Antibody-cytokine grafted proteins and methods of use in the treatment of cancer

Abstract

To provide IL2 engrafted into CDR sequences of an antibody having preferred therapeutic profiles over molecules known and used in the clinic.SOLUTION: An antibody cytokine engrafted protein comprises: (a) an IgG class heavy chain comprising an IgG class heavy chain variable region (VH), comprising Complementarity Determining Regions (CDR) HCDR1, HCDR2, HCDR3, wherein HCDR comprises 0-1 substitution; (b) an IgG class light chain comprising an IgG class light chain variable region (VL), comprising LCDR1, LCDR2, LCDR3, wherein LCDR comprises 0-1 substitution; and (c) a mutated Interleukin 2 (IL2) molecule engrafted into HCDR1, wherein the mutated IL2 molecule comprises mutations that reduce affinity of the mutated IL2 molecule for a high-affinity IL2 receptor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 510,533, filed May 24, 2017. This application claims the benefit of US Provisional Patent Application No. 2006 / 0119994, the contents of which are incorporated herein by reference in their entirety. It is used.

[0002] The present invention relates to an antibody-cytokine that binds to the interleukin-2 (IL2) low affinity receptor. The present invention relates to grafted proteins and methods for treating cancer.

[0003] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on April 5, 2018, is incorporated herein by reference. It is called T057462-WO-PCT_SL.txt and is 69,489 bytes in size. be. [Background technology]

[0004] IL2 was first cloned in 1983 (Taniguchi et al. ,Nature 1983,302:305-310,Devos et al.,Nu Cleic Acid Res.1983,11(13):4307-4323, Mae da et al.,Biochem.Biophys.Res.Comm.1983, 115:1040-1047). The IL2 protein contains a signal peptide consisting of amino acids 1-20. It is 153 amino acids long, including the thiol, and folds into four antiparallel amphipathic α-helices. (Smith KA, Science 1988, 240:1169 -1176).

[0005] IL2 exerts its biological effects by signaling through high- or low-affinity receptors. (Kreig et al., PNAS 2010, 107(26)119 06-11911). The high affinity receptors are IL2-Rα (CD25) and IL2-Rβ (CD The low-affinity receptor is a trimer consisting of I It is a dimer consisting of only the IL2-Rβ (CD122) and IL2-Rγ (CD132) chains. The low-affinity receptor binds IL2, but the affinity is 10-15 times that of the high-affinity receptor, which is a trimer. Although IL2-Rα (CD25) is important for increasing affinity, It is suggested that IL2 is not a signal transmission component (Kreig et al., supra). Receptor expression is also distinctive. The high-affinity IL2 receptor is expressed on activated T cells and CD4+ / Foxp3+ regulatory T cells (Tregs) express low-affinity IL2 receptors. is found in CD8+ T effector cells and natural killer (NK) cells.

[0006] Recombinant IL2 (rhIL2) was originally approved for clinical use in 1992 (Coven try et al.,Cancer Mgt Res.2012 4:215-221 Proleukin® (aldesleukin) is a non-glycosylated, N Modified IL lacking the terminal alanine and substituting serine for cysteine ​​at amino acid 125 2. Proleukin® was initially used as a treatment for malignant melanoma and renal cell carcinoma. It was first indicated as a forensic drug but has been used in other cancer types such as colorectal cancer, breast cancer, lung cancer and mesothelioma. (Coventry, op. cit.) 259 kidney In a study of patients with uterine carcinoma, 23 patients had a complete response and 30 had a partial response. It was found that (Klapper et al., Cancer 2008 113 (2):293-301), which corresponds to an overall objective response rate of 20%, 7% of patients with renal cell carcinoma. % achieved complete tumor regression (Klapper et al., supra).

[0007] However, cancer treatment with IL2 was not without adverse effects. Studies of patients with thrombus formation have shown capillary / vascular leakage, vasodilation, and oliguria. Grade 3 and Grade 4 neutrophil dysfunction, both in infectious and general infections There were also 4 infections (Klapper et al., op. cit.). The literature on Proleukin® is based on the following: Crohn's disease, scleroderma, thyroiditis , inflammatory arthritis, diabetes mellitus, ophthalmo-bulbar myasthenia gravis, crescentic IgA glomeruli Nephritis, cholecystitis, cerebral vasculitis, Stevens-Johnson syndrome, and bullous pemphigoid It has been linked to the exacerbation of autoimmune and inflammatory disorders.

[0008] Treg cells constitutively express high-affinity IL2 receptors and are dependent on IL2 for survival and function. The finding that the drug was dependent on the steroids suggests why this side effect was observed (D' Cruz et al., Nat. Immuno. 2005, 6: 1152-1159) It has improved pharmacokinetics and activates Treg cells via a high-affinity receptor. It is selective for activating CD8+ T cells via low-affinity receptors without This allows for the treatment of cancer without the unwanted side effects seen with Proleukin®. This indicates the need for IL2 therapeutic agents that can achieve this. Summary of the Invention [Means for solving the problem]

[0009] explanation The present disclosure provides compounds that have a favorable therapeutic profile compared to known clinically used molecules. The present invention provides an IL2 grafted onto the CDR sequences of an antibody. The cytokine-grafted protein composition reduces the activity of Treg cells while inhibiting C In addition, the provided compositions increase or maintain D8+ T effector cells. The composition is an improvement over recombinant human IL2 preparations such as Proleukin®. The present disclosure provides a method for binding to the IL2 high affinity receptor. IL2 binds to and promotes favorable signal transduction through the low-affinity receptor with reduced activity. The present invention provides an antibody-cytokine grafted protein that promotes the expression of IgG. (i) A heavy chain variable region (VH) and (ii) an immunoglobulin heavy chain sequence comprising a light chain variable region (VL). and an antibody-cytokine grafted protein comprising the VH of the antibody. Alternatively, an IL2 molecule is grafted into the complementarity determining region (CDR) of the VL.

[0010] An embodiment of the present disclosure comprises: (a) heavy chain variable region, including complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3; Area (VH); and (b) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3; and (c) an interleukin 2 (IL2) molecule grafted into the CDR of VH or VL; The present invention provides an antibody-cytokine grafted protein comprising:

[0011] Antibody-cytokine grafted proteins containing IL2 molecules grafted onto heavy chain CDRs Quality.

[0012] The IL2 molecule has complementarity-determining region 1 (HCDR1), complementarity-determining region 2 (HCDR2) or is grafted onto a region selected from complementarity determining region 3 (HCDR3), Itokine-grafted protein.

[0013] Antibody-cytokine grafted proteins containing IL2 molecules grafted into HCDR1 Quality.

[0014] Antibody-cytokine grafted proteins containing IL2 molecules grafted onto light chain CDRs Quality.

[0015] The IL2 molecule has complementarity-determining region 1 (LCDR1), complementarity-determining region 2 (LCDR2), or an antibody having a region selected from complementarity determining region 3 (LCDR3) grafted thereon. Cytokine-grafted proteins.

[0016] IL2 containing a mutation that reduces the affinity of the IL2 molecule for the high-affinity IL2 receptor Antibody-cytokine-grafted protein containing molecules.

[0017] Stimulation of CD8 T cell effector proliferation by antibody-cytokine grafted proteins antibody cytokine group, whose activity is greater than that of recombinant IL2 or Proleukin® Rafted proteins.

[0018] Stimulation of Treg cell proliferation by antibody-cytokine grafted proteins is enhanced by recombinant IL2 or antibody-cytokine grafted proteins smaller than Proleukin® Quality.

[0019] Stimulation of NK cell proliferation by antibody-cytokine grafted proteins is associated with the development of recombinant IL2 or Antibody-cytokine grafted protein larger than Proleukin® .

[0020] The antibody-cytokine grafted protein is recombinant IL2 or Proleukin (registered trademark) An antibody-cytokine grafted protein with a longer half-life than IgG1A (BioNutrition) or IgG2A (BioNutrition) (Trademark).

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

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

[0023] An antibody-cytokine grafted protein comprising an IgG class antibody heavy chain.

[0024] an antibody cytokine graft, wherein IgG is selected from IgG1, IgG2, or IgG4; Fused proteins.

[0025] The binding specificity of the CDRs to the target was increased by 10%, 2% by the grafted IL2 molecule. 0%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 9 9% or 100% reduction in antibody-cytokine grafted proteins.

[0026] The binding specificity of the CDRs to the target is 10% in the presence of the grafted IL2 molecule, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% retention of antibody-cytokine grafted proteins.

[0027] Antibody-cytokine grafting in which the binding specificity of the CDR differs from that of the IL2 molecule protein.

[0028] Antibody-cytokine-grafted target, wherein the binding specificity of the CDRs is for a non-human target. Protein.

[0029] An antibody-cytokine grafted protein in which the non-human antigen is a virus.

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

[0031] The RSV is selected from RSV subgroup A and RSV subgroup B. Tocain-grafted proteins.

[0032] an antibody, wherein the antibody scaffold portion of the antibody-cytokine grafted protein is humanized or human; Cytokine-grafted proteins.

[0033] An embodiment of the present disclosure provides: (i) (a) HCDR1 of SEQ ID NO: 13; (b) HCDR1 of SEQ ID NO: 14; (c) a heavy chain variable region comprising an HCDR2 of SEQ ID NO: 15, and (d) an HCDR3 of SEQ ID NO: 2 9, (e) LCDR2 of SEQ ID NO: 30, and (f) LCDR of SEQ ID NO: 31 3; or (ii) a light chain variable region comprising (a) an HCDR1 of SEQ ID NO: 45, (b) an HCDR1 of SEQ ID NO: 46, (c) a heavy chain variable region comprising an HCDR2 of SEQ ID NO: 46, an HCDR3 of SEQ ID NO: 47; and (d) a heavy chain variable region comprising an HCDR2 of SEQ ID NO: 46, an HCDR3 of SEQ ID NO: 47, and (e) LCDR1 of sequence number 61, (f) LCDR2 of sequence number 62, and (g) LCDR3 of sequence number 63. Antibody-cytokine-grafted proteins containing a light chain variable region containing LCDR3 are provided. .

[0034] An embodiment of the present disclosure includes (i) a heavy chain variable region (VH) comprising SEQ ID NO: 19, and a 35; or (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 51. ), and an antibody cytokine-grafted template comprising a light chain variable region (VL) comprising SEQ ID NO: 67. Provides protein.

[0035] Antibody cytokines, wherein the antibody comprises a modified Fc region corresponding to reduced effector function Grafted proteins.

[0036] The modified Fc region includes D265A, P329A, P329G, N297A, L234A, and and L235A. Protein.

[0037] Modified Fc region is D265A / P329A, D265A / N297A, L234 / L2 35A, P329A / L234A / L235A, and P329G / L234A / L235 Antibody-cytokine grafting comprising a combination of mutations selected from one or more of A. protein.

[0038] An embodiment of the present disclosure includes HCDR1 of SEQ ID NO: 13, HCDR2 of SEQ ID NO: 14, HCDR3 of SEQ ID NO: 15, HCDR4 of SEQ ID NO: 16, HCDR5 of SEQ ID NO: 17, HCDR6 of SEQ ID NO: 18, HCDR7 of SEQ ID NO: 19, HCDR8 of SEQ ID NO: 20, HCDR9 of SEQ ID No. 15, LCDR1 of SEQ ID NO: 29, LCDR2 of SEQ ID NO: 30, 31 LCDR3, an antibody sequence containing a modified Fc region containing the mutations D265A / P329A and a cytokine-grafted protein, wherein the antibody-cytokine-grafted protein lower Treg cell counts compared to recombinant IL2 or Proleukin® Stimulates activation.

[0039] An embodiment of the present disclosure includes HCDR1 of SEQ ID NO: 45, HCDR2 of SEQ ID NO: 46, No. 47, LCDR1 of SEQ ID NO: 61, LCDR2 of SEQ ID NO: 62, 63 LCDR3, an antibody sequence containing a modified Fc region containing mutations D265A / P329A and a cytokine-grafted protein, wherein the antibody-cytokine-grafted protein lower Treg cell counts compared to recombinant IL2 or Proleukin® Stimulates activation.

[0040] An embodiment of the present disclosure is a nucleic acid sequence comprising: (i) a heavy chain of SEQ ID NO: 22 and / or a light chain of SEQ ID NO: 38; or (ii) an antibody cytokine graft comprising a heavy chain of SEQ ID NO: 54 and / or a light chain of SEQ ID NO: 70; An isolated nucleic acid encoding a fused protein is provided.

[0041] Embodiments of the present disclosure include heavy and light chain polypeptides of a protein, and optionally a secretory sequence. and antibody-cytokine-grafted proteins comprising the nucleic acids disclosed herein encoding the signal. Recombinant host cells suitable for the production of proteins are provided.

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

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

[0044] Embodiments of the present disclosure include the use of antibody-cytokine grafted proteins as disclosed herein. and one or more pharmaceutically acceptable carriers.

[0045] Embodiments of the present disclosure provide a method of treating cancer in an individual in need thereof, comprising: The method comprises administering to a subject a therapeutic agent comprising the antibody-cytokine grafted protein or pharmaceutical composition disclosed herein. The method includes administering a therapeutically effective amount to an individual.

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

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

[0048] A method of treating cancer wherein the therapeutic agent is another antibody cytokine grafted protein.

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

[0050] Immune checkpoints include PD-1, PD-L1, PD-L2, TIM3, and CTLA- 4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIG Therapeutic use of cancer Treatment method.

[0051] Embodiments of the present disclosure provide for the expansion of CD8 T effector cells in patients in need thereof. The present invention provides a method for the preparation of a human antibody-cytokine grafted protein comprising administering the antibody-cytokine grafted protein disclosed herein to a subject. The method includes administering the protein or pharmaceutical composition to the patient.

[0052] CD8 T effector cells are expanded, but Treg cells are not expanded, Methods for expanding T effector cells.

[0053] CD8 T effector cells are expanded and NK cells are not expanded, How to expand cytokeratocytes.

[0054] and expanding CD8 T effector cells, further including administration of immune checkpoint inhibitors. How to do it.

[0055] Immune checkpoints include PD-1, PD-L1, PD-L2, TIM3, and CTLA- 4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIG IT, LAIR1, CD160, 2B4 and TGFR, selected from the group consisting of CD8 Methods for expanding T effector cells.

[0056] Embodiments of the present disclosure provide a method for treating cancer, comprising administering to a patient a therapeutically effective amount of: (i) (a) HCDR1 of SEQ ID NO: 13, (b) HCDR2 of SEQ ID NO: 14, (c) HCDR3 of SEQ ID NO: 15, (d) HCDR4 of SEQ ID NO: 16, (e) HCDR5 of SEQ ID NO: 17, (f) HCDR6 of SEQ ID NO: b) a heavy chain variable region comprising an HCDR2 of SEQ ID NO: 14, (c) an HCDR3 of SEQ ID NO: 15, and and (d) LCDR1 of SEQ ID NO: 29, (e) LCDR2 of SEQ ID NO: 30, and (f) sequence (ii) a light chain variable region comprising an LCDR3 of SEQ ID NO: 31; and (ii) (a) an HCDR of SEQ ID NO: 45. 1, (b) an HCDR2 of SEQ ID NO: 46, and (c) an HCDR3 of SEQ ID NO: 47. region; and (d) LCDR1 of SEQ ID NO: 61, (e) LCDR2 of SEQ ID NO: 62, and ( f) antibody cytokines in the treatment of cancers containing a light chain variable region comprising LCDR3 of SEQ ID NO: 63 Uses of the grafted proteins are provided.

[0057] The antibody-cytokine grafted protein is administered in combination with another therapeutic agent, Use of tocain-grafted proteins.

[0058] The therapeutic agent is an antibody cytokine graft, which is an antagonist of immune checkpoint inhibitors. Use of fusoflated proteins.

[0059] Immune checkpoint inhibitor antagonists target PD-1, PD-L1, and PD-L2 , TIM3, CTLA-4, LAG-3, CEACAM-1, CEACAM-5, VIS A group consisting of TA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFR Selected from, use.

[0060] In certain embodiments, the antibody-cytokine grafted protein is an IgG class antibody. In particular embodiments, the immunoglobulin is an IgG1, IgG2, or is selected from the IgG4 subclass Fc region. The antibody, antibody fragment, or antigen-binding molecule is Optionally, modulate (i.e., increase or decrease) binding of the antibody or antibody fragment to an Fc receptor. The immunoglobulin heavy chain optionally contains at least one modified phenotype. In particular embodiments, the immunoglobulin heavy chain may include modifications that confer vector function. are D265A, P329A, P329G, N297A, D265A / P329A, D2 65A / N297A, L234 / L235A, P329A / L234A / L235A, and and P329G / L234A / L235A. The mutation may include a mutation that confers

[0061] In some embodiments, the antibody-cytokine grafted protein also comprises the molecule IL-1. This includes bipartite mutations. These mutations can be single amino acid changes, single amino acid deletions, or multiple amino acid deletions. The IL2 cytokine portion of the molecule may have amino acid changes and multiple amino acid deletions. These changes were due to the antibody cytokine grafted protein against the high affinity IL2 receptor. The affinity can be reduced.

[0062] Furthermore, the present disclosure provides an antibody-cytokine grafted protein as described herein. and providing a polynucleotide encoding at least a heavy chain and / or a light chain protein of the protein. In another related embodiment, the host cell is an antibody site as described herein. Host cells suitable for the production of kinin-grafted proteins are provided. The host cell then expresses the light and / or heavy chain polypeptides of the antibody cytokine grafted protein. In yet another embodiment, the antibody-cytokine grafted protein comprises a nucleic acid encoding the antibody-cytokine grafted protein. A method for producing a protein is provided, the method comprising the steps of: a provided host cell as described herein under conditions suitable for expression, formation, and secretion of and recovering the antibody-cytokine-grafted protein from the culture. In a further aspect, the present disclosure provides an antibody cytokine as described herein. Further provided is a kit containing the grafted protein.

[0063] In another related aspect, the present disclosure provides an antibody cytokine as described herein. The present invention further provides a composition comprising the grafted protein and a pharmaceutically acceptable carrier. In some embodiments, the present disclosure provides an antibody-cytokine grafted antibody-antibody complex for administration to an individual. A pharmaceutical composition comprising the protein is provided.

[0064] In another embodiment, there is provided a method of treating cancer in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of the compound described herein. administering to an individual a therapeutically effective amount of an antibody-cytokine grafted protein as described in In a further embodiment, for use in treating or preventing cancer in an individual. An antibody-cytokine grafted protein of the formula:

[0065] In some embodiments, the patient has a cell proliferative disorder or cancer, e.g., melanoma, lung cancer, colorectal cancer, He has bowel cancer, prostate cancer, breast cancer and lymphoma.

[0066] definition "Antibody" refers to a molecule of the immunoglobulin family that contains a tetrameric structural unit. Each tetramer contains two identical pairs of polypeptide chains, each pair held together by a disulfide bond. It has two "light" chains (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The immunoglobulin genes known include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes; The light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn determine the structure of immunoglobulins. The classes are IgG, IgM, IgA, IgD, and IgE, respectively. isotype / class (e.g., IgG, IgM, IgA, IgD, and IgE), or Any subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgA3, IgA4, IgA5, IgA6, IgA7, IgA8, IgA9, IgA10, IgA11, IgA12, IgA13, IgA14, IgA15, IgA16, IgA17, IgA18, IgA gA2).

[0067] Both the light and heavy chains are divided into regions of structural and functional homology. The N-terminus of each chain is primarily involved in antigen recognition. Defines a variable (V) region or domain of about 100-110 amino acids or more that contributes to The term variable light chain (V L ) and variable heavy chain (V H ) are these regions of the light and heavy chains, respectively. V H and V L These V regions pair together to form a single antigen-binding site. Both the heavy and light chains contain constant (C) regions or domains. The region comprises three C domains CH1, CH2, CH3, optionally CH4 (Cμ), and The membrane-bound immunoglobulin C region also consists of a membrane domain and an intracellular domain. Each light chain has a V L followed by a constant domain (C) at the other end The constant domains of the light chain (CL) and heavy chain (CH1, CH2 or CH3) are secreted, They confer important biological properties such as transplacental transfer, Fc receptor binding, and complement fixation. The constant region domains are numbered in increasing order of their distal position 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 domains contain the carboxy-terminal domains of the heavy and light chains, respectively. VL is aligned with VH. The CL aligns with the first constant domain of the heavy chain. The term "antibody" encompasses conventional antibody structures and variant antibodies. cytokine-grafted proteins, full-length antibodies, chimeric antibodies, humanized antibodies, human antibodies , and antibody fragments thereof.

[0068] Antibodies exist as intact immunoglobulin chains or as fragments that are bound to various peptidases. Antibody fragments exist as a number of well-characterized antibody fragments produced by digestion with The term "antibody fragment" as used herein refers to an antibody fragment that retains all six CDRs. Thus, for example, pepsin binds to the disulfide bonds in the hinge region. When the antibody is digested, the light chains themselves are separated by disulfide bonds into V H -C H Concatenated to 1 The resulting Fab' dimer, F(ab)'2, is produced. F(ab)'2 is reduced under mild conditions. This breaks the disulfide bond in the hinge region, resulting in the F(ab)'2 dimer being converted into Fa The Fab' monomer is essentially an F Fab' monomer with a portion of the hinge region. ab (Paul, Fundamental Immunology 3rd ed. (1993)). Various antibody fragments have been defined in terms of digestion of the intact antibody. However, those skilled in the art will appreciate that such fragments may be obtained chemically or by using recombinant DNA methods. It will be understood that the antibody fragment may be synthesized. are produced by modification of whole antibodies or synthesized de novo using recombinant DNA methods. refers to one or more portions of an antibody that retain binding specificity and functional activity, either Examples of antibody fragments include Fv fragments, single-chain antibodies (ScFv), and Fab, Fab', Fd (Vh and CH1 domains), dAb (Vh and isolated CDRs); and multimeric forms of these fragments (e.g., F(ab')2). The grafted proteins may also be modified to include the antibody fragments necessary to achieve the desired binding specificity and activity. It may also include pieces.

