RU2815389C2 - Antibody-based proteins with cytokine grafts and methods of their use in cancer treatment - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to proteins based on antibodies with a grafted cytokine to activate immune cells, and can be used in medicine to treat cancer. The following is proposed: a fusion protein design based on an antibody containing a heavy chain variable region (VH) and a light chain variable region (VL), in which a mutant interleukin 2 (IL2) molecule is grafted onto HCDR1.

EFFECT: invention provides for production of an immunocytokine with selectivity for activation of CD8+ T-cells with a decrease in the activation of Treg-cells, an increased half-life and effectiveness in the treatment of cancer.

29 cl, 8 dwg, 2 tbl, 8 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit from U.S. Provisional Patent Application No. 62/510533, filed May 24, 2017, the contents of which are hereby incorporated by reference in their entirety.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[002] The present invention relates to cytokine-grafted antibody proteins that bind the low-affinity interleukin 2 (IL2) receptor and methods for treating cancer.

LIST OF SEQUENCES

[003] This application contains a sequence listing that was filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy in question, created on April 5, 2018, is named PAT057462-WO-PCT_SL.txt and is 69489 bytes in size.

BACKGROUND OF THE INVENTION

[004] IL2 was first cloned in 1983 (Taniguchi et al., Nature 1983, 302:305-310, Devos et al., Nucleic Acid Res. 1983, 11(13):4307-4323, Maeda et al. , Biochem Biophys Res Comm 1983, 115:1040-1047). The IL2 protein is 153 amino acids long with a signal peptide of amino acids 1-20 and folds into a structure of 4 antiparallel amphipathic alpha helices (Smith K.A., Science 1988, 240:1169-1176).

[005] IL2 mediates its biological effect by signaling through a high-affinity or low-affinity receptor (Kreig et al., PNAS 2010, 107(26)11906-11911). The high-affinity receptor is trimeric, consisting of IL2-Rα (CD25), IL2-Rβ (CD122) and IL2-Rγ (CD132). The low-affinity receptor is dimeric, consisting only of IL2-Rβ (CD122) and IL2-Rγ (CD132) chains. The low-affinity receptor binds IL2, but with 10- to 100-fold lower affinity than the trimeric high-affinity receptor, indicating that IL2-Rα (CD25) is important for increasing affinity but is not a component involved in signal transduction ( Kreig et al., supra). The expression of IL2 receptors also differs. The high-affinity IL2 receptor is expressed on activated T cells and CD4+/Foxp3+ regulatory T cells (Tregs). In contrast, the low-affinity IL2 receptor is found on CD8+ effector T cells and natural killer (NK) cells.

[006] Recombinant IL2 (rhIL2) was initially approved for clinical use in 1992 (Coventry et al., Cancer Mgt Res. 2012 4:215-221). Proleukin® (aldesleukin) is a modified IL2 that is aglycosylated, lacks an N-terminal alanine, and has a serine-substituted cysteine at amino acid position 125. Proleukin® was originally indicated as a therapeutic drug used in malignant melanoma and renal cell carcinoma, however, it has been used in other types of cancer such as colorectal cancer, breast cancer, lung cancer and mesothelioma (Coventry, supra). In a study of 259 patients with renal cell carcinoma conducted from 1986 to 2006, it was found that 23 patients had a complete response and 30 had a partial response (Klapper et al., Cancer 2008 113(2):293- 301). Overall, this represented a cumulative objective response rate of 20%, with complete tumor regression observed in 7% of patients with renal cell carcinoma (Klapper et al., supra).

[007] However, treating cancer with IL2 is not without side effects. In a study of 259 patients, capillary leakage/transudation, vasodilatation, and oliguria were noted. There were also grade 3 and grade 4 infections, both due to the use of catheters, and a general infection associated with neutrophil dysfunction (Klapper et al., supra). The literature on Proleukin® has noted that Proleukin® has been associated with exacerbation of autoimmune diseases and inflammatory disorders such as Crohn's disease, scleroderma, thyroiditis, inflammatory arthritis, diabetes mellitus, oculobulbar myasthenia gravis, crescentic IgA glomerulonephritis, cholecystitis, cerebral vasculitis, syndrome Stevens-Johnson and bullous pemphigoid.

[008] The discovery that Treg cells constitutively expressed the high affinity IL2 receptor and were dependent on IL2 for their survival and function pointed to the reason for the observation of this side effect (D'Cruz et al., Nat. Immuno. 2005, 6:1152- 1159). This illustrates the need for IL2-based therapeutics with improved pharmacokinetic properties and selective activation of CD8+ T cells through the low-affinity receptor without activating Treg cells through the high-affinity receptor, as this allows the treatment of cancer without the unwanted side effects observed with Proleukin®.

DESCRIPTION

[009] The present invention provides IL2 grafted onto the CDR sequence of an antibody having preferential therapeutic profiles compared to molecules known and used in clinical practice. In particular, the provided cytokine-grafted antibody protein compositions provide an increase or maintenance of CD8+ effector T cell levels while decreasing Treg cell activity. In addition, the contemplated compositions exhibit improved half-life, stability, and manufacturing feasibility compared to recombinant human IL2 formulations such as Proleukin®. Thus, the present invention provides cytokine-grafted antibody proteins that bind to and promote preferential signaling through the low-affinity IL2 receptor with reduced binding to the high-affinity IL2 receptor. Cytokine-grafted antibody proteins are provided comprising (i) an immunoglobulin heavy chain sequence containing a heavy chain variable region (VH), and (ii) an immunoglobulin light chain sequence containing a light chain variable region (VL), wherein an IL2 molecule is grafted onto complementarity determining region (CDR), VH or VL of the antibody.

[0010] Embodiments of the present invention provide cytokine-grafted antibody proteins comprising:

(a) a heavy chain variable region (VH) containing the complementarity determining regions (CDRs), HCDR1, HCDR2, HCDR3; And

(b) a light chain variable region (VL) containing LCDR1, LCDR2, LCDR3; And

(c) an interleukin 2 (IL2) molecule grafted onto the VH or VL CDR.

[0011] A cytokine-grafted antibody protein comprising an IL2 molecule grafted onto a heavy chain CDR.

[0012] A cytokine-grafted antibody protein, wherein the IL2 molecule is grafted to a region selected from complementarity-determining region 1 (HCDR1), complementarity-determining region 2 (HCDR2), or complementarity-determining region 3 (HCDR3).

[0013] A cytokine-grafted antibody protein containing an IL2 molecule grafted onto HCDR1.

[0014] A cytokine-grafted antibody protein comprising an IL2 molecule grafted onto a light chain CDR.

[0015] A cytokine-grafted antibody protein, wherein the IL2 molecule is grafted to a region selected from complementarity-determining region 1 (LCDR1), complementarity-determining region 2 (LCDR2), or complementarity-determining region 3 (LCDR3).

[0016] A cytokine-grafted antibody protein containing an IL2 molecule containing a mutation that causes a decrease in the affinity of the IL2 molecule for the high-affinity IL2 receptor.

[0017] A cytokine-grafted antibody protein, wherein the cytokine-grafted antibody protein stimulates the proliferation of CD8+ effector T cells to a greater extent than recombinant IL2 or Proleukin®.

[0018] A cytokine-grafted antibody protein, wherein the cytokine-grafted antibody protein stimulates Treg cell proliferation to a lesser extent than recombinant IL2 or Proleukin®.

[0019] A cytokine-grafted antibody protein, wherein the cytokine-grafted antibody protein stimulates NK cell proliferation to a greater extent than recombinant IL2 or Proleukin®.

[0020] A cytokine-grafted antibody protein, wherein the cytokine-grafted antibody protein has a longer half-life than recombinant IL2 or Proleukin®.

[0021] A cytokine-grafted antibody protein, wherein the IL2 molecule consists of SEQ ID NO:4.

[0022] A cytokine-grafted antibody protein, wherein the IL2 molecule consists of SEQ ID NO:6.

[0023] A cytokine-grafted antibody protein containing an IgG antibody heavy chain.

[0024] A cytokine-grafted antibody protein, wherein the IgG is selected from IgG1, IgG2 and IgG4.

[0025] A cytokine-grafted antibody protein wherein the CDR target binding specificity is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98 %, 99%, or 100% due to the grafted IL2 molecule.

[0026] A cytokine-grafted antibody protein wherein the CDR target binding specificity is maintained at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98 %, 99%, or 100% in the presence of the grafted IL2 molecule.

[0027] A cytokine-grafted antibody protein wherein the binding specificity of the CDR is different from the binding specificity of the IL2 molecule.

[0028] A cytokine-grafted antibody protein wherein the binding specificity of the CDR is directed to a non-human target.

[0029] A cytokine-grafted antibody protein where the non-human antigen is a virus.

[0030] A cytokine-grafted antibody protein, wherein the virus is respiratory syncytial virus (RSV).

[0031] A cytokine-grafted antibody protein, wherein the RSV is selected from subgroup A RSV and subgroup B RSV.

[0032] A cytokine-grafted antibody protein, wherein the backbone portion of the cytokine-grafted antibody protein is humanized or human.

[0033] Embodiments of the present invention provide cytokine-grafted antibody proteins comprising: (i) a heavy chain variable region that contains (a) HCDR1 of SEQ ID NO: 13, (b) HCDR2 of SEQ ID NO: 14 , (c) HCDR3 of SEQ ID NO:15, and a light chain variable region which contains (d) LCDR1 of SEQ ID NO:29, (e) LCDR2 of SEQ ID NO:30, and (f) LCDR3 of SEQ ID NO: :31; or (ii) a heavy chain variable region which comprises (a) HCDR1 of SEQ ID NO:45, (b) HCDR2 of SEQ ID NO:46, (c) HCDR3 of SEQ ID NO:47; and a light chain variable region which contains (d) LCDR1 of SEQ ID NO:61, (e) LCDR2 of SEQ ID NO:62 and (f) LCDR3 of SEQ ID NO:63.

[0034] Embodiments of the present invention provide cytokine-grafted antibody proteins comprising: (i) a heavy chain variable region (VH), which contains SEQ ID NO:19, and a light chain variable region (VL), which contains SEQ ID NO: 35; or (ii) a heavy chain variable region (VH) that contains SEQ ID NO: 51, and a light chain variable region (VL) that contains SEQ ID NO: 67.

[0035] A cytokine-grafted antibody protein, wherein the antibody contains a modified Fc region corresponding to reduced effector function.

[0036] A cytokine-grafted antibody protein, wherein the modified Fc region contains a mutation selected from one or more of D265A, P329A, P329G, N297A, L234A, and L235A.

[0037] A cytokine-grafted antibody protein, wherein the modified Fc region contains a combination of mutations selected from one or more of D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[0038] Embodiments of the present invention provide cytokine-grafted antibody proteins comprising HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, HCDR3 of SEQ ID NO: 15, LCDR1 of SEQ ID NO: 29, LCDR2 under SEQ ID NO: 30, LCDR3 under SEQ ID NO: 31, a modified Fc region containing the D265A/P329A mutation, where the cytokine-grafted antibody protein stimulates Treg cell activation to a lesser extent compared to recombinant IL2 or Proleukin ®.

[0039] Embodiments of the present invention provide cytokine-grafted antibody proteins comprising HCDR1 of SEQ ID NO: 45, HCDR2 of SEQ ID NO: 46, HCDR3 of SEQ ID NO: 47, LCDR1 of SEQ ID NO: 61, LCDR2 under SEQ ID NO: 62, LCDR3 under SEQ ID NO: 63, a modified Fc region containing the D265A/P329A mutation, where the cytokine-grafted antibody protein stimulates Treg cell activation to a lesser extent compared to recombinant IL2 or Proleukin ®.

[0040] Embodiments of the present invention provide isolated nucleic acids encoding a cytokine-grafted antibody protein comprising: (i) a heavy chain of SEQ ID NO: 22 and/or a light chain of SEQ ID NO: 38 or (ii) the heavy chain of SEQ ID NO: 54 and/or the light chain of SEQ ID NO: 70.

[0041] Embodiments of the present invention provide recombinant host cells suitable for producing a cytokine-grafted antibody protein comprising nucleic acids disclosed herein encoding polypeptide heavy and light chains of the protein and optionally a secretion signal.

[0042] A recombinant host cell that is a mammalian cell line.

[0043] A recombinant host cell, wherein the mammalian cell line is a CHO cell line.

[0044] Embodiments of the present invention provide pharmaceutical compositions comprising a cytokine-grafted antibody protein disclosed herein and one or more pharmaceutically acceptable carriers.

[0045] Embodiments of the present invention provide methods for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a cytokine-grafted antibody protein or pharmaceutical composition disclosed herein.

[0046] A method of treating cancer, wherein the cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer and lymphoma.

[0047] A method of treating cancer, wherein the cytokine-grafted antibody protein or pharmaceutical composition is administered in combination with another therapeutic agent.

[0048] A method of treating cancer, wherein the therapeutic agent is another protein-based antibody grafted with a cytokine.

[0049] A method of treating cancer, wherein the therapeutic agent is an immune checkpoint inhibitor.

[0050] A method of treating cancer, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA , BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0051] Embodiments of the present invention provide methods for promoting expansion of CD8+ effector T cells in a patient in need thereof, comprising administering to the patient a cytokine-grafted antibody protein or pharmaceutical composition disclosed herein.

[0052] A method for promoting expansion of CD8+ effector T cells, wherein CD8+ effector T cells are expanded and Treg cells are not.

[0053] A method for promoting expansion of CD8+ effector T cells, wherein CD8+ effector T cells are expanded and NK cells are not.

[0054] A method of promoting expansion of CD8+ effector T cells, further comprising administering an immune checkpoint inhibitor.

[0055] A method for promoting expansion of CD8+ effector T cells, wherein the immune checkpoint is selected from the group consisting of PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM -5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0056] Embodiments of the present invention provide methods for using in the treatment of cancer a cytokine-grafted antibody protein comprising: (i) a heavy chain variable region that contains (a) HCDR1 of SEQ ID NO: 13, (b) HCDR2 of SEQ ID NO: 14, (c) HCDR3 of SEQ ID NO: 15, and a light chain variable region that contains (d) LCDR1 of SEQ ID NO: 29, (e) LCDR2 of SEQ ID NO: 30, and (f) LCDR3 under SEQ ID NO: 31; and (ii) a heavy chain variable region which contains (a) HCDR1 of SEQ ID NO: 45, (b) HCDR2 of SEQ ID NO: 46, (c) HCDR3 of SEQ ID NO: 47; and a light chain variable region which contains (d) LCDR1 of SEQ ID NO: 61, (e) LCDR2 of SEQ ID NO: 62 and (f) LCDR3 of SEQ ID NO: 63, in the treatment of cancer.

[0057] Use of a cytokine-grafted antibody protein in the treatment of cancer, wherein the cytokine-grafted antibody protein is administered in combination with another therapeutic agent.

[0058] Use of a cytokine-grafted antibody protein, wherein the therapeutic agent is an immune checkpoint inhibitor antagonist.

[0059] Application wherein the immune checkpoint inhibitor antagonist is selected from the group consisting of PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR.

[0060] In certain embodiments, the cytokine-grafted antibody protein comprises the Fc region of an IgG antibody. In specific embodiments, the immunoglobulin is selected from the Fc region of the IgG1, IgG2, or IgG4 subclass. The antibody, antibody fragment, or antigen-binding molecule optionally contains at least one modification that modulates (ie, increases or decreases) the binding of the antibody or antibody fragment to the Fc receptor. The immunoglobulin heavy chain may optionally contain a modification conferring a modified effector function. In specific embodiments, the immunoglobulin heavy chain may contain a mutation conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A /L235A.

[0061] In some embodiments, the cytokine-grafted antibody protein also contains modifications to the IL2 portion of the molecule. The modifications may be single amino acid changes, single amino acid deletions, multiple amino acid changes, and multiple amino acid deletions. Such changes in the IL2 cytokine portion of the molecule may provide a decrease in the affinity of the cytokine-grafted antibody protein for the high-affinity IL2 receptor.

[0062] In addition, the present invention provides polynucleotides encoding at least a protein heavy chain and/or a light chain of a cytokine-grafted antibody protein described herein. In another related aspect, host cells are provided that are suitable for producing the cytokine-grafted antibody protein described herein. In specific embodiments, the host cells contain nucleic acids encoding a cytokine-grafted antibody polypeptide light chain and/or heavy chain protein. In yet another aspect, methods are provided for producing cytokine-grafted antibody proteins, comprising culturing the host cells provided herein under conditions suitable for expression, production, and secretion of the cytokine-grafted antibody protein, and recovering the cytokine-grafted antibody protein. antibodies grafted with cytokine from culture. In a further aspect, the present invention further provides kits comprising the cytokine-grafted antibody protein described herein.

[0063] In another related aspect, the present invention further provides compositions comprising a cytokine-grafted antibody protein described herein and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides pharmaceutical compositions comprising a cytokine-grafted antibody protein for administration to an individual.

[0064] In another aspect, methods are provided for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a cytokine-grafted antibody protein described herein. In a further aspect, a cytokine-grafted antibody protein is provided for use in the treatment or prevention of cancer in an individual.

[0065] In some embodiments, the patient has a cell proliferation disorder or cancer, such as melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, and lymphoma.

DEFINITIONS

[0066] "Antibody" refers to a molecule from the immunoglobulin family containing a tetrameric structural unit. Each tetramer consists of two identical pairs of polypeptide chains, with each pair having one "light" chain (approximately 25 kDa) and one "heavy" chain (approximately 50-70 kDa) joined by a disulfide bond. Well-known immunoglobulin genes include the κ, λ, α, γ, δ, ε and μ constant region genes, as well as a large number of immunoglobulin variable region genes. Light chains are classified as κ or λ. Heavy chains are classified as γ, μ, α, δ, or ε, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. Antibodies can be of any isotype/class (eg, IgG, IgM, IgA, IgD, and IgE) or any subclass (eg, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2).

[0067] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used in a structural sense and a functional sense. The N-terminus of each chain defines a variable (V) region or domain of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The terms "light chain variable region" ( _VL ) and "heavy chain variable region" ( _VH ) respectively refer to these regions of the light and heavy chains. When V _H and V _L pair with each other, one antigen-binding site is formed. In addition to V regions, both heavy chains and light chains contain a constant (C) region or domain. The secreted form of the immunoglobulin C region consists of three C domains CH1, CH2, CH3, optionally CH4 (Cμ) and a hinge region. The membrane-bound form of the immunoglobulin C region also has membrane and intracellular domains. Each light chain has a V _L at its N-terminus, followed by a constant domain (C) at its other end. The light chain (CL) and heavy chain constant domains (CH1, CH2 or CH3) confer important biological properties such as secretion, placental translocation, Fc receptor binding, complement fixation, etc. It is generally accepted that constant region domain numbers increase as they move away from the antigen-binding site or amino terminus of the antibody. The N-terminus is the variable region, and the C-terminus is the constant region; The CH3 and CL domains actually contain the carboxy-terminal heavy and light chain domains, respectively. VL is aligned with VH, and CL is aligned with the first heavy chain constant domain. As used herein, “antibody” includes traditional antibody structures and antibody variations. Thus, cytokine-grafted antibody proteins, full-length antibodies, chimeric antibodies, humanized antibodies, human antibodies, and fragments of such antibodies are included within the scope of this concept.

[0068] Antibodies exist as intact immunoglobulin chains or as a series of well-studied antibody fragments resulting from cleavage by various peptidases. The term "antibody fragment" as used herein refers to one or more portions of an antibody in which the six CDRs are retained. Thus, for example, pepsin cleaves the antibody below the disulfide bonds in the hinge region to form F(ab)' ₂ , a Fab' dimer, which as such is a light chain linked to V _{H -CH} 1 by a disulfide bond. F(ab)' ₂ can be reduced under mild conditions to break the disulfide bond at the hinge region, resulting in the conversion of the F(ab)' ₂ dimer to Fab' monomer. A Fab' monomer is essentially a Fab with part of the hinge region (Paul, Fundamental Immunology 3d ed. (1993)). Although various antibody fragments are defined in terms of cleavage of the intact antibody, one skilled in the art will appreciate that such fragments can be synthesized de novo chemically or using recombinant DNA technology. As used herein, “antibody fragment” refers to one or more parts of an antibody produced by modification of whole antibodies or synthesized de novo using recombinant DNA technologies that retain binding specificity and functional activity. Examples of antibody fragments include Fv fragments, single chain antibodies (ScFv), Fab, Fab', Fd (VH and CH1 domains), dAb (VH and dedicated CDR); as well as multimeric variants of these fragments (eg, F(ab') ₂ ) with the same binding specificity. Cytokine-grafted antibody proteins may also contain antibody fragments necessary to achieve the required binding specificity and activity.

[0069] A "Fab" domain, as used herein, comprises a heavy chain variable domain, a CH1 constant region domain, a light chain variable domain, and a light chain constant region CL domain. The interaction of the domains is stabilized by a disulfide bond between the CH1 and CL domains. In some embodiments, the heavy chain domains in the Fab are arranged from N-terminus to the C-terminus in the order VH-CH, and the light chain domains in the Fab are arranged from the N-terminus to the C-terminus in the order VL-CL. In some embodiments, the heavy chain domains in the Fab are arranged from N-terminus to the C-terminus in the order CH-VH, and the light chain domains in the Fab are arranged in the order CL-VL. Although the Fab fragment has historically been identified by digestion of intact immunoglobulin with papain, in the context of the present invention, a "Fab" is typically generated recombinantly by any method. Each Fab fragment is monovalent with regard to antigen binding, i.e. it has one antigen-binding site.

[0070] “Complementarity determining domains” or “complementarity determining regions” (“CDRs”) refer interchangeably to the V _L and V _H hypervariable regions. CDRs are the region where antibody chains bind to a target protein and have specificity for that target protein. Each human V _L or V _H has three CDRs (CDR1-3, numbered sequentially from the N-terminus), comprising approximately 15-20% of the variable domains. CDRs are structurally complementary to the epitope of the target protein, and thus are directly responsible for binding specificity. The remaining stretches in V _L or V _H , the so-called framework regions (FR), are characterized by less variability in the amino acid sequence (Kuby, Immunology, 4th ed., Chapter 4. WH Freeman & Co., New York, 2000).

[0071] The positions of the CDRs and framework regions can be determined using various definitions well known in the art, for example, according to Kabat, Chothia and AbM (see, for example, Kabat et al. 1991 Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Johnson et al., Nucleic Acids Res., 29:205-206 (2001); Chothia and Lesk, J. Mol. Biol., 196:901- 917 (1987); Chothia et al., Nature, 342:877-883 (1989); Chothia et al., J. Mol. Biol., 227:799-817 (1992); Al-Lazikani et al., J Mol. Biol., 273:927-748 (1997)). Definitions of antigen-binding active sites are also described in the following sources: Ruiz et al., Nucleic Acids Res., 28:219-221 (2000); and Lefranc, M.P., Nucleic Acids Res., 29:207-209 (2001); (ImMunoGeneTics (IMGT) numbering) Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003); MacCallum et al., J. Mol. Biol., 262:732-745 (1996); and Martin et al., Proc. Natl. Acad. Sci. USA, 86:9268-9272 (1989); Martin et al., Methods Enzymol., 203:121-153 (1991); and Rees et al., in Sternberg M.J.E. (ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

[0072] According to Kabat, the amino acid residues of the CDRs in V _H are numbered 31-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3); and the CDR amino acid residues in V _L are numbered 24-34 (LCDR1), 50-56 (LCDR2) and 89-97 (LCDR3). According to Chothia, the amino acid CDRs in _VH are numbered 26-32 (HCDR1), 52-56 (HCDR2) and 95-102 (HCDR3); and the amino acid residues in V _L are numbered 26-32 (LCDR1), 50-52 (LCDR2) and 91-96 (LCDR3). When combining the Kabat and Chothia definitions of CDRs, CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2) and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 ( LCDR2) and 89-97 (LCDR3) in human VL.

