Antibody-cytokine engrafted proteins and methods of use in treatment of cancer

Abstract

To provide IL2 engrafted into the CDR sequences of an antibody having preferred therapeutic profiles over molecules known and used in the clinic.SOLUTION: The present invention provides an antibody cytokine engrafted protein comprising an antibody having a specific sequence and IL-2. The antibody cytokine engrafted protein composition increases or maintains CD8+ T effector cells while reducing the activity of Treg cells. Additionally, the provided composition conveys improved half-life, stability and productivity.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 / 0133636, 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 ingrafted proteins and methods of 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 tide, and is folded 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 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 has been 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+ is expressed in regulatory T cells (Tregs). In contrast, the low affinity IL2 receptor is found in CD8+ T effector and natural killer (NK) cells.

[0006] Recombinant IL2 (rhIL2) was originally approved for clinical use in 1992 (Covenant et al., 2003). try et al.,Cancer Mgt Res.2012 4:215-221 Proleukin® (aldesleukin) is a non-glycosylated, N A modified IL lacking the terminal alanine and replacing the cysteine ​​at amino acid 125 with serine. 2 Proleukin® was initially approved as a treatment for melanoma and renal cell carcinoma. It has been approved as a forensic drug but has not been used in other cancer types, such as colorectal cancer, breast cancer, lung cancer, and mesothelioma. (Coventry, op. cit.) 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). This corresponds to an overall objective response rate of 20%, which is 7 out of 10 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 pulmonary embolism have shown capillary / vascular leakage, vasodilation, and oliguria. Grade 3 and Grade 4 neutrophil dysfunction, both in patients with pulmonary tuberculosis and in those with general infections There was also an infection with Proleukin 4 (Klapper et al., supra). The literature on Proleukin® indicates that Proleukin® is effective in treating Crohn's disease, scleroderma, and 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 the high-affinity IL2 receptor and are critical for survival and function in the control of IL2 The finding that the drug was dependent on 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 any This allows 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. In particular, the antibody sequence provided is an antibody having an IL2 grafted thereto. The cytokine-grafted protein composition reduces the activity of Treg cells while suppressing C In addition, the compositions provided herein are intended to increase or maintain D8+ T effector cells. The composition is an improvement over recombinant human IL2 preparations such as Proleukin®. The present disclosure thus provides a method for the production of IL2-binding proteins that bind to the high affinity IL2 receptor. IL2 binds to and promotes favorable signaling through the low affinity receptor with reduced activity. The present invention provides an antibody-cytokine grafted protein that promotes the expression of a cellular signaling pathway. (i) A heavy chain variable region (VH) (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 regions (CDRs) 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 CDRs of VH or VL; The present invention provides an antibody-cytokine grafted protein comprising:

[0011] Antibody cytokine grafted protein comprising an IL2 molecule grafted onto a heavy chain CDR 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 protein containing an IL2 molecule grafted to HCDR1 Quality.

[0014] Antibody cytokine grafted protein comprising an IL2 molecule grafted onto a light chain CDR 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) thereof. Cytokine-grafted proteins.

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

[0017] Stimulation of CD8 T cell effector proliferation by antibody-cytokine grafted proteins antibody cytokine group, which has a higher activity than recombinant IL2 or Proleukin® Rafted proteins.

[0018] Stimulation of Treg cell proliferation by antibody-cytokine grafted protein enhances the expression of recombinant IL2 or an antibody-cytokine grafted protein smaller than Proleukin® Quality.

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

[0020] The antibody-cytokine grafted protein is recombinant IL2 or Proleukin (registered An antibody-cytokine grafted protein having a longer half-life than IgG1.

[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] The antibody cytokine gram-positive antibody, 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 protein.

[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 is different from that of the IL2 molecule protein.

[0028] The binding specificity of the CDRs is for a non-human target. Compact quality.

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

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

[0031] The antibody sequence of claim 1, wherein the RSV is selected from RSV subgroup A and RSV subgroup B. Tocainine-grafted proteins.

[0032] The antibody scaffold portion of the antibody cytokine grafted protein is humanized or human. Cytokine-grafted proteins.

[0033] An embodiment of the present disclosure includes (i) (a) HCDR1 of SEQ ID NO: 13, (b) HCDR2 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: 3. (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: 47, an HCDR3 of SEQ ID NO: 48, and (e) LCDR1 of sequence number 61, (f) LCDR2 of sequence number 62, and (g) LCDR3 of sequence number 63. Provided is an antibody cytokine grafted protein comprising a light chain variable region comprising LCDR3 .

[0034] An embodiment of the disclosure includes a nucleic acid sequence comprising: (i) a heavy chain variable region (VH) comprising SEQ ID NO: 19; and 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. Compact quality.

[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: 19, HCDR7 of SEQ ID NO: 20, HCDR8 of SEQ ID NO: 21, 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 The present invention provides an antibody-cytokine grafted protein, IL2 or Proleukin® reduced Treg cell counts Stimulates activation.

[0039] An embodiment of the present disclosure includes HCDR1 of SEQ ID NO: 45, HCDR2 of SEQ ID NO: 46, HCDR3 of SEQ ID NO: 47, HCDR4 of SEQ ID NO: 49, HCDR5 of SEQ ID NO: 50, HCDR6 of SEQ ID NO: 51, HCDR7 of SEQ ID NO: 52, HCDR8 of SEQ ID NO: 53, HCDR9 of SEQ ID No. 47, LCDR1 of SEQ ID NO: 61, LCDR2 of SEQ ID NO: 62, The antibody sequence contains 63 LCDR3, a modified Fc region containing the mutations D265A / P329A. The present invention provides an antibody-cytokine grafted protein, IL2 or Proleukin® reduced Treg cell counts Stimulates activation.

[0040] An embodiment of the disclosure includes (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 fusogenic protein is provided.

[0041] Embodiments of the present disclosure include heavy and light chain polypeptides of a protein, and optionally a secretory system. and an antibody-cytokine grafted protein comprising a nucleic acid as disclosed herein encoding a signal. A recombinant host cell suitable for producing the protein is provided.

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

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

[0044] An embodiment of the present disclosure is directed to the antibody-cytokine grafted protein disclosed herein. and one or more pharma- ceutical compositions comprising the compound of formula (I) and one or more pharma- ceutical acceptable carriers are provided.

[0045] Embodiments of the present disclosure provide a method of treating cancer in an individual in need thereof, comprising administering to said individual The method includes 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 The present invention relates to a method for treating cancer, comprising administering to said patient a therapeutically effective amount of a compound selected from the group consisting of IT, LAIR1, CD160, 2B4 and TGFR. Treatment method.

[0051] Embodiments of the present disclosure provide a method for expanding CD8 T effector cells in a patient in need thereof. The present invention provides a method for the preparation of a medicament for the treatment of a cancer, comprising administering to the patient an antibody-cytokine grafted protein as disclosed herein. The method includes administering the protein or pharmaceutical composition to a patient.

[0052] CD8 T effector cells are expanded and 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 cytor cells.

[0054] Further treatment options include administration of immune checkpoint inhibitors to expand CD8 T effector cells. 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, Methods for expanding T effector cells.

[0056] An embodiment of the present disclosure relates to a method for treating cancer, comprising administering to a patient a compound comprising: (i) (a) HCDR1 of SEQ ID NO: 13, b) a heavy chain variable region comprising an HCDR2 of SEQ ID NO: 14, and (c) an HCDR3 of SEQ ID NO: 15; and (d) LCDR1 of SEQ ID NO: 29, (e) LCDR2 of SEQ ID NO: 30, and (f) the sequence (ii)(a) a light chain variable region comprising an LCDR3 of SEQ ID NO:31; and 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 that contain a light chain variable region that contains LCDR3 of SEQ ID NO: 63 Uses of the ingrafted 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 granulocyte colony-stimulating agent that is an antagonist of immune checkpoint inhibitors. Use of fusogenic 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 comprises an IgG1, IgG2, or is selected from the IgG4 subclass Fc region. The antibody, antibody fragment, or antigen-binding molecule comprises 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 IFN-γ fragment. In particular embodiments, the immunoglobulin heavy chain may include a modification that confers vector function. are D265A, P329A, P329G, N297A, D265A / P329A, D2 65A / N297A, L234 / L235A, P329A / L234A / L235A, and and P329G / L234A / L235A. The present invention may include a mutation that confers

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

[0062] Further, the present disclosure relates to an antibody-cytokine grafted protein as described herein. The present invention provides a polynucleotide encoding at least a heavy and / or light chain protein of a protein. In another related embodiment, the host cell comprises an antibody site as described herein. A host cell suitable for the production of kine-grafted proteins is 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 is 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 relates to an antibody cytokine as described herein. The present invention further provides a kit comprising 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 pharma- ceutically acceptable carrier. In some embodiments, the present disclosure provides an antibody-cytokine grafted template 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 invention is provided.