[0069] A "Fab" domain, as used in this context, refers to a heavy chain variable domain, a constant region CH The light chain constant region comprises a CL domain, a light chain variable domain, and a light chain constant region CL domain. The interaction between the CH1 and CL domains is stabilized by a disulfide bond. In some embodiments, the heavy chain domain of a Fab is, from N-terminus to C-terminus, VH-C The light chain domains of Fab are ordered VL-CL from N-terminus to C-terminus. In some embodiments, the heavy chain domain of the Fab is in the order CH-VH from N-terminus to C-terminus. The light chain domains of Fab are in the order CL-VL. Fab fragments have historically been Although identified by papain digestion of tactic immunoglobulins, in the context of this disclosure The "Fab" fragments are typically recombinantly produced by any method. Each Fab fragment binds to an antigen. It is monovalent with respect to binding, i.e., it has a single antigen-binding site.

[0070] "Complementarity determining domain" or "complementarity determining region" ("CDR") is used interchangeably with V. L Reach BiV H CDRs are the hypervariable regions of an antibody chain that are specific for the target protein. Each human VL or VH contains three CDRs (CDRs). R1-R3, numbered consecutively from the N-terminus), which make up approximately 15-20% of the variable domain The CDRs are structurally complementary to the epitope of the target protein and Therefore, it is directly involved in binding specificity. The framework region (FR) shows little variation in amino acid sequence (Kuby, Immuno logy,4th ed.,Chapter 4.WHFreeman & Co. ,New York,2000).

[0071] The locations of CDRs and framework regions may be defined by various well-known definitions in the art, e.g. For example, Kabat, Chothia, and AbM (e.g., Kabat et al. 991 Sequences of Proteins of Immunologic al Interest, Fifth Edition,USDepartmen of Health and Human Services,NIH Publi cation No.91-3242,Johnson et al.,Johnson et al., Nucleic Acids Res.,29:205-206(20 01);Chothia and Lesk, J. Mol. Biol., 196:901 -917(1987);Chothia et al.,Nature,342:877 -883(1989);Chothia et al., J.Mol.Biol.,22 7:799-817(1992);Al-Lazikani et al., J.Mol , 273:927-748 (1997)). The definition of the original binding site is also described in Ruiz et al., Nuclei c Acids Res., 28:219-221(2000); and Lefranc, MP, Nucleic Acids Res.,29:207-209(2001) ;(ImMunoGenTics(IMGT)numbering)Lefranc,M .-P.,The Immunologist,7,132-136(1999);Le franc,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);Mar tin et al.,Methods Enzymol.,203:121-153( 1991); and Rees et al., In Sternberg MJE ( ed.), Protein Structure Prediction, Oxford University Press, Oxford, 141-172 (1996).

[0072] According to Kabat, V H The CDR amino acid residues are 31-35 (HCDR1), 50 ~65 (HCDR2), and 95~102 (HCDR3); and V L CDR The amino acid residues are 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Based on Chothia, V H The CDR amino acids are 26 ~32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3) Numbered; and V L The amino acid residues of LCDR1 are 26-32, and the amino acid residues of LCDR2 are 50-52. 2), and 91-96 (LCDR3). By combining these CDR definitions, the CDR is located at amino acid residue 26 in human VH. ~35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3), and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2) in human VL. ), and 89 to 97 (LCDR3).

[0073] "Antibody variable light chain" or "antibody variable heavy chain" as used herein refers to V L or V H refers to a polypeptide comprising an endogenous V L The gene segments V (variable) and J (linked) Encoded by endogenous V H is coded by V, D (diversity), and J V Lor V H Each of the CDRs comprises a CDR as well as a framework region (FR). "V region" or "V region" are synonymous with FR1-CDR1-FR2-CDR2-FR3-CDR1. V regions refer to heavy or light chains containing DR3-FR4. V regions may be naturally occurring, recombinant, or synthetic. In this application, antibody light chains and / or antibody heavy chains are sometimes collectively referred to as "antibody light chains." As provided and further described herein, an "antibody chain" may be referred to as an "antibody molecule." "Antibody variable light chain" or "antibody variable heavy chain" and / or "variable region" and / or "antibody chain" are optional and comprises a cytokine polypeptide sequence incorporated into the CDRs.

[0074] As used herein, the C-terminus of an immunoglobulin heavy chain, including, for example, the CH2 and CH3 domains The terminal portion is the "Fc" domain. As used herein, the "Fc region" refers to the first The constant region of an antibody excluding the immunoglobulin domain (CH1). , IgA, IgD, and the last two constant region immunoglobulin domains of IgG, IgE and the last three constant region immunoglobulin domains of IgM, as well as the Refers to the flexible hinge N-terminus. For IgA and IgM, Fc may include the J chain. Ig For G, Fc is the immunoglobulin domains Cγ2 and Cγ3, and Cγ1 and Cγ The boundaries of an Fc region may vary in the art; However, the human IgG heavy chain Fc region usually consists of residues C226 or P230 to its carboxyl (The numbering used is from Kabat et al. (1991, NIH Public ation 91-3242, National Technical Informa EU Index at the tion Service, Springfield, Va. It is understood that the term "Fc region" is defined to include this region alone. or may refer to this region in the context of an antibody or antibody fragment. "Fc region" refers to the naturally occurring portion of an Fc region, including modifications that modulate, for example, effector function. The Fc region also contains allelic variants that affect biological functions, for example, in the CH2 and CH3 regions. For example, the N-terminus or One or more amino acids are deleted from the C-terminus without substantial loss of biological function. For example, in certain embodiments, the C-terminal lysine is modified, replaced, or removed. In particular embodiments, one or more C-terminal residues of the Fc region are altered or removed. In some embodiments, one or more C-terminal residues of the Fc (eg, terminal lysine) are deleted. In certain other embodiments, one or more C-terminal residues of Fc are replaced with another amino acid (e.g., (e.g., the terminal lysine is replaced). Such variants have minimal effect on activity. The nucleotide sequence is selected according to general rules known in the art (e.g., Bowie , et al., Science 247:306-1310, 1990) The Fc domain is the domain of immunoglobulin (Ig) that is recognized by cellular receptors such as FcR. The 5' end of the CH2 exon is where the complement-activating protein C1q binds. The lower hinge region, encoded by the fragment, provides flexibility within the antibody for binding to the FcR receptor. to provide.

[0075] A "chimeric antibody" is an antibody that (a) has a different or altered class, effector function, and / or The constant regions of the various species, or an entirely different molecule, e.g., an enzyme, that confers novel properties to the chimeric antibody. The antigen-binding site (variable region) is linked to proteins, toxins, hormones, growth factors, and drugs. (b) the constant region, or a portion thereof, has been altered, replaced, or exchanged; or The variable region, or a portion thereof, may be modified by a variable region having a different or altered antigen specificity. An antibody molecule that has been altered, replaced, or exchanged.

[0076] "Humanized" antibodies are antibodies that are less immunogenic in humans but still retain the reactivity of non-human antibodies (e.g. This is an antibody that retains the CDR region of the non-human CDR. This can be achieved by retaining the human portion of the antibody and replacing the remainder with a human counterpart. 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,Mole c.Immun.,28:489-498(1991);Padlan,Molec.I See mmun., 31(3):169-217 (1994).

[0077] The term "human antibody" refers to an antibody in which both the framework and CDR regions contain sequences of human origin. Furthermore, when the antibody contains a constant region, the constant region Also, such human sequences, e.g., human germline sequences, or amplified versions of human germline sequences, may be used. Mutants, or, for example, Knappik et al., J. Mol. Biol. 296: 57-86, 2000) obtained from human framework sequence analysis. Human antibodies are derived from antibodies containing consensus framework sequences. may include unencoded amino acid residues (e.g., in vitro random or site-specific by specific mutation, by somatic mutation in vivo, or by stable or is a mutation introduced by conservative substitution to facilitate production).

[0078] The term "corresponding human germline sequence" refers to a human germline immunoglobulin variable region sequence. The reference variable region is compared to all other known variable region amino acid sequences encoded by the sequence. The human soluble amino acid sequence having the highest determined amino acid sequence identity with the region amino acid sequence or subsequence The term "variable region" refers to a nucleic acid sequence that encodes a variable region amino acid sequence or subsequence. The alignment also shows the relative position of the reference variable region amino acid sequence compared to all other evaluated variable region amino acid sequences. The human variable region amino acid sequence with the highest amino acid sequence identity to the amino acid sequence or subsequence The corresponding human germline sequence may refer to only the framework regions, SDR only, framework and complementarity determining regions, variable segment (as defined above) Sequence identity may be any combination of sequences or subsequences that contain variable regions. The identity can be determined using, for example, BLAST, ALIGN, or other alignment techniques known in the art. Align the two sequences using the alignment algorithm, using the methods described herein. The corresponding human germline nucleic acid or amino acid sequence can be determined by using the reference variable region nucleic acid or At least about 90%, 91%, 92%, 93%, 94%, 95%, 96% %, 97%, 98%, 99%, or 100% sequence identity.

[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 to one target molecule or a specific site on a target molecule. When a polypeptide contains two or more target binding sites, each target binding site may be the same or can specifically bind to different molecules (e.g., different molecules, e.g., different antigens, or the same molecule) (It can bind to different epitopes on the molecule.) Conventional antibodies, for example, have two binding sites: "Trivalent" and "tetravalent" refer to antibody molecules with three and four binding sites, respectively. The antibody-cytokine grafted protein is monovalent (i.e., They may be bivalent (i.e., bind to one target molecule), bivalent, or multivalent (i.e., bind to two or more target molecules). It could be.

[0080] The phrases "specifically bind" or "binding specificity" refer to the binding specificity between a target (e.g., a protein) and an antibody. Used in the context of describing the interaction between cytokines and grafted proteins When a heterogeneous population of proteins and other biological materials is present, for example, in a biological sample, e.g., blood refers to a binding reaction that determines the presence of a target in a serum, plasma, or tissue sample. Under specified conditions, antibody-cytokine grafted proteins with specific binding specificities A quality is a measure of the binding of a specific target to at least twice the background level and to other targets present in the sample. In one embodiment, the antibody does not bind to the target in any significant amount under specified conditions. In this study, antibody-cytokine grafted proteins with specific binding specificity were targeted to specific antigens. Binds at least 10 times background and does not bind significantly to other targets present in the sample Under such conditions, the antibody-cytokine grafted protein For specific binding to proteins, antibodies, cytokine-grafted proteins bind to specific target proteins. As used herein, the antibody may need to be selected for its specificity for Specific binding is an antibody that selectively binds to the human IL2 low affinity receptor. These proteins interact with other cytokine receptor superfamily members, e.g. In some embodiments, the antibody-cytokine grafted protein does not contain a differentially reactive antibody. , which selectively binds to the human IL2 low affinity receptor and to the non-human primate IL2R (e.g., crab Antibody-cytokine grafted proteins that cross-react with the macaque IL2R are selected. In some embodiments, a compound that selectively binds to the human IL2 low affinity receptor and is capable of binding to a further target Antibody-grafted proteins that react with specific target proteins are selected. Various formats are used to select antibody-cytokine grafted proteins that show reactivity. For example, solid-phase ELISA immunoassays can be used to detect antibodies that specifically immunoreact with proteins. are routinely used to select antibodies of a specific nature (e.g., to determine specific immunoreactivity). For a description of immunoassay formats and conditions that may be used, see Harlow & Lan e,Using Antibodies,A Laboratory Manual(1 998). Typically, specific or selective binding reactions are performed using a background At least 2x the signal, more typically at least 10-10 above background 0 times, will generate a signal.

[0081] The term "equilibrium dissociation constant (KD, M)" refers to the dissociation rate constant (kd, time-1) The equilibrium dissociation constant is the value obtained by dividing the equilibrium dissociation constant by the rate constant (kA, time-1, M-1). The antibody-cytokine grafted protein may be measured using any known method in the field. The substance is generally about 10 -7 or 10 -8 Less than m, e.g., about 10 -9 M or 10 -10 Less than M, In one embodiment, about 10 -11 M, 10 -12 M or 10 -13 have an equilibrium dissociation constant less than M It will.

[0082] As used herein, the term "epitope" or "binding region" refers to the region between an antibody CDR and an antibody CDR. It refers to the domain in an antigen protein that is involved in specific binding with the original protein.

[0083] As used herein, the term "receptor-cytokine binding region" refers to a grafted The specific binding between a selected cytokine and its receptor (e.g., IL2 low affinity receptor) The grafted cytokine portion of the antibody-cytokine grafted protein Each antibody-cytokine grafted protein contains at least one There are receptor-cytokine binding domains, each of which may be identical to the others, or may be different.

[0084] The term "agonist" is used interchangeably with "agonist" to mean an agent that activates a receptor and induces full or partial receptor mediation. It refers to an antibody that has the ability to induce a mediated response. For example, an agonist of the IL2 low affinity receptor It binds to the IL2 low affinity receptor and mediates IL2-mediated intracellular signaling, cell activation, and and / or induces proliferation of CD8+ T effector cells and NK cells. Rafted protein agonists have low IL-2 affinity in some respects, similar to the natural IL2 ligand. When IL2 binds to the IL2 low affinity receptor, it stimulates signal transduction through the receptor. Induction of Jak1 and Jak2 activation, which leads to STAT5 phosphorylation In some embodiments, the antibody-cytokine grafted protein agonist is an IL 2 low affinity receptors and phosphorylates STAT5 and / or CD8+ T effectors These antibodies can be identified by their ability to induce proliferation of NK cells or NK cells.

[0085] The term "IL2" or "interleukin 2" or "interleukin-2" or "IL "-2" is synonymous with the alpha helix, a naturally occurring protein that functions in the regulation and maintenance of inflammatory processes. IL2 is a member of the IL-1 cytokine family. The properties of IL2 are that the N- and C-termini are open. The IL2 cytokine protein is therefore closely related to the antibody graft. In the context of agonist antibody cytokine grafted proteins, A full-length native human IL2 containing residues 21 to 153 is utilized. The human IL2 has at least about 90%, 91%, and 100% identical amino acid sequence to SEQ ID NO:2 over its entire length. %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% and an antibody-cytokine grafted template as described herein. It retains preferential agonist activity of the protein and is available under GenBank accession number NP_00057 7. SEQ ID NO: 1 is the human IL2 cDNA sequence. The human IL2 nucleic acid encoding the IL2 protein as shown is A nucleic acid sequence identical to the nucleic acid sequence of SEQ ID NO: 1, which has at least about 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, 99%, or 100% sequence identity, and It was published under accession number NM_000586.

[0086] The term "antibody cytokine grafted protein" or "antibody cytokine graft" or "Grafted" means that at least one cytokine is directly incorporated into the CDR of the antibody. Cytokines have HCDR1, HCDR2, and HCDR3. It can be loaded into HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3. Cytokines are composed of HCDR1, HCDR2, HCDR3, LCDR1, and LCDR2. or into LCDR3, towards the N-terminal sequence of the CDR, or towards the C-terminal sequence of the CDR The cytokine incorporated into the CDR can be used to bind to the original target protein. The specific binding of the antibody moiety to the antibody may be disrupted, or the antibody-cytokine grafted tag may be The protein may retain its specific binding to its target protein. 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, I FN-β, IFN-γ, GM-CSF, MIP-1α, MIP-1β, TGF-β, TN F-α, and TNF-β. The antibody can be grafted onto a specific CDR and another different cytokine can be grafted onto the other "arm" of the antibody. For example, IL2 can be grafted onto one of the "arms" of an antibody. IL-7 is grafted onto the HCDR1 of the antibody cytokine grafted protein. When grafted onto LCDR1 in the other "arm" of the protein, a bifunctional antibody cytokine graft is generated. It is possible to produce fused proteins.

[0087] The term "isolated," when applied to a nucleic acid or protein, refers to the nucleic acid or protein. This means that it is essentially free of other cellular components with which it is naturally associated. is preferably in a homogeneous state. This may be either dry or in an aqueous solution. Purity and homogeneity is typically analyzed by techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. Determined using analytical chemistry techniques. Proteins that are the predominant species present in a preparation are substantially Specifically, an isolated gene is a gene that is specifically purified from the genome of interest and is adjacent to the gene. Separate from open reading frames encoding other proteins. "Purified" means that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In particular, this means that the nucleic acid or protein is at least 85% pure, more preferably Preferably at least 95% pure, and most preferably at least 99% pure. means.

[0088] The term "nucleic acid" or "polynucleotide" refers to a nucleic acid molecule in either single-stranded or double-stranded form. It refers to oxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers. Unless otherwise limited, the term also includes any nucleic acid that has similar binding properties to the reference nucleic acid and that is naturally occurring. Nucleic acids containing known analogues of natural nucleotides that are metabolized in the same way as natural nucleotides are Unless otherwise indicated, a particular nucleic acid sequence also refers only to the sequence explicitly indicated. and its conservatively modified variants (e.g., degenerate codon substitutions), alleles, or alleles. Solologs, SNPs, and complementary sequences are also implicitly included. Specifically, degenerate codon substitutions are The third position of one or more selected (or all) codons may contain mixed bases and / or deoxynucleotides. This can be achieved by creating sequences in which the α-amino acid residues are substituted with α-amino acid residues (Batzer et al. al.,Nucleic Acid Res.19:5081(1991);Ohts uka et al.,J.Biol.Chem.260:2605-2608(198 5); and Rossolini et al., Mol. Cell. Probes 8: 91-98(1994)).

[0089] The terms "polypeptide," "peptide," and "protein" are used herein to refer to a compound having an amino acid sequence. These terms are used interchangeably to refer to a polymer of one or more amino acid residues. amino acid polymers that are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as and applies to naturally occurring and non-naturally occurring amino acid polymers. do.

[0090] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acids that function similarly to natural amino acids. Naturally occurring amino acids are those amino acids that are encoded by the genetic code. as well as amino acids that are later modified, e.g., hydroxyproline, γ -carboxyglutamic acid, and O-phosphoserine. Compounds that have the same basic chemical structure as carboxylic acids, i.e., hydrogen, carboxyl groups, amino groups, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl This refers to the α-carbon bonded to the methylsulfonium. Such analogs are also available with modified R groups ( norleucine) or a modified peptide backbone but with the same groups as natural amino acids. Amino acid mimetics are compounds that have a structure that differs from the general chemical structure of an amino acid. It refers to compounds that have the same structure as natural amino acids but function similarly to natural amino acids.

[0091] "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to nucleic acid sequences, conservatively modified variants are those that contain identical or essentially identical amino acids. a nucleic acid encoding an amino acid sequence, or, if the nucleic acid does not encode an amino acid sequence, an essentially identical Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids are possible for any given gene. For example, the codons GCA, GCC, GCG, and GCU all encode amino acids. Therefore, alanine is present at all positions specified by the codon. In this position, a codon may be substituted with the corresponding codon as described without altering the encoded polypeptide. Such nucleic acid changes are "silent mutations", which , a type of conservatively modified mutation. Every nucleic acid sequence also describes every possible silent variation of the nucleic acid. Each codon in the The codons (except for TGG, which is the only codon for the nucleotide sequence) are such that they yield functionally identical molecules. It will be appreciated that the nucleic acid encoding the polypeptide may be modified in any manner. Each silent variation of is implicit in each described sequence.

[0092] For amino acid sequences, a single amino acid or a small portion of the amino acids in the coding sequence to the nucleic acid, peptide, polypeptide, or protein sequence to be changed, added, or deleted. Each substitution, deletion, or addition to an amino acid is performed by replacing the amino acid with a chemically similar amino acid. Those skilled in the art will recognize that "conservatively modified variants" result in substitutions. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants include polymorphic variants, interspecies homologs, and Each of the following eight groups is in addition to, but does not exclude, the following alleles: contains amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G) 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine 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, e.g., Creighton, Proteins (1984) (See

[0093] "Percentage of sequence identity" is the result of comparing two optimally aligned sequences. The polynucleotide sequences are determined by comparing the polynucleotide sequences across the The part within the window does not contain additions or deletions for optimal alignment of the two sequences. Contains additions or deletions (i.e., gaps) when compared to a reference sequence (e.g., polypeptide) that does not contain The percentages are the positions where the same nucleic acid base or amino acid residue appears in both sequences. Determine the number of matching positions by determining the number of matching positions, and then compare the number of matching positions in the comparison window. Divide by the total number of positions and multiply the result by 100 to get the percentage sequence identity. It is calculated as follows.

