Novel IL2 agonists and their usage

Abstract

This provides a novel IL2 therapy with improved therapeutic efficacy and safety profile. [Solution] An IL2 agonist is provided, comprising (a) an Fc domain and (b) an IL2 portion at the C-terminus of the Fc domain, wherein the IL2 portion comprises an IL2 domain and an IL2-Rα domain.

Description

[Technical Field]

[0001] This invention relates to novel IL2 agonists and methods for using them. 1. Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 62 / 951,831, filed on 20 December 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] 2. Sequence Listing This application includes an electronically submitted sequence listing in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on December 18, 2020, is named RGN-006C-WO_SL and has a size of 162,034 bytes. [Background technology]

[0003] Interleukin-2 (IL-2 or IL2) is mainly CD4 + It is a pluripotent cytokine produced by helper T cells. It stimulates the proliferation and differentiation of T cells, induces the generation of cytotoxic T lymphocytes (CTLs) and the differentiation of peripheral blood lymphocytes into cytotoxic cells and lymphokine-activated killer (LAK) cells, promotes the expression of cytokines and cytolytic molecules by T cells, facilitates the proliferation and differentiation of B cells and the synthesis of immunoglobulins by B cells, and stimulates the generation, proliferation, and activation of natural killer (NK) cells (see Non-Patent Documents 1 and 2).

[0004] IL-2 has three different receptors: high-affinity receptors, intermediate-affinity receptors, and low-affinity receptors. The high-affinity receptor has three subunits: interleukin-2 receptor alpha (IL-2Rα; CD25), interleukin-2 receptor beta (IL-2Rβ; CD122), and interleukin-2 receptor gamma (IL-2Rγ; CD132; common gamma chain). The low-affinity receptor is IL-2Rα, which is a 55kD polypeptide (p55) that appears when T cells are activated and was originally called the Tac (T-activating) antigen. IL-2Rα is approximately 10 -8 M's K D It binds to IL-2. Binding of IL-2 to cells expressing only IL-2Rα does not elicit any detectable biological response.

[0005] Intermediate affinity receptors consist of IL-2Rβ and IL-2Rγ. IL-2Rβ is a member of the type I cytokine receptor family, characterized by two cysteine / WSXWS motifs (SEQ ID NO: 1). IL-2Rγ, a 64kD polypeptide, is shared by several cytokine receptors, including those for interleukin-4 and interleukin-7, and is therefore also known as the common gamma chain. Intermediate affinity receptors also mediate interleukin-15 (IL-15 or IL15) signaling.

[0006] It is believed that resting immune cells express only intermediate affinity receptors. For example, IL-2Rα is rapidly expressed upon activation of antigen receptor-mediated immune cells by resting T cells. When IL-2Rα binds to IL-2, it subsequently binds to IL-2Rβ and IL-2Rγ. IL-2 binding by the IL-2Rαβγ complex leads to signaling via STAT5 and IL-2-mediated growth stimulation in effector T cells, particularly those that destroy virus-infected cells and tumor cells.

[0007] In addition to its stimulating effect on effector T cells, IL-2 mediates activation-induced cell death (AICD) in T cells (Non-Patent Literature 3). AICD is a process in which fully activated T cells undergo programmed cell death, resulting in resistance not only to established normal autoantigens but also to persistent antigens such as tumor antigens.

[0008] IL2 is a peripheral CD4 cell, also known as a suppressor T cell. + CD25 + Adjustability T(T reg They are also involved in the maintenance of cells (see, for example, Non-Patent Document 4). They suppress effector T cells from destroying (self) targets either by intercellular contact through the assistance and inhibition of T cell activation, or by the release of immunosuppressive cytokines such as IL-10 or TGFβ. reg Cell depletion has been shown to enhance IL2-induced antitumor immunity (Non-Patent Literature 5).

[0009] Due to its multifaceted effects, IL-2 is not optimal for inhibiting tumor growth. The use of IL-2 as an antitumor agent has been limited by the severe toxicity associated with doses required for tumor response. Proleukin® (marketed by Prometheus Laboratories, San Diego, Calif.) is a recombinant IL-2 approved for the treatment of metastatic melanoma and metastatic renal cell carcinoma, but its use is only recommended in hospital settings where intensive care can be provided due to its extremely severe side effects. Patients receiving high-dose IL-2 therapy frequently experience severe cardiovascular, pulmonary, renal, hepatic, gastrointestinal, neurological, cutaneous, hematological, and systemic adverse events requiring intensive monitoring and hospitalization. The main side effect of IL-2 therapy is vascular leak syndrome (VLS), which causes interstitial fluid accumulation in the lungs and liver, leading to pulmonary edema and liver damage. There is no treatment for VLS other than discontinuation of IL-2. Low-dose IL-2 regimens have been tested in patients to avoid VLS, but the treatment outcomes are not optimal. IL2-induced pulmonary edema has been shown to be caused by the direct binding of IL2 to pulmonary endothelial cells that express low to intermediate levels of functionally high-affinity IL2 receptors (Non-Patent Literature 6).

[0010] Various IL2 variants have been generated with the aim of reducing the toxicity of IL2 cancer therapy. A common approach is to generate an IL2 molecule that allows IL2 to bind to the intermediate affinity receptor but is reluctant to bind to CD25 (hence called CD122 bias). The rationale for this approach consists of two elements. First, CD25 is a sink, and excess CD25 can act as a sink, thereby potentially depriving effector T cells of IL2. regIt is prominently expressed on cells. Secondly, endothelial cells that mediate VLS express CD25. Theoretically, reducing binding to CD25 (IL-2Rα) would redirect the IL2 signal to effector T cells, improving the antitumor effect, reducing VLS, and thereby reducing toxicity. Strategies for obtaining CD122-biased IL2 formulations include, for example, CD122-oriented IL2 complexes in which anti-IL2 monoclonal antibodies encapsulate the CD25 binding site, IL2 muteins with mutations in the CD25 binding site, and IL2-transporting polyethylene glycol (PEG) groups with CD25 binding sites. Non-patent document 7.

[0011] Despite being generally accepted in the field of CD122-targeted IL2 therapy developed for cancer treatment, it has been surprisingly found that such molecules have a low therapeutic index in cancer therapy and require high toxic doses to produce a moderate anti-cancer effect. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Waldmann, 2009, Nat Rev Immunol, 6:595-601 [Non-Patent Document 2] Malek, 2008, Annu Rev Immunol, 26:453-79 [Non-Patent Document 3] Lenardo et al., 1991, Nature, 353:858-61 [Non-Patent Document 4] Fontenot et al., 2005, Nature Immunol, 6:1142-51 [Non-Patent Document 5] Imai et al., 2007, Cancer Sci, 98:416-23 [Non-Patent Document 6] Krieg et al., 2010, Proc Nat Acad Sci USA, 107:11906-11 [Non-Patent Document 7] Onur and Arenas-Ramirez, 2019, Swiss Med Wkly 149:w14697

Summary of the Invention

Problems to be Solved by the Invention

[0013] Therefore, in the art, there is a need for a novel IL2 therapy with improved therapeutic effects and safety profiles.

Means for Solving the Problems

[0014] The present disclosure starts from several discoveries regarding the activity of IL2 molecules that affect the antitumor effect. As shown herein, the CD122-directed IL2 molecule surprisingly has only a moderate antitumor effect even at high doses that cause obvious toxicity. This can be explained by several other discoveries. First, the inventors found that, in contrast to wild IL2, which preferentially expands CD8 + T cells in tumors but not peripherally, the CD122-directed IL2 molecule is designed to preferentially expand NK and CD8 + T cells against Treg cells, but they were found to expand mainly in the blood and spleen and to a very low extent in tumors. In addition, the CD8 + T cells expanded by the CD122-biased IL2 molecule are mostly CD44 + CD62L + central memory-like T cells that lack specificity for tumor cells, while wild-type IL2 is CD44 + CD62L - effector CD8 +T cells can be enlarged. Therefore, the antitumor effect of these molecules is reduced compared to wild-type IL2. Furthermore, the inventors discovered that tumor Treg cells are less responsive to IL2 than blood and splenic Treg cells. Thus, the adverse effects of non-CD122-directed IL2 cancer therapy can be mitigated by directing such non-CD122-directed IL2 to the tumor, where Tregs are less responsive to IL2 and IL2 preferentially enlarges CD8+ T cells.

[0015] Furthermore, the inventors have discovered that the therapeutic indices of various IL2 molecules, with varying degrees of receptor attenuation, can be improved by appropriately balancing the ability of IL2 agonists to localize to a target site or cell type (e.g., generally the tumor environment or specifically tumor-responsive T cells) and by regulating the level activity of IL2 components until they reach the target site and / or cell type.

[0016] Based on the aforementioned findings, the inventors have developed an IL2 molecule (referred to herein as an IL2 agonist) that is considered to be more effective than the CD122-directed IL2 molecules currently under development for the treatment of cancer. The IL2 agonists of this disclosure are not directed to CD122, and in some cases, CD122 binding is reduced or minimized. The IL2 agonists of this disclosure have an IL2 moiety, as well as (1) an optional tumor-targeting moiety and / or (2) an optional multimerization, e.g., a dimerization moiety and / or (3) an optional stabilization moiety. The tumor-targeting moiety, e.g., the antigen-binding domain ("ABD") of an antibody, can bind, for example, to target molecules present on the tumor surface (e.g., tumor-associated antigens), the tumor microenvironment, and / or tumor-reactive lymphocytes.

[0017] The IL2 portions that can be used in the IL2 agonists of this disclosure are described in Section 6.3. The targeting portions that can be used with the IL2 agonists of this disclosure are described in Section 6.4.

[0018] The multimerized portions that can be used in the IL2 agonists of this disclosure are described in Section 6.5. The stabilization portions that can be used in the IL2 agonists of this disclosure are described in Section 6.6.

[0019] Various exemplary configurations of the IL2 agonist of this disclosure are described in the following specific embodiments 1-166, 199-258, and 267-322. Linkers that can be used to connect different components of the IL2 agonist of this disclosure are described in Section 6.7.

[0020] This disclosure further provides nucleic acids encoding the IL2 agonists of this disclosure. The nucleic acids encoding the IL2 agonists may be a single nucleic acid (e.g., a vector encoding all polypeptide chains of the IL2 agonist) or multiple nucleic acids (e.g., two or more vectors encoding different polypeptide chains of the IL2 agonist). This disclosure further provides host cells and cell lines engineered to express the nucleic acids and IL2 agonists of this disclosure. This disclosure further provides methods for producing the IL2 agonists of this disclosure. Exemplary nucleic acids, host cells and cell lines, and methods for producing IL2 agonists are described in Section 6.8 below, as well as in Specific Embodiments 167-169, 259-261, and 323-325.

[0021] This disclosure further provides pharmaceutical compositions comprising the IL2 agonists of this disclosure. Exemplary pharmaceutical compositions are described in Section 6.9 below, as well as in specific embodiments 170, 262, and 326.

[0022] For example, methods of using the IL2 agonists and pharmaceutical compositions of this disclosure to treat cancer and immunodeficiencies are further provided herein. Exemplary methods are described in Section 6.10. The IL2 agonists of this disclosure are useful in combination therapy, for example, as adjuncts to CART therapy. Exemplary combination therapy methods are disclosed in 6.11. Specific embodiments of the therapeutic methods of this disclosure are described in the following specific embodiments 171-198, 263-266, and 327-346. [Brief explanation of the drawing]

[0023] [Figure 1] The signaling pathways of IL2 and IL15, which have their own unique receptor subunits but share a common β / γ receptor subunit, are illustrated. [Figure 2A] The presentation of antigens to T cells by antigen-presenting cells via class I (Figure 2A) and class II (Figure 2B) MHC complexes, as well as the activation of T cell receptor complexes by MHC-peptide complexes (Figures 2C-2D), are illustrated. [Figure 2B] Same as above. [Figure 2C] Same as above. [Figure 2D] Same as above. [Figure 3A] This shows the equilibrium between different structures formed from two different species of IL-2Rα-containing IL2 mutein. [Figure 3B] Same as above. [Figure 4A] This shows the activity of IL2M1 against human PBMCs. IL2M1 exhibits reduced activity against IL-2Rα+ Tregs (Figure 4A), but maintains full activity against IL-2Rα-CD8+ T cells (Figure 4B) and NK cells (Figure 4C). [Figure 4B] Same as above. [Figure 4C] Same as above. [Figure 5A]In vivo, IL2M1 selectively expands NK cells (Figure 5B) and CD8+ T cells (Figures 5C-5D) compared to Treg cells (Figure 5A), but IL2M0 (referred to as IL2-Fc in the figures) preferentially expands Treg cells (Figures 5A-5D). [Figure 5B] Same as above. [Figure 5C] Same as above. [Figure 5D] Same as above. [Figure 6A] The mean tumor volume (mm³ + SD) (Figure 6A) and Kaplan-Meier survival curves (Figure 6B) of mice treated with IL2 mutein and / or anti-PD1 antibody are shown. [Figure 6B] Same as above. [Figure 7A] This shows that IL2M0 (referred to as IL2-Fc in the figure) induces an antitumor response in a dose-dependent manner. [Figure 7B] Same as above. [Figure 7C.1] Same as above. [Figure 7C.2] Same as above. [Figure 7C.3] Same as above. [Figure 7C.4] Same as above. [Figure 7C.5] Same as above. [Figure 8A] It exhibits moderate antitumor effects against IL2M1 (Figures 8A, 8B) and associated apparent toxicity (Figure 8C). [Figure 8B] Same as above. [Figure 8C] Same as above. [Figure 9A] The images show moderate antitumor effects against IL15M1 (Figure 9A) and associated apparent toxicity (Figure 9B), as well as peripheral immune cell profiles induced by IL15M1 (Figures 9C.1-C.4). [Figure 9B] Same as above. [Figure 9C.1] Same as above. [Figure 9C.2] Same as above. [Figure 9C.3] Same as above. [Figure 9C.4] Same as above. [Figure 10A.1]This shows the activity of IL2M0 (referred to as IL2-Fc in the figure) and IL2M1 on tumor and peripheral lymphocyte proliferation. [Figure 10A.2] Same as above. [Figure 10A.3] Same as above. [Figure 10A.4] Same as above. [Figure 10A.5] Same as above. [Figure 10B] Same as above. [Figure 10C.1] Same as above. [Figure 10C.2] Same as above. [Figure 10C.3] Same as above. [Figure 10C.4] Same as above. [Figure 10C.5] Same as above. [Figure 10C.6] Same as above. [Figure 10C.7] Same as above. [Figure 10D] Same as above. [Figure 10E] Same as above. [Figure 10F] Same as above. [Figure 10G] Same as above. [Figure 10H] Same as above. [Figure 10I] Same as above. [Figure 10J] Same as above. [Figure 10K] Same as above. [Figure 10L] Same as above. [Figure 10M] Same as above. [Figure 10N] Same as above. [Figure 10O] Same as above. [Figure 10P] Same as above. [Figure 10Q] Same as above. [Figure 10R] Same as above. [Figure 11A] They exhibit different IL2 responses in splenic and tumor-infiltrating Treg cells, as well as CD8+ T cells. [Figure 11B] Same as above. [Figure 12A]This study shows that IL2M2 and IL2M3 exhibit reduced activity (Figures 12A–12C) and decreased in vivo toxicity (Figure 12D) across multiple lymphocyte populations of human PBMCs. The results in Figures 12A–12C were obtained for IL2M2 and IL2M3 proteins, while Figure 12D is based on in vivo administration of IL2M2 and IL2M3 coding nucleic acids via hydrodynamic DNA delivery ("HDD"). [Figure 12B] Same as above. [Figure 12C] Same as above. [Figure 12D] Same as above. [Figure 13A] This study demonstrates that IL2M2 exhibits antitumor effects as a monotherapy in multiple syngeneic mouse tumor models. IL2M2 was delivered via hydrodynamic DNA delivery (HDD) (Figures 13A and 13B) or as purified protein (Figure 13C). [Figure 13B] Same as above. [Figure 13C] Same as above. [Figure 14A] This study demonstrates the activity of IL2M4 and IL2M5 against different lymphocyte populations of human PBMCs. [Figure 14B] Same as above. [Figure 14C] Same as above. [Figure 15A] This demonstrates the antitumor effect of IL2M5. [Figure 15B] Same as above. [Figure 15C] Same as above. [Figure 15D] Same as above. [Figure 16A] The results of SEC-MALS studies for IL2M2 (Figure 16A), which mainly consists of higher-order oligomers, and IL2M3 (Figure 16B), which mainly exists as an Fc homodimer (thought to be a result of the orientation of the IL2 domain relative to the Fc domain and / or the length of the linker connecting the domains), are shown. [Figure 16B] Same as above. [Figure 17] The results of FACS binding of anti-PD1 and T1-IL2M3 to HEK293-mPD1 cells are shown. [Figure 18A]T1-IL2M3 showed superior antitumor effects compared to the combination of anti-PD1 and untargeted IL2M3 (Figures 18A-18B.4). T1-IL2M3 enlarged PD1+ effector CD8+ T cells but enlarged Tregs less than untargeted IL2M3 (Figures 18C.1-18E.2). [Figure 18B.1] Same as above. [Figure 18B.2] Same as above. [Figure 18B.3] Same as above. [Figure 18B.4] Same as above. [Figure 18C.1] Same as above. [Figure 18C.2] Same as above. [Figure 18D.1] Same as above. [Figure 18D.2] Same as above. [Figure 18E.1] Same as above. [Figure 18E.2] Same as above. [Figure 19] The diagram shows (a) IL2 binding by the IL-2Rαβγ complex resulting in STAT5-mediated signaling and potent cell activation (left portion of the figure), (b) low-level binding of anti-PD1-IL2 mutein 3 fusions to the IL-2Rαβγ complex on lymphocytes and endothelial cells in the absence of PD-1 expressed on the cell surface (low or no activation of these cells) (center portion of the figure between the two dotted lines), and (c) a schematic diagram of the binding of anti-PD1-IL2 mutein 3 fusions to the IL-2Rαβγ complex on tumor-specific T cells with PD-1 expressed on the cell surface, resulting in signaling and sustained T cell activation. [Figure 20A] The antitumor effects of the anti-mPD1-IL2 mutein 3 fusion are demonstrated in mouse syngeneic tumor models: lung (Figure 20A), skin (Figure 20B), mammary gland (Figure 20C), and colon (Figure 20D). [Figure 20B] Same as above. [Figure 20C] Same as above. [Figure 20D] Same as above. [Figure 21A]The antitumor effects of the anti-LAG3-IL2 mutein 3 fusion (Figure 21A) and the antitumor effects of the anti-LAG3-IL2 mutein 3 fusion combined with an anti-mPD1 antibody (Figure 21B) are shown. [Figure 21B] Same as above. [Figure 22A.1] Embodiments of homodimers and heterodimers of single-chain peptide-MHC-targeted IL2 mutein (Figures 22A.1-22A.2), as well as research results demonstrating that single-chain peptide-MHC-targeted IL2 mutein fusions enable selective stimulation of antigen-specific mouse CD8+ T cells (Figure 22B), are shown. [Figure 22A.2] Same as above. [Figure 22B] Same as above. [Figure 23A] This shows the activity of single-chain peptide MHC-targeted IL2 muteins against non-CD8+ T cells that do not express antigen-specific TCRs. Different muteins show indistinguishable activity. [Figure 23B] Same as above. [Figure 24] This demonstrates the selective stimulation of CMV antigen-specific human CD8+ T cells by single-chain IL2 mutein containing a peptide-MHC targeting moiety. [Figure 25A] This specification shows a schematic diagram (Figure 25A) of a chimeric antigen receptor (CAR) comprising a peptide-MHC targeting moiety referred to as T3, a human CD8a hinge and transmembrane (TM) domain, a 4-1BB costimulatory domain, a CD3z signaling domain, and a VL-VH scFv that recognizes a P2A:eGFP sequence for tracking CAR-transduced T cells, as well as the frequency and composition of viable CAR-T cells after expansion (Figures 25B-25C). [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 26A] The structures of the bivalent targeted IL2 mutains T2-IL2M6 and T3-IL2M6 (Figure 26A), and the monovalent targeted IL2 mutains T7-IL2M7 and T8-IL2M7 (Figure 26B) (both possessing peptide-MHC targeting moieties) are shown. [Figure 26B] Same as above. [Figure 27A] Figures 26A and 26B illustrate STAT5 stimulation of CAR-expressing CD4+ (Figure 27A) and CD8+ (Figure 27B) T cells by peptide-MHC-targeted IL2 mutein. [Figure 27B] Same as above. [Figure 28A.1] This shows selective enrichment of CAR-T cells by monovalent IL2 mutein having a targeting moiety recognized by CAR scFV. Figures 28A.1–28A.16 show that the frequency of CAR-T cells is enriched during expansion in response to monovalent single-chain peptide-carrying HLA-A2, as determined by flow cytometry. All biologics were maintained at a concentration of 3.3 × 10⁻¹⁰ M. [Figure 28A.2] Same as above. [Figure 28A.3] Same as above. [Figure 28A.4] Same as above. [Figure 28A.5] Same as above. [Figure 28A.6] Same as above. [Figure 28A.7] Same as above. [Figure 28A.8] Same as above. [Figure 28A.9] Same as above. [Figure 28A.10] Same as above. [Figure 28A.11] Same as above. [Figure 28A.12] Same as above. [Figure 28A.13] Same as above. [Figure 28A.14] Same as above. [Figure 28A.15] Same as above. [Figure 28A.16] Same as above. [Figure 28B]This shows selective enrichment of CAR-T cells by monovalent IL2 mutein having a targeting region recognized by CAR scFV. Figures 28B.1-28B.4 show a selective increase in the CARPos / CARNeg ratio during expansion in response to monovalent single-chain peptide-carrying HLA-A2 fused to attenuated IL(2m) (white circles). Recombinant IL2 (aldesleukin) results in the largest total T cell number, but the degree of CAR-T enrichment is less compared to single-chain peptide-carrying HLA-A2 fused to attenuated IL2(2m). [Figure 28C] This shows selective enrichment of CAR-T cells by monovalent IL2 mutain having a targeting moiety recognized by the scFV of the CAR. Figure 28C shows that expansion of CAR-T cells (white circles) using single-chain peptide-carrying HLA-A2 fused to attenuated IL2 (2m) results in the maximum number of viable CAR-T cells. [Figure 29] This report presents the results of in vivo pharmacokinetic evaluation of monovalent and bivalent targeted attenuated IL2 muteins in immunocompetent (C57BL / 6J) and immunodeficient (NOD.Scid.IL2Rgnull) mice. Both bivalent and monovalent muteins exhibit delayed clearance against hIL2 fused to Fc (IL2-Fc). Mice were injected with each bioagent, and blood samples were collected 2, 24, 48, and 72 hours after administration. Pharmacokinetics of Fc-fusion proteins in plasma were examined by Western blotting. The protein of interest is indicated by a black box. [Modes for carrying out the invention]

