sequences of the heavy chain and light chain of the Her-2/neu antagonist antibody.

SEQ ID NO: 160-161 are the amino acid sequences of the heavy chain and light chain of the EGFR antagonist antibody.

SEQ ID NO: 162 is the amino acid sequence of the CH1-CH2-CH3 domains of human IgG1 with reduced/abstracted Fc effector function.

SEQ ID NO: 163 is the amino acid sequence of the Knob chain of the CH1-CH2-CH3 domains of human IgG1 with reduced/abstracted Fc effector function.

SEQ ID NO: 164 is the amino acid sequence of the Hole chain of the CH1-CH2-CH3 domains of human IgG1 with reduced/abstracted Fc effector function.

SEQ ID NO: 165-169 are the amino acid sequences of fusion proteins of various IL-2 variants with an antibody.

SEQ ID NO: 170 is the amino acid sequence of the Sushi domain of human IL-2 receptor alpha.

SEQ ID NOs: 171-174 are the amino acid sequences of the Fc fusion proteins IL-2 and IL-2RSushi.

SEQ ID NOs: 175-181 are the amino acid sequences of the Knob chains of fusion proteins of various IL-2 variants with a human PD-1 antagonist antibody.

SEQ ID NOs: 182-184 are the amino acid sequences of the fusion protein of the reference variant IL-2 with the antibody.

SEQ ID NO: 185-187 are the amino acid sequences of the anti-mouse PD-1 surrogate antibody with heterodimeric heavy chains.

SEQ ID NO: 188 is the amino acid sequence of a reference variant of IL-2.

SEQ ID NOs: 189-191 are the amino acid sequences of the Knob chains of fusion proteins of various IL-2 variants with a human PD-1 antagonist antibody.

SEQUENCE LIST

Human IL-2 precursor sequence

Naturally occurring sequence of the mature form of human IL-2

Sequence of the mature form of wild-type human IL-2

Sequence of the 8125I variant of human IL-2

Sequence of the extracellular domain of human IL-2Rα (CD25)

Fc human IgG1

Human IgG1 Fc with reduced/aborted effector function

Human IgG1 Fc with reduced/abstracted effector function and increased half-life

Knob-Fc human IgG with reduced/eliminated effector function

Hole-Fc human IgG with reduced/eliminated effector function

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Peptide linker sequence

Sequence of F42A/S125I-Variant IL-2

Sequence of the R38F/S125I variant of IL-2

Sequence of the R38G/S125I variant of IL-2

Sequence of the R38A/S125I variant of IL-2

Sequence of the T41A/S125I variant of IL-2

Sequence of the T 41G/S125I variant of IL-2

Sequence of the T41V/S125I variant of IL-2

Sequence of the F44G/S125I variant of IL-2

Sequence of the F44V/S125I variant of IL-2

Sequence of the E62A/S125I variant of IL-2

Sequence of the E62F/S125I variant of IL-2

Sequence of the E62H/S125I variant of IL-2

Sequence of the E62L/S125I variant of IL-2

Sequence of the P65G/S125I variant of IL-2

Sequence of the P65E/S125I variant of IL-2

Sequence of the P65H/8125I variant of IL-2

Sequence of the P65K/8125I variant of IL-2

Sequence of the P65A/8125I variant of IL-2

Sequence of the P65K/S125I variant of IL-2

Sequence of the P651CH/8125I variant of IL-2

Sequence of the P65Q/S125I variant of IL-2

Sequence of the E68A/S125I variant of IL-2

Sequence of the E68F/S125I variant of IL-2

Sequence of the E68H/S125I variant of IL-2

Sequence of the E68L/S125I variant of IL-2

Sequence of the E68P/8125I variant of IL-2

Sequence of Y107G/S125I variant of IL-2

Sequence of Y107H/S125I variant of IL-2

Sequence of Y107L/S125I variant of IL-2

Sequence of Y107V/S125I variant of IL-2

Sequence of F42A/E62F/S125I variant of IL-2

Sequence of F42A/E62A/S125I variant of IL-2

Sequence of F42A/E62H/S125I variant of IL-2

Sequence of F42A/P65H/S125I variant of IL-2

Sequence of F42A/P65R/S125I variant of IL-2

Sequence of F42A/P65A/S125I variant of IL-2

R-0250

R-0531

R-0613

R-0614

R-0615

R-0602

R-0603

R-0604

R-0605

R-0606

R-0607

R-0624

R-0625

R-0626

R-0627

R-0608

R-0633

R-0634

R-0635

R-0628

R-0629

R-0630

R-0631

R-0632

R-0609

R-0610

R-0611

R-0612

R-0551

Knob-chain P-0704

Knob-chain P-0706

Knob-chain P-0707

Knob-chain P-0708

Knob-chain P-0709

Knob-chain P-0702

Knob-chain P-0766

Knob-chain P-0767

Knob-chain P-0703

Knob-chain P-0705

Knob-chain P-0765

Knob-chain P-0689

Reference Knob chain

Sequence of the extracellular domain of human IL-2Rβ (CD122)

Sequence of the extracellular domain of human common subunit gamma γ _c (CD 132)

Sequence of L19H/P65K/8125I variant of IL-2

Sequence of the L19Q/P65R/S125I variant of IL-2

Sequence of the L19Y/P65R/S125I variant of IL-2

Sequence of the L19H/P65Q/S125I variant of IL-2

Sequence of the L19H/P65H/S125I variant of IL-2

Sequence of the L19H/P65N/S125I variant of IL-2

Sequence of the L19Q/P65Q/S125I variant of IL-2

Sequence of the L19Q/P65H/S125I variant of IL-2

Sequence of the L19Q/P65N/S125I variant of IL-2

Sequence of the P65R/S125I/Q126E variant of IL-2

Knob-chain P-0731

Knob-chain P-0759

Knob-chain P-0761

Knob-chain P-0811

Knob-chain P-0812

Knob-chain P-0813

Knob-chain P-0814

Knob-chain P-0815

Knob-chain P-0816

Knob-chain P-0732

R-0758

R-0760

R-0762

Knob-Fc with increased half-life in vivo

Hole-Fc with increased half-life in vivo

Humanized anti-FAP antibody heavy chain

Humanized anti-FAP antibody kappa light chain

Human PD-1 antagonist antibody heavy chain

Lλ antibody antagonist of human PD-1

NS of humanized PD-1 antagonist antibody

Lκ humanized PD-1 antagonist antibody

NS of humanized PD-1 antagonist antibody

Lκ humanized PD-1 antagonist antibody

NS of humanized FD-1 antagonist antibody

Lκ humanized FD-1 antagonist antibody

NS human PD-1 antagonist antibody

Lκ human PD-1 antagonist antibody

NS of humanized PD-L1 antagonist antibody

Lκ humanized PD-L1 antagonist antibody

NS human CTLA-4 antagonist antibody

Lκ human CTLA-4 antagonist antibody

NS human CD40 agonist antibody

Lκ human CD40 agonist antibody

NS humanized anti-fibronectin antibody

Lκ humanized anti-fibronectin antibody

NS of chimeric antibody against CD20

Lκ chimeric antibody against CD20

NS humanized antibody against Her2

Lκ humanized antibody against Her2

NS of chimeric antibody against EGFR

Lκ chimeric antibody against EGFR

CH1-CH2-CH3 domains of human IgG1 with reduced/aborted effector function

Knob chain of CH1-CH2-CH3 domains of human IgG1 with reduced/eliminated effector function