[0094] "Identical" or "percent identical" in the context of two or more nucleic acid or polypeptide sequences The term "identity" refers to the degree to which two or more sequences or subsequences are the same sequence. Alignment and inspection by hand, using one of the sequence comparison algorithms The two sequences have the greatest similarity over a comparison window, or indicated region, as determined by inspection. Amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence. The target sequence has a specified percentage of the target sequence (i.e., over a specified region of the reference sequence, or If not specified, at least 85%, 90%, 91%, or 92% across the entire sequence , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Two sequences are "substantially identical" if they have the same or similar structure (e.g., nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, The respective polypeptides or polynucleotides (e.g., SEQ ID NO: 19, SEQ ID NO: 35) , SEQ ID NO: 51, or SEQ ID NO: 67. The identity is at least 1 / 2 of the reference sequence. over a region of about 15, 25 or 50 nucleotides in length, or more preferably 100 to 5 over a region of 0 or 1000 nucleotides or more in length, or over the entire length With respect to amino acid sequences, identity or substantial identity means that there are at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length The amino acid sequence may be a region of the amino acid sequence, or the entire length of the amino acid sequence. For example, for amino acid sequences of 20 amino acids or less, one or two amino acid residues may be Substantial identity exists when conservative substitutions are made according to the definition of conservative substitutions. .

[0095] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer. A subsequence is specified, and, if necessary, sequence algorithm program parameters are specified. Default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then performs a sequence comparison on the reference sequences based on the program parameters. The percent sequence identity for the test sequence compared to the alignment is calculated.

[0096] As used herein, a "comparison window" refers to the region over which two sequences are optimally aligned. After this, the sequence can be compared to a reference sequence at the same number of consecutive positions, typically 20-600. from about 50 to about 200, more usually from about 100 to about 150, Contains reference to any one segment of the number of positions. Alignment of sequences for comparison Methods for alignment are well known in the art. For example, Smith and Waterman, (1970) Adv.Ap Needleman by the partial homology algorithm in pl.Math.2:482c and Wunsch, (1970) J. Mol. Biol. 48:443 The alignment algorithm, Pearson and Lipman, (198 8) Proc. Nat'l. Acad. Sci. USA 85:2444 similarity search method Therefore, computerized implementations of these algorithms (Wisconsin Ge genetics Software Package,Genetics Compute r Group, 575 Science Dr., Madison, WI P, BESTFIT, FASTA, and TFASTA) or by manual alignment. Measurement and visual inspection (e.g., Ausubel et al., Current Practice See Protocols in Molecular Biology (1995 Supplement) )

[0097] Two examples of suitable algorithms for determining percent sequence identity and sequence similarity are , BLAST, and BLAST 2.0 algorithms, which are Alts chul et al(1977)Nuc.Acids Res.25:3389-34 02,; and Altschul et al (1990) J.Mol.Biol.215 :403-410. Software for performing BLAST analysis is available from N ational Center for Biotechnology Informa This algorithm is published by the National Institute of Standards and Technology (NITS) in 2009. If it is aligned with a word, it will meet some positive threshold score. Meets or meets the e-valued threshold score Therefore, by identifying short word lengths W in the query sequence, high-scoring sequence pairs (HS T is called the neighborhood word score threshold (Altsch These initial neighborhood word hits are then analyzed to find the loci containing them. They act as seeds to start searches to find new HSPs. extended in both directions along each sequence for as far as the cumulative alignment score can be increased. The cumulative score is calculated based on the parameter M (the number of matching residues per pair) for nucleotide sequences. reward score for mismatching residues; always >0) and N (penalty score for mismatching residues; always <0) For amino acid sequences, the scoring matrix is ​​used to calculate the cumulative score. The extension of the word hit in each direction stops when If the cumulative alignment score decreases by an amount X from its maximum achieved value; or if the cumulative score becomes zero or less due to the accumulation of multiple negatively scoring residue alignments. or when the end of either sequence is reached. BLAST Algorithm Parameters W, T, and X determine the sensitivity and speed of the alignment. BLASTN program (For nucleotide sequences) defaults to a word length (W) of 11, a period of 10, For the amino acid sequence, BL The ASTP program defaults to a word length of 3, an expectation (E) of 10, and and 50 BLOSUM62 scoring matrices (Henikoff and He nikoff,(1989)Proc.Natl.Acad.Sci.USA 89:1 (See 0915) Alignment s (B), Expectation of 10 (E), M = 5, N = -4, and and a comparison of both strands is used.

[0098] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., K arlin and Altschul(1993)Proc.Natl.Acad.S ci.USA 90:5873-5787, see the BLAST algorithm. One measure of similarity provided is the degree to which a match between two nucleotide or amino acid sequences is by chance. The minimum sum probability (P(N)) provides an indication of the probability that the test nucleic acid and the reference nucleic acid will occur. The minimum sum probability in comparison with nucleic acids is less than about 0.2, more preferably less than about 0.01. Most preferably, a nucleic acid is considered similar to a reference sequence if its nucleotide sequence is less than about 0.001. can be.

[0099] Other indicators that two nucleic acid sequences or polypeptides are substantially identical include the use of the nucleotide sequence ... Thus, the polypeptide encoded by the first nucleic acid is a polypeptide encoded by the second nucleic acid. The main objective of the present invention is to immunologically cross-react with antibodies raised against the polypeptide. For example, where two peptides differ only by conservative substitutions, the polypeptides are typically The nucleic acid sequence of the polypeptide is substantially identical to the second polypeptide. Another indicator is that the two molecules or their complements react under stringent conditions, as described below. The term "hybridize" refers to the fact that two nucleic acid sequences are substantially identical. A further indication is that the same primers can be used to amplify the sequence.

[0100] The term "linked" refers to how the binding domains are linked within the antibody-cytokine grafted proteins of the invention. When used in the context of describing how regions are connected, Many binding domains are frequently covalently linked (e.g. They are linked by chemical bonds, such as peptide bonds or disulfide bonds, or non-covalent bonds. This can be done by direct binding (i.e., no linker between the two binding regions) or indirect binding ( i.e., with the aid of at least one linker molecule between two or more binding regions It could be either.

[0101] The terms "subject," "patient," and "individual" are used interchangeably to refer to a mammal, e.g., a human or non-human. Primate mammals. Mammals also include laboratory mammals, such as mice, rats, and rabbits. In some embodiments, the mammal is an agricultural mammal ( horses, sheep, cattle, pigs, camels) or domestic mammals (e.g., dogs, rats, It may also be (ko).

[0102] As used herein, "treating" or "treating" any disease or disorder means The term "treatment" or "treatment" refers, in one embodiment, to improving (or ameliorating) a disease or disorder. That is, slowing or halting the development of the disease or at least one of its clinical symptoms. In another embodiment, "treat," "treating," or "alleviating" refers to "Treatment" refers to the measurement of at least one physical parameter, including those not perceptible by the patient. In yet another embodiment, "treating" or "treating" refers to reducing or ameliorating the symptoms of "Treatment" or "treatment" means the physical treatment of a disease or disorder (e.g., relief of discernible symptoms). regulating physiologically (e.g., stabilizing a physical parameter), or both In yet another embodiment, "treat," "treating," or "treatment" refers to " refers to preventing or delaying the onset or development or progression of a disease or disorder.

[0103] The term "therapeutically acceptable amount" or "therapeutically effective dose" refers to a dose that is sufficient to achieve the desired result (or i.e., reduction of inflammation, inhibition of pain, prevention of inflammation, inhibition or prevention of the inflammatory response In certain embodiments, a therapeutically acceptable amount is desirably Therapeutically acceptable doses are those that are administered at low doses initially. by administering a small amount and then gradually increasing the dose until the desired effect is achieved. "Prophylactically Effective Doses" of IL2 Antibody Cytokine Grafted Proteins That Can Be Determined and "therapeutically effective dose" refers to a dose that effectively treats symptoms of a disease, including symptoms associated with immune-related disorders. It may prevent the onset or result in a reduction in its severity.

[0104] The term "co-administered" refers to the simultaneous presence of two (or more) active agents in an individual. Co-administered active agents can be delivered simultaneously or sequentially.

[0105] As used herein, the phrase "consisting essentially of" "essentially of" refers to the genus or species of the pharmaceutically active agent included in the method or composition; and any inert carrier or excipient for the intended purpose of the method or composition. In some embodiments, the phrase "consisting essentially of" is used. "essentially of") is one other than the IL2 antibody cytokine grafted protein In some embodiments, the phrase " "consisting essentially of" means Further additional active agents other than the L2 antibody cytokine grafted protein and second co- Explicitly excludes the inclusion of administered medications.

[0106] The terms "a," "an," and "the" refer to plural unless the context clearly dictates otherwise. Includes referent. [Brief explanation of the drawings]

[0107] [Figure 1] 1 is a table summarizing exemplary IL2 antibody cytokine grafted proteins and their activity on CD8 T effector cells. [Figure 2] 1 shows that IgG.IL2R67A.H1 has a longer half-life than Proleukin®. As shown in the graph, IgG.IL2R67A.H1 has a half-life of 12-14 hours, while Proleukin® has a T1 / 2 of less than 4 hours and cannot be shown on the graph. [Figure 3A] Figures 3A-C demonstrate that IgG.IL2R67A.H1 expands CD8+ T effector cells more effectively and with less toxicity than Proleukin® or IL2-Fc fusion molecules in C57BL / 6 mice at 100 μg equivalent doses on days 4, 8, and 11. [Figure 3B] (As mentioned above.) [Figure 3C] (As mentioned above.) [Figure 3D] Figures 3D-F: Demonstrates that IgG.IL2R67A.H1 expands CD8+ T effector cells more effectively and with less toxicity than Proleukin® or IL2-Fc fusion molecules in C57BL / 6 mice at 500 μg equivalent doses on days 4, 8, and 11. [Figure 3E] (As mentioned above.) [Figure 3F] (As mentioned above.) [Figure 4A]Figure 1 shows that IgG.IL2R67A.H1 selectively expands CD8 T effectors and is better tolerated than Proleukin® in NOD mice. [Figure 4B] 1 shows a table illustrating the increased activity of IgG.IL2R67A.H1 and IgG.IL2F71A.H1 against CD8 T effectors in NOD mice. [Figure 5] 1 shows a graph of the single agent efficacy of IgG.IL2R67A.H1 in the CT26 tumor model. [Figure 6] Data are presented for IgG.IL2R67A.H1 either as a single agent or in combination with antibodies in the B16 melanoma mouse model. The graph shows that IgG.IL2R67A.H1 in combination with the anti-TRP1 antibody TA99 is more effective than TA99 alone, the IL2-Fc fusion molecule alone, and TA99 + IL2-Fc fusion. Synergy was observed with 100 and 500 μg doses of TA99 and IgG.IL2R67A.H1. [Figure 7] 1 shows a graph containing values ​​monitoring pSTAT5 activity in a panel of human cells comparing IgG.IL2R67A.H1 and IgG.IL2F71A.H1 to Proleukin®. [Figure 8] This graph shows ELISA data demonstrating that when IL2 is grafted onto CDRH1 of an anti-RSV antibody (IgG.IL2R67A.H1), RSV binding is maintained. However, when IL2 is grafted onto CDRL3 or CDRH3, binding to RSV is reduced. When IL2 is grafted onto a different antibody scaffold (Xolair), there is no binding to RSV. DETAILED DESCRIPTION OF THE INVENTION

[0108] Antibody-cytokine-grafted proteins targeting the IL2 low-affinity receptor The present invention includes an IL2 molecule grafted into the complementarity determining region (CDR) of an antibody. Protein constructs are provided. The antibody-cytokine grafted proteins of the present disclosure are human. exhibiting properties suitable for use in patients, e.g., immunostimulatory properties similar to natural or recombinant human IL2 However, negative effects, such as stimulation of Treg cells, are reduced. Other activities and characteristics are also demonstrated throughout this specification. Therapeutic protease activity compared to IL2 and modified IL2 therapeutic agents, such as Proleukin® The file includes an improved antibody-cytokine grafted protein and the antibody site provided. Methods of using tokine-grafted proteins in cancer treatment are provided.

[0109] Thus, the present disclosure provides agonists of the IL2 low affinity receptor with selective activity profiles. The present invention provides an antibody-cytokine grafted protein that is a target of immunoglobulin heavy chain sequence. and an immunoglobulin light chain sequence. Each immunoglobulin heavy chain sequence comprises a heavy chain variable region (VH) and a heavy chain constant region (CH), Here, the heavy chain constant region consists of CH1, CH2, and CH3 constant regions. The light chain sequence includes a light chain variable region (VL) and a light chain constant region (CL). In the grafted protein, the IL2 molecule is composed of the complementarity-determining regions (CDRs) of VH or VL. is taken in.

[0110] In some embodiments, the antibody-cytokine grafted protein comprises a heavy chain CDR. In a specific embodiment, the IL2 molecule comprises a heavy chain complementarity determining region (CDR) of In a specific embodiment, the IL2 molecule is incorporated into heavy chain complementarity determining region 1 (HCDR1). In certain embodiments, the IL2 molecule is incorporated into heavy chain complementarity region 2 (HCDR2). It is incorporated into the HCDR3.

[0111] In some embodiments, the antibody-cytokine grafted protein comprises a nucleotide sequence encoding a nucleotide sequence encoding a nucleotide sequence of ... In a specific embodiment, the IL2 molecule comprises a light chain complementarity determining region (CDR) of the IL2 molecule. In a specific embodiment, the IL2 molecule is incorporated into the light chain complementarity determining region 1 (LCDR1). In a specific embodiment, the IL2 molecule is incorporated into light chain complementarity region 2 (LCDR2). It is captured in decision region 3 (LCDR3).

[0112] In some embodiments, the grafted antibody cytokine is incorporated into the CDR. The insertion comprises an IL2 sequence inserted into the CDR sequence. N-terminal region or its vicinity, the central region of the CDR, or the C-terminal region of the CDR or its vicinity In another embodiment, the antibody cytokine grafting is carried out in the CDR. The IL2 sequence frameshifts the CDR sequence. In another embodiment, the grafted antibody cytokine has no CDRs. The IL2 molecule is incorporated into the IL2 gene, whereby the IL2 sequence is substituted for all or part of the CDR sequence. The replacement may be in the N-terminal region of the CDR, the central region of the CDR, or the C-terminal region of the CDR. The substitution may be as little as one or two amino acids in the CDR sequence. It may be a single CDR or the entire CDR sequence.

[0113] In some embodiments, the IL2 molecule is grafted directly onto the CDR without a peptide linker. and there are no additional amino acids between the CDR sequences and the IL2 sequence.

[0114] In some embodiments, the antibody-cytokine grafted protein is an IgG class antibody heavy chain. In certain embodiments, the IgG heavy chain is an immunoglobulin heavy chain, such as IgG1, Ig It is either G2 or IgG4 subclass.

[0115] In some embodiments, the antibody-cytokine grafted protein is a compound having known clinical utility. The immunoglobulin sequences comprise heavy and light chain immunoglobulin sequences selected from those used in In certain embodiments, the antibody-cytokine grafted protein is a humanized sequence. In another specific embodiment, the antibody comprises a heavy and a light chain immunoglobulin sequence. The grafted protein contains heavy and light chain immunoglobulin sequences that are human sequences.

[0116] In some embodiments, the antibody-cytokine grafted protein is a germline immune graft. The invention includes heavy and light chain immunoglobulin sequences selected from the group consisting of:

[0117] In some embodiments, the antibody-cytokine grafted protein inhibits binding of an IL2 molecule. A heavy chain having a binding specificity of an immunoglobulin variable domain for a target different from the specificity of the heavy chain and In some embodiments, the immunoglobulin variable domain comprises a light chain immunoglobulin sequence. The binding specificity to its target was 10%, 20% in the presence of grafted cytokines. %, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99 In certain embodiments, the retained binding specificity is greater than that of the non-human target. In certain embodiments, the binding specificity that is retained is directed against a virus, e.g. For example, for RSV. In another embodiment, the binding specificity is used in conjunction with IL2 therapy. In certain embodiments, immunoglobulins are directed against human targets that have therapeutic utility. Targeting the binding specificity of IL-2 confers additional therapeutic benefit to the IL-2 component. In certain embodiments, the binding specificity of the immunoglobulin for its target is IL2. Provides synergistic activity.

[0118] In yet another embodiment, the binding specificity of the immunoglobulin is determined by grafting an IL2 molecule. By doing so, the rate can be increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, Decreased by 90%, 95%, 98%, 99%, or 100%.

[0119] The provided antibody-cytokine grafted proteins include those having a VH or VL complementarity determining region. In some embodiments, the IL2 sequence comprises an IL2 molecule grafted into the CDRs (CDRs). has at least 85%, 89%, 90%, 91%, 92%, or 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity In some embodiments, the IL2 molecule comprises the sequence of SEQ ID NO: 4. In one embodiment, the IL2 molecule consists of the sequence of SEQ ID NO: 4. The grafted protein is a complementarity determination of VH or VL of the antibody cytokine grafted protein. In some embodiments, the IL2 molecule comprises an IL2 sequence incorporated into the CDRs. The sequence is at least 85%, 90%, 91%, 92%, 93% identical to the amino acid sequence of SEQ ID NO:4. , 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity In some embodiments, the IL2 molecule comprises the sequence of SEQ ID NO: 4. In this case, the IL2 molecule consists of the sequence of SEQ ID NO:4.

[0120] The provided antibody-cytokine grafted proteins include those having a VH or VL complementarity determining region. In some embodiments, the IL2 sequence comprises an IL2 molecule grafted into the CDRs (CDRs). has at least 85%, 89%, 90%, 91%, 92%, or 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity In some embodiments, the IL2 molecule comprises the sequence of SEQ ID NO:6. In one embodiment, the IL2 molecule consists of the sequence of SEQ ID NO:6.

[0121] In some embodiments, the antibody-cytokine grafted protein comprises human IL2, Superior to recombinant human IL2, Proleukin®, or IL2 fused to Fc The antibody-cytokine grafted protein confers immunomodulatory properties. , compared with recombinant human IL2, Proleukin® or IL2 fused to Fc When administered intracellularly, it confers increased activity on CD8 T effector cells, while inhibiting Treg activity. This results in a decrease in

[0122] In some embodiments, the antibody-cytokine grafted protein comprises a modified effector In certain embodiments, the modified immunoglobulin IgG has a modified Fc that confers a specific function. The modified Fc region is D265A, P329A, P329G, N297A, L234A and L235A. The immunoglobulin heavy chain consists of D265A, P329A, P329G, N297A, and D265A / P329A, D265A / N297A, L234 / L235A, P329A / L234A / L235A, and P329G / L234A / L235A The gene may contain a mutation or combination of mutations that confers reduced vector function.

[0123] In some embodiments, the antibody-cytokine grafted protein comprises (i) SEQ ID NO: 19 heavy chain variable regions and at least 85%, 89%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity and (ii) a heavy chain variable region having at least 85%, 89%, or more of the light chain variable region of SEQ ID NO: 35. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, or 100% amino acid sequence identity. is an IgG class selected from IgG1, IgG2, or IgG4. In the form, the immunoglobulin optionally contains D265A, P329A, P329G, N 297A, D265A / P329A, D265A / N297A, L234 / L235A, P329A / L234A / L235A and P329G / L234A / L235A A mutation or combination of mutations that confers reduced effector function selected from the following: This includes.

[0124] In some embodiments, the antibody-cytokine grafted protein comprises (i) SEQ ID NO: 51 heavy chain variable regions and at least 85%, 89%, 90%, 91%, 92%, 93%, 9 4%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity and (ii) a heavy chain variable region having at least 85%, 89%, or more of the light chain variable region of SEQ ID NO: 67. %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 %, or 100% amino acid sequence identity. is an IgG class selected from IgG1, IgG2, or IgG4. In the form, the immunoglobulin optionally contains D265A, P329A, P329G, N 297A, D265A / P329A, D265A / N297A, L234 / L235A, P329A / L234A / L235A and P329G / L234A / L235A A mutation or combination of mutations that confers reduced effector function selected from the following: This includes.

[0125] Engineered and modified antibody-cytokine grafted proteins In certain embodiments, the antibody cytokine grafted construct comprises an IL2 sequence linked to an immunoglobulin. Both heavy and light chain immunoglobulins are created by grafting the CDR regions of the phospholipid scaffold. Immunoglobulin chains are produced to produce the final antibody-grafted protein. Kine-grafted proteins confer favorable therapeutic activity to CD8 T effector cells. The antibody-cytokine grafted protein may be derived from natural or recombinant human IL2 (rhIL2 or compared to IL2 fused to Proleukin® or Fc. The activity is reduced.

[0126] To engineer antibody-cytokine grafted proteins, specific muteins containing I The L2 sequence (SEQ ID NO: 4 or SEQ ID NO: 6) is inserted into the CDR loop of the immunoglobulin chain scaffold protein. The grafted constructs are inserted into various known grafts that are utilized in clinical settings. Immunoglobulin sequences, known immunoglobulin sequences currently in drug discovery and / or clinical development, human Prepared using both germline antibody sequences as well as novel antibody immunoglobulin chain sequences Constructs may be prepared using standard recombinant DNA techniques that utilize the relevant sequences. IL-1 in an exemplary scaffold called GFTX3b is produced using molecular biology methods. The sequences of 2 are shown in Table 2. Insertion points were selected based on available structural or homology model data. The insertion point was chosen to be the midpoint of the CDR loop, however, the insertion point was chosen to be either N-terminal or It can be adjusted towards the C-terminal end.