[0024] 6. Detailed explanation 6.1.Definition Approximately, roughly: Terms such as “approximately” and “roughly” are used throughout this specification before numbers to indicate that the numbers are not necessarily exact (e.g., to account for fractions, variations in measurement accuracy, and / or accuracy, timing, etc.). It should be understood that a disclosure of “approximately X” or “roughly X” where X is a number is also a disclosure of “X”. Thus, for example, a disclosure of an embodiment in which one sequence has “approximately X% sequence identity” with respect to another sequence is also a disclosure of an embodiment in which that sequence has “X% sequence identity” with respect to another sequence.

[0025] And, or: Unless otherwise specified, the conjunction "or" is intended to be used in the correct sense as a Boolean logical operator, encompassing both the selection of an alternative feature (A or B, the selection of A is mutually exclusive from B) and the selection of a feature in combination (A or B, both A and B are selected). In some parts of the text, the terms "and / or" are used for the same purpose, but should not be interpreted as meaning that "or" is used to refer to mutually exclusive alternatives.

[0026] Antigen-binding domain or ABD: As used herein, the terms “antigen-binding domain” or “ABD” refer to a portion of a targeting moiety capable of specific, non-covalent, and reversible binding to a targeting molecule.

[0027] Related: In the context of IL2 agonists or their components (e.g., targeted moieties such as antibodies), the term “associated” refers to a functional relationship between two or more polypeptide chains. In particular, the term “associated” means that two or more polypeptides associate with each other, for example, non-covalently through molecular interactions or covalently through one or more disulfide crosslinks or chemical crosslinks, in order to produce a functional IL2 agonist. Examples of associations that may exist in the IL2 agonists of this disclosure include, but are not limited to, associations between homodimer or heterodimer Fc domains in the Fc region, associations between VH and VL regions in Fab or scFv, associations between CH1 and CL in Fab, and associations between CH3 and CH3 in domain-substituted Fab.

[0028] Divalent: As used herein with respect to the IL2 moiety and / or targeting moiety in an IL2 agonist, the term “divalent” means an IL2 agonist having two IL2 moieties and / or targeting moieties, respectively. Typically, an IL2 agonist that is divalent with respect to the IL2 moiety and / or targeting moiety is a dimer (either a homodimer or a heterodimer).

[0029] Cancer: The term "cancer" refers to a disease characterized by the uncontrolled (and often rapid) growth of abnormal cells. Cancer cells can spread locally or to other parts of the body via the bloodstream and lymphatic system. Examples of various cancers described herein, but not limited to, include breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, adrenal cancer, autonomic ganglion cancer, biliary tract cancer, bone cancer, endometrial cancer, eye cancer, fallopian duct cancer, reproductive tract cancer, colorectal cancer, meningeal cancer, esophageal cancer, peritoneal cancer, pituitary cancer, penile cancer, placental cancer, pleural cancer, salivary gland cancer, small intestine cancer, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, upper respiratory tract and gastrointestinal cancer, urinary tract cancer, vaginal cancer, vulvar cancer, lymphoma, leukemia, and lung cancer.

[0030] Complementarity-Determining Regions or CDRs: As used herein, the term “complementarity-determining region” or “CDR” refers to the sequence of amino acids within the antibody variable region that confer antigen specificity and binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, HCDR-H3), and each light chain variable region has three CDRs (CDR1-L1, CDR-L2, CDR-L3). Exemplary rules that can be used to identify the boundaries of CDRs include, for example, the Kabat definition, the Chothia definition, the ABM definition, and the IMGT definition. For example, see Kabat, 1991, “Sequences of Proteins of Immunological Interest,” National Institutes of Health, Bethesda, Md. (Kabat numbering scheme), Al-Lazikani et al., 1997, J.Mol.Biol.273:927-948 (Chothia numbering scheme), Martin et al., 1989, Proc.Natl.Acad.Sci.USA 86:9268-9272 (ABM numbering scheme), and Lefranc et al., 2003, Dev.Comp.Immunol.27:55-77 (IMGT numbering scheme). Public databases are also available to identify CDR sequences within antibodies.

[0031] EC50: The term "EC50" refers to the semi-maximal effective concentration of a molecule (such as an IL2 agonist) that induces a response midway between baseline and maximum after a specific exposure time. Essentially, EC50 represents the concentration of an antibody or IL2 agonist at which 50% of its maximum effect is observed. In certain embodiments, the EC50 value is equal to the concentration of the IL2 agonist that gives up to half the maximum STAT5 activation in the assay, as described in Section 7.1.2.

[0032] Epitope: An epitope, or antigenic determinant, is a portion of an antigen (e.g., a target molecule) recognized by an antibody or other antigen-binding moiety described herein. Epitopes can be linear or conformated.

[0033] Fab: In the context of the targeted portion of this disclosure, the term “Fab” refers to a pair of polypeptide chains, the first comprising the variable heavy (VH) domain of the antibody at the N-terminus of a first constant domain (referred to herein as C1), and the second comprising the variable light (VL) domain of the antibody at the N-terminus of a second constant domain that can pair with the first constant domain (referred to herein as C2). In natural antibodies, VH is the N-terminus of the first constant domain (CH1) of the heavy chain, and VL is the N-terminus of the constant domain of the light chain (CL). The Fabs of this disclosure may be arranged according to their natural orientation, or may involve domain substitutions or exchanges to facilitate correct VH and VL pair formation. For example, the CH1 and CL domain pair of the Fab can be replaced with a CH3 domain pair to facilitate correct modified Fab chain pair formation of heterodimer molecules. CH1 and CL can also be reversed, with CH1 attached to VL and CL attached to VH, a configuration commonly known as Crossmab.

[0034] Fc domains and Fc regions: The term "Fc domain" refers to a portion of a heavy chain that pairs with a corresponding portion of another heavy chain. The term "Fc region" refers to a region of an antibody-based binding molecule formed by the association of two heavy chain Fc domains. Two Fc domains within an Fc region may be identical or different. In natural antibodies, Fc domains are typically identical, but one or both Fc domains may be advantageously modified to enable heterodimerization, for example, via knob-in-hole interactions.

[0035] Host Cells: As used herein, the term “host cells” refers to cells into which the nucleic acids of this disclosure have been introduced. The terms “host cells” and “recombinant host cells” are used interchangeably herein. Such terms are understood to refer to specific target cells and their offspring or potential offspring. Such offspring may not be identical to the parent cells in practice, as certain modifications may occur in the next generation either by mutation or environmental influence, but are still included within the scope of the terms used herein. Typical host cells are eukaryotic host cells, such as mammalian host cells. Exemplary eukaryotic host cells include yeast and mammalian cells, and vertebrate cells such as mouse, rat, monkey, or human cell lines, e.g., HKB11 cells, PER.C6 cells, HEK cells, or CHO cells.

[0036] IL2 mutein: A variant IL2 molecule possessing IL2 activity. The variant may be an IL2 fusion protein (e.g., IL2 fused to IL-2Rα) and / or a mutant IL2 having one or more amino acid substitutions compared to wild-type IL2. IL2 mutein may have modified functions (e.g., receptor binding, affinity, cytokine activity) and / or modified pharmacokinetics compared to wild-type IL2. In the context of IL2 agonists as described herein, the term "IL2 mutein" may refer to the non-targeting component (and associated linker moiety) of the IL2 molecule, and it should be understood that the term "IL2 mutein" encompasses the IL2 molecule with or without the targeting moiety and with or without the multimerized moiety, unless otherwise indicated in the context.

[0037] Major Histocompatibility Complex (MHC): These terms refer to naturally occurring MHC molecules, individual chains of MHC molecules (e.g., MHC class I α(heavy) chain, β2 microglobulin, MHC class II α chain, and MHC class II β chain), individual subunits of such chains of MHC molecules (e.g., α1, α2, and / or α3 subunits of MHC class I α chain, α1-α2 subunits of MHC class II α chain, β1-β2 subunits of MHC class II β chain), as well as parts (e.g., peptide bonding parts, e.g., peptide bonding grooves), variants, and their various derivatives (including fusion proteins), such parts, variants, and derivatives that retain the ability to present antigenic peptides for recognition by T cell receptors (TCRs), e.g., antigen-specific TCRs. MHC class I molecules contain peptide bonding grooves formed by the α1 and α2 domains of the heavy chain, which can accommodate peptides of about 8-10 amino acids. Despite the fact that both classes of MHC bind to a core of approximately nine amino acids (e.g., 5–17 amino acids) within a peptide, the MHC class II peptide binding groove (the α1 domain of class II MHC, which is a polypeptide that associates with the β1 domain of class II MHC β polypeptides) allows for a wider range of peptide lengths. Peptides that bind to MHC class II typically vary between 13–17 amino acids in length, although shorter or longer lengths are not uncommon. As a result, peptides can shift within the MHC class II peptide binding groove, potentially altering which 9-mer is directly located within the groove at any given time. Conventional identification of specific MHC variants is used herein. This term includes "human leukocyte antigen" or "HLA".

[0038] Monovalent: As used herein with respect to the IL2 moiety and / or targeting moiety in an IL2 agonist, the term "monovalent" means an IL2 agonist having only a single IL2 moiety and / or targeting moiety, respectively. Typically, an IL2 agonist that is monovalent with respect to the IL2 moiety and / or targeting moiety is a monomer or heterodimer.

[0039] Operable linkage: As used herein, the term “operable linkage” refers to a functional relationship between two or more regions of a polypeptide chain that are linked together to produce a functional polypeptide, or two or more nucleic acid sequences, for example, to result in an in-frame fusion of two polypeptide components or to link a regulatory sequence to a coding sequence.

[0040] Peptide-MHC complex, pMHC complex, peptide groove: (i) MHC domain (e.g., human MHC molecule or a portion thereof (e.g., its peptide-binding groove and e.g., its extracellular portion)), (ii) antigenic peptide, and optionally, (iii) β2 microglobulin domain (e.g., human β2 microglobulin or a portion thereof), where the MHC domain, antigenic peptide, and optionally β2 microglobulin domain are complexed in such a manner that specific binding to the T cell receptor is possible. In some embodiments, the pMHC complex includes at least the extracellular domain of a human HLA class I / human β2 microglobulin molecule and / or a human HLA class II molecule.

[0041] Single-chain Fv or scFv: As used herein, the terms "single-chain Fv" or "scFv" refer to a polypeptide chain containing the VH and VL domains of an antibody, where these domains are present in a single polypeptide chain.

[0042] Specific (or selective) binding: As used herein, the term “specific (or selective) binding” means that the targeting moiety, for example, an antibody or its antigen-binding domain ("ABD"), forms a complex with a target molecule that is relatively stable under physiological conditions. Specific binding is approximately 5 × 10⁻¹⁶. -2 M or less (for example, 5 x 10 -2 Less than M, 10 -2 Less than M, 5 x 10 -3 Less than M, 10 -3 Less than M, 5 x 10 -4 Less than M, 10 -4 Less than M, 5 x 10 -5 Less than M, 10 -5Less than M, 5 x 10 -6 Less than M, 10 -6 Less than M, 5 x 10 -7 Less than M, 10 -7 Less than M, 5 x 10 -8 Less than M, 10 -8 Less than M, 5 x 10 -9 Less than M, 10 -9 Less than M, or 10 -10 K (less than M) Dを特徴とし得る。 Methods for determining the binding affinity of a target portion of an antibody or antibody fragment, such as an IL2 agonist or component, to a target molecule are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance (e.g., Biacore assay), and fluorescence-activated cell sorting (FACS) binding assays. An IL2 agonist of this disclosure, comprising a targeting portion or its ABD that specifically binds to a target molecule from one species, may, however, exhibit cross-reactivity to target molecules from one or more other species.

[0043] Subject: The term “subject” includes humans and non-human animals. Non-human animals include all vertebrates, such as non-human primates, mammals and non-mammals such as sheep, dogs, cattle, chickens, amphibians, and reptiles. Unless otherwise specified, the terms “patient” and “subject” are used interchangeably herein.

[0044] Target Molecules: As used herein, the term “target molecule” refers to any biomolecule expressed on the cell surface or in the extracellular matrix that can be specifically bound by the targeting site of the IL2 agonist of this disclosure (e.g., proteins, carbohydrates, lipids, or combinations thereof).

[0045] Targeting moiety: As used herein, the term “targeting moiety” refers to the site where the IL2 agonist of this disclosure is localized, for example, on a tumor cell or lymphocyte in the tumor microenvironment, any molecule or its binding portion (e.g., immunoglobulin or antigen-binding fragment) that can bind to a cell surface or extracellular matrix molecule. In addition to localizing the IL2 agonist to a specific site, the targeting moiety may also have functional activity. For example, a targeting moiety that is an anti-PD1 antibody or its antigen-binding portion may also exhibit antitumor activity by inhibiting PD1 signaling or enhance the antitumor activity of IL2 mutein.