Hole chain of CH1-CH2-CH3 domains of human IgG1 with reduced/aborted effector function

IL-2 variant with NS humanized antibody antagonist FD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Hole chain HC humanized IgG1 antibody ela antagonist a PD-1

Sushi domain sequence of human IL-2Rα

R-0327

R-0422

Hole chain P-0482

Knob-chain P-0482

Hole chain HC humanized IgG1 antibody ela- PD-1 antagonist

Knob chain of IL-2 variant with NS humanized antibody-antagonist of a PD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Hole chain of the reference variant with NS of the FD-1 antagonist antibody

Knob chain of the reference variant of IL-2 with the HC of the FD-1 antagonist antibody

Reference variant with Lλ antibody antagonist FD-1

Chain 1 of the surrogate antibody antagonist of mouse FD-1

Chain 2 of the surrogate antibody antagonist of mouse FD-1

LC surrogate antibody antagonist of murine PD-1

Reference variant IL-2

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

Knob chain of IL-2 variant with HC humanized antibody-antagonist a FD-1

--->

SEQUENCE LISTING

<110> Cugene Inc

<120> NOVEL INTERLEUKIN-2 VARIANTS FOR THE TREATMENT OF CANCER

<130>CACCG1.0004WO

<160> 191

<170> PatentIn version 3.5

<210> 1

<211> 153

<212> PRT

<213> Homo sapiens

<400> 1

Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu

1 5 10 15

Val Thr Asn Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

20 25 30

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

35 40 45

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

50 55 60

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

65 70 75 80

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

85 90 95

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

100 105 110

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

115 120 125

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

130 135 140

Cys Gln Ser Ile Ile Ser Thr Leu Thr

145 150

<210> 2

<211> 133

<212> PRT

<213> Homo sapiens

<400> 2

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 3

<211> 133

<212> PRT

<213> Homo sapiens

<400> 3

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 4

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the human IL-2 S125I variant

<400> 4

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 5

<211> 219

<212> PRT

<213> Homo sapiens

<400> 5

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly Glu Met Glu Thr Ser Gln Phe Pro Gly Glu Glu

165 170 175

Lys Pro Gln Ala Ser Pro Glu Gly Arg Pro Glu Ser Glu Thr Ser Cys

180 185 190

Leu Val Thr Thr Thr Asp Phe Gln Ile Gln Thr Glu Met Ala Ala Thr

195 200 205

Met Glu Thr Ser Ile Phe Thr Thr Glu Tyr Gln

210 215

<210> 6

<211> 226

<212> PRT

<213> Homo sapiens

<400> 6

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

1 5 10 15

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 7

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Reduced/Abolished Human IgG1 Fc Sequence

effector function

<400> 7

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 8

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Reduced/Abolished Human IgG1 Fc Sequence

effector function

and an increased half-life

<400> 8

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 9

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Knob-Fc human IgG with reduced/aborted effector function

<400> 9

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 10

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc human IgG with reduced/aborted effector function

<400> 10

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 11

<211> 12

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 11

Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser

1 5 10

<210> 12

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 12

Gly Gly Gly Ser

1

<210> 13

<211> 13

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 13

Gly Ser Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly

1 5 10

<210> 14

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 14

Gly Ser Ser Gly Th

1 5

<210> 15

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 15

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 16

<211> 17

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 16

Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys

1 5 10 15

Ala

<210> 17

<211> 20

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 17

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 18

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 18

Gly Gly Gly Ser Gly Gly Gly Ser

1 5

<210> 19

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 19

GlySerGlySerThr

1 5

<210> 20

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 20

Gly Gly Ser Ser

1

<210> 21

<211> 5

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 21

Gly Gly Gly Gly Ser

1 5

<210> 22

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 22

Gly Gly Ser Gly

1

<210> 23

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 23

Ser Gly Gly Gly

1

<210> 24

<211> 4

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 24

Gly Ser Gly Ser

1

<210> 25

<211> 6

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 25

Gly Ser Gly Ser Gly Ser

1 5

<210> 26

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 26

Gly Ser Gly Ser Gly Ser Gly Ser

1 5

<210> 27

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 27

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 28

<211> 12

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 28

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser

1 5 10

<210> 29

<211> 10

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 29

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10

<210> 30

<211> 15

<212> PRT

<213> Artificial

<220>

<223> Peptide linker

<400> 30

Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly Gly Ser

1 5 10 15

<210> 31

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the F42A/S125I variant of IL-2

<400> 31

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 32

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the R38F/S125I variant of IL-2

<400> 32

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 33

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the R38G/S125I variant of IL-2

<400> 33

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Gly Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 34

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the R38A/S125I variant of IL-2

<400> 34

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Ala Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 35

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the T41A/S125I variant of IL-2

<400> 35

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Ala Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 36

<211> 133

<212> PRT

<213> Artificial

<220>

<223> APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLGFKFYMPKKATELKHLQCLEEELKP

LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFIQSIIS

TLT

<400> 36

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Gly Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 37

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the T41V/S125I variant of IL-2

<400> 37

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Val Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 38

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the F44G/S125I variant of IL-2

<400> 38

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Gly Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 39

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the F44V/S125I variant of IL-2

<400> 39

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Val Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 40

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the E62A/S125I variant of IL-2

<400> 40

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 41

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the E62F/S125I variant of IL-2

<400> 41

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Phe Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 42

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the E62H/S125I variant of IL-2

<400> 42

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu His Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 43

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the E62L/S125I variant of IL-2

<400> 43

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Leu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 44

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the P65G/S125I variant of IL-2

<400> 44

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Gly Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 45

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the P65E/S125I variant of IL-2

<400> 45

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 46

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the P65H/S125I variant of IL-2

<400> 46

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 47

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the P65R/S125I variant of IL-2

<400> 47

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 48

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the P65A/S125I variant of IL-2

<400> 48

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 49

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the P65K/S125I variant of IL-2

<400> 49

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 50

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the P65N/S125I variant of IL-2

<400> 50

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 51

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the P65Q/S125I variant of IL-2

<400> 51

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 52

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the E68A/S125I variant of IL-2

<400> 52

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 53

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the E68F/S125I variant of IL-2

<400> 53

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 54

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the E68H/S125I variant of IL-2

<400> 54

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 55

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the E68L/S125I variant of IL-2

<400> 55

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 56

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of the E68P/S125I variant of IL-2

<400> 56

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 57

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of Y107G/S125I variant of IL-2

<400> 57

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Val Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Gly Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 58