[0127] Thus, the present disclosure provides methods for the preparation of xenogenic antibody proteins or polypeptides such that grafted proteins are produced. Specific for the low affinity IL2 receptor containing the IL2 protein recombinantly inserted into the polypeptide In particular, the present disclosure provides an antibody or fragment thereof that specifically binds to the antibody or fragment thereof described herein. An antibody or antigen-binding fragment of an antibody or any other relevant scaffold antibody polypeptide (e.g. , complete antibody immunoglobulin protein, Fab fragment, Fc fragment, Fv fragment, F(ab)2 fragments, VH domains, VH CDRs, VL domains, VL CDRs, etc.) and heterologous cytokines. Grafted proteins containing an IL-1 protein, polypeptide, or peptide, such as IL2. The protein, polypeptide, or peptide is fused or combined with an antibody or antibody fragment. Methods for conjugating ribonucleotides are known in the art. See, for example, U.S. Pat. Specification No. 336,603, Specification No. 5,622,929, No. 5,359,046 The specification, the specification of the same patents 5,349,053, the specification of the same patents 5,447,851, and the specification of the same patents 5,349,053, ... EP 5,112,946; EP 307,434 and EP 367,16 No. 6 specification; International Publication Nos. 96 / 04388 and 91 / 06570 Ashkenazi et al.,1991,Proc.Natl.Ac ad.Sci.USA 88:10535-10539;Zheng et al.,1 995, J. Immunol. 154:5590-5600; and Vil et al. ,1992,Proc.Natl.Acad.Sci.USA 89:11337-11 See 341. In addition, antibody-cytokine grafted proteins have been shown to be useful in gene shutting. shuffling, motif shuffling, exon shuffling, and / or codon shuffling It can be created by the technique of flinging (collectively known as "DNA shuffling"). DNA shuffling can be used to prepare grafted protein constructs and / or alters the activity of the antibody or fragment thereof (e.g., higher affinity and lower dissociation rate). Generally, U.S. Patent No. 5,605, ,793 specification, 5,811,238 specification, 5,830,721 specification , 5,834,252, and 5,837,458; Patte n et al.,1997,Curr.Opinion Biotechnol.8: 724-33;Harayama,1998,Trends Biotechnol.1 6(2):76-82;Hansson,et al.,1999,J.Mol.Bio l.287:265-76; and Lorenzo and Blasco, 1998, B Biotechniques 24(2):308-313. or the encoded antibody or fragment thereof may be subjected to error-prone PCR, By subjecting the cells to random mutagenesis by random nucleotide insertion or other methods. The antibody or its fragment that specifically binds to the antigen protein of interest can be The polynucleotide encoding the antibody cytokine gene as provided herein. For the preparation of a homogenized protein, one or more heterologous cytokine molecules, e.g., one or more of IL2, The above components, motifs, sections, parts, domains, fragments, etc. may be recombined.

[0128] The antibody Fab contains six CDs that can serve as potential insertion sites for cytokine proteins. R-loops, three in the light chain (CDRL1, CDRL2, CDRL3) and three in the heavy chain (CDR H1, CDRH2, CDRH3). Any one or more CDR loops may contain a cytokine. The decision to insert a CDR loop takes into account structural and functional considerations. Because the size and conformation of CDRs vary greatly between different antibodies, the optimal CDRs for insertion are: This can be determined experimentally for each specific antibody / protein combination. Since the tokine protein is inserted into the CDR loop, as discussed in Example 1, This may impose further constraints on the structure of cytokine proteins.

[0129] CDRs of immunoglobulin chains are well known in the art, including those described herein. For example, the CDRs are determined by the well-known numbering system: (1) Kabat et al. et al. (1991), “Sequences of Proteins of Im munological Interest,”5th Ed.Public Heal th Service, National Institutes of Health ,Bethesda, MD ("Kabat" numbering scheme), NIH publicat ion No. 91-3242; and (2) using the numbering system described in Chothia. It is identified and defined by Al-Lazikani et al., (19 97) “Standard conformations for the canon ical structures of immunoglobulins,”J.Mo See I. Biol. 273:927-948. For CDR amino acid sequences, the desired specific binding and / or agonist activity may still be retained. However, one or two conservative amino acid residue substitutions may be tolerated.

[0130] The antibody-cytokine grafted protein may further be modified to have specificity from the starting antibody-grafted protein. A method for engineering modified antibody-cytokine grafted proteins with altered properties. As the source material, the CDRs and / or VH and / or VL shown herein (e.g., Table 2) Alternatively, modified antibodies can be prepared using antibodies having one or more of the sequences. Any known antibody sequence can be used as a scaffold for engineering cytokine-grafted proteins. For example, any known clinically used antibody may be used as an antibody-grafted protein. Known antibodies and corresponding immunoglobulins may be used as starting material scaffolds for the preparation of proteins. Examples of the purine sequence include palivizumab, alirocumab, mepolizumab, and necitumab. nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, penicillin-resistant Staphylococcus aureus Brolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab tuzumab, raxibacumab, pertuzumab, belimumab, ipilimumab, denosumab, Silizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certoliz Mab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, bevacizumab Basizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritum Mabtiuxetan, adalimumab, alemtuzumab, gemtuzumab, infliximab, Basiliximab, daclizumab, rituximab, abciximab, muromonab, or any of these Known antibody and immunoglobulin sequences also include germline modifications. Framework sequences can also be derived from publicly available antibody sequences, including germline antibody gene sequences. DNA sequences can be obtained from public DNA databases or published references. For example, For the germline DNA sequences of heavy and light chain variable region genes, refer to "VBase Human Germline sequence database and Kabat, EA, et al., 1991 Se Quences of Proteins of Immunological Int erest,Fifth Edition,USDepartment of He alth and Human Services,NIH Publication No.91-3242;Tomlinson,IM,et al.,1992 J. fol. Biol. 227:776-798; and Cox, J.P. Let al., See 1994 Eur. J Immunol. 24:827-836. In still other instances, antibodies from other known entities may be in early drug discovery and / or drug development. The antibody and corresponding immunoglobulin sequence are similarly modified antibody cytokine grafted proteins. It may be suitable as a starting material for engineering quality.

[0131] The resulting polypeptide is adapted for uptake of cytokines (e.g., IL2). A wide variety of antibody / immunoglobulin frameworks, as long as they contain at least one binding domain Such frameworks or scaffolds may be prepared from human immunoglobulins. or fragments thereof, and may be derived from other animal species, preferably humanized and / or immunoglobulins with human aspects. Novel antibodies, frameworks, scaffolds and fragments are continually discovered and developed by those skilled in the art.

[0132] Antibodies can be produced using methods known in the art. Any technique known in the art can be used to prepare a cloned antibody (e.g., For example, Kohler & Milstein, Nature 256:495-497 1975);Kozbor et al., Immunology Today 4:7 2(1983);Cole et al., Monoclonal Antibodies s and Cancer Therapy,pp.77-96.Alan R.Lis s, Inc. 1985). Techniques for producing single chain antibodies (see U.S. Patent No. 4,946,7 No. 78) is a method for producing antibodies for use in antibody-cytokine grafted proteins. It can also be adapted to other animals such as transgenic mice or other mammals. The organisms may be used to express and identify primatized or humanized antibodies or human antibodies. Alternatively, antibody-cytokine grafted proteins can be generated using phage display technology. Identifying antibodies and heteromeric Fab fragments that specifically bind to an antigen of choice for use in (See, e.g., McCafferty et al., supra; Marks et al., supra). See t al., Biotechnology, 10:779-783, (1992). (This refers to

[0133] Methods for primatizing or humanizing non-human antibodies are well known in the art. Therefore, a primatized or humanized antibody has one or more antibodies introduced into it from a non-primate or non-human source. These non-primate or non-human amino acid residues are often transferred This is typically referred to as the import residue of the imported variable domain. (import variable domain). Humanization is essentially According to the method of Winter and coworkers (e.g., Jones et al., Natl. ure 321:522-525(1986);Riechmann et al.,N ature 332:323-327(1988);Verhoeyen et al. ,Science 239:1534-1536(1988) and Presta, Cur r. Op. Struct. Biol. 2:593-596 (1992)), This can be done by substituting rodent CDR or CDR sequences for the corresponding sequences of a human antibody. Therefore, such humanized antibodies are chimeric antibodies (see U.S. Pat. No. 4,816,567). (see text), in which substantially less than intact human variable domains are fused to counterparts from non-human species. In practice, primatized or humanized antibodies typically Primate or human antibodies, including some complementarity determining region ("CDR") residues and optionally Some framework ("FR") residues confer binding specificity and are therefore These are substituted by residues from analogous sites in a human antibody (rodent antibody).

[0134] Alternatively or additionally, the antibody may be modified to have the same or better binding characteristics as the non-human antibody. In one embodiment, a non-human antibody variable region is replaced with a human variable region in the antibody while providing similar binding characteristics. In vivo methods may be used to convert non-human antibodies into engineered human antibodies. For example, Patent Application Publication No. 20050008625, U.S. Patent Application Publication No. 2005 / 025 See, e.g., US Pat. No. 5,552, 5552. Alternatively, human V segments can be synthesized by sequential cassette replacement. A library can be created in which initially only a portion of the reference antibody V-segments are humanized. then in the context of the remaining reference antibody amino acid sequence Identified human "cassettes" that support binding were recombined in a second library screen. These are used to generate fully human V segments (U.S. Patent Application Publication No. 2006 / 01340 (See specification No. 98).

[0135] Various antibody or antigen binding proteins for use in preparing antibody-cytokine grafted proteins Antibody fragments may be produced by enzymatic or chemical modification of intact antibodies, or They can be synthesized de novo using recombinant DNA methods (e.g., single-chain Fv) or can be synthesized by phage These can be identified using display libraries (e.g., McCafferty (See, for example, Wang et al., Nature 348:552-554, 1990). For example, minibodies have been described in the art, e.g., by Vaughan and Solla zzo,Comb Chem High Throughput Screen.4:4 Bispecific antibodies can be produced using the methods described in J. Immunol. 17-30 2001. These may be produced by a variety of methods, including grafting hybridomas or linking Fab' fragments. For example, Songsivilai & Lachmann, Cli n.Exp.Immunol.79:315-321(1990);Kostelny See, e.g., et al., J. Immunol. 148, 1547-1553 (1992). Single-chain antibodies can be generated using phage display libraries or ribosome display libraries. Such libraries can be identified using gene shuffling libraries. can be constructed from synthetic, semi-synthetic or natural and immunocompetent sources. In preparing antibody-cytokine grafted protein constructs as provided herein may utilize the immunoglobulin sequence of choice.

[0136] As used in the present disclosure, an antibody, antigen-binding molecule, or antibody-cytokine-grafted molecule is a bispecific antibody. Bispecific or bifunctional antibodies further include antibodies that contain two different heavy / light chain pairs and An artificial hybrid antibody has two different binding sites. Other antigen-binding fragments or antibodies The part is a bivalent scFv (diabody), an antibody molecule that recognizes two different epitopes. Examples include bispecific scFv antibodies, single binding domains (dAbs), and minibodies that recognize Thus, the antibody-cytokine grafted proteins as provided herein In preparing the antibody construct, the immunoglobulin sequence of choice can be utilized.

[0137] Antibody fragments can be synthesized using antigen-binding fragments of antibodies, such as Fab fragments and scFv fragments, as building blocks. Tokine-grafted proteins can be constructed, optionally including multivalent forms. In some embodiments, such multivalent molecules comprise a constant region (e.g., Fc) of an antibody. .

[0138] The antibody-cytokine grafted protein may comprise one or both variable regions (i.e., V modifying one or more residues within the H and / or VL, e.g., within one or more CDR regions and such adapted VH and / or VL region sequences can be engineered using It can be used for grafting cytokines or for preparing cytokine grafts. Antibodies are primarily composed of amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). Therefore, the amino acid sequences within the CDRs vary widely among individual antibodies. CDR sequences are involved in most antibody-antigen interactions. , specific antibodies grafted onto framework sequences from different antibodies with different properties Mimicking the properties of specific antibodies by constructing expression vectors containing CDR sequences from It is possible to express recombinant antibodies (see, e.g., 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.,USA86:10029-1003 3; U.S. Patent No. 5,225,539 to Winter, and Queen et al. U.S. Patent Nos. 5,530,101; 5,585,089; (See US Pat. Nos. 5,693,762 and 6,180,370). In certain instances, the antigen-binding ability of an antibody can be improved by mutating residues within the framework regions. Maintaining or increasing the strength of the Patent No. 5,530,101; Patent No. 5,585,089; Patent No. 5,693 (See US Pat. Nos. 6,180,370 and 6,762).

[0139] In some embodiments, improving one or more binding characteristics (e.g., affinity) of an antibody of interest VH and / or VL CDR1, CDR2, and and / or mutation of amino acid residues within the CDR3 region, e.g., cytokine-grafted proteins This may be beneficial for optimizing the antigen binding of antibodies in the context of the protein. Natural mutagenesis or PCR-mediated mutagenesis may be performed to introduce one or more mutations. and their effect on antibody binding or other desired functional properties are described herein. In vitro or in vivo assays described herein and / or alternatives known in the art Conservative modifications can be introduced and evaluated in alternative or additional assays. The mutations may be amino acid substitutions, additions or deletions. Furthermore, typically, the CDR regions No more than 1, 2, 3, 4 or 5 residues within the region are changed.

[0140] The engineered antibody or antibody fragment may have additional modifications to framework residues within VH and / or VL, e.g. In some embodiments, such antibodies may be modified to improve their properties. Framework modifications are made to reduce the immunogenicity of antibodies. For example, one approach is the alteration of one or more framework residues to the corresponding germline sequence. Specifically, antibodies that have undergone somatic mutations may have sequences that differ from the germline sequence from which they are derived. Such residues may include framework residues that contribute to the formation of antibody framework sequences. The framework region sequences can be identified by comparing them with the germline sequences from which they are derived. Somatic mutations can be removed, e.g., by site-directed mutagenesis, to restore the original germline configuration. Additional framework modifications can be "backmutated" to the germline sequence. , by varying one or more residues in the framework regions, or even in one or more CDR regions. to remove T cell epitopes, thereby reducing the potential immunogenicity of the antibody. This technique is also called "deimmunization" and is described in U.S. Patent Application Publication No. 2002 / 0022990 by Carr et al. This is described in further detail in patent application 0030153043.

[0141] The constant region of the antibody or antibody fragment used to prepare the antibody-cytokine grafted protein The area may be of any type or subtype, as appropriate, and the subject to be treated by the present method species (e.g., humans, non-human primates, or other mammals, e.g., agricultural mammals (e.g., horses, sheep, cattle, pigs, camels), domestic animal mammals (e.g., dogs, cats) or geese The mammalian species may be selected from rodents (e.g., rats, mice, hamsters, and rabbits). In some embodiments, the antibody utilized in the antibody-cytokine grafted protein is a human Humanized or Humaneered® antibodies are engineered to produce In some embodiments, the antibody utilized in the antibody-cytokine grafted protein is human. In some embodiments, the antibody constant region isotype is IgG, e.g., I In certain embodiments, the constant region The antibody-cytokine grafted protein is of the IgG1 type. In some embodiments, the antibody-cytokine grafted protein comprises an IgG. In some embodiments, the antibody-cytokine-grafted protein comprises an IgG1 Fc. The protein contains IgG2 Fc.

[0142] In addition to, or instead of, modifications made within the framework or CDR regions, antibody sequences may be modified. The antibody or antibody fragment used to prepare the cytokine-grafted protein is typically an antibody one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antibody activity. The Fc region may be engineered to contain modifications to alter antigen-dependent cytotoxicity, etc. Furthermore, antibodies, antibody fragments thereof, or antibody-cytokine grafted proteins are also useful in this case. In this case, the antibody-cytokine grafted protein may also be modified to alter one or more functional properties. Therefore, the antibody can be chemically modified (e.g., by adding one or more chemical moieties to the antibody). or can be modified to alter its glycosylation.

[0143] In one embodiment, the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. For example, this technique was further developed by Bodmer et al. No. 5,677,425, which discloses, for example, the light and heavy chain amino acids To promote assembly or to increase the stability of antibody-cytokine grafted proteins The number of cysteine ​​residues in the hinge region of CH1 was varied to increase or decrease the In another embodiment, the biological half-life of the antibody-cytokine grafted protein is altered. The Fc hinge region of the antibody is mutated to enhance the expression of the antibody cytokine group. Staphylococcal protein A, a rafted protein tein A (SpA) binding was impaired compared to native Fc-hinge domain SpA binding. one or more amino acids at the CH2-CH3 domain junction region of the Fc-hinge fragment, Mutations are introduced. This technique is described in U.S. Pat. No. 6,165, by Ward et al. This is described in further detail in US Pat. No. 745.

[0144] The present disclosure provides an antibody that specifically binds to the IL2 low affinity receptor and has a long in vivo half-life. In another embodiment, a cytokine-grafted protein is provided. The grafted protein is modified to increase its biological half-life. Antibody-cytokine grafted proteins with increased in vivo half-lives are also available. or one or more amino acid modifications (i.e., substitutions, insertions, or deletions) in the IgG constant domain, or The FcRn-binding fragment (preferably an Fc or hinge-Fc domain fragment) is introduced into the FcRn-binding fragment. For example, see U.S. Patent No. 6,277,375 to Ward. As described above, one or more of the following mutations can be introduced: T252L , T254S, T256F. For example, International Publication No. 98 / 23289; International Publication No. 97 / 34631; and U.S. Pat. No. 6,277,375 Alternatively, antibody-cytokine grafted antibodies may be used to increase biological half-life. Proteins are disclosed in U.S. Patent Nos. 5,869,046 and 6,869,046 by Presta et al. As described in the specification of 121,022, two CH2 domains of the Fc region of IgG The CH1 or CL region was designed to contain a salvage receptor binding epitope taken from a loop of In yet another embodiment, at least one amino acid residue is varied within a different amino acid. The Fc region was altered by replacing it with an acid residue to form an antibody-cytokine grafted protein. Alters the effector function of proteins, e.g., their affinity for effector ligands Antibody-cytokine grafted proteins that have altered binding sites but retain the antigen-binding ability of the parent antibody. One or more amino acids can be replaced with different amino acid residues to form a protein. The effector ligand to which affinity is changed can be, for example, an Fc receptor (FcR ) or the C1 component of complement. Both of these methods are described in Winter et al. Further, U.S. Patent Nos. 5,624,821 and 5,648,260 by It is described in detail.

[0145] In another embodiment, the antibody-cytokine grafted protein has altered C1q binding and and / or selected from amino acid residues so as to reduce or eliminate complement-dependent cytotoxicity (CDC). One or more amino acids that are present in the sequence can be replaced with different amino acid residues. See U.S. Patent No. 6,194,551 to Idusogie et al. for further details. is described in.

[0146] Antibodies mediated by antibody-cytokine grafted proteins containing such mutations Antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) is reduced or completely eliminated. In some embodiments, amino acid residues L234 and L235 of the IgG1 constant region are In some embodiments, the amino acids of the IgG1 constant region are substituted with Ala234 and Ala235. The amino acid residue N267 is replaced by Ala267.

[0147] In another embodiment, one or more amino acid residues are altered to thereby enhance the antibody cytokine This alters the complement-fixing ability of the grafted protein. and is further described in WO 94 / 29351 by r et al.

[0148] In yet another embodiment, the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) is increased. and / or antibodies against Fcγ receptors. The Fc region is modified by modifying one or more amino acids to increase affinity. Further details on the methodology are available in WO 00 / 42072 by Presta. Furthermore, the binding of FcγRI, FcγRII, FcγRIII, and FcRn has been described. The binding site on human IgG1 has been mapped and mutants with improved binding have been described. (Shields, RLet et al., 2001 J. Biol. Chen. 276:6591-6604).

[0149] In yet another embodiment, the glycosylation of the antibody cytokine grafted protein For example, to create a non-glycosylated antibody-cytokine grafted protein. (i.e., the antibody-cytokine grafted protein lacks glycosylation. Glycosylation is a process that involves altering the affinity of an antibody for an "antigen," for example. Such carbohydrate modifications can be made, for example, by modifying one or more glycosylation sites within the antibody sequence. This can be achieved by, for example, altering one or more of the variable region framework regions. one or more cosylation sites, resulting in removal of the glycosylation site, thereby removing glycosylation at that site The above amino acid substitutions can be made. Such aglycosylation can reduce the affinity of the antibody for the antigen. Such an approach may be described in U.S. Patent No. 5,714,350 to Co et al. Nos. 6,350,861 and 6,350,861.

[0150] Additionally or alternatively, hypofucosylated antibody cytokines having reduced amounts of fucosyl residues. Altered types of antibodies, such as grafted proteins or antibodies with increased bisecting GlcNac structures Antibody-cytokine grafted proteins with glycosylation can be produced. The altered glycosylation pattern increases the antibody-dependent cellular cytotoxicity (ADCC) ability of the antibody. Such carbohydrate modifications have been demonstrated to result in, for example, altered glycosylation machinery. This is achieved by expressing antibody-cytokine grafted proteins in host cells. Cells with altered glycosylation machinery have been described in the art. , expressing a recombinant antibody-cytokine grafted protein, thereby improving glycosylation Use as host cells to produce altered antibody-cytokine grafted proteins For example, European Patent No. 1,176,195 by Hang et al. We describe a cell line with a disrupted FUT8 gene, which mediates fucosylation. transferases and thus expressed in such cell lines. The grafted protein exhibits low fucosylation. No. 03 / 035835 describes the addition of fucose to Asn(297)-linked carbohydrates. We describe a mutant CHO cell line, Lecl3 cells, that has a reduced ability to host the host cells. Resulting in hypofucosylation of antibody-cytokine grafted proteins expressed in host cells (Shields,RLet al.,2002 J.Biol.Chem.27 7:26733-26740). Umana et al., International Publication No. 99 / 010999 No. 54342 pamphlet describes glycoprotein-modifying glycosyltransferases (e.g., , β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) ), and the results of the experiments using engineered cell lines The antibody-cytokine grafted proteins exhibited increased bisecting GlcNac structures, and This results in an increase in the ADCC activity of the antibody (Umana et al., 1999 9 Nat. Biotech. 17:176-180).