[0046] To treat, to treat, to treat: As used herein, the terms “to treat,” “to treat,” and “to treat” mean a reduction or improvement in the progression, severity, and / or duration of a proliferative disorder, or improvement of one or more symptoms (preferably one or more identifiable symptoms) of a proliferative disorder resulting from the administration of one or more IL2 agonists of the Disclosure. In certain embodiments, the terms “to treat,” “to treat,” and “to treat” mean improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which is not necessarily identifiable by the patient. In other embodiments, the terms “to treat,” “to treat,” and “to treat” mean inhibition of the progression of a proliferative disorder, either physically, by stabilization of an identifiable symptom, or physiologically, by stabilization of a physical parameter, for example. In other embodiments, the terms “to treat,” “to treat,” and “to treat” mean a reduction or stabilization of tumor size or cancer cell number.

[0047] Tumor: The term “tumor” is used herein as interchangeable with the term “cancer,” and both terms, for example, encompass solid and liquid tumors, such as diffuse or circulating tumors. As used herein, the terms “cancer” or “tumor” include precancerous and malignant cancers and tumors.

[0048] Tumor-associated antigens: The term “tumor-associated antigens” or “TAA” refers to molecules (typically proteins, carbohydrates, lipids, or combinations thereof) that are expressed on the surface of cancer cells, either completely or as fragments (e.g., MHC / peptides), and are useful for preferential targeting of drugs to cancer cells. In some embodiments, TAAs are markers expressed by both normal and cancer cells, e.g., lineage markers, e.g., CD19 on B cells. In some embodiments, TAAs are cell surface molecules that are overexpressed in cancer cells compared to normal cells, e.g., 1x overexpression, 2x overexpression, 3x overexpression, or more compared to normal cells. In some embodiments, TAAs are cell surface molecules that are improperly synthesized in cancer cells, e.g., molecules containing deletions, additions, or mutations compared to molecules expressed on normal cells. In some embodiments, TAAs are expressed only on the cell surface of cancer cells, either completely or as fragments (e.g., MHC / peptides), and not synthesized or expressed on the surface of normal cells. Therefore, the term "TAA" encompasses antigens specific to cancer cells and is sometimes known in the art as tumor-specific antigen ("TSA").

[0049] Universal Light Chain: As used herein in the context of the targeted moiety, the term “universal light chain” refers to a light chain polypeptide that can pair with the heavy chain region of the targeted moiety and with other heavy chain regions. Universal light chains are also known as “common light chains.”

[0050] VH: The term "VH" refers to the variable region of the immunoglobulin heavy chain of an antibody, which includes the scFv or Fab heavy chain. VL: The term "VL" refers to the variable region of an immunoglobulin light chain, including the scFv or Fab light chain.

[0051] 6.2. IL2 Agonist This disclosure provides an IL2 agonist comprising an IL2 portion, an optional multimerized portion, and an optional targeted portion.

[0052] The IL2 agonists of this disclosure may be monomers or multimers, e.g., dimers (homodimers or heterodimers) or higher-order complexes. For convenience, IL2 agonists that are homodimers (or higher-order multimers of the same polypeptide) are described by the monomers of their constituents, but homodimer (or higher-order multimer) molecules can be produced by recombinant expression of the monomers of the components in a suitable cell line.

[0053] In various embodiments, the IL2 agonist is (a) monovalent or divalent with respect to the IL2 portion and / or (b) monovalent or divalent with respect to the targeted portion (if present).

[0054] The IL2 agonists of this disclosure and / or the IL2 muteins in the IL2 agonists of this disclosure are typically not CD122-oriented; for example, they do not have amino acid substitutions or other modifications (e.g., pegylation) on the IL2 moiety that would preferentially bind them to IL-2Rβ compared to IL-2Rα with respect to wild-type IL2. However, in certain embodiments, the IL2 agonists of this disclosure may be CD122-oriented, for example, when the IL2 agonist is pMHC-targeted (e.g., as described in Section 6.4.3) and / or used in combination with CAR Treg therapy for autoimmune diseases, as described in Sections 6.11.1 and 6.11.1.4. In some embodiments, such IL2 agonists may have a binding affinity of up to 1000-fold reduced to IL-2Rα and a binding affinity of up to 50-fold reduced to IL-2Rβ.

[0055] The IL2 agonists of the present disclosure and / or the IL2 muteins in the IL2 agonists of the present disclosure may be CD25-oriented, for example, having one or more amino acid substitutions or other modifications on the IL2 moiety that cause them to preferentially bind to IL-2Rα compared to wild-type IL2 and compared to IL-2Rβ.

[0056] Therefore, the IL2 agonists of the present disclosure and / or the IL2 muteins in the IL2 agonists of the present disclosure may have amino acid modifications that result in a decrease in IL-2Rβ and / or IL-2Rα binding affinity.

[0057] Overall, the IL2 agonists of the present disclosure and / or IL2 muteins in the IL2 agonists of the present disclosure may have normal or attenuated binding (i.e., reduced affinity) to intermediate and / or high affinity IL2 receptors (e.g., up to 10-fold, up to 50-fold, up to 100-fold, up to 200-fold, up to 500-fold, up to 1,000-fold, or up to 5,000-fold). In some embodiments, although the binding is attenuated, the preference for intermediate affinity versus high affinity IL2 receptors is similar to that for wild-type IL2.

[0058] The preferential binding of one receptor to the other can be evaluated by measuring the difference in binding affinity between high-affinity receptor (IL-2Rαβγ)-expressing cells and intermediate-affinity receptor (IL-2Rβγ)-expressing cells, and comparing the ratio to the corresponding ratio for wild-type IL-2.

[0059] In certain embodiments, the IL2 agonists of this disclosure have one or more amino acid substitutions in the IL2 moiety that reduce binding to IL-2Rβ. For example, in some embodiments, the IL2 moiety may have binding to human IL-2Rβ reduced by up to 100 to 1,000 times compared to wild-type human IL2.

[0060] The IL2 moiety with reduced binding to IL-2Rβ retains its affinity for IL-2Rα, or its binding to IL-2Rα is reduced. For example, in some embodiments, the IL2 moiety may have binding to human IL2-Rα that is attenuated by up to 50, 100, 500, 1,000, or 5,000 times compared to wild-type human IL2.

[0061] Binding affinities to IL-2Rα, IL-2Rβ, intermediate affinity receptors, and high affinity receptors can be assayed using surface plasmon resonance (SPR) techniques (analyzed with a BIAcore instrument) (Liljeblad et al., 2000, Glyco J 17:323-329).

[0062] Examples of IL2 portions suitable for use in the IL2 agonists of this disclosure are described in Section 6.2. IL2 agonists may be fusion proteins comprising an IL2 moiety and a multimerizing moiety and / or targeting moiety. An exemplary multimerizing moiety is described in Section 6.5 and includes an Fc domain that confers homodimerization or heterodimerization ability to the IL2 agonist. Free IL2 has very low pharmacokinetics (serum half-life less than 10 minutes), and without being constrained by theory, the inclusion of a multimerizing domain such as an Fc domain is thought to improve serum stability and the pharmacokinetic profile of the IL2 agonist. Therefore, the Fc domain may be a dual-purpose domain conferring stabilizing properties to the stabilizing moiety, as described in Section 6.6.

[0063] Occasionally, for convenience, the IL2 moiety and optional multimerized and / or stabilized moieties are referred to herein as IL2 muteins, but the term “mutein” also encompasses molecules having a targeting moiety. Exemplary targeting moieties are described in Section 6.4 and include antigen-binding domains (e.g., scFv or Fab) that bind to tumor-associated antigens, tumor microenvironment antigens, or tumor lymphocytes, as well as peptide-MHC complexes that recognize tumor lymphocytes.

[0064] Furthermore, each of the IL2 moiety, the multimerized moiety, and the targeting moiety can itself be a fusion protein. For example, the IL2 moiety may include an IL2 or IL2 variant domain and an IL-2Rα domain.

[0065] In various embodiments, the IL2 agonist does not contain (a) cytokines other than IL2, (b) anti-IL2 antibodies or antibody fragments, (c) anti-DNA antibodies or antibody fragments, (b) unbound antibody variable domains, or any combination of any two, three, or four of the above.

[0066] IL2 agonists may contain one or more linker sequences that connect different components of the molecule, such as different domains present in the fusion protein. Exemplary linkers are described in Section 6.7.

[0067] In certain embodiments, the IL2 agonists of this disclosure increase the ratio of CD8+ T cells to Treg cells in a tumor after administration to a subject (e.g., a patient with cancer or a tumor-bearing mouse).

[0068] The following are some exemplary orientations of the IL2 muteins and agonists of this disclosure in N-terminal orientation: (1) Orientation 1: IL2 portion - optional linker - multimerized and / or stabilized portion.

[0069] (2) Orientation 2: Multimerized and / or stabilized portion - optional linker - IL2 portion. (3) Orientation 3: Targeting portion - optional linker - multimerized and / or stabilizing portion - optional linker - IL2 portion.

[0070] (4) Orientation 4: Heterodimer containing the following two polypeptides A and B: • Polypeptide A: IL2 portion - optional linker - multimerization portion, and • Polypeptide B: Targeting moiety - Optional linker - Multimerization moiety.

[0071] In the IL2 agonists of this disclosure, if the targeting moiety is the antigen-binding domain ("ABD") of an antibody, each IL2 molecule may consist of two polypeptide chains (one having a heavy chain variable region and the other having a light chain variable region). Thus, in orientations 3 and 4, the targeting moiety itself may contain heavy and light chain variable domains on separate polypeptide chains. For example, with respect to the IL2 agonist of orientation 4, polypeptide B may consist of two polypeptide chains, B-1 and B-2. Polypeptide chain B-1 may contain the heavy chain variable domain of the targeting moiety—an optional linker—a multimerized moiety, and polypeptide chain B-2 may contain the light chain variable domain of the targeting moiety. The polypeptide A-polypeptide B heterodimer may associate via pairing of Fc heterodimerized variants, as described in Section 6.5.1.2.

[0072] Alternatively, scFv can be used in which the heavy chain and light chain variable regions are fused together in a single polypeptide. An exemplary embodiment of an IL2 agonist with orientation 1 includes the following in the N-to-C-terminus orientation: a) IL2 portion including the following: i) With or without substitution at C125 to reduce aggregation (e.g., C125S, C125A, or C125V), the IL2 or IL2 variant (e.g., IL2 N88D) domain, ii) Linkers (for example, as described in Section 6.7), and iii) The IL2 binding portion of IL-2Rα, b) Linkers (for example, as described in Section 6.7), and c) Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections).

[0073] The IL2 agonists referred to as IL2M0 and IL2M2 in Section 7 are specific embodiments of the IL2 agonist with orientation 1. An exemplary embodiment of an IL2 agonist with orientation 2 includes the following in the N-to-C-terminal orientation: a) Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections), b) Linkers (for example, as described in Section 6.7), and c) IL2 or IL2 variant (e.g., IL2 N88D) domains, with or without substitution at C125 to reduce aggregation (e.g., C125S, C125A, or C125V).

[0074] The IL2 agonists referred to as IL2M4 and IL2M5 are specific embodiments of these orientation 2 IL2 agonists. Another exemplary embodiment of the IL2 agonist with orientation 2 includes the following in the N-to-C-terminal orientation: a) Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections), b) Linkers (for example, as described in Section 6.7), and c) IL2 portion including the following: i) With or without substitution at C125 to reduce aggregation (e.g., C125S, C125A, or C125V), the IL2 or IL2 variant (e.g., IL2 N88D) domain, ii) Linkers (for example, as described in Section 6.7), and iii) The IL2 binding portion of IL-2Rα.

[0075] The IL2 agonist referred to as IL2M3 in Section 7 is a specific embodiment of this orientation 2 IL2 agonist. An exemplary embodiment of an IL2 agonist with orientation 3 includes the following in the N-to-C-terminal orientation: a) heavy chain variable region of scFv or Fab (associating with the corresponding light chain variable region on a separate polypeptide) (for example, as described in Section 6.4.2 and its subsections), b) Linker (for example, as described in Section 6.7), c) Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections), d) Linkers (for example, as described in Section 6.7), and e) IL2 portion including the following: i) With or without substitution at C125 to reduce aggregation (e.g., C125S, C125A, or C125V), IL2 or IL2 variant (e.g., IL2 N88D) domains, ii) Linkers (for example, as described in Section 6.7), and iii) The IL2 binding portion of IL-2Rα (for example, as described in Section 6.3).

[0076] The T1-targeted version of the IL2 agonist, referred to as IL2M3 in Section 7, is a specific embodiment of this orientation 3 IL2 agonist. Another exemplary embodiment of the IL2 agonist with orientation 3 includes the following in the N-to-C-terminal orientation: a) Peptide-MHC complexes containing the following (e.g., those described in Section 6.4.3): i) MHC peptides, ii) Linkers (for example, as described in Section 6.7 or its subsections, for example, Section 6.7.1), iii) Optional β2-microglobulin (β2m), iv) Optional linkers (e.g., those described in Section 6.7 or its subsections, e.g., Section 6.7.1), and v) MHC, b) Optional linkers (e.g., those described in Section 6.7), c) Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections), d) Linker (for example, as described in Section 6.7), e) IL2 portion including the following: i) With or without substitution at C125 to reduce aggregation (e.g., C125S, C125A, or C125V), IL2 or IL2 variant (e.g., IL2 N88D) domains, ii) Linkers (for example, as described in Section 6.7), and iii) The IL2 binding portion of IL-2Rα (for example, as described in Section 6.3).

[0077] The T2 and T3 targeted versions of the IL2 agonist, referred to as IL2M3 in Section 7, are specific embodiments of this orientation 3 IL2 agonist. In an exemplary embodiment, the 4-orientation IL2 agonist comprises two polypeptides, A and B, in an N-to-C-terminal orientation: In a particular embodiment, the 4-orientation IL2 agonist comprises two polypeptides, A and B, in an N-to-C-terminal orientation: Polypeptide A: a) Targeting moieties, e.g., peptide-MHC complexes (e.g., as described in Section 6.4.3), Fab domains (e.g., as described in Section 6.4.2.2) (e.g., a third polypeptide containing the light chain of Fab, the heavy chain of Fab on polypeptide A associating with polypeptide C), or scFv domains (e.g., as described in Section 6.4.2.1), b) Optional linkers (e.g., those described in Section 6.7), and c) The first Fc domain.

[0078] Polypeptide B: d) IL2 moiety containing IL2 or IL2 variant domains (e.g., IL2 domains with substitutions at C125 to reduce aggregation (e.g., C125S, C125A, or C125V), also called IL2(2m), e) Optional linkers (e.g., those described in Section 6.7), and f) A second Fc domain that is not identical to the first Fc domain but can be heterodimerized (e.g., as described in Section 6.5.1.2).

[0079] In some embodiments, the IL2 agonist is a heterodimer that is monovalent with respect to the targeting moiety and monovalent with respect to the IL2 moiety. A specific embodiment of this orientation 4 IL2 agonist includes: Polypeptide A: a) Peptide-MHC complexes containing the following (e.g., those described in Section 6.4.3): i) MHC peptides, ii) Linker (for example, as described in Section 6.7), iii) Optional β2-microglobulin (β2m), iv) Optional linkers (e.g., those described in Section 6.7), and v) MHC, b) Optional linkers (e.g., those described in Section 6.7), and c) The first Fc domain.

[0080] Polypeptide B: d) IL2 portion containing IL2 or an IL2 variant (also known as IL2 H16A, F42A, IL2(2m)) with or without substitution with C125 to reduce aggregation (e.g., C125S, C125A, or C125V), e) Optional linkers (e.g., those described in Section 6.7), and f) A second Fc domain that is not identical to the first Fc domain but can be heterodimerized (e.g., as described in Section 6.5.1.2).

[0081] The first and second Fc domains may be, for example, IgG1 or IgG4 Fc domains, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections.

[0082] An exemplary IL2 agonist with orientation 4 is shown in Figure 22A.2. Including β2-microglobulin in peptide-MHC complexes, such as the exemplary orientation 3 and orientation 4 agonists described above, is thought to stabilize human cell surface MHC I molecules and facilitate their loading by exogenous peptides. See, for example, Shields et al., 1998, J Biol Chem. 273(43):28010-8 and Obermann et al., 2007, Immunology 122(1):90-7.

[0083] In certain embodiments, the IL2 agonist has the configuration of IL2M0, IL2M1, IL2M2, IL2M3, IL2M4, IL2M5, IL2M6, or IL2M7 and includes, for example, an IL2 mutein having an optional targeting moiety and an optional linker at the N-terminus.

[0084] The IL2 agonists of this disclosure generally have improved therapeutic indices. In certain embodiments, the therapeutic index may be measured as the ratio of the dose that causes toxicity (e.g., weight loss) in tumor-bearing mice to the minimum antitumor effective dose of the IL2 agonist as a whole or its non-targeted component (e.g., a molecule comprising the IL2 moiety and a multimerized moiety, which for convenience is referred herein as the “IL2 mutein” or “IL2M” component), as illustrated in Section 7. The data in Section 7 demonstrate the following therapeutic indices:

[0085] [Table 1]

[0086] In certain embodiments, the IL2 agonist of the Disclosure or its IL2M component has a therapeutic index greater than 1, preferably greater than 2, and more preferably greater than 10. In certain embodiments, the therapeutic index is about 10, about 20, about 100, or about 200.