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of Y107H/S125I variant of IL-2

<400> 58

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Val Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu His Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 59

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of Y107L/S125I variant of IL-2

<400> 59

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Val Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Leu Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 60

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of Y107V/S125I variant of IL-2

<400> 60

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Val Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Val Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 61

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of F42A/E62F/S125I variant of IL-2

<400> 61

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Phe Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 62

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of F42A/E62A/S125I variant of IL-2

<400> 62

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Ala Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 63

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of F42A/E62H/S125I variant of IL-2

<400> 63

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu His Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 64

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of F42A/P65H/S125I variant of IL-2

<400> 64

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 65

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of F42A/P65R/S125I variant of IL-2

<400> 65

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 66

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Sequence of F42A/P65A/S125I variant of IL-2

<400> 66

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 67

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0250

<400> 67

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 68

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0531

<400> 68

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 69

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0613

<400> 69

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 70

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0614

<400> 70

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Phe

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 71

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0615

<400> 71

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Gly

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 72

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0602

<400> 72

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Ala

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 73

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0603

<400> 73

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Ala Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 74

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0604

<400> 74

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Gly Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 75

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0605

<400> 75

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Val Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 76

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0606

<400> 76

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Gly Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 77

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0607

<400> 77

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Val Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 78

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0624

<400> 78

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Ala Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 79

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0625

<400> 79

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Phe Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 80

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0626

<400> 80

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu His Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 81

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0627

<400> 81

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Leu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 82

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0608

<400> 82

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gly Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 83

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0633

<400> 83

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Glu Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 84

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0634

<400> 84

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys His Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 85

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0635

<400> 85

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 86

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0628

<400> 86

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Ala Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 87

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0629

<400> 87

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Phe Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 88

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0630

<400> 88

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu His Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 89

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0631

<400> 89

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Leu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 90

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0632

<400> 90

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Pro Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 91

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0609

<400> 91

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Val Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Gly Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 92

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0610

<400> 92

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Val Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu His Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 93

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0611

<400> 93

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Val Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Leu Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 94

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0612

<400> 94

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Val Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Val Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 95

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0551

<400> 95

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Ala Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Gly Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 96

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0704

<400> 96

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 97

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob chain P-0706

<400> 97

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Ala Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 98

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0707

<400> 98

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Lys Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 99

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0708

<400> 99

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Asn Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 100

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0709

<400> 100

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gln Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 101

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0702

<400> 101

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Phe Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 102

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob chain P-0766

<400> 102

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Ala Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 103

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob chain P-0767

<400> 103

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu His Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 104

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0703

<400> 104

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys His Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 105

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0705

<400> 105

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 106

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0765

<400> 106

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Ala Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 107

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0689

<400> 107

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 108

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Reference Knob Chain

<400> 108

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Ala Lys Phe Ala Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Gly Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 109

<211> 214

<212> PRT

<213> Homo sapiens

<400> 109

Ala Val Asn Gly Thr Ser Gln Phe Thr Cys Phe Tyr Asn Ser Arg Ala

1 5 10 15

Asn Ile Ser Cys Val Trp Ser Gln Asp Gly Ala Leu Gln Asp Thr Ser

20 25 30

Cys Gln Val His Ala Trp Pro Asp Arg Arg Arg Trp Asn Gln Thr Cys

35 40 45

Glu Leu Leu Pro Val Ser Gln Ala Ser Trp Ala Cys Asn Leu Ile Leu

50 55 60

Gly Ala Pro Asp Ser Gln Lys Leu Thr Thr Val Asp Ile Val Thr Leu

65 70 75 80

Arg Val Leu Cys Arg Glu Gly Val Arg Trp Arg Val Met Ala Ile Gln

85 90 95

Asp Phe Lys Pro Phe Glu Asn Leu Arg Leu Met Ala Pro Ile Ser Leu

100 105 110

Gln Val Val His Val Glu Thr His Arg Cys Asn Ile Ser Trp Glu Ile

115 120 125

Ser Gln Ala Ser His Tyr Phe Glu Arg His Leu Glu Phe Glu Ala Arg

130 135 140

Thr Leu Ser Pro Gly His Thr Trp Glu Glu Ala Pro Leu Leu Thr Leu

145 150 155 160

Lys Gln Lys Gln Glu Trp Ile Cys Leu Glu Thr Leu Thr Pro Asp Thr

165 170 175

Gln Tyr Glu Phe Gln Val Arg Val Lys Pro Leu Gln Gly Glu Phe Thr

180 185 190

Thr Trp Ser Pro Trp Ser Gln Pro Leu Ala Phe Arg Thr Lys Pro Ala

195 200 205

Ala Leu Gly Lys Asp Thr

210

<210> 110

<211> 240

<212> PRT

<213> Homo sapiens

<400> 110

Leu Asn Thr Thr Ile Leu Thr Pro Asn Gly Asn Glu Asp Thr Thr Ala

1 5 10 15

Asp Phe Phe Leu Thr Thr Met Pro Thr Asp Ser Leu Ser Val Ser Thr

20 25 30

Leu Pro Leu Pro Glu Val Gln Cys Phe Val Phe Asn Val Glu Tyr Met

35 40 45

Asn Cys Thr Trp Asn Ser Ser Ser Glu Pro Gln Pro Thr Asn Leu Thr

50 55 60

Leu His Tyr Trp Tyr Lys Asn Ser Asp Asn Asp Lys Val Gln Lys Cys

65 70 75 80

Ser His Tyr Leu Phe Ser Glu Glu Ile Thr Ser Gly Cys Gln Leu Gln

85 90 95

Lys Lys Glu Ile His Leu Tyr Gln Thr Phe Val Val Gln Leu Gln Asp

100 105 110

Pro Arg Glu Pro Arg Arg Gln Ala Thr Gln Met Leu Lys Leu Gln Asn

115 120 125

Leu Val Ile Pro Trp Ala Pro Glu Asn Leu Thr Leu His Lys Leu Ser

130 135 140

Glu Ser Gln Leu Glu Leu Asn Trp Asn Asn Arg Phe Leu Asn His Cys

145 150 155 160

Leu Glu His Leu Val Gln Tyr Arg Thr Asp Trp Asp His Ser Trp Thr

165 170 175

Glu Gln Ser Val Asp Tyr Arg His Lys Phe Ser Leu Pro Ser Val Asp

180 185 190

Gly Gln Lys Arg Tyr Thr Phe Arg Val Arg Ser Arg Phe Asn Pro Leu

195 200 205

Cys Gly Ser Ala Gln His Trp Ser Glu Trp Ser His Pro Ile His Trp

210 215 220

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

225 230 235 240

<210> 111

<211> 133

<212> PRT

<213> Artificial

<220>

<223> L19H/P65R/S125I sequence of IL-2 variant

<400> 111

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu His Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 112

<211> 133

<212> PRT

<213> Artificial

<220>

<223> L19Q/P65R/S125I sequence of IL-2 variant

<400> 112

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Gln Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 113