[0065] In some embodiments, the patient has a cell proliferation disorder or cancer, e.g., melanoma, lung cancer, colorectal cancer, 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 consisting of one or more amino acids linked together by disulfide bonds. It has one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The immunoglobulin genes that have been identified include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes; The light chains are classified as either κ or λ. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn determine the structure of the immunoglobulin linkages. 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 responsible for antigen recognition. The variable (V) region or domain is about 100-110 amino acids or more long. 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 In addition to the V region, the heavy Both the hematopoietic 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 is also composed 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 the heavy chain (CH1, CH2 or CH3) are involved in secretion, They confer important biological properties, such as transplacental transfer, Fc receptor binding, and complement fixation. The constant region domains are numbered increasing in number as they become more distal 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. and CL aligns with the first constant domain of the heavy chain. The term "antibody" encompasses conventional antibody structures as well as 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 bound to various peptidases. Antibody fragments exist as a number of well-characterized fragments produced by digestion with The term "antibody fragment" as used herein refers to an antibody fragment that retains the six CDRs. Thus, for example, pepsin binds to the disulfide bonds in the hinge region. When an 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, which causes the F(ab)'2 dimer to form Fa. The Fab' monomer essentially consists of an F β monomer with a portion of the hinge region. ab (Paul, Fundamental Immunology 3d ed. (1993)). Various antibody fragments have been defined in terms of the digestion of the intact antibody. However, those skilled in the art will appreciate that such fragments may be prepared chemically or by recombinant DNA techniques. It will be understood that the antibody fragment may be polysynthesized. are produced by modification of whole antibodies or synthesized de novo using recombinant DNA methods. The term "antibody" 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 polymeric forms of these fragments (e.g., F(ab')2). The grafted proteins also contain 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 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 H, and the light chain domains of Fab are ordered VL-CL from N-terminus to C-terminus. In some embodiments, the heavy chain domain of a Fab has 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" is typically produced recombinantly by any method. Each Fab fragment is 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"), interchangeably, refers to a V L Reach BiV H CDRs refer to the hypervariable regions of the antibody chain that are specific for the target protein. Each human VL or VH has three CDRs (CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR20, CDR21, CDR22, CDR23, CDR24, CDR2 R1-3, numbered consecutively from the N-terminus), which make up approximately 15-20% of the variable domain The CDRs are structurally complementary to an epitope of a target protein and Therefore, it is directly involved in the binding specificity. The work 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 determined 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 .Biol., 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, H The CDR amino acid residues of ~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). According to 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, the amino acid residues of LCDR2 are 50-52, and the amino acid residues of LCDR3 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] An "antibody variable light chain" or an "antibody variable heavy chain" as used herein, respectively, are L Or V H refers to a polypeptide comprising an endogenous V L The gene segments V (variable) and J (linked) (conclusion) and endogenous V H is coded by V, D (diversity), and J. V LOr V H Each of the CDRs comprises a framework region (FR). "V region" or "V region" are synonymous with FR1-CDR1-FR2-CDR2-FR3 ...CDR2-CDR3-CDR1-CDR2-CDR3-CDR1-CDR2-CDR3- V-regions refer to heavy or light chains that contain DR3-FR4. V-regions may be naturally occurring, recombinant, or synthetic. In this application, the antibody light chain and / or the antibody heavy chain are sometimes referred to collectively as "antibody As provided and further described herein, an "antibody chain" may be referred to as an "antibody molecule." "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] The C-terminus of an immunoglobulin heavy chain, e.g., including the CH2 and CH3 domains, The terminal portion is the "Fc" domain. As used herein, an "Fc region" refers to a first The constant region of an antibody excluding the constant region (CH1) immunoglobulin domain of Fc. , 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 For IgA and IgM, Fc may include the J chain. Ig For G, Fc is the immunoglobulin domains Cγ2 and Cγ3, as well as the domains Cγ1 and Cγ The boundaries of an Fc region may vary in the art, but include the hinge between the Fc domain and the ribosome. However, the human IgG heavy chain Fc region usually consists of residues C226 or P230 to their carboxyl groups. (The numbering used is from Kabat et al. (1991, NIH Public ation 91-3242, National Technical Informa The EU Index at the EU Trade Commission (EU Trade 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 functions. The Fc region also contains allelic variants that have 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 one embodiment, one or more C-terminal residues of 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 that binds immunoglobulins (Ig) and is recognized by cellular receptors such as FcR. The 5' end of the CH2 exon is a portion of the complement activating protein C1q that binds to the CH2 exon. The lower hinge region, encoded by the portion, 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 entirely different molecules, e.g., enzymes, that confer novel properties to the chimeric antibody. The antigen-binding site (variable region) is linked to antigens, 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 retain the reactivity of non-human antibodies (e.g. This is an antibody that retains the same binding specificity and activity as the non-human CDR region. 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, can be used. Mutants, or, for example, those described in Knappik et al., J. Mol. Biol. 296: 57-86, 2000) 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 are mutations introduced by conservative substitutions 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 IgG1 gene has the highest determined amino acid sequence identity with the amino acid sequence or subsequence of the human IgG1 gene. The term refers to a nucleic acid sequence that encodes a variant amino acid sequence or subsequence. The sequence also ranks the reference variable region amino acid sequence by comparing it to all other evaluated variable region amino acid sequences. The human variable region amino acid sequence having 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, the complementary SDR only, framework and complementarity determining regions, variable segments (as defined above) Such as, or other combinations of sequences or subsequences that include the variable regions. The quality can be determined, for example, using BLAST, ALIGN, or other alignment techniques known in the art. The alignment algorithm is used to align the two sequences 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 to 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) (A conventional antibody, for example, has two binding sites: "Trivalent" and "tetravalent" refer to antibody molecules that have three and four binding sites, respectively. The antibody-cytokine grafted protein is monovalent (i.e., can 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 a cytokine and a grafted protein In a heterogeneous population of proteins and other biological materials, for example, 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, the antibody-cytokine grafted protein has a particular binding specificity. A quality control is one that binds to a specific target at least twice as much as background and does not bind to any other target 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, we investigated whether the antibody-cytokine grafted protein with a specific binding specificity binds to a specific antigen. Binds at least 10-fold above background and does not bind significant amounts of other targets present in the sample Under such conditions, the antibody-cytokine grafted protein does not bind to the antibody. Specific binding to proteins involves the binding of antibodies, cytokine-grafted proteins to specific target proteins. As used herein, the antibody may need to be selected for its specificity for Specific binding refers to an antibody that selectively binds to the human IL2 low affinity receptor. These include proteins that interact with other cytokine receptor superfamily members, e.g. In some embodiments, the antibody does not include a cytokine grafted protein that reacts with the antibody. , selectively binds to the human IL2 low affinity receptor and to non-human primate IL2R (e.g., crab Antibody-cytokine grafted proteins that cross-react with the macaque IL2R are selected. In some embodiments, a target that selectively binds to the human IL2 low affinity receptor and is An antibody-grafted protein that reacts with a target is selected. Various formats are used to select antibody-cytokine grafted proteins that show a positive response. For example, solid-phase ELISA immunoassays can be used to detect antibodies that specifically immunoreact with a protein. 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 method to reduce background At least twice 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) of a molecule. The equilibrium dissociation constant is the value divided by the rate constant (ka, time-1, M-1). The antibody, cytokine, grafted protein, and / or the like can be measured using any method known in the art. Generally, the quality of the material is 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 Has an equilibrium dissociation constant less than M would.

[0082] As used herein, the term "epitope" or "binding region" refers to the region that is the CDR and / or CDR of an antibody. 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 Regarding the specific binding between a selected cytokine and its receptor (e.g., the 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 is identical to the others, may vary.

[0084] The term "agonist" is used interchangeably with "agonist" to describe an agent that activates a receptor and induces complete or partial receptor mediation. It refers to an antibody that has the ability to induce a inflammatory response. For example, it is an agonist of the IL2 low affinity receptor. IL2 binds to the low affinity receptor for IL2 and mediates intracellular signaling, cell activation and and / or induce proliferation of CD8+ T effector cells and NK cells. Rafted protein agonists have low IL-affinity in some respects similar to the natural IL2 ligand. When IL2 binds to the IL2 low affinity receptor, it stimulates signaling through the IL2 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. Binds to low affinity receptors and phosphorylates STAT5 and / or CD8+ T effectors The antibodies can be identified by their ability to induce proliferation of IL-1 or NK cells.

[0085] The term "IL2" or "interleukin 2" or "interleukin-2" or "IL "-2" is synonymous with alpha-helix, a naturally occurring protein that functions in the regulation and maintenance of inflammatory processes. It is a member of the IL-2 cytokine family. The properties of IL-2 are that it has empty N- and C-termini. The IL2 cytokine protein is therefore in close proximity 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. The antibody has preferential agonist activity against 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 At least about 90%, 91%, 92%, 93%, 94%, 95% identical to the nucleic acid sequence of SEQ ID NO:1 , 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" refers to the incorporation of at least one cytokine directly into the CDRs of an antibody. Cytokines have HCDR1, which is inserted into the CDR sequence. It can be loaded into HCDR2, HCDR3, LCDR1, LCDR2 or LCDR3. Cytokines can bind HCDR1, HCDR2, HCDR3, LCDR1, and LCDR2. or into LCDR3, toward the N-terminal sequence of the CDR, or toward the C-terminal sequence of the CDR The cytokine incorporated into the CDR can be expressed as a nucleotide sequence of 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. It is possible to graft a specific CDR onto the antibody and attach a different cytokine to the other "arm" of the antibody. For example, IL2 can be grafted onto one of the "arms" of an antibody. The antibody cytokine grafted protein is grafted to the HCDR1 of the antibody cytokine grafted protein. When grafted onto the LCDR1 of the other "arm" of the protein, a bifunctional antibody cytokine graft is generated. It is possible to produce fusogenic proteins.

[0087] The term "isolated," as 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 performed using techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. It is determined using analytical chemistry techniques. The protein that is the predominant species present in the preparation is essentially Specifically, an isolated gene is a gene that is substantially purified from the genome of interest. Separate from open reading frames that code for proteins other than the nucleic acid. "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 any 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 includes any nucleic acid that has similar binding properties to the reference nucleic acid and is naturally occurring. Nucleic acids that contain 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 includes only the sequence explicitly indicated. and its conservatively modified variants (e.g., degenerate codon substitutions), alleles, or oligonucleotides. Solologs, SNPs, and complementary sequences are also implicitly included. The third position of one or more selected (or all) codons is a mixed base and / or a deoxyribonucleic acid. This can be achieved by creating a sequence in which 3-aminobutyric acid residues have been substituted with 3-aminobutyric 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 polypeptide consisting of amino acids. These terms are used interchangeably to refer to a polymer of one or more amino acid residues. However, amino acid polymers that are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as and to naturally occurring and non-naturally occurring amino acid polymers. do.

[0090] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acids that function similarly to the naturally occurring amino acids. Naturally occurring amino acids are those that are encoded by the genetic code and are known as amino acid analogs and amino acid mimetics. as well as amino acids that are subsequently modified, e.g., hydroxyproline, γ The amino acid analogs are O-carboxyglutamic acid, O-phosphoserine, and O-phosphoserine. Compounds that have the same basic chemical structure as cyclic acids, i.e. hydrogen, carboxyl groups, amino groups, and R groups, e.g., homoserine, norleucine, methionine sulfoxide, methionine methionine This refers to the α-carbon bonded to the thylsulfonium. Such analogs are represented by modified R groups ( norleucine) or modified peptide backbones but with the same groups as natural amino acids. An amino acid mimetic is a compound that has a structure that is different 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 tag. For example, the codons GCA, GCC, GCG, and GCU all encode amino acids that encode amino acids that encode amino acids. Thus, alanine is encoded by all positions specified by the codon. In this position, the codons may be substituted with the corresponding codons described without altering the encoded polypeptide. Such a nucleic acid change is a "silent mutation", which means , a conservatively modified variant. Every nucleic acid sequence also describes every possible silent variation of the nucleic acid. Each codon in the sequence (usually AUG, which is the only codon for methionine, and usually The codons (except for TGG, the only codon for the nucleotide sequence) are identical to those of the nucleotide sequence, resulting in functionally identical molecules. It will be appreciated that the nucleic acid encoding the polypeptide may be modified in any manner. Each silent variation is implicit in each described sequence.