[0087] Further details of the components of the IL2 agonist in this disclosure are presented below. 6.3.IL2 part The IL2 portion of the IL2 antagonists of this disclosure comprises a wild-type or variant IL2 domain, which is optionally fused to the IL2-binding domain of IL-2Rα via a linker (as described, e.g., in Section 6.7). If present, the IL2-binding domain of IL-2Rα may be the N-terminus or C-terminus of the wild-type or variant IL2 domain.

[0088] In eukaryotic cells, human IL-2 is synthesized as a precursor polypeptide of 153 amino acids, from which 20 amino acids are removed to produce mature secreted IL-2 (Taniguchi et al., 1983, Nature 302(5906):305-10). Mature human IL-2 has the following amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(Sequence ID 2) The IL2 portions of this disclosure are typically not CD122-oriented; for example, they do not have amino acid substitutions in the IL2 domain that would preferentially bind them to IL-2Rβ compared to IL-2Rα.

[0089] The IL2 portions of this disclosure may be CD25-oriented, and for example, they have one or more amino acid substitutions in the IL2 domain that preferentially bind them to IL-2Rα compared to IL-2Rβ.

[0090] In certain embodiments, the IL2 agonists of this disclosure have one or more amino acid substitutions in the IL2 moiety that reduce binding to IL-2Rβ. For example, in some embodiments, the IL2 moiety can reduce binding to human IL-2Rβ by up to 50-fold (and up to 100-fold in some embodiments) to 1,000-fold compared to wild-type human IL2.

[0091] The IL2 moiety with reduced binding to IL-2Rβ may retain its affinity for IL-2Rα, or its binding to IL-2Rα may be reduced. For example, in some embodiments, the IL2 moiety may have up to 50-fold reduced binding to human IL2-Rα compared to wild-type human IL2.

[0092] Other characteristics of useful IL2 variants may include the ability to induce proliferation of IL-2Rα-possessing CD8+ T cells in tumors, the ability to induce IL2 signaling in IL-2Rα-possessing CD8+ T cells in tumors, and improvement of the therapeutic index.

[0093] In one embodiment, the IL2 portion includes one or more amino acid substitutions that reduce affinity for IL-2Rβ while maintaining affinity for IL2-Rα. An exemplary amino acid substitution is N88D. Other amino acid substitutions that reduce or eliminate IL2's affinity for IL-2Rβ include D20T, N88R, N88D, or Q126D (see, for example, U.S. Patent Application Publication No. 2007 / 0036752).

[0094] In one embodiment, the IL2 moiety includes one or more amino acid substitutions that reduce affinity for IL2-Rα, maintain affinity for IL-2Rβ, or reduce affinity to a lower degree, resulting in a CD122-targeted IL2 moiety. Such IL2 moieties are particularly useful for incorporation into IL2 agonists having peptide-MHC targeting moieties, as disclosed in Section 6.11.1.4, and / or for use as adjunctive therapy in CAR Treg therapy. Exemplary CD122-targeted IL2 moieties include both H16A and F42A substitutions, as exemplified in IL2M6 and IL2M7. Thus, in some embodiments, the IL2 moiety includes an amino acid sequence of human IL2 having H16A and F42A substitutions, as shown below: SAPTSSSTKKTQLQLEALLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(Sequence ID 124) In certain embodiments, the IL2 moiety includes an amino acid substitution that eliminates the O-glycosylation site of IL2 at a position corresponding to residue 3 of human IL2. Exemplary amino acid substitutions in T3 are T3A, T3G, T3Q, T3E, T3N, T3D, T3R, T3K, and T3P. In certain embodiments, the substitution is T3A.

[0095] The IL2 portion is preferably an essentially full-length IL2 molecule, such as a human IL2 molecule. In certain embodiments, the IL2 portion is a human IL-2 molecule. As described in U.S. Patent No. 4,518,584, C125 can be replaced with S, V, or A to reduce protein aggregation.

[0096] As described there, it is also possible to obtain des-A1 IL2 by deleting the N-terminal alanine residue of IL2. Furthermore, the IL2 portion may include substitution of methionine 104 with a neutral amino acid such as alanine, as described in U.S. Patent No. 5,206,344.

[0097] Accordingly, the IL2 portion of this disclosure may have amino acid deletions and / or substitutions selected from des-A1 M104A IL2, des-A1 M104A C125S IL2, M104A IL2, M104A C125A IL2, des-A1 M104A C125A IL2, or M104A C125S IL2, in addition to other variations that modify the binding of IL2 to its receptor. These and other variants can be found in U.S. Patent No. 5,116,943 and Weiger et al., 1989, Eur J Biochem 180:295-300.

[0098] In various embodiments, any of the aforementioned IL2 portions comprises an amino acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with mature human IL2.

[0099] The IL2 portion of this disclosure may further include the IL2-binding domain of IL-2Rα (referred to for convenience as the "IL2-Rα domain"), for example, optionally, the extracellular domain of IL-2Rα fused at the C-terminus or N-terminus of IL2 via a linker as described in Section 6.7. The sequence of the mature human IL-2Rα extracellular domain (corresponding to amino acids 22-272 of human IL-Rα) is as follows:

[0100] [ka]

[0101] The sequence of the IL2 binding region of the human IL-2Rα extracellular domain (including two "sushi" domains corresponding to amino acids 22-186 of human IL-2Rα) is as follows:

[0102] [ka]

[0103] The sequence of the IL2 binding region, which is a substitute for the extracellular domain of human IL-2Rα and corresponds to amino acids 22-240 of human IL-2Rα, is as follows:

[0104] [ka]

[0105] The IL2-Rα domain or the IL2 binding portion of the IL-2Rα extracellular domain preferably has an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any of the above sequences, i.e., amino acids 22-186, amino acids 22-240, or amino acids 22-272 of IL-2Rα, or any of the IL2 binding portions.

[0106] In certain embodiments, the IL2-Rα domain or IL2 binding portion contains or may contain an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with respect to the IL2 binding portion of human IL-2Rα, and optionally, the binding portion has an amino acid sequence of (a) at least 160 amino acids, at least 161 amino acids, at least 162 amino acids, at least 164 amino acids, or at least 165 amino acids, and / or (b) up to 251, up to 240, up to 230, up to 220, up to 210, up to 200, up to 190, up to 180, or up to 170 amino acids of the extracellular domain of human IL2-Rα. In certain embodiments, the human IL-2Rα portion is conjugated by either (a) or (b) of the preceding paragraph, for example, at least 160 and up to 180 amino acids derived from human IL-2Rα, at least 162 and up to 200 amino acids derived from human IL-2Rα, at least 160 and up to 220 amino acids derived from human IL-2Rα, at least 164 and up to 190 amino acids derived from human IL-2Rα, and so on.

[0107] In some embodiments, the IL2-Rα domain or IL2 binding portion comprises or consists of an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with amino acids 22-186, with or without up to 5, at least 10, at least 15, at least 20, at least 30, or at least 40 additional amino acids at the C-terminus of amino acid residue 186 of IL2-Rα.

[0108] As shown in FIGS. 3A to 3B, the fusion of the N-terminal Fc domain of IL2 to the C-terminus of the IL2-binding portion of IL-2Rα results in an IL2 agonist that does not form a higher-order structure, similar to the IL2-IL-2Rα extracellular domain-Fc-containing IL2 agonist in the N-to-C direction. Thus, the IL-2Rα-containing IL2 mutein of the present disclosure preferably has an IL-2Rα extracellular domain at the N-terminus of IL2. An exemplary mutein of this orientation is IL2M3.

[0109] In certain embodiments, the IL2-Rα domain or IL-2Rα extracellular domain has at least one fewer O-glycosylation and / or N-glycosylation compared to the extracellular domain of natural IL-2Rα, for example, by substitution of one or more of the amino acids N49, N68, T74, T85, T197, T203, T208, and T216. In some embodiments, one or more substitutions are from asparagine to an amino acid selected from the group consisting of alanine, threonine, serine, arginine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tryptophan, tyrosine, and valine. In some embodiments, one or more substitutions are substitutions of threonine with an amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, tryptophan, tyrosine, and valine. In some embodiments, one or more substitutions are amino acids S50 (e.g., S50P), amino acids S51 (e.g., S51R, S51N, S51D, S51C, S51Q, S51E, S51G, S51H, S51I, S51L, S51K, S51M, S51F, S51P, S51W, S51Y, or S51V), amino acids T69 (e.g., T69P), amino acids T70 (e.g., T70R, T70N, T70D, T70C, T70Q, T70E, T70G, T70H, T70I, T70L, T70K, T70M, T70F, T70P, T70W, T70Y, or T70V, amino acid C192 (e.g., C192R, C192N, C192D, C192Q, C192E, C192G, C192H, C192I, C192L, C192K, C192M, C192F, C192P, C192W, C192Y, or C192V), or any combination thereof.

[0110] 6.4. Targeting part By incorporating a targeting moiety into the IL2 agonist of the present disclosure, delivery of high concentrations of IL2 to the tumor microenvironment or tumor-reactive lymphocytes (including CART lymphocytes) is enabled, while at the same time systemic exposure is reduced and there are fewer side effects than those obtained using wild-type IL2.

[0111] Suitable formats for the targeting moiety are described in Section 6.4.2. The targeting moiety is preferably an antigen-binding moiety, for example, an antibody or an antigen-binding portion of an antibody, such as an scFv described in Section 6.4.2.1, or a Fab described in Section 6.4.2.2.

[0112] Antibodies and antigen-binding portions generally bind to specific epitopes and can direct the IL2 agonist to a target site, such as a specific tumor cell type or tumor stroma bearing the epitope. Exemplary target molecules recognized by the targeting moieties of the present disclosure are described in Section 6.4.1.

[0113] In other embodiments, the targeting moiety is a peptide-MHC complex described in 6.4.3, for example, a peptide-MHC complex recognized by tumor lymphocytes. In some embodiments, the targeting moiety is in the form of an Fc fusion protein, as exemplified in structures referred to as T7 and T8 in the Examples, for example. The Fc portion of the targeting moiety polypeptide can be used to multimerize with the Fc domain of a separate polypeptide chain containing the IL2 portion.

[0114] 6.4.1. Target Molecules The target molecules recognized by the targeting moiety of the IL2 agonists of this disclosure are generally found, for example, on the surface of activated T cells, on the surface of tumor cells, on the surface of virus-infected cells, on the surface of other affected cells, in a free state in serum, in the extracellular matrix (ECM), or on immune cells present at the target site, such as tumor-reactive lymphocytes. When immune cells are administered exogenously (e.g., chimeric antigen receptors "CARs" expressed on T cells), the targeting moiety can recognize the chimeric antigen receptors (CARs) or other molecules found on the surface of CAR T cells. In various embodiments, the CARs include CDR or VH and VL sequences (e.g., in the form of scFv) that specifically recognize TAA or pMHC complexes.

[0115] Examples of target molecules include fibroblast-activating protein (FAP), the A1 domain of tenascin-C (TNC A1), and the A2 domain of tenascin-C (TNC A2) Fibronectin extradomain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophyllin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) and its immunogenic epitopes PSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, MAGE-tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAG E-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-tumor antigen family (e.g., GAGE-1, GAGE-2, GAGE- 3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, and γ-catenin, p120ctn, gp100Viral products such as Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2, and GD2 ganglioside, human papillomavirus proteins, Smad family of tumor antigens, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, cerebral glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), and CD3 (associated with TCR). (heteromultimer), CD22 (B cell receptor), CD23 (low affinity IgE receptor), CD30 (cytokine receptor), CD33 (bone marrow cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R- (IL-6 receptor), CD20, MCSP, PDGFβR (β-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA These include IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) and extra domain B (EDB) of fibronectin, and the A1 domain (TnC A1) of tenascin-C.

[0116] Non-specific examples of viral antigens include EBV antigen (e.g., Epstein-Barr virus LMP-1), hepatitis C virus antigen (e.g., hepatitis C virus E2 glycoprotein), HIV antigen (e.g., HIV gp160 and HIV gp120), CMV antigen, HPV-specific antigen, or influenza virus antigen (e.g., influenza virus hemagglutinin).

[0117] Non-exclusive examples of ECM antigens include syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch, tenascin, collagen, and matrixin.

[0118] Other target molecules include cell surface molecules of tumor or viral lymphocytes, such as T cell costimulatory proteins like CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0119] In certain embodiments, the target molecule is a checkpoint inhibitor, such as CTLA-4, PD1, PDL1, PDL2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, or CHK2. In certain embodiments, the target molecule is PD1. In other embodiments, the target molecule is LAG3.

[0120] 6.4.2. Format of the Targeted Section In certain embodiments, the targeting moiety may be any type of antibody or fragment thereof that maintains specific binding to an antigenic determinant. In one embodiment, the antigen-binding moiety is a full-length antibody. In one embodiment, the antigen-binding moiety is an immunoglobulin molecule, particularly an IgG class immunoglobulin molecule, more specifically an IgG1 or IgG4 immunoglobulin molecule. The antibody fragment may contain VH (or V H) Fragment, VL (or V L This includes, but is not limited to, ) fragments, Fab fragments, F(ab')2 fragments, scFv fragments, Fv fragments, minibodies, diabodies, triabodies, and tetrabodies.

[0121] 6.2.1.scFv Single-chain Fv or "scFv" antibody fragments contain the VH and VL domains of an antibody in a single polypeptide chain and can be expressed as single-chain polypeptides, retaining the specificity of the intact antibody from which they are derived. Generally, scFv polypeptides further include a polypeptide linker between the VH and VL domains, enabling the scFv to form a desired structure for target binding. Examples of suitable linkers for connecting the VH and VL chains of scFv are the linkers identified in Section 6.4.3.

[0122] Unless otherwise specified, as used herein, scFv may have the VL and VH variable regions in either order, for example, with respect to the N-terminus and C-terminus of a polypeptide, scFv may contain VL-linker-VH or VH-linker-VL.

[0123] scFv may contain VH and VL sequences from any suitable species, such as mouse, human, or humanized VH and VL sequences. To construct scFv-coding nucleic acids, the VH and VL coding DNA fragments are operably linked to another fragment encoding a linker, for example, a linker as described in Section 6.4.3 (typically an amino acid glycine and serine, e.g., a repeat of a sequence containing the amino acid sequence (Gly4~Ser)3 (SEQ ID NO: 6)), and the VH and VL sequences can be expressed as a continuous single-chain protein, with the VL and VH regions joined by a flexible linker (see, e.g., Bird et al., 1988, Science 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., 1990, Nature 348:552-554).

[0124] 6.4.2.2.Fab Traditionally, Fab domains have been produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain. In the IL2 agonists of this disclosure, the Fab domain is typically recombinantly expressed as part of the IL2 agonist.

[0125] The Fab domain can contain constant domains and variable region sequences from any suitable species and can therefore be mouse, chimeric, human, or humanized. The Fab domain typically contains a CH1 domain attached to the VH domain, which pairs with a CL domain attached to the VL domain. In wild-type immunoglobulin, the VH domain pairs with the VL domain to form the Fv region, and the CH1 domain pairs with the CL domain to further stabilize the binding module. Disulfide bonds between the two constant domains can further stabilize the Fab domain.

[0126] For the IL2 agonists of the present disclosure, particularly when the light chains are not common or universal light chains, it is advantageous to use a Fab heterodimerization strategy to enable the correct association of Fab domains belonging to the same ABD and minimize the abnormal pairing of Fab domains belonging to another ABD. For example, the Fab heterodimerization strategy shown in Table 1 below can be used:

[0127] [Table 2]

[0128] Thus, in certain embodiments, the correct association between the two polypeptides of the Fab is promoted, for example, by exchanging the VL and VH domains of the Fab or by exchanging the CH1 and CL domains of the Fab, as described in WO2009 / 080251.

[0129] Correct Fab pairing can also be promoted by introducing one or more amino acid modifications in the CH1 domain and one or more amino acid modifications in the CL domain of the Fab, and / or one or more amino acid modifications in the VH domain and one or more amino acid modifications in the VL domain. The amino acids to be modified are typically part of the VH:VL and CH1:CL interfaces such that the Fab components preferentially pair with each other rather than with the components of other Fabs.

[0130] In one embodiment, the one or more amino acid modifications are limited to conserved framework residues of the variable (VH, VL) and constant (CH1, CL) domains indicated by the Kabat numbering of the residues. Almagro, 2008, Frontiers In Bioscience 13:1619-1633 provides definitions of framework residues based on the Kabat, Chothia, and IMGT numbering schemes.

[0131] In one embodiment, modifications introduced to the VH and CH1 domains and / or the VL and CL domains are complementary to each other. Complementarity at the heavy-light chain interface can be achieved based on steric and hydrophobic contact, electrostatic / charge interactions, or various combinations of interactions. Complementarity between protein surfaces has been widely described in the literature in terms of lock-and-key fit, knob-into-hole, protrusion and cavity, donor and acceptor, all of which refer to the structural and chemical matching properties between two interacting surfaces.

[0132] In one embodiment, one or more introduced modifications introduce new hydrogen bonds across the interface of the Fab components. In one embodiment, one or more introduced modifications introduce new salt bridges across the interface of the Fab components. Exemplary substitutions are described in WO2014 / 150973 and WO2014 / 082179, the contents of which are incorporated herein by reference.

[0133] In some embodiments, the Fab domain includes a 192E substitution in the CH1 domain, as well as 114A and 137K substitutions in the CL domain, which introduces a salt bridge between the CH1 and CL domains (see, for example, Golay et al., 2016, J Immunol 196:3199-211).

[0134] In some embodiments, the Fab domain includes 143Q and 188V substitutions in the CH1 domain, as well as 113T and 176V substitutions in the CL domain, which helps to exchange the hydrophobic and polar regions of the contact between the CH1 and CL domains (see, e.g., Golay et al., 2016, J Immunol 196:3199-211).