[0073] “Antibody light chain variable region” or “antibody heavy chain variable region” as used herein refers to a polypeptide containing V _L or V _H , respectively. Endogenous V _L is encoded by the V (variable) and J (junction) gene segments, and endogenous V _H is encoded by V, D (additional) and J. Each of the V _L or V _H contains CDRs as well as framework regions (FR). The term "variable region" or "V region" interchangeably refers to the heavy or light chain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The V region can be naturally occurring, recombinant or synthetic. In this application, light chains of antibodies and/or heavy chains of antibodies in some cases may be collectively referred to as "antibody chains". As provided and further described herein, “antibody light chain variable region” or “antibody heavy chain variable region” and/or “variable region” and/or “antibody chain” optionally comprises a cytokine polypeptide sequence included in the CDR.

[0074] The C-terminal region of an immunoglobulin heavy chain, containing, for example, CH2 and CH3 domains, is referred to herein as an "Fc" domain. "Fc region" as used herein refers to the constant region of an antibody, excluding the first immunoglobulin constant region domain (CH1). Fc refers to the last two immunoglobulin constant region domains of IgA, IgD and IgG, the last three immunoglobulin constant region domains of IgE and IgM, and the flexible hinge region in the N-terminal direction of these domains. In the case of IgA and IgM, the Fc may contain a J chain. In the case of IgG, the Fc contains the Cγ2 and Cγ3 immunoglobulin domains and the hinge region between Cγ1 and Cγ. It will be appreciated in the art that the boundaries of the Fc region may vary, however, the human IgG heavy chain Fc region is generally defined to contain residues C226 or P230 at its carboxyl terminus when using EU index numbering as set forth in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, Va.). "Fc region" may refer to the region in an isolated state or to the region as a constituent of an antibody or antibody fragment. "Fc region" includes naturally occurring allelic variants of the Fc region, for example, the CH2 and CH3 regions, including, for example, those having modifications that provide modulation of effector function. Fc regions also include variants that do not result in changes in biological function. For example, one or more amino acids are removed from the N-terminus or C-terminus of the Fc region of an immunoglobulin without significant loss of biological function. For example, in certain embodiments, the C-terminal lysine is modified, substituted, or removed. In specific embodiments, one or more C-terminal residues in the Fc region are altered or deleted. In certain embodiments, one or more C-terminal residues in the Fc (eg, terminal lysine) are removed. In certain other embodiments, one or more C-terminal residues in the Fc are replaced with an alternative amino acid (eg, the terminal lysine is replaced). Such variants are selected according to general rules known in the art so that the effect on activity is minimal (see, eg, Bowie, et al., Science 247:306-1310, 1990). The Fc domain is the portion of immunoglobulin (Ig) that is recognized by cellular receptors such as FcR and to which complement-activating protein C1q binds. The lower hinge region, which is encoded in the 5' portion of the CH2 exon, provides flexibility for the antibody to bind to FcR receptors.

[0075] A "chimeric antibody" is an antibody molecule in which (a) a constant region or portion thereof is altered, substituted, or exchanged such that an antigen-binding region (variable region) is linked to a constant region of a different or altered class having a different or altered effector function and/or derived from a different or altered species, or with an entirely different molecule that imparts new properties to the chimeric antibody, such as an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region or part thereof is altered, replaced or exchanged for a variable region having a different or altered antigenic specificity.

[0076] A “humanized” antibody is an antibody that retains the reactivity (eg, binding specificity, activity) of a non-human antibody, but is less immunogenic in humans. This can be achieved, for example, by retaining non-human CDR regions and replacing the remaining portions of the antibody with human counterparts. See, for example, Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988); Padlan, Molec. Immun., 28:489-498 (1991); Padlan, Molec. Immun., 31(3):169–217 (1994).

[0077] "Human antibody" includes antibodies having variable regions in which both framework regions and CDR regions are derived from sequences of human origin. In addition, if the antibody contains a constant region, then the constant region is also derived from such human sequences, for example, human germline sequences, or mutant variants of human germline sequences, or an antibody containing consensus framework sequences obtained from analysis of human framework sequences areas, for example as described in Knappik et al., J. Mol. Biol. 296:57-86, 2000. Human antibodies may contain amino acid residues that are not encoded by human sequences (eg, mutations introduced through random or site-directed mutagenesis in vitro or through somatic mutations in vivo, or a conservative substitution that promotes stability or production).

[0078] The term “corresponding human germline sequence” refers to a nucleic acid sequence encoding a human variable region amino acid sequence or subsequence that has the highest established amino acid sequence identity to a reference amino acid sequence or variable region subsequence compared to all other known amino acid sequences variable region encoded by the human germline immunoglobulin variable region gene sequences. A corresponding human germline sequence may also refer to the human variable region amino acid sequence or subsequence having the highest amino acid sequence identity to a reference variable region amino acid sequence or subsequence compared to all other variable region amino acid sequences evaluated. The corresponding human germline sequence may be framework regions only, complementarity determining regions only, framework regions and complementarity determining regions, a variable segment (defined above), or other combinations of sequences or subsequences that contain a variable region. Sequence identity can be determined using methods described herein, for example, by aligning two sequences using BLAST, ALIGN, or other alignment algorithm known in the art. The corresponding human nucleic acid sequence or germline amino acid sequence may have at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity sequences with a reference nucleic acid sequence or a variable region amino acid sequence.

[0079] The term "valency" as used herein refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or a specific site within a target molecule. If a polypeptide contains more than one target binding site, then each target binding site may specifically bind the same or different molecules (eg, may bind to different molecules, eg, different antigens, or different epitopes on the same molecule). A traditional antibody, for example, has two binding sites and is bivalent; "trivalent" and "tetravalent" refer to the presence, respectively, of three binding sites and four binding sites in the antibody molecule. Cytokine-grafted antibody proteins can be monovalent (ie, bind one target molecule), bivalent, or polyvalent (ie, bind more than one target molecule).

[0080] The phrase “specifically binds” or “binding specificity,” when used in the context of describing the interaction between a target (e.g., a protein) and a cytokine-coupled antibody protein, refers to the binding reaction that is determinative of the presence of the target in a heterogeneous population of proteins and other biological substances, for example, in a biological sample, such as a sample of blood, serum, blood plasma or tissue. Thus, under certain specified conditions, a cytokine-grafted antibody protein with a particular binding specificity binds to a particular target at a level that is at least twice the background level and does not substantially bind in significant amounts to other targets present in the sample . In one embodiment, under established conditions, a cytokine-grafted antibody protein with a defined binding specificity binds to a particular antigen at a level at least ten (10) times the background level and does not substantially bind to other targets, present in the sample. Specific binding to a cytokine-grafted antibody protein under such conditions may require that the cytokine-grafted antibody protein be selected for its specificity for a particular target protein. As used herein, specific binding involves cytokine-grafted antibody proteins that selectively bind to the low-affinity human IL2 receptor, and does not involve cytokine-grafted antibody proteins that cross-react, for example, with other members of the cytokine receptor superfamily. In some embodiments, such cytokine-coupled antibody proteins are selected that selectively bind to the low-affinity human IL2 receptor and cross-react with non-human IL2R (eg, cynomolgus IL2R). In some embodiments, such antibody graft proteins are selected that selectively bind to the low affinity human IL2 receptor and react with an additional target. A variety of formats can be used to select proteins based on cytokine-grafted antibodies that are specifically reactive to a particular target protein. For example, ELISA enzyme-linked immunoassays are commonly used to screen for antibodies having specific immunoreactivity for a protein (see, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual (1998), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). A specific or selective binding reaction will typically produce a signal that is at least two times the background signal, and more typically at least 10-100 times the background signal.

[0081] The term "equilibrium dissociation constant (K _D , M)" refers to the dissociation rate constant (k _d , time ^-1 ) divided by the association rate constant ( _ka , time ^-1 , M ^-1 ). Equilibrium dissociation constants can be measured using any method known in the art. Typically, cytokine-grafted antibody proteins will have an equilibrium dissociation constant of less than about 10 ^-7 or 10 ^-8 M, such as less than about 10 ^-9 M or 10 ^-10 M, in some embodiments less than approximately 10 ^-11 M, 10 ^-12 M or 10 ^-13 M.

[0082] As used herein, the term “epitope” or “binding region” refers to a domain in a protein antigen that is responsible for specific binding between the antibody CDR and the protein antigen.

[0083] As used herein, the term “cytokine receptor-binding region” refers to a domain in a portion of a cytokine-grafted antibody protein that is a grafted cytokine that is responsible for the specific binding between the grafted cytokine and its receptor (eg, the low-affinity IL2 receptor). There is at least one such cytokine receptor binding region present in each cytokine-grafted antibody protein, and each of the binding regions may be identical to or different from the others.

[0084] The term "agonist" interchangeably refers to an antibody capable of activating a receptor to induce a complete or partial receptor-mediated response. For example, a low-affinity IL2 receptor agonist binds to the low-affinity IL2 receptor and induces IL2-mediated intracellular signaling, cell activation, and/or proliferation of CD8+ effector T cells and NK cells. The agonist, which is a cytokine-grafted antibody protein, stimulates signaling through the low-affinity IL receptor in some respects similar to the native IL2 ligand. Binding of IL2 to the low-affinity IL2 receptor induces activation of Jak1 and Jak2, leading to phosphorylation of STAT5. In some embodiments, the agonist, which is a cytokine-grafted antibody protein, can be identified by its ability to bind to the low-affinity IL2 receptor and induce STAT5 phosphorylation and/or proliferation of CD8+ effector T cells or NK cells.

[0085] The terms "IL2" or "interleukin 2" or "interleukin-2" or "IL-2" interchangeably refer to a member of the alpha-helical cytokine family, where the native protein functions in regulating and maintaining inflammatory processes. A feature of IL2 is that the N- and C-termini are located close to each other in space, which makes the IL2 cytokine protein suitable for grafting onto an antibody. IL2, containing residues 21-153 of the full-length native human protein, is used as a component of agonists, which are cytokine-grafted antibody proteins. Human IL2 disclosed herein is characterized over its entire length to be at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2 and retains the preferred agonistic activity of the cytokine-grafted antibody proteins described herein, and has been published under GenBank accession number NP_000577. SEQ ID NO: 1 is the cDNA sequence of human IL2. The human IL2 nucleic acid encoding the IL2 protein disclosed herein is characterized over its entire length by at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, or 100% sequence identity to the nucleic acid sequence SEQ ID NO:1, and was published under GenBank accession number NM_000586.

[0086] The term “cytokine-grafted antibody protein” or “cytokine-grafted antibody” or “grafted” means that at least one cytokine is included directly in the CDR of the antibody, interrupting the CDR sequence. The cytokine may be included in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3. The cytokine may be included in HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3 and is included near the N-terminal CDR sequence or near the C-terminal CDR sequence. The cytokine included in the CDR may disrupt the specific binding of a portion of the antibody to the original target protein, or the cytokine-grafted antibody protein may retain its specific binding to its target protein. Exemplary cytokines include, but are not limited to, IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-12, IFN-α , IFN-β, IFN-γ, GM-CSF, MIP-1α, MIP-1β, TGF-β, TNF-α, and TNF-β. It is also possible to graft a cytokine onto a specific CDR of one antibody arm and graft another distinct cytokine onto a CDR of another antibody arm. For example, grafting IL2 onto HCDR1 of one arm of an antibody and grafting IL-7 onto LCDR1 of another arm of a cytokine-grafted antibody protein can create a bifunctional cytokine-grafted antibody protein.

[0087] The term “isolated” when applied to a nucleic acid or protein means that the nucleic acid or protein is substantially free of other cellular components with which it is naturally associated. They are preferably in a homogeneous state. They can be in a dry state or in an aqueous solution. Purity and uniformity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein, which is the predominant molecular species present in the preparation, is essentially purified. In particular, the isolated gene is separated from open reading frames that flank the gene and encode a protein different from that encoded by the gene of interest. The term "purified" means that the nucleic acid or protein produces essentially a single band on an electrophoretic gel. In particular, this means that the nucleic acid or protein is at least 85% pure, more preferably at least 95% pure, and most preferably at least 99% pure.

[0088] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acids (DNA) or ribonucleic acids (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term covers nucleic acids containing known analogues of naturally occurring nucleotides that have binding properties similar to those of the reference nucleic acid and are metabolized by a mechanism similar to that of naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly includes variants thereof with conservative modifications (eg, degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as the explicitly stated sequence. In particular, degenerate codon substitutions can be accomplished by creating sequences in which the third position of one or more selected (or all) codons is replaced with any of the canonical bases and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0089] The terms "polypeptide", "peptide" and "protein" are used interchangeably herein and refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to polymers of naturally occurring amino acids and a polymer of non-naturally occurring amino acids.

[0090] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics, which function similarly to naturally occurring amino acids. Naturally occurring amino acids include amino acids encoded by the genetic code, as well as those that have been subsequently modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogues refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e. have an α-carbon atom, which is bonded to a hydrogen atom, a carboxyl group, an amino group and an R-group, for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogues have modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of an amino acid, but that function similarly to a naturally occurring amino acid.

[0091] “Conservatively modified variants” refer to both amino acid sequences and nucleic acid sequences. With respect to specific nucleic acid sequences, variants with conservative modifications refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, substantially identical sequences. Due to the degeneracy of the genetic code, any given protein is encoded by a large number of functionally identical nucleic acids. For example, the codons GCA, GCC, GCG and GCU all code for the amino acid alanine. Thus, at each position at which the codon specifies alanine, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variants are “silent variants,” which are one type of variant with conservative modifications. Herein, each nucleic acid sequence that encodes a polypeptide also describes each possible silent nucleic acid variant. One skilled in the art will appreciate that every codon in a nucleic acid (except AUG, which is typically the only codon for methionine, and TGG, which is typically the only codon for tryptophan) can be modified to produce a functionally identical molecule. Accordingly, each silent nucleic acid variant that encodes a polypeptide is implicitly defined in each sequence described.

[0092] With respect to amino acid sequences, one skilled in the art will recognize that individual substitutions, deletions, or additions in a nucleic acid, peptide, polypeptide, or protein sequence that alter, add, or delete one amino acid or a small percentage of amino acids in the encoded sequences are classified as a “conservative modification variant” if the change results in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables that list functionally similar amino acids are well known in the art. Such variants with conservative modifications complement and do not exclude polymorphic variants, interspecific homologues and alleles. Each of the following eight groups contains amino acids that are conservative substitutions for each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine (C), methionine (M) (see, for example, Creighton, Proteins (1984)).

[0093] "Percentage sequence identity" is determined by comparing two optimally aligned sequences in a comparison window, wherein to optimally align the two sequences, a portion of the polynucleotide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence (for example, a polypeptide) that does not contain additions or deletions. The percentage is calculated by determining the number of positions at which an identical nucleic acid base or amino acid residue occurs in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. .

[0094] The terms “identical” or “percent identity” when applied to two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same sequence. Two sequences are "substantially identical" if the two sequences have a certain percentage of amino acid residues or nucleotides that are the same (i.e., at least 85%, 90%, 91%, 92%, 93%, 94% identical) , 95%, 96%, 97%, 98%, 99%, or 100% sequence identity in a defined region or, if not specified, in the entire sequence of the reference sequence) when compared and aligned to achieve maximum fit within the comparison window or defined region when measured by one of the following sequence comparison algorithms or by manual alignment and visual inspection. The present invention provides polypeptides or polynucleotides that are substantially identical, respectively, to the polypeptides or polynucleotides exemplified herein (e.g., the variable regions exemplified by any of SEQ ID NO: 19, SEQ ID NO: 35, SEQ ID NO: 51 or SEQ ID NO: 67). The identity occurs in a region having a length of at least about 15, 25 or 50 nucleotides, or more preferably in a region having a length of 100-500 or 1000 or more nucleotides, or over the entire length of the reference sequence. With respect to amino acid sequences, identity or substantial identity may exist in a region having a length of at least 5, 10, 15 or 20 amino acids, optionally having a length of at least about 25, 30, 35, 40, 50, 75 or 100 amino acids , optionally having a length of at least about 150, 200 or 250 amino acids, or the entire length of the reference sequence. For shorter amino acid sequences, such as amino acid sequences of 20 amino acids or fewer, significant identity exists if one or two amino acid residues are conservatively substituted in accordance with the conservative substitutions defined herein.

[0095] When comparing sequences, typically one sequence serves as a reference sequence to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into the computer, the coordinates of the subsequences are set, if necessary, and the software parameters of the algorithm for sequence analysis are set. The default program parameters can be used, or alternative parameters can be set. The sequence comparison algorithm then calculates percent sequence identity values for the test sequences compared to the reference sequence based on the software parameters.

[0096] A "comparison window" as used herein includes a reference to a segment of any number of contiguous positions selected from the group consisting of from 20 to 600, typically from about 50 to about 200, more commonly from about 100 to about 150 , in which a sequence can be compared to a reference sequence with the same number of adjacent positions after the two sequences have been subjected to an optimal alignment. Methods for aligning sequences for comparison purposes are well known in the art. Optimal sequence alignment for comparison purposes can be achieved, for example, using the Smith-Waterman local homology search algorithm, (1970) Adv. Appl. Math. 2:482c, using the Needleman-Wunsch homology region alignment algorithm, (1970) J. Mol. Biol. 48:443, using the Pearson-Lipman similarity search method, (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, using computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA as part of the Wisconsin Genetics software package, Genetics Computer Group, 575 Science Dr., Madison, WI) or using manual alignment and visual inspection (See, for example, Ausubel et al., Current Protocols in Molecular Biology (supplement to the 1995 edition)).

[0097] Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described respectively in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402 and Altschul et al. (1990) J. Mol. Biol. 215:403-410. Software for performing BLAST analyzes is publicly available from the National Center for Biotechnology Information. At the first stage, this algorithm involves the identification of sequence pairs with a high similarity score (HSP) by identifying short words of length W in the query sequence that either match or satisfy some threshold score T with a positive value when aligned with a word of the same length in the database sequence data. T is called the threshold similarity score for neighboring words (Altschul et al., supra). These initial matches of neighboring words act as "seeds" to begin searches designed to find longer HSPs containing them. Word matches are extended in both directions along each sequence as long as the total alignment score can increase. For nucleotide sequences, total scores are calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatched residues; always < 0). In the case of amino acid sequences, a substitution matrix is used to calculate the total score. The extension of word matches in each direction stops when the total alignment score decreases by an amount X relative to its maximum value achieved; the total score tends to zero or lower due to the accumulation of one or more residual alignments with negative scores; or the end of any of the sequences is reached. The W, T, and X parameters of the BLAST algorithm determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectancy value (E) of 10, M=5, N = -4, and comparison of both strands as default parameters. For amino acid sequences, BLASTP uses the default parameters for a word length of 3 and an expectation (E) value of 10, as well as the BLOSUM62 substitution matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci USA 89:10915), number of alignments (B) being 50, expectation value (E) being 10, M=5, N=-4 and comparison of both strands.

[0098] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the lowest cumulative probability (P(N)), which indicates the probability with which a match between two nucleotide or amino acid sequences occurred by chance. For example, a nucleic acid is considered similar to a reference sequence if the lowest overall probability when comparing the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0099] An indication that two nucleic acid sequences or two polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid immunologically cross-reacts with antibodies induced by the polypeptide encoded by the second nucleic acid, as described below. Thus, the polypeptide is typically substantially identical to the second polypeptide, for example, if the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complementary strands hybridize to each other under stringent conditions, as described below. Another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequence.

[00100] The term “linkage,” when used in the context of describing how binding regions are connected in a cytokine-grafted antibody protein of the present invention, includes all possible means for physically linking these regions. A large number of binding regions are often linked through chemical bonds, such as a covalent bond (such as a peptide bond or disulfide bond) or a non-covalent bond, which can be a direct bond (i.e., no linker between two binding regions) or an indirect bond ( i.e., using at least one linker molecule between two or more binding regions).

[00101] The terms “subject,” “patient,” and “individual” interchangeably refer to a mammal, such as a human or non-human primate mammal. The mammal may also be a laboratory mammal, such as a mouse, rat, rabbit, hamster. In some embodiments, the mammal may be a farm mammal (eg, horse, sheep, bull, pig, camel) or a domestic mammal (eg, dog, cat).

[00102] As used herein, the terms “treat,” “treat,” or “cure” a disease or disorder, in one embodiment, refer to reducing the severity of the disease or disorder (i.e., slowing, stopping, or mitigating development of the disease or at least one of its clinical symptoms). In another embodiment, “treat,” “treat,” or “treat” refer to alleviating or reducing the severity of at least one physical parameter, including those that may not be apparent to the patient. In yet another embodiment, “treat,” “treat,” or “treat” refers to modulating a disease or disorder either physically (eg, by stabilizing an overt symptom) or physiologically (eg, by stabilizing a physical parameter), or both. to others. In yet another embodiment, “treat,” “treat,” or “treat” refers to preventing or delaying the onset, or development, or progression of a disease or disorder.

[00103] The term "therapeutically acceptable amount" or "therapeutically effective dose" interchangeably refers to an amount sufficient to achieve a desired result (ie, reducing inflammation, inhibiting pain, preventing inflammation, inhibiting or preventing an inflammatory response). In some embodiments, the therapeutically acceptable amount does not induce or cause unwanted side effects. The therapeutically acceptable amount can be determined by initially administering a low dose and then gradually increasing that dose until the desired effect is achieved. A "prophylactically effective dose" and a "therapeutically effective dose" of an IL2 cytokine-grafted antibody protein may, respectively, prevent the onset of or result in a reduction in the severity of disease symptoms, including symptoms associated with cancer and cancer treatment.

[00104] The term “co-administration” refers to the simultaneous presence of two (or more) active agents in the body of an individual. Active agents that are coadministered may be delivered simultaneously or sequentially.

[00105] As used herein, the phrase “consisting essentially of” refers to the species or types of active pharmaceutical agents included in the method or composition, as well as any inactive carriers or excipients for the intended purpose of using the methods or compositions. In some embodiments, the phrase “consisting essentially of” specifically excludes the inclusion of one or more additional active agents other than the IL2 cytokine-grafted antibody protein. In some embodiments, the phrase “consisting essentially of” specifically excludes the inclusion of more additional active agents other than the IL2 cytokine-grafted antibody protein and the second agent co-administered.

[00106] Singular noun forms include plural forms unless the context clearly indicates otherwise.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[00107] Figure 1 is a table that summarizes exemplary IL2 cytokine-grafted antibody proteins and their patterns of activity against CD8+ effector T cells.

[00108] Figure 2 shows that IgG.IL2R67A.H1 has a longer half-life than Proleukin®. IgG.IL2R67A.H1 has a half-life of 12-14 hours as shown on the graph, whereas Proleukin® has a T1/2 of less than 4 hours and cannot be shown on the graph.