[0092] For amino acid sequences, a single amino acid or a small portion of an amino acid in a coding sequence The nucleic acid, peptide, polypeptide, or protein sequence to be changed, added, or deleted Each substitution, deletion, or addition to an amino acid is considered to be an amino acid that is chemically similar to the 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, and 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 win-win of comparing two optimally aligned sequences. The polynucleotide sequences are determined by comparing the sequence of the comparison The portion within the window does not include additions or deletions for optimal alignment of the two sequences. The polypeptide may contain additions or deletions (i.e., gaps) when compared to a reference sequence (e.g., a polypeptide) that is not 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 in the comparison window. Divide by the total number of positions and multiply the result by 100 to get the percentage of 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 have the same sequence. Alignment by one of the sequence comparison algorithms of The two sequences have the largest 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 to the nearest match. The target sequence has a specified percentage of the octide (i.e., over a specified region of the reference sequence, or Unless otherwise specified, at least 85%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity Two sequences are "substantially identical" if they have the following structure: Each of the 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 one amino acid sequence of the reference sequence. Over a region of about 15, 25 or 50 nucleotides in length, or more preferably 100 to 50 over a region of 00 or 1000 nucleotides or more in length, or over the entire length With respect to amino acid sequences, identity or substantial identity is defined as at least one amino acid sequence that is identical to the reference sequence. 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 the amino acid sequence is conservatively substituted according to the conservative substitutions defined in .

[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 the comparison of 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 sequencing, the sequence can be compared to a reference sequence at the same number of consecutive positions, typically 20 to 600. From about 50 to about 200, more usually from about 100 to about 150, Contains a 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, see 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 is based on the Pearson and Lipman (198 8) Proc. Nat'l. Acad. Sci. USA 85:2444 similarity search method Therefore, a computerized implementation of these algorithms (Wisconsin Ge genetics Software Package,Genetics Compute GA at r Group, 575 Science Dr., Madison, WI P, BESTFIT, FASTA, and TFASTA) or manually. Measurement and visual inspection (e.g., Ausubel et al., Current P See protocols in Molecular Biology (1995 supplement). )

[0097] Two examples of algorithms suitable 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, 2002. Software for performing BLAST analyses is available from N ational Center for Biotechnology Informa This algorithm is published by the National Institute of Standards and Technology (NITS) in the journal Nature. If the word is aligned with some positive threshold score, e-valued threshold score) T By identifying short word lengths W in the query sequence, we can find high-scoring sequence pairs (HS P), where T is called the neighborhood word score threshold (Altsch These initial neighborhood word hits are then analyzed to find the exact sequence of longer words containing them. They act as seeds to initiate searches to find new HSPs. are extended in both directions along each sequence as far as the cumulative alignment score can be increased. The cumulative score is calculated based on the parameter M for nucleotide sequences, which is the number of matching residues per pair. reward score for matching; always >0) and N (penalty score for mismatched residues; always <0 For amino acid sequences, the scoring matrix is ​​calculated as the cumulative score: The extension of the word hit in each direction is stopped when 1. If the cumulative alignment score decreases by an amount X from its maximum achieved value; or due to the accumulation of multiple negatively scoring residue alignments, the cumulative score becomes zero or less. 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) the default is a word length (W) of 11, a term 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 below.) By the BLAST algorithm One measure of similarity provided is the degree to which a match between two nucleotide or amino acid sequences would be coincidental. The minimum sum probability (P(N)) provides an indication of the probability that the test nucleic acid and the reference nucleic acid The minimum sum probability in comparison with a nucleic acid 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 α is less than about 0.001. can be.

[0099] Additional 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 encoded by the second nucleic acid. The main objective of the present invention is to provide a method for the preparation of a polypeptide that is immunologically cross-reactive with antibodies raised against the polypeptide. For example, where the two peptides differ only by conservative substitutions, the polypeptides are typically 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. It is the ability of two nucleic acid sequences to hybridize to one another at substantially the same position. 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 in the antibody-cytokine grafted protein of the invention. When used in the context of describing how regions are connected, Many binding domains are frequently covalently linked (e.g. The molecules are linked by chemical bonds, such as peptide bonds or disulfide bonds, or non-covalent bonds. This can be achieved by direct attachment (i.e., no linker between the two binding regions) or indirect attachment ( 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, etc.) It may also be (ko).

[0102] As used herein, "treating" or "treating" any disease or disorder. The term "treatment" or "treatment" refers, in one embodiment, to improving a disease or disorder. That is, slowing or halting the onset of the disease or at least one of its clinical symptoms. In another embodiment, "treat," "treating," or "alleviating" refers to "Treatment" refers to the determination of at least one physical parameter, including those that are not perceptible by the patient. In yet another embodiment, "treat" or "treat "Treatment" or "treatment" refers to the treatment of a disease or disorder, either physically (e.g., by alleviating 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 slowing 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 a desired result. i.e., reducing inflammation, inhibiting pain, preventing inflammation, inhibiting or preventing the inflammatory response. In certain embodiments, a therapeutically acceptable amount is desirably A therapeutically acceptable amount is one that is 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 is effective to treat symptoms of a disease, including symptoms associated with immune-related disorders. It may prevent its 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 "sentially of" refers to the genus or species of medicament that is included in the method or composition; as well as any inert carrier or excipient for the intended purpose of the method or composition. In some embodiments, the phrase "consisting essentially of "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 the referent. [Brief description 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. [Diagram 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 depicted on the graph. [Figure 3A] Figures 3A-C: 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 days 4, 8, and 11 at a 100 μg equivalent dose. [Figure 3B] (As stated above.) [Figure 3C] (As stated 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 days 4, 8, and 11 at a 500 μg equivalent dose. [Figure 3E] (As stated above.) [Figure 3F] (As stated above.) [Figure 4A]1 shows that IgG.IL2R67A.H1 selectively expands CD8 T effectors in NOD mice and is better tolerated than Proleukin®. [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. [Diagram 5] 1 shows a graph of single agent efficacy of IgG.IL2R67A.H1 in the CT26 tumor model. [Figure 6] Data is 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 TA99, an anti-TRP1 antibody, is more effective than TA99 alone, IL2-Fc fusion molecule alone, and TA99+IL2-Fc fusion. Synergy was seen 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] Graph of ELISA data showing 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 PREFERRED EMBODIMENTS

[0108] Antibody-cytokine-grafted proteins targeting the IL2 low affinity receptor The present invention relates to a method for the preparation of a method for the preparation of a 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. It exhibits suitable properties for use in patients, e.g., immune stimulation similar to natural or recombinant human IL2. It retains its activity. However, negative effects, such as stimulation of Treg cells, are reduced. Other activities and characteristics are also demonstrated throughout the specification. Therapeutic prostaglandins compared to IL2 and modified IL2 therapeutic agents, such as Proleukin® The file includes an improved antibody cytokine grafted protein and an antibody site provided. Methods of using the tokine-grafted proteins in the treatment of cancer 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 which 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 In a particular embodiment, the IL2 molecule is incorporated into heavy chain complementarity determining region 1 (HCDR1). In a particular embodiment, the IL2 molecule is incorporated into heavy chain complementarity region 2 (HCDR2). It is incorporated into the heterotypic determining region 3 (HCDR3).

[0111] In some embodiments, the antibody cytokine grafted protein comprises a light chain CDR. In a specific embodiment, the IL2 molecule comprises a light chain complementarity determining region (CDR) of In a particular embodiment, the IL2 molecule is incorporated into the light chain complementarity determining region 1 (LCDR1). In a particular 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 that is 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 in the CDR. The IL2 molecule contains an inserted IL2 sequence, which frameshifts the CDR sequence. In another embodiment, the grafted antibody cytokine has no CDRs. The present invention includes an IL2 molecule that is incorporated into a human IL2 gene, whereby an IL2 sequence is placed in all or part of the CDR sequence. The replacement may be in the N-terminal region of the CDR, in the central region of the CDR, or in the C-terminal region of the CDR. The replacement may be as little as one or two amino acids in the CDR sequence. It may be absent or the entire CDR sequence.

[0113] In some embodiments, the IL2 molecule is grafted directly into the CDRs 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 IgG1, an Ig It is either G2 or IgG4 subclass.

[0115] In some embodiments, the antibody-cytokine grafted protein is an antibody that has a known clinical benefit. The immunoglobulin sequences include heavy and light chain immunoglobulin sequences selected from the immunoglobulin sequences currently used in In certain embodiments, the antibody-cytokine grafted protein is a humanized sequence. In another specific embodiment, the antibody comprises a cytokine. The grafted protein contains heavy and light chain immunoglobulin sequences which are human sequences.

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

[0117] In some embodiments, the antibody-cytokine grafted protein inhibits the binding of IL2 molecules. A heavy chain having a binding specificity of an immunoglobulin variable domain for a target different from the specificity thereof and In some embodiments, the immunoglobulin variable domain comprises a light chain immunoglobulin sequence. The binding specificity for the target was 10%, 20%, and 40% in the presence of grafted cytokines. %, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99 In certain embodiments, the binding specificity is retained relative to the non-human target. In certain embodiments, the binding specificity that is retained is directed against a virus, e.g. For example, against RSV. In another embodiment, the binding specificity is In certain embodiments, the 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 Decreased by 90%, 95%, 98%, 99%, or 100%.

[0119] The antibody-cytokine grafted protein provided comprises a VH or VL complementarity determining region. In some embodiments, the IL2 molecule comprises a CDR grafted into the IL2 sequence. 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 complementation determination of the 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 corresponds to the amino acid sequence of SEQ ID NO:4 at least 85%, 90%, 91%, 92%, 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 this regard, the IL2 molecule consists of the sequence of SEQ ID NO:4.

[0120] The antibody-cytokine grafted protein provided comprises a VH or VL complementarity determining region. In some embodiments, the IL2 molecule comprises a CDR grafted into the IL2 sequence. 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 is human IL2, Superior to recombinant human IL2, Proleukin® or IL2 fused to Fc The antibody-cytokine grafted protein confers immunomodulatory properties. , compared to recombinant human IL2, Proleukin® or IL2 fused to Fc When activated, it confers increased activity against CD8 T effector cells, whereas 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 In particular embodiments, the immune system comprises a mutation selected from one or more of: L235A, L236A, and L237A. The immunoglobulin heavy chain is D265A, P329A, P329G, N297A, 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 preferably at least 90%, a 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 of the immunoglobulin, optionally, 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 preferably at least 90%, a 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 of the immunoglobulin, optionally, 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 the heavy and light chains are produced by grafting the CDR regions of the phospho-scaffold. Immunoglobulin chains are then 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 with IL2 fused to Proleukin® or Fc, which inhibited Treg Activity is decreasing.