[0135] In some embodiments, the Fab domain may include modifications in some or all of the VH, CH1, VL, and CL domains to introduce orthogonal Fab interfaces that facilitate the correct assembly of the Fab domain (Lewis et al., 2014 Nature Biotechnology 32:191-198). In one embodiment, the 39K, 62E modification is introduced into the VH domain, the H172A, F174G modification is introduced into the CH1 domain, the 1R, 38D, (36F) modification is introduced into the VL domain, and the L135Y, S176W modification is introduced into the CL domain. In another embodiment, the 39Y modification is introduced into the VH domain, and the 38R modification is introduced into the VL domain.

[0136] The Fab domain can also be modified to replace the natural CH1:CL disulfide bond with an engineered disulfide bond, thereby improving the efficiency of Fab component pair formation. For example, the engineered disulfide bond can be introduced by introducing 126C into the CH1 domain and 121C into the CL domain (see, e.g., Mazor et al., 2015, MAbs 7:377-89).

[0137] Fab domains can also be modified by replacing the CH1 and CL domains with alternative domains that facilitate correct assembly. For example, Wu et al., 2015, MAbs 7:364-76 describe replacing the CH1 domain with the constant domain of the T cell receptor and the CL domain with the β domain of the T cell receptor, and pairing these domain substitutions with additional charge-charge interactions between the VL and VH domains by introducing a 38D modification to the VL domain and a 39K modification to the VH domain.

[0138] Instead of, or in addition to, the use of Fab heterodimerization strategies to promote correct VH-VL pair formation, a common light chain (also called a universal light chain) VL can be used in each Fab VL region of the IL2 agonist of this disclosure. In various embodiments, using a common light chain as described herein reduces the number of inappropriate species of IL2 agonist compared to using the original congeneral VL. In various embodiments, the VL domain of the IL2 agonist is identified from a monospecific antibody containing a common light chain. In various embodiments, the VH region of the IL2 agonist contains a human heavy chain variable gene segment that is rearranged in vivo in mouse B cells pre-engineered to express a limited human light chain repertoire, or a single human light chain congeneral to a human heavy chain, and in response to exposure to the antigen of interest, generates an antibody repertoire containing multiple human VHs congeneral to one of one or two possible human VLs, the antibody repertoire being specific to the antigen of interest. The common light chain is derived from a rearranged human Vκ1-39Jκ5 sequence or a rearranged human Vκ3-20Jκ1 sequence, including somatic mutation (e.g., affinity maturation) versions. See, for example, U.S. Patent No. 10,412,940.

[0139] 6.4.3.MHC-peptide fusions The targeting portion of the IL2 agonist of this disclosure may be a peptide-MHC complex ("pMHC complex"), for example, a peptide complexed with an MHC class I domain, or a peptide complexed with an MHC class II domain and optionally with a β2 microglobulin domain.

[0140] Spontaneously occurring MHCs are encoded by clusters of genes on human chromosome 6. MHCs include, but are not limited to, HLA specificities such as A (e.g., A1-A74), B (e.g., B1-B77), C (e.g., C1-C11), D (e.g., D1-D26), DR (e.g., DR1-DR8), DQ (e.g., DQ1-DQ9), and DP (e.g., DP1-DP6). HLA specificities include A1, A2, A3, All, A23, A24, A28, A30, A33, B7, B8, B35, B44, B53, B60, B62, DR1, DR2, DR3, DR4, DR7, DR8, and DR11.

[0141] Spontaneously occurring MHC class I molecules bind to peptides derived from proteins degraded by proteolysis. The resulting small peptides are then transported to the endoplasmic reticulum, as shown in Figure 3A, where they associate with newly synthesized MHC class I molecules and are subsequently presented on the cell surface via the Golgi apparatus for recognition by cytotoxic T lymphocytes.

[0142] Spontaneously occurring MHC class I molecules consist of an α(heavy) chain that associates with β2-microglobulin. The heavy chain consists of subunits α1-α3. The β2-microglobulin protein and the α3 subunit of the heavy chain are associated. In certain embodiments, the β2-microglobulin and the α3 subunit are associated by covalent bonds. In certain embodiments, the β2-microglobulin and the α3 subunit are associated non-covalently. The α1 and α2 subunits of the heavy chain fold to form grooves for peptides, such as antigenic determinants, that are presented and recognized by the TCR.

[0143] Class I molecules generally associate, for example, with peptides that are about 8-9 amino acids long (e.g., 7-11 amino acids). Every human has 3-6 different class I molecules, each of which can bind to many different types of peptides. In one particular embodiment, a class I MHC polypeptide is a human class I MHC polypeptide selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and HLA-G.

[0144] In some embodiments, the targeting moiety comprises an MHC class I α heavy chain extracellular domain (human α1, α2, and / or α3 domain) without a transmembrane domain. In some embodiments, the class I α heavy chain polypeptide is HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, or HLA-L. In some embodiments, the HLA-A sequence may be an HLA-A*0201 sequence.

[0145] The peptide in the pMHC complex may have an amino acid sequence of a peptide that can be presented by associating with an MHC class I molecule, for example. In certain embodiments, the sequence may consist of 6 to 20 consecutive amino acids. In certain embodiments, the peptide sequence may be a sequence of a protein fragment, the protein of which may be derived from a part of a cellular protein, such as a protein that associates with cancer or cancer-causing antigens, and the peptide may be bound to an MHC class I heavy chain.

[0146] In some embodiments, the pMHC complex targeting moiety comprises (i) an antigenic peptide, (ii) a class I MHC polypeptide, or a fragment, variant, or derivative thereof (e.g., an extracellular domain), and optionally, (iii) a β2 microglobulin polypeptide, or a fragment, variant, or derivative thereof. For example, the pMHC complex may comprise (i) an antigenic peptide, (ii) a β2M sequence, and (iii) a class Iα (heavy) chain sequence from the N-terminus to the C-terminus. Alternatively, the pMHC complex may comprise (i) an antigenic peptide, (ii) a class Iα (heavy) chain sequence, and (iii) a β2M sequence from the N-terminus to the C-terminus.

[0147] In one particular embodiment, the antigenic peptide and the MHC sequence and / or the MHC sequence and the β2M domain are linked to each other via a peptide linker, for example, as described in Section 6.7. In some embodiments, the single-chain pMHC complex may include a first flexible linker between the peptide segment and the β2 microglobulin segment. For example, the linker may extend from the carboxyl terminus of the peptide and connect to the amino terminus of the β2 microglobulin segment. In some embodiments, the linker is structured to allow the peptide to fold into a binding groove to result in a functional pMHC complex. In some embodiments, this linker may contain at least three amino acids and up to about fifteen amino acids (e.g., 20 amino acids). The pMHC linker may include a second flexible linker inserted between the β2 microglobulin and the MHC I heavy chain segment. For example, the linker may extend from the carboxyl terminus of the β2 microglobulin segment and connect to the amino terminus of the MHC I heavy chain segment. In certain embodiments, β2-microglobulin and MHC I heavy chains can fold into binding grooves, resulting in molecules that can function in promoting T cell expansion.

[0148] In the presence of β2M, the pMHC complex contains mutations in both β2M and the MHC class I α heavy chain domain, allowing for the formation of a disulfide bond between them. Exemplary amino acid pairs that can be substituted with cysteine ​​to enable a disulfide bond between the two domains are identified in Table 2 below, or as described in PCT Publication 2015 / 195531, which is incorporated herein in its entirety by reference.

[0149] [Table 3]

[0150] In further embodiments, the single-chain pMHC complex may include a peptide covalently attached to an MHC class Iα(heavy) chain via a disulfide crosslink (i.e., a disulfide bond between two cysteines). See, for example, U.S. Patents 8,992,937 and 8,895,020, each of which is incorporated in whole by reference. In certain embodiments, the disulfide bond includes a first cysteine ​​located within a linker extending from the carboxyl terminus of the peptide, and a second cysteine ​​located within the MHC class I heavy chain (e.g., an MHC class Iα(heavy) chain having a non-covalent site of the antigen peptide). In certain embodiments, the second cysteine ​​may be a mutation (addition or substitution) in the MHC class Iα(heavy) chain. Preferably, the pMHC complex may include one consecutive polypeptide chain and a disulfide crosslink. Alternatively, the pMHC complex may include two consecutive polypeptide chains attached via a disulfide crosslink as the sole covalent bond. In some embodiments, the linking sequence may contain at least one amino acid in addition to cysteine, comprising one or more glycines, one or more alanines, and / or one or more serines. In some embodiments, the single-chain molecule comprises, from N-terminus to C-terminus, an MHC class I peptide (e.g., an antigenic peptide), a first linker containing a first cysteine, a β2-microglobulin sequence, a second linker, and an MHC class I heavy chain sequence containing a second cysteine, wherein the first and second cysteines comprise a disulfide crosslink. In some embodiments, the second cysteine ​​is an amino acid substitution of the MHC class I heavy chain selected from the group consisting of T80C, Y84C, and N86C (Y84C refers to a mutation at position 108 of the mature protein, where the mature protein lacks a signal sequence; or, if the protein still contains a 24-mer signal sequence, that position is instead called Y108C).

[0151] In a particular embodiment, if the pMHC complex contains a first cysteine ​​in a glycerin linker extending between the C-terminus of the peptide and β2-microglobulin, and a second cysteine ​​at an adjacent heavy chain position, the disulfide bridge can link the peptide within the class I groove of the pMHC complex.

[0152] If present, the β2-microglobulin sequence may include a full-length (human or non-human) β2-microglobulin sequence. In certain embodiments, the β2-microglobulin sequence lacks a leader peptide sequence. Therefore, the β2-microglobulin sequence may contain approximately 99 amino acids. An exemplary human β2-microglobulin sequence is Genbank accession number AF072097.1.

[0153] As an alternative to type I MHC-based pMHC complexes, the IL2 agonists of this disclosure may include a class II MHC-based pMHC complex as the targeting moiety. Class II MHC-based pMHC complexes generally comprise a class I MHC polypeptide, or a fragment, variant, or derivative thereof. In one particular embodiment, the MHC comprises α and β polypeptides of a class II MHC molecule, or a fragment, variant, or derivative thereof. In one particular embodiment, the α and β polypeptides are linked by a peptide linker. In one particular embodiment, the MHC comprises α and β polypeptides of a human class II MHC molecule selected from the group consisting of HLA-DP, HLA-DR, HLA-DQ, HLA-DM, and HLA-DO.

[0154] MHC class II molecules generally consist of two polypeptide chains, α and β. These chains can originate from the DP, DQ, or DR gene family. Approximately 40 different human MHC class II molecules are known. While their basic structure is the same, their molecular structures differ slightly. MHC class II molecules bind to peptides that are 13 to 18 amino acids long.

[0155] In some embodiments, the pMHC complex comprises one or more MHC class IIα chains or their extracellular components. In some embodiments, the class IIα chain is HLA-DMA, HLA-DOA, HLA-DPA, HLA-DQA, or HLA-DRA.

[0156] In other embodiments, the pMHC complex comprises one or more MHC class IIβ chains or their extracellular components. In some embodiments, the class IIβ chain is HLA-DMB, HLA-DOB, HLA-DPB, HLA-DQB, or HLA-DRB.

[0157] The peptides in the pMHC complex can be any peptides that can bind to MHC proteins in such a way that the pMHC complex can bind to the TCR, for example, in a specific manner.

[0158] Examples include peptides produced by hydrolysis, and synthetically produced peptides, most typically including randomly generated peptides, specifically designed peptides, and peptides in which at least some amino acid positions are conserved among several peptides, with the remaining positions being random.

[0159] In nature, peptides produced by hydrolysis undergo hydrolysis before the antigen can bind to the MHC protein. Class I MHC typically presents peptides derived from proteins actively synthesized in the cytoplasm of the cell. In contrast, Class II MHC typically presents peptides derived from either exogenous proteins that enter the cell's endocytosis pathway or proteins synthesized in the ER. Intracellular transport allows peptides to associate with MHC proteins.

[0160] The binding of peptides to the MHC peptide binding groove can control the spatial arrangement of MHC and / or peptide amino acid residues recognized by the TCR, or pMHC-binding proteins produced by genetically modified animals as disclosed herein. Such spatial control is partly due to hydrogen bonds formed between the peptide and the MHC protein. Based on knowledge of how peptides bind to various MHCs, it is possible to determine the major MHC anchor amino acids and the surface-exposed amino acids that differ between different peptides. In some embodiments, the length of MHC-binding peptides is 5–40 amino acid residues, e.g., 6–30 amino acid residues, e.g., 8–20 amino acid residues, e.g., 9–11 amino acid residues, and peptides of any size with a length of 5–40 amino acids are included in integer units (i.e., 5, 6, 7, 8, 9...40). Naturally, MHC class II-binding peptides vary up to about 9–40 amino acids, and in almost all cases, peptides can be shortened to a 9–11 amino acid core without losing MHC-binding activity or T cell recognition.

[0161] The peptides in the pMHC complexes of this disclosure are typically proteins of infectious agents (e.g., bacteria, viruses, or parasites), allergens, and at least some tumor-associated proteins, such as antigenic determinants. Preferably, the pMHC complexes contain antigenic determinants of cancer cells. Exemplary antigenic determinants of cancer cells include LCMV-derived peptides gp33-41, APF (126-134), BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100 (209-217), HBV core (18-27), Her2 / neu (369-377; V2v9); HPV E7 (11-20), HPV E7 (11-19), HPV This includes E7(82-90), KLK4(11-19), LMP1(125-133), MAG-A3(112-120), NYESO1(157-165, C165A), NYES1(157-165, C165V), p54 WT(264-272), PAP-3(136-143), PSMA(4-12), PSMA(135-145), Survivin(96-014), Tyrosinase(369-377, 371D), and WT1(126-134). An exemplary HPV E7(11-19) peptide sequence is YMLDLQPET(SEQ ID NO: 7), SEQ ID NO: 537 in International Patent Publication 2019 / 005897. An exemplary HPV E7(82-90) peptide sequence is LLMGTLGIV(SEQ ID NO: 8), SEQ ID NO: 538 in International Patent Publication 2019 / 005897. The contents of International Patent Publication 2019 / 005897 are incorporated herein by reference in their entirety.

[0162] Other cancer cell antigenic determinants suitable for incorporation into the pMHC targeting portion of this disclosure are the neoplastic antigens and their corresponding HLA alleles listed in Table 3 below. The neoplastic antigens include any mutations from the wild-type allele, or mutations resulting from the expression of a new open reading frame in cancer cells, as shown in Table 1 of Fritsch et al., 2014, Cancer Immunol Res 2:522-529 (and incorporated herein by reference in its entirety).

[0163] [Table 4]

[0164] 6.5. Multimerized portion 6.5.1. Fc Domain The IL2 agonists of this disclosure may include an Fc region derived from any preferred species. In one embodiment, the Fc region is derived from a human Fc domain. In a preferred embodiment, the IL2 domain is fused to an IgG Fc molecule.

[0165] The IL2 domain can be fused to the N-terminus or C-terminus of the IgG Fc region. As shown in the examples, fusion to the C-terminus of the IgG Fc region maintains IL2 domain activity to a greater extent than fusion to the N-terminus of IgG Fc.

[0166] One embodiment of the present disclosure relates to a dimer comprising two Fc-fusion polypeptides prepared by fusing an IL2 domain to the Fc region of an antibody. The dimer can be prepared, for example, by inserting a gene fusion encoding a fusion protein into a suitable expression vector, expressing the gene fusion in host cells transformed with a recombinant expression vector, and assembling the expressed fusion protein like an antibody molecule, thereby forming an interchain bond between the Fc portions and obtaining a dimer.

[0167] The Fc domain can be derived from any suitable class of antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In one embodiment, the Fc domain is derived from IgG1, IgG2, IgG3, or IgG4. In one embodiment, the Fc domain is derived from IgG1. In one embodiment, the Fc domain is derived from IgG4.

[0168] Two Fc domains within an Fc region may be identical or different from one another. In natural antibodies, the Fc domains are typically identical, but for the purpose of producing multispecific binding molecules, such as the IL2 agonists of this disclosure, the Fc domains may be advantageously different, as described in Section 6.5.1.2 below, enabling heterodimerization.

[0169] In natural antibodies, the heavy chain Fc domains of IgA, IgD, and IgG consist of two heavy chain constant domains (CH2 and CH3), while the heavy chain Fc domains of IgE and IgM consist of three heavy chain constant domains (CH2, CH3, and CH4). These dimerize to form the Fc region.

[0170] In the IL2 agonists of this disclosure, the Fc region, and / or the Fc domain within it, may contain heavy chain constant domains derived from one or more different classes of antibodies, e.g., one, two, or three different classes.

[0171] In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG1. In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG2.

[0172] In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG3. In one embodiment, the Fc region includes CH2 and CH3 domains derived from lgG4.

[0173] In one embodiment, the Fc region includes a CH4 domain from IgM. The IgM CH4 domain is typically located at the C-terminus of the CH3 domain. In one embodiment, the Fc region includes CH2 and CH3 domains derived from IgG, as well as a CH4 domain derived from IgM.

[0174] It will be understood that the heavy chain constant domains for use in producing the Fc region for the IL2 agonist of this disclosure may include variants of the naturally occurring constant domains described above. Such variants may include one or more amino acid variations compared to the wild-type constant domain. In one example, the Fc region of this disclosure includes at least one constant domain whose sequence differs from that of the wild-type constant domain. It will be understood that the variant constant domain may be longer or shorter than the wild-type constant domain. Preferably, the variant constant domain is at least 60% identical or similar to the wild-type constant domain. In another example, the variant constant domain is at least 70% identical or similar. In yet another example, the variant constant domain is at least 80% identical or similar. In yet another example, the variant constant domain is at least 90% identical or similar. In yet another example, the variant constant domain is at least 95% identical or similar.