[00109] Figures 3A-3C demonstrate that IgG.IL2R67A.H1 promotes expansion of CD8+ effector T cells more efficiently and with less toxicity than Proleukin® or an IL2-Fc fusion molecule in C57BL/6 mice at an equivalent dose of 100 μg at time points day 4, day 8 and day 11.

[00110] Figures 3D-3F demonstrate that IgG.IL2R67A.H1 promotes expansion of CD8+ effector T cells more efficiently and with less toxicity than Proleukin® or an IL2-Fc fusion molecule in C57BL/6 mice at an equivalent dose of 500 μg at time points day 4, day 8 and day 11.

[00111] Figure 4A shows that IgG.IL2R67A.H1 selectively expands CD8+ effector T cells and is better tolerated than Proleukin® in NOD mice.

[00112] Figure 4B is a table showing the increased activity of IgG.IL2R67A.H1 and IgG.IL2F71A.H1 on CD8+ effector T cells in NOD mice.

[00113] Figure 5 shows a graph of the effectiveness of IgG.IL2R67A.H1 as a monotherapy in a CT26 cell tumor model.

[00114] Figure 6 presents data on IgG.IL2R67A.H1 as a monotherapy or in combination with an antibody in a B16 cell melanoma mouse model. The graph shows that IgG.IL2R67A.H1 in combination with the anti-TRP1 antibody TA99 is more effective than TA99 alone, IL2-Fc fusion alone, TA99 together with IL2-Fc fusion. Synergy was observed using TA99 and IgG.IL2R67A.H1 at doses of 100 and 500 μg.

[00115] Figure 7 shows a graph of values that track pSTAT5 activity in a panel of human cells comparing IgG.IL2R67A.H1 and IgG.IL2F71A.H1 with Proleukin®.

[00116] Figure 8 is a plot of ELISA data showing that when IL2 is grafted onto CDRH1 with an anti-RSV antibody (IgG.IL2R67A.H1), binding to RSV is maintained. At the same time, binding to RSV is reduced when IL2 is grafted onto CDRL3 or CDRH3. If IL2 is grafted onto another antibody backbone (Xolair), there is no binding to RSV.

Cytokine-grafted antibody-based proteins targeting the low-affinity IL2 receptor

[00117] Provided herein are protein constructs comprising an IL2 molecule grafted onto a complementarity determining region (CDR) of an antibody. The cytokine-grafted antibody proteins of the present invention exhibit suitable properties for use in human patients, for example, they retain immunostimulatory activity similar to that of native or recombinant human IL2. At the same time, negative effects are reduced. For example, there is less stimulation of Treg cells. Other activities and characteristics are also demonstrated throughout this specification. Thus, provided are cytokine-grafted antibody proteins having an improved therapeutic profile compared to previously known IL2 and modified IL2-based therapeutics, such as Proleukin®, and methods of using the provided cytokine-grafted antibody proteins in the treatment of cancer.

[00118] Accordingly, the present invention provides cytokine-grafted antibody proteins that are low-affinity IL2 receptor agonists with selective activity profiles. The provided cytokine-grafted antibody proteins contain an immunoglobulin heavy chain sequence and an immunoglobulin light chain sequence. Each immunoglobulin heavy chain sequence contains a heavy chain variable region (VH) and a heavy chain constant region (CH), where the heavy chain constant region consists of CH1, CH2 and CH3 constant regions. Each immunoglobulin light chain sequence contains a light chain variable region (VL) and a light chain constant region (CL). In each antibody-cytokine graft protein, an IL2 molecule is included in the complementarity determining region (CDR), VH or VL.

[00119] In some embodiments, the cytokine-grafted antibody protein comprises an IL2 molecule included in a heavy chain CDR. In certain embodiments, the IL2 molecule is included in the heavy chain complementarity determining region 1 (HCDR1). In certain embodiments, the IL2 molecule is included in the heavy chain complementarity determining region 2 (HCDR2). In certain embodiments, the IL2 molecule is included in the heavy chain complementarity determining region 3 (HCDR3).

[00120] In some embodiments, the cytokine-grafted antibody protein comprises an IL2 molecule included in a light chain CDR. In certain embodiments, the IL2 molecule is included in the light chain complementarity determining region 1 (LCDR1). In certain embodiments, the IL2 molecule is included in the light chain complementarity determining region 2 (LCDR2). In certain embodiments, the IL2 molecule is included in the light chain complementarity determining region 3 (LCDR3).

[00121] In some embodiments, the cytokine-grafted antibody protein comprises an IL2 sequence included in a CDR, wherein the IL2 sequence is inserted into the CDR sequence. The insertion may occur in or near the N-terminal CDR region, in the middle CDR region, or in or near the C-terminal CDR region. In other embodiments, the cytokine-grafted antibody protein comprises an IL2 molecule included in a CDR, wherein the IL2 sequence does not frameshift the CDR sequence. In other embodiments, the cytokine-grafted antibody protein comprises an IL2 molecule included in a CDR, wherein the IL2 sequence replaces all or a portion of the CDR sequence. The substitution may occur at or near the N-terminal CDR region, the middle CDR region, or the C-terminal CDR region. The substitution may affect only one or two amino acids of the CDR sequence or the entire CDR sequence.

[00122] In some embodiments, the IL2 molecule is grafted directly onto the CDR without a peptide linker, without additional amino acids between the CDR sequence and the IL2 sequence.

[00123] In some embodiments, the cytokine-grafted antibody proteins comprise immunoglobulin heavy chains, which are IgG antibody heavy chains. In certain embodiments, the IgG heavy chain is any of the IgG1, IgG2, or IgG4 subclasses.

[00124] In some embodiments, the cytokine-grafted antibody proteins comprise immunoglobulin heavy and light chain sequences selected from a known immunoglobulin sequence used in clinical practice. In certain embodiments, the cytokine-grafted antibody proteins comprise immunoglobulin heavy and light chain sequences that are humanized sequences. In other certain embodiments, the cytokine-grafted antibody proteins comprise immunoglobulin heavy and light chain sequences that are human sequences.

[00125] In some embodiments, the cytokine-grafted antibody proteins comprise immunoglobulin heavy and light chain sequences selected from germline immunoglobulin sequences.

[00126] In some embodiments, cytokine-grafted antibody proteins comprise immunoglobulin heavy and light chain sequences having a target binding specificity of the immunoglobulin variable domains that is different from the binding specificity of the IL2 molecule. In some embodiments, the binding specificity of the immunoglobulin variable domain to its target is maintained at 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% in the presence of the grafted cytokine. In certain embodiments, the retained binding specificity is directed to a non-human target. In certain embodiments, the retained binding specificity is directed to a virus, such as RSV. In other embodiments, the binding specificity is directed to a human target having therapeutic utility in IL2 therapy. In certain embodiments, targeting immunoglobulin binding specificity provides an additional therapeutically beneficial effect of the IL2 component. In certain embodiments, the binding specificity of the immunoglobulin to its target represents synergistic activity with IL2.

[00127] In still some other embodiments, the immunoglobulin binding specificity is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% as a result of grafting the IL2 molecule.

[00128] Provided cytokine-grafted antibody proteins comprise an IL2 molecule grafted to a complementarity determining region (CDR), VH or VL. In some embodiments, the IL2 sequence has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:4. In some embodiments, the IL2 molecule contains the sequence of SEQ ID NO:4. In some embodiments, the IL2 molecule consists of the sequence of SEQ ID NO:4.

[00129] Provided cytokine-grafted antibody proteins comprise an IL2 molecule grafted to a complementarity determining region (CDR), VH or VL. In some embodiments, the IL2 sequence has at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the amino acid sequence of SEQ ID NO:6. In some embodiments, the IL2 molecule contains the sequence of SEQ ID NO:6. In some embodiments, the IL2 molecule consists of the sequence of SEQ ID NO:6.

[00130] In some embodiments, the cytokine-grafted antibody protein provides pro-immunomodulatory properties superior to those of human IL2, recombinant human IL2, Proleukin®, or Fc-fused IL2. The cytokine-grafted antibody protein confers increased activity on CD8+ effector T cells while providing reduced Treg activity compared to human IL2, recombinant human IL2, Proleukin® or Fc-fusion IL2.

[00131] In some embodiments, the cytokine-grafted antibody proteins comprise a modified IgG immunoglobulin having a modified Fc conferring a modified effector function. In certain embodiments, the modified Fc region contains a mutation selected from one or more of D265A, P329A, P329G, N297A, L234A, and L235A. In specific embodiments, the immunoglobulin heavy chain may contain a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/L234A/L235A.

[00132] In some embodiments, the cytokine-grafted antibody proteins comprise (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the heavy chain variable region of SEQ ID NO:19, and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the light chain variable region of SEQ ID NO:35. The immunoglobulin chain is of the IgG class, selected from IgG1, IgG2 or IgG4. In certain embodiments, the immunoglobulin optionally contains a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/ L234A/L235A.

[00133] In some embodiments, the cytokine-grafted antibody proteins comprise (i) a heavy chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with the heavy chain variable region of SEQ ID NO:51, and (ii) a light chain variable region having at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to the light chain variable region of SEQ ID NO:67. The immunoglobulin chain is of the IgG class, selected from IgG1, IgG2 or IgG4. In certain embodiments, the immunoglobulin optionally contains a mutation or combination of mutations conferring reduced effector function selected from any of D265A, P329A, P329G, N297A, D265A/P329A, D265A/N297A, L234/L235A, P329A/L234A/L235A, and P329G/ L234A/L235A.

Engineered and modified cytokine-grafted antibody-based proteins

[00134] In certain embodiments, cytokine-grafted antibody constructs are created by grafting an IL2 sequence onto a CDR region of an immunoglobulin backbone. Both immunoglobulin heavy and light chains are produced to create the final antibody-based graft proteins. Cytokine-grafted antibody proteins confer preferential therapeutic activity on CD8+ effector T cells, and cytokine-grafted antibody proteins have reduced Treg activity compared to native or recombinant human IL2 (rhIL2 or Proleukin®) or Fc-fusion IL2.

[00135] To construct cytokine-grafted antibody proteins, IL2 sequences containing specific muteins (SEQ ID NO:4 or SEQ ID NO:6) are inserted into the CDR loop of the immunoglobulin backbone protein. Graft constructs can be generated using any of a variety of known immunoglobulin sequences that have been used in clinical settings, known immunoglobulin sequences currently in discovery and/or clinical development, germline human antibody sequences, as well as novel antibody chain immunoglobulin sequences. . The constructs were obtained using standard molecular biology techniques by using recombinant DNA encoding the corresponding sequences. The IL2 sequences in an exemplary backbone called GFTX3b are displayed in Table 2. Insertion points were chosen to represent the midpoint of the loop based on available structural or homology modeling data, but insertion points could be adjusted to were located near the N- or C-terminus of the CDR loop.

[00136] Thus, the present invention provides antibodies or fragments thereof that specifically bind to the low-affinity IL2 receptor, comprising IL2 protein recombinantly inserted into a heterologous antibody protein or polypeptide to create graft proteins. In particular, the present invention provides graft proteins comprising an antibody or antigen binding fragment of an antibody described herein, or any other appropriate antibody core polypeptide (e.g., immunoglobulin protein, complete antibody, Fab fragment, Fc fragment, Fv- fragment, F(ab)2 fragment, VH domain, CDR VH, VL domain, CDR VL, etc.) and a heterologous protein, polypeptide or cytokine peptide, for example IL2. Methods for fusing or conjugating proteins, polypeptides or peptides with an antibody or antibody fragment are known in the art. See, for example, US Patent Nos. 5336603, 5622929, 5359046, 5349053, 5447851 and 5112946; European patents Nos. EP 307434 and EP 367166; international publications Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337–11341. In addition, cytokine-grafted antibody proteins can be generated using gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling techniques (collectively referred to as “DNA shuffling”). DNA shuffling can be used to produce grafted protein constructs and/or to alter the activity of antibodies or fragments thereof (eg, antibodies or fragments thereof with higher affinities and lower dissociation rates). See generally US Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, and 5,837,458; Patten et al., 1997, Curr. Opinion Biotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson, et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313. Antibodies or fragments thereof, or encoded antibodies or fragments thereof, can be altered by subjecting them to random mutagenesis using error-injection PCR, random nucleotide insertion, or other means prior to recombination. A polynucleotide encoding an antibody or fragment thereof that specifically binds to an antigenic protein of interest may be subject to recombination with one or more components, motifs, segments, portions, domains, fragments, etc. one or more heterologous cytokine molecules, for example, IL2, to produce the cytokine-grafted antibody proteins provided herein.

[00137] An antibody Fab contains six CDR loops, 3 in the light chain (CDRL1, CDRL2, CDRL3) and 3 in the heavy chain (CDRH1, CDRH2, CDRH3), which can act as potential cytokine protein insertion sites. Structural and functional aspects are taken into account to determine which CDR loop(s) the cytokine should be inserted into. Because the size and conformation of CDR loops varies greatly among antibodies, the optimal CDR for insertion can be determined empirically for each specific antibody/protein combination. Additionally, since the cytokine protein will be inserted into the CDR loop, this may place further constraints on the structure of the cytokine protein, as discussed in Example 1.

[00138] The CDRs of immunoglobulin chains are determined using well-known numbering systems known in the art, including those described herein. For example, CDRs were identified and defined by (1) applying the numbering system described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (Kabat numbering schemes), NIH publication No. 91-3242; and (2) Chothia, see Al-Lazikani et al., (1997) "Standard conformations for the canonical structures of immunoglobulins", J.Mol.Biol. 273:927–948. In the case of identified CDR amino acid sequences that are less than 20 amino acids in length, one or more conservative amino acid residue substitutions may be acceptable as long as the desired specific binding and/or agonist activity is still retained.

[00139] A cytokine-grafted antibody protein can also be produced using an antibody having one or more of the CDR and/or VH and/or VL sequences shown herein (e.g., Table 2) as starting material for constructing a modified antibody-based protein with a grafted cytokine, which may have altered properties compared to the original grafted antibody-based protein. Alternatively, any known antibody sequences can be used as a backbone to construct a modified cytokine-grafted antibody protein. For example, any known antibody used in clinical practice can be used as the backbone of the starting material to produce an antibody graft protein. Known antibodies and corresponding immunoglobulin sequences include, for example, palivizumab, alirocumab, mepolizumab, necitumumab, nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab, raxibacumab, pert uzumab, belimumab, ipilimumab, denosumab, tocilizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certolizumab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritumomab tiuxetan, adalimumab, and lemtuzumab, gemtuzumab, infliximab, basiliximab, daclizumab, rituximab , abciximab, muromonab or their modifications. Known antibodies and immunoglobulin sequences also include germline antibody sequences. Framework sequences can be obtained from publicly available DNA databases or published literature sources that contain germline antibody gene sequences. For example, germline DNA sequences for the human heavy and light chain variable region genes can be found in the human germline sequence database "VBase" and also in Kabat, E. A., et al., 1991 Sequences of Proteins of Immunological Interest, Fifth Edition. U.S. Department of Health and Human Services, NIH Publication No. 91-3242; Tomlinson, I. M., et al., 1992 J. Mol. Biol. 227:776-798; and Cox, J. P. L. et al., 1994 Eur. J Immunol. 24:827-836. In yet other examples, antibodies and corresponding immunoglobulin sequences from other known structures that may be in early drug discovery and/or development can similarly be adapted as starting material for the construction of a modified cytokine-grafted antibody protein.

[00140] A wide variety of antibody/immunoglobulin frameworks or scaffolds can be used as long as the resulting polypeptide contains at least one binding region that is adapted to include a cytokine (eg, IL2). Such scaffolds include the 5 major idiotypes of human immunoglobulins or fragments thereof and include immunoglobulins from other animal species, preferably having humanized and/or human moieties. Experts in the field continue to research and develop new antibodies, scaffolds, scaffolds and fragments.

[00141] Antibodies can be created using methods that are known in the art. To obtain monoclonal antibodies, any technique known in the art can be used (see, for example, Kohler & Milstein, Nature 256:495-497 (1975); Kozbor et al., Immunology Today 4:72 (1983); Cole et al. ., Monoclonal Antibodies and Cancer Therapy, pp. 77-96 (Alan R. Liss, Inc. 1985). Single chain antibody production techniques (US Pat. No. 4,946,778) can be adapted to produce antibodies for use in cytokine-grafted antibody proteins. In addition, transgenic mice or other organisms, such as other mammals, can be used to express and identify primatized or humanized or human antibodies. Alternatively, phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens for use in cytokine-grafted antibody proteins (see, e.g., McCafferty et al., supra; Marks et al., supra; al., Biotechnology, 10:779-783, (1992)).

[00142] Methods for primatizing or humanizing non-human antibodies are well known in the art. Typically, a primatized or humanized antibody has one or more amino acid residues introduced into it from a non-primate or non-human source. Such non-primate or non-human amino acid residues are often referred to as imported residues, which are typically taken from an imported variable domain. Humanization can be performed essentially according to the method of Winter and co-workers (see, for example, Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al. , Science 239:1534-1536 (1988), and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992)) by replacing the corresponding human antibody CDR sequences or rodent CDR sequences. Accordingly, such humanized antibodies are chimeric antibodies (US Pat. No. 4,816,567) in which substantially less than the intact human variable domain has been replaced by a corresponding sequence from a non-human species. In practice, primatized or humanized antibodies are typically primate or human antibodies in which some of the complementarity determining region ("CDR") residues, and possibly some of the framework region ("FR") residues, are replaced with residues from similar regions in species from which they originate (eg, rodent antibodies) to impart binding specificity.

[00143] Alternatively or additionally, a method of replacing a non-human antibody variable region with a human variable region in an antibody in vivo while maintaining the same or providing better binding characteristics compared to those of a non-human antibody can be used to convert non-human antibodies into engineered human antibodies. See, for example, US Patent Application Publication No. 20050008625, US Patent Application Publication No. 2005/0255552. Alternatively, libraries of human V segments can be generated by sequential cassette replacement, in which only a portion of the V segment of the reference antibody is initially replaced with a library of human sequences; and the identified human binding cassettes surrounding the residual amino acid sequences of the reference antibody are then recombined in a second library screen to generate fully human V segments (see US Patent Application Publication No. 2006/0134098).

[00144] Various antibodies or antigen-binding fragments thereof for use in the production of cytokine-grafted antibody proteins can be produced by enzymatic or chemical modification of intact antibodies, or synthesized de novo using recombinant DNA technologies (e.g., single-stranded Fv), or identified using phage display libraries (see, for example, McCafferty et al., Nature 348:552-554, 1990). For example, miniantibodies can be produced using methods described in the prior art, for example, Vaughan and Sollazzo, Comb. Chem. High Throughput Screen 4:417-30 2001. Bispecific antibodies can be produced using a variety of methods, including grafting with hybridomas or linking Fab' fragments. See, for example, Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148, 1547-1553 (1992). Single chain antibodies can be identified using phage display libraries or ribosomal display libraries, shuffled gene libraries. Such libraries can be constructed from synthetic, semi-synthetic, or native and immunocompetent sources. The selected immunoglobulin sequences can thus be used in the preparation of the cytokine-grafted antibody protein constructs provided herein.

[00145] Antibodies, antigen binding molecules or cytokine grafted antibody proteins for use in the present invention further include bispecific antibodies. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Other antigen binding fragments or antibody portions include bivalent scFvs (diabodies), bispecific scFv antibodies in which the antibody molecule recognizes two different epitopes, single domain binding antibodies (dAbs), and miniantibodies. The selected immunoglobulin sequences can thus be used in the preparation of the cytokine-grafted antibody protein constructs provided herein.

[00146] Antigen-binding antibody fragments, eg, Fab fragment, scFv, can be used as building blocks for the construction of cytokine-grafted antibody proteins, and may optionally include multivalent formats. In some embodiments, such multivalent molecules comprise an antibody constant region (eg, Fc).

[00147] Cytokine-grafted antibody proteins can be constructed by modifying one or more residues in one or both variable regions (i.e., VH and/or VL) of the antibody, for example, in one or more CDR regions, and such adapted VH and/or VL region sequences are used for cytokine grafting or in preparation for cytokine grafting. Antibodies interact with target antigens primarily through amino acid residues that are located in the six complementarity determining regions (CDRs) of the heavy and light chains. For this reason, amino acid sequences within the CDR are more diverse among individual antibodies than sequences outside the CDR. Because CDR sequences are responsible for most antibody-antigen interactions, it is possible to express recombinant antibodies that mimic the properties of a particular antibody by constructing expression vectors that contain CDR sequences from a particular antibody grafted onto framework sequences from another antibody with different properties (see, for example, Riechmann, L. et al., 1998 Nature 332:323-327; Jones, P. et al., 1986 Nature 321:522-525; Queen, C. et al., 1989 Proc. Natl. Acad., U.S.A. 86:10029-10033; US Patent No. 5225539 by Winter and US Patent Nos. 5530101; 5585089; 5693762 and 6180370 by Queen et al.). In certain cases, it is advantageous to mutate residues within the framework regions to maintain or increase the antigen-binding ability of the antibody (see, for example, US Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al).

[00148] In some aspects, mutation of amino acid residues in the CDR1, CDR2, and/or CDR3 regions of VH and/or VL to thereby improve one or more binding properties (e.g., affinity) of the antibody of interest, known as “affinity maturation” " may be beneficial, for example, to optimize antigen binding by an antibody given that it is present as part of a cytokine-grafted protein. Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutation(s), and the effect on antibody binding or other functional property of interest can be assessed in in vitro or in vivo assays described herein, and/or alternative or additional assays known in the art. Conservative modifications can be made. Mutations may be amino acid substitutions, additions or deletions. Moreover, typically no more than one, two, three, four or five residues within the CDR region are changed.

[00149] Engineered antibodies or antibody fragments include antibodies or fragments thereof in which modifications have been made to residues of the framework regions within the VH and/or VL, for example, to improve the properties of the antibody. In some embodiments, such modifications to the framework regions are made to reduce the immunogenicity of the antibody. For example, one approach is to change one or more residues of the framework region to the corresponding germline sequence. More specifically, an antibody that has undergone a somatic mutation may contain framework region residues that differ from the germline sequence from which the antibody is derived. Such residues can be identified by comparing the sequences of the framework regions of the antibody with the germline sequences from which the antibody is derived. To return the framework sequences to their germline configuration, "reverse" somatic mutations can be introduced to restore the germline sequence through, for example, site-directed mutagenesis. Further modification of the framework region involves mutation of one or more residues within the framework region, or even within one or more CDR regions, to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This approach is also called "deimmunization" and is described in more detail in US Patent Application Publication No. 20030153043 by Carr et al.

[00150] The constant regions of the antibodies or antibody fragments used to produce the cytokine-grafted antibody protein may be of any type or subtype as appropriate and may be selected to be from the species of the subject to be treated by the methods of the present invention (e.g., a human, non-human primate, or other mammal, e.g., a farm mammal (e.g., horse, sheep, bovine, pig, camel), domestic mammal (e.g., dog, cat), or rodent (e.g. rat, mouse, hamster, rabbit)). In some embodiments, the antibodies used in cytokine-grafted antibody proteins are engineered to create humanized or Humaneered® antibodies. In some embodiments, the antibodies used in the cytokine-grafted antibody proteins are human antibodies. In some embodiments, the isotype of the antibody constant region is IgG, for example, IgG1, IgG2, IgG3, IgG4. In certain embodiments, the constant region isotype is IgG ₁ . In some embodiments, the cytokine-grafted antibody proteins comprise IgG. In some embodiments, the cytokine-grafted antibody proteins comprise an IgG1 Fc. In some embodiments, the cytokine-grafted antibody proteins comprise an IgG2 Fc.