[0126] To engineer antibody-cytokine grafted proteins, specific muteins were included in the I The L2 sequence (SEQ ID NO: 4 or SEQ ID NO: 6) is a CDR loop of an immunoglobulin chain scaffold protein. The grafted constructs are inserted into various known grafts that have been 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 The constructs may be prepared using standard techniques utilizing recombinant DNA encoding the relevant sequences. The IL-1 in an exemplary scaffold called GFTX3b is produced using molecular biology methods. The sequences of the two are shown in Table 2. The 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 not located on the N-terminal or It can be adjusted toward the C-terminus.

[0127] Thus, the present disclosure provides methods for the preparation of grafted proteins using xenogenous antibody proteins or polypeptides. 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 An antibody or an 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 fragment, VH domain, VH CDR, VL domain, VL CDR, etc.) and a heterologous Grafted proteins comprising an IL2 polypeptide or peptide, such as The protein, polypeptide, or peptide is fused or linked to an antibody or antibody fragment. Methods for conjugating such compounds are known in the art. See, for example, U.S. Patent No. 5,333,636. Specification No. 336,603, Specification No. 5,622,929, No. 5,359,046 The specification, the specification of the same patent No. 5,349,053, the specification of the same patent No. 5,447,851, and the specification of the same patent No. No. 5,112,946; European Patent Nos. 307,434 and 367,16 No. 6; International Publication No. 96 / 04388 and International Publication No. 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 In addition, antibody-cytokine grafted proteins have been shown to be useful in the treatment of cancer. Fling, motif shuffling, exon shuffling, and / or codon shuffling They can be created using 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. 5,605,625 may be used to detect the presence of a cytochrome P454 antibody or a fragment thereof. ,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 See Biotechniques 24(2):308-313. or the encoded antibodies or fragments thereof may be subjected to error-prone PCR, By subjecting the sample to random nucleotide insertion or other random mutagenesis methods. The antibody or its fragment that specifically binds to the antigen protein of interest can be The polynucleotide encoding the antibody-cytokine graph 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 CD4s that could 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 of the CDR loops may contain a cytochrome P450 amino acid sequence. The decision to insert a CDR loop involves 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] The CDRs of an immunoglobulin chain are well known in the art, including those described herein. For example, the CDRs are numbered according to the well-known numbering system. 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. These are 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. The CDR amino acid sequences may be modified to still retain the desired specific binding and / or agonist activity. However, one or two conservative amino acid residue substitutions may be tolerated.

[0130] The antibody-cytokine grafted protein may further comprise a unique A method for engineering modified antibodies, cytokines, and grafted proteins with altered properties. As the source material, the CDRs and / or VH and / or VL shown herein (e.g., Table 2) can be used. 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. The known antibodies and corresponding immunoglobulins may be used as starting materials for the preparation of proteins. Examples of the purine sequence include palivizumab, alirocumab, mepolizumab, and necitumab. nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, penicillin Brolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, trastuzumab Tuzumab, raxibacumab, pertuzumab, belimumab, ipilimumab, denosumab, Silizumab, ofatumumab, canakinumab, golimumab, ustekinumab, certoliz Mab, catumaxomab, eculizumab, ranibizumab, panitumumab, natalizumab, ben Basizumab, cetuximab, efalizumab, omalizumab, tositumomab, ibritum Mabtiuxetan, adalimumab, alemtuzumab, gemtuzumab, infliximab, Basiliximab, daclizumab, rituximab, abciximab, muromonab, or Known antibody and immunoglobulin sequences also include germline variants. 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, Germline DNA sequences of heavy and light chain variable region genes were obtained from "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 yet other instances, antibodies from other known entities may be in early drug discovery and / or drug development. The antibody and the corresponding immunoglobulin sequence are similarly modified antibody cytokine grafted protein. 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 can be used, such as human immunoglobulins. or fragments thereof, and can be used in combination with other animal species, preferably humanized and / or immunoglobulins with human aspects. Novel antibodies, frameworks, scaffolds and fragments are continually being discovered and developed by those skilled in the art.

[0132] Antibodies can be produced using methods known in the art. To prepare a cloned antibody, any technique known in the art can be used (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 (See U.S. Patent No. 4,946,747, Inc. 1985). No. 78 discloses the preparation of 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 produced using phage display technology. To identify antibodies and heteromeric Fab fragments that specifically bind to an antigen of choice for use in (e.g., McCafferty et al., supra; Marks et al., supra). See, e.g., 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. Thus, a primatized or humanized antibody has one or more components introduced therein from a non-primate or non-human source. Such non-primate or non-human amino acid residues are often This is typically referred to as the import residue in 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). In particular, the present invention relates to a method for the preparation of a human variable domain having a substantially intact human variable domain, which is fused to a counterpart from a non-human species. In practice, primatized or humanized antibodies typically have 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 replaced by residues from analogous sites in a mouse antibody (e.g., a rodent antibody).

[0134] Alternatively or additionally, the antibody may be modified to have the same or better binding characteristics as the non-human antibody. The invention relates to an antibody that replaces non-human antibody variable regions with human variable regions while providing similar binding characteristics. In vivo methods may be used to convert non-human antibodies into engineered human antibodies. Published patent application No. 20050008625, published U.S. patent application No. 2005 / 025 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 then in the context of the remaining reference antibody amino acid sequence The identified human "cassettes" that supported binding were recombined in a second library screen. These are then 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 can 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 (see, 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 can be produced by a variety of methods, including grafting of hybridomas or linking of 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 produced using phage display libraries or ribosome display libraries. In particular, gene shuffling libraries can be used to identify gene-shuffling targets. The antibodies can be synthetic, semi-synthetic or constructed from natural and immunologically competent sources. In preparing an antibody-cytokine grafted protein construct as provided herein may utilize the immunoglobulin sequence of choice.

[0136] The antibody, antigen-binding molecule, or antibody-cytokine grafted molecule used in the present disclosure is a bispecific antibody. Bispecific or bifunctional antibodies further include antibodies that are specific to the 2 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. These include bispecific scFv antibodies, single binding domains (dAbs), and minibodies that recognize Thus, the antibody-cytokine grafted protein as provided herein In preparing the antibody construct, the immunoglobulin sequence of choice can be utilized.

[0137] Antibody fragments that bind to antigens, such as Fab fragments and scFv fragments, can be used as building blocks to generate antibody fragments. Tokine-grafted proteins can be constructed, optionally including multivalent forms. In some embodiments, such a multivalent molecule comprises 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 H and / or VL), e.g., modifying one or more residues in one or more CDR regions. and such adapted VH and / or VL region sequences can be It can be used for grafting cytokines or for the preparation of cytokine grafts. Antibodies are primarily composed of amino acid residues located in the six heavy and light chain complementarity determining regions (CDRs). For this reason, the amino acid sequences within the CDRs vary widely between individual antibodies. CDR sequences are involved in most antibody-antigen interactions. , a specific antibody 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. Pat. No. 5,225,539 to Winter, and Queen et al. U.S. Patent Nos. 5,530,101 to 5,585,089 to (See US Pat. Nos. 5,693,762 and 6,180,370). In certain instances, the antigen-binding ability of an antibody is improved by mutating residues within the framework regions. It is beneficial to maintain or increase 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. To achieve this, the VH and / or VL CDR1, CDR2, and and / or mutation of amino acid residues in the CDR3 region, e.g., cytokine grafted proteins It may be beneficial to optimize the antigen binding of antibodies in the context of the protein. mutagenesis or PCR-mediated mutagenesis to introduce one or more mutations. and their effects on antibody binding or other functional properties of interest are described herein. In vitro or in vivo assays described herein and / or alternatives known in the art Conservative modifications can be introduced. 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 altered.

[0140] The engineered antibody or antibody fragment may be modified by modifying framework residues within VH and / or VL, e.g. In some embodiments, such modifications may be made to improve the properties of the antibody. Framework modifications are made to reduce the immunogenicity of antibodies. For example, one approach is to change one or more framework residues to the corresponding germline sequence. Specifically, antibodies that have undergone somatic mutations have sequences that differ from the germline sequence from which they are derived. Such residues may include framework residues that bind the antibody framework sequence to the The framework region sequences can be identified by comparing them to the germline sequences from which they are derived. Somatic mutations can be removed, for example by site-directed mutagenesis, to restore the original germline configuration. Additional framework modifications can be made to the germline sequence by "backmutating" the , by suddenly varying one or more residues in the framework regions, or even in one or more CDR regions. By altering the T-cell epitopes, the antibody can be made less immunogenic. This technique is also called "deimmunization" and is described in U.S. Patent Application Publication No. 2003 / 0133335 by Carr et al. This is described in further detail in US Pat. No. 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 animal mammals (e.g., horses, sheep, cattle, pigs, camels), domestic animal mammals (e.g. dogs, cats) or geese The mouse 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 can be 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 identifier is In some embodiments, the antibody cytokine grafted protein is 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, the antibody sequence may 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 binding. The Fc region may be engineered to contain modifications to alter antigen-dependent cytotoxicity, etc. Furthermore, the antibody, the antibody fragment thereof, or the antibody-cytokine grafted protein may be used in this case. In some cases, the antibody-cytokine grafted protein may be modified to alter one or more functional properties. For example, the antibody may be chemically modified (e.g., by adding one or more chemical moieties to the antibody). or its glycosylation can be altered.

[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 is incorporated herein by reference, in which, for example, light and heavy chain amino acids are 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 altered to increase or decrease In another embodiment, the biological half-life of the antibody-cytokine grafted protein is altered. More specifically, the Fc hinge region of the antibody is mutated to enhance the expression of the antibody cytokine group. Staphylococcal protein A, a raft-forming protein tein A (SpA) binding was impaired compared to native Fc-hinge domain SpA binding. The Fc-hinge fragment has one or more amino acids at the CH2-CH3 domain junction region, Mutations are introduced. This technique is described in U.S. Pat. No. 6,165,333, by Ward et al. Further details are provided in US Pat. No. 745.

[0144] The present disclosure provides an antibody that specifically binds to the IL2 low affinity receptor that has a long in vivo half-life. In another embodiment, an antibody 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 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 to prepare 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. See, for example, International Publication No. 98 / 23289; International Publication No. 97 / 34631; and U.S. Pat. No. 6,277,375. Alternatively, antibody-cytokine grafted proteins may be used to increase biological half-life. Proteins are disclosed in U.S. Patents 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 domain is designed to contain a salvage receptor binding epitope taken from a loop of In yet another embodiment, at least one amino acid residue is changed to a different amino acid. The Fc region was altered by replacing it with an acid residue to produce an antibody-cytokine grafted protein. Alters the effector function of a protein, e.g., its affinity for an effector ligand Antibody-cytokine grafted proteins that have altered function but retain the antigen-binding ability of the parent antibody. One or more amino acids can be replaced with different amino acid residues to produce a protein. The effector ligand for which affinity is changed can be, for example, an Fc receptor (FcR ) or the C1 component of complement. Both of these techniques are described in Winter et al. Nos. 5,624,821 and 5,648,260. 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 of the amino acids that are present in the sequence can be replaced with different amino acid residues. For further details, see U.S. Patent No. 6,194,551 to Idusogie et al. It is described in.