[0151] In some embodiments, one or more domains of the antibody-cytokine grafted protein The domain or region may be linked by a linker, such as a peptide linker, such as those known in the art. (e.g., Holliger, P., et al. (1993) Proc.Natl.Acad.Sci.USA 90:6444-6448;Polj ak,RJ.,et al.(1994)Structure 2:1121-1123 (See, for example, the linker may be 1 to 1. The linker can be 0 to 50 amino acids long, typically the linker is 5 to 50 amino acids long, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 3 3, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 , 47, 48, 49, or 50 amino acids in length.

[0152] In some embodiments, IL2 is optionally linked using one or more peptide linker sequences. In certain embodiments, one or more peptide phosphorylation sites are grafted onto the CDR sequences. Carr is (Gly n -Ser) m Sequence (SEQ ID NO: 71), (Gly n -Ala) m Array (Array No. 72), or (Gly n -Ser) m / (Gly n -Ala) m Any combination of arrays (SEQ ID NOs: 71 to 72) [wherein each n is independently an integer of 1 to 5, and each m is independently an integer of 0 to 1 0. Examples of linkers include, but are not limited to: Glycine-based linkers or gly / ser linkers G / S, e.g., (G m S) n [In the ceremony , n is a positive integer equal to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, and m is 0 , 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10] (SEQ ID NO: 73 In certain embodiments, one or more linkers include G4S (SEQ ID NO: 74 ) repeats, e.g., Gly-Ser linkers (G4S) n [wherein n is a positive integer of 1 or more] For example, n=1, n=2, n=3, n=4, n=5 and n=6 (SEQ ID NO: 74). n=7, n=8, n=9 and n=10. In some embodiments, Ser is Al a, for example, (G m A) n [Wherein, n is 1, 2, 3, 4, 5, 6, is a positive integer equal to 7, 8, 9, or 10, and m is 0, 1, 2, 3, 4, 5, 6, 7, is an integer equal to 8, 9, or 10] (SEQ ID NO: 75). In certain embodiments, one or more linkers comprise a G4A (SEQ ID NO: 76) repeat, (G4 A) n [Wherein, n is a positive integer of 1 or more (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 comprises multiple repeats of the linker. In this case, the linker is a combination or multiple of G4S (SEQ ID NO: 74) and G4A (SEQ ID NO: 7 6).

[0153] Other examples of linkers include those that minimize immunogenicity caused by the linker and junction. Examples include those based on the flexible linker sequence naturally occurring in antibodies. For example, there is a natural flexibility between the variable domain and the CH1 constant domain of an antibody molecule. This natural linkage is made up of 4-6 residues from the C-terminus of the V domain and a CH Each domain contains approximately 10 to 12 amino acid residues, of which 4 to 6 residues contribute from the N-terminus. The mitochondrial grafted protein may be, for example, a 5-6 amino acid residue at the end of CH1 or a 1 Linkers incorporating 1 to 12 amino acid residues as a linker can be used. The N-terminal residues of the domain, especially the first 5-6 amino acids, form a loop without significant secondary structure. The CH1 domain can adopt various conformations and thus act as a flexible linker. The N-terminal residue of the amino acid sequence is a natural extension of the variable domain as it is part of the Ig sequence. and therefore eliminates any immunogenicity potentially caused by the linker and junction. In some embodiments, the linker sequence is based on the hinge sequence. The modified peptide sequence includes a

[0154] Furthermore, the antibody-cytokine grafted protein is It may contain a marker sequence, such as a peptide, to facilitate purification of the protein. In an embodiment, the marker amino acid sequence is incorporated into, inter alia, a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1) A tag such as a hexa-histidine peptide (SEQ ID NO: 121), Many are commercially available. Gentz ​​et al., 1989, Proc. Natl. A For example, hexahistidine, as described in cad. Sci. USA 86:821-824. The cysteine ​​(SEQ ID NO: 78) provides convenient purification of the grafted protein. Other peptide tags of use include, but are not limited to, influenza hemagglutinin. Hemagglutinin ("HA") tag (Wilson et al.) corresponding to an epitope derived from protein et al., 1984, Cell 37:767), and the "flag" tag. can be.

[0155] The antibody may also be attached to a solid support, which may be used in immunoassays or purification of the target antigen. Such solid supports include, but are not limited to, glass, cellulose, , polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene Examples include:

[0156] Assay of antibody-cytokine grafted protein activity Assays for identifying antibody-cytokine grafted proteins are known in the art. Agonist antibody cytokine grafted protein Proteins bind to the IL2 low affinity receptor and promote, induce, and stimulate intracellular signaling to induce CD 8 Promotes T effector cell proliferation as well as other immunostimulatory effects.

[0157] Binding of the antibody-cytokine grafted protein to the IL2 low affinity receptor is This can be determined using any method known in the art. For example, IL2-low parenteral Binding to affinity receptors can be measured by, without limitation, ELISA, Western blot, surface plasmon resonance imaging (SPFM), or other methods. known methods, including stationary phase resonance (SPR) (e.g., BIAcore), and flow cytometry. This can be determined using techniques.

[0158] Intracellular signaling through the IL2 low affinity receptor can be mediated by any of the methods known in the art. For example, the activation of IL2 low affinity receptors by IL2 can be measured by any method. Activation promotes STAT5 activation and signal transduction. Methods for measuring STAT5 activation include: The phosphorylation status of the STAT5 protein, (e.g., reporter gene assay, downstream signaling assay, etc.) through the IL2 low affinity receptor Activation of CD8 T effector cells expands them, and thus CD8 T effector cells The absolute number of CD8 T effector cells can be assayed, or the ratio of CD8 T effector cells to Tregs can be assayed. Methods for measuring cell proliferation are standard in the art. (for example, 3 H-thymidine incorporation assay (CFSE-labeled). Methods for measuring cytokine production Methods are well known in the art (e.g., ELISA assays, ELISpot assays, etc.). In performing the in vitro assay, antibody-cytokine grafted proteins Test cells or culture supernatant from test cells contacted with the substrate and antibody-cytokine grafted substrate Control cells not exposed to the protein or culture supernatant from control cells and / or recombinant human contact with IL2 (e.g., Proleukin®) or an IL2-FC fusion molecule It can be compared with what has been done.

[0159] The activity of the antibody-cytokine grafted protein may also be assessed ex vivo and / or in vivo. In some embodiments, the activity of untreated control animals and / or Prole Antibody cytokine grafts compared to animals similarly treated with ukin® STAT5 activation across various cell types ex vivo from animals treated with phospholipid-containing proteins The differential activity of agonist antibody-grafted proteins between cell types was investigated using a method that measures the activity of agonist antibodies. Preferred agonist antibody-cytokine grafted proteins are It has the ability to activate and expand CD8 T effector cells. For example, In vivo activation and expansion of effector cells can be achieved by any method known in the art. The activity can be measured using methods such as flow cytometry. Antibody-cytokine grafted proteins are useful in the treatment of cancer, e.g., melanoma, lung cancer, colorectal cancer. may be therapeutically useful in the prevention, reduction, amelioration or treatment of prostate cancer, breast cancer and lymphoma. The efficacy of the antibody-cytokine grafted protein is determined by the therapeutically effective amount of the antibody-cytokine. administering the grafted protein to a subject and administering an antibody cytokine to the subject before administering the grafted protein; This can be determined by comparing the levels of antibodies, cytokines, and grafted proteins in the control group. The efficacy of the protein was also evaluated by administering a therapeutically effective amount of the antibody-cytokine grafted protein to the test subjects. administering the antibody to a test subject and comparing the test subject to a control subject who has not received the antibody; and / or The results may also be determined by comparison with subjects similarly treated with ®.

[0160] Polynucleotides encoding antibody-cytokine-grafted proteins In another embodiment, the heavy and light chain proteins of the antibody cytokine grafted protein An isolated nucleic acid encoding an antibody-cytokine grafted protein is provided, including, but not limited to, Although not widely known, there are numerous techniques known in the art, including recombinant expression, chemical synthesis, and enzymatic digestion of antibody tetramers. Recombinant expression can be produced by any means known in the art. Any suitable host cell known in the art, for example, a mammalian host cell, a bacterial host cell, a yeast host cell, The host cell may be from a cell, an insect host cell, or the like.

[0161] Any of SEQ ID NO: 20, SEQ ID NO: 36, SEQ ID NO: 52, and SEQ ID NO: 68 is herein Polynucleotides encoding any one of the exemplary variable regions are provided.

[0162] Thus, the present disclosure provides a method for the production of a light chain of an antibody-cytokine grafted protein as described herein. and / or a polynucleotide encoding a heavy chain polypeptide, such as those described herein. A fragment encoding a light or heavy chain variable region or segment containing the complementarity determining regions as described above. In some embodiments, a polynucleotide encoding a heavy chain variable region is provided. The nucleotide is a polynucleotide selected from the group consisting of SEQ ID NO: 20 and SEQ ID NO: 52. Chid and at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, Includes sequences with 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain variable region is SEQ ID NO: 36 and SEQ ID NO: 68. 9%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 9 9%, or 100% nucleic acid sequence identity.

[0163] In some embodiments, the polynucleotide encoding the heavy chain is the polynucleotide of SEQ ID NO:22. Nucleotides and at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain is the polynucleotide of SEQ ID NO: 38. Reotide and at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity.

[0164] In some embodiments, the polynucleotide encoding the heavy chain is the polynucleotide of SEQ ID NO:54. Nucleotides and at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity. In some embodiments, the polynucleotide encoding the light chain is selected from SEQ ID NO: 70. Polynucleotides with at least 85%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 11 have 4%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity do.

[0165] The polynucleotide encodes only the variable region sequences of the antibody cytokine grafted protein. Polynucleotides can also be used to encode antibody-cytokine grafted proteins. Some polynucleotide sequences may encode both variable and constant regions of the polypeptide. The columns represent the variable domains of both the heavy and light chains of one of the antibody-cytokine grafted proteins. Other polynucleotides encode polypeptides containing antibody cytokines. and a heavy chain and a light chain variable region substantially identical to the heavy chain and light chain variable regions of one of the grafted proteins, respectively. It encodes two polypeptide segments:

[0166] In certain embodiments, the polynucleotide or nucleic acid comprises DNA. In the present context, polynucleotides or nucleic acids include RNA, which may be single-stranded or double-stranded. may be.

[0167] In some embodiments, one or more immunoglobulins of the antibody-cytokine grafted protein A recombinant host comprising a nucleic acid encoding a globulin protein chain and, optionally, a secretion signal. In certain embodiments, the recombinant host cell is a host cell expressing one immunoglobulin tag. In certain other embodiments, the vector includes a vector encoding a protein chain and a secretion signal. The recombinant host cells express two immunoglobulin proteins, the antibody cytokine grafted protein. In some embodiments, the vectors include one or more vectors encoding the protein chain and a secretion signal. The recombinant host cells express two immunoglobulins of the antibody cytokine grafted protein. In some embodiments, the vector encodes a protein chain and a secretion signal. In the recombinant host cell, one of the heavy chain immunoglobulins of the antibody cytokine grafted protein is expressed. one encoding the immunoglobulin protein chains and the other encoding the light immunoglobulin protein chains The recombinant host cell may be a prokaryotic or eukaryotic cell. In some embodiments, the host cell is a eukaryotic cell line. In embodiments, the host cell is a mammalian cell line. In some embodiments, the host cell line is A CHO cell line for antibody production.

[0168] Additionally, methods for producing antibody-cytokine grafted proteins are provided. In one embodiment, the method comprises: (i) preparing an immunoglobulin of an antibody-cytokine grafted protein; Host cells containing one or more vectors encoding phosphoprotein chains are cultured in an antibody cytokine lab. (ii) culturing the culture under conditions suitable for the expression, formation, and secretion of rafted proteins; ) recovering the antibody-cytokine-grafted protein.

[0169] The polynucleotide sequence encodes the polypeptide chain of the antibody cytokine grafted protein. De novo solid-phase DNA synthesis or can be generated by PCR mutagenesis. Direct chemical synthesis of nucleic acids is known as Narang e phosphotriester method of T. et al., Meth. Enzymol. 68:90, 1979 Brown et al., Meth. Enzymol. 68:109, 1979 Phosphodiester method; Beaucage et al., Tetra. Lett., 22: 1859, 1981 diethyl phosphoramidite method; and U.S. Pat. No. 4,458,066 This can be done by methods known in the art, such as the solid support method of US Pat. No. 6,499,499. Introduction of mutations into polynucleotide sequences by PCR is known, for example, from PCR Technology ology:Principles and Applications for DN A Amplification, HA Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide t o Methods and Applications,Innis et al.( Ed.), Academic Press, San Diego, CA, 1990;Ma ttila et al., Nucleic Acids Res.19:967,19 91; and Eckert et al., PCR Methods and Applications This can be done as described in Cations 1:17, 1991.

[0170] Also, expression to produce the antibody-cytokine grafted protein described above. Vectors and host cells are also provided in the present disclosure. For expressing polynucleotides encoding immunoglobulin polypeptide chains or fragments A variety of expression vectors can be used to express immunoglobulins in mammalian host cells. Both viral-based and non-viral expression vectors are available for protein production. Non-viral vectors and systems typically include those that deliver proteins. or plasmids, episomal vectors, and and human artificial chromosomes (e.g., Harrington et al., Nat Genet 15:345, 1997). For example, mammalian (e.g., human) cells Expression of antibody-cytokine-grafted protein polynucleotides and polypeptides in cells Currently available non-viral vectors include pThioHis A, B & C, and pcDN A3.1 / His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors and related techniques for expressing other proteins. Many other vectors are known in the art. Useful viral vectors include: , retroviruses, adenoviruses, adeno-associated viruses, and herpesvirus-based vectors vectors, SV40-based vectors, papillomavirus, HBP Epstein-Barr virus Examples include vaccinia virus vectors, vaccinia virus vectors, and Semliki Forest virus (SFV). Brent et al., supra; Smith, Annu. Rev. Microbiol .49:807, 1995; and Rosenfeld et al., Cell 68: 143, 1992.

[0171] The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, the expression vector encodes an immunoglobulin protein of an antibody cytokine grafted protein. a promoter and other regulatory elements operably linked to the polynucleotide encoding the protein; In one embodiment, the promoter comprises an inducible promoter sequence (e.g., an enhancer sequence). is used to prevent expression of the inserted sequence except under inducing conditions. Examples of promoters include arabinose, lacZ, metallothionein promoters, and A heat shock promoter is one example. A culture of the transformed organism is then grown in a medium containing the expression product. under non-inducing conditions without biasing the population towards coding sequences that are better tolerated by host cells. In addition to the promoter, other regulatory elements may also be included in the antibody cytokine gene. required for efficient expression of immunoglobulin chains or fragments of the target protein These elements typically include the ATG initiation codon and adjacent It may contain a ribosome binding site or other sequences. Furthermore, the efficiency of expression may be affected by the cell line in use. It can be increased by the inclusion of appropriate enhancers (e.g., Scharf et al. ,Results Probl.Cell Differ.20:125,1994; and Bittner et al.,Meth.Enzymol.,153:516,19 For example, the SV40 enhancer or CMV enhancer may be used in mammalian hosts. It can be used to increase expression in cells.

[0172] The expression vector also contains an antibody cytokine gene that is exported from the cell into the culture medium. A secretory signal sequence may also be provided to form a secreted protein. In embodiments, the immunoglobulin sequence of the antibody cytokine grafted protein to be inserted The immunoglobulin light and heavy chain variable fragments are linked to a signal sequence before inclusion in the vector. The vectors used to receive the sequences encoding the domains sometimes also contain constant regions or Such vectors may also encode a grafted protein containing a constant region. The antibody-cytokine grafted template allows expression of all variable regions. Typically, such constant regions are human.

[0173] Host cells for carrying and expressing antibody cytokine grafted protein chains include prokaryotic E. coli can be either eukaryotic or eukaryotic. is one prokaryotic host useful for cloning and expressing the Microbial hosts include bacilli such as Bacillus subtilis, and Salmonella, Serratia, and various serovars Other Enterobacteriaceae species include Pseudomonas species. In prokaryotic hosts, expression control sequences compatible with the host cell (e.g., origin of replication, It is also possible to prepare an expression vector containing the lactose promoter system, tryptophan (trp) promoter system, β-lactamase promoter system, or lambda Any number of different well-known promoters exist, including promoter systems derived from phage. The promoter is typically, optionally with an operator sequence. Ribosome binding site sequences for controlling expression and initiating and completing transcription and translation Other microorganisms, such as yeast, also have antibodies, cytokines, and grafted proteins. Insect cells in combination with baculovirus vectors can also be used to express It can be done.

[0174] In some preferred embodiments, the antibody-cytokine grafted protein polypeptide Mammalian host cells are used for expression and production, e.g., those containing exogenous expression vectors. These may be any mammalian cell line that is capable of producing cells of any normal, dead or normal young variety. or abnormal immortal animal or human cells, e.g., CHO cell lines, various Cos cells, including strains, HeLa cells, myeloma cell lines, transformed B cells and hybridomas. Several suitable host cell lines capable of secreting immunoglobulins have been developed. The use of mammalian tissue cell culture to express polypeptides is described, for example, in Winn et al. acker,From Genes to Clones,VCH Publisher s, NY, NY, 1987. For mammalian host cells Expression vectors contain expression control sequences such as an origin of replication, a promoter, and an enhancer (e.g., Queen et al., Immunol. Rev. 89:49-68, 1986 (see ), as well as ribosome binding sites, RNA splice sites, polyadenylation sites, and These sequences may contain necessary processing information sites such as a transcription terminator sequence. Modern vectors usually contain promoters derived from mammalian genes or mammalian viruses. Suitable promoters may be constitutive, cell type specific, stage specific, and / or regulatable. Useful promoters include, but are not limited to, metallothionein. Onein promoter, constitutive adenovirus major late promoter, dexamethasone-induced Conductive MMTV promoter, SV40 promoter, MRPpolIII promoter, Constitutive MPSV promoter, tetracycline-inducible CMV promoter (human immediate early promoter) CMV promoter), constitutive CMV promoter, and promoters known in the art Examples of promoter-enhancer combinations include:

[0175] Methods for introducing expression vectors containing polynucleotide sequences of interest include For example, calcium chloride transfection varies depending on the type of cell host. Although calcium phosphate treatment or electroporation is commonly used for , may be used for other cellular hosts (see generally Sambrook et al., supra). Other methods include electroporation and calcium phosphate treatment. , liposome-mediated transformation, injection and microinjection, ballistic method , virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virion, grafting onto the herpesvirus structural protein VP22 (Elliot et al. and O'Hare, Cell 88:223, 1997), drug-facilitated uptake of DNA These include ex vivo transduction and ex vivo transduction. For example, stable expression of antibodies, cytokines, and proteins is often desirable. Cell lines stably expressing protein chains are constructed by incorporating a viral origin of replication or endogenous expression elements. It can be prepared by using an expression vector containing a selectable marker gene. Cells are allowed to grow for 1-2 days in an enriched media before being switched to a selective media. The purpose of the marker is to confer resistance to selection, and its presence is essential for the selection of This allows the growth of cells that successfully express the introduced sequence. The transplanted cells can be grown using tissue culture techniques appropriate to the cell type.

[0176] Compositions Comprising Antibody-Cytokine-Grafted Proteins The antibody-cytokine grafted protein is formulated with a pharmaceutically acceptable carrier. Optionally, the pharmaceutical composition is useful for the treatment or prevention of a given disorder. The pharmaceutical composition may further comprise other suitable therapeutic agents. Pharmaceutically acceptable carriers include those that are physiologically acceptable. Compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delay agents Examples include agents.

[0177] The pharmaceutical compositions of the present disclosure can be administered by a variety of methods known in the art. The route and / or mode of administration will vary depending on the desired results. (e.g., intravenous, intramuscular, intraperitoneal, intrathecal, intraarterial, or subcutaneous) Preferably, the drug is administered by a pharmaceutically acceptable carrier or in the vicinity of the target site. The body may be administered intravenously, intramuscularly, intraperitoneally, intrathecally, intraarterially, subcutaneously, intranasally, by inhalation, spinal or epidermal injection. The administration route is suitable for administration by any one or more of the following routes: Accordingly, the active compound, for example, the antibody-cytokine grafted protein, is resistant to the action of acid and its In addition, the compound may be coated with a material that protects it from natural conditions that may inactivate it. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In an embodiment, the pharmaceutical composition is formulated for subcutaneous administration.