[00151] As an addition or alternative to modifications made within framework regions or CDR regions, antibodies or antibody fragments used in the production of cytokine-grafted antibody proteins can be designed to include modifications within the Fc -region, typically to alter one or more functional properties of the antibody, such as, for example, serum half-life, complement fixation, Fc receptor binding and/or antigen-dependent cellular cytotoxicity. In addition, the antibody, antibody fragment, or cytokine-grafted antibody protein may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter its glycosylation pattern, again to alter one or more several functional properties of a protein based on a cytokine-grafted antibody.

[00152] In one embodiment, the CH1 hinge region is modified such that the number of cysteine residues in the hinge region is changed, for example, increased or decreased. For example, this approach is further described in US Pat. No. 5,677,425 to Bodmer et al., in which the number of cysteine residues in the CH1 hinge region is varied, for example, to facilitate light and heavy chain assembly or to increase or decrease the stability of a cytokine-grafted antibody protein. . In another embodiment, the Fc hinge region of the antibody is mutated to alter the biological half-life of the cytokine-grafted antibody protein. More specifically, one or more amino acid mutations are introduced into the border region between the CH2-CH3 domains of the Fc-hinge fragment, whereby the cytokine-grafted antibody protein exhibits impaired binding to staphylococcal protein A (SpA) compared to binding of the native Fc-hinge domain with SpA. This approach is described in more detail in US patent No. 6165745 by Ward et al.

[00153] The present invention provides cytokine-grafted antibody proteins that specifically bind to the low-affinity IL2 receptor, which have an extended half-life in vivo. In another embodiment, the cytokine-grafted antibody protein is modified to increase its biological half-life. Various approaches are possible. Cytokine-grafted antibody proteins having an extended half-life in vivo can also be generated by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into the IgG constant domain or its FcRn-binding fragment (preferably Fc- fragment or hinge-Fc domain fragment). For example, one or more of the following mutations may be introduced: T252L, T254S, T256F, as described in US Pat. No. 6,277,375 to Ward. See, for example, international publication No. WO 98/23289; International Publication No. WO 97/34631 and US Patent No. 6277375. Alternatively, to increase the biological half-life, the cytokine-grafted antibody protein is modified within the CH1 or CL region so that it contains the salvage receptor binding epitope derived from the two loops of the CH2 domain of the IgG Fc region, as described in US patent Nos. 5869046 and 6121022 by Presta et al. In still several other embodiments, the Fc region is modified by replacing at least one amino acid residue with another amino acid residue to alter the effector functions of the cytokine-grafted antibody protein. For example, one or more amino acids can be replaced with another amino acid residue such that the cytokine-grafted antibody protein has altered affinity for the effector ligand but retains the antigen-binding ability of the original antibody. The effector ligand for which the affinity is changed may be, for example, an Fc receptor (FcR) or a component C1 of the complement system. This approach is described in detail, for example, in US patent Nos. 5624821 and 5648260, both by Winter et al.

[00154] In another embodiment, one or more amino acids selected from amino acid residues can be replaced with another amino acid residue such that the cytokine-grafted antibody protein exhibits altered C1q binding and/or reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in more detail in US patent No. 6194551 by Idusogie et al.

[00155] Cytokine-grafted antibody proteins containing such mutations mediate or do not mediate reduced antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). In some embodiments, amino acid residues L234 and L235 in the IgG1 constant region are replaced with Ala234 and Ala235. In some embodiments, amino acid residue N267 in the IgG1 constant region is replaced with Ala267.

[00156] In another embodiment, one or more amino acid residues are altered to thereby alter the complement fixation ability of the cytokine-grafted antibody protein. This approach is further described in PCT publication WO 94/29351 by Bodmer et al.

[00157] In yet another embodiment, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the cytokine-grafted antibody protein for the Fcγ receptor through modification of one or more amino acids. This approach is further described in PCT publication WO 00/42072 by Presta. Moreover, the binding sites on human IgG1 for FcγRI, FcγRII, FcγRIII and FcRn have been mapped, and variants with improved binding have been described (see Shields, R. L. et al., 2001 J. Biol. Chen. 276:6591-6604).

[00158] In yet another embodiment, the glycosylation pattern of a protein is modified based on a cytokine-grafted antibody. For example, an aglycosylated cytokine-grafted antibody protein (ie, a cytokine-grafted antibody protein that lacks glycosylation) can be produced. The nature of glycosylation can be changed, for example, to increase the affinity of the antibody for the "antigen". Such modifications of carbohydrates can be accomplished, for example, by changing one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions may be made that result in the elimination of one or more glycosylation sites in the framework regions of the variable region, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. This approach is described in detail, for example, in US patent Nos. 5714350 and 6350861 by Co et al.

[00159] As an addition or alternative, a cytokine-grafted antibody protein may be produced that is characterized by an altered glycosylation pattern, such as a hypofucosylated cytokine-grafted antibody protein having a reduced number of fucosyl residues, or an antibody having an increased content of GlcNAc structures in branch points. This altered glycosylation pattern has been demonstrated to enhance the ability of antibodies to exhibit antibody-dependent cellular cytotoxicity (ADCC). Such modifications of carbohydrates can be accomplished, for example, by expressing a cytokine-grafted antibody protein in a host cell with an altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which recombinant cytokine-grafted antibody proteins are expressed, thereby producing cytokine-grafted antibody proteins with altered glycosylation patterns. For example, in EP 1176195 by Hang et al. describes a cell line with a functional disruption of the FUT8 gene, which encodes a fucosyltransferase, whereby cytokine-grafted antibody proteins expressed in such a cell line exhibit hypofucosylation. PCT publication WO 03/035835 by Presta describes a variant of the CHO cell line, Lecl3 cells, with a reduced ability to attach fucose to Asn(297)-linked carbohydrates, which also leads to hypofucosylation of cytokine-grafted antibody proteins expressed in such a host cell (see also Shields, R.L. et al., 2002 J. Biol. Chem. 277:26733-26740). In the publication according to PCT WO 99/54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta-(1,4)-N-acetylglucosaminyltransferase III (GnTIII)), whereby cytokine-grafted antibody proteins expressed in the engineered cell lines are enriched GlcNAc structures at branch points, which leads to increased ADCC activity in antibodies (see also Umana et al., 1999 Nat. Biotech. 17:176-180).

[00160] In some embodiments, one or more domains or regions of the cytokine-grafted antibody protein are linked by a linker, e.g., a peptide linker, such as linkers well known in the art (see, e.g., Holliger, P., et al (1993) Proc Natl Acad Sci USA 90:6444-6448; Poljak, RJ, et al (1994) Structure 2:1121-1123). The length of the peptide linker may vary, for example the linker may be 1-100 amino acids long, typically the linker will be five to 50 amino acids long, for example 5, 6, 7, 8, 9, 10, 11, 12, 13 long , 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 , 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[00161] In some embodiments, IL2 is grafted onto a CDR sequence, optionally with one or more peptide linker sequences. In certain embodiments, one or more peptide linkers are independently selected from a (Gly _n -Ser) _m sequence (SEQ ID NO: 71), a (Gly _n -Ala) _m sequence (SEQ ID NO: 72), or any combination of (Gly _n -Ser) _m /(Gly _n -Ala) _m (SEQ ID NO: 71-72), where each n is independently an integer from 1 to 5, and each m is independently an integer from 0 to 10. Examples of linkers include, but are not limited to, glycine-based linkers or Gly/Ser G/S linkers such as (G _m S) _n , where n is a positive integer equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is an integer equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 73). In certain embodiments, one or more linkers comprise G ₄ S repeats (SEQ ID NO: 74), for example, a Gly-Ser (G ₄ S) _n linker, where n is a positive number equal to or greater than 1 (SEQ ID NO: 74). For example, n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In some embodiments, Ser may be replaced by Ala, for example, in G/A linkers such as (G _m A) _n , where n is a positive integer equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is an integer equal to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (SEQ ID NO: 75). In certain embodiments, one or more linkers comprise G ₄ A (SEQ ID NO: 76), (G ₄ A) _n repeats, where n is a positive number equal to or greater than 1 (SEQ ID NO: 76). For example, n=1, n=2, n=3, n=4, n=5 and n=6, n=7, n=8, n=9 and n=10. In some embodiments, the linker contains multiple linker repeats. In other embodiments, the linker contains combinations and pluralities of G ₄ S (SEQ ID NO: 74) and G ₄ A (SEQ ID NO: 76).

[00162] Other examples of linkers include linkers based on flexible linker sequences that occur naturally in antibodies to minimize immunogenicity due to linkers and junctions. For example, in the molecular structure of an antibody, there is a naturally occurring flexible link between the variable domain and the CH1 constant domain. This natural linkage contains approximately 10-12 amino acid residues, consisting of 4-6 residues from the C-terminus of the V domain and 4-6 residues from the N-terminus of the CH1 domain. Cytokine-grafted antibody proteins, for example, may use linkers that include the terminal 5-6 amino acid residues or 11-12 amino acid residues of CH1 as a linker. The N-terminal residues of the CH1 domain, in particular the first 5-6 amino acid residues, adopt a loop conformation without stable secondary structure and thus can act as a flexible linker. The N-terminal residues of the CH1 domain are a natural extension of variable domains since they are part of the Ig sequences and therefore largely minimize any immunogenicity potentially due to linkers and junctions. In some embodiments, the linker sequence comprises a modified peptide sequence based on the hinge region sequence.

[00163] In addition, cytokine-grafted antibody proteins may contain marker sequences, such as a peptide, to facilitate purification of cytokine-grafted antibody proteins. In preferred embodiments, the marker amino acid sequence is a hexahistidine peptide (SEQ ID NO: 78), such as, among others, the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA 91311), many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, for example, hexahistidine (SEQ ID NO: 78) provides convenient purification of the graft protein. Other peptide tags useful for purification include, but are not limited to, a hemagglutinin (“HA”) tag, which corresponds to an epitope derived from the influenza virus hemagglutinin protein (Wilson et al., 1984, Cell 37:767), and a “FLAG” tag.

[00164] Antibodies can also be attached to solid supports, which are particularly useful for immunoassays or target antigen purification. Such solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride, or polypropylene.

Cytokine-Grafted Antibody Protein Activity Assays

[00165] Assays for identifying proteins based on cytokine-grafted antibodies are known in the art and are described herein. Cytokine-grafted antibody-based agonist proteins bind to the low-affinity IL2 receptor and promote, induce, stimulate intracellular signal transduction leading to the proliferation of CD8+ effector T cells, as well as other immunostimulatory effects.

[00166] The binding of cytokine-grafted antibody proteins to the low-affinity IL2 receptor can be determined using any method known in the art. For example, binding to the low affinity IL2 receptor can be determined using known techniques, including but not limited to ELISA, Western blotting, surface plasmon resonance (SPR) (eg, BIAcore) and flow cytometry.

[00167] Intracellular signaling through the low affinity IL2 receptor can be measured using any method known in the art. For example, activation of the low-affinity IL2 receptor by IL2 promotes STAT5 activation and signal transduction through it. Methods for measuring STAT5 activation are standard in the art (eg, determination of STAT5 protein phosphorylation status, reporter gene assays, downstream signaling assays, etc.). Activation by the low-affinity IL2 receptor allows expansion of CD8+ effector T cells so that the absolute number of CD8+ effector T cells or the ratio of CD8+ effector T cells to Tregs can be analyzed. Methods for measuring cell proliferation are standard in the art (eg, ³ H-thymidine incorporation assays, CFSE labeling). Methods for measuring cytokine production are well known in the art (eg, ELISA assays, ELISpot assays). When performing in vitro assays, test cells or test cell culture supernatant that are contacted with cytokine-grafted antibody proteins can be compared with control cells or control cell culture supernatant samples that have not been contacted with the antibody protein. with the grafted cytokine and/or which have been brought into contact with recombinant human IL2 (eg, Proleukin®) or IL2-Fc fusion molecule.

[00168] The activity of cytokine-grafted antibody proteins can also be measured ex vivo and/or in vivo. In some aspects, methods of measuring STAT5 activation in various cell types ex vivo from animals that received treatment with cytokine-grafted antibody proteins compared to control animals that were not treated and/or animals that were similarly treated with Proleukin®, can be used to demonstrate the differential activity of cytokine-grafted antibody agonist proteins in different cell types. Preferred cytokine-grafted antibody proteins have the ability to mediate the activation and expansion of CD8+ effector T cells. For example, the activation and expansion of CD8+ T cells in vivo can be measured using any method known in the art, such as flow cytometry. Preferred cytokine-grafted antibody agonist proteins may be therapeutically useful for the prevention, amelioration, amelioration, or treatment of cancer, such as melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, and lymphoma. The effectiveness of cytokine-grafted antibody proteins can be determined by administering a therapeutically effective amount of the cytokine-grafted antibody protein to a subject and comparing the subject before and after administration of the cytokine-grafted antibody protein. The effectiveness of cytokine-grafted antibody proteins can also be determined by administering a therapeutically effective amount of cytokine-grafted antibody protein to a test subject and comparing the test subject with a control subject who was not administered the antibody and/or comparison with a subject who was similarly treated with Proleukin®.

Polynucleotides encoding cytokine-grafted antibody proteins

[00169] In another aspect, isolated nucleic acids encoding the protein heavy and light chains of cytokine-grafted antibody proteins are provided. Cytokine-grafted antibody proteins can be produced by any methods known in the art, including, but not limited to, recombinant expression, chemical synthesis, and enzymatic digestion of tetrameric antibodies. Recombinant expression can be carried out in any suitable host cells known in the art, for example, mammalian host cells, bacterial host cells, yeast host cells, insect host cells, etc. d.

[00170] Provided herein are polynucleotides that encode the variable regions exemplified by any of SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, and SEQ ID NO: 68.

[00171] The present invention thus provides polynucleotides encoding polypeptide light and/or heavy chains of cytokine-grafted antibody proteins described herein, for example, polynucleotides encoding variable regions or light or heavy chain segments containing regions complementarity determinants described in this document. In some embodiments, the polynucleotide encoding the heavy chain variable regions contains a sequence characterized by at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO:20 and SEQ ID NO:52. In some embodiments, the polynucleotide encoding the light chain variable regions contains a sequence characterized by at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from the group consisting of SEQ ID NO:36 and SEQ ID NO:68.

[00172] In some embodiments, the polynucleotide encoding the heavy chain is characterized by at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with the polynucleotide under SEQ ID NO:22. In some embodiments, the polynucleotide encoding the light chain is characterized by at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with the polynucleotide of SEQ ID NO:38.

[00173] In some embodiments, the polynucleotide encoding the heavy chain is characterized by at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% nucleic acid sequence identity with the polynucleotide under SEQ ID NO:54. In some embodiments, the polynucleotide encoding the light chain is characterized by at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity with a polynucleotide selected from SEQ ID NO:70.

[00174] Polynucleotides may encode only the variable region sequence of a cytokine-grafted antibody protein. They may also encode both the variable region and the constant region of a cytokine-grafted antibody protein. Some of the polynucleotide sequences encode a polypeptide that contains variable regions of both the heavy chain and the light chain of one of the cytokine-grafted antibody proteins. Certain other polynucleotides encode two polypeptide segments that are substantially identical to the heavy chain and light chain variable regions, respectively, of one of the cytokine-grafted antibody proteins.

[00175] In certain embodiments, the polynucleotides or nucleic acids comprise DNA. In other embodiments, the polynucleotides or nucleic acids comprise RNA, which may be single-stranded or double-stranded.

[00176] In some embodiments, a recombinant host cell is provided comprising nucleic acids encoding one or more immunoglobulin protein chains in a cytokine-grafted antibody protein and optionally secretion signals. In certain embodiments, the recombinant host cell contains a vector encoding a single immunoglobulin protein chain and secretion signals. In certain other embodiments, the recombinant host cell contains one or more vectors encoding two chains of immunoglobulin proteins in a cytokine-grafted antibody protein and secretion signals. In some embodiments, the recombinant host cell contains a single vector encoding two chains of immunoglobulin proteins in a cytokine-grafted antibody protein and secretion signals. In some embodiments, the recombinant host cell contains two vectors, one encoding an immunoglobulin protein heavy chain and the other encoding an immunoglobulin protein light chain in a cytokine-grafted antibody protein, each containing secretion signals. The recombinant host cell may be a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a eukaryotic cell line. In some embodiments, the host cell is a mammalian cell line. In some embodiments, the host cell line is a CHO cell line for antibody production.

[00177] In addition, methods are provided for producing cytokine-grafted antibody proteins. In some embodiments, the method includes the steps of (i) culturing a host cell containing one or more vectors encoding immunoglobulin protein chains in the cytokine-grafted antibody protein under conditions suitable for expression, production, and secretion of the cytokine-grafted antibody protein. cytokine, and (ii) protein extraction based on the cytokine-grafted antibody.

[00178] Polynucleotide sequences can be obtained by solid phase de novo DNA synthesis or by PCR mutagenesis of an existing sequence (eg, the sequences described herein) encoding the polypeptide chain of a cytokine-grafted antibody protein. Direct chemical synthesis of nucleic acids can be carried out using methods known in the art, such as the phosphotriester method from Narang et al., Meth. Enzymol. 68:90, 1979; phosphodiester method from Brown et al., Meth. Enzymol. 68:109, 1979; diethylphosphoramidite method from Beaucage et al., Tetra. Lett., 22:1859, 1981; and the solid support method of US Pat. No. 4,458,066. The introduction of mutations into a polynucleotide sequence by PCR can be accomplished as described, for example, in PCR Technology: Principles and Applications for DNA Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1:17, 1991.

[00179] The present invention also provides expression vectors and host cells for the production of the cytokine-grafted antibody proteins described above. Various expression vectors can be used to express polynucleotides encoding immunoglobulin polypeptide chains or fragments thereof in cytokine-grafted antibody proteins. Both viral and non-viral expression vectors can be used to produce immunoglobulin proteins in a mammalian host cell. Non-viral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for protein or RNA expression, and artificial human chromosomes (see, for example, Harrington et al., Nat Genet. 15:345, 1997). For example, non-viral vectors useful for expressing polynucleotides and polypeptides corresponding to cytokine-grafted antibody proteins in mammalian (eg, human) cells include pThioHis A, B and C, pcDNA3.1/His, pEBVHis A, B and C (Invitrogen, San Diego, Calif.), MPSV-based vectors, and numerous other vectors known in the art for the expression of other proteins. Useful viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, SV40, papillomavirus, Epstein-Barr HBP, vaccinia virus, and Semliki Forest virus (SFV) vectors. See Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68:143, 1992.

[00180] The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, expression vectors contain a promoter and other regulatory sequences (eg, enhancers) that are operably linked to polynucleotides encoding an immunoglobulin protein in a cytokine-grafted antibody protein. In some embodiments, an inducible promoter is used to prevent the inserted sequences from being expressed under conditions other than inducing conditions. Inducible promoters include, for example, the arabinose promoter, lacZ, metallothionein gene promoter, or heat shock protein gene promoter. Cultures of transformed organisms can be propagated under non-inducing conditions without a population bias toward coding sequences whose expression products are better tolerated by host cells. For efficient expression of an immunoglobulin chain or fragment thereof in cytokine-grafted antibody proteins, other regulatory elements in addition to promoters may also be necessary or desirable. These elements typically include an ATG start codon and an adjacent ribosome binding site or other sequences. In addition, the efficiency of expression can be increased by including enhancers appropriate to the cell system used (see, for example, Scharf et al., Results Probl. Cell Differ. 20:125, 1994; and Bittner et al., Meth. Enzymol., 153 :516, 1987). For example, the SV40 enhancer or the CMV enhancer can be used to increase expression in mammalian host cells.

[00181] Expression vectors may also provide a position for a secretion signal sequence to produce a cytokine-grafted antibody protein that is exported from the cell and into the culture medium. In certain aspects, inserted immunoglobulin sequences in cytokine-grafted antibody proteins are coupled to signal sequences prior to incorporation into a vector. Vectors to be used to obtain sequences encoding the variable domains of the light and heavy chains of immunoglobulins sometimes also encode constant regions or parts thereof. Such vectors allow variable regions to be expressed as grafted proteins with constant regions, thereby resulting in the production of intact cytokine-grafted antibody proteins or fragments thereof. Typically, such constant regions are human.

[00182] The host cells carrying and expressing the cytokine-grafted antibody protein chains may be prokaryotic or eukaryotic. E. coli is one prokaryotic host useful for cloning and expression of the polynucleotides of the present invention. Other suitable microorganism hosts include bacilli, such as Bacillus subtilis, and other members of Enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. For these prokaryotic hosts, it is also possible to create expression vectors, which typically contain expression control sequences compatible with the host cell (eg, origin of replication). In addition, any number of various well known promoters will be present, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or the lambda phage promoter system. Typically, promoters control expression, optionally by an operator sequence, and have ribosome binding site sequences, etc., to initiate and terminate transcription and translation. Other microorganisms, such as yeast, can also be used to express polypeptides in cytokine-grafted antibody proteins. Insect cells can also be used in combination with baculovirus vectors.

[00183] In some preferred embodiments, mammalian host cells are used to express and produce polypeptides in cytokine-grafted antibody proteins. For example, they may refer to any mammalian cell line containing an exogenous expression vector. They cover any normal mortal or normal or abnormal immortal animal cells or human cells. For example, a number of suitable host cell lines capable of secreting intact immunoglobulins have been developed, including CHO cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. The use of mammalian tissue culture for polypeptide expression is discussed generally in, for example, Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells may contain expression control sequences , such as the origin of replication, promoter and enhancer (see, for example, Queen et al., Immunol. Rev. 89:49-68, 1986), and necessary sites carrying information for processing, such as ribosome binding sites, sites RNA splicing, polyadenylation sites and transcription termination sequences. These expression vectors typically contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type specific, developmental stage specific, and/or modulated or regulated. Useful promoters include, but are not limited to, metallothionein gene promoter, constitutive adenovirus major late promoter, dexamethasone-inducible MMTV promoter, SV40 promoter, polIII MRP promoter, constitutive MPSV promoter, tetracycline-inducible CMV promoter (such as the human CMV immediate-early promoter), constitutive promoter CMV and promoter-enhancer combinations known in the art.

[00184] Methods for introducing expression vectors containing polynucleotide sequences of interest vary depending on the type of host cell. For example, calcium chloride transfection is typically used for prokaryotic cells, while calcium phosphate treatment or electroporation can be used for other host cells (see generally Sambrook et al., supra). Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, grafting onto the VP22 structural protein of the herpes virus (Elliot and O'Hare, Cell 88:223, 1997), chemically enhanced DNA uptake and ex vivo transduction. To ensure long-term production of recombinant proteins in high yield, stable expression will often be required. For example, cell lines that stably express immunoglobulin chains in cytokine-grafted antibody proteins can be generated using expression vectors disclosed herein that contain viral origins of replication or endogenous expression elements and a selectable marker gene. After vector injection, cells can be allowed to grow for 1-2 days in enriched media before being transferred to selective media. The purpose of a selectable marker is to confer resistance to selection factors, and its presence allows cells that successfully express the introduced sequences to grow in a selective environment. Proliferation of stable, stably transfected cells can be achieved using tissue culture techniques appropriate to the cell type.