[0146] Antibodies mediated by antibody cytokine grafted proteins containing such mutations ADCC or CDC are reduced or absent. 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 no 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 expression. This technique alters the complement-fixing ability of the grafted protein. This 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 the parent of the antibody cytokine grafted protein against the Fcγ receptor. The Fc region is modified by one or more amino acid modifications to increase affinity. Further details on the methodology are provided in WO 00 / 42072 by Presta. Furthermore, the binding of FcγRl, FcγRII, FcγRIII, and FcRn has been described. The binding site on human IgG1 for IgG1 IgG1 has been mapped and mutants with improved binding have been described. (Shields, RLet al., 2001 J. Biol. Chen. 276:6591-6604).

[0149] In yet another embodiment, the glycosylation of the antibody cytokine grafted protein For example, by making a non-glycosylated antibody-cytokine grafted protein. (i.e., the antibody-cytokine grafted protein lacks glycosylation. Glycosylation can be modified, for example, to increase the affinity of an antibody for an "antigen." Such carbohydrate modifications can be made, for example, by modifying one or more glycosylation sites within the antibody sequence. This can be achieved, for example, by altering one or more of the variable region framework regions. one or more cosylation sites, thereby eliminating glycosylation at said sites The above amino acid substitutions can be made. Such aglycosylation reduces 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, a hypofucosylated antibody cytokine having a reduced amount 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. Altered glycosylation patterns increase the antibody-dependent cellular cytotoxicity (ADCC) ability of antibodies Such carbohydrate modifications have been demonstrated to result in, for example, altered glycosylation machinery. This is achieved by expressing an antibody-cytokine grafted protein in a host cell. Cells with altered glycosylation machinery have been described in the art. , expressing a recombinant antibody cytokine grafted protein, thereby Use as host cells to produce altered antibody-cytokine grafted proteins For example, European Patent No. 1,176,195 to Hang et al. We describe a cell line carrying a disrupted FUT8 gene, which mediates fucosyltransferase expression. The antibody cytokine expressed in such cell lines encodes a transferase. The grafted protein will exhibit low fucosylation. International Publication by Presta No. 03 / 035835 describes a method for adding fucose to Asn(297)-linked carbohydrates. We describe a mutant CHO cell line, Lecl3 cells, that has a reduced ability to host Providing 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 / 013662. No. 54342 describes glycoprotein-modifying glycosyltransferases (e.g. , β(1,4)-N-acetylglucosaminyltransferase III (GnTIII) The authors described a cell line engineered to express the . The antibody-cytokine grafted proteins exhibited an increase in bisecting GlcNac structures, and This results in increased 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, as 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, US Pat. No. 6,393,962, and US Pat. No. 6,393,962). The peptide linker may vary in length, e.g., the linker may be 1 to 1 00 amino acids in length, typically the linker is 5 to 50 amino acids in length, 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 phospholipids 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 sequences (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 [In the formula, 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 [In the formula, 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, 8, 9, or 10] (SEQ ID NO: 75). In certain embodiments, one or more linkers include G4A (SEQ ID NO: 76) repeats, (G4 A) n [In the formula, 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 the present invention, the linker is a combination or a plurality of G4S (SEQ ID NO: 74) and G4A (SEQ ID NO: 7 6) is included.

[0153] Other examples of linkers include those that minimize immunogenicity caused by the linker and the conjugate. Examples of such linker sequences include those based on the flexible linker sequences naturally occurring in antibodies. For example, there is a natural mobility between the variable domain and the CH1 constant domain of the antibody molecule structure. 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 are contributed from the N-terminus. The mitokine-grafted protein may be, for example, a 5-6 amino acid residue at the end of CH1 or a 1 A linker that incorporates 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 with no significant secondary structure. The CH1 domain can adopt a conformation and thus act as a flexible linker. The N-terminal residue of the nucleotide is a natural extension of the variable domain as it is part of the Ig sequence. and therefore any immunogenicity potentially caused by the linker and conjugate. 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 The polypeptide may include a marker sequence, such as a peptide, to facilitate purification of the polypeptide. In an embodiment, the marker amino acid sequence is, inter alia, incorporated into a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 9131 1) A hexahistidine peptide (SEQ ID NO: 121), such as the tag provided in 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 for convenient purification of the grafted protein. Other peptide tags of interest include, but are not limited to, influenza hemagglutinin protein. A hemagglutinin ("HA") tag corresponding to an epitope derived from a protein (Wilson 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 Some examples include:

[0156] Assay of antibody-cytokine grafted protein activity Assays for identifying antibody-cytokine grafted proteins are known in the art. Agonist antibodies, cytokines, grafted proteins, and the like are known and described herein. Proteins bind to the IL2 low affinity receptor and promote, induce, and stimulate intracellular signaling to induce CD4+ 8 Induces T effector cell proliferation as well as other immune stimulatory effects.

[0157] Binding of the antibody-cytokine grafted protein to the IL2 low affinity receptor is Any method known in the art can be used to determine IL2 hyperplasia. Binding to compatible receptors can be measured using techniques including, but not limited to, ELISA, Western blot, surface plasmon resonance (SPRF) assays. known methods, including stationary plasmon 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 method known in the art. For example, the activation of the IL2 low affinity receptor 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 cells expands the CD8 T effector cells, and thus 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 labeling. 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, the antibody-cytokine grafted protein The test cells or culture supernatant from the test cells contacted with the substrate and the antibody-cytokine grafted substrate are Control cells or culture supernatants from control cells not exposed to the protein and / or recombinant human Contact with IL2 (e.g., Proleukin®) or an IL2-FC fusion molecule. It is possible to compare the results with those obtained by

[0159] The activity of the antibody-cytokine grafted protein may also be evaluated ex vivo and / or in vivo. In some embodiments, the levels of the IL-1 protein in untreated control animals and / or in Prole Antibody-cytokine grafts compared to animals similarly treated with ukin® STAT5 activation across a variety of cell types ex vivo from animals treated with activated proteins The differential activity of agonist antibody-grafted proteins between cell types was measured using a method that measures A preferred agonist antibody cytokine grafted protein is It has the ability to activate and expand CD8 T effector cells. For example, In vivo activation and expansion of effector cells can be performed by any method known in the art. The agonist activity can be measured using methods such as flow cytometry. The antibody-cytokine grafted protein is useful for treating cancer, e.g., melanoma, lung cancer, colorectal cancer. , may be therapeutically useful in preventing, reducing, ameliorating or treating 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. The grafted protein is administered to a subject, and an antibody cytokine is administered prior to administration of the grafted protein. The antibody cytokine grafted protein can be determined by comparing the antibody cytokine grafted protein with the control after administration. The efficacy of the protein was also evaluated by administering a therapeutically effective amount of the antibody-cytokine grafted protein to the test subjects. and comparing the test subjects to control subjects who have not been administered 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 the antibody-cytokine grafted protein is provided, including, but not limited to, Although not disclosed herein, 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 the production of microbial cells, such as mammalian host cells, bacterial host cells, yeast host cells, The host cell may be from a host cell, an insect host cell, etc.

[0161] Any of SEQ ID NO:20, SEQ ID NO:36, SEQ ID NO:52, and SEQ ID NO:68 is herein incorporated by reference. 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%, This 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 at least 85%, 85%, or 85% of a polynucleotide selected from the group consisting of 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. Leotide 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 that are at least 85%, 89%, 90%, 91%, 92%, 93%, or 9 have 4%, 95%, 96%, 97%, 98%, 99%, or 100% nucleic acid sequence identity do.

[0165] The polynucleotide encodes only the variable region sequence of the antibody cytokine grafted protein. The polynucleotides can also be used to encode antibodies, cytokines, and grafted proteins. Some polynucleotide sequences may also encode both variable and constant regions of the polypeptide. The columns represent the variable regions of both the heavy and light chains of one of the antibody-cytokine grafted proteins. Some other polynucleotides encode polypeptides that contain antibody cytokines. The heavy and light chain variable regions of one of the grafted proteins are substantially identical to each other. The polypeptide segments are encoded by:

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

[0167] In some embodiments, one or more of the immunoglobulins of the antibody-cytokine grafted protein are 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 that expresses 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 comprises a single vector encoding the protein chain and a secretion signal. In the recombinant host cell, one expresses the heavy chain immunoglobulin of the antibody cytokine grafted protein. one encoding the IgG1 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 includes: (i) detecting an immunoglobulin of an antibody-cytokine grafted protein; Host cells containing one or more vectors encoding phosphoprotein chains are cultured using antibody cytokine digestion. (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 of a pre-existing sequence (e.g., a sequence described herein) encoding can be generated by PCR mutagenesis. Direct chemical synthesis of nucleic acids is known as 68:90, 1979. Brown et al., Meth. Enzymol. 68:109, 1979 Sulfodiester method; Beaucage et al., Tetra. Lett., 22: 1859, 1981 diethyl phosphoramidite method; and U.S. Pat. No. 4,458,06 This can be done by methods known in the art, such as the solid support method of US Pat. No. 6,993,636. Introduction of mutations into a polynucleotide sequence by PCR is described, for example, in the PCR Technique. 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 performed as described in Cations 1:17,1991.

[0170] Also, the expression to produce the antibody-cytokine grafted protein described above. Vectors and host cells are also provided in the present disclosure. To express a polynucleotide encoding an immunoglobulin polypeptide chain or fragment, 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 incorporate the protein or RNA expression cassettes, such as 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, Calif., 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 herpes viruses vector, SV40-based vector, 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: See 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 is an immunoglobulin protein of an antibody cytokine grafted protein. A promoter and other regulatory control sequences 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, or A culture of the transformed organism is then grown to host 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 be included in the antibody cytokine graph. required for efficient expression of immunoglobulin chains or fragments of the target protein These elements typically include the ATG initiation codon and adjacent In addition, the efficiency of expression may vary depending on the cell line in use. This can be enhanced by the inclusion of appropriate enhancers (see, 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 the CMV enhancer may be expressed in a mammalian host. It can be used to increase expression in cells.