[0178] The antibody-cytokine grafted protein may be used alone or in combination with other suitable components. and may be made into an aerosol formulation for administration by inhalation (i.e., it is "nebulized"). Aerosol formulations may be prepared using a variety of aerosols, including dichlorodifluoromethane, propane, nitrogen, etc. , may be placed into a pressurized acceptable propellant.

[0179] In some embodiments, the pharmaceutical composition is sterile and fluid. For example, the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersions, and the use of an interfacial This can be maintained by the use of active ingredients. Often, isotonic agents, such as sugars, are added to the composition. polyhydric alcohols such as mannitol or sorbitol, and sodium chloride Agents that delay absorption, such as aluminum monostearate or gelatin, are preferred. Prolonged absorption of the injectable compositions can be achieved by including cin in the composition. In certain embodiments, the composition is lyophilized using a suitable excipient (e.g., sucrose). The composition can be prepared for storage in the form of a pharmaceutical preparation.

[0180] Pharmaceutical compositions can be prepared according to conventional methods well known in the art. Acceptable carriers will depend, in part, on the particular composition being administered, as well as the nature of the agent used to administer the composition. The type of formulation that will be used will depend on the particular method used. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions. Formulation of antibody-cytokine grafted proteins and determination of appropriate administration and schedule For specific applicable methods, see, for example, Remington: The Science and Practice of Pharmacy, 21 st Ed.,Univer City of the Sciences in Philadelphia,Eds ., Lippincott Williams & Wilkins(2005); and Martindale:The Complete Drug Reference,S weetman,2005,London:Pharmaceutical Press ., and Martindale, Martindale: The Extra Phar macopoeia,31st Edition.,1996,Amer Pharma ceutical Assn, and Sustained and Controlled Release Drug Delivery Systems,JRRobin son, ed., Marcel Dekker, Inc., New York, 1978 Pharmaceutical compositions are preferably manufactured under GMP conditions. In the pharmaceutical composition, a therapeutically effective dose or an effective dose of an antibody-cytokine-grafted protein is contained. The antibody-cytokine grafted protein can be prepared by conventional methods known to those skilled in the art. The dosage regimen is determined based on the desired response (e.g., In determining a therapeutically or prophylactically effective dose, In this case, a low dose can be administered, followed by the desired response with minimal or no undesired side effects. For example, a single bolus may be administered, Several divided doses may be administered over time or as indicated by the exigencies of the therapeutic situation. Doses may be proportionally increased or decreased accordingly, particularly for ease of administration and uniformity of dosage. Therefore, it is advantageous to formulate parenteral compositions in dosage unit form. As used herein, a physically discrete unit suited as a unitary dosage for treatment is a compound. Each unit is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Contains a fixed amount of active compound.

[0181] The actual dosage level of the active ingredient present in the pharmaceutical composition is non-toxic to the patient and is not of active ingredients effective to achieve the desired therapeutic response for the appropriate patient, composition, and mode of administration. The dosage level selected may be varied depending on the particular composition used. the activity, route of administration, time of administration, details of use of the compound, or its ester, salt or amide The excretion rate of the compound, duration of treatment, use in combination with other drugs, and the specific composition used the compound and / or material being treated, the age, sex, weight, condition, general health and The dose depends on various pharmacokinetic factors, including the patient's age and previous medical history.

[0182] Products / Kits In some embodiments, the antibody-cytokine grafted protein is provided as an article of manufacture (i.e., a kit). The antibody-cytokine grafted proteins provided are generally packaged in vials or The product is therefore in the container and the label on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and solutions. Optionally, the antibody-cytokine grafted protein may be in liquid or dry form. The container may be in a liquid form (e.g., lyophilized form). The container may be in a liquid form by itself or in combination with another composition. The combination provides a composition effective for preparing a composition for treating, preventing and / or ameliorating cancer. The label or package insert shall state that the composition is used for treating, preventing, and / or ameliorating cancer. Indicated. Comprising an antibody-cytokine grafted protein as described herein. The article of manufacture (kit) optionally includes one or more additional agents. In some embodiments, The product (kit) comprises an antibody-cytokine grafted protein and a pharmaceutically acceptable diluent. In some embodiments, the antibody-cytokine grafted protein is in the same formulation (e.g., as a mixture) with one or more additional active agents In some embodiments, the antibody-cytokine grafted protein is a product ( provided in a separate formulation (e.g., in a separate container) with a second or third agent in a kit In certain embodiments, the product (kit) comprises an antibody-cytokine grafted protein. In some embodiments, the aliquot provides one or more doses. Multiple dose aliquots are provided in which the doses are uniform or variable. In particular embodiments, the varying dosing regimen is either escalating or tapering, as appropriate. In some embodiments, the antibody-cytokine grafted protein and the second agent The dosages may be independently uniform or independently variable. In certain embodiments, the product ( The kit may contain additional agents, such as anti-cancer drugs or immune checkpoint molecules. The choice of additional agent will depend on dosage, delivery, and the disease state to be treated. do.

[0183] Methods for treating cancer and uses of therapeutic compositions thereof Conditions to be treated or prevented The antibody-cytokine grafted proteins find use in the treatment, amelioration, or prevention of cancer. In one aspect, the disclosure provides a method for treating cancer in an individual in need thereof, The method of claim 1, further comprising administering to a subject a therapeutically effective amount of an antibody-cytokine grafted protein as described herein. In some embodiments, the method comprises administering to the individual an effective amount of a compound selected from the group consisting of benzodiazepines, ... and an antibody-cytokine grafted protein for use as a therapeutic agent in In a further aspect, the present disclosure provides a method for treating or ameliorating cancer in an individual in need thereof. and a composition comprising such an antibody-cytokine grafted protein for use in .

[0184] Conditions that may be treated include various cancer diseases. For treatment purposes, an individual is diagnosed with cancer. For protective or prophylactic purposes, the individual may be in remission from cancer or may be at risk of future onset. In some embodiments, the patient has or is suspected of having cancer. or in remission from cancer. Treatment with antibody-cytokine grafted proteins The cancers targeted by the method are generally those mediated by IL2 low affinity receptor signaling, as described herein. Cancer diseases that may be treated include, but are not limited to: melanoma, These include lung cancer, colorectal cancer, prostate cancer, breast cancer and lymphoma.

[0185] Administration of antibody-cytokine-grafted proteins A physician or veterinarian should be aware of the use of antibody-cytokine-grafted proteins in pharmaceutical compositions. The dose should be initiated at a level lower than that required to achieve the desired therapeutic effect, and should be increased until the desired effect is achieved. Dosage can be gradually increased until the desired effect is achieved. Generally, an effective dose of the composition is The specific disease or condition to be treated, the means of administration, the target site, the physiological state of the patient, Whether human or animal, other medications being administered, and whether the treatment is prophylactic or Therapeutic dosages vary depending on many different factors, including whether the drug is safe and effective. Titration is typically required to optimize the antibody-cytokine grafted protein. For administration with steroids, the dosage ranges from about 0.0001 to 100 mg / kg of the host body weight, and more commonly The usual range is 0.01 to 5 mg / kg. For example, the dosage is 1 mg / kg body weight or 10 mg g / kg body weight or in the range of 1 to 10 mg / kg. This may be daily, weekly, biweekly, monthly, or more or less frequently as needed. Suggested treatment regimes will necessarily involve weekly, biweekly, monthly, or every 3-6 months. It involves multiple doses.

[0186] The antibody-cytokine grafted protein can be administered in a single dose or in divided doses. Antibody-cytokine grafted proteins are usually administered on multiple occasions. The interval between the amounts may be weekly, biweekly, monthly, or yearly, as needed or desired. Also, by measuring blood levels of antibody-cytokine grafted proteins in patients May be administered irregularly as directed. In some methods, dosages range from 1 to 1000 μg / ml, and in some methods, 25-300 μg / ml of plasma antibody cytokine grafting The protein concentration is adjusted to achieve the desired concentration. Proteins can be administered as sustained release formulations, in which case less frequent administration is required. Dosage and frequency will depend on the half-life of the antibody-cytokine grafted protein in the patient. Generally, the antibody-grafted protein is derived from natural IL2 or Proleukin (IL2). It exhibits a longer half-life than recombinant cytokines such as CYP2000-10000 (registered trademark). The dosage and frequency of administration are This can vary depending on whether the treatment is preventative or therapeutic. Some patients experience Generally, for prophylactic applications, lower dosages are used at lower frequency. Generally, for therapeutic use, the drug is administered at intervals over a long period of time, with the goal of reducing or halting disease progression. and preferably until the patient shows partial or complete improvement in the symptoms of the disease. Relatively high dosages at relatively short intervals are sometimes required. The patient then begins a prophylactic regime. This may be done.

[0187] Coadministration with a second drug The term "combination therapy" refers to the use of two therapeutic agents to treat a condition or disorder described in this disclosure. Such administration refers to the administration of a single capsule having a fixed ratio of active ingredients. Alternatively, this includes co-administration of these therapeutic agents in a substantially simultaneous manner, such as by administering the same to the patient. Such administration may involve multiple or separate containers (e.g., capsules, powders) for each active ingredient. The powder and / or liquid may be adjusted to the desired dose prior to administration. Furthermore, such administration may be simultaneous, at approximately the same time, or at different times. It also encompasses the use of each type of therapeutic agent in a continuous manner. provides a beneficial effect of the drug combination in treating the conditions or disorders described herein. do.

[0188] Combination therapy can provide a "synergistic effect" and is "synergistic," i.e., the active ingredients The effects achieved when used together are greater than those achieved when the compounds are used individually. The synergistic effect may be found to be higher than the sum of the active ingredients (1) when co-formulated and administered; (2) when delivered simultaneously in separate or combined unit dosage formulations; or (3) by any other regimen, When delivered in an alternating administration regimen, a synergistic effect can be achieved when the compounds are administered in separate serial doses, e.g. This can be achieved when the two drugs are administered or delivered sequentially by different injections in a syringe. During alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e., serially, and In combination therapy, effective dosages of two or more active ingredients are administered together.

[0189] In one aspect, the present disclosure provides a method for treating rheumatoid arthritis, including, but not limited to, EGFR inhibitors, Her2 inhibitors, H one or more tyrosine kinase inhibitors, including er3 inhibitors, IGFR inhibitors, and Met inhibitors Antibody-cytokine grafted proteins in combination with inhibitors to subjects in need The present invention provides a method for treating cancer by administering

[0190] For example, tyrosine kinase inhibitors include, but are not limited to, erlotinib hydrochloride ( Tarceva®; Linifanib (N-[4-(3-amino-1H-indole]); [N'-(2-fluoro-5-methylphenyl)-4-(4-phenyl-4-ylphenyl)urea], ABT 869, available from Genentech); sunitinib malate (Sute nt®); bosutinib (4-[(2,4-dichloro-5-methoxyphenyl) Amino]-6-methoxy-7-[3-(4-methylpiperazin-1-yl)propoxy] Quinoline-3-carbonitrile, also known as SKI-606, and U.S. Patent No. 6,780,999 No. 6); dasatinib (Sprycel®); pazopanib ( Votrient®; sorafenib (Nexavar®); Za ctima (ZD6474); nilotinib (Tasigna®); regorafenib nib (Stivarga®) and imatinib or imatinib mesylate (Gil vec® and Gleevec®).

[0191] Epidermal growth factor receptor (EGFR) inhibitors include, but are not limited to, erlotinib salts. methicillin-resistant Staphylococcus aureus (Tarceva®), gefitinib (Iress a (registered trademark); N-[4-[(3-chloro-4-fluorophenyl)amino]-7- [[(3"S")-tetrahydro-3-furanyl]oxy]-6-quinazolinyl]-4( dimethylamino)-2-butenamide, Tovok®); vandetanib (Ca prelsa®); 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 (BMS69 0514;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 Neratinib (HKI-272); N-[4-[[1-[(3- Fluorophenyl)methyl]-1H-indazol-5-yl]amino]-5-methylpi Carbohydrate [2,1-f][1,2,4]triazin-6-yl]-carbamic acid, (3S)- 3-morpholinylmethyl ester (BMS599626); N-(3,4-dichloro-2 -fluorophenyl)-6-methoxy-7-[[(3aα,5β,6aα)-octahydro 2-methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazoline amine (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) is an example.

[0192] EGFR antibodies include, but are not limited to, cetuximab (Erbitux® )); panitumumab (Vectibix®); matuzumab (EMD-7200 0); nimotuzumab (hR3); zalutumumab; TheraCIM h-R3; MDX0 447 (CAS 339151-96-1); and ch806 (mAb-806, CAS 946414-09-1).

[0193] Human epidermal growth factor receptor 2 (HER2 receptor) (also known as Neu, ErbB-2, CD34 0 or p185) inhibitors include, but are not limited to, trastuzumab (Hercep tin®); 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 and WO 05 / 028443 lapatinib or lapatinib ditosylate (Tykerb®) );(3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pi 1,2,4]triazin-5-yl)methyl)piperidin-3-ol (2E)-N-[4-[(3-chloro-4-fluorophenyl)-2-methyl-2-propanol] (3S)-tetrahydro-3-furanyloxy]-6-quina zolinyl]-4-(dimethylamino)-2-butenamide (BIBW-2992, CAS 850140-72-6);N-[4-[[1-[(3-fluorophenyl)methyl] -1H-indazol-5-yl]amino]-5-methylpyrrolo[2,1-f][1,2 ,4]triazin-6-yl]-carbamic acid, (3S)-3-morpholinylmethyl ester Terephthalate (BMS 599626, CAS 714971-09-2); Canertinib dihydrochloride salt (PD183805 or CI-1033); and N-(3,4-dichloro-2-fluoro (3aα,5β,6aα)-octahydro-2- Methylcyclopenta[c]pyrrol-5-yl]methoxy]-4-quinazolinamine (X L647, CAS 781613-23-8).

[0194] HER3 inhibitors include, but are not limited to, LJM716, MM-121, AMG- 888, RG7116, REGN-1400, AV-203, MP-RM-1, MM-1 11, and MEHD-7945A.

[0195] MET inhibitors include, but are not limited to, cabozantinib (XL184, CAS 8 49217-68-1); foretinib (GSK1363089, formerly XL880, CAS 849217-64-7); tivantinib (ARQ197, CAS 1000873- 98-2; 1-(2-hydroxy-2-methylpropyl)-N-(5-(7-methoxyphenyl)- Quinolin-4-yloxy)pyridin-2-yl)-5-methyl-3-oxo-2-phenanthroline Nyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458); Crizotinib (Xalkori®, PF-0234) 1066);(3Z)-5-(2,3-dihydro-1H-indol-1-ylsulfonyl yl)-3-({3,5-dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl -1H-pyrrol-2-yl}methylene)-1,3-dihydro-2H-indole- 2-one (SU11271); (3Z)-N-(3-chlorophenyl)-3-({3,5 -Dimethyl-4-[(4-methylpiperazin-1-yl)carbonyl]-1H-pyrrole -2-yl}methylene)-N-methyl-2-oxoindoline-5-sulfonamide (S U11274);(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-[di Fluoro[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-pyrazo [1-[(2-methyl-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]ci Clohepta[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(S GX523, 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-pyrrole [2-yl]methylene]-1,3-dihydro-2H-indol-2-one (PHAS6 65752, CAS 477575-56-7).

[0196] IGF1R inhibitors include, but are not limited to, BMS-754807 and XL-228 , OSI-906, GSK0904529A, A-928605, AXL1717, KW -2450, MK0646, AMG479, IMCA12, MEDI-573, and BI 836845. For reviews, see, for example, Yee, JNCI, 104; See 975(2012).

[0197] In another aspect, the present disclosure provides compounds that are useful in treating rheumatoid arthritis, including, but not limited to, MEK inhibitors, Braf inhibitors, P one or more F inhibitors, including an I3K / Akt inhibitor, an SHP2 inhibitor, and also an mTor inhibitor The antibody-cytokine grafted protein was administered in combination with a GF downstream signaling pathway inhibitor. The present invention provides a method for treating cancer by administering the compound to a subject in need thereof.

[0198] For example, mitogen-activated protein kinase (MEK) inhibitors include, but are not limited to: However, XL-518 (also known as GDC-0973, CAS number 1029872-29-4) , available from ACC Corp.); 2-[(2-chloro-4-iodophenyl)amine] o]-N-(cyclopropylmethoxy)-3,4-difluorobenzamide (also known as CI -1040 or PD184352 and International Publication No. 2000035436 N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro 2-[(2-fluoro-4-iodophenyl)amino]-benzamide (also known as PD0 325901 and International Publication No. 2002006213);2, 3-bis[amino[(2-aminophenyl)thio]methylene]butanedinitrile (also known as U0126 and U.S. Pat. No. 2,779,780);N-[3,4- Difluoro-2-[(2-fluoro-4-iodophenyl)amino]-6-methoxyphenyl [(2R)-2,3-dihydroxypropyl]-cyclopropanesulfonyl Mido (also known as RDEA119 or BAY869766 and International Publication No. 2007014011 Described in the pamphlet No.); (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 the pamphlet of International Publication No. 2003076424) 2'-amino-3'-methoxyflavone (also known as PD98059 Biaffin Gm bH & Co.,KG, Germany); vemurafenib (PLX-4 032, CAS 918504-65-1; (R)-3-(2,3-dihydroxypropanediol) Pyr)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methyl Pyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione (TAK-733, C AS 1035555-63-5; Pimasertib (AS-703026, CAS 12 04531-26-9); and trametinib dimethyl sulfoxide (GSK-11202 12, CAS 1204531-25-80).

[0199] Phosphoinositide 3-kinase (PI3K) inhibitors include, but are not limited to, 4- [2-(1H-indazol-4-yl)-6-[[4-(methylsulfonyl)piperazine [3,2-d]pyrimidin-4-yl]morpholine (also known as GDC 0941 and International Publication No. 09 / 036082 and International Publication No. 9 / 055730 pamphlet); 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-B EZ 235 and described in WO 06 / 122806);4- (Trifluoromethyl)-5-(2,6-dimorpholinopyrimidin-4-yl)pyridine -2-amine (also known as BKM120 or NVP-BKM120, and International Publication No. 2007 / 002366) 084786 brochure); tozasertib (VX680 or MK-04 57, 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-dimethyl-cyclopenta[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).

[0200] mTor inhibitors include, but are not limited to, temsirolimus (Torisel) trademark); ridaforolimus (officially known as deforolimus) , (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-diox Sa-4-azatricyclo[30.3.1.0 4,9 ]Hexatriaconta-16,24,2 6,28-tetraen-12-yl]propyl]-2-methoxycyclohexyldimethyl Phosphinate, also known as AP23573 and MK8669, and WO 03 / 0643 everolimus (Afinitor® or RAD001); rapamycin (AY22989, Sirolimus®); Semapimo simapimod (CAS 164301-51-3); (5-{2,4-bis [(3S)-3-Methylmorpholin-4-yl]pyrido[2,3-d]pyrimidine-7- 2-amino-8-[(2-amino-2-phenyl)methyl]-2-methoxyphenylmethanol (AZD8055); trans-4-(2-hydroxyethoxy)cyclohexyl]-6-(6-methoxy-3- pyridinyl)-4-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one (PF 04691502, CAS 1013101-36-4); and N 2 -[1,4-diox So-4-[[4-(4-oxo-8-phenyl-4H-1-benzopyran-2-yl)mo [4-(4-aminopropyl)methyl]-L-arginylglycyl-L-α-ascorbic acid aryl L-serine-("L-arginylglycyl-L- α-Aspartyl L-serine-), inner salt (SF1126, CAS 936487-67 -1) are listed.

[0201] In yet another aspect, the present disclosure provides compounds that are useful in treating cancer, including, but not limited to, IAP inhibitors, Bcl2 inhibitors, One or more pro-apoptotic drugs, including MCl1 inhibitors, Trail agents, and Chk inhibitors The antibody-cytokine grafted protein is administered in combination with a substance to a subject in need thereof. The present invention provides a method for treating cancer by administering

[0202] For example, IAP inhibitors include, but are not limited to, NVP-LCL161, GDC- 0917, AEG-35156, AT406, and TL32711. Other examples of inhibitors include, but are not limited to, those described in WO 04 / 005284. , International Publication No. 04 / 007529, International Publication No. 05 / 097791 Brochure, International Publication No. 05 / 069894 Brochure, International Publication No. 05 / 069 No. 888, International Publication No. 05 / 094818, U.S. Patent Application Publication No. Publication No. 2006 / 0014700, U.S. Patent Application Publication No. 2006 / 0025347 No. 2006 / 069063, WO 06 / 01011 8, WO 06 / 017295, and WO 08 Examples include those described in brochure No. 134679.

[0203] BCL-2 inhibitors include, but are not limited to, 4-[4-[[2-(4-chlorophenoxyethanol) Nyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperazinium -N-[[4-[[(1R)-3-(4-morpholinyl)-1-[(phenylthio) Methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]sulfonyl [Sulfonyl]benzamide (also known as ABT-263 and WO 09 / 155386 (described in FRET); tetrocarcin A; antimycin; gossypol ((-)BL -193;Ovatoclax;Ethyl-2-amino-6-cyclopentyl-4-(1- Cyano-2-ethoxy-2-oxoethyl)-4Hchromone-3-carboxylate(H A14-1); Oblimersen (G3139, Genasense®); Ba k BH3 peptide; (-)-gossypol acetic 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, CA S 852808-04-9); and navitoclax (ABT-263, CAS 923 564-51-6).