Compositions containing cytokine-grafted antibody proteins

[00185] Pharmaceutical compositions are provided comprising a cytokine-grafted antibody protein formulated together with a pharmaceutically acceptable carrier. The pharmaceutical compositions optionally further contain other therapeutic agents that are suitable for treating or preventing the specified disorder. Pharmaceutically acceptable carriers enhance or stabilize the composition or facilitate the preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption retarding agents, and the like, which are physiologically compatible.

[00186] The pharmaceutical composition of the present invention can be administered using a variety of methods known in the art. The route and/or route of administration varies depending on the desired results. Preferably, the administration is parenteral (eg, selected from any of intravenous, intramuscular, intraperitoneal, intrathecal, intra-arterial or subcutaneous) or the administration is near the target site. The pharmaceutically acceptable carrier is suitable for administration by any one or more of intravenous, intramuscular, intraperitoneal, intrathecal, intraarterial, subcutaneous, intranasal, inhalation, spinal, or epidermal administration (eg, injection). Depending on the route of administration, the active compound, such as a cytokine-grafted antibody protein, may be coated with a material to protect the compound from acids and other natural conditions that may inactivate the compound. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration.

[00187] The cytokine-grafted antibody protein, alone or in combination with other suitable components, can be formulated into aerosol formulations (ie, they can be "nebulized") for administration by inhalation. Aerosol formulations may be carried in suitable compressed propellants such as dichlorodifluoromethane, propane, nitrogen, etc.

[00188] In some embodiments, the pharmaceutical composition is sterile and liquid. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion, and by the use of surfactants. In many cases, it is preferred to include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that retards absorption, for example aluminum monostearate or gelatin. In certain embodiments, the compositions may be prepared for storage in lyophilized form using appropriate excipients (eg, sucrose).

[00189] Pharmaceutical compositions can be prepared in accordance with methods well known and commonly practiced in the art. Pharmaceutically acceptable carriers are determined in part by the particular composition administered, as well as the particular method used to administer the composition. Accordingly, a wide variety of suitable pharmaceutical compositions are available. Applicable methods for formulating a cytokine-grafted antibody protein and determining the appropriate dose and dosing schedule can be found, for example, in Remington: The Science and Practice of Pharmacy, ^21st Ed., University of the Sciences in Philadelphia, Eds., Lippincott Williams & Wilkins (2005); and in Martindale: The Complete Drug Reference, Sweetman, 2005, London: Pharmaceutical Press., and in Martindale, Martindale: The Extra Pharmacopoeia, 31st Edition., 1996, Amer Pharmaceutical Assn., and Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured in accordance with GMP regulations. Typically, pharmaceutical compositions utilize a therapeutically effective dose or an effective dose of a cytokine-grafted antibody protein. The cytokine-grafted antibody protein is formulated into a pharmaceutically acceptable dosage form using conventional methods known to those skilled in the art. Dosage regimens are adjusted to provide the desired response (eg, therapeutic response). When administering a therapeutically or prophylactically effective dose, a low dose may be administered and then gradually increased until the desired response is achieved with minimal or no unwanted side effects. For example, a single bolus dose may be administered, multiple divided doses may be administered over time, or the dose may be proportionally decreased or increased according to the needs of the therapeutic situation. It is particularly advantageous to formulate compositions for parenteral use in a single dosage form for ease of administration and uniformity of dosage. Unit dosage form, as used herein, refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect, in combination with the required pharmaceutical carrier.

[00190] Actual dosage levels of active ingredients in pharmaceutical compositions can be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and route of administration without being toxic to the patient. The dose level selected depends on a variety of pharmacokinetic factors, including the potency of the particular compositions used or their ester, salt or amide, route of administration, time of administration, elimination rate of the particular compound used, duration of treatment, other drugs, compounds and/or materials, used in combination with the particular compositions used, the age, sex, body weight, condition, general health and medical history of the patient receiving treatment, and similar factors.

Products/sets

[00191] In some aspects, the cytokine-grafted antibody protein is provided in an article of manufacture (ie, a kit). The provided antibody or cytokine graft protein is typically contained in a vial or container. Thus, the product includes a container and a label or package insert on or with the container. Suitable containers include, for example, a bottle, vial, syringe, solution bag, etc. If desired, the cytokine-grafted antibody protein may be in liquid or dried (eg, lyophilized) form. The container contains a composition that, alone or in combination with another composition, is effective for producing a composition for the treatment, prevention and/or amelioration of cancer. The label or instructions for use of the drug indicate that the composition is used to treat, prevent and/or reduce the severity of cancer. Products (kits) containing the cytokine-grafted antibody protein described herein optionally contain one or more additional agents. In some embodiments, the product (kit) contains a cytokine-grafted antibody protein and a pharmaceutically acceptable diluent. In some embodiments, the cytokine-grafted antibody protein is provided in an article (kit) with one or more additional active agents in the same composition (eg, in the form of mixtures). In some embodiments, the cytokine-grafted antibody protein is provided in an article (kit) with a second or third agent in separate formulations (eg, in separate containers). In certain embodiments, the product (kit) contains aliquots of a cytokine-grafted antibody protein, where the aliquot comprises one or more doses. In some embodiments, aliquots are provided for multiple administrations, wherein the doses are the same or different. In specific embodiments, the various dosage regimens provide for increasing or decreasing the dosage as needed. In some embodiments, the dosages of the cytokine-grafted antibody protein and the second agent are independently the same or independently different. In certain embodiments, the product (kit) contains an additional agent, such as an antitumor agent or an immune checkpoint molecule. The choice of one or more additional agents will depend on the dose, delivery, and disease state being treated.

Methods of treatment and use of compositions for the treatment of cancer

Conditions to be treated or prevented

[00192] Cytokine-grafted antibody proteins find use in the treatment, mitigation, or prevention of cancer. In one aspect, the present invention provides methods for treating cancer in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of a cytokine-grafted antibody protein described herein. In some embodiments, a cytokine-grafted antibody protein is provided for use as a therapeutic agent for treating or preventing cancer in an individual. In a further aspect, the present invention provides a composition comprising such a cytokine-grafted antibody protein for use in treating or ameliorating cancer in an individual in need thereof.

[00193] Conditions to be treated include various cancer indications. For therapeutic purposes, an individual has been diagnosed with cancer. For preventive or prophylactic purposes, the individual may be in remission from cancer or may expect the disease to appear in the future. In some embodiments, the patient has cancer, is suspected of having cancer, or is in remission from cancer. For cancers to be treated with a cytokine-grafted antibody protein, activation of low-affinity IL2 receptor signaling as described herein is generally beneficial. Cancer indications to be treated include, but are not limited to, melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer and lymphoma.

Administration of Cytokine-Grafted Antibody Proteins

[00194] The physician or veterinarian may begin dosing the cytokine-grafted antibody protein used in the pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect has been achieved. In general, effective dosages of the compositions will vary depending on many different factors, including the specific disease or condition being treated, route of administration, target site, physiological condition of the patient, whether the patient is human or animal, and other drugs administered. and whether the treatment is preventive or therapeutic. Treatment dosages typically need to be adjusted to optimize safety and efficacy. When administering a cytokine-grafted antibody protein, the dose ranges from about 0.0001 to 100 mg/kg, and more commonly from 0.01 to 5 mg/kg of the body weight of the patient receiving it. For example, doses may be 1 mg/kg body weight or 10 mg/kg body weight, or be in the range of 1-10 mg/kg. Dosing may occur daily, weekly, biweekly, monthly, or more frequently or less frequently as needed or desired. An exemplary treatment regimen is administered once a week, once every two weeks, or once a month, or once every 3-6 months.

[00195] The cytokine-grafted antibody protein can be administered in a single dose or in divided doses. The cytokine-grafted antibody protein is typically administered multiple times. The intervals between individual doses may be, if necessary or desired, a week, two weeks, a month or a year. The intervals may also be irregular, as indicated by measuring cytokine-coupled antibody protein levels in the patient's blood. In some methods, the dose is adjusted to achieve a plasma concentration of the cytokine-grafted antibody protein of 1-1000 μg/ml, and in some methods 25-300 μg/ml. Alternatively, the cytokine-grafted antibody protein can be administered as a sustained release formulation, in which case less frequent administration is required. The dose and frequency vary depending on the half-life of the cytokine-grafted antibody protein from the patient. In general, cytokine-grafted antibody proteins have a longer half-life than native IL2 or recombinant cytokines such as Proleukin®. The dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. Typically, in prophylactic routes of administration, a relatively low dose is administered at relatively large intervals over a long period of time. Some patients continue to receive treatment throughout their lives. In general, in therapeutic routes of administration, it is sometimes necessary to administer a relatively high dose at relatively short intervals until disease progression has subsided or is complete, and preferably until the patient has experienced partial or complete relief of symptoms of the disease. . The patient can then be administered according to the prophylactic regimen.

Co-administration with a second drug

[00196] The term “combination therapy” refers to the administration of two or more therapeutic agents to treat a therapeutic condition or disorder described in the present invention. Such administration includes co-administration of these therapeutic agents substantially simultaneously, for example, in a single capsule having a fixed ratio of active ingredients. Alternatively, such administration includes co-administration using multiple or separate containers (eg, capsules, powders and liquids) for each active ingredient. Powders and/or liquids may be reconstituted or diluted to the required dose before administration. In addition, such administration also covers the use of each type of therapeutic agent in a sequential manner at approximately the same time or at different times. In any case, the treatment regimen will provide the beneficial effects of the drug combination in treating the conditions or disorders described herein.

[00197] Combination therapy may provide "synergism" and appear to be "synergistic", i.e. the effect achieved by using the active ingredients together exceeds the sum of the effects that result from using the compounds separately. A synergistic effect can be achieved if the active ingredients are: (1) formulated together and administered or delivered simultaneously as a single-dose combination formulation; (2) delivered in sequence or in parallel as separate trains, or (3) some other mode is used. When delivered as a sequential therapy, a synergistic effect may be achieved if the compounds are administered or delivered sequentially, for example, through different injections in separate syringes. Typically, during alternating therapy, effective doses of each active ingredient are administered sequentially, i.e. serially, whereas in combination therapy, effective doses of two or more active ingredients are administered together.

[00198] In one aspect of the present invention, there is provided a method of treating cancer by administering to a subject in need thereof a cytokine-grafted antibody protein in combination with one or more tyrosine kinase inhibitors, including, but not limited to, EGFR inhibitors, Her2 inhibitors, Her3 inhibitors, IGFR inhibitors and Met inhibitors.

[00199] For example, tyrosine kinase inhibitors include, but are not limited to, erlotinib hydrochloride (Tarceva®); linifanib (N-[4-(3-amino-1H-indazol-4-yl)phenyl]-N'-(2-fluoro-5-methylphenyl)urea, also known as ABT 869, available from Genentech); sunitinib malate (Sutent®); bosutinib (4-[(2,4-dichloro-5-methoxyphenyl)amino]-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy]quinolin-3-carbonitrile, also known as SKI- 606 and described in US Pat. No. 6,780,996); dasatinib (Sprycel®); pazopanib (Votrient®); sorafenib (Nexavar®); zaktimu (ZD6474); nilotinib (Tasigna®); regorafenib (Stivarga®) and imatinib or imatinib mesylate (Gilvec® and Gleevec®).

[00200] Epidermal growth factor receptor (EGFR) inhibitors include, but are not limited to, erlotinib hydrochloride (Tarceva®), gefitinib (Iressa®); N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3''S'')-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4(dimethylamino)- 2-butenamide (Tovok®); vandetanib (Caprelsa®); lapatinib (Tykerb®); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3 -ol (BMS690514); canertinib dihydrochloride (CI-1033); 6-[4-[(4-ethyl-1-piperazinyl)methyl]phenyl]-N-[(1R)-1-phenylethyl]-7H-pyrrolo[2,3-d]pyrimidin-4-amine (AEE788, CAS 497839-62-0); mubritinib (TAK165); pelitinib (EKB569); afatinib (BIBW2992); neratinib (HKI-272); (3S)-3-morpholinylmethyl ester N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1 ,2,4]triazin-6-yl]-carbamic acid (BMS599626); N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8) and 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol (PKI166, CAS 187724-61-4).

[00201] Anti-EGFR antibodies include, but are not limited to, cetuximab (Erbitux®); panitumumab (Vectibix®); matuzumab (EMD-72000); nimotuzumab (hR3); zalutumumab; TheraCIM h-R3; MDX0447 (CAS 339151-96-1) and ch806 (mAb-806, CAS 946414-09-1).

[00202] Inhibitors of human epidermal growth factor receptor 2 (HER2 receptor) (also known as Neu, ErbB-2, CD340 or p185) include, but are not limited to, trastuzumab (Herceptin®); pertuzumab (Omnitarg®); neratinib (HKI-272, (2E)-N-[4-[[3-chloro-4-[(pyridin-2-yl)methoxy]phenyl]amino]-3-cyano-7-ethoxyquinolin-6-yl] -4-(dimethylamino)but-2-enamide, described in PCT publication No. WO 05/028443); lapatinib or lapatinib ditosylate (Tykerb®); (3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyrrolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3 -ol (BMS690514); (2E)-N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[[(3S)-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4-(dimethylamino)- 2-butenamide (BIBW-2992, CAS 850140-72-6); (3S)-3-morpholinylmethyl ester N-[4-[[1-[(3-fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1 ,2,4]triazin-6-yl]-carbamic acid (BMS 599626, CAS 714971-09-2); canertinib dihydrochloride (PD183805 or CI-1033) and N-(3,4-dichloro-2-fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro-2-methylcyclopenta[c]pyrrol- 5-yl]methoxy]-4-quinazolinamine (XL647, CAS 781613-23-8).

[00203] HER3 inhibitors include, but are not limited to, LJM716, MM-121, AMG-888, RG7116, REGN-1400, AV-203, MP-RM-1, MM-111, and MEHD-7945A.

[00204] Met inhibitors include, but are not limited to, cabozantinib (XL184, CAS 849217-68-1); foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); tivantinib (ARQ197, CAS 1000873-98-2); 1-(2-hydroxy-2-methylpropyl)-N-(5-(7-methoxyquinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenyl-2,3-dihydro -1H-pyrazole-4-carboxamide (AMG 458); crizotinib (Xalkori®, PF-02341066); (3Z)-5-(2,3-dihydro-1H-indol-1-ylsulfonyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrole -2-yl}methylene)-1,3-dihydro-2H-indol-2-one (SU11271); (3Z)-N-(3-chlorophenyl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrol-2-yl}methylene)-N- methyl 2-oxoindoline-5-sulfonamide (SU11274); (3Z)-N-(3-chlorophenyl)-3-{[3,5-dimethyl-4-(3-morpholin-4-ylpropyl)-1H-pyrrol-2-yl]methylene}-N-methyl-2 -oxoindoline-5-sulfonamide (SU11606); 6-[difluoro[6-(1-methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]methyl]-quinoline (JNJ38877605, CAS 943540 -75-8); 2-[4-[1-(quinolin-6-ylmethyl)-1H-[1,2,3]triazolo[4,5-b]pyrazin-6-yl]-1H-pyrazol-1-yl]ethanol ( PF04217903, CAS 956905-27-4); N-((2R)-1,4-dioxan-2-ylmethyl)-N-methyl-N'-[3-(1-methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[ 4,5]cyclohepta[1,2-b]pyridin-7-yl]sulfamide (MK2461, CAS 917879-39-1); 6-[[6-(1-Methyl-1H-pyrazol-4-yl)-1,2,4-triazolo[4,3-b]pyridazin-3-yl]thio]-quinoline (SGX523, CAS 1022150- 57-7) and (3Z)-5-[[(2,6-dichlorophenyl)methyl]sulfonyl]-3-[[3,5-dimethyl-4-[[(2R)-2-(1-pyrrolidinylmethyl) -1-pyrrolidinyl]carbonyl]-1H-pyrrol-2-yl]methylene]-1,3-dihydro-2H-indol-2-one (PHA665752, CAS 477575-56-7).

[00205] IGF1R inhibitors include, but are not limited to, BMS-754807, XL-228, OSI-906, GSK0904529A, A-928605, AXL1717, KW-2450, MK0646, AMG479, IMCA12, MEDI-573, and BI836845. For an overview see, for example, Yee, JNCI, 104; 975 (2012).

[00206] In another aspect of the present invention, there is provided a method of treating cancer by administering to a subject in need thereof a cytokine-loaded antibody protein in combination with one or more inhibitors of FGF downstream signaling pathways, including, but not limited to, MEK inhibitors, Braf inhibitors, PI3K/Akt inhibitors, SHP2 inhibitors, and mTor inhibitors.

[00207] For example, mitogen-activated protein kinase (MEK) inhibitors include, but are not limited to, XL-518 (also known as GDC-0973, Cas No. 1029872-29-4, available from ACC Corp.); 2-[(2-chloro-4-iodophenyl)amino]-N-(cyclopropylmethoxy)-3,4-difluorobenzamide (also known as CI-1040 or PD184352 and described in PCT Publication No. WO2000035436); N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]benzamide (also known as PD0325901 and described in PCT Publication No. WO2002006213); 2,3-bis[amino[(2-aminophenyl)thio]methylene]butane dinitrile (also known as U0126 and described in US Pat. No. 2,779,780); N-[3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl]-1-[(2R)-2,3-dihydroxypropyl]cyclopropanesulfonamide (also known as RDEA119 or BAY869766 and described in publication according to PCT No. WO2007014011); (3S,4R,5Z,8S,9S,11E)-14-(ethylamino)-8,9,16-trihydroxy-3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine- 1,7(8H)-dione] (also known as E6201 and described in PCT Publication No. WO2003076424); 2'-amino-3'-methoxyflavone (also known as PD98059, available from Biaffin GmbH & Co., KG, Germany); vemurafenib (PLX-4032, CAS 918504-65-1); (R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H) -dione (TAK-733, CAS 1035555-63-5);

pimasertib (AS-703026, CAS 1204531-26-9) and trametinib dimethyl sulfoxide (GSK-1120212, CAS 1204531-25-80).

[00208] Phosphoinositide 3-kinase (PI3K) inhibitors include, but are not limited to, 4-[2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazin-1-yl]methyl]thieno[3 ,2-d]pyrimidin-4-yl]morpholine (also known as GDC 0941 and described in PCT publication nos. WO 09/036082 and WO 09/055730); 2-methyl-2-[4-[3-methyl-2-oxo-8-(quinolin-3-yl)-2,3-dihydroimidazo[4,5-c]quinolin-1-yl]phenyl]propionitrile ( also known as BEZ 235 or NVP-BEZ 235 and described in PCT publication No. WO 06/122806); 4-(trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridin-2-amine (also known as BKM120 or NVP-BKM120 and described in PCT Publication No. WO2007/084786); tosasertib (VX680 or MK-0457, CAS 639089-54-6); (5Z)-5-[[4-(4-pyridinyl)-6-quinolinyl]methylene]-2,4-thiazolidinedione (GSK1059615, CAS 958852-01-2); (1E,4S,4aR,5R,6aS,9aR)-5-(acetyloxy)-1-[(di-2-propenylamino)methylene]-4,4a,5,6,6a,8,9,9a-octahydro -11-hydroxy-4-(methoxymethyl)-4a,6a-dimethylcyclopenta[5,6]naphtho[1,2-c]pyran-2,7,10(1H)-trione (PX866, CAS 502632-66-8 ) and 8-phenyl-2-(morpholin-4-yl)-chromen-4-one (LY294002, CAS 154447-36-6).

[00209] mTOR inhibitors include, but are not limited to, temsirolimus (Torisel®); ridaforolimus (formerly known as deferolimus, (1R,2R,4S)-4-[(2R)-2-[(1R,9S,12S,15R,16E,18R,19R,21R,23S,24E,26E,28Z, 30S,32S,35R)-1,18-dihydroxy-19,30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa -4-azatricyclo[30.3.1.0 ^4,9 ]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethylphosphinate, also known as AP23573 and MK8669 and described in PCT publication No. WO 03 /064383); everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); simapimod (CAS 164301-51-3); (5-{2,4-bis[(3S)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl}-2-methoxyphenyl)methanol (AZD8055); 2-amino-8-[trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3-pyridinyl)-4-methylpyrido[2,3-d]pyrimidin-7(8H)-one ( PF04691502, CAS 1013101-36-4) and N ² -[1,4-dioxo-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)morpholinium-4-yl ]methoxy]butyl]-L-arginylglycyl-L-α-aspartyl-L-serine (“L-arginylglycyl-L-α-aspartyl-L-serine” is disclosed under SEQ ID NO: 77) in the form of internal salt (SF1126, CAS 936487-67-1).

[00210] In another aspect of the present invention, there is provided a method of treating cancer by administering to a subject in need thereof a cytokine-grafted antibody protein in combination with one or more pro-apoptotic agents, including, but not limited to, IAP inhibitors, Bcl2 inhibitors, MCL1 inhibitors , TRAIL-based agents, Chk inhibitors.

[00211] For example, IAP inhibitors include, but are not limited to, NVP-LCL161, GDC-0917, AEG-35156, AT406, and TL32711. Other examples of IAP inhibitors include, but are not limited to, those disclosed in WO04/005284, WO 04/007529, WO05/097791, WO 05/069894, WO 05/069888, WO 05/094818, US2006/0014700, US2006/0025347, WO 06/06 9063, WO 06/010118, WO 06/017295 and WO08/134679.

[00212] BCL-2 inhibitors include, but are not limited to, 4-[4-[[2-(4-chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperazinyl]-N-[ [4-[[(1R)-3-(4-morpholinyl)-1-[(phenylthio)methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl]benzamide (also known as ABT-263 and described in publication according to PCT No. WO 09/155386); tetrocarcin A; antimycin; gossypol ((-)BL-193); obatoclax; ethyl 2-amino-6-cyclopentyl-4-(1-cyano-2-ethoxy-2-oxoethyl)-4H-chromone-3-carboxylate (HA14-1); oblimersen (G3139, Genasense®); Bak BH3 peptide; (-)-gossypoacetic acid (AT-101); 4-[4-[(4'-chloro[1,1'-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)- 1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]sulfonyl]-benzamide (ABT-737, CAS 852808-04-9) and navitoclax (ABT-263, CAS 923564-51-6).

[00213] Pro-apoptotic receptor agonists (PARA), including DR4 (TRAILR1) and DR5 (TRAILR2), include, but are not limited to, dulanermine (AMG-951, RhApo2L/TRAIL); mapatumumab (HRS-ETR1, CAS 658052-09-6); lexatumumab (HGS-ETR2, CAS 845816-02-6); Apomab®; conatumumab (AMG655, CAS 896731-82-1) and tigatuzumab (CS1008, CAS 946415-34-5, available from Daiichi Sankyo).

[00214] Checkpoint kinase (CHK) inhibitors include, but are not limited to, 7-hydroxystaurosporine (UCN-01); 6-bromo-3-(1-methyl-1H-pyrazol-4-yl)-5-(3R)-3-piperidinylpyrazolo[1,5-a]pyrimidin-7-amine (SCH900776, CAS 891494-63-6 ); 5-(3-fluorophenyl)-3-ureidothiophene-2-carboxylic acid N-[(S)-piperidin-3-yl]amide (AZD7762, CAS 860352-01-8); 4-[((3S)-1-azabicyclo[2.2.2]oct-3-yl)amino]-3-(1H-benzimidazol-2-yl)-6-chloroquinolin-2(1H)-one (CHIR 124 , CAS 405168-58-3); 7-aminodactinomycin (7-AAD), isogranulatimide, debromohymenialdizine; N-[5-bromo-4-methyl-2-[(2S)-2-morpholinylmethoxy]phenyl]-N'-(5-methyl-2-pyrazinyl)urea (LY2603618, CAS 911222-45-2); sulforaphane (CAS 4478-93-7, 4-methylsulfinylbutyl isothiocyanate); 9,10,11,12-tetrahydro-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1, 6]benzodiazocine-1,3(2H)-dione (SB-218078, CAS 135897-06-2) and TAT-S216A (Sha et al., Mol. Cancer. Ther 2007; 6(1):147-153) , as well as CBP501.