[0172] The expression vector also contains an antibody / cytokine graph that is exported from the cell into the culture medium. A secretion signal sequence may also be provided to form a target protein. In an embodiment, the immunoglobulin sequence of the antibody cytokine grafted protein to be inserted The immunoglobulin light and heavy chain variable The vectors used to receive the sequences encoding the domains sometimes also contain constant regions or Such vectors also encode a grafted protein containing a constant region. The antibody-cytokine grafted template is a grafted antibody that allows expression of all variable regions of the antibody. 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. One prokaryotic host useful for cloning and expressing the Microbial hosts include bacilli such as Bacillus subtilis, Salmonella, Serratia, and various strains of Other Enterobacteriaceae species include Pseudomonas species. In prokaryotic hosts, typically 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 There are any number of different well-known promoters available, including promoter systems derived from phage. The promoter typically, optionally together 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, grafted proteins, polypeptides. 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 For expression and production, mammalian host cells are used, e.g., those that carry an exogenous expression vector. These may be any mammalian cell line capable of producing a culture of any normal dead or normal young cell. or abnormal immortal animal or human cells, e.g., CHO cell lines, various Cos cells, In vitro assays, including IgG1, IgG2, IgG3, IgG4, IgG5, IgG6, IgG7, IgG8, IgG1, IgG1, IgG4, IgG1, IgG4, IgG5, IgG1, IgG1, IgG2, IgG4 ...4, IgG1, 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 For mammalian host cells, An expression vector contains expression control sequences, such as an origin of replication, a promoter, and an enhancer. Queen et al., Immunol. Rev. 89:49-68, 1986 (see ), as well as ribosome binding sites, RNA splice sites, polyadenylation sites, and These expression sequences may include 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 CMV promoter), constitutive CMV promoter, and those known in the art Examples of such promoter-enhancer combinations include:

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

[0176] Compositions Comprising Antibody-Cytokine-Grafted Proteins The antibody-cytokine grafted protein is formulated with a pharma- ceutically acceptable carrier. Optionally, the pharmaceutical composition is useful for the treatment or prevention of a given disorder. The composition may further include other suitable therapeutic agents. A pharma- ceutically acceptable carrier may enhance or stabilize the composition. 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) It is preferred that the drug be administered by injection or proximal to the target site. The body may be administered intravenously, intramuscularly, intraperitoneally, intrathecally, intraarterially, subcutaneously, intranasally, by inhalation, spinal cord or epidermal injection. The present invention is suitable for administration by any one or more of the following routes: Accordingly, the active compound, e.g., the antibody-cytokine-grafted protein, is resistant to the action of acid and In addition, the compound may be coated with a material that protects it from natural conditions that may inactivate the compound. 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 via inhalation (i.e., it is referred to as "nebulized" Aerosol formulations may be prepared using aerosols such as 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 application of surface active agents. This can be maintained by the use of active agents. Often, isotonic agents, such as sugars, are included in the composition. polyhydric alcohols such as mannitol or sorbitol, and sodium chloride It is preferred to use agents that delay absorption, such as aluminum monostearate or gelatin. Prolonged absorption of the injectable compositions can be brought about by including a 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] The 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 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.,University 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 The 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 produced by conventional methods known to those skilled in the art. The dosage regimen is adjusted to provide the desired response (e.g., In determining a therapeutically or prophylactically effective dose, In some cases, a low dose may be administered, followed by the desired response with minimal or no undesirable 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. The dosage 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 that is Each unit is calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. It contains a fixed amount of active compound.

[0181] The actual dosage levels of the active ingredients present in the pharmaceutical compositions should be such that they are not toxic to the patient and are not A composition of active ingredients effective to achieve a desired therapeutic response for a given patient, composition, and mode of administration. The dosage level selected may vary depending on the particular composition used. The activity, route of administration, time of administration, details of the compound or its ester, salt or amide used The rate of excretion 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 dosage depends on a variety of pharmacokinetic factors, including the dosage and previous medical history.

[0182] Products / Kits In some embodiments, the antibody-cytokine grafted protein is 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 a container and a label on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, solutions, etc. Bags, etc. 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). In combination, the compositions are effective for the preparation of a composition for the treatment, prevention and / or amelioration of cancer. The label or package insert shall state that the composition is used for treating, preventing, and / or ameliorating cancer. The antibody-cytokine grafted protein as described herein is included. 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 pharma- ceutical 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 In some embodiments, the antibody-cytokine grafted protein is In a separate formulation (e.g., in a separate container) with a second or third agent in a kit In a particular embodiment, the product (kit) comprises an antibody-cytokine grafted protein. In some embodiments, the aliquot provides one or more doses. In the present invention, multiple dose aliquots are provided, where the dose is uniform or variable. In particular embodiments, the varying dosing regimen is either incremental or incremental, as appropriate. In some embodiments, the antibody-cytokine grafted protein and the second agent The dosages are independently uniform or independently variable. In certain embodiments, the products ( The kit may include an additional agent, such as an anti-cancer agent or an immune checkpoint molecule. 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 Condition 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, comprising: The method of the present invention comprises the step of administering an antibody-cytokine grafted protein as described herein. In some embodiments, the method comprises administering to the individual an effective amount of a compound that is capable of treating or preventing cancer in the individual. 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] The 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 prevented from future onset. In some embodiments, the patient has or is suspected of having cancer. or are in remission from cancer. Treatment with antibody-cytokine grafted protein The cancer to be treated is generally a cancer that is 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 may be advised to use the antibody-cytokine grafted protein in a pharmaceutical composition. 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 therapeutic 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 The therapeutic dose will vary depending on many different factors, including whether the therapeutic agent is safe and effective. Titration is typically required to optimize the antibody-cytokine grafted protein. For administration with the present invention, the dosage is about 0.0001 to 100 mg / kg of the host body weight, and more commonly The dosage is usually in the range of 0.01 to 5 mg / kg. For example, the dosage is 1 mg / kg body weight or 10 mg / kg. The dosage may be in the range of 1 to 10 mg / kg body weight or 1 to 10 mg / kg. This may be daily, weekly, biweekly, monthly, or more or less frequently as appropriate. Suggested treatment regimes will necessarily involve once a week, once every 2 weeks, or once a month or once every 3-6 months. It involves administration once.

[0186] The antibody-cytokine grafted protein can be administered in a single dose or in divided doses. The antibody-cytokine grafted protein is usually administered on multiple occasions. The interval between the amounts may be weekly, biweekly, monthly or yearly, as needed or desired. In addition, by measuring blood levels of antibody-cytokine grafted proteins in patients May be taken irregularly as indicated. 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 a natural IL2 or Proleukin ( These cytokines exhibit a longer half-life than recombinant cytokines such as IL-13, IL-13C, IL-13D, IL-13E, IL-13F, IL-13G, IL-13H ... This can vary depending on whether the treatment is preventative or therapeutic. Some patients will experience Generally, for prophylactic applications, lower dosages are administered at lower frequency. Generally, for therapeutic applications, therapies are administered at intervals over a long period of time to reduce or halt 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 receives a prophylactic regime. This may be done.

[0187] Coadministration with a second agent The term "combination therapy" refers to the use of two therapeutic agents to treat a therapeutic condition or disorder described in this disclosure. Such administration refers to administration of one or more therapeutic agents in a single capsule having a fixed ratio of active ingredients. Alternatively, this method includes the co-administration of these therapeutic agents in a substantially simultaneous manner, such as by administering Such administration may involve multiple or separate containers (e.g., capsules, powders, etc.) for each active ingredient. The powder and / or liquid may be adjusted to the desired dose prior to administration. Moreover, such administration may be simultaneous, at about the same time, or at different times. It also includes 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 the active ingredients are coformulated and administered; (2) when delivered simultaneously in separate formulations 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., consecutively. In combination therapy, effective dosages of two or more active ingredients are administered together.

[0189] In one aspect, the present disclosure relates to a method for treating cancer including, but not limited to, EGFR inhibitors, Her2 inhibitors, H One or more tyrosine kinase inhibitors, including er3 inhibitors, IGFR inhibitors, and Met inhibitors Delivering 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 [4-(2-fluoro-5-methylphenyl)urea]-N'-(2-fluoro-4-ylphenyl)urea, ABT 869, available from Genentech); sunitinib malate (Sute nt (registered trademark); 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®); 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. tert-butyl phosphate (Tarceva®), gefitnib (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 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 Mine (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)buta-2-enamide and WO 05 / 028443 lapatinib or lapatinib ditosylate (Tykerb®) );(3R,4R)-4-amino-1-((4-((3-methoxyphenyl)amino)pyridine Rolo[2,1-f][1,2,4]triazin-5-yl)methyl)piperidin-3-ol (2E)-N-[4-[(3-chloro-4-fluorophenyl) (3S)-tetrahydro-3-furanyl)oxy]-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-phenyl Nyl-2,3-dihydro-1H-pyrazole-4-carboxamide (AMG 458); Crizotinib (Xalkori®, PF-0234) 1066);(3Z)-5-(2,3-dihydro-1H-indol-1-ylsulfonyl 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-yl]ethanol (PF04217903, CAS 956905-27-4) ;N-((2R)-1,4-dioxane-2-ylmethyl)-N-methyl-N'-[3- (1-Methyl-1H-pyrazol-4-yl)-5-oxo-5H-benzo[4,5]ci Chlohepta[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-pyrrolo [2-yl]methylene]-1,3-dihydro-2H-indol-2-one (PHA6 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 therapeutic agents 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 In combination with GF downstream signaling pathway inhibitors, antibody-cytokine grafted proteins are The present invention provides a method for treating cancer by administering 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 CI cyclopropylmethoxy-3,4-difluorobenzamide -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 WO 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 Nyl]-1-[(2R)-2,3-dihydroxypropyl]-cyclopropanesulfonyl mido (also known as RDEA119 or BAY869766 and International Publication No. 2007014011 Listed 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-benzoxacyclotetradecyne-1,7(8H)-dione ] (also known as E6201 and described in WO 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-dihydroxypropane 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 WO 09 / 036082 and WO 0 9 / 055730 brochure); 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 as 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 / 0133625). 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 (formally 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 Phosphinates, also known as AP23573 and MK8669, and WO 03 / 0643 83); everolimus (Afinitor® or RAD001; rapamycin (AY22989, Sirolimus (registered trademark); 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-methoxyphenyl)-1,2-diamino-2-phenyl-1,2-diamino-2-phenyl-1-methyl- lance-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)mono [4-sulfolinium-4-yl]methoxy]butyl]-L-arginylglycyl-L-α-aspartate Pyryl L-serine-("L-arginylglycyl-L- α-Aspartyl L-serine-), inner salt (SF1126, CAS 936487-67) -1) are some examples.