<211> 133

<212> PRT

<213> Artificial

<220>

<223> L19Y/P65R/S125I sequence of IL-2 variant

<400> 113

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Tyr Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 114

<211> 133

<212> PRT

<213> Artificial

<220>

<223> L19H/P65Q/S125I sequence of IL-2 variant

<400> 114

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu His Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 115

<211> 133

<212> PRT

<213> Artificial

<220>

<223> L19H/P65H/S125I sequence of IL-2 variant

<400> 115

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu His Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 116

<211> 133

<212> PRT

<213> Artificial

<220>

<223> L19H/P65N/S125I sequence of IL-2 variant

<400> 116

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu His Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 117

<211> 133

<212> PRT

<213> Artificial

<220>

<223> L19Q/P65Q/S125I sequence of IL-2 variant

<400> 117

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Gln Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 118

<211> 133

<212> PRT

<213> Artificial

<220>

<223> L19Q/P65H/S125I sequence of IL-2 variant

<400> 118

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Gln Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 119

<211> 133

<212> PRT

<213> Artificial

<220>

<223> L19Q/P65N/S125I sequence of IL-2 variant

<400> 119

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Gln Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 120

<211> 133

<212> PRT

<213> Artificial

<220>

<223> P65R/S125I/Q126E sequence of IL-2 variant

<400> 120

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Glu Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 121

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob chain P-0731

<400> 121

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu His Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 122

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0759

<400> 122

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Gln Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 123

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob chain P-0761

<400> 123

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Tyr Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 124

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob chain P-0811

<400> 124

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu His Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gln Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 125

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob chain P-0812

<400> 125

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu His Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys His Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 126

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0813

<400> 126

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu His Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Asn Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 127

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0814

<400> 127

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Gln Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gln Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 128

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0815

<400> 128

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Gln Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys His Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 129

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0816

<400> 129

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Gln Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Asn Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 130

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Knob chain P-0732

<400> 130

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Glu Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 131

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0758

<400> 131

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu His Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 132

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0760

<400> 132

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Gln Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 133

<211> 367

<212> PRT

<213> Artificial

<220>

<223> P-0762

<400> 133

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Tyr Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 134

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Knob-Fc with an increased half-life in vivo

<400> 134

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 135

<211> 226

<212> PRT

<213> Artificial

<220>

<223> Hole-Fc with an increased half-life in vivo

<400> 135

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly

225

<210> 136

<211> 449

<212> PRT

<213> Artificial

<220>

<223> Humanized anti-FAP antibody heavy chain

<400> 136

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 Glu Asn

20 25 30

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

35 40 45

Gly Trp Phe His Pro Gly Ser Gly Ser Ile Lys Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg His Gly Gly Thr Gly Arg Gly Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

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

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 137

<211> 218

<212> PRT

<213> Artificial

<220>

<223> Humanized anti-FAP antibody kappa light chain

<400> 137

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Arg Ser Ile Ser Thr Ser

20 25 30

Ala Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro

35 40 45

Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ser

50 55 60

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

65 70 75 80

Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln His Ser Arg

85 90 95

Glu Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

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

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

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

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 138

<211> 448

<212> PRT

<213> Artificial

<220>

<223> Human PD-1 antagonist antibody heavy chain

<400> 138

Glu 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 Arg Phe Thr Ser Tyr

20 25 30

Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu

50 55 60

Gln Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Ala Asp Tyr Ser Ser Gly Ser Gly Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

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

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

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

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

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

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 139

<211> 214

<212> PRT

<213> Artificial

<220>

<223> L? human PD-1 antagonist antibody

<400> 139

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

1 5 10 15

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

20 25 30

Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Met Val Ile Tyr

35 40 45

Lys Asp Thr Glu Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser

50 55 60

Ser Ser Gly Thr Lys Val Thr Leu Thr Ile Ser Gly Val Gln Ala Glu

65 70 75 80

Asp Glu Ala Asp Tyr Tyr Cys Gln Ser Ala Asp Asn Ser Ile Thr Tyr

85 90 95

Arg Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu Gly Gln Pro Lys

100 105 110

Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln

115 120 125

Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly

130 135 140

Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly

145 150 155 160

Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala

165 170 175

Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser

180 185 190

Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val

195 200 205

Ala Pro Thr Glu Cys Ser

210

<210> 140

<211> 446

<212> PRT

<213> Artificial

<220>

<223> HC humanized PD-1 antagonist antibody

<400> 140

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 141

<211> 214

<212> PRT

<213> Artificial

<220>

<223>L humanized PD-1 antagonist antibody

<400> 141

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

1 5 10 15

Glu Pro Ala Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Glu Thr Val

20 25 30

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

35 40 45

Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala

65 70 75 80

Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr Ser Arg Tyr Pro Trp

85 90 95

Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 142

<211> 452

<212> PRT

<213> Artificial

<220>

<223> HC humanized PD-1 antagonist antibody

<400> 142

Gln Gly 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 Asp Tyr

20 25 30

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

35 40 45

Gly Val Ile Glu Ser Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Glu Gly Ile Thr Thr Val Ala Thr Thr Tyr Tyr Trp Tyr Phe

100 105 110

Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

115 120 125

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser

130 135 140

Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

145 150 155 160

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

165 170 175

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

180 185 190

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys

195 200 205

Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

210 215 220

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

225 230 235 240

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser

325 330 335

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val

355 360 365

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

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Leu Gly Lys

450

<210> 143

<211> 219

<212> PRT

<213> Artificial

<220>

<223>L humanized PD-1 antagonist antibody

<400> 143

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

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

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

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

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 144

<211> 447

<212> PRT

<213> Artificial

<220>

<223> HC humanized PD-1 antagonist antibody

<400> 144

Gln Val Gln Leu Val Gln Ser Gly Val 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 Asn Tyr

20 25 30

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

35 40 45

Gly Gly Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Lys Ser Leu Gln Phe Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg Asp Tyr Arg Phe Asp Met Gly Phe Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro

210 215 220

Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu

260 265 270

Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

435 440 445

<210> 145

<211> 218

<212> PRT

<213> Artificial

<220>

<223>L humanized PD-1 antagonist antibody

<400> 145

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Lys Gly Val Ser Thr Ser

20 25 30

Gly Tyr Ser Tyr Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Tyr Leu Glu Ser Gly Val Pro Ala

50 55 60

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

65 70 75 80

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Ser Arg

85 90 95

Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg

100 105 110

Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

115 120 125

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

130 135 140

Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

145 150 155 160

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

165 170 175

Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys

180 185 190

His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro

195 200 205

Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 146

<211> 440

<212> PRT

<213> Artificial

<220>

<223> HC human PD-1 antagonist antibody

<400> 146

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser

20 25 30

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

35 40 45

Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser

100 105 110

Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser

115 120 125

Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp

130 135 140

Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr

145 150 155 160

Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

165 170 175

Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys

180 185 190

Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp

195 200 205

Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala

210 215 220

Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro

225 230 235 240

Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val

245 250 255

Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val

260 265 270

Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln

275 280 285

Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln

290 295 300

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

305 310 315 320

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

325 330 335

Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr

340 345 350

Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser

355 360 365

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

370 375 380

Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr

385 390 395 400

Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe

405 410 415

Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys

420 425 430

Ser Leu Ser Leu Ser Leu Gly Lys

435 440

<210> 147

<211> 214

<212> PRT

<213> Artificial

<220>

<223> L human PD-1 antagonist antibody

<400> 147

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 148

<211> 448

<212> PRT

<213> Artificial

<220>

<223> HC humanized PD-L1 antagonist antibody

<400> 148

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Ser

20 25 30

Trp Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Trp Ile Ser Pro Tyr Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Arg His Trp Pro Gly Gly Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