[0204] including, but not limited to, dulanermin (AMG-951, RhApo2L / TRAIL) ;Mapatumumab (HRS-ETR1, CAS 658052-09-6);Lexatumumab (HGS-ETR2, CAS 845816-02-6);Apomab (Apoma b(R)); conatumumab (AMG655, CAS 896731-82-1); and tigatuzumab (CS1008, CAS 946415-34-5, Daiichi Sankyo Co., Ltd. DR4 (TRAILR1) and DR5 (available from Ichi Sankyo) Proapoptotic receptor agonists (PARAs), including (TRAILR2).

[0205] Checkpoint kinase (CHK) inhibitors include, but are not limited to, 7-hydroxybenzoates, Xystaurosporine (UCN-01); 6-bromo-3-(1-methyl-1H-pyrazoline) (4-yl)-5-(3R)-3-piperidinyl-pyrazolo[1,5-a]pyrimidinyl 5-(3- (S)-piperidinyl)-3-ureidothiophene-2-carboxylic acid N-[(fluorophenyl) 4-[((-3-yl)amide (AZD7762, CAS 860352-01-8); (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-Ami nodactinomycin (7-AAD), isogranulatimide, debromohymeni Ardisine;N-[5-bromo-4-methyl-2-[(2S)-2-morpholinylmethacrylate si]-phenyl]-N'-(5-methyl-2-pyrazinyl)urea (LY2603618, CAS 911222-45-2; sulforaphane (CAS 4478-93-7, 4 -Methylsulfinylbutylisothiocyanate);9,10,11,12-tetrahydro 1,2,3-fg:3',2',1'-k l]pyrrolo[3,4-i][1,6]benzodiazocin-1,3(2H)-dione (SB -218078, CAS 135897-06-2); and TAT-S216A (Sha et al.,Mol.Cancer.Ther 2007;6(1):147-15 3), and CBP501.

[0206] In one aspect, the present disclosure provides an antibody-cytokine glucanase inhibitor in combination with one or more FGFR inhibitors. The present invention provides a method for treating cancer by administering rafted proteins to a subject in need thereof. 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); bar Gatef (BIBF1120, CAS 928326-83-4); Dovitinib dilactic acid (T KI258, CAS 852433-84-2; 3-(2,6-dichloro-3,5-di methoxy-phenyl)-1-{6-[4-(4-ethyl-piperazin-1-yl)-phenyl nylamino]-pyrimidin-4-yl}-1-methyl-urea (BGJ398, CAS 8 72511-34-7; danusertib (PHA-739358); and (PD1730 74, CAS 219580-11-7). The disclosure relates to 3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6((4-( 4-Ethylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)-1-methyl methylurea (also known as BGJ-398); or 4-amino-5-fluoro-3-(5-(4-methyl- (1-yl)-1H-benzo[d]imidazol-2-yl)quinoline-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 [Diemthyl]-4-(3R,5S)-dimethylpiperazine )-1-yl)benzamide), ponatinib (AP24534; Gozgit et al. l.,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 In combination with FGFR2 inhibitors such as benzodiazepines, CAS 943319-70-8, etc. Methods for treating cancer by administering antibody drug conjugates to a subject in need thereof to provide.

[0207] Antibody-cytokine grafted proteins have also been used in combination with immune checkpoint inhibitors. In one embodiment, the antibody-cytokine grafted protein may be administered. PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEA CAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD an immune checkpoint molecule selected from one or more of 160, 2B4, or TGFR In one embodiment, the compound can be administered in combination with an inhibitor of immune checkpoint inhibitors. The inhibitor is an anti-PD-1 antibody, where the anti-PD-1 antibody is nivolumab, pembrolizumab, In some embodiments, the anti-PD-1 antibody molecule is selected from nivolumab or pidilizumab. Nivolumab is also known as MDX-1106, MDX-1106-04, In some embodiments, the antibody may be selected from the group consisting of ONO-4538, ONO-4538, and BMS-936558. The PD-1 antibody is nivolumab (CAS registration number: 946414-94-4). Mab is a fully human IgG4 monoclonal antibody that specifically blocks PD1. Mab (clone 5C4) and other human monoclonal antibodies that specifically bind to PD1 The body is a material as described in U.S. Pat. No. 8,008,449 and WO 2006 / 121168 is disclosed in the brochure.

[0208] In some embodiments, the anti-PD-1 antibody is pembrolizumab. lambrolizumab (also known as MK-3475, MK03475, SCH-900475, or KEYTRUDA® (Merck) is a humanized IgG4 antibody that binds to PD-1. It is a monoclonal antibody. Pembrolizumab and other humanized anti-PD-1 antibodies: Hamid, O. et al. (2013) New England Journal of Medicine 369(2):134-44, U.S. Patent No. 8,354,509 and WO 2009 / 114335.

[0209] In some embodiments, the anti-PD-1 antibody is pidilizumab. -011 (Cure Tech) is a humanized IgG1k monoclonal antibody that binds to PD1. Pidilizumab and other humanized anti-PD-1 monoclonal antibodies are being studied internationally. This is disclosed in Publication No. 2009 / 101611.

[0210] Other anti-PD1 antibodies include AMP 514 (Amplimmune) and, for example, U.S. Patent No. 8,609,089, U.S. Patent Application Publication No. 2010 / 028330 The specification, and / or the specification of U.S. Patent Application Publication No. 2012 / 0114649 and U.S. Patent Examples include the anti-PD1 antibodies disclosed in Application Publication No. 2016 / 0108123. In some embodiments, the antibody-cytokine grafted protein is a compound according to the method of the present invention, as described in U.S. Pat. It is also possible to administer the compound in combination with the anti-Tim3 antibody disclosed in the specification of 2015 / 0218274. In another embodiment, the antibody-cytokine grafted protein is prepared as described in U.S. Pat. Durva, an anti-PD-L1 antibody disclosed in Patent Publication No. 2016 / 0108123 lumab® (MEDI4736), Atezolizumab® (MPDL3280A) or Avelumab® . [Example]

[0211] Example 1: Creation of IL2 antibody cytokine grafted protein The IL2 sequence was engineered into the CDR regions of various immunoglobulin scaffolds to produce antibody sizes. Tokine-grafted proteins were then prepared, and both heavy and light immunoglobulin chains were then added. The final antibody-cytokine protein was generated using the antibody-cytokine grafting method. The protein confers the favorable therapeutic properties of IL2; however, the antibody cytokine group Rafted proteins have desirable effects such as increased Treg cell activity compared to rhIL2. The effectiveness of this treatment has decreased.

[0212] To create antibody-cytokine grafted proteins, IL2 sequences containing muteins ( SEQ ID NO: 4 or 6) was inserted into the CDR loop of the immunoglobulin chain scaffold. Various known immunoglobulin sequences and germline antibody sequences are used in An antibody-cytokine grafted protein was prepared using the antibody-cytokine grafted protein. The sequence of IL2 in the target scaffold is shown in Table 2. The insertion points were determined based on available structural or homology models. Based on the data, the midpoint of the loop was selected. Proteins are isolated using standard molecular biology methods using recombinant DNA encoding the relevant sequences. This was generated.

[0213] The choice of which CDR to select for cytokine grafting depends on the following parameters: Selected: desired biological and biophysical properties and favorable development profile. In predicting which positions within the DRs and CDRs will provide the desired parameters, The modeling software was only partially useful, therefore all six possible antibody samples were not included. The grafts are then prepared and evaluated in biological assays. If this is achieved, then the antibody-cytokine grafted molecule binds to the respective cytokine receptor. and biophysical properties such as structural decomposition of how they interact with each other.

[0214] Regarding the IL2 antibody cytokine grafted molecule, we first considered cytokine grafting. The structure of the antibody candidate was solved. From this structure, it was found that the paratope is located at the N-terminus of the antibody "arm." The fact that the cytokines are located at the tip of the nucleus and that grafting them to this location will activate their respective receptors It was found that cytokines would be presented to the grafting technique. Each antibody IL2 grafted protein contains CDR loops of different lengths, sequences and structural environments. Therefore, LCDR-1, LCDR-2, LCDR-3 and HCDR-1 IL2 was grafted onto all six CDRs corresponding to HCDR-2 and HCDR-3. From this table in Figure 1, the IL2 grafted onto the light chain of CDR2 (IgG.IL2.L2 ) was not expressed, and the antibody-cytokine grafted protein was not It is clear that there are differences in the Fc function. It was also observed that the L2 antibody cytokine graft had a better profile.

[0215] The IL2 points that had the best combination of properties (biophysical and biological) and were included HCDR-1 was chosen because the mutation enhanced the desired biological properties. For selection, the structural center of the CDR loop is the largest on either side (linear size 3. The spacing was chosen based on what could be achieved by any one theory (8 Å × number of residues). Although not constrained by this, this may independently allow for easier folding of IL2. This provided a stable molecule. The structure of the graft scaffold GFTX3b was already known, Therefore, the structural center of each CDR was also known. This was determined using the Chothia numbering system. As mentioned above, the CDR loop sequence of the IL-2 graft was The insertion point can be shifted away from the center of the CDR loop towards either the N-terminal or C-terminal end. However, shifting IL2 within the CDR loop did not significantly affect biological activity. No significant difference occurred.

[0216] In summary, grafting into the CDRs provides a level of steric hindrance to the IL2 receptor. Based on the structure, the CDRs in each subunit are The insertion point was selected. The final decision on which CDR graft is best for a particular cytokine was made. The selection was based on the desired biological and biophysical properties. Cytokine / receptor interactions and signaling mechanisms also played a role, and This is done by comparing each individual antibody cytokine molecule with respect to its respective properties. It was.

[0217] [Table 1-1]

[0218] [Table 1-2]

[0219] [Table 2-1]

[0220] [Table 2-2]

[0221] [Table 2-3]

[0222] [Table 2-4]

[0223] [Table 2-5]

[0224] [Table 2-6]

[0225] [Table 2-7]

[0226] [Table 2-8]

[0227] Example 2: IgG.IL2R67A.H1 inhibits Proleukin® (IL-2 ) has a long half-life compared to Naive CD-1 mice were IP-administered and the doses were measured at 1 hour, 3, 7, 24, and 3 hours after administration. All animals were bled at 1, 48, 55, and 72 hours. Blood samples were centrifuged and plasma samples were collected. The obtained plasma samples were transferred to single polypropylene tubes and frozen at -80°C. All samples were analyzed and plasma concentrations of IgG.IL2R67A.H1 were determined by immunoassay. Pharmacokinetic parameters such as half-life were calculated. Each sample was prepared in duplicate. Each duplicate run required 5 μL of sample diluted 1:20. Capture: goat anti-human IL-2 biotinylated antibody (R&D Systems BAF202 ) Detection: Alexa 647 anti-human IL-2, clone MQ1-17H12 (Biole All immunoassays were performed using Gyrolab® Bio The results were obtained using the Oaffy200 in conjunction with the Gyros CD-200. As shown, the half-life of IgG.IL2R67A.H1 is approximately 12 hours, followed by It decays over 48 hours. The half-life of Proleukin® is approximately 4 Because of the time involved, this could not be shown on the graph.

[0228] Example 3: IgG.IL2R67A.H1 inhibits CD8 T effector responses in normal B6 mice selectively expands the target and is more potent than IL-2 Fc or Proleukin® Well tolerated IgG.IL2R67A.H1 inhibited Proleukin® compared to Tregs It increases CD8 T effector activity without causing adverse events associated with administration. After administration to mice on day 1, CD8 T effector expansion was observed on days 4, 8, and 11. At each time point, the CD8 T effector cell population expanded significantly, and Tregs expanded. This is because the mortality and morbidity observed with equimolar doses of IL-2 were similar to those observed with Proleu This was in contrast to the IL-2Fc fusions.

[0229] B6 female mice were treated with Proleukin® (5 times a week), IL-2 Fc and Ig G.IL2R67A.H1 (once a week) was administered at equimolar concentrations. Eight days after the first treatment, Spleens were processed to obtain single cell suspensions and washed with RPMI (10% FBS). The cells were lysed in red blood cell lysis buffer (Sigma #R7757) and analyzed for cell number and viability. Cells were counted using FACS buffer (1x PBS + 0.5 mL) according to standard protocols. FACS staining was performed using 0.1% BSA + 0.05% sodium azide. Surface antibody: Rat anti-mouse CD3-efluor450 (Ebioscience #48 -0032), rat anti-mouse CD4-Pacific Blue (BD Pharminge n #558107), rat anti-mouse CD8-PerCp (BD Pharminge n #553036), rat anti-mouse CD44 FITC (Pharmingen # 553133), rat anti-mouse CD25-APC (Ebioscience #17- 0251), rat anti-mouse Nk1.1 (Ebioscience #95-5941) Staining with , followed by fixation / permeabilization and anti-mouse / rat FoxP3 staining set PE (Eb Cells were stained with FoxP3 according to the method described in Bioscience #72-5775. n-Dickinson LSR Fortessa® or Becton-D Analysis was performed on a Chickinson FACS LSR II and data were analyzed using FlowJo (registered trademark) ) software was used for analysis.

[0230] Figures 3A-3C show the effect of Proleukin (registered trademark) (five times a week), IL2-Fc, and I gG.IL2R67A.H1 (once per week) at equimolar concentrations with Proleukin® (IgG.IL2R67A.H1 and IL2-Fc 100 μg approx. 1 nmol IL2 Preferential expansion of CD8 T effector cells in B6 female mice after administration of 100 mg of IFN-γ-α (100 mg / kg bw) at 100 mg / kg bw (100 mg / kg bw) The data in these graphs demonstrate that CD8 T effector cells induce similar proliferation of Tregs. These data demonstrate that CD8 T effectors and This contrasts with Proleukin®, which expanded both IgG and Tregs. IL2R67A.H1 significantly increased the absolute number of CD8 T effector cells expanded and the CD8 T effector Note that the IL2-Fc fusion construct was superior in both terms of the IL2:Treg ratio. The IgG.IL2R67A.H1 antibody cytokine grafted protein is Figures 3D-3F show that IgG.IL2R67 A. The beneficial effects of H1 are more pronounced at higher doses. When B6 mice were administered IgG.IL2R67A.H1 at a dose of 100 mg / kg (IL2 equivalent), Similarly, there was a preferential expansion of CD8 T effector cells compared with Treg cells. However, in the IL2-Fc treated group, at higher levels, mice showed only a slight increase after a single dose. These results suggest that IgG.IL2R6 7A.H1 has a higher therapeutic index and a wider dosage range than IL2-Fc fusion constructs It has been pointed out that it can be safely administered at

[0231] Example 4: IgG.IL2R67A.H1 inhibits CD8 T effector -Selectively expands cells and is better tolerated than Proleukin® Non-obese diabetic (NOD) mice spontaneously develop type 1 diabetes and are an animal model of human type 1 diabetes. The same protocol for B6 mice described in Example 3 was used. Using the col, IgG.IL2R67A.H1, IL2-Fc and Proleukin ( (registered trademark) was administered to NOD mice at an equimolar amount to Proleukin (registered trademark). Again, as shown in the graph in Figure 4A, this dose of IgG.IL2R67A Administration of .H1 preferentially expanded CD8 T effector cells compared with Tregs. Therefore, administration of IgG.IL2R67A.H1 did not cause any adverse events in NOD mice. However, there were 5 moribund mice and 2 deaths in the Proleukin® treatment group. Figure 4B reports the dosage, fold change in cells, and cell types from the NOD mouse model. This is a graph.

[0232] Example 5: IgG.IL2R67A.H1 inhibits single-agent IL2R67A in the CT26 colon tumor mouse model Demonstrate effectiveness After examining the safety of IgG.IL2R67A.H1, we investigated its efficacy as a single agent in the CT26 mouse model. Mouse CT26 cells have been used in over 500 published studies. are rapidly growing grade IV colon cancer cells, a cell type often used in drug development. CT26 (ATCC CRL-2638) cells are one of the cell lines and models. The cells were grown under sterile conditions in a 37°C incubator for 2 h. The cells were cultured in 10% FBS supplemented medium. The cells were cultured in RPMI 1640 medium supplemented with HCl. The cells were subcultured every 3-4 days. Cells were harvested (passage 11) and cultured at 2.5 × 10 6 The cells were resuspended at a concentration of 1 / ml. Cells were Radil tested for mycoplasma and mouse viruses. A total of 0.25 × 10 6 Cells were injected into a 28g needle (100µl injection volume) After implantation, when tumors became palpable, the animals were The specimens were measured with a caliper and weighed three times a week. The caliper measurements were calculated using (L × W × W) / 2. Mice were fed a normal diet and maintained in accordance with the Guide for the Care and Use of Laboratory Animals (Guide for and Use of Laboratory Animals and Animal Experiments Committee (Institutional Animal Care and Use Co. The animals were kept in an SPF animal facility in accordance with the regulations of the International Animal Welfare Agency (IHA).

[0233] The tumor is approximately 100 mm 3 When the IgG-IL2 concentration reached 12.5-100 μg, the mice were R67A.H1 was administered by intraperitoneal route. Tumors were measured twice weekly. Prism 5 Mean tumor volumes were plotted using GraphPad® software. The tumor size is 1000mm 3 The efficacy study endpoint is achieved when a volume of After injection, the mice were also closely monitored for signs of clinical deterioration. However, for some reason, symptoms of respiratory distress, hunched posture, decreased activity, complete paralysis of the hind legs, and pleural effusion signs of any medical condition, including tachypnea, weight loss approaching 20% ​​or 15% plus other signs Mice are considered safe if they show signs of fainting or if their ability to perform normal activities (feeding, movement) is impaired. He was put to death mercifully.

[0234] In a 20-day study, four doses of IgG.IL2R67A.H1 were administered over 17 days. IgG.IL2R67A.H1 was effective in the CT26 mouse model at 12.5 μg to 10 The tumor volume curves shown in Figure 5 show that tumor volumes were effective at doses ranging from 15 to 10 μg. It was kept below 200mm for five days, then below 400mm for the remaining five days. This demonstrates the efficacy of IgG.IL2R67A.H1 in this study.

[0235] Example 6: IgG.IL2R67A.H1 and additional cancer therapeutics in the B16 mouse model Demonstrates effectiveness in To evaluate the efficacy of IgG.IL2R67A.H1 in combination with other cancer therapeutics, The 6F10 melanoma mouse model was used. B16F10 cells (ATCC CRL-6 475) were grown in a 5% CO2, 37°C incubator under sterile conditions for 2 weeks. B16F10 cells were cultured in DMEM + 10% FBS. The cells were harvested and cultured in FBS-free medium. Concentrate 1 x 10 ml of DMEM 6 B16F10 cells were resuspended at a concentration of 1 / 100 μl. Radil tested for plasma and mouse viruses. 28-gauge needle (100 μl injection) The cells were implanted into the right flank of B6 mice using a 1000 μg / ml (1000 μg / ml) ... Mice were measured with calipers and weighed twice a week. Caliper measurements were calculated using the formula (L × W × W) / 2. and calculated it.

[0236] In this study, IgG.IL2R67A.H1 was administered as a single agent or in B16F10 cells. It was used in combination with the TA99 antibody, which binds to the expressed antigen, Trp1. The fusions were administered as single agents or in combination with TA99 antibody. was administered as a single agent.

[0237] Surprisingly, in this model, IgG.IL-1 was significantly increased when administered as a single agent at a 500 μg dose. 2R67A.H1 was the most effective treatment (Figure 6). The next best treatment was IgG.IL The combination was 2R67A.H1 (100 μg) and TA99. of IgG.IL2F71A.H1 as a single agent, 500 μg of IgG.IL2F71A. TA99 in combination with H1, and as a single agent or in IL2-Fc / TA99 combination TA99 was more effective than IL2-Fc when administered alone. The mean tumor volume was similar to that of untreated controls. This data supports the results of IgG.IL2R67A. H1 is effective as a single agent in melanoma mouse tumor models, but it is also It has also proven effective when combined with other anti-cancer drugs.

[0238] Example 7: IgG.IL2R67A.H1 and IgG.IL2F71A in human cells .H1 activity Testing the activity of IgG.IL2R67A.H1 against human CD8 T effectors To investigate this, human peripheral blood mononuclear cells (PBMCs) were assayed for pSTAT5 activity. C cells were placed in serum-free test medium and plated. PBMCs were incubated with IgG and IL2R. 67A.H1, IgG.IL2F71A.H1 or Proleukin® The cells were then incubated at 37°C for 20 minutes. After 20 minutes, the cells were soaked in 1.6% formaldehyde. After 30 minutes at room temperature, the samples were washed, resuspended, and stained with surface markers. The cell pellet was permeabilized with -20°C methanol, washed, and transfected with pSTAT5 and DNA. Cells were stained for intercalator. Cells were run on Cytof® and data were analyzed by F pSTAT5 activity levels were determined by analysis with lowJo® software. The table in Figure 7 shows the effect of IgG.IL2R67A.H1 on CD8 T effector cells. demonstrate preferential activation of Treg cells against Tregs and minimize activation of Treg cells.