[00215] In one aspect of the present invention, there is provided a method of treating cancer by administering to a subject in need thereof a cytokine-grafted antibody protein in combination with one or more FGFR inhibitors. For example, FGFR inhibitors include, but are not limited to, brivanib alaninate (BMS-582664, (S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5- methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate); vargatef (BIBF1120, CAS 928326-83-4); dovitinib dilactate (TKI258, CAS 852433-84-2); 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-{6-[4-(4-ethylpiperazin-1-yl)-phenylamino]-pyrimidin-4-yl}-1-methylurea (BGJ398, CAS 872511-34-7); danusertib (PHA-739358) and (PD173074, CAS 219580-11-7). In a specific aspect, the present invention provides a method of treating cancer by administering to a subject in need thereof an antibody-drug conjugate in combination with an FGFR2 inhibitor such as 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-( 6((4-(4-ethylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-urea (also known as BGJ-398) or 4-amino-5-fluoro-3-(5- (4-methylpiperazin-1-yl)-1H-benzo[d]imidazol-2-yl)quinolin-2(1H)-one (also known as dovitinib or TKI-258), AZD4547 (Gavine et al., 2012, Cancer Research 72, 2045-56, N-[5-[2-(3,5-dimethoxyphenyl)ethyl]-2H-pyrazol-3-yl]-4-(3R,5S)-dimethylpiperazin-1-yl)benzamide ), ponatinib (AP24534; Gozgit et al., 2012, Mol Cancer Ther., 11; 690-99; 3-[2-(imidazo[1,2-b]pyridazin-3-yl)ethynyl]-4-methyl -N-{4-[(4-methylpiperazin-1-yl)methyl]-3-(trifluoromethyl)phenyl}benzamide, CAS 943319-70-8).

[00216] Cytokine-grafted antibody proteins can also be administered in combination with an immune checkpoint inhibitor. In one embodiment, cytokine-grafted antibody proteins may be administered in combination with an inhibitor of an immune checkpoint molecule selected from one or more of PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 or TGFR. In one embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody, wherein the anti-PD-1 antibody is selected from nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the anti-PD-1 antibody molecule is nivolumab. Alternative names for nivolumab include MDX-1106, MDX-1106-04, ONO-4538, or BMS-936558. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registration number: 946414-94-4). Nivolumab is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Nivolumab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 are disclosed in US 8008449 and WO 2006/121168.

[00217] In some embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab (also called lambrolizumab, MK-3475, MK03475, SCH-900475, or KEYTRUDA®; Merck) is a humanized IgG4 monoclonal antibody that binds to PD-1. Pembrolizumab and other humanized anti-PD-1 antibodies are disclosed in Hamid, O. et al. (2013) New England Journal of Medicine 369(2): 134-44, US 8354509 and WO 2009/114335.

[00218] In some embodiments, the anti-PD-1 antibody is pidilizumab. Pidilizumab (CT-011; Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are disclosed in WO 2009/101611.

[00219] Other anti-PD1 antibodies include AMP 514 (Amplimmune) and, for example, the anti-PD1 antibodies disclosed in US 8609089, US 2010/028330 and/or US 2012/0114649 and US 2016/0108123.

In some embodiments, cytokine-grafted antibody proteins can be administered with the anti-Tim3 antibody disclosed in US 2015/0218274. In other embodiments, cytokine-grafted antibody proteins can be administered with the anti-PD-L1 antibody disclosed in US 2016/0108123, Durvalumab® (MEDI4736), Atezolizumab® (MPDL3280A) or Avelumab®.

EXAMPLES

Example 1. Creation of proteins based on antibodies grafted with the cytokine IL2

[00220] Cytokine-grafted antibody proteins were prepared by inserting the IL2 sequence into the CDR regions of various immunoglobulin backbones, and then both the heavy and light chains of the immunoglobulins were used to produce the final antibody and cytokine proteins. Cytokine-grafted antibody proteins confer preferential therapeutic properties to IL2; however, cytokine-grafted antibody proteins exhibit reduced off-target effects, such as increased Treg cell activity, compared to rhIL2.

[00221] To create cytokine-grafted antibody proteins, IL2 sequences containing muteins (SEQ ID NO:4 or 6) were inserted into the CDR loops of the immunoglobulin backbone. Cytokine-grafted antibody proteins were generated using a variety of known immunoglobulin sequences that have been used in clinical settings, as well as germline antibody sequences. The sequences of IL2 in an exemplary backbone called GFTX3b are displayed in Table 2. Insertion points were selected to represent the midpoint of the loop based on available structural or homology modeling data. Cytokine-grafted antibody proteins were produced using standard molecular biology techniques by using recombinant DNA encoding the corresponding sequences.

[00222] The choice of which CDR to select for cytokine grafting was made based on parameters of desired biological and biophysical properties and favorable development profile. The modeling software was only partially useful in predicting which CDR and which position in the CDR would provide the required parameters, so all six possible cytokine-grafted antibody molecules were thus generated and then evaluated in biological assays. Once the desired biological activity has been achieved, biophysical properties, such as structural resolution, are then determined regarding how the cytokine-grafted antibody molecule interacts with the corresponding cytokine receptor.

[00223] In the case of IL2 cytokine grafted antibody molecules, the structure of the candidate antibody being considered for cytokine grafting was initially determined. From this structure, it was noted that the paratope was at the extreme position of the N-terminal arm of the antibody, and that a cytokine grafted at this position would present the cytokine to its corresponding receptor. Thanks to the grafting technology, each IL2-grafted antibody protein is constrained by a CDR loop with a different length, sequence, and structural environment. Therefore, IL2 was grafted onto all six CDRs corresponding to LCDR-1, LCDR-2, LCDR-3 and HCDR-1, HCDR-2 and HCDR-3. Based on the table in Figure 1, it is apparent that the cytokine-grafted antibody proteins differ in their activity, including that IL2 grafted onto the light chain CDR2 (IgG.IL2.L2) was not expressed. It was also observed that antibody molecules grafted with the cytokine IL2 with altered Fc function (eg, Fc silenced) had a better profile.

[00224] HCDR-1 was selected because it had the best combination of properties (biophysical and biological) and the IL2 point mutations that were included provided an enhancement of the desired biological properties. When selecting the insertion point, the structural center of the CDR loop was chosen because it provided the largest amount of space on each side (linear size 3.8Å x number of residues) and, without being limited by theory, it provided a stable molecule, allowing IL2 to be more free to lay independently. Since the structure of the GFTX3b grafting backbone was already known, the structural center of each CDR was also known. It matched the center of the CDR loop sequence determined using the Chothia numbering format. As mentioned previously, the insertion point of the grafted IL-2 molecules was shifted from the center towards the N- or C-terminal part of the CDR loop. At the same time, the displacement of IL2 within the CDR loop did not cause a significant difference in biological activity.

[00225] In summary, the insertion point in each CDR was chosen on a structural basis, based on the hypothesis that grafting into the CDR would provide some level of steric mismatch of the individual IL2 receptor subunits. The final selection of which grafting CDR was most effective for a particular cytokine was based on the desired biological and biophysical properties. The nature of the cytokine receptor, cytokine/receptor interactions and signal transduction mechanism also played a role, and this selection was made by comparing each individual antibody and cytokine molecule for its respective properties.

TABLE 1

SEQ ID NO:1 DNA encoding wild-type IL2 agttccctatcactctctttaatcactactcacagtaacctcaactcctgccacaatgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacaaacagtgcacctacttcaagttctacaaagaaaacacagctacaactggagcatttactgctggatttacagatgattttgaatggaattaataattacaagaatccca aactcaccaggatgctcacatttaagttttacatgcccaagaaggccacagaactgaaacatcttcagtgtctagaagaagaactcaaacctctggaggaagtgctaaatttagctcaaagcaaaaactttcacttaagacccaggacttaatcagcaatatcaacgtaatagttctggaactaaagggatctgaaacaacattcatgtgtgaatatgctgatgaga cagcaaccattgtagaatttctgaacagatggattaccttttgtcaaagcatcatctcaacactgacttgataattaagtgcttcccacttaaaacatatcaggccttctatttatttaaatatttaaattttatatttattgttgaatgtatggtttgctacctattgtaactattattcttaatcttaaaactataaatatggatcttttatgattctttttg taagccctaggggctctaaaatggtttcacttatttatcccaaaatatttattattatgttgaatgttaaatatagtatctatgtagattggttagtaaaactatttaataaatttgataaatataaaaaaaaaaaaaaaaaaaaaaaaa SEQ ID NO:2 IL2 protein myrmqllscialslalvtnsaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistlt SEQ ID NO:3 DNA encoding IL2 mutein CAAGTCACACTGCGTGAAAGCGGCCCTGCCCTGGTCAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCAGCCTGGCCCCTACCTCCTCCAGCACCAAGAAAACCCAGCTGCAGCTCGAACATCTGCTGCTGGACCTGCAGATGATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGACCCGGATGCTGACCGCCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAACAT CTGCAGTGCCTGGAAGAGGAACTGAAGCCCCTGGAAGAAGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTGAGGCCTCGGGACCTGATCTCCAACATCAACGTGATCGTGCTGGAACTGAAGGGCTCCGAGACAACCTTCATGTGCGAGTACGCCGACGAGACAGCCACCATCGTGGAATTTCTGAACCGGTGGATCACCTTCTGCCAGTCCATCTCCACCCTGACCTCCACCTCCGGCATG TCCGTGGGCTGGATCCGGCAGCCTCCTGGCAAGGCCCTGGAGTGGCTGGCCGACATTTGGTGGGACGACAAGAAGGACTACAACCCCAGCCTGAAGTCCCGGCTGACCATCTCCAAGGACACCTCCAAGAACCAAGTGGTGCTGAAAGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCCGGTCCATGATCACCAACTGGTACTTCGACGTGTGGGGCGCTGGCACCACCGTGACCG TGTCCTCT SEQ ID NO:4 IL2 mutein protein, mutant amino acid in bold and underlined APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO:5 DNA encoding IL2 mutein GCCCCTACCTCCTCCAGCACCAAGAAAACCCAGCTGCAGCTCGAACATCTGCTGCTGGACCTGCAGATGATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGACCCGGATGCTGACCGCCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAACATCTGCAGTGCCTGGAAGAGGAACTGAAGCCCCTGGAAGAAGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTGAGGCCTCGG GACCTGATCCCAACATCAACGTGATCGTGCTGGAACTGAAGGGCTCCGAGACAACCTTCATGTGCGAGTACGCCGACGAGACAGCCACCATCGTGGAATTTCTGAACCGGTGGATCACCTTCTGCCAGTCCATCATCTCCACCCCTGACC SEQ ID NO:6 IL2 mutein protein, mutant amino acid in bold and underlined APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

TABLE 2

IgG.IL2R67A.H1 SEQ ID NO:7 (combined) HCDR1 GFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMSVG SEQ ID NO:8 (combined) HCDR2 DIWWDDKKDYNPSLKS SEQ ID NO:9 (combined) HCDR3 SMITNWYFDV SEQ ID NO:10 (Kabat) HCDR1 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMSVG SEQ ID NO:11 (Kabat) HCDR2 DIWWDDKKDYNPSLKS SEQ ID NO:12 (Kabat) HCDR3 SMITNWYFDV SEQ ID NO:13(Chothia) HCDR1 GFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGM SEQ ID NO:14 (Chothia) HCDR2 WWDDK SEQ ID NO:15 (Chothia) HCDR3 SMITNWYFDV SEQ ID NO:16 (IMGT) HCDR1 GFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMS SEQ ID NO:17 (IMGT) HCDR2 IWWDDKK SEQ ID NO:18 (IMGT) HCDR3 ARSMITNWYFDV SEQ ID NO:19 VH QVTLRESGPALVKPTQTLTLTCTFSGFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLT ISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSS SEQ ID NO:20 DNA encoding VH CAAGTCACACTGCGTGAAAGCGGCCCTGCCCTGGTCAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCAGCCTGGCCCCTACCTCCTCCAGCACCAAGAAAACCCAGCTGCAGCTCGAACATCTGCTGCTGGACCTGCAGATGATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGACCGCCATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAACAT CTGCAGTGCCTGGAAGAGGAACTGAAGCCCCTGGAAGAAGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTGAGGCCTCGGGACCTGATCTCCAACATCAACGTGATCGTGCTGGAACTGAAGGGCTCCGAGACAACCTTCATGTGCGAGTACGCCGACGAGACAGCCACCATCGTGGAATTTCTGAACCGGTGGATCACCTTCTGCCAGTCCATCTCCACCCTGACCTCCACCTCCGGCATG TCCGTGGGCTGGATCCGGCAGCCTCCTGGCAAGGCCCTGGAGTGGCTGGCCGACATTTGGTGGGACGACAAGAAGGACTACAACCCCAGCCTGAAGTCCCGGCTGACCATCTCCAAGGACACCTCCAAGAACCAAGTGGTGCTGAAAGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCCGGTCCATGATCACCAACTGGTACTTCGACGTGTGGGGCGCTGGCACCACCGTGACCG TGTCCTCT SEQ ID NO:21 Heavy chain QVTLRESGPALVKPTQTLTLTCTFSGFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLT ISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:22 DNA encoding the heavy chain CAAGTCACACTGCGTGAAAGCGGCCCTGCCCTGGTCAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCAGCCTGGCCCCTACCTCCTCCAGCACCAAGAAAACCCAGCTGCAGCTCGAACATCTGCTGCTGGACCTGCAGATGATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGACCGCCATGCTGACCTTCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAACAT CTGCAGTGCCTGGAAGAGGAACTGAAGCCCCTGGAAGAAGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTGAGGCCTCGGGACCTGATCTCCAACATCAACGTGATCGTGCTGGAACTGAAGGGCTCCGAGACAACCTTCATGTGCGAGTACGCCGACGAGACAGCCACCATCGTGGAATTTCTGAACCGGTGGATCACCTTCTGCCAGTCCATCTCCACCCTGACCTCCACCTCCGGCATG TCCGTGGGCTGGATCCGGCAGCCTCCTGGCAAGGCCCTGGAGTGGCTGGCCGACATTTGGTGGGACGACAAGAAGGACTACAACCCCAGCCTGAAGTCCCGGCTGACCATCTCCAAGGACACCTCCAAGAACCAAGTGGTGCTGAAAGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCCGGTCCATGATCACCAACTGGTACTTCGACGTGTGGGGCGCTGGCACCACCGTGACCG TGTCCTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGAC CTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAACTGCTGGGCGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGG TACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGGCCGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACC AAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTC CCTGTCTCCCGGCAAG SEQ ID NO:23 (merged) LCDR1 KAQLSVGYMH SEQ ID NO:24 (merged) LCDR2 DTSKLAS SEQ ID NO:25 (combined) LCDR3 FQGSGYPFT SEQ ID NO:26 (Kabat) LCDR1 KAQLSVGYMH SEQ ID NO:27 (Kabat) LCDR2 DTSKLAS SEQ ID NO:28 (Kabat) LCDR3 FQGSGYPFT SEQ ID NO:29 (Chothia) LCDR1 QLSVGY SEQ ID NO:30 (Chothia) LCDR2 DTS SEQ ID NO:31 (Chothia) LCDR3 GSGYPF SEQ ID NO:32 (IMGT) LCDR1 LSVGY SEQ ID NO:33 (IMGT) LCDR2 DTS SEQ ID NO:34 (IMGT) LCDR3 FQGSGYPFT SEQ ID NO:35 VL DIQMTQSPSTLSASVGDRVTTITCKAQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIK SEQ ID NO:36 DNA encoding VL GACATCCAGATGACCCAGAGCCCCTCCACCCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACTTGCAAGGCCCAGCTGTCCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTCACCCTGACCATCTCCAGCCTGCAGCCCGACGACTTCG CCACCTACTACTGTTTTCAAGGCTCCGGCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG SEQ ID NO:37 Light chain DIQMTQSPSTLSASVGDRVTITCKAQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:38 DNA encoding the light chain GACATCCAGATGACCCAGAGCCCCTCCACCCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACTTGCAAGGCCCAGCTGTCCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTCACCCTGACCATCTCCAGCCTGCAGCCCGACGACTTCG CCACCTACTACTGTTTTCAAGGCTCCGGCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAG CAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC IgG.IL2F71A.H1 SEQ ID NO:39 (merged) HCDR1 GFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMSVG SEQ ID NO:40 (combined) HCDR2 DIWWDDKKDYNPSLKS SEQ ID NO:41 (merged) HCDR3 SMITNWYFDV SEQ ID NO:42 (Kabat) HCDR1 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMSVG SEQ ID NO:43 (Kabat) HCDR2 DIWWDDKKDYNPSLKS SEQ ID NO:44 (Kabat) HCDR3 SMITNWYFDV SEQ ID NO:45 (Chothia) HCDR1 GFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGM SEQ ID NO:46 (Chothia) HCDR2 WWDDK SEQ ID NO:47 (Chothia) HCDR3 SMITNWYFDV SEQ ID NO:48 (IMGT) HCDR1 GFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMS SEQ ID NO:49 (IMGT) HCDR2 IWWDDKK SEQ ID NO:50 (IMGT) HCDR3 ARSMITNWYFDV SEQ ID NO:51 VH QVTLRESGPALVKPTQTLTLTCTFSGFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLT ISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSS SEQ ID NO:52 DNA encoding VH CAAGTCACACTGCGTGAAAGCGGCCCTGCCCTGGTCAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCAGCCTGGCCCCTACCTCCTCCAGCACCAAGAAAACCCAGCTGCAGCTCGAACATCTGCTGCTGGACCTGCAGATGATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGACCCGGATGCTGACCGCCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAACAT CTGCAGTGCCTGGAAGAGGAACTGAAGCCCCTGGAAGAAGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTGAGGCCTCGGGACCTGATCTCCAACATCAACGTGATCGTGCTGGAACTGAAGGGCTCCGAGACAACCTTCATGTGCGAGTACGCCGACGAGACAGCCACCATCGTGGAATTTCTGAACCGGTGGATCACCTTCTGCCAGTCCATCTCCACCCTGACCTCCACCTCCGGCATG TCCGTGGGCTGGATCCGGCAGCCTCCTGGCAAGGCCCTGGAGTGGCTGGCCGACATTTGGTGGGACGACAAGAAGGACTACAACCCCAGCCTGAAGTCCCGGCTGACCATCTCCAAGGACACCTCCAAGAACCAAGTGGTGCTGAAAGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCCGGTCCATGATCACCAACTGGTACTTCGACGTGTGGGGCGCTGGCACCACCGTGACCG TGTCCTCT SEQ ID NO:53 Heavy chain QVTLRESGPALVKPTQTLTLTCTFSGFSLAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLTSTSGMSVGWIRQPPGKALEWLADIWWDDKKDYNPSLKSRLT ISKDTSKNQVVLKVTNMDPADTATYYCARSMITNWYFDVWGAGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:54 DNA encoding the heavy chain CAAGTCACACTGCGTGAAAGCGGCCCTGCCCTGGTCAAGCCCACCCAGACCCTGACCCTGACCTGCACCTTCTCCGGCTTCAGCCTGGCCCCTACCTCCTCCAGCACCAAGAAAACCCAGCTGCAGCTCGAACATCTGCTGCTGGACCTGCAGATGATCCTGAACGGCATCAACAACTACAAGAACCCCAAGCTGACCCGGATGCTGACCGCCAAGTTCTACATGCCCAAGAAGGCCACCGAGCTGAAACAT CTGCAGTGCCTGGAAGAGGAACTGAAGCCCCTGGAAGAAGTGCTGAACCTGGCCCAGTCCAAGAACTTCCACCTGAGGCCTCGGGACCTGATCTCCAACATCAACGTGATCGTGCTGGAACTGAAGGGCTCCGAGACAACCTTCATGTGCGAGTACGCCGACGAGACAGCCACCATCGTGGAATTTCTGAACCGGTGGATCACCTTCTGCCAGTCCATCTCCACCCTGACCTCCACCTCCGGCATG TCCGTGGGCTGGATCCGGCAGCCTCCTGGCAAGGCCCTGGAGTGGCTGGCCGACATTTGGTGGGACGACAAGAAGGACTACAACCCCAGCCTGAAGTCCCGGCTGACCATCTCCAAGGACACCTCCAAGAACCAAGTGGTGCTGAAAGTGACCAACATGGACCCCGCCGACACCGCCACCTACTACTGCGCCCGGTCCATGATCACCAACTGGTACTTCGACGTGTGGGGCGCTGGCACCACCGTGACCG TGTCCTCTGCTAGCACCAAGGGCCCCTCCGTGTTCCCTCTGGCCCCTTCCAGCAAGTCTACCTCCGGCGGCACAGCTGCTCTGGGCTGCCTGGTCAAGGACTACTTCCCTGAGCCTGTGACAGTGTCCTGGAACTCTGGCGCCCTGACCTCTGGCGTGCACACCTTCCCTGCCGTGCTGCAGTCCTCCGGCCTGTACTCCCTGTCCTCCGTGGTCACAGTGCCTTCAAGCAGCCTGGGCACCCAGAC CTATATCTGCAACGTGAACCACAAGCCTTCCAACACCAAGGTGGACAAGCGGGTGGAGCCTAAGTCCTGCGACAAGACCCACACCTGTCCTCCCTGCCCTGCTCCTGAACTGCTGGGCGGCCCTTCTGTGTTCCTGTTCCCTCCAAAGCCCAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACCTGCGTGGTGGTGGCCGTGTCCCACGAGGATCCTGAAGTGAAGTTCAATTGG TACGTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCTCGGGAGGAACAGTACAACTCCACCTACCGGGTGGTGTCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAGTACAAGTGCAAAGTCTCCAACAAGGCCCTGGCCGCCCCTATCGAAAAGACAATCTCCAAGGCCAAGGGCCAGCCTAGGGAACCCCAGGTGTACACCCTGCCACCCAGCCGGGAGGAAATGACC AAGAACCAGGTGTCCCTGACCTGTCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAGTGGGAGTCTAACGGCCAGCCTGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAACTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCCCTGTC CCTGTCTCCCGGCAAG SEQ ID NO:55 (merged) LCDR1 KAQLSVGYMH SEQ ID NO:56 (merged) LCDR2 DTSKLAS SEQ ID NO:57 (merged) LCDR3 FQGSGYPFT SEQ ID NO:58 (Kabat) LCDR1 KAQLSVGYMH SEQ ID NO:59 (Kabat) LCDR2 DTSKLAS SEQ ID NO:60 (Kabat) LCDR3 FQGSGYPFT SEQ ID NO:61 (Chothia) LCDR1 QLSVGY SEQ ID NO:62 (Chothia) LCDR2 DTS SEQ ID NO:63 (Chothia) LCDR3 GSGYPF SEQ ID NO:64 (IMGT) LCDR1 LSVGY SEQ ID NO:65 (IMGT) LCDR2 DTS SEQ ID NO:66 (IMGT) LCDR3 FQGSGYPFT SEQ ID NO:67 VL DIQMTQSPSTLSASVGDRVTTITCKAQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIK SEQ ID NO:68 DNA encoding VL GACATCCAGATGACCCAGAGCCCCTCCACCCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACTTGCAAGGCCCAGCTGTCCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTCACCCTGACCATCTCCAGCCTGCAGCCCGACGACTTCG CCACCTACTACTGTTTTCAAGGCTCCGGCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAG SEQ ID NO:69 Light chain DIQMTQSPSTLSASVGDRVTITCKAQLSVGYMHWYQQKPGKAPKLLIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCFQGSGYPFTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKV YACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:70 DNA encoding the light chain GACATCCAGATGACCCAGAGCCCCTCCACCCTGTCCGCCTCCGTGGGCGACAGAGTGACCATCACTTGCAAGGCCCAGCTGTCCGTGGGCTACATGCACTGGTATCAGCAGAAGCCCGGCAAGGCCCCTAAGCTGCTGATCTACGACACCTCCAAGCTGGCCTCCGGCGTGCCCTCCAGATTCTCCGGCTCTGGCTCCGGCACCGAGTTCACCCTGACCATCTCCAGCCTGCAGCCCGACGACTTCG CCACCTACTACTGTTTTCAAGGCTCCGGCTACCCCTTCACCTTCGGCGGAGGCACCAAGCTGGAAATCAAGCGTACGGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAG CAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCATAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACAGGGGCGAGTGC

Example 2: IgG.IL2R67A.H1 has a longer half-life compared to Proleukin® (IL-2)

[00226] Naïve CD-1 mice were administered I.P. dose, and blood was collected from all animals predose, 1 hour, 3, 7, 24, 31, 48, 55, and 72 hours postdose. Blood samples were centrifuged, and blood plasma samples were obtained. The resulting blood plasma samples were transferred into one polypropylene tube and frozen at -80ºC. All samples were analyzed and plasma IgG.IL2R67A.H1 concentrations were measured using immunoassays. Pharmacokinetic parameters such as half-life were calculated. Each sample was analyzed in duplicate, with each duplicate assay requiring 5 μL of sample, which was diluted 1:20. Capture: goat biotinylated anti-human IL-2 antibody (R&D Systems BAF202). Identification: Alexa 647-conjugated anti-human IL-2 antibody, clone MQ1-17H12 (Biolegend #500315). All immunoassays were performed using Gyrolab® Bioaffy200 with Gyros CD-200. As shown in the graph in Figure 2, the half-life of IgG.IL2R67A.H1 is approximately 12 hours and then decreases over the next 48 hours. The half-life of Proleukin® could not be shown on this graph because its half-life is approximately 4 hours.