[0175] IgM and IgA occur spontaneously in humans as covalent multimers of a common H2L2 antibody unit. IgM occurs as a pentamer when a J chain is incorporated, and as a hexamer when the J chain is absent. IgA occurs in monomeric and dimer forms. The heavy chains of IgM and IgA have an 18-amino acid extension to a C-terminal constant domain, known as the tailpiece. The tailpiece contains cysteine ​​residues that form disulfide bonds between the heavy chains in the polymer and is thought to play a crucial role in polymerization. The tailpiece also contains glycosylation sites. In certain embodiments, the IL2 agonists of this disclosure do not contain a tailpiece.

[0176] The Fc domain incorporated into the IL2 agonist of this disclosure may include one or more modifications that alter the functional properties of the protein, such as binding to Fc receptors such as FcRn or leukocyte receptors, binding to complement, modified disulfide bond structures, or modified glycosylation patterns. Exemplary Fc modifications that alter effector function are described in Section 6.5.1.1.

[0177] The Fc domain can also be modified to include modifications that improve the manufacturability of asymmetric IL2 agonists, for example, by enabling heterodimerization, which is the preferential pairing of non-identical Fc domains to identical Fc domains. Heterodimerization allows for the production of IL2 agonists in which different polypeptide components are linked together by Fc regions containing Fc domains with different sequences. Examples of heterodimerization strategies are illustrated in Section 6.5.1.2.

[0178] It will be understood that any of the above modifications can be combined in any preferred manner to achieve the desired functional characteristics, and / or combined with other modifications to alter the properties of the IL2 agonist.

[0179] 6.5.1.1. Fc domain with modified effector functionality In some embodiments, the Fc domain includes one or more amino acid substitutions that reduce binding to the Fc receptor and / or effector function.

[0180] In certain embodiments, the Fc receptor is an Fcγ receptor. In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is an activated Fc receptor. In specific embodiments, the Fc receptor is an activated human Fcγ receptor, more specifically human FcγRIIIa, FcγRI, or FcγRIIa, most specifically human FcγRIIIa. In one embodiment, the effector function is one or more selected from complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent phagocytosis (ADCP), and cytokine secretion. In certain embodiments, the effector function is ADCC.

[0181] In one embodiment, the Fc region includes an amino acid substitution at a position selected from the group E233, L234, L235, N297, P331, and P329 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc region includes an amino acid substitution at a position selected from the group L234, L235, and P329 (numbered according to the Kabat EU index). In some embodiments, the Fc region includes amino acid substitutions L234A and L235A (numbered according to the Kabat EU index). In one such embodiment, the Fc region is an Igd Fc region, particularly a human Igd Fc region. In one embodiment, the Fc region includes an amino acid substitution at position P329. In a more specific embodiment, the amino acid substitution is P329A or P329G, particularly P329G (numbered according to the Kabat EU index). In one embodiment, the Fc region includes an amino acid substitution at position P329, and further amino acid substitutions at positions selected from E233, L234, L235, N297, and P331 (numbered according to the Kabat EU index). In a more specific embodiment, the further amino acid substitutions are E233P, L234A, L235A, L235E, N297A, N297D, or P331S. In a particular embodiment, the Fc region includes amino acid substitutions at positions P329, L234, and L235 (numbered according to the Kabat EU index). In a more specific embodiment, the Fc region includes amino acid mutations L234A, L235A, and P329G ("P329G LALA", "PGLALA", or "LALAPG").

[0182] Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region contains the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), namely, in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (Kabat EU index numbering).

[0183] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. Typically, the same one or more amino acid substitutions are present in each of the two Fc domains of the Fc region. Thus, in certain embodiments, each Fc domain of the Fc region contains the amino acid substitutions L234A, L235A, and P329G (Kabat EU index numbering), namely, in each of the first and second Fc domains of the Fc region, the leucine residue at position 234 is replaced by an alanine residue (L234A), the leucine residue at position 235 is replaced by an alanine residue (L235A), and the proline residue at position 329 is replaced by a glycine residue (P329G) (Kabat EU index numbering).

[0184] In one embodiment, the Fc domain is an IgG1 Fc domain, particularly a human IgG1 Fc domain. In some embodiments, the IgG1 Fc domain is a variant IgG1 containing the D265A, N297A mutation (EU numbered) which reduces effector function.

[0185] In another embodiment, the Fc domain is an IgG4 Fc domain having reduced binding to the Fc receptor. An exemplary IgG4 Fc domain with reduced binding to the Fc receptor may include an amino acid sequence selected from Table 4 below: In some embodiments, the Fc domain includes only the bolded portion of the sequence shown below:

[0186] [Table 5-1]

[0187] [Table 5-2]

[0188] [Table 5-3]

[0189] In certain embodiments, IgG4 having reduced effector function includes the bolded portion of the amino acid sequence of Sequence ID No. 31 of WO2014 / 121087, which may be referred to herein as IgG4 or hIgG4.

[0190] Regarding the heterodimer Fc region, it is possible to incorporate a combination of the variant IgG4 Fc sequences described above, for example, an Fc region containing a combination of sequence number 30 (or its bolded portion) of WO2014 / 121087 and sequence number 37 (or its bolded portion) of WO2014 / 121087, or an Fc region containing a combination of sequence number 31 (or its bolded portion) of WO2014 / 121087 and sequence number 38 (or its bolded portion) of WO2014 / 121087.

[0191] 6.5.1.2. Fc heterodimerized variants Certain IL2 agonists, unlike native immunoglobulins, are operably linked to non-identical N-terminal regions, for example, one Fc domain is connected to Fab and the other to the IL2 portion, involving dimerization between the two Fc domains. Improper heterodimerization of the two Fc regions forming the Fc domain can be an obstacle to increasing the yield of the desired heterodimer molecule and represents a purification challenge. For example, as disclosed in EP1870459A1, U.S. Patent Nos. 5,582,996, 5,731,168, 5,910,573, 5,932,448, 6,833,441, 7,183,076, U.S. Patent Publication No. 2006 / 204493A1, and PCT Publication No. 2009 / 089004A1, various approaches available in the art can be used to enhance the dimerization of Fc domains that may be present in the IL2 agonist of this disclosure.

[0192] This disclosure provides IL2 agonists containing Fc heterodimers, i.e., Fc regions containing heterogeneous, non-identical Fc domains. Typically, each Fc domain in the Fc heterodimer contains a CH3 domain of an antibody. The CH3 domain is derived from the constant region of an antibody of any isotype, class, or subclass, preferably an IgG (IgG1, IgG2, IgG3, and IgG4) class, as described in the previous section.

[0193] Heterodimerization of two different heavy chains at the CH3 domain yields the desired IL2 agonist, while homodimerization of the same heavy chain reduces the yield of the desired IL2 agonist. Therefore, in preferred embodiments, the polypeptides that associate to form the IL2 agonist of this disclosure contain a CH3 domain having modifications favorable for heterodimer association with respect to the unmodified Fc domain.

[0194] In specific embodiments, the modification that promotes the formation of Fc heterodimers is a so-called "knob-into-hole" or "knob-in-hole" modification, which includes a "knob" modification in one of the Fc domains and a "hole" modification in the other Fc domain. Knob-into-hole techniques are described, for example, in U.S. Patent No. 5,731,168, U.S. Patent No. 7,695,936, Ridgway et al., 1996, Prot Eng 9:617-621, and Carter, 2001, Immunol Meth 248:7-15. Generally, the method involves introducing a protrusion ("knob") at the interface of a first polypeptide and a corresponding cavity ("hole") at the interface of a second polypeptide, and the protrusion may be positioned within the cavity to promote heterodimer formation and prevent homodimer formation. The protrusion is constructed by replacing a small amino acid side chain from the interface of the first polypeptide with a larger side chain (e.g., tyrosine or tryptophan). Compensatory cavities of the same or similar size as the protrusions are created at the interface of the second polypeptide by replacing larger amino acid side chains with smaller ones (e.g., alanine or threonine).

[0195] Accordingly, in some embodiments, an amino acid residue in the CH3 domain of the first subunit of the Fc domain is replaced with an amino acid residue having a larger side-chain volume, thereby generating a protrusion within the CH3 domain of the first subunit, which can be positioned within a cavity in the CH3 domain of the second subunit, and an amino acid residue in the CH3 domain of the second subunit of the Fc domain is replaced with an amino acid residue having a smaller side-chain volume, thereby generating a cavity within the CH3 domain of the second subunit, to which the protrusion within the CH3 domain of the first subunit can be positioned. Preferably, the amino acid residue having a larger side-chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W). Preferably, the amino acid residue having a smaller side-chain volume is selected from the group consisting of alanine (A), serine (S), threonine (T), and valine (V). The protrusion and cavity can be produced, for example, by modifying the nucleic acid encoding the polypeptide by site-directed mutagenesis or by peptide synthesis. An exemplary substitution is Y470T.

[0196] In certain such embodiments, in the first Fc domain, the threonine residue at position 366 is replaced with a tryptophan residue (T366W), in the Fc domain, the tyrosine residue at position 407 is replaced with a valine residue (Y407V), optionally, the threonine residue at position 366 is replaced with a serine residue (T366S), and the leucine residue at position 368 is replaced with an alanine residue (L368A) (numbered according to the Kabat EU index). In further embodiments, in the first Fc domain, the serine residue at position 354 is additionally replaced by a cysteine ​​residue (S354C), or the glutamic acid residue at position 356 is replaced by a cysteine ​​residue (E356C) (in particular, the serine residue at position 354 is replaced by a cysteine ​​residue), and in the second Fc domain, the tyrosine residue at position 349 is additionally replaced by a cysteine ​​residue (Y349C) (numbered according to the Kabat EU index). In certain embodiments, the first Fc domain includes amino acid substitutions S354C and T366W, and the second Fc domain includes amino acid substitutions Y349C, T366S, L368A, and Y407V (numbered according to the Kabat EU index).

[0197] In some embodiments, electrostatic steering (e.g., described in Gunasekaran et al., 2010, J Biol Chem 285(25):19637-46) can be used to facilitate the association of the first and second Fc domains of the Fc region.

[0198] As an alternative to, or in addition to, the use of modified Fc domains to promote heterodimerization, the Fc domain can be modified to enable purification strategies that allow for the selection of Fc heterodimers. In one such embodiment, a polypeptide comprises a modified Fc domain that invalidates its binding to protein A, thus enabling a purification method that yields a heterodimeric protein. See, for example, U.S. Patent No. 8,586,713. As such, an IL2 agonist comprises a first CH3 domain and a second Ig CH3 domain, where the first and second Ig CH3 domains differ from each other by at least one amino acid, and this difference of at least one amino acid reduces the binding of the IL2 agonist to protein A compared to a corresponding IL2 agonist lacking the amino acid difference. In one embodiment, the first CH3 domain binds to protein A, and the second CH3 domain contains a mutation / modification that reduces or eliminates protein A binding, such as the H95R modification (according to IMGT exon numbering, H435R in EU numbering). The second CH3 may further include the Y96F modification (by IMGT, which is Y436F in the EU). Therefore, this class of modification is referred to as the “star-shaped” mutation in this specification.

[0199] In some embodiments, Fc may contain one or more mutations (e.g., knob and hole mutations) to facilitate heterodimerization, as well as star-shaped mutations to facilitate purification.

[0200] 6.6. Stabilization part The IL2 agonists of this disclosure may contain a stabilizing moiety that can extend the serum half-life of the molecule in vivo. The serum half-life is often divided into alpha and beta phases. By adding an appropriate stabilizing moiety, either or both phases can be significantly improved. For example, a stabilizing moiety can increase the serum half-life of an IL2 agonist by 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 400, 600, 800, 1000%, or more compared to a corresponding IL2 agonist that does not contain a stabilizing moiety. For the purposes of this disclosure, serum half-life may refer to the half-life in humans or other mammals (e.g., mice or non-human primates).

[0201] Wild-type IL2 has a serum half-life of less than 10 minutes. The IL2 agonists of this disclosure preferably have a serum half-life of at least about 2 hours, at least about 4 hours, at least about 6 hours, or at least about 8 hours in humans and / or mice. In some embodiments, the IL2 agonists of this disclosure have a serum half-life of at least 10 hours, at least 12 hours, at least 15 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, or at least 72 hours.

[0202] The stabilizing portion includes a polyoxyalkylene moiety (e.g., polyethylene glycol), a sugar (e.g., sialic acid), and a highly tolerable protein moiety (e.g., Fc, and its fragments and variants, transferrin, or serum albumin).

[0203] Other stabilizing moieties that may be used with the IL2 agonists of this disclosure include those described in Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76. Such stabilizing moieties include, but are not limited to, human serum albumin fusions, human serum albumin conjugates, human serum albumin binders (e.g., adnectin PKE, AlbudAb, ABD), XTEN fusions, PAS fusions (i.e., recombinant PEG mimics based on three amino acids: proline, alanine, and serine), carbohydrate conjugates (e.g., hydroxyethyl starch (HES)), glycosylation, polysialic acid conjugates, and fatty acid conjugates.

[0204] Therefore, in some embodiments, the present disclosure provides IL2 agonists comprising a stabilizing moiety that is a high molecular weight sugar. Serum albumin can also be involved in half-life extension via modules with the ability to interact with albumin non-covalently. Therefore, the IL2 agonists of this disclosure may include albumin-binding proteins as stabilizing portions. Albumin-binding proteins can be conjugated to or genetically fused to one or more other components of the IL2 agonists of this disclosure. Proteins with albumin-binding activity are known from certain bacteria. For example, Streptococcus protein G contains several small albumin-binding domains consisting of approximately 50 amino acid residues (6 kDa). Additional examples of serum albumin-binding proteins include those described in U.S. Publications 2007 / 0178082 and 2007 / 0269422. Fusing albumin-binding domains to proteins significantly extends their half-life (see Kontermann et al., 2011, Current Opinion in Biotechnology 22:868-76).

[0205] In other embodiments, the stabilizing portion is human serum albumin. In other embodiments, the stabilizing portion is transferrin. In some embodiments, the stabilizing portion is an Fc domain, for example, one of the Fc domains described in Section 6.5.1 and its subsections, which are incorporated herein by reference. The Fc domains described in Section 6.5.1 are generally dimerizable. However, for stabilization purposes, the Fc domain may be a soluble monomeric Fc domain with reduced self-associating ability. See, for example, Helm et al., 1996, J. Biol. Chem. 271:7494-7500 and Ying et al., 2012, J Biol Chem. 287(23):19399-19408. An example of a soluble monomeric Fc domain includes amino acid substitutions at positions corresponding to T366 and / or Y407 of CH3, as described in U.S. Patent Application Publication No. 2019 / 0367611. The monomeric Fc domain is any Ig subtype and may include additional substitutions that reduce effector function, as described in Section 6.5.1 and its subsections.

[0206] In yet another embodiment, the stabilizing portion is a polyethylene glycol portion or another polymer, as described in Section 6.6.1 below. The stabilization portion may be connected to one or more other components of the IL2 agonist of this disclosure via a linker, for example, as described in Section 6.7 below.

[0207] 6.6.1. Polyethylene glycol In some embodiments, the IL2 agonist includes polyethylene glycol (PEG) or another hydrophilic polymer as a stabilizing portion, such as ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (either homopolymer or random copolymer), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymer, prolipropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. The polymer may have any molecular weight and may be branched or unbranched.

[0208] PEG is a well-known water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). The term "PEG" is widely used to encompass any polyethylene glycol molecule, regardless of size or modification at the PEG terminus, and can be represented by the following formula: X--O(CH2CH2O) n -1CH2CH2OH, where n is between 20 and 2300, and X is H or a terminal modification, e.g., C 1-4 It is alkyl. PEG may contain further chemical groups that are necessary for bonding reactions resulting from the chemical synthesis of the molecule or that function as spacers for optimal distances between molecular parts. In addition, such PEG may consist of one or more PEG side chains linked together. PEG having two or more PEG chains is called multi-armed or branched PEG. Branched PEG is described, for example, in European Patent Application No. 473084A and U.S. Patent No. 5,932,462.

[0209] One or more PEG molecules can be attached to different positions on an IL2 agonist, and such attachment can be achieved by reaction with an amine, thiol, or other suitable reactive group. The amine moiety may be, for example, a primary amine found at the N-terminus of the IL2 agonist (or its components), or an amine group present in an amino acid such as lysine or arginine. In some embodiments, the PEG moiety is attached to a) the N-terminus, b) between the N-terminus and the most N-terminal alpha-helix, c) a loop located on the surface of IL2 that binds to IL2-Rβ, d) between the C-terminus and the most C-terminal alpha-helix, e) a loop connecting two alpha-helices, and / or f) the C-terminus of the IL2 agonist.

[0210] PEGylation can be achieved by site-directed PEGylation, in which a suitable reactive group is introduced into the protein to create a site where PEGylation preferentially occurs. In some embodiments, the IL2 agonist is modified to introduce a cysteine ​​residue at a desired location, enabling site-directed PEGylation on cysteine. A cysteine ​​residue can be generated by introducing a mutation into the coding sequence of the IL2 agonist of this disclosure. This can be achieved, for example, by mutating one or more amino acid residues to cysteine. Preferred amino acids for mutation into cysteine ​​residues include serine, threonine, alanine, and other hydrophilic residues. Preferably, the residue mutated to cysteine ​​is a surface-exposed residue. Algorithms for predicting the surface accessibility of residues based on primary sequence or three-dimensional structure are well known in the art. The three-dimensional structure of IL2 is described, for example, in Wang et al., 2005, Science 310(5751):1159-63, and can be used to identify surface-exposed residues that may mutate to cysteine. Mutations may be selected to avoid disruption of the interaction between IL2 and one or more of its receptors, but in some embodiments (e.g., when biased binding to the receptor is desired), amino acid substitution with cysteine ​​and subsequent pegylation are designed to reduce binding to one or more of the receptors (e.g., IL2-Rα or IL2-Rβ). PEGylation of the cysteine ​​residue can be carried out using, for example, PEG-maleimide, PEG-vinyl sulfone, PEG-iodoacetamide, or PEG-orthopyridyl disulfide.