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

340 345 350

Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 149

<211> 214

<212> PRT

<213> Artificial

<220>

<223>L humanized PD-L1 antagonist antibody

<400> 149

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Ser Thr Ala

20 25 30

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

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Leu Tyr His Pro Ala

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 150

<211> 448

<212> PRT

<213> Artificial

<220>

<223> HC human CTLA-4 antagonist antibody

<400> 150

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

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

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Ile Tyr Tyr Cys

85 90 95

Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

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

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 151

<211> 215

<212> PRT

<213> Artificial

<220>

<223> L human CTLA-4 antagonist antibody

<400> 151

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

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro

85 90 95

Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

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

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 152

<211> 452

<212> PRT

<213> Artificial

<220>

<223> HC human CD40 agonist antibody

<400> 152

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 Gly Tyr

20 25 30

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

35 40 45

Gly Trp Ile Asn Pro Asp Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

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

65 70 75 80

Met Glu Leu Asn Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys

85 90 95

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

100 105 110

Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser

115 120 125

Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr

130 135 140

Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro

145 150 155 160

Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val

165 170 175

His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser

180 185 190

Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr Tyr Thr

195 200 205

Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Thr Val

210 215 220

Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro Pro Val

225 230 235 240

Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

245 250 255

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

260 265 270

His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu

275 280 285

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr

290 295 300

Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn

305 310 315 320

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro

325 330 335

Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln

340 345 350

Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val

355 360 365

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ser Val

370 375 380

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

385 390 395 400

Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

405 410 415

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

420 425 430

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

435 440 445

Ser Pro Gly Lys

450

<210> 153

<211> 214

<212> PRT

<213> Artificial

<220>

<223> L human CD40 agonist antibody

<400> 153

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Ser Trp

20 25 30

Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu Ile

35 40 45

Tyr Thr Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ile Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 154

<211> 445

<212> PRT

<213> Artificial

<220>

<223> HC humanized anti-fibronectin antibody

<400> 154

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe

20 25 30

Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Lys Asn Thr Leu Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro

115 120 125

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val

130 135 140

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala

145 150 155 160

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly

165 170 175

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly

180 185 190

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys

195 200 205

Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys

210 215 220

Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu

225 230 235 240

Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

245 250 255

Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys

260 265 270

Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

275 280 285

Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu

290 295 300

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

305 310 315 320

Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys

325 330 335

Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

340 345 350

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

355 360 365

Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

370 375 380

Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

385 390 395 400

Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln

405 410 415

Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

420 425 430

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 155

<211> 215

<212> PRT

<213> Artificial

<220>

<223> L humanized anti-fibronectin antibody

<400> 155

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro

85 90 95

Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala

100 105 110

Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser

115 120 125

Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu

130 135 140

Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser

145 150 155 160

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

165 170 175

Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val

180 185 190

Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys

195 200 205

Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 156

<211> 451

<212> PRT

<213> Artificial

<220>

<223> HC chimeric anti-CD20 antibody

<400> 156

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

1 5 10 15

Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu Glu Trp Ile

35 40 45

Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly

100 105 110

Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser

115 120 125

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

130 135 140

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

145 150 155 160

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

165 170 175

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

180 185 190

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

195 200 205

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys

210 215 220

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

225 230 235 240

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

245 250 255

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

260 265 270

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

275 280 285

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

290 295 300

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

305 310 315 320

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

325 330 335

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

340 345 350

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

355 360 365

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

370 375 380

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

385 390 395 400

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

405 410 415

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

420 425 430

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

435 440 445

Pro Gly Lys

450

<210> 157

<211> 213

<212> PRT

<213> Artificial

<220>

<223>L chimeric anti-CD20 antibody

<400> 157

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr

115 120 125

Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys

130 135 140

Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu

145 150 155 160

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

165 170 175

Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala

180 185 190

Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe

195 200 205

AsnArgGlyGluCys

210

<210> 158

<211> 450

<212> PRT

<213> Artificial

<220>

<223> HC humanized anti-Her2 antibody

<400> 158

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 159

<211> 214

<212> PRT

<213> Artificial

<220>

<223> L humanized anti-Her2 antibody

<400> 159

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

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

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

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 160

<211> 449

<212> PRT

<213> Artificial

<220>

<223> HC chimeric anti-EGFR antibody

<400> 160

Gln Val Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln

1 5 10 15

Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asn Tyr

20 25 30

Gly Val His Trp Val Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Ser Gly Gly Asn Thr Asp Tyr Asn Thr Pro Phe Thr

50 55 60

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

65 70 75 80

Lys Met Asn Ser Leu Gln Ser Asn Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Arg Ala Leu Thr Tyr Tyr Asp Tyr Glu Phe Ala Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

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

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

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

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

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

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

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

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 161

<211> 214

<212> PRT

<213> Artificial

<220>

<223>L chimeric anti-EGFR antibody

<400> 161

Asp Ile Leu Leu Thr Gln Ser Pro Val Ile Leu Ser Val Ser Pro Gly

1 5 10 15

Glu Arg Val Ser Phe Ser Cys Arg Ala Ser Gln Ser Ile Gly Thr Asn

20 25 30

Ile His Trp Tyr Gln Gln Arg Thr Asn Gly Ser Pro Arg Leu Leu Ile

35 40 45

Lys Tyr Ala Ser Glu Ser Ile Ser Gly Ile Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Ser Ile Asn Ser Val Glu Ser

65 70 75 80

Glu Asp Ile Ala Asp Tyr Tyr Cys Gln Gln Asn Asn Asn Trp Pro Thr

85 90 95

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

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

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 162

<211> 329

<212> PRT

<213> Artificial

<220>

<223> CH1-CH2-CH3 domains of human IgG1 with reduced/abstracted

effector function

<400> 162

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

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

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu

225 230 235 240

Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 163

<211> 329

<212> PRT

<213> Artificial

<220>

<223> Knob chain of CH1-CH2-CH3 domains of human IgG1 with

reduced/eliminated effector function

<400> 163

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

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

210 215 220

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

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 164

<211> 329

<212> PRT

<213> Artificial

<220>

<223> Hole chain of CH1-CH2-CH3 domains of human IgG1 with

reduced/eliminated effector function

<400> 164

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys

100 105 110

Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro

115 120 125

Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys

130 135 140

Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp

145 150 155 160

Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu

165 170 175

Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu

180 185 190

His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn

195 200 205

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

210 215 220

Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Glu Glu

225 230 235 240

Met Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr

245 250 255

Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn

260 265 270

Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe

275 280 285

Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn

290 295 300

Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr

305 310 315 320

Gln Lys Ser Leu Ser Leu Ser Pro Gly

325

<210> 165

<211> 587

<212> PRT

<213> Artificial

<220>

<223> IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 165

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Ala His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr

450 455 460

Gln Leu Gln Leu Glu His Leu Leu His Asp Leu Gln Met Ile Leu Asn

465 470 475 480

Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe

485 490 495

Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys

500 505 510

Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu Asn Leu Ala Gln

515 520 525

Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn

530 535 540

Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu

545 550 555 560

Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile

565 570 575

Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

580 585

<210> 166

<211> 587

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 166

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr

450 455 460

Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn

465 470 475 480

Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe

485 490 495

Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys

500 505 510

Leu Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu Asn Leu Ala Gln

515 520 525

Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn

530 535 540

Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu

545 550 555 560

Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile

565 570 575

Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

580 585

<210> 167

<211> 587

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD 1 antagonist antibody

<400> 167

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr

450 455 460

Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn

465 470 475 480

Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe

485 490 495

Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys

500 505 510

Leu Glu Glu Glu Leu Lys Gln Leu Glu Glu Val Leu Asn Leu Ala Gln

515 520 525

Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn

530 535 540

Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu

545 550 555 560

Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile

565 570 575

Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

580 585

<210> 168

<211> 587

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 168

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr

450 455 460

Gln Leu Gln Leu Glu His Leu Leu Gln Asp Leu Gln Met Ile Leu Asn

465 470 475 480

Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe

485 490 495

Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys

500 505 510

Leu Glu Glu Glu Leu Lys Gln Leu Glu Glu Val Leu Asn Leu Ala Gln

515 520 525

Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn

530 535 540

Val Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu

545 550 555 560

Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile

565 570 575

Thr Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

580 585

<210> 169

<211> 446

<212> PRT

<213> Artificial

<220>

<223> Hole chain HC humanized IgG1 antibody antagonist of PD-1

<400> 169

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

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

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 170

<211> 165

<212> PRT

<213> Artificial

<220>

<223> Sequence of the Sushi domain of human IL-2R?

<400> 170

Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe Lys

1 5 10 15

Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys Arg

20 25 30

Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr Gly

35 40 45

Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser Ser

50 55 60

Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu Gln

65 70 75 80

Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val Asp

85 90 95

Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu Asn

100 105 110

Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val Tyr

115 120 125

Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala Glu

130 135 140

Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro Gln

145 150 155 160

Leu Ile Cys Thr Gly

165

<210> 171

<211> 554

<212> PRT

<213> Artificial

<220>

<223> P-0327

<400> 171

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

130 135 140

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe

245 250 255

Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys

260 265 270

Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr

275 280 285

Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser

290 295 300

Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu

305 310 315 320

Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val

325 330 335

Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu

340 345 350

Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val

355 360 365

Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala

370 375 380

Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro

385 390 395 400

Gln Leu Ile Cys Thr Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

405 410 415

Gly Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln

420 425 430

Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly

435 440 445

Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys

450 455 460

Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu

465 470 475 480

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

485 490 495

Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val

500 505 510

Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr

515 520 525

Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr

530 535 540

Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr

545 550

<210> 172

<211> 548

<212> PRT

<213> Artificial

<220>

<223> P-0422

<400> 172

Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

130 135 140

Gly Gly Gly Ser Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His

145 150 155 160

Ala Thr Phe Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys

165 170 175

Glu Cys Lys Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met

180 185 190

Leu Cys Thr Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln

195 200 205

Cys Thr Ser Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln

210 215 220

Pro Glu Glu Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met

225 230 235 240

Gln Pro Val Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro

245 250 255

Pro Trp Glu Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly

260 265 270

Gln Met Val Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg

275 280 285

Gly Pro Ala Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp

290 295 300

Thr Gln Pro Gln Leu Ile Cys Thr Gly Gly Gly Gly Ser Gly Gly Gly

305 310 315 320

Gly Ser Gly Gly Gly Gly Ser Cys Pro Pro Cys Pro Ala Pro Glu Ala

325 330 335

Ala Gly Ala Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr

340 345 350

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

355 360 365

Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val

370 375 380

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser

385 390 395 400

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

405 410 415

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala

420 425 430

Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

435 440 445

Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln

450 455 460

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

465 470 475 480

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

485 490 495

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu

500 505 510

Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser

515 520 525

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

530 535 540

Leu Ser Pro Gly

545

<210> 173

<211> 367

<212> PRT

<213> Artificial

<220>

<223> Hole-chain P-0482

<400> 173

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Tyr Thr Leu Pro Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Ser Gly Gly Gly Ser Ala Pro Thr Ser Ser Ser

225 230 235 240

Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln

245 250 255

Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg

260 265 270

Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys

275 280 285

His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu

290 295 300

Asn Leu Ala Gln Ser Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile

305 310 315 320

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

325 330 335

Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu

340 345 350

Asn Arg Trp Ile Thr Phe Ser Gln Ser Ile Ile Ser Thr Leu Thr

355 360 365

<210> 174

<211> 405

<212> PRT

<213> Artificial

<220>

<223> Knob-chain P-0482

<400> 174

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly

1 5 10 15

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

20 25 30

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

35 40 45

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

50 55 60

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

65 70 75 80

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

85 90 95

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile

100 105 110

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

115 120 125

Cys Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser

130 135 140

Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

145 150 155 160

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

165 170 175

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

180 185 190

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

195 200 205

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

210 215 220

Pro Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

225 230 235 240

Ser Glu Leu Cys Asp Asp Asp Pro Pro Glu Ile Pro His Ala Thr Phe

245 250 255

Lys Ala Met Ala Tyr Lys Glu Gly Thr Met Leu Asn Cys Glu Cys Lys

260 265 270

Arg Gly Phe Arg Arg Ile Lys Ser Gly Ser Leu Tyr Met Leu Cys Thr

275 280 285

Gly Asn Ser Ser His Ser Ser Trp Asp Asn Gln Cys Gln Cys Thr Ser

290 295 300

Ser Ala Thr Arg Asn Thr Thr Lys Gln Val Thr Pro Gln Pro Glu Glu

305 310 315 320

Gln Lys Glu Arg Lys Thr Thr Glu Met Gln Ser Pro Met Gln Pro Val

325 330 335

Asp Gln Ala Ser Leu Pro Gly His Cys Arg Glu Pro Pro Pro Trp Glu

340 345 350

Asn Glu Ala Thr Glu Arg Ile Tyr His Phe Val Val Gly Gln Met Val

355 360 365

Tyr Tyr Gln Cys Val Gln Gly Tyr Arg Ala Leu His Arg Gly Pro Ala

370 375 380

Glu Ser Val Cys Lys Met Thr His Gly Lys Thr Arg Trp Thr Gln Pro

385 390 395 400

Gln Leu Ile Cys Thr

405

<210> 175

<211> 446

<212> PRT

<213> Artificial

<220>

<223> Hole chain HC humanized IgG1 antibody antagonist of PD-1

<400> 175

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro

340 345 350

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

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

<210> 176

<211> 594

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL 2 variant with HC humanized PD-1 antagonist antibody