[0201] In yet another aspect, the present disclosure provides therapeutic agents including, but not limited to, IAP inhibitors, Bcl2 inhibitors , one or more pro-apoptotic agents, 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 Brochure, International Publication No. 05 / 097791 Brochure, International Publication No. WO 05 / 069894 Brochure, International Publication No. WO 05 / 069 No. 888, International Publication No. 05 / 094818, U.S. Patent Application Publication No. Publication No. 2006 / 0014700, Publication No. 2006 / 0025347 No. 06 / 069063, WO 06 / 01011 No. 8, International Publication No. 06 / 017295, and International Publication No. 08 Examples include those described in pamphlet No. 134679.

[0203] BCL-2 inhibitors include, but are not limited to, 4-[4-[[2-(4-chlorophenoxy) Nyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl]-1-piperaziny -N-[[4-[[(1R)-3-(4-morpholinyl)-1-[(phenylthio) Methyl]propyl]amino]-3-[(trifluoromethyl)sulfonyl]phenyl]s

[0023] [sulfonyl] benzamide (also known as ABT-263 and WO 09 / 155386 described in FRET); tetrocarcin A; antimycin; gossypol ((-)BL -193;Obatoclax;Ethyl-2-amino-6-cyclopentyl-4-(1- Cyano-2-ethoxy-2-oxoethyl)-4Hchromone-3-carboxylate (H A14-1; Oblimersen (G3139, Genasense (registered trademark)); 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 duranermin (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 (TRAILR6) are available from Proapoptotic receptor agonists (PARAs), including (TRAILR2).

[0205] Checkpoint kinase (CHK) inhibitors include, but are not limited to, 7-hydroxytryptamine, 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-carboxylate 4-[(phenyl-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 Argine;N-[5-bromo-4-methyl-2-[(2S)-2-morpholinylmethacrylate [Ci]-phenyl]-N'-(5-methyl-2-pyrazinyl)urea (LY2603618, CAS 911222-45-2; Sulforaphane (CAS 4478-93-7, 4 -Methylsulfinylbutylisothiocyanate;9,10,11,12-tetrahydrofuran 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 glucagon-binding protein (AGG) antibody 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); Gatef (BIBF1120, CAS 928326-83-4); Dovitinib dilactate (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 4-amino-5-fluoro-3-(5-(4-methyl- Tylpiperazin-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 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) Methods for treating cancer by administering antibody drug conjugates to a subject in need thereof - Patent Application 20070123333 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 can be administered. PD-1, PD-L1, PD-L2, TIM3, CTLA-4, LAG-3, CEA CAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD Immune checkpoint molecules selected from one or more of 160, 2B4, or TGFR. In one embodiment, the present invention 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 the group consisting of nivolumab, nivolumab, and pidilizumab. Nivolumab is also known by other names such as MDX-1106, MDX-1106-04, In some embodiments, the antibody is selected from the group consisting of ONO-4538, BMS-936558, and / or 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 disclosed 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 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 investigated 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. Pat. Examples include the anti-PD1 antibodies disclosed in Application Publication No. 2016 / 0108123. In some embodiments, the antibody-cytokine grafted protein is It is also possible to administer the compound in combination with the anti-Tim3 antibody disclosed in US Pat. No. 2015 / 0218274. In another embodiment, the antibody-cytokine grafted protein is a polypeptide as described in U.S. Pat. The anti-PD-L1 antibody, Durva, disclosed in Patent Publication No. 2016 / 0108123 Lumab® (MEDI4736), Atezolizumab® (MPDL3280A) or Avelumab® . EXAMPLES

[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 sequences. Tokine-grafted proteins were then prepared, and both the heavy and light immunoglobulin chains were then synthesized. The final antibody-cytokine protein was generated using the antibody-cytokine grafting The protein confers the favorable therapeutic properties of IL2; however, the antibody cytokine group Rafted proteins have been shown to have desirable effects such as increased Treg cell activity when compared to rhIL2. The effectiveness of this treatment has decreased.

[0212] To create an antibody-cytokine grafted protein, the IL2 sequence ( SEQ ID NO: 4 or 6) was inserted into the CDR loop of the immunoglobulin chain scaffold. The present study uses various known immunoglobulin sequences and germline antibody sequences that are used in An antibody-cytokine grafted protein was prepared using the antibody-cytokine grafted protein GFTX3b. 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 from the antibody-cytokine graft protein The protein is then isolated using standard molecular biology techniques using recombinant DNA encoding the relevant sequence. This was generated.

[0213] The choice of which CDR to choose for cytokine grafting should be based on the following parameters: The selected compound has the desired biological and biophysical properties and a 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 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 solving biophysical properties such as structural resolution of how they interact with each other.

[0214] Regarding the IL2 antibody cytokine grafting molecule, first consider 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 result in their respective receptors It was found that cytokines would be presented to the Each antibody IL2 graft protein contains CDR loops of different length, sequence and structural environment. Therefore, LCDR-1, LCDR-2, LCDR-3 and HCDR-1 IL2 was grafted into all six CDRs corresponding to HCDR-2 and HCDR-3. From this table in Figure 1, IL2 grafted onto the light chain of CDR2 (IgG.IL2.L2 ) was not expressed, and the antibody-cytokine grafted protein was not shown to be effective in terms of its activity. It is clear that there are differences in I 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 to yield a spacing of 8 Å × number of residues. Although not constrained by this, this may independently allow 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. This coincided with the center of the CDR loop sequence as defined. The insertion point was shifted away from the center of the CDR loop toward either the N-terminal or C-terminal part. 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 hypothesis that the CDRs can be provided to the individual subunits of 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. This is done by comparing each individual antibody-cytokine molecule with respect to its respective properties. I did.

[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 results were analyzed at pre-administration, 1 hour, 3, 7, 24, and 3 hours post-administration. All animals were bled at 1, 48, 55 and 72 hours. Blood samples were centrifuged and plasma samples were The 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 assessed by immunoassay. Pharmacokinetic parameters such as half-life were calculated. Each sample was 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 by using the oaffy200 together 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, it was not possible to plot this on a graph.

[0228] Example 3: IgG.IL2R67A.H1 inhibits CD8 T effector transcription 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 Increases CD8 T effector activity without causing adverse events seen with administration. 1 After administration to mice on day 1, expansion of CD8 T effectors was observed on days 4, 8, and 11. At each time point, the CD8 T effector cell population expanded significantly, and Treg expansion This is because the mortality and morbidity observed with equimolar doses of IL-2 was not significantly different from that observed with Proleu This was in contrast to kin® and IL-2Fc fusions.

[0229] B6 female mice were treated with Proleukin® (five 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 in RPMI (10% FBS). The cells were lysed in red blood cell lysis buffer (Sigma #R7757) and analyzed for cell number and viability. The 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. 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 Bectonoscience protocol (#72-5775). n-Dickinson LSR Fortessa® or Becton-D Analysis was performed on a Fischer FACS LSR II and data were analyzed using FlowJo ( ) software was used for analysis.

[0230] 3A-3C show the effect of Proleukin® (five times a week), IL2-Fc and I gG.IL2R67A.H1 (once per week) in equimolar concentrations with Proleukin® (IgG.IL2R67A.H1 and IL2-Fc 100μg approx. 1nmol IL2 Preferential expansion of CD8 T effector cells in B6 female mice following administration of 100 mg of 10 ... The data in these graphs demonstrate that CD8 T effector cells promote similar proliferation of Tregs. These data demonstrate that CD8 T effector and This is in contrast to 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 Treg:Treg ratio. To achieve this, we developed a structure for the IgG.IL2R67A.H1 antibody cytokine grafted protein. These results demonstrate that there is a critical and functional basis for the expression of IgG.IL2R67. The beneficial effects of A.H1 are more evident at higher doses. When B6 mice were administered IgG.IL2R67A.H1 at a dose of 100 mg / kg / day (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. This suggests that IgG.IL2R6 7A.H1 has a higher therapeutic index and a broader dosage range than IL2-Fc fusion constructs It has been noted that it can be safely administered at

[0231] Example 4: IgG.IL2R67A.H1 inhibits CD8 T effector agonists in NOD mice -Selectively expands cells and is better tolerated than Proleukin® Nonobese diabetic (NOD) mice spontaneously develop type 1 diabetes and serve as an animal model of human type 1 diabetes. The same protocol for B6 mice described in Example 3 was used. Using 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 cell change, and cell type 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. It is a fast-growing grade IV colon cancer cell line that is often used in drug development. CT26 (ATCC CRL-2638) cells were grown in 5% CO The cells were grown in sterile conditions in a 37°C incubator for 2 h. The cells were supplemented with 10% FBS. The cells were cultured in RPMI 1640 medium supplemented with 100 mM NaCl. The cells were subcultured every 3 to 4 days. Cells were harvested (passage 11) and cultured at 2.5 × 10 6 The cells were resuspended at a concentration of 100 / ml. The cells were Radil tested for mycoplasma and mouse viruses. For each mouse, 0.25 × 10 6 Cells were injected into a 28g needle (100μl injection volume) After implantation, when the tumors were palpable, the animals were They were measured with calipers and weighed three times a week. Caliper measurements were calculated using (L x W x 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 a SPF animal facility in accordance with the regulations of the International Animal Welfare Center (IHC).

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

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

[0235] Example 6: IgG.IL2R67A.H1 and additional cancer therapeutics in the B16 mouse model Demonstrated effectiveness in To evaluate the efficacy of IgG.IL2R67A.H1 in combination with other cancer therapy drugs, The 6F10 melanoma mouse model was used. B16F10 cells (ATCC CRL-6 475) were grown in a 37°C incubator with 5% CO2 under axenic 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 in 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 100 μg / ml of 1000 mM NaCl. After implantation, tumors became palpable. Mice were then measured and weighed twice weekly. 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 combination with IL2-Fc / TA99 It was more effective than IL2-Fc. TA99 was ineffective when administered as a single agent, and Average tumor volume was similar to untreated controls. This data supports the findings of IgG.IL2R67A. H1 is effective as a single agent in melanoma mouse tumor models, but it is also It has also demonstrated efficacy when combined with other anticancer 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 the pSTAT5 activity in human peripheral blood mononuclear cells (PBMCs), we assayed pSTAT5 activity in PBMCs. C cells were placed in serum-free test medium and plated. PBMCs were incubated with IgG.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 min at room temperature, the samples were washed, resuspended, and stained with surface markers. The cell pellet was permeabilized with -20°C methanol, washed, and incubated with pSTAT5 and DNA immunoglobulins. Cells were stained for intercalator. The cells were run on Cytof® and the data were analyzed using F pSTAT5 activity levels were determined by analysis with lowJo® software. The table in Figure 7 shows that IgG.IL2R67A.H1 binds to CD8 T effector cells. The present invention demonstrates preferential activation of Treg cells over T cells that minimizes activation of Treg cells.