[0211] PEGs are typically activated with suitable activating groups appropriate for coupling to a desired site on a polypeptide. PEGylation methods are well known in the art and are further described in Zalipsky et al., “Use of Functionalized Poly(Ethylene Glycols) for Modification of Polypeptides” in Polyethylene Glycol Chemistry: Biotechnical and Biomedical Applications, JM Harris, Plenus Press, New York (1992) and Zalipsky, 1995, Advanced Drug Reviews 16:157-182.

[0212] The PEG portion can vary considerably in molecular weight and can be branched or linear. Typically, the weight-average molecular weight of PEG ranges from about 100 daltons to about 150,000 daltons. Exemplary weight-average molecular weights of PEG include about 20,000 daltons, about 40,000 daltons, about 60,000 daltons, and about 80,000 daltons. In certain embodiments, the molecular weight of PEG is 40,000 daltons. Branched versions of PEG having any of the aforementioned total molecular weights can also be used. In some embodiments, PEG has two branches. In other embodiments, PEG has four branches. In yet another embodiment, PEG is bis-PEG (NOF Corporation, DE-200MA) in which two IL2-containing polypeptide chains are conjugated.

[0213] PEGylated IL2 agonists can be purified using conventional separation and purification techniques known in the art, such as size exclusion (e.g., gel filtration) and ion exchange chromatography. Products can also be separated using SDS-PAGE. Products that can be separated include mono, di, tri, poly, and non-PEGylated IL2 agonists, as well as free PEG. The percentage of mono-PEG conjugate can be controlled by increasing the percentage of mono-PEG in the composition by pooling a broad fraction around the elution peak. Approximately 90% mono-PEG conjugate represents a good balance of yield and activity.

[0214] In some embodiments, the pegylated IL2 agonist preferably retains at least about 25%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100% of the biological activity associated with the unmodified IL2 agonist. In some embodiments, the biological activity is K D , k on , or k off This refers to its ability to bind to high- or intermediate-affinity IL2 receptors, as assessed by [specific criteria].

[0215] 6.7. Linker In certain embodiments, the disclosure provides IL2 agonists in which two or more components of an IL2 agonist are linked to one another by a peptide linker. Examples, but not limited to, that a linker may be used to link (a) an IL2 moiety and a multimerization moiety, (b) an IL2 moiety and a targeting moiety, (c) a targeting moiety and a multimerization moiety (e.g., a Fab domain and an Fc domain), (d) different domains within an IL2 moiety (e.g., an IL2 domain and an IL-Rα domain), or (e) different domains within a targeting moiety (e.g., different components of a peptide-MHC complex or VH and VL domains within scFv).

[0216] The peptide linker may be in the range of 2 to 60 amino acids or more, and in certain embodiments, the peptide linker may be in the range of lengths of 3 to 50 amino acids, 4 to 30 amino acids, 5 to 25 amino acids, 10 to 25 amino acids, 10 to 60 amino acids, 12 to 20 amino acids, 20 to 50 amino acids, or 25 to 35 amino acids.

[0217] In certain embodiments, the peptide linker is at least 5 amino acids long, at least 6 amino acids long, or at least 7 amino acids long, and optionally at most 30 amino acids long, at most 40 amino acids long, at most 50 amino acids long, or at most 60 amino acids long.

[0218] In some of the embodiments described above, the linker is in the length range of 5 to 50 amino acids, for example, in the length range of 5 to 50, 5 to 45, 5 to 40, 5 to 35, 5 to 30, 5 to 25, or 5 to 20 amino acids. In other embodiments described above, the linker is in the length range of 6 to 50 amino acids, for example, in the length range of 6 to 50, 6 to 45, 6 to 40, 6 to 35, 6 to 30, 6 to 25, or 6 to 20 amino acids. In yet another embodiment described above, the linker is in the length range of 7 to 50 amino acids, for example, in the length range of 7 to 50, 7 to 45, 7 to 40, 7 to 35, 7 to 30, 7 to 25, or 7 to 20 amino acids.

[0219] Charged linkers (e.g., charged hydrophilic linkers) and / or flexible linkers are particularly preferred. Examples of flexible linkers that can be used with the IL2 agonists of this disclosure include those disclosed in Chen et al., 2013, Adv Drug Deliv Rev. 65(10):1357-1369 and Klein et al., 2014, Protein Engineering, Design & Selection 27(10):325-330. Particularly useful flexible linkers are glycine and serine repeats, e.g., G nThe linker is a monomer or multimer of S (SEQ ID NO: 55) or SGn (SEQ ID NO: 56), or contains them, where n is an integer from 1 to 10, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the linker is G4S, for example, (GGGGS) n (SEQ ID NO: 57) is a monomer or multimer of the repeat, or contains them.

[0220] Polyglycine linkers can be suitably used in the IL2 agonists of this disclosure. In some embodiments, the peptide linker includes two consecutive glycine molecules (2Gly), three consecutive glycine molecules (3Gly), four consecutive glycine molecules (4Gly) (SEQ ID NO: 58), five consecutive glycine molecules (5Gly) (SEQ ID NO: 59), six consecutive glycine molecules (6Gly) (SEQ ID NO: 60), seven consecutive glycine molecules (7Gly) (SEQ ID NO: 61), eight consecutive glycine molecules (8Gly) (SEQ ID NO: 62), or nine consecutive glycine molecules (9Gly) (SEQ ID NO: 63).

[0221] 6.7.1. pMHC Linker For pMHC complexes, preferred linkers may range from 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, or 7 amino acids. In addition to the above linkers, pMHC linkers include glycine polymers (G)n, glycine-serine polymers (e.g., (GS)n, (GSGGS)n (SEQ ID NO: 64), and (GGGS)n (SEQ ID NO: 65), where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used. Since both Gly and Ser are relatively unstructured, they can function as neutral tethers between components. Glycine polymers can be used. Glycine has access to far more phi-psi spaces than alanine and is far less restrictive than residues with longer side chains (see Scheraga, 1992, Rev. Computational Chem. 1 1173-142, the whole of which is incorporated herein by reference). Exemplary linkers may include, but are not limited to, amino acid sequences such as GGSG (SEQ ID NO: 66), GGSGG (SEQ ID NO: 67), GGSSG (SEQ ID NO: 68), GSGGG (SEQ ID NO: 69), GGGSG (SEQ ID NO: 70), GSSSG (SEQ ID NO: 71), GCGASGGGSGGGGS (SEQ ID NO: 72), GGGGSGGGGS (SEQ ID NO: 73), GGGASGGGGSGGGGS (SEQ ID NO: 74), GGGGSGGGGSGGGGS (SEQ ID NO: 6), GGGASGGGGS (SEQ ID NO: 75), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 76), GCGGS (SEQ ID NO: 77), etc. In some embodiments, the linker polypeptide includes a cysteine ​​residue that can form a disulfide bond with a cysteine ​​residue present in another part of the pMHC complex. In certain embodiments, the linker includes the amino acid sequence GCGGS (SEQ ID NO: 77).The substitution of glycine with cysteine ​​in the G4S linker (SEQ ID NO: 57) can lead to the formation of an MHC targeting moiety with a disulfide bond, such as the corresponding cysteine ​​substitution in HLA.A2 that stabilizes MHC peptides within the MHC complex.

[0222] 6.7.2. Hinge Arrangement In other embodiments, the IL2 agonists of this disclosure include a linker which is a hinge region. In particular, if the IL2 agonist contains an immunoglobulin-based targeting moiety, the hinge can be used to connect the targeting moiety, e.g., the Fab domain, to a multimerization domain, e.g., the Fc domain. The hinge region may be a native or modified hinge region. The hinge region is typically found at the N-terminus of the Fc domain. Unless otherwise indicated by context, the term “hinge region” in the context of a single or monomeric polypeptide chain refers to a naturally occurring hinge sequence which is a monomeric hinge domain, and in the context of a dimer polypeptide (e.g., a homodimer or heterodimer IL2 agonist formed by the association of two Fc domains) may include two associated hinge sequences on separate polypeptide chains.

[0223] The native hinge region is the hinge region typically found between the Fab domain and the Fc domain in naturally occurring antibodies. A modified hinge region is any hinge that differs in length and / or composition from the native hinge region. Such hinges may include hinge regions from other species, such as human, mouse, rat, rabbit, shark, pig, hamster, camel, llama, or goat hinge regions. Other modified hinge regions may include complete hinge regions derived from antibodies of a different class or subclass than those in the heavy chain Fc region. Alternatively, a modified hinge region may include a native hinge or a portion of a repeating unit, where each unit in the repeat originates from the native hinge region. Further alternatives include modifying the native hinge region by converting one or more cysteine ​​or other residues to neutral residues such as serine or alanine, or by converting suitably positioned residues to cysteine ​​residues. By such means, the number of cysteine ​​residues in the hinge region may be increased or decreased. Other modified hinge regions may be entirely synthetic and can be designed to have desired properties such as length, cysteine ​​composition, and flexibility.

[0224] Several modified hinge regions have already been described, for example, in U.S. Patent Nos. 5,677,425, WO99 / 15549, WO2005 / 003170, WO2005 / 003169, WO2005 / 003170, WO98 / 25971, and WO2005 / 003171, which are incorporated herein by reference.

[0225] In one embodiment, the IL2 agonist of the present disclosure comprises an Fc region in which one or both Fc domains have an intact hinge region at their N-terminus. In various embodiments, positions 233–236 within the hinge region may be G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, and the positions are numbered by EU numbering.

[0226] In some embodiments, the IL2 agonists of this disclosure include a modified hinge region that reduces the binding affinity for the Fcγ receptor compared to the wild-type hinge region of the same isotype (e.g., human IgG1 or human IgG4).

[0227] In one embodiment, the IL2 agonist of this disclosure comprises an Fc domain in which each Fc domain has an intact hinge region at its N-terminus, each Fc domain and hinge region originating from lgG4, and each hinge region containing the modified sequence CPPC (SEQ ID NO: 78). The core hinge region of human lgG4 contains the sequence CPSC (SEQ ID NO: 79) compared to lgG1 which contains the sequence CPPC (SEQ ID NO: 78). The serine residue present in the lgG4 sequence results in increased flexibility in this region, and therefore a portion of the molecule forms a disulfide bond within the same protein chain (intrachain disulfide) rather than crosslinking to other heavy chains in the IgG molecule to form interchain disulfides. (Angel et al., 1993, Mol Immunol 30(1):105-108). Replacing the serine residue with proline to obtain the same core sequence as lgG1 allows for the complete formation of interchain disulfides in the lgG4 hinge region, thus reducing heterogeneity in the purified product. This modified isotype is called lgG4P.

[0228] 6.7.2.1. Chimera Hinge Array The hinge region may be a chimeric hinge region. For example, a chimeric hinge may include an "upper hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region, combined with an "lower hinge" sequence derived from the human IgG1, human IgG2, or human IgG4 hinge region.

[0229] In certain embodiments, the chimeric hinge region comprises the amino acid sequence EPKSCDKTHTCPPCPAPPVA (SEQ ID NO: 80) (which is incorporated herein in its entirety by reference, previously disclosed as SEQ ID NO: 8 in WO2014 / 121087) or ESKYGPPCPPCPAPPVA (SEQ ID NO: 81) (which was previously disclosed as SEQ ID NO: 9 in WO2014 / 121087). Such a chimeric hinge sequence can be suitably ligated to the IgG4 CH2 region (for example, by ligating to the IgG4 Fc domain, e.g., a human or mouse Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, as described, for example, in Section 6.5.1.1).

[0230] 6.7.2.2. Hinge arrangement with reduced effector functionality In further embodiments, the hinge region can be modified to reduce its effector function, for example, as described in WO2016 / 161010A2, which is incorporated herein in its entirety by reference. In various embodiments, positions 233–236 of the modified hinge region are G, G, G, and unoccupied; G, G, unoccupied, and unoccupied; G, unoccupied, unoccupied, and unoccupied; or all unoccupied, with the positions numbered by EU numbering (as shown in Figure 1 of WO2016 / 161010A2). These segments can be represented as GGG-, GG-, G---, or ----, where "-" indicates an unoccupied position.

[0231] Position 236 is unoccupied in standard human IgG2 but is occupied in other standard human IgG isotypes. Positions 233–235 are occupied by non-G residues in all four human isotypes (as shown in Figure 1 of WO2016 / 161010A2).

[0232] Hinge modifications within positions 233-236 can be combined with the occupation of position 228 by P. Position 228 is naturally occupied by P in human IgG1 and IgG2, but by S in human IgG4 and R in human IgG3. The S228P mutation in IgG4 antibodies is advantageous for stabilizing IgG4 antibodies and reducing heavy-light chain pair exchange between exogenous and endogenous antibodies. Preferably, positions 226-229 are occupied by C, P, P, and C, respectively.

[0233] Exemplary hinge regions, sometimes called intermediate (or core) and lower hinges, have residues 226-236 and are occupied by modified hinge sequences called GGG-(233-236), GG--(233-236), G---(233-236), and G-less(233-236). Optionally, the hinge domain amino acid sequence may include CPPCPAPGG-GPSVF (SEQ ID NO: 82) (previously disclosed as SEQ ID NO: 1 in WO2016 / 161010A2), CPPCPAPGG--GPSVF (SEQ ID NO: 83) (previously disclosed as SEQ ID NO: 2 in WO2016 / 161010A2), CPPCPAP---GPSVF (SEQ ID NO: 84) (previously disclosed as SEQ ID NO: 3 in WO2016 / 161010A2), or CPPCPAP----GPSVF (SEQ ID NO: 85) (previously disclosed as SEQ ID NO: 4 in WO2016 / 161010A2).

[0234] The modified hinge regions described above can be incorporated into heavy chain constant regions, which typically include CH2 and CH3 domains and may have additional hinge segments (e.g., upper hinges) adjacent to the designated region. Such additional constant region segments that exist are typically of the same isotype, preferably human isotype, but may be hybrids of different isotypes. The isotype of such additional human constant region segments is preferably human IgG4, but may also be human IgG1, IgG2, or IgG3, or hybrids of those in which the domains are of different isotypes. Exemplary sequences of human IgG1, IgG2, and IgG4 are shown in Figures 2-4 of WO2016 / 161010A2.

[0235] In specific embodiments, the modified hinge sequence can be linked to the IgG4 CH2 region (for example, by incorporating it into the IgG4 Fc domain, e.g., a human or mouse Fc domain, which can be further modified in the CH2 and / or CH3 domains to reduce effector function, as described, for example, in Section 6.5.1.1).

[0236] 6.8. Nucleic acids and host cells In another embodiment, the Disclosure provides nucleic acids encoding the IL2 agonists of the Disclosure. In some embodiments, the IL2 agonist is encoded by a single nucleic acid. In other embodiments, for example, in the case of a heterodimer molecule or a molecule containing a targeting moiety composed of two or more polypeptide chains, the IL2 agonist may be encoded by multiple (e.g., two, three, four or more) nucleic acids.

[0237] A single nucleic acid can encode an IL2 agonist containing a single polypeptide chain, an IL2 agonist containing two or more polypeptide chains, or a portion of an IL2 agonist containing three or more polypeptide chains (for example, a single nucleic acid can encode two polypeptide chains of an IL2 agonist containing three, four or more polypeptide chains, or three polypeptide chains of an IL2 agonist containing four or more polypeptide chains). For the control of distinct expression, an open reading frame encoding two or more polypeptide chains can be placed under the control of distinct transcriptional regulators (e.g., promoters and / or enhancers). An open reading frame encoding two or more polypeptides can also be controlled by the same transcriptional regulator and separated by an internal ribosome entry site (IRES) sequence, allowing translation into distinct polypeptides.

[0238] In some embodiments, an IL2 agonist containing two or more polypeptide chains is encoded by two or more nucleic acids. The number of nucleic acids encoding the IL2 agonist may be less than or equal to the number of polypeptide chains in the IL2 agonist (for example, if two or more polypeptide chains are encoded by a single nucleic acid).

[0239] The nucleic acids in this disclosure may be DNA or RNA (e.g., mRNA). In another aspect, the Disclosure provides host cells and vectors containing the nucleic acids of the Disclosure. The nucleic acids may be present in a single vector or in separate vectors present in the same host cell or in separate host cells, as described in more detail below herein.

[0240] 6.8.1. Vectors This disclosure provides vectors comprising nucleotide sequences encoding IL2 agonists or IL2 agonist components described herein, such as one or two polypeptide chains of semi-antibodies. Vectors may include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs).

[0241] Numerous vector systems can be used. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV, or MOMLV), or SV40 virus. Another class of vectors utilizes RNA elements derived from RNA viruses such as Semlik Forest virus, Eastern equine encephalitis virus, and flavivirus.

[0242] Additionally, cells in which DNA has been stably incorporated into their chromosomes can be selected by introducing one or more markers that enable selection of the transfected host cell. These markers may provide, for example, prototrophicity to a trophic host, biocide resistance (e.g., antibiotics), or resistance to heavy metals such as copper. The selectable marker gene can be directly ligated to the expressed DNA sequence or introduced into the same cell by co-transformation. Optimal mRNA synthesis may require additional elements. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.

[0243] Once an expression vector or DNA sequence containing a construct is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this, various techniques may be used, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene guns, lipid-based transfection, or other conventional techniques. Methods and conditions for culturing the resulting transfected cells and recovering the expressed polypeptide are known to those skilled in the art and may be modified or optimized based on this description depending on the specific expression vector and mammalian host cell used.