<400> 176

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr

450 455 460

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu His

465 470 475 480

Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys

485 490 495

Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr

500 505 510

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu

515 520 525

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

530 535 540

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

545 550 555 560

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

565 570 575

Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr

580 585 590

Leu Th

<210> 177

<211> 594

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL 2 variant with HC humanized PD-1 antagonist antibody

<400> 177

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr

450 455 460

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu

465 470 475 480

Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys

485 490 495

Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr

500 505 510

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu

515 520 525

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

530 535 540

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

545 550 555 560

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

565 570 575

Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr

580 585 590

Leu Th

<210> 178

<211> 594

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 178

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr

450 455 460

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Gln

465 470 475 480

Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys

485 490 495

Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr

500 505 510

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gln Leu Glu

515 520 525

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

530 535 540

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

545 550 555 560

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

565 570 575

Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr

580 585 590

Leu Th

<210> 179

<211> 594

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 179

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr

450 455 460

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Gln

465 470 475 480

Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys

485 490 495

Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr

500 505 510

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Arg Leu Glu

515 520 525

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

530 535 540

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

545 550 555 560

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

565 570 575

Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr

580 585 590

Leu Th

<210> 180

<211> 594

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 180

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr

450 455 460

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu His

465 470 475 480

Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys

485 490 495

Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr

500 505 510

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Gln Leu Glu

515 520 525

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

530 535 540

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

545 550 555 560

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

565 570 575

Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr

580 585 590

Leu Th

<210> 181

<211> 594

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 181

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Pro Thr

450 455 460

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Gln

465 470 475 480

Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys

485 490 495

Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys Lys Ala Thr

500 505 510

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Asn Leu Glu

515 520 525

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

530 535 540

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

545 550 555 560

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

565 570 575

Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile Gln Ser Ile Ile Ser Thr

580 585 590

Leu Th

<210> 182

<211> 449

<212> PRT

<213> Artificial

<220>

<223> Hole chain of the reference variant with HC of the PD-1 antagonist antibody

<400> 182

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

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

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 183

<211> 597

<212> PRT

<213> Artificial

<220>

<223> Knob chain of the reference variant of IL-2 with HC of the PD-1 antagonist antibody

<400> 183

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu

355 360 365

Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

450 455 460

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

465 470 475 480

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

485 490 495

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys

500 505 510

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

515 520 525

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

530 535 540

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

545 550 555 560

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

565 570 575

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

580 585 590

Ile Ser Thr Leu Thr

595

<210> 184

<211> 449

<212> PRT

<213> Artificial

<220>

<223> Reference variant with L? PD-1 antagonist antibody

<400> 184

Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Ser Tyr

20 25 30

Thr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Val Leu Leu Thr Gly Arg Val Tyr Phe Ala Leu Asp Ser Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

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

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Gly Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys

340 345 350

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

355 360 365

Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly

<210> 185

<211> 443

<212> PRT

<213> Artificial

<220>

<223> HC chain 1 of mouse PD-1 antagonist surrogate antibody

<400> 185

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ser

20 25 30

Tyr Arg Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Arg Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Ser Ala Gly Ile Ser Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Arg Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Val Asn Ser Val Thr Thr Glu Asp Ala Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Ser Asp Asn Met Gly Thr Thr Pro Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

180 185 190

Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala

195 200 205

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

210 215 220

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

245 250 255

Thr Cys Val Val Val Ala Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

260 265 270

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro

275 280 285

Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

290 295 300

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

305 310 315 320

Asn Ser Ala Ala Phe Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

340 345 350

Lys Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn

355 360 365

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

370 375 380

Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Lys Thr Asp Gly Ser

385 390 395 400

Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

405 410 415

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

420 425 430

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

<210> 186

<211> 443

<212> PRT

<213> Artificial

<220>

<223> HC chain 2 of mouse PD-1 antagonist surrogate antibody

<400> 186

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

1 5 10 15

Ser Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Ser

20 25 30

Tyr Arg Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Arg Leu Glu Trp

35 40 45

Met Gly Tyr Ile Asn Ser Ala Gly Ile Ser Asn Tyr Asn Pro Ser Leu

50 55 60

Lys Arg Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe

65 70 75 80

Leu Gln Val Asn Ser Val Thr Thr Glu Asp Ala Ala Thr Tyr Tyr Cys

85 90 95

Ala Arg Ser Asp Asn Met Gly Thr Thr Pro Phe Thr Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val

115 120 125

Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr

130 135 140

Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr

145 150 155 160

Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser

180 185 190

Ser Thr Trp Pro Ser Gln Thr Val Thr Cys Asn Val Ala His Pro Ala

195 200 205

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

210 215 220

Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe

225 230 235 240

Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val

245 250 255

Thr Cys Val Val Val Ala Ile Ser Lys Asp Asp Pro Glu Val Gln Phe

260 265 270

Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln Thr Lys Pro

275 280 285

Arg Glu Glu Gln Ile Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro

290 295 300

Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val

305 310 315 320

Asn Ser Ala Ala Phe Gly Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr

325 330 335

Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys

340 345 350

Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn

355 360 365

Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro

370 375 380

Ala Glu Asn Tyr Asp Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser

385 390 395 400

Tyr Phe Val Tyr Ser Asp Leu Asn Val Gln Lys Ser Asn Trp Glu Ala

405 410 415

Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His

420 425 430

His Thr Glu Lys Ser Leu Ser His Ser Pro Gly

435 440

<210> 187

<211> 218

<212> PRT

<213> Artificial

<220>

<223> LC of a surrogate antibody antagonist of murine PD-1

<400> 187

Asp Ile Val Met Thr Gln Gly Thr Leu Pro Asn Pro Val Pro Ser Gly

1 5 10 15

Glu Ser Val Ser Ile Thr Cys Arg Ser Ser Lys Ser Leu Leu Tyr Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Tyr Leu Gln Arg Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Trp Met Ser Thr Arg Ala Ser Gly Val Ser

50 55 60

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

65 70 75 80

Ser Gly Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Gln Gln Gly

85 90 95

Leu Glu Phe Pro Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg

100 105 110

Thr Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln

115 120 125

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

130 135 140

Pro Arg Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln

145 150 155 160

Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr

165 170 175

Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg

180 185 190

His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro

195 200 205

Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

210 215

<210> 188

<211> 133

<212> PRT

<213> Artificial

<220>

<223> Reference variant IL-2

<400> 188

Ala Pro Ala Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

1 5 10 15

Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

20 25 30

Asn Pro Lys Leu Thr Arg Met Leu Thr Ala Lys Phe Ala Met Pro Lys

35 40 45

Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

50 55 60

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

65 70 75 80

Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val Leu Glu Leu

85 90 95

Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala

100 105 110

Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ala Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr

130

<210> 189

<211> 586

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 189

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Lys Thr Gln

450 455 460

Leu Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly

465 470 475 480

Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys

485 490 495

Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu

500 505 510

Glu Glu Glu Leu Lys Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser

515 520 525

Lys Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val

530 535 540

Ile Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr

545 550 555 560

Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr

565 570 575

Phe Ile Gln Ser Ile Ile Ser Thr Leu Thr

580 585

<210> 190

<211> 585

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 190

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Thr Gln Leu

450 455 460

Gln Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile

465 470 475 480

Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe

485 490 495

Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu

500 505 510

Glu Glu Leu Lys Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys

515 520 525

Asn Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile

530 535 540

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

545 550 555 560

Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe

565 570 575

Ile Gln Ser Ile Ile Ser Thr Leu Thr

580 585

<210> 191

<211> 584

<212> PRT

<213> Artificial

<220>

<223> Knob chain of IL-2 variant with HC humanized PD-1 antagonist antibody

<400> 191

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

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr

20 25 30

Asp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

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

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ser Pro Asp Ser Ser Gly Val Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Ala Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Cys Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly

435 440 445

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Leu Gln

450 455 460

Leu Glu His Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn

465 470 475 480

Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr

485 490 495

Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu

500 505 510

Glu Leu Lys Arg Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn

515 520 525

Phe His Leu Arg Pro Arg Asp Leu Ile Ser Asn Ile Asn Val Ile Val

530 535 540

Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp

545 550 555 560

Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Ile

565 570 575

Gln Ser Ile Ile Ser Thr Leu Thr

580

<---

Claims (15)

1. An isolated IL-2 (interleukin-2) variant polypeptide that exhibits reduced binding to IL-2Rα (interleukin-2 receptor alpha) with reduced Treg (regulatory T cell) activity compared to the polypeptide shown in SEQ ID NO:3, but retains the ability to bind to and activate the IL-2Rβγ (interleukin-2 receptor beta gamma) complex, and wherein the IL-2 variant polypeptide comprises only two amino acid substitutions at amino acid residue positions P65 and S125 of SEQ ID NO:3; wherein the amino acid substitution is selected from the group consisting of a P65H, P65K, P65N, P65Q, P65R substitution at position 65, and a S125I substitution at position SEQ ID NO:3; or wherein the IL-2 variant polypeptide comprises only three amino acid substitutions at amino acid residue positions L19, P65 and S125 of SEQ ID NO:3, wherein the amino acid substitution is selected from the group consisting of a L19D, L19H, L19Q, L19Y substitution at position 19, a P65H, P65K, P65N, P65Q, P65R substitution at position 65, and a S125I substitution at position 125 of SEQ ID NO:3; or wherein the IL-2 variant polypeptide comprises only four amino acid substitutions at amino acid residue positions L19, P65, S125 and Q126 of SEQ ID NO: 3; wherein the amino acid substitution is selected from the group consisting of substitution L19H, L19Q, L19Y at position 19, substitution P65H, P65K, P65N, P65Q, P65R at position 65, substitution S125I at position 125, and substitution Q126E at position 126 of SEQ ID NO: 3. 2. The IL-2 variant polypeptide of claim 1, comprising only two amino acid substitutions at amino acid residue positions P65 and S125 of SEQ ID NO:3, which are a S125I substitution and one substitution selected from the group consisting of P65H, P65K, P65N, P65Q and P65R. 3. The IL-2 variant polypeptide of claim 1, comprising only three amino acid substitutions at amino acid residue positions L19, P65 and S125 of SEQ ID NO:3, which are substitution S125I, one substitution selected from the group consisting of substitution L19H, L19Q, L19Y, and one substitution selected from the group consisting of P65H, P65K, P65N, P65Q and P65R. 4. The IL-2 variant polypeptide of claim 1, comprising only four amino acid substitutions at amino acid residue positions L19, P65, S125 and Q126 of SEQ ID NO: 3, which are S125I and Q126E, one substitution selected from the group consisting of substitution L19H, L19Q and L19Y, and one substitution selected from the group consisting of P65H, P65K, P65N, P65Q and P65R. 5. The IL-2 variant polypeptide of any one of claims 1-4, comprising an amino acid sequence selected from the group consisting of the amino acid sequences presented in SEQ ID NO:44-51 and SEQ ID NO:111-120. 6. An isolated fusion protein having reduced binding to IL 2Rα with reduced activity against Tregs compared to the polypeptide shown in SEQ ID NO:3, but retaining the ability to bind to and activate the IL 2Rβγ complex, comprising (1) an IL-2 variant polypeptide according to any one of claims 1-5 and (2) a heterologous protein, wherein said fusion protein is in the form of a monomer or a dimer, wherein the heterologous protein is a targeting moiety in the form of an antibody, a heavy chain or a light chain of an antibody, or an antibody fragment, targeting a tumor-associated antigen (TAA). 7. An isolated fusion protein according to claim 6, wherein said IL-2 variant polypeptide is fused at its N-terminal amino acid to a C-terminal amino acid of a heterologous protein, possibly via a peptide linker, or wherein said IL-2 variant polypeptide is fused at its C-terminal amino acid to an N-terminal amino acid of a heterologous protein, possibly via a peptide linker. 8. The isolated fusion protein of claim 6, wherein the heterologous protein increases the half-life of the IL-2 variant polypeptide from circulation. 9. The isolated fusion protein of claim 6, wherein the heterologous protein is an antibody or antibody fragment to an immune checkpoint modulator. 10. The isolated fusion protein of claim 9, wherein the antibody is an antagonist antibody or fragment of an antagonist antibody to programmed cell death protein 1 (PD-1). 11. The isolated fusion protein of claim 10, wherein the antibody is an antagonist humanized PD-1 antibody selected from an antibody comprising the heavy chain and light chain amino acid sequences shown in SEQ ID NOs: 138 and 139; the heavy chain and light chain amino acid sequences shown in SEQ ID NOs: 140 and 141; the heavy chain and light chain amino acid sequences shown in SEQ ID NOs: 142 and 143; the heavy chain and light chain amino acid sequences shown in SEQ ID NOs: 144 and 145; and the heavy chain and light chain amino acid sequences shown in SEQ ID NOs: 146 and 147. 12. A pharmaceutical composition having reduced binding to IL 2Rα with reduced activity against Tregs compared to the polypeptide presented in SEQ ID NO:3, but retaining the ability to bind to the IL 2Rβγ complex and activate it, comprising an effective amount of the IL-2 variant polypeptide according to any one of claims 1-5 or the isolated fusion protein according to any one of claims 6-11 in a mixture with a pharmaceutically acceptable carrier. 13. Use of a pharmaceutical composition according to claim 12 in the treatment of cancer in a subject, comprising administering to the subject a therapeutically effective amount of said pharmaceutical composition. 14. Use of a pharmaceutical composition according to claim 12 in the treatment of cancer in a subject, comprising administering to the subject a therapeutically effective amount of said pharmaceutical composition, wherein the treatment further comprises administering a second therapeutic agent or method of action capable of treating cancer in the subject.