[0239] Example 8: Binding of antibody-cytokine grafted proteins The IL2 sequence containing the mutein (SEQ ID NO: 4) was inserted into the CDR loop 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 grouped into the CDR of this antibody. To determine whether rafting reduced or abolished binding to RSV, 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 the 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 can retain the binding to the original target of the antibody scaffold, or These results demonstrate that the effect of

[0240] The examples and embodiments described herein are for illustrative purposes only and various modifications thereto may be made. or modifications may be suggested to those skilled in the art and are within the spirit and scope of this application and the scope of the appended claims. It is understood that the scope of the present invention is to be encompassed by the following publications, sequence accession numbers, and the like cited herein. All issues, patents, and patent applications are hereby incorporated by reference in their entirety for all purposes. This is used as a reference.

Claims

1. (a) a heavy chain complementary determining region (CDR) including HCDR1, HCDR2, and HCDR3; Variable domain (VH); and (b) a light chain variable region (VL) comprising LCDR1, LCDR2, LCDR3; and (c) an interleukin 2 (IL2) molecule grafted into the CDRs of VH or VL; An antibody-cytokine grafted protein comprising:

2. The antibody cytokine of claim 1, wherein the IL2 molecule is grafted into a heavy chain CDR. Ingrafted proteins.

3. Heavy chain CDRs are complementarity determining region 1 (HCDR1), complementarity determining region 2 (HCDR2), and complementarity determining region 3 (HCDR3). Grafted proteins.

4. The antibody cytokine of claim 3, wherein the IL2 molecule is grafted to HCDR1. Ingrafted proteins.

5. The antibody cytokine of claim 1, wherein the IL2 molecule is grafted into a light chain CDR. Ingrafted proteins.

6. The light chain CDRs are complementary determining region 1 (LCDR1), complementary determining region 2 (LCDR2), 6. The antibody site of claim 5, wherein the antibody site is selected from the complementarity determining region 3 (LCDR3) and the complementarity determining region 4 (LCDR5). Cain-grafted protein.

7. The IL2 molecule reduces the affinity of the IL2 molecule for the high affinity IL2 receptor. The antibody-cytokine grafted polypeptide according to any one of claims 1 to 6, comprising a mutation Compact quality.

8. Inhibition of CD8 T effector cell proliferation by the antibody-cytokine grafted protein The stimulation of the antibody of claims 1 to 7 is greater than that of recombinant IL2 or Proleukin®. An antibody-cytokine grafted protein according to any one of claims 1 to 4.

9. Stimulation of CD4 T regulatory cell proliferation by the antibody-cytokine grafted protein Any of claims 1 to 8, which is smaller than recombinant IL2 or Proleukin®. An antibody-cytokine grafted protein according to any one of claims 1 to 4.

10. Stimulation of NK cell proliferation by the antibody-cytokine grafted protein was achieved by recombinant IL2 or greater than Proleukin® according to any one of claims 1 to 9. Antibody-cytokine-grafted proteins.

11. The antibody-cytokine grafted protein is selected from the group consisting of native IL2 and Proleukin ( The antibody site according to any one of claims 1 to 10, which has a half-life longer than that of the antibody Cain-grafted protein.

12. The antibody subunit of any one of claims 1 to 11, wherein the IL2 molecule consists of SEQ ID NO:

4. Itokine-grafted protein.

13. The antibody subunit of any one of claims 1 to 11, wherein the IL2 molecule consists of SEQ ID NO:

6. Itokine-grafted protein.

14. The antibody site according to any one of claims 1 to 13, further comprising an IgG class antibody heavy chain. Cain-grafted protein.

15. The IgG class heavy chain is selected from IgG1, IgG2, and IgG4.

15. An antibody-cytokine grafted protein according to claim 14.

16. The binding specificity of the CDRs for the target is increased by the grafted IL2 molecule for 10 ％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、９５％、９８ %, 99%, or 100% reduction in the antibody site of any one of claims 1 to 15. Cain-grafted protein.

17. The binding specificity of the CDRs for a target is determined in the presence of the grafted IL2 molecule. ０％、２０％、３０％、４０％、５０％、６０％、７０％、８０％、９０％、９５％、９ The antibody sample according to any one of claims 1 to 15, wherein the antibody sample is retained at 8%, 99%, or 100%. Itokine-grafted protein.

18. 18. Any of claims 1 to 17, wherein the binding specificity of said CDRs is different from the binding specificity of said IL2 molecule. An antibody-cytokine grafted protein described in any one of claims 1 to 4.

19. Any of claims 1 to 18, wherein the binding specificity of the CDRs is for a non-human antigen. The antibody-cytokine grafted protein according to claim 1.

20. The antibody-cytokine-grafted antigen of claim 19, wherein the non-human antigen is a virus. Compact quality.

21. The antibody sequence of claim 20, wherein the virus is respiratory syncytial virus (RSV). Tocainine-grafted proteins.

22. The RSV is selected from RSV subgroup A and RSV subgroup B. Item 22. An antibody-cytokine grafted protein according to item 21.

23. The antibody scaffold portion of the antibody cytokine grafted protein is humanized or human. The antibody-cytokine grafted protein according to any one of claims 1 to 22.

24. (i) (a) HCDR1 of SEQ ID NO: 13, (b) HCDR2 of SEQ ID NO: 14, (c) (d) a heavy chain variable region comprising an HCDR3 of sequence number 15, and (e) an LCDR1 of sequence number 29; (f) a light chain variable region comprising an LCDR2 of SEQ ID NO: 30, and (f) an LCDR3 of SEQ ID NO:

31. or (ii) (a) HCDR1 of SEQ ID NO: 45, (b) HCDR2 of SEQ ID NO: 46, (c) (d) a heavy chain variable region comprising a HCDR3 of SEQ ID NO: 47; and (e) a LCDR1 of SEQ ID NO:

61. (e) a light chain variable region comprising an LCDR2 of SEQ ID NO: 62, and (f) an LCDR3 of SEQ ID NO:

63. region 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 comprising SEQ ID NO: 35 region (VL); 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 Area (VL) An antibody-cytokine grafted protein comprising:

26. Any of claims 1 to 25, further comprising a modified Fc region corresponding to a reduced effector function. An antibody-cytokine grafted protein according to any one of claims 1 to 4.

27. The modified Fc region is D265A, P329A, P329G, N297A, L234A 27. The antibody of claim 26, comprising a mutation selected from one or more of: L235A, L236B, and L237C. Itokine-grafted protein.

28. The modified Fc region is D265A / P329A, D265A / N297A, L234 / L235A, P329A / L234A / L235A, and P329G / L234A / L2 28. The antibody of claim 27, comprising a combination of mutations selected from one or more of: Cytokine-grafted proteins.

29. 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, Antibody cytokine grafted with a modified Fc region containing the natural mutations D265A / P329A Proteins that produce lower Treg cell counts compared to Proleukin® Antibody-cytokine-grafted proteins that stimulate activation.

30. 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, Antibody cytokine grafted with a modified Fc region containing the natural mutations D265A / P329A Proteins that produce lower Treg cell counts compared to Proleukin® Antibody-cytokine-grafted proteins that stimulate activation.

31. (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 an isolated antibody cytokine grafted protein encoding the antibody comprising the light chain of SEQ ID NO: 70 Nucleic acid.

32. A recombinant host cell suitable for producing an antibody-cytokine grafted protein, comprising: Claims encoding heavy and light chain polypeptides of an antibody cytokine grafted protein.

32. A recombinant host cell comprising the nucleic acid of 31 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. The antibody-cytokine grafted protein according to any one of claims 1 to 30 and a pharmaceutical and a physiologically acceptable carrier.

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

37. The cancer is selected from the group consisting of melanoma, lung cancer, colorectal cancer, prostate cancer, breast cancer and lymphoma.

37. The method of claim 36, wherein

38. The antibody-cytokine-grafted protein or pharmaceutical composition is administered in combination with another therapeutic agent.

38. The method of claim 36 or 37, wherein

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

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

41. The immune checkpoints are PD-1, PD-L1, PD-L2, TIM3, and CTL. A-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, T The present invention relates to a method for treating cancer, comprising administering to a patient a cancer-related gene that is selected from the group consisting of IGIT, LAIR1, CD160, 2B4 and TGFR. The method according to claim 40.

42. 1. A method for expanding CD8 T effector cells in a patient in need thereof, comprising: The antibody-cytokine grafted protein according to any one of claims 1 to 30.

36. A method comprising administering to the patient the pharmaceutical composition of claim 35.

43. The CD8 T effector cells are expanded and Treg cells are not expanded.

43. The method according to claim 42.

44. 5. The method of claim 4, wherein the CD8 T effector cells are expanded and the NK cells are not expanded.

2. The method according to claim 2.

45. The method according to any one of claims 42 to 44, further comprising administering an immune checkpoint inhibitor. Method of posting.

46. The immune checkpoints are PD-1, PD-L1, PD-L2, TIM3, and CTL. A-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, T The present invention relates to a method for treating cancer, comprising administering to a patient a cancer-related gene that is selected from the group consisting of IGIT, LAIR1, CD160, 2B4 and TGFR. The method according to claim 45.

47. In the treatment of cancer (i) (a) HCDR1 of SEQ ID NO: 13, (b) HCDR2 of SEQ ID NO: 14, (c) (d) a heavy chain variable region comprising an HCDR3 of sequence number 15, and (e) an LCDR1 of sequence number 29; (f) a light chain variable region comprising an LCDR2 of SEQ ID NO: 30, and (f) an LCDR3 of SEQ ID NO:

31. or (ii) (a) HCDR1 of SEQ ID NO: 45, (b) HCDR2 of SEQ ID NO: 46, (c) (d) a heavy chain variable region comprising a HCDR3 of SEQ ID NO: 47; and (e) a LCDR1 of SEQ ID NO:

61. (e) a light chain variable region comprising an LCDR2 of SEQ ID NO: 62, and (f) an LCDR3 of SEQ ID NO:

63. region Use of an antibody-cytokine grafted protein comprising:

48. The antibody-cytokine grafted protein is administered in combination with another therapeutic agent.

48. The use according to item 47.

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

50. The immune checkpoints are PD-1, PD-L1, PD-L2, TIM3, and CTL. A-4, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, T The present invention relates to a method for treating cancer, comprising administering to a patient a cancer-related gene that is selected from the group consisting of IGIT, LAIR1, CD160, 2B4 and TGFR. The use according to claim 49.

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