[0239] Example 8: Conjugation of antibody-cytokine grafted proteins The IL2 sequence (SEQ ID NO: 4) containing the mutein was inserted into the CDR loops of the immunoglobulin chain scaffold. Antibody-cytokine grafted proteins are being utilized in clinical settings. A variety of known immunoglobulin sequences as well as germline antibody sequences were used. One of the antibodies has RSV as its antigen. IL2 is labeled into the CDR of this antibody. To determine whether rafting reduced or abolished binding to RSV, PBS or ELISA assays for RSV proteins were performed in either carbonate buffer. As shown in Figure 8, this is influenced by which CDRs are chosen for IL2 grafting. For example, IgG.IL2R67A.H1 appears to be the original, ungrafted (unmodified) In contrast, IL2 binds to the light chain of CDR3 (CDR-L3) and has similar RSV binding to the antibody. Grafting IL2 onto CDR-H3 or CDR-H4 reduces binding. When grafted onto an antibody (Xolair), no binding occurs. This is because the antibody cytokine The grafted protein may retain the binding of the antibody scaffold to its original target, or this result This demonstrates that the correlation can be reduced.

[0240] It is understood that the examples and embodiments described herein are for illustrative purposes, and that various modifications or variations therein may be suggested to those skilled in the art and are to be encompassed within the spirit and scope of this application and the appended claims. All publications, sequence accession numbers, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. The present invention may include the following aspects. [1] (a) a heavy chain variable region (VH) including complementarity determining regions (CDRs) HCDR1, HCDR2, and HCDR3; and (b) a light chain variable region (VL) comprising LCDR1, LCDR2, and LCDR3; and (c) An antibody cytokine grafted protein comprising an interleukin 2 (IL2) molecule grafted into the CDR of VH or VL. [2] The antibody-cytokine grafted protein of claim 1, wherein the IL2 molecule is grafted into the heavy chain CDR. [3] The antibody-cytokine grafted protein of claim 2, wherein the heavy chain CDRs are selected from complementarity determining region 1 (HCDR1), complementarity determining region 2 (HCDR2), and complementarity determining region 3 (HCDR3). [4] 4. The antibody-cytokine grafted protein of claim 3, wherein the IL2 molecule is grafted into HCDR1. [5] The antibody-cytokine grafted protein of claim 1, wherein the IL2 molecule is grafted into the light chain CDR. [6] The antibody-cytokine grafted protein of claim 5, wherein the light chain CDR is selected from complementarity determining region 1 (LCDR1), complementarity determining region 2 (LCDR2), and complementarity determining region 3 (LCDR3). [7] 7. The antibody-cytokine-grafted protein of claim 1, wherein the IL2 molecule comprises a mutation that reduces the affinity of the IL2 molecule for the high-affinity IL2 receptor. [8] 8. The antibody-cytokine-grafted protein according to claim 1, wherein stimulation of CD8 T effector cell proliferation by the antibody-cytokine-grafted protein is greater than that by recombinant IL2 or Proleukin®. [9] 9. The antibody-cytokine-grafted protein according to claim 1, wherein the stimulation of CD4 T regulatory cell proliferation by the antibody-cytokine-grafted protein is less than that by recombinant IL2 or Proleukin®.

[10] 10. The antibody-cytokine-grafted protein according to claim 1, wherein stimulation of NK cell proliferation by the antibody-cytokine-grafted protein is greater than that by recombinant IL2 or Proleukin (registered trademark).

[11] 11. The antibody-cytokine-grafted protein according to claim 1, wherein the antibody-cytokine-grafted protein has a longer half-life than native IL2 or Proleukin®.

[12] 12. The antibody-cytokine-grafted protein according to claim 1, wherein the IL2 molecule consists of SEQ ID NO:4.

[13] 12. The antibody-cytokine-grafted protein according to claim 1, wherein the IL2 molecule consists of SEQ ID NO:6.

[14] 14. The antibody-cytokine-grafted protein according to claim 1, further comprising an IgG class antibody heavy chain.

[15] The antibody-cytokine-grafted protein of claim 14, wherein the IgG class heavy chain is selected from IgG1, IgG2, and IgG4.

[16] 16. The antibody-cytokine-grafted protein of claim 1, wherein the binding specificity of the CDR for the target is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or 100% by the grafted IL2 molecule.

[17] 16. The antibody-cytokine-grafted protein of claim 1, wherein the binding specificity of the CDR for the target is retained by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or 100% in the presence of the grafted IL2 molecule.

[18] 18. The antibody-cytokine-grafted protein according to claim 1, wherein the binding specificity of the CDR is different from the binding specificity of the IL2 molecule.

[19] 19. The antibody-cytokine-grafted protein according to claim 1, wherein the binding specificity of the CDR is for a non-human antigen.

[20] 20. The antibody-cytokine-grafted protein of claim 19, wherein the non-human antigen is a virus.

[21] 21. The antibody-cytokine-grafted protein of claim 20, wherein the virus is respiratory syncytial virus (RSV).

[22] The antibody-cytokine grafted protein of claim 21, wherein the RSV is selected from RSV subgroup A and RSV subgroup B.

[23] 23. The antibody-cytokine-grafted protein of claim 1, wherein the antibody scaffold portion of the antibody-cytokine-grafted protein is humanized or human.

[24] (i) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 13, (b) an HCDR2 of SEQ ID NO: 14, (c) an HCDR3 of SEQ ID NO: 15, and (d) an LCDR1 of SEQ ID NO: 29, (e) an LCDR2 of SEQ ID NO: 30, and (f) an LCDR3 of SEQ ID NO: 31; or (ii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 45, (b) an HCDR2 of SEQ ID NO: 46, (c) an HCDR3 of SEQ ID NO: 47; and (d) an LCDR1 of SEQ ID NO: 61, (e) an LCDR2 of SEQ ID NO: 62, and (f) an LCDR3 of SEQ ID NO: 63. An antibody-cytokine grafted protein comprising:

[25] (i) a heavy chain variable region (VH) comprising SEQ ID NO: 19, and a light chain variable region (VL) comprising SEQ ID NO: 35; or (ii) a heavy chain variable region (VH) comprising SEQ ID NO: 51 and a light chain variable region (VL) comprising SEQ ID NO: 67 An antibody-cytokine grafted protein comprising:

[26] 26. The antibody-cytokine-grafted protein according to any one of claims 1 to 25, further comprising a modified Fc region corresponding to a reduced effector function.

[27] 27. The antibody cytokine grafted protein of claim 26, wherein the modified Fc region comprises a mutation selected from one or more of D265A, P329A, P329G, N297A, L234A, and L235A.

[28] 28. The antibody cytokine grafted protein of claim 27, wherein the modified Fc region comprises a combination of mutations selected from one or more of: D265A / P329A, D265A / N297A, L234 / L235A, P329A / L234A / L235A, and P329G / L234A / L235A.

[29] An antibody cytokine-grafted protein 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 of SEQ ID NO: 30, LCDR3 of SEQ ID NO: 31, and a modified Fc region containing mutations D265A / P329A, which stimulates lower activation of Treg cells compared to Proleukin®.

[30] An antibody cytokine-grafted protein 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 of SEQ ID NO: 62, LCDR3 of SEQ ID NO: 63, and a modified Fc region containing mutations D265A / P329A, which stimulates lower activation of Treg cells compared to Proleukin®.

[31] An isolated nucleic acid encoding an antibody cytokine-grafted protein comprising: (i) a heavy chain of SEQ ID NO: 22 and a light chain of SEQ ID NO: 38; or (ii) a heavy chain of SEQ ID NO: 54 and a light chain of SEQ ID NO: 70.

[32] 32. A recombinant host cell suitable for producing an antibody cytokine grafted protein, comprising the nucleic acid of claim 31 encoding the heavy and light chain polypeptides of said antibody cytokine grafted protein, and optionally a secretion signal.

[33] 33. The recombinant host cell of claim 32, which is a mammalian cell line.

[34] 34. The recombinant host cell of claim 33, wherein the mammalian cell line is a CHO cell line.

[35] 31. A pharmaceutical composition comprising the antibody-cytokine-grafted protein according to any one of claims 1 to 30 and a pharmaceutically acceptable carrier.

[36] 36. A method for treating cancer in an individual in need thereof, comprising administering to said individual a therapeutically effective amount of an antibody-cytokine-grafted protein according to any one of claims 1 to 30 or a pharmaceutical composition according to claim 35.

[37] 37. The method of claim 36, wherein the cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer, and lymphoma.

[38] 38. The method of claim 36 or 37, wherein the antibody-cytokine-grafted protein or pharmaceutical composition is administered in combination with another therapeutic agent.

[39] 39. The method of claim 38, wherein the therapeutic agent is another antibody-cytokine grafted protein.

[40] 39. The method of claim 38, wherein the therapeutic agent is an immune checkpoint inhibitor.

[41] 41. The method of claim 40, 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.

[42] 36. A method for expanding CD8 T effector cells in a patient in need thereof, comprising administering to said patient an antibody-cytokine-grafted protein according to any one of claims 1 to 30 or a pharmaceutical composition according to claim 35.

[43] 43. The method of claim 42, wherein the CD8 T effector cells are expanded and the Treg cells are not expanded.

[44] 43. The method of claim 42, wherein the CD8 T effector cells are expanded and the NK cells are not expanded.

[45] The method according to any one of claims 42 to 44, further comprising administration of an immune checkpoint inhibitor.

[46] 46. ​​The method of claim 45, 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.

[47] In the treatment of cancer, (i) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 13, (b) an HCDR2 of SEQ ID NO: 14, (c) an HCDR3 of SEQ ID NO: 15, and (d) an LCDR1 of SEQ ID NO: 29, (e) an LCDR2 of SEQ ID NO: 30, and (f) an LCDR3 of SEQ ID NO: 31; or (ii) a heavy chain variable region comprising (a) an HCDR1 of SEQ ID NO: 45, (b) an HCDR2 of SEQ ID NO: 46, (c) an HCDR3 of SEQ ID NO: 47; and (d) an LCDR1 of SEQ ID NO: 61, (e) an LCDR2 of SEQ ID NO: 62, and (f) an LCDR3 of SEQ ID NO: 63. Use of an antibody-cytokine grafted protein comprising:

[48] 48. The use of claim 47, wherein the antibody-cytokine-grafted protein is administered in combination with another therapeutic agent.

[49] 49. The use of claim 48, wherein the therapeutic agent is an immune checkpoint inhibitor.

[50] 50. The use of claim 49, 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.

Claims

1. (a) an IgG class heavy chain comprising an IgG class heavy chain variable region (VH) including complementarity determining regions (CDRs) HCDR1 having a grafted mutant interleukin 2 (IL2) molecule comprising SEQ ID NO: 7, HCDR2 having SEQ ID NO: 8, and HCDR3 having SEQ ID NO: 9; and an IgG class light chain comprising an IgG class light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 23, an LCDR2 comprising SEQ ID NO: 24, and an LCDR3 comprising SEQ ID NO: 25; (b) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO:26, an LCDR2 comprising SEQ ID NO:27, and an LCDR3 comprising SEQ ID NO:28; (c) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 13, an HCDR2 comprising SEQ ID NO: 14, and an HCDR3 comprising SEQ ID NO: 15; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO:29, an LCDR2 comprising SEQ ID NO:30, and an LCDR3 comprising SEQ ID NO:31; (d) an IgG class heavy chain comprising an IgG class VH comprising a mutated IL2 molecule grafted with HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17, and HCDR3 comprising SEQ ID NO: 18; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 32, an LCDR2 comprising SEQ ID NO: 33, and an LCDR3 comprising SEQ ID NO: 34; (e) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 39, an HCDR2 comprising SEQ ID NO: 40, and an HCDR3 comprising SEQ ID NO: 41; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 55, an LCDR2 comprising SEQ ID NO: 56, and an LCDR3 comprising SEQ ID NO: 57; (f) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 42, an HCDR2 comprising SEQ ID NO: 43, and an HCDR3 comprising SEQ ID NO: 44; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 58, an LCDR2 comprising SEQ ID NO: 59, and an LCDR3 comprising SEQ ID NO: 60; (g) an IgG class heavy chain comprising an IgG class VH comprising a mutated IL2 molecule grafted with HCDR1 comprising SEQ ID NO: 45, HCDR2 comprising SEQ ID NO: 46, and HCDR3 comprising SEQ ID NO: 47; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 61, an LCDR2 comprising SEQ ID NO: 62, and an LCDR3 comprising SEQ ID NO: 63; or (h) an IgG class heavy chain comprising an IgG class VH comprising a mutated IL2 molecule grafted with HCDR1 comprising SEQ ID NO: 48, HCDR2 comprising SEQ ID NO: 49, and HCDR3 comprising SEQ ID NO: 50; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 64, an LCDR2 comprising SEQ ID NO: 65, and an LCDR3 comprising SEQ ID NO: 66; an antibody-cytokine grafted protein comprising: An antibody-cytokine grafted protein, wherein the mutated IL2 molecule comprises a mutation that reduces the affinity of the mutated IL2 molecule for the high affinity IL2 receptor.

2. (a) an IgG class heavy chain comprising an IgG class heavy chain variable region (VH) including complementarity determining regions (CDRs) HCDR1 having a grafted mutant interleukin 2 (IL2) molecule comprising SEQ ID NO: 7, HCDR2 having SEQ ID NO: 8, and HCDR3 having SEQ ID NO: 9; and an IgG class light chain comprising an IgG class light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 23, an LCDR2 comprising SEQ ID NO: 24, and an LCDR3 comprising SEQ ID NO: 25; (b) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO:26, an LCDR2 comprising SEQ ID NO:27, and an LCDR3 comprising SEQ ID NO:28; (c) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 13, an HCDR2 comprising SEQ ID NO: 14, and an HCDR3 comprising SEQ ID NO: 15; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO:29, an LCDR2 comprising SEQ ID NO:30, and an LCDR3 comprising SEQ ID NO:31; (d) an IgG class heavy chain comprising an IgG class VH comprising a mutated IL2 molecule grafted with HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17, and HCDR3 comprising SEQ ID NO: 18; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 32, an LCDR2 comprising SEQ ID NO: 33, and an LCDR3 comprising SEQ ID NO: 34; (e) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 39, an HCDR2 comprising SEQ ID NO: 40, and an HCDR3 comprising SEQ ID NO: 41; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 55, an LCDR2 comprising SEQ ID NO: 56, and an LCDR3 comprising SEQ ID NO: 57; (f) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 42, an HCDR2 comprising SEQ ID NO: 43, and an HCDR3 comprising SEQ ID NO: 44; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 58, an LCDR2 comprising SEQ ID NO: 59, and an LCDR3 comprising SEQ ID NO: 60; (g) an IgG class heavy chain comprising an IgG class VH comprising a mutated IL2 molecule grafted with HCDR1 comprising SEQ ID NO: 45, HCDR2 comprising SEQ ID NO: 46, and HCDR3 comprising SEQ ID NO: 47; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 61, an LCDR2 comprising SEQ ID NO: 62, and an LCDR3 comprising SEQ ID NO: 63; or (h) an IgG class heavy chain comprising an IgG class VH comprising a mutated IL2 molecule grafted with HCDR1 comprising SEQ ID NO: 48, HCDR2 comprising SEQ ID NO: 49, and HCDR3 comprising SEQ ID NO: 50; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 64, an LCDR2 comprising SEQ ID NO: 65, and an LCDR3 comprising SEQ ID NO: 66; an antibody-cytokine grafted protein comprising: An antibody-cytokine grafted protein, wherein the antibody-cytokine grafted protein preferentially expands T effector cells over regulatory T cells. (a) an IgG class heavy chain comprising an IgG class heavy chain variable region (VH) comprising complementarity determining regions (CDRs) HCDR1 having a grafted mutant interleukin 2 (IL2) molecule comprising SEQ ID NO:7, HCDR2 having SEQ ID NO:8, and HCDR3 having SEQ ID NO:9; and an IgG class light chain comprising an IgG class light chain variable region (VL) comprising an LCDR1 comprising SEQ ID NO: 23, an LCDR2 comprising SEQ ID NO: 24, and an LCDR3 comprising SEQ ID NO: 25; (b) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 10, an HCDR2 comprising SEQ ID NO: 11, and an HCDR3 comprising SEQ ID NO: 12; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO:26, an LCDR2 comprising SEQ ID NO:27, and an LCDR3 comprising SEQ ID NO:28; (c) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 13, an HCDR2 comprising SEQ ID NO: 14, and an HCDR3 comprising SEQ ID NO: 15; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO:29, an LCDR2 comprising SEQ ID NO:30, and an LCDR3 comprising SEQ ID NO:31; (d) an IgG class heavy chain comprising an IgG class VH comprising a mutated IL2 molecule grafted with HCDR1 comprising SEQ ID NO: 16, HCDR2 comprising SEQ ID NO: 17, and HCDR3 comprising SEQ ID NO: 18; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 32, an LCDR2 comprising SEQ ID NO: 33, and an LCDR3 comprising SEQ ID NO: 34; (e) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 39, an HCDR2 comprising SEQ ID NO: 40, and an HCDR3 comprising SEQ ID NO: 41; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 55, an LCDR2 comprising SEQ ID NO: 56, and an LCDR3 comprising SEQ ID NO: 57; (f) an IgG class heavy chain comprising an IgG class VH comprising a HCDR1 grafted with a mutant IL2 molecule comprising SEQ ID NO: 42, an HCDR2 comprising SEQ ID NO: 43, and an HCDR3 comprising SEQ ID NO: 44; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 58, an LCDR2 comprising SEQ ID NO: 59, and an LCDR3 comprising SEQ ID NO: 60; (g) an IgG class heavy chain comprising an IgG class VH comprising a mutated IL2 molecule grafted with HCDR1 comprising SEQ ID NO: 45, HCDR2 comprising SEQ ID NO: 46, and HCDR3 comprising SEQ ID NO: 47; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 61, an LCDR2 comprising SEQ ID NO: 62, and an LCDR3 comprising SEQ ID NO: 63; or (h) an IgG class heavy chain comprising an IgG class VH comprising a mutated IL2 molecule grafted with HCDR1 comprising SEQ ID NO: 48, HCDR2 comprising SEQ ID NO: 49, and HCDR3 comprising SEQ ID NO: 50; and an IgG class light chain comprising an IgG class VL comprising an LCDR1 comprising SEQ ID NO: 64, an LCDR2 comprising SEQ ID NO: 65, and an LCDR3 comprising SEQ ID NO: 66; an antibody-cytokine grafted protein comprising: An antibody-cytokine-grafted protein, wherein the antibody-cytokine-grafted protein stimulates one or more of CD8+ T effector cell proliferation, natural killer (NK) cell proliferation, or a combination thereof, to a greater extent than recombinant IL2 or aldesleukin.

4. 2. The antibody-cytokine grafted protein of claim 1, wherein the VH comprises SEQ ID NO: 19 or SEQ ID NO: 51, and the VL comprises SEQ ID NO: 35 or SEQ ID NO:

67.

5. The antibody-cytokine grafted protein of claim 1 , wherein the IgG class heavy chain is selected from IgG1, IgG2, and IgG4.

6. The antibody-cytokine grafted protein of claim 1, wherein the IgG class heavy chain comprises SEQ ID NO: 21 or SEQ ID NO:

53.

7. The antibody-cytokine grafted protein of claim 1, wherein the IgG class light chain comprises SEQ ID NO: 69 and the IgG class heavy chain comprises SEQ ID NO:

53.

8. The antibody-cytokine grafted protein of claim 1, wherein the IgG class light chain comprises SEQ ID NO: 37 and the IgG class heavy chain comprises SEQ ID NO:

21.

9. The antibody-cytokine grafted protein of claim 1, wherein the IgG class light chain comprises SEQ ID NO: 69 and the IgG class heavy chain comprises SEQ ID NO:

21.

10. The antibody-cytokine grafted protein of claim 1, wherein the IgG class light chain comprises SEQ ID NO: 37 and the IgG class heavy chain comprises SEQ ID NO:

53.

11. A pharmaceutical composition comprising the antibody-cytokine-grafted protein according to any one of claims 1 to 10 and a pharmaceutically acceptable carrier.

12. A pharmaceutical composition for treating cancer, comprising the antibody-cytokine-grafted protein according to any one of claims 1 to 10.

13. 13. The pharmaceutical composition of claim 12, wherein the cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer and lymphoma.

14. The pharmaceutical composition of claim 12, wherein the antibody-cytokine grafted protein is administered in combination with another therapeutic agent.

15. 15. The pharmaceutical composition of claim 14, wherein the therapeutic agent is a second antibody-cytokine-grafted protein.

16. The pharmaceutical composition of claim 14, wherein the therapeutic agent is an immune checkpoint molecule inhibitor.

17. 17. The pharmaceutical composition of claim 16, wherein the immune checkpoint molecule 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. 15. The pharmaceutical composition of claim 14, wherein the therapeutic agent is a tyrosine kinase inhibitor.

19. Use of the antibody-cytokine-grafted protein according to any one of claims 1 to 10 or the pharmaceutical composition according to claim 11 in the manufacture of a medicament for treating cancer.

20. 20. The use according to claim 19, wherein the cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer and lymphoma.

21. 20. The use of claim 19, wherein the antibody-cytokine-grafted protein or pharmaceutical composition is used in combination with another therapeutic agent.

22. 22. The use of claim 21, wherein the therapeutic agent is a second antibody-cytokine grafted protein.

23. 22. The use of claim 21, wherein the therapeutic agent is an immune checkpoint molecule inhibitor.

24. 24. The use of claim 23, wherein the immune checkpoint molecule 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.

25. 22. The use of claim 21, wherein the therapeutic agent is a tyrosine kinase inhibitor.

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