Example 3: IgG.IL2R67A.H1 selectively expands CD8+ effector T cells and is better tolerated than IL2-Fc or Proleukin® in normal B6 mice

[00227] IgG.IL2R67A.H1 provides an increase in the number of CD8+ effector T cells compared to Tregs, without causing the adverse events observed with the administration of Proleukin®. After dosing mice on day 1, expansion of CD8+ effector T cells was monitored on day 4, day 8, and day 11. At each time point, the CD8+ effector T cell population showed significant expansion without expansion of Tregs. This was in contrast to that observed with Proleukin® and the IL2-Fc fusion, for which mortality and morbidity were observed at equimolar doses of IL-2.

[00228] Female B6 mice were administered Proleukin® (5x/week), IL2-Fc and IgG.IL2R67A.H1 (1x/week) at equimolar concentrations. Eight days after the first round of treatment, spleens were processed into a single cell suspension and washed in RPMI (10% FBS). RBCs were lysed with erythrocyte lysis buffer (Sigma, no. R7757), and cell number and cell viability were determined. FACS staining was performed according to standard protocols using FACS buffer (1xPBS+0.5% BSA+0.05% sodium azide). Cells were stained with surface antibodies: rat anti-mouse CD3 conjugated to eFluor 450 (Ebioscience no. 48-0032), anti-mouse CD4 conjugated to Pacific Blue (BD Pharmingen no. 558107), rat anti-mouse CD8 conjugated to PerCp (BD Pharmingen #553036), FITC-conjugated rat anti-mouse CD44 (Pharmingen #553133), APC-conjugated rat anti-mouse CD25 (Ebioscience #17-0251), rat anti-mouse Nk1.1 (Ebioscience #95-5941 ) and then fixed/permeabilized and stained for FoxP3 using a PE-conjugated anti-mouse/rat FoxP3 antibody staining kit (Ebioscience #72-5775). Cells were analyzed on a Becton-Dickinson LSR Fortessa® or Becton-Dickinson FACS LSR II, and data were analyzed using FlowJo® software.

[00229] Figures 3A-3C show preferential expansion of CD8+ effector T cells in female B6 mice following administration of Proleukin® (5x/week), IL2-Fc and IgG.IL2R67A.H1 (1x/week) at concentrations equimolar to Proleukin® (100 μg IgG.IL2R67A.H1 and IL2-Fc ~ equivalent to 1 nmol IL2). Data in the graphs demonstrate that CD8+ effector T cells proliferate without similar Treg proliferation. In contrast to these data, Proleukin® promoted expansion of both CD8+ effector T cells and Tregs. Of note, IgG.IL2R67A.H1 was superior to the IL2-Fc fusion construct in both the absolute number of proliferating CD8+ effector T cells and the CD8+ effector T cell:Treg ratio, demonstrating that IgG.IL2R67A.H1 is structural and functional a cytokine-grafted antibody protein backbone. Figures 3D-3F show that the beneficial effect of IgG.IL2R67A.H1 was more pronounced at higher doses. When B6 mice were administered 500 μg (equivalent to 5 nmol IL2) IgG.IL2R67A.H1, a preferential expansion of CD8+ effector T cells over Treg cells was observed, similar to lower doses. However, in the IL2-Fc treatment group, mice were found to die after just a single dose at a higher level (data not shown). This indicates that IgG.IL2R67A.H1 has a higher therapeutic index than IL2-Fc fusion constructs and can be safely administered over a wider dose range.

Example 4: IgG.IL2R67A.H1 selectively expands CD8+ effector T cells and is better tolerated than Proleukin® in NOD mice

[00230] Nonobese diabetic (NOD) mice spontaneously develop type 1 diabetes and are often used as an animal model for type 1 diabetes in humans. According to the same protocol used for B6 mice described in Example 3, IgG.IL2R67A.H1, IL2-Fc and Proleukin® were administered to NOD mice in equimolar equivalents of Proleukin®. In this case, administration of IgG.IL2R67A.H1 at this dose also provided preferential expansion of CD8+ effector T cells compared to Tregs, as shown in the graph in Figure 4A. In addition, when administered IgG.IL2R67A.H1, NOD mice showed no side effects, whereas in the Proleukin® treatment group, 5 mice were in agony and 2 mice died. Figure 4B is a graph showing dose, fold change in cell number, and cell type in a mouse model of NOD.

Example 5 IgG.IL2R67A.H1 Demonstrates Efficacy as Monotherapy in a CT26 Cell Colon Tumor Mouse Model

[00231] After studying the safety of IgG.IL2R67A.H1, its effectiveness as a monotherapy was tested in a mouse model of CT26 cells. Murine CT26 cells are rapidly growing stage IV colon carcinoma cells used in more than 500 published studies and are one of the most widely used cell lines and models in drug development. CT26 cells (ATCC CRL-2638) were grown under sterile conditions in an incubator at 37 ^° C with 5% CO ₂ . Cells were cultured in RPMI 1640 medium supplemented with 10% FBS. Cells were passaged every 3-4 days. On the day of injection, cells were harvested (passage 11) and resuspended in HBSS at a concentration of 2.5×10 ⁶ /ml. Cells were tested with Radil against mycoplasma and murine viruses. Balbc mice were used. For each mouse, 0.25 x 10 ⁶ cells were implanted by subcutaneous injection into the right flank using a 28 G needle (injection volume 100 μl). After implantation, animals were measured with calipers and weighed 3 times per week after tumors became palpable. Caliper measurements were used in calculations according to the expression (LxWxW)/2. Mice were fed a normal diet and housed in an SPF animal facility in accordance with the Guide for the Care and Use of Laboratory Animals and Institutional Animal Care and Use Committee regulations.

[00232] When tumors reached approximately 100 mm ³ , mice were injected intraperitoneally with 12.5-100 μg of IgG.IL2R67A.H1. Tumors were measured twice a week. Mean tumor volumes were plotted using Prism 5 software (GraphPad®). The end point in efficacy studies was reached when the tumor size reached 1000 mm ³ . After injection, the mice were also closely monitored for signs of clinical deterioration. If for any reason the mice showed any signs of morbidity, including respiratory distress, hunched posture, decreased activity, paralysis of the hind limbs, tachypnea as a sign of pleural effusion, loss of body weight of approximately 20% or 15% in combination with other signs, or their ability to carry out normal activities (feeding, locomotion) was impaired, the mice were sacrificed.

[00233] IgG.IL2R67A.H1 was effective in a CT26 cell mouse model at doses ranging from 12.5 μg to 100 μg, with 4 administrations of IgG.IL2R67A.H1 occurring over 17 days in a 20-day study . The tumor volume curves shown in Figure 5 demonstrate the effectiveness of IgG.IL2R67A.H1 in this study, as tumor volumes were maintained at up to 200 mm for 15 days and then up to 400 mm for the remaining 5 days.

Example 6 IgG.IL2R67A.H1 and Additional Anti-Cancer Therapeutics Demonstrate Efficacy in a B16 Cell Mouse Model

[00234] A B16F10 cell melanoma mouse model was used to evaluate the effectiveness of IgG.IL2R67A.H1 in combination with other anticancer therapeutics. B16F10 cells (ATCC CRL-6475) were grown under sterile conditions in an incubator at 37^oC with 5% CO₂ in two weeks. B16F10 cells were cultured in DMEM+10% FBS. Cells were harvested and resuspended in FBS-free DMEM at a concentration of 1 × 10⁶/100 µl. B16F10 cells were tested with Radil against mycoplasma and murine viruses. Cells were implanted into the right flank of B6 mice using a 28-gauge needle (injection volume 100 μL). After implantation, mice were measured with calipers and weighed twice a week after tumors became palpable. Caliper measurements were used in calculations according to the expression (L × W × W)/2.

[00235] In this study, IgG.IL2R67A.H1 was used as a monotherapy or in combination with the TA99 antibody, which binds the Trp1 antigen expressed on B16F10 cells. The IL2-Fc fusion construct was administered as monotherapy or in combination with the TA99 antibody. For control, TA99 antibody was administered as monotherapy.

[00236] Surprisingly, it was found that IgG.IL2R67A.H1, when administered as monotherapy at a dose of 500 μg, was the most effective treatment in this model (Figure 6). The next most effective treatment was the combination of IgG.IL2R67A.H1 (100 μg) and TA99. This combination was more effective than IgG.IL2F71A.H1 as monotherapy at 100 μg, TA99 in combination with IgG.IL2F71A.H1 at 500 μg, and IL2-Fc as monotherapy or as part of the IL2-Fc/TA99 combination. When administered as monotherapy, TA99 had no effect and mean tumor volume was similar to that observed in the untreated control group. These data demonstrate that IgG.IL2R67A.H1 is effective as a monotherapy in a murine melanoma tumor model, but is also effective when paired with another antitumor agent.

Example 7. Activity of IgG.IL2R67A.H1 and IgG.IL2F71A.H1 in human cells

[00237] To test the activity of IgG.IL2R67A.H1 against human CD8+ effector T cells, human peripheral blood mononuclear cells (PBMCs) were assayed for pSTAT5 activity. PBMCs were maintained in serum-free testing medium and plated. IgG.IL2R67A.H1, IgG.IL2F71A.H1 or Proleukin® were added to PBMC and incubated for 20 minutes at 37°C. After 20 minutes, cells were fixed with 1.6% formaldehyde, washed, and stained for surface markers. After 30 minutes at room temperature, samples were washed and resuspended cell pellets were permeabilized with methanol at −20°C, washed, and stained for pSTAT5 and DNA intercalators. Cells were analyzed on Cytof® and data analyzed using FlowJo® software to quantify the level of pSTAT5 activity. The table in Figure 7 demonstrates that when using IgG.IL2R67A.H1, preferential activation of CD8+ effector T cells was observed, and Treg cell activation was minimized.

Example 8 Binding of Antibody Proteins to a Grafted Cytokine

[00238] IL2 sequences containing mutein (SEQ ID NO:4) were inserted into the CDR loops of the immunoglobulin chain backbone. Cytokine-grafted antibody proteins were produced using a variety of known immunoglobulin sequences that have been used in clinical settings, as well as germline antibody sequences. The antigen of one of the antibodies used was RSV. To determine whether grafting of IL2 onto the CDR of a given antibody resulted in reduced or eliminated binding to RSV, an ELISA assay was performed against RSV proteins in either PBS buffer or carbonate buffer. As shown in Figure 8, this appeared to be influenced by which CDR was selected for IL2 grafting. For example, IgG.IL2R67A.H1 exhibits binding to RSV similar to that of the ungrafted (unmodified) parent antibody. In contrast, grafting IL2 onto light chain CDR3 (CDR-L3) or CDR-H3 resulted in decreased binding. As expected, grafting IL2 onto an irrelevant antibody (Xolair) resulted in no binding. This demonstrates that cytokine-grafted antibody proteins may retain binding to the original target antibody backbone, or this binding may be reduced.

[00239] It should be understood that the examples and embodiments described herein are intended for illustrative purposes, and that various modifications or changes in light thereof will be suggested to those skilled in the art and should be included within the spirit and scope of this application and scope the attached claims. All publications, sequence accession numbers, patents and patent applications mentioned herein are incorporated herein by reference in their entirety for all purposes.

--->

LIST OF SEQUENCES

<110> NOVARTIS AG

<120> ANTIBODY-BASED PROTEINS WITH CYTOKINE GRAFTED AND METHODS FOR THEM

APPLICATIONS IN CANCER TREATMENT

<130> PAT057462-WO-PCT

<140>

<141>

<150> 62/510,533

<151> 2017-05-24

<160> 78

<170> PatentIn version 3.5

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tacttcaagt tctacaaaga aaacacagct acaactggag catttactgc tggatttaca 180

gatgattttg aatggaatta ataattacaa gaatcccaaa ctcaccagga tgctcacatt 240

taagttttac atgcccaaga aggccacaga actgaaacat cttcagtgtc tagaagaaga 300

actcaaacct ctggaggaag tgctaaattt agctcaaagc aaaaactttc acttaagacc 360

caggactta atcagcaata tcaacgtaat agttctggaa ctaaagggat ctgaaacaac 420

attcatgtgt gaatatgctg atgagacagc aaccattgta gaatttctga acagatggat 480

taccttttgt caaagcatca tctcaacact gacttgataa ttaagtgctt cccacttaaa 540

acatatcagg ccttctattt atttaaatat ttaaatttta tatttattgt tgaatgtatg 600

gtttgctacc tattgtaact attattctta atcttaaaac tataaatatg gatcttttat 660

gattcttttt gtaagcccta ggggctctaa aatggtttca cttatttatc ccaaaatatt 720

tattattatg ttgaatgtta aatatagtat ctatgtagat tggttagtaa aactatttaa 780

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<213> Homo sapiens

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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

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Cys Gln Ser Ile Ile Ser Thr Leu Thr

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<210> 3

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<223> /note="Description of the artificial sequence:

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caagtcacac tgcgtgaaag cggccctgcc ctggtcaagc ccacccagac cctgaccctg 60

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ctgcagctcg aacatctgct gctggacctg cagatgatcc tgaacggcat caacaactac 180

aagaacccca agctgacccg gatgctgacc gccaagttct acatgcccaa gaaggccacc 240

gagctgaaac atctgcagtg cctggaagag gaactgaagc ccctggaaga agtgctgaac 300

ctggcccagt ccaagaactt ccacctgagg cctcgggacc tgatctccaa catcaacgtg 360

atcgtgctgg aactgaaggg ctccgagaca accttcatgt gcgagtacgc cgacgagaca 420

gccaccatcg tggaatttct gaaccggtgg atcaccttct gccagtccat catctccacc 480

ctgacctcca cctccggcat gtccgtgggc tggatccggc agcctcctgg caaggccctg 540

gagtggctgg ccgacatttg gtgggacgac aagaaggact acaaccccag cctgaagtcc 600

cggctgacca tctccaagga cacctccaag aaccaagtgg tgctgaaagt gaccaacatg 660

gaccccgccg acaccgccac ctactactgc gcccggtcca tgatcaccaa ctggtacttc 720

gacgtgtggg gcgctggcac caccgtgacc gtgtcctct 759

<210> 4

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<212> PROTEIN

<213> Artificial sequence

<220>

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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

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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

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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> 5

<211> 399

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

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<400> 5

gcccctacct cctccagcac caagaaaacc cagctgcagc tcgaacatct gctgctggac 60

ctgcagatga tcctgaacgg catcaacaac tacaagaacc ccaagctgac ccggatgctg 120

accgccaagt tctacatgcc caagaaggcc accgagctga aacatctgca gtgcctggaa 180

gaggaactga agcccctgga agaagtgctg aacctggccc agtccaagaa cttccacctg 240

aggcctcggg acctgatctc caacatcaac gtgatcgtgc tggaactgaa gggctccgag 300

acaaccttca tgtgcgagta cgccgacgag acagccacca tcgtggaatt tctgaaccgg 360

tggatcacct tctgccagtc catcatctcc accctgacc 399

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<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

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<400> 6

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

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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

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

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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> 7

<211> 145

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

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<400> 7

Gly Phe Ser Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

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

100 105 110

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

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

130 135 140

Gly

145

<210> 8

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

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<400> 8

Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 9

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

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<400> 9

Ser Met Ile Thr Asn Trp Tyr Phe Asp Val

1 5 10

<210> 10

<211> 141

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

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<400> 10

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

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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

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Ile Ser Thr Leu Thr Ser Thr Ser Gly Met Ser Val Gly

130 135 140

<210> 11

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 11

Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 12

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

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<400> 12

Ser Met Ile Thr Asn Trp Tyr Phe Asp Val

1 5 10

<210> 13

<211> 142

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 13

Gly Phe Ser Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Cys Gln Ser Ile Ile Ser Thr Leu Thr Ser Thr Ser Gly Met

130 135 140

<210> 14

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

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<400> 14

Trp Trp Asp Asp Lys

15

<210> 15

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

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<400> 15

Ser Met Ile Thr Asn Trp Tyr Phe Asp Val

1 5 10

<210> 16

<211> 143

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 16

Gly Phe Ser Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Cys Gln Ser Ile Ile Ser Thr Leu Thr Ser Thr Ser Gly Met Ser

130 135 140

<210> 17

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 17

Ile Trp Trp Asp Asp Lys Lys

15

<210> 18

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 18

Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val

1 5 10

<210> 19

<211> 253

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 19

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ala Pro Thr

20 25 30

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu

35 40 45

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

50 55 60

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

65 70 75 80

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu

85 90 95

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

100 105 110

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

115 120 125

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

130 135 140

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

145 150 155 160

Leu Thr Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro

165 170 175

Gly Lys Ala Leu Glu Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys

180 185 190

Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr

195 200 205

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

210 215 220

Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe

225 230 235 240

Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

245 250

<210> 20

<211> 759

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 20

caagtcacac tgcgtgaaag cggccctgcc ctggtcaagc ccacccagac cctgaccctg 60

acctgcacct tctccggctt cagcctggcc cctacctcct ccagcaccaa gaaaacccag 120

ctgcagctcg aacatctgct gctggacctg cagatgatcc tgaacggcat caacaactac 180

aagaacccca agctgaccgc catgctgacc ttcaagttct acatgcccaa gaaggccacc 240

gagctgaaac atctgcagtg cctggaagag gaactgaagc ccctggaaga agtgctgaac 300

ctggcccagt ccaagaactt ccacctgagg cctcgggacc tgatctccaa catcaacgtg 360

atcgtgctgg aactgaaggg ctccgagaca accttcatgt gcgagtacgc cgacgagaca 420

gccaccatcg tggaatttct gaaccggtgg atcaccttct gccagtccat catctccacc 480

ctgacctcca cctccggcat gtccgtgggc tggatccggc agcctcctgg caaggccctg 540

gagtggctgg ccgacatttg gtgggacgac aagaaggact acaaccccag cctgaagtcc 600

cggctgacca tctccaagga cacctccaag aaccaagtgg tgctgaaagt gaccaacatg 660

gaccccgccg acaccgccac ctactactgc gcccggtcca tgatcaccaa ctggtacttc 720

gacgtgtggg gcgctggcac caccgtgacc gtgtcctct 759

<210> 21

<211> 583

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 21

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ala Pro Thr

20 25 30

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu

35 40 45

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

50 55 60

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

65 70 75 80

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu

85 90 95

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

100 105 110

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

115 120 125

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

130 135 140

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

145 150 155 160

Leu Thr Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro

165 170 175

Gly Lys Ala Leu Glu Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys

180 185 190

Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr

195 200 205

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

210 215 220

Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe

225 230 235 240

Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

245 250 255

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

260 265 270

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

275 280 285

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

290 295 300

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

305 310 315 320

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

325 330 335

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

340 345 350

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

355 360 365

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

370 375 380

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

385 390 395 400

Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

405 410 415

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

420 425 430

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

435 440 445

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

450 455 460

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

465 470 475 480

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

485 490 495

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

500 505 510

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

515 520 525

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

530 535 540

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

545 550 555 560

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

565 570 575

Leu Ser Leu Ser Pro Gly Lys

580

<210> 22

<211> 1749

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 22

caagtcacac tgcgtgaaag cggccctgcc ctggtcaagc ccacccagac cctgaccctg 60

acctgcacct tctccggctt cagcctggcc cctacctcct ccagcaccaa gaaaacccag 120

ctgcagctcg aacatctgct gctggacctg cagatgatcc tgaacggcat caacaactac 180

aagaacccca agctgaccgc catgctgacc ttcaagttct acatgcccaa gaaggccacc 240

gagctgaaac atctgcagtg cctggaagag gaactgaagc ccctggaaga agtgctgaac 300

ctggcccagt ccaagaactt ccacctgagg cctcgggacc tgatctccaa catcaacgtg 360

atcgtgctgg aactgaaggg ctccgagaca accttcatgt gcgagtacgc cgacgagaca 420

gccaccatcg tggaatttct gaaccggtgg atcaccttct gccagtccat catctccacc 480

ctgacctcca cctccggcat gtccgtgggc tggatccggc agcctcctgg caaggccctg 540

gagtggctgg ccgacatttg gtgggacgac aagaaggact acaaccccag cctgaagtcc 600

cggctgacca tctccaagga cacctccaag aaccaagtgg tgctgaaagt gaccaacatg 660

gaccccgccg acaccgccac ctactactgc gcccggtcca tgatcaccaa ctggtacttc 720

gacgtgtggg gcgctggcac caccgtgacc gtgtcctctg ctagcaccaa gggcccctcc 780

gtgttccctc tggccccttc cagcaagtct acctccggcg gcacagctgc tctgggctgc 840

ctggtcaagg actacttccc tgagcctgtg acagtgtcct ggaactctgg cgccctgacc 900

tctggcgtgc acaccttccc tgccgtgctg cagtcctccg gcctgtactc cctgtcctcc 960

gtggtcacag tgccttcaag cagcctgggc acccagacct atatctgcaa cgtgaaccac 1020

aagccttcca acaccaaggt ggacaagcgg gtggagccta agtcctgcga caagacccac 1080

acctgtcctc cctgccctgc tcctgaactg ctgggcggcc cttctgtgtt cctgttccct 1140

ccaaagccca aggacaccct gatgatctcc cggacccctg aagtgacctg cgtggtggtg 1200

gccgtgtccc acgaggatcc tgaagtgaag ttcaattggt acgtggacgg cgtggaggtg 1260

cacaacgcca agaccaagcc tcgggaggaa cagtacaact ccacctaccg ggtggtgtcc 1320

gtgctgaccg tgctgcacca ggactggctg aacggcaaag agtacaagtg caaagtctcc 1380

aacaaggccc tggccgcccc tatcgaaaag acaatctcca aggccaaggg ccagcctagg 1440

gaaccccagg tgtacaccct gccacccagc cgggaggaaa tgaccaagaa ccaggtgtcc 1500

ctgacctgtc tggtcaaggg cttctaccct tccgatatcg ccgtggagtg ggagtctaac 1560

ggccagcctg agaacaacta caagaccacc cctcctgtgc tggactccga cggctccttc 1620

ttcctgtact ccaaactgac cgtggacaag tcccggtggc agcagggcaa cgtgttctcc 1680

tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct gtccctgtct 1740

cccggcaag 1749

<210> 23

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 23

Lys Ala Gln Leu Ser Val Gly Tyr Met His

1 5 10

<210> 24

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 24

Asp Thr Ser Lys Leu Ala Ser

15

<210> 25

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 25

Phe Gln Gly Ser Gly Tyr Pro Phe Thr

15

<210> 26

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 26

Lys Ala Gln Leu Ser Val Gly Tyr Met His

1 5 10

<210> 27

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 27

Asp Thr Ser Lys Leu Ala Ser

15

<210> 28

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 28

Phe Gln Gly Ser Gly Tyr Pro Phe Thr

15

<210> 29

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 29

Gln Leu Ser Val Gly Tyr

15

<210> 30

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 30

Asp Thr Ser

1

<210> 31

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 31

Gly Ser Gly Tyr Pro Phe

15

<210> 32

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 32

Leu Ser Val Gly Tyr

15

<210> 33

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 33

Asp Thr Ser

1

<210> 34

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 34

Phe Gln Gly Ser Gly Tyr Pro Phe Thr

15

<210> 35

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 35

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 36

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 36

gacatccaga tgacccagag cccctccacc ctgtccgcct ccgtgggcga cagagtgacc 60

atcacttgca aggcccagct gtccgtgggc tacatgcact ggtatcagca gaagcccggc 120

aaggccccta agctgctgat ctacgacacc tccaagctgg cctccggcgt gccctccaga 180

ttctccggct ctggctccgg caccgagttc accctgacca tctccagcct gcagcccgac 240

gacttcgcca cctactactg ttttcaaggc tccggctacc ccttcacctt cggcggaggc 300

accaagctgg aaatcaag 318

<210> 37

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 37

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe 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

Asn Arg Gly Glu Cys

210

<210> 38

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 38

gacatccaga tgacccagag cccctccacc ctgtccgcct ccgtgggcga cagagtgacc 60

atcacttgca aggcccagct gtccgtgggc tacatgcact ggtatcagca gaagcccggc 120

aaggccccta agctgctgat ctacgacacc tccaagctgg cctccggcgt gccctccaga 180

ttctccggct ctggctccgg caccgagttc accctgacca tctccagcct gcagcccgac 240

gacttcgcca cctactactg ttttcaaggc tccggctacc ccttcacctt cggcggaggc 300

accaagctgg aaatcaagcg tacggtggcc gctcccagcg tgttcatctt cccccccagc 360

gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc 420

cgggaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480

agcgtcaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctgaccctg 540

agcaaggccg actacgagaa gcataaggtg tacgcctgcg aggtgaccca ccagggcctg 600

tccagccccg tgaccaagag cttcaacagg ggcgagtgc 639

<210> 39

<211> 145

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 39

Gly Phe Ser Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

Gly

145

<210> 40

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 40

Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 41

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 41

Ser Met Ile Thr Asn Trp Tyr Phe Asp Val

1 5 10

<210> 42

<211> 141

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<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 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 Cys Gln Ser Ile

115 120 125

Ile Ser Thr Leu Thr Ser Thr Ser Gly Met Ser Val Gly

130 135 140

<210> 43

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 43

Asp Ile Trp Trp Asp Asp Lys Lys Asp Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 44

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 44

Ser Met Ile Thr Asn Trp Tyr Phe Asp Val

1 5 10

<210> 45

<211> 142

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 45

Gly Phe Ser Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Cys Gln Ser Ile Ile Ser Thr Leu Thr Ser Thr Ser Gly Met

130 135 140

<210> 46

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 46

Trp Trp Asp Asp Lys

15

<210> 47

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 47

Ser Met Ile Thr Asn Trp Tyr Phe Asp Val

1 5 10

<210> 48

<211> 143

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 48

Gly Phe Ser Leu Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Cys Gln Ser Ile Ile Ser Thr Leu Thr Ser Thr Ser Gly Met Ser

130 135 140

<210> 49

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 49

Ile Trp Trp Asp Asp Lys Lys

15

<210> 50

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 50

Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe Asp Val

1 5 10

<210> 51

<211> 253

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 51

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ala Pro Thr

20 25 30

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu

35 40 45

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

50 55 60

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

65 70 75 80

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu

85 90 95

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

100 105 110

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

115 120 125

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

130 135 140

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

145 150 155 160

Leu Thr Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro

165 170 175

Gly Lys Ala Leu Glu Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys

180 185 190

Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr

195 200 205

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

210 215 220

Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe

225 230 235 240

Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser

245 250

<210> 52

<211> 759

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 52

caagtcacac tgcgtgaaag cggccctgcc ctggtcaagc ccacccagac cctgaccctg 60

acctgcacct tctccggctt cagcctggcc cctacctcct ccagcaccaa gaaaacccag 120

ctgcagctcg aacatctgct gctggacctg cagatgatcc tgaacggcat caacaactac 180

aagaacccca agctgacccg gatgctgacc gccaagttct acatgcccaa gaaggccacc 240

gagctgaaac atctgcagtg cctggaagag gaactgaagc ccctggaaga agtgctgaac 300

ctggcccagt ccaagaactt ccacctgagg cctcgggacc tgatctccaa catcaacgtg 360

atcgtgctgg aactgaaggg ctccgagaca accttcatgt gcgagtacgc cgacgagaca 420

gccaccatcg tggaatttct gaaccggtgg atcaccttct gccagtccat catctccacc 480

ctgacctcca cctccggcat gtccgtgggc tggatccggc agcctcctgg caaggccctg 540

gagtggctgg ccgacatttg gtgggacgac aagaaggact acaaccccag cctgaagtcc 600

cggctgacca tctccaagga cacctccaag aaccaagtgg tgctgaaagt gaccaacatg 660

gaccccgccg acaccgccac ctactactgc gcccggtcca tgatcaccaa ctggtacttc 720

gacgtgtggg gcgctggcac caccgtgacc gtgtcctct 759

<210> 53

<211> 583

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 53

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

1 5 10 15

Thr Leu Thr Leu Thr Cys Thr Phe Ser Gly Phe Ser Leu Ala Pro Thr

20 25 30

Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His Leu Leu Leu

35 40 45

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

50 55 60

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

65 70 75 80

Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu

85 90 95

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

100 105 110

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

115 120 125

Glu Thr Thr Phe Met Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val

130 135 140

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

145 150 155 160

Leu Thr Ser Thr Ser Gly Met Ser Val Gly Trp Ile Arg Gln Pro Pro

165 170 175

Gly Lys Ala Leu Glu Trp Leu Ala Asp Ile Trp Trp Asp Asp Lys Lys

180 185 190

Asp Tyr Asn Pro Ser Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Thr

195 200 205

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

210 215 220

Thr Ala Thr Tyr Tyr Cys Ala Arg Ser Met Ile Thr Asn Trp Tyr Phe

225 230 235 240

Asp Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr

245 250 255

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

260 265 270

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

275 280 285

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

290 295 300

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

305 310 315 320

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

325 330 335

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu

340 345 350

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

355 360 365

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

370 375 380

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

385 390 395 400

Ala Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

405 410 415

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

420 425 430

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

435 440 445

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

450 455 460

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

465 470 475 480

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

485 490 495

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

500 505 510

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

515 520 525

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

530 535 540

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

545 550 555 560

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

565 570 575

Leu Ser Leu Ser Pro Gly Lys

580

<210> 54

<211> 1749

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 54

caagtcacac tgcgtgaaag cggccctgcc ctggtcaagc ccacccagac cctgaccctg 60

acctgcacct tctccggctt cagcctggcc cctacctcct ccagcaccaa gaaaacccag 120

ctgcagctcg aacatctgct gctggacctg cagatgatcc tgaacggcat caacaactac 180

aagaacccca agctgacccg gatgctgacc gccaagttct acatgcccaa gaaggccacc 240

gagctgaaac atctgcagtg cctggaagag gaactgaagc ccctggaaga agtgctgaac 300

ctggcccagt ccaagaactt ccacctgagg cctcgggacc tgatctccaa catcaacgtg 360

atcgtgctgg aactgaaggg ctccgagaca accttcatgt gcgagtacgc cgacgagaca 420

gccaccatcg tggaatttct gaaccggtgg atcaccttct gccagtccat catctccacc 480

ctgacctcca cctccggcat gtccgtgggc tggatccggc agcctcctgg caaggccctg 540

gagtggctgg ccgacatttg gtgggacgac aagaaggact acaaccccag cctgaagtcc 600

cggctgacca tctccaagga cacctccaag aaccaagtgg tgctgaaagt gaccaacatg 660

gaccccgccg acaccgccac ctactactgc gcccggtcca tgatcaccaa ctggtacttc 720

gacgtgtggg gcgctggcac caccgtgacc gtgtcctctg ctagcaccaa gggcccctcc 780

gtgttccctc tggccccttc cagcaagtct acctccggcg gcacagctgc tctgggctgc 840

ctggtcaagg actacttccc tgagcctgtg acagtgtcct ggaactctgg cgccctgacc 900

tctggcgtgc acaccttccc tgccgtgctg cagtcctccg gcctgtactc cctgtcctcc 960

gtggtcacag tgccttcaag cagcctgggc acccagacct atatctgcaa cgtgaaccac 1020

aagccttcca acaccaaggt ggacaagcgg gtggagccta agtcctgcga caagacccac 1080

acctgtcctc cctgccctgc tcctgaactg ctgggcggcc cttctgtgtt cctgttccct 1140

ccaaagccca aggacaccct gatgatctcc cggacccctg aagtgacctg cgtggtggtg 1200

gccgtgtccc acgaggatcc tgaagtgaag ttcaattggt acgtggacgg cgtggaggtg 1260

cacaacgcca agaccaagcc tcgggaggaa cagtacaact ccacctaccg ggtggtgtcc 1320

gtgctgaccg tgctgcacca ggactggctg aacggcaaag agtacaagtg caaagtctcc 1380

aacaaggccc tggccgcccc tatcgaaaag acaatctcca aggccaaggg ccagcctagg 1440

gaaccccagg tgtacaccct gccacccagc cgggaggaaa tgaccaagaa ccaggtgtcc 1500

ctgacctgtc tggtcaaggg cttctaccct tccgatatcg ccgtggagtg ggagtctaac 1560

ggccagcctg agaacaacta caagaccacc cctcctgtgc tggactccga cggctccttc 1620

ttcctgtact ccaaactgac cgtggacaag tcccggtggc agcagggcaa cgtgttctcc 1680

tgctccgtga tgcacgaggc cctgcacaac cactacaccc agaagtccct gtccctgtct 1740

cccggcaag 1749

<210> 55

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 55

Lys Ala Gln Leu Ser Val Gly Tyr Met His

1 5 10

<210> 56

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 56

Asp Thr Ser Lys Leu Ala Ser

15

<210> 57

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 57

Phe Gln Gly Ser Gly Tyr Pro Phe Thr

15

<210> 58

<211> 10

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 58

Lys Ala Gln Leu Ser Val Gly Tyr Met His

1 5 10

<210> 59

<211> 7

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 59

Asp Thr Ser Lys Leu Ala Ser

15

<210> 60

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 60

Phe Gln Gly Ser Gly Tyr Pro Phe Thr

15

<210> 61

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 61

Gln Leu Ser Val Gly Tyr

15

<210> 62

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 62

Asp Thr Ser

1

<210> 63

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 63

Gly Ser Gly Tyr Pro Phe

15

<210> 64

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 64

Leu Ser Val Gly Tyr

15

<210> 65

<211> 3

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 65

Asp Thr Ser

1

<210> 66

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 66

Phe Gln Gly Ser Gly Tyr Pro Phe Thr

15

<210> 67

<211> 106

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 67

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105

<210> 68

<211> 318

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 68

gacatccaga tgacccagag cccctccacc ctgtccgcct ccgtgggcga cagagtgacc 60

atcacttgca aggcccagct gtccgtgggc tacatgcact ggtatcagca gaagcccggc 120

aaggccccta agctgctgat ctacgacacc tccaagctgg cctccggcgt gccctccaga 180

ttctccggct ctggctccgg caccgagttc accctgacca tctccagcct gcagcccgac 240

gacttcgcca cctactactg ttttcaaggc tccggctacc ccttcacctt cggcggaggc 300

accaagctgg aaatcaag 318

<210> 69

<211> 213

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 69

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

1 5 10 15

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

20 25 30

His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr

35 40 45

Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp

65 70 75 80

Asp Phe Ala Thr Tyr Tyr Cys Phe Gln Gly Ser Gly Tyr Pro Phe 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

Asn Arg Gly Glu Cys

210

<210> 70

<211> 639

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<400> 70

gacatccaga tgacccagag cccctccacc ctgtccgcct ccgtgggcga cagagtgacc 60

atcacttgca aggcccagct gtccgtgggc tacatgcact ggtatcagca gaagcccggc 120

aaggccccta agctgctgat ctacgacacc tccaagctgg cctccggcgt gccctccaga 180

ttctccggct ctggctccgg caccgagttc accctgacca tctccagcct gcagcccgac 240

gacttcgcca cctactactg ttttcaaggc tccggctacc ccttcacctt cggcggaggc 300

accaagctgg aaatcaagcg tacggtggcc gctcccagcg tgttcatctt cccccccagc 360

gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc 420

cgggaggcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480

agcgtcaccg agcaggacag caaggactcc acctacagcc tgagcagcac cctgaccctg 540

agcaaggccg actacgagaa gcataaggtg tacgcctgcg aggtgaccca ccagggcctg 600

tccagccccg tgaccaagag cttcaacagg ggcgagtgc 639

<210> 71

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (7)..(11)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (13)..(17)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (19)..(23)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (25)..(29)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (31)..(35)

<223> /note="This area may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (37)..(41)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (43)..(47)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (49)..(53)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (55)..(59)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (1)..(60)

<223> /note="This sequence can span 0-10

repeating units '(Gly)n-Ser', where n=1-5"

<400> 71

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

<210> 72

<211> 60

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> MISC_FEATURE

<222> (1)..(5)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (7)..(11)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (13)..(17)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (19)..(23)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (25)..(29)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (31)..(35)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (37)..(41)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (43)..(47)

<223> /note="This area may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (49)..(53)

<223> /note="This region may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (55)..(59)

<223> /note="This area may cover 1-5 residues"

<220>

<221> MISC_FEATURE

<222> (1)..(60)

<223> /note="This sequence can span 0-10

repeating units '(Gly)n-Ala', where n=1-5"

<400> 72

Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly

1 5 10 15

Gly Ala Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Ala Gly Gly

20 25 30

Gly Gly Gly Ala Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Ala

35 40 45

Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Ala

50 55 60

<210> 73

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (12)..(21)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (23)..(32)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (34)..(43)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (45)..(54)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (56)..(65)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (67)..(76)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (78)..(87)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (89)..(98)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (100)..(109)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (1)..(110)

<223> /note="This sequence may span 1-10

repeating units '(Gly)n-Ser', where n=0-10"

<400> 73

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

<210> 74

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<220>

<221> source

<223> /note="See submitted description for detailed description

substitutions and preferred embodiments"

<400> 74

Gly Gly Gly Gly Ser

15

<210> 75

<211> 110

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> MISC_FEATURE

<222> (1)..(10)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (12)..(21)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (23)..(32)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (34)..(43)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (45)..(54)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (56)..(65)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (67)..(76)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (78)..(87)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (89)..(98)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (100)..(109)

<223> /note="This area may cover 0-10 residues"

<220>

<221> MISC_FEATURE

<222> (1)..(110)

<223> /note="This sequence may span 1-10

repeating units '(Gly)n-Ala', where n=0-10"

<400> 75

Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly

1 5 10 15

Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly

20 25 30

Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly

35 40 45

Gly Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly

50 55 60

Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala Gly Gly Gly

65 70 75 80

Gly Gly Gly Gly Gly Gly Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly

85 90 95

Gly Gly Ala Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ala

100 105 110

<210> 76

<211> 5

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<220>

<221> source

<223> /note="See submitted description for detailed description

substitutions and preferred embodiments"

<400> 76

Gly Gly Gly Gly Ala

15

<210> 77

<211> 4

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 77

Arg Gly Asp Ser

1

<210> 78

<211> 6

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

Synthetic 6xHis tag"

<400> 78

His His His His His

15

<---

Claims (35)

1. An antibody-based protein with a grafted cytokine to activate immune cells, containing: (a) a heavy chain variable region (VH) containing the HCDR1 complementarity determining regions (CDRs) containing SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16, SEQ ID NO: 39 , SEQ ID NO: 42, SEQ ID NO: 45 or SEQ ID NO: 48, HCDR2 containing SEQ ID NO: 8, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 17, SEQ ID NO: 40, SEQ ID NO: 46 or SEQ ID NO: 49, and HCDR3 containing SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 18, SEQ ID NO: 41, SEQ ID NO: 44, SEQ ID NO: 47, or SEQ ID NO: 50, and wherein the HCDRs include 0 to 1 substitution; And (b) a light chain variable region (VL) containing LCDR1, containing SEQ ID NO: 23, SEQ ID NO: 26, SEQ ID NO: 29, SEQ ID NO: 32, SEQ ID NO: 55, SEQ ID NO: 58 , SEQ ID NO: 61 or SEQ ID NO: 64, LCDR2 containing SEQ ID NO: SEQ ID NO: 24, SEQ ID NO: 27, SEQ ID NO: 30, SEQ ID NO: 33, SEQ ID NO: 56, SEQ ID NO: 59, SEQ ID NO: 62, or SEQ ID NO: 65, and LCDR3 containing SEQ ID NO: 25, SEQ ID NO: 28, SEQ ID NO: 31, SEQ ID NO: 34, SEQ ID NO: 57, SEQ ID NO: 60, SEQ ID NO: 63, or SEQ ID NO: 66, and wherein the LCDRs include 0 to 1 substitution; And (c) a mutant interleukin 2 (IL2) molecule or fragment thereof grafted onto HCDR1, and wherein the mutant IL2 molecule or fragment thereof contains a mutation that reduces the affinity of the mutant IL2 molecule for the high affinity IL2 receptor. 2. The cytokine-grafted antibody protein of claim 1, wherein the cytokine-grafted antibody protein preferentially increases effector T cells over regulatory T cells. 3. Protein based on an antibody with a grafted cytokine according to claim 1, wherein the cytokine-grafted antibody protein stimulates one or more CD8+ T effector cells, NK cell proliferation, or a combination thereof to a greater extent than recombinant IL2 or aldesleukin. 4. The cytokine-grafted antibody protein of claim 1, wherein the VH comprises SEQ ID NO: 19 or SEQ ID NO: 51. 5. An antibody-based protein with a grafted cytokine according to claim 1, additionally containing an IgG class heavy chain and an IgG class light chain. 6. The cytokine-grafted antibody protein of claim 5, wherein the heavy chain of the IgG class is selected from IgG1, IgG2 and IgG4. 7. The cytokine-grafted antibody protein of claim 5, wherein the IgG heavy chain comprises SEQ ID NO: 21 or SEQ ID NO: 53. 8. The cytokine-grafted antibody protein of claim 5, wherein the IgG light chain contains SEQ ID NO: 69 and the IgG heavy chain contains SEQ ID NO: 53. 9. The cytokine-grafted antibody protein of claim 5, wherein the IgG light chain contains SEQ ID NO: 37 and the IgG heavy chain contains SEQ ID NO: 21. 10. The cytokine-grafted antibody protein of claim 5, wherein the IgG light chain contains SEQ ID NO: 69 and the IgG heavy chain contains SEQ ID NO: 21. 11. The cytokine-grafted antibody protein of claim 5, wherein the IgG light chain contains SEQ ID NO: 37 and the IgG heavy chain contains SEQ ID NO: 53. 12. A pharmaceutical composition for the treatment of cancer comprising an effective amount of the cytokine-grafted antibody protein of claim 1 and a pharmaceutically acceptable carrier. 13. The pharmaceutical composition according to claim 12, wherein the cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer and lymphoma. 14. A pharmaceutical composition for the treatment of cancer comprising an effective amount of the cytokine-grafted antibody protein of claim 1 or the pharmaceutical composition of claim 12 in combination with an effective amount of a therapeutic agent. 15. The pharmaceutical composition according to claim 14, wherein the therapeutic agent is a second cytokine-grafted antibody protein. 16. The pharmaceutical composition according to claim 14, wherein the therapeutic agent is an immune checkpoint inhibitor. 17. The pharmaceutical composition according to claim 16, where the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR. 18. The pharmaceutical composition according to claim 14, wherein the therapeutic agent is a tyrosine kinase inhibitor. 19. A method of preferentially expanding T effector cells over regulatory T cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the cytokine-grafted antibody protein of claim 1. 20. The method of claim 19, wherein the effector T cells are expanded and the Treg cells are not expanded. 21. A method of preferentially expanding T effector cells over regulatory T cells in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the cytokine-grafted antibody protein of claim 1 and a therapeutically effective amount of an immune checkpoint inhibitor. 22. The method of claim 21, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR. 23. Use of a cytokine-grafted antibody protein according to claim 1 for the production of a drug for the treatment of cancer. 24. Use of a cytokine-grafted antibody protein according to claim 1 in combination with a therapeutic agent to produce a medicament for the treatment of cancer. 25. Use according to claim 24, wherein the therapeutic agent is an immune checkpoint inhibitor. 26. The use of claim 25, wherein the immune checkpoint is selected from the group consisting of: PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4 and TGFR. 27. An expression vector containing an isolated nucleic acid molecule that encodes a cytokine-grafted antibody protein for activating immune cells, the expression vector comprising: (i) the nucleic acid sequence shown in SEQ ID NO: 22 encoding the heavy chain and the nucleic acid sequence shown in SEQ ID NO: 38 encoding the light chain; or (ii) the nucleic acid sequence shown in SEQ ID NO: 54 encoding the heavy chain and the nucleic acid sequence shown in SEQ ID NO: 70 encoding the light chain. 28. A recombinant host cell for producing a cytokine-grafted antibody protein containing an expression vector according to claim 27. 29. A mammalian cell for producing a protein based on a cytokine-grafted antibody, containing an expression vector according to claim 27.

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