[0244] 6.8.2.Cells This disclosure also provides host cells containing the nucleic acids of this disclosure. In one embodiment, a host cell is genetically engineered to contain one or more nucleic acids described herein.

[0245] In one embodiment, host cells are genetically engineered by using an expression cassette. The phrase “expression cassette” refers to a nucleotide sequence that can influence the expression of a gene in a host of such sequence compatibility. Such a cassette may include a promoter, an open reading frame with or without introns, and a termination signal. Additional factors necessary or useful for bringing about expression, such as an inducible promoter, may also be used.

[0246] This disclosure also provides host cells containing the vectors described herein. The cells may be eukaryotic cells, bacterial cells, insect cells, or human cells, but are not limited to these. Suitable eukaryotic cells include, but are not limited to, Vero cells, HeLa cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but are not limited to, Sf9 cells.

[0247] 6.9. Pharmaceutical Compositions 6.9.1. Pharmaceutical compositions containing IL2 agonist polypeptides The IL2 agonists of this disclosure may be in the form of a composition comprising the IL2 agonist and one or more carriers, excipients, and / or diluents. The composition may be formulated for specific uses, such as veterinary use or pharmacokinetic use in humans. The form of the composition (e.g., dry powder, liquid formulation, etc.), and the excipients, diluents, and / or carriers used, depend on the intended use of the IL2 agonist and, for therapeutic use, the mode of administration.

[0248] For therapeutic use, the composition may be supplied as part of a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier. This composition may be in any preferred form (depending on the desired method of administration to the patient). The pharmaceutical composition may be administered to the patient by various routes, such as orally, transdermally, subcutaneously, intranasally, intravenously, intramuscularly, intratumorally, intrathecally, topically, or locally. The most preferred route of administration in any given case will depend on the specific antibody, the target, as well as the nature and severity of the disease and the patient's physical condition. Typically, the pharmaceutical composition will be administered intravenously or subcutaneously.

[0249] Pharmaceutical compositions can be conveniently presented in unit dosage forms containing a predetermined amount of the IL2 agonist of this disclosure per dose. The amount of IL2 agonist contained in a unit dose will depend on the disease being treated and other factors well known in the art. Such unit doses may be in the form of a lyophilized dry powder containing an amount of IL2 agonist suitable for a single dose, or in liquid form. Dry powder unit dosage forms may be packaged in kits comprising a syringe, a suitable amount of diluent, and / or other components useful for administration. Unit doses in liquid form may be conveniently supplied in the form of syringes pre-filled with an amount of IL2 agonist suitable for a single dose.

[0250] The pharmaceutical composition also contains an amount of IL2 agonist suitable for multiple doses, and can therefore be supplied in large quantities. Pharmaceutical compositions can be prepared for storage as lyophilized formulations or aqueous solutions by mixing an IL2 agonist of desired purity with any choice of pharmaceutically acceptable carriers, excipients, or stabilizers (all of which are referred to herein as “carriers”) typically used in the art, namely buffers, stabilizers, preservatives, isotonic agents, nonionic cleaning agents, antioxidants, and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such additives should be nontoxic to the recipient at the dosage and concentration used.

[0251] Buffers help maintain a pH range close to physiological conditions. They can exist in a wide variety of concentrations, but typically they will be in the range of about 2 mM to about 50 mM. Suitable buffers for use in this disclosure include both organic and inorganic acids, as well as their salts, e.g., citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citrate-trisodium citrate mixture, citrate-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumarate-monosodium fumarate mixture, fumarate These include disodium fumarate mixtures, monosodium fumarate-disodium fumarate mixtures, etc., gluconate buffers (e.g., gluconic acid-sodium glycoside mixtures, gluconic acid-sodium hydroxide mixtures, gluconic acid-potassium glycoside mixtures, etc.), oxalate buffers (e.g., oxalic acid-sodium oxalate mixtures, oxalic acid-sodium hydroxide mixtures, oxalic acid-potassium oxalate mixtures, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixtures, lactic acid-sodium hydroxide mixtures, lactic acid-potassium lactate mixtures, etc.), and acetate buffers (e.g., acetic acid-sodium acetate mixtures, acetic acid-sodium hydroxide mixtures, etc.). Additionally, phosphate buffers, histidine buffers, and trimethylamine salts such as Tris can be used.

[0252] Preservatives may be added to slow microbial growth and may be added in amounts ranging from about 0.2% to 1% (w / v). Suitable preservatives for use in this disclosure include phenol, benzyl alcohol, metacresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkylparabens, e.g., methyl or propylparaben, catechol, resorcinol, cyclohexanol, and 3-pentanol. Isotonic agents, known as “stabilizers,” may be added to ensure the isotonicity of the liquid compositions of this disclosure and include polyhydric sugar alcohols, e.g., trihydric or higher sugar alcohols such as glycerin, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers refer to a broad category of excipients whose functions can range from volume extenders to additives that help solubilize therapeutic agents or prevent denaturation or adhesion to container walls. Typical stabilizers include polyhydric sugar alcohols (listed above); amino acids, e.g., arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, e.g., lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myo-inititol, galactitol, glycerol, etc. (including cyclitol, e.g., inositol); polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, e.g., urea, glutamic acid. The materials may include thion, thioctic acid, sodium thioglycolate, thioglycerol, α-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (e.g., peptides with 10 or fewer residues); proteins, e.g., human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone monosaccharides, e.g., xylose, mannose, fructose, glucose; disaccharides, e.g., lactose, maltose, sucrose, and trehalose; trisaccharides, e.g., raffinose; and polysaccharides, e.g., dextran.The stabilizer may be present in an amount ranging from 0.5% to 10% by weight of the IL2 agonist.

[0253] Nonionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize glycoproteins and protect them from aggregation induced by agitation, which also allows the formulation to be exposed to a stressed shear surface without causing protein denaturation. Suitable nonionic surfactants include polysorbates (20, 80, etc.), polyoxomers (184, 188, etc.), and Pluronic® polyols. Nonionic surfactants may be present in concentrations ranging from about 0.05 mg / mL to about 1.0 mg / mL, for example, from about 0.07 mg / mL to about 0.2 mg / mL.

[0254] Various additional excipients include bulking agents (e.g., starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbic acid, methionine, vitamin E), and co-solvents.

[0255] 6.9.2. Pharmaceutical compositions for the delivery of nucleic acid-encoding IL-2 agonists The IL2 agonists of this disclosure can be delivered by any method useful for gene therapy, for example, as mRNA, or via a viral vector encoding the IL2 agonist under the control of a suitable promoter.

[0256] Exemplary gene therapy vectors include adenovirus or AAV-based therapeutics.Non-limiting examples of adenovirus-based or AAV-based therapeutics for use in the methods, uses, or compositions herein include: rAd-p53, for example, a recombinant adenovirus vector encoding wild-type human tumor suppressor protein p53 for use in the treatment of cancer (also known as Gendicine®, Genkaxin®, Qi et al., 2006, Modern Oncology, 14:1295-1297); Ad5_d11520, an adenovirus lacking the E1B gene for inactivating host p53 (also known as H101 or ONYX-015, see, for example, Russell et al., 2012, Nature Biotechnology 30:658-670); and AD5-D24-GM-CSF, for example, an adenovirus containing the cytokine GM-CSF for use in the treatment of cancer (see Cerullo et al., 2010, Cancer Res.70:4297); rAd-HSVtk, for example, a replication-deficient adenovirus having the HSV thymidine kinase gene for the treatment of cancer (developed as Cerepro®, Ark Therapeutics, see, for example, U.S. Patent No. 6,579,855; developed by Advantagene as ProstAtak®; International PCT Application No. 2005 / 049094); rAd-TNFα, for example, a replication-deficient adenovirus vector expressing human tumor necrosis factor alpha (TNFα) under the control of a chemoradiation-inducible EGR-1 promoter for the treatment of cancer (TNFerade®, GenVec; Rasmussen et al., 2002, Cancer Gene Ther.9:951-7; Ad-IFNβ, for example, adenovirus serotype 5 vectors in which the E1 and E3 genes are deleted and the human interferon-beta gene is expressed under the direction of the cytomegalovirus (CMV) initial promoter (BG00001 and H5.110CMVhIFN-β, Biogen; Sterman et al., 2010, Mol.Ther.18:852-860) for treating cancer, are examples of such vectors.

[0257] Nucleic acid molecules (e.g., mRNA) or viruses can be formulated as the sole pharmaceutically active ingredient in a pharmaceutical composition, or in combination with other activators for a specific disorder to be treated. Optionally, other drugs, pharmaceuticals, carriers, adjuvants, and diluents may be included in the compositions provided herein. For example, wetting agents, emulsifiers, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as any one or more of colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives, antioxidants, chelating agents, and inert gases may also be present in the compositions. Other exemplary agents and excipients that can be included in the composition include, for example, water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium pyrosulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and α-tocopherol; and metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0258] When used as an adjunct therapy to adoptive cell transplantation, for example, CAR-expressing cell therapy as described in Section 6.11.1, the cell therapy, e.g., CAR-expressing cells, may be engineered to express the IL2 agonist of this disclosure. The IL2 agonist may target a specific genomic locus, e.g., the endogenous IL2 locus, or another locus that is active in activated or dysfunctional lymphocytes, e.g., the PD-1 locus, or inserted into a nonspecific genomic locus. Targeting a specific genomic locus can be achieved, for example, through gene editing using zinc finger proteins, CRISPR / Cas9 systems, etc.

[0259] 6.10. Indications and Treatment Methods The IL2 agonists of this disclosure are useful for treating medical conditions in which stimulation of the host immune system is beneficial, particularly conditions in which an enhanced cellular immune response is desirable. These may include medical conditions in which the host immune response is inadequate or absent. Medical conditions in which the IL2 agonists of this disclosure can be administered include, for example, tumors or infections in which the cellular immune response is a key immune mechanism in certain cases. Specific medical conditions in which the IL2 agonists of this disclosure can be used include cancers, e.g., renal cell carcinoma or melanoma, and immunodeficiency, particularly in HIV-positive patients, immunosuppressed patients, and chronic infections. The IL2 agonists of this disclosure can be administered on their own or in any suitable pharmaceutical composition.

[0260] In one embodiment, an IL2 agonist of the present disclosure is provided for use as a pharmaceutical agent. In a further embodiment, an IL2 agonist of the present disclosure is provided for use in the treatment of a disease. In a particular embodiment, an IL2 agonist of the present disclosure is provided for use in a therapeutic method. In one embodiment, the present disclosure provides an IL2 agonist described herein for use in a subject requiring treatment of a disease. In a particular embodiment, the present disclosure provides an IL2 agonist for use in a method of treating a subject having a disease, comprising administering a therapeutically effective amount of the IL2 agonist to the subject. In a particular embodiment, the disease to be treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In a particular embodiment, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent, for example, an anticancer agent, if the disease to be treated is cancer, to the subject. In a further embodiment, the present disclosure provides an IL2 agonist for use in stimulating the immune system. In certain embodiments, the Disclosure provides an IL-2 agonist for use in a method of stimulating the immune system in a subject, which includes administering an effective amount of the IL-2 agonist to the subject to stimulate the immune system. The “subject” in any of the above embodiments is a mammal, preferably a human. The “stimulation of the immune system” in any of the above embodiments may include one or more of the following: a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, or an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity.

[0261] In further embodiments, the Disclosure provides the use of the IL2 agonist of the Disclosure in the manufacture or preparation of a drug for a subject in need of treatment for a disease. In one embodiment, the drug is for use in a method of treating a disease, comprising administering a therapeutically effective amount of the drug to a subject having the disease. In a particular embodiment, the disease to be treated is a proliferative disorder. In a preferred embodiment, the disease is cancer. In one such embodiment, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent, for example, an anticancer agent if the disease to be treated is cancer. In further embodiments, the drug is for stimulating the immune system. In further embodiments, the drug is for use in a method of stimulating the immune system in a subject, comprising administering to the subject an effective amount of the drug to stimulate the immune system. The “subject” in any of the above embodiments may be a mammal, preferably a human. The “stimulation of the immune system” according to any of the above embodiments may include one or more of the following: a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, or an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity.

[0262] In further embodiments, the Disclosure provides a method for treating a disease in a subject, comprising administering a therapeutically effective amount of the IL2 agonist of the Disclosure to the subject. In one embodiment, a composition comprising the IL2 agonist of the Disclosure in a pharmaceutically acceptable form is administered to the subject. In certain embodiments, the disease to be treated is a proliferative disorder. In preferred embodiments, the disease is cancer. In certain embodiments, the method further comprises administering a therapeutically effective amount of at least one additional therapeutic agent, for example, an anticancer agent, to the subject if the disease to be treated is cancer. In further embodiments, the Disclosure provides a method for stimulating the immune system in a subject, comprising administering a therapeutically effective amount of the IL2 agonist to the subject to stimulate the immune system. The “subject” in any of the above embodiments may be a mammal, preferably a human. The “stimulation of the immune system” according to any of the above embodiments may include one or more of the following: a general increase in immune function, an increase in T cell function, an increase in B cell function, a restoration of lymphocyte function, an increase in IL-2 receptor expression, an increase in T cell responsiveness, or an increase in natural killer cell activity or lymphokine-activated killer (LAK) cell activity.

[0263] In certain embodiments, the disease being treated is a proliferative disorder, preferably cancer. Non-limiting examples of cancer include bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, stomach cancer, prostate cancer, hematological cancer, skin cancer, squamous cell carcinoma, bone cancer, and kidney cancer. Other proliferative disorders that can be treated with the IL2 agonists of this disclosure include, but are not limited to, neoplasms located in the abdomen, bone, breast, digestive system, liver, pancreas, peritoneum, endocrine glands (adrenal glands, parathyroid glands, pituitary gland, testes, ovaries, thymus, thyroid gland), eye, head and neck, nervous system (central and peripheral), lymphatic system, pelvis, skin, soft tissue, spleen, thoracic region, and genitourinary system. Precancerous conditions or lesions and cancer metastases are also included. In certain embodiments, cancer is selected from the group consisting of renal cell carcinoma, skin cancer, lung cancer, colorectal cancer, breast cancer, brain cancer, and head and neck cancer. Similarly, other cell proliferation disorders can also be treated with the IL2 agonists of this disclosure. Examples of such cell proliferation disorders include, but are not limited to, hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemia, purpura, sarcoidosis, Sézary syndrome, Waldenström hypergammaglobulinemia, Gaucher disease, histiocytic proliferative disorders, and any other cell proliferation disorders other than neoplasms located in the organ systems listed above. In other embodiments, the disease is associated with autoimmunity, transplant rejection, post-traumatic immune responses, and infections (e.g., HIV). More specifically, IL2 agonists may be used to eliminate cells involved in immune cell-mediated disorders, including lymphoma, autoimmunity, transplant rejection, graft-versus-host disease, ischemia, and stroke. Those skilled in the art readily recognize that, in many cases, IL-2 agonists may not result in a cure, but may provide only partial benefit. In some embodiments, physiological changes that provide some benefit are also considered therapeutically beneficial. Therefore, in some embodiments, the amount of IL-2 agonist that produces a physiological change is considered the “effective dose” or “therapeutic effective dose.” The subject, patient, or individual requiring treatment is typically a mammal, more specifically a human.

[0264] For the prevention or treatment of disease, the appropriate dose of the IL2 agonist of this disclosure (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease being treated, the route of administration, the patient's weight, the specific IL2 agonist, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous or concurrent therapeutic interventions, the patient's medical history and response to the IL2 agonist, and the discretion of the attending physician. The practicing physician responsible for administration will, in any case, determine the concentration of the active ingredient in the composition and the appropriate dose for each individual subject. Various administration schedules are contemplated herein, including but not limited to single or multiple doses, bolus administration, and pulse infusion at various time points.

[0265] A single dose of unconjugated IL2 can range from approximately 50,000 IU / kg to over 1,000,000 IU / kg, more typically around 600,000 IU / kg of IL2. This may be repeated several times a day (e.g., 2-3 times) for several days (e.g., approximately 3-5 consecutive days), followed by one or more repetitions after a rest period (e.g., approximately 7-14 days). Therefore, a therapeutically effective dose may consist of a single dose over a period of time or multiple doses (e.g., approximately 20-30 individual doses of approximately 600,000 IU / kg of IL2, each given over a period of approximately 10-20 days).

[0266] Similarly, IL-2 agonists are preferably administered to patients as a single dose or over a series of treatments. Depending on the type and severity of the disease, an IL-2 agonist of approximately 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be the initial candidate dose for administration to a patient, for example, by one or more separate doses or by continuous infusion. A typical daily dose may range from approximately 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. In the case of repeated administration over several days or more, treatment will generally be continued, depending on the condition, until the desired suppression of disease symptoms occurs. One exemplary dose of an IL-2 agonist may range from approximately 0.005 mg / kg to approximately 10 mg / kg. In other non-limiting examples, doses may also include approximately 1 μg / kg / body weight, approximately 5 μg / kg / body weight, approximately 10 μg / kg / body weight, approximately 50 μg / kg / body weight, approximately 100 μg / kg / body weight, approximately 200 μg / kg / body weight, approximately 350 μg / kg / body weight, approximately 500 μg / kg / body weight, approximately 1 mg / kg / body weight, approximately 5 mg / kg / body weight, approximately 10 mg / kg / body weight, approximately 50 mg / kg / body weight, approximately 100 mg / kg / body weight, approximately 200 mg / kg / body weight, approximately 350 mg / kg / body weight, approximately 500 mg / kg / body weight, approximately 1000 mg / kg / body weight, or more, and any range deriveable therefrom. In non-limiting examples of the derivable range from the values ​​listed herein, ranges such as approximately 5 mg / kg / body weight to approximately 100 mg / kg / body weight, and approximately 5 μg / kg / body weight to approximately 500 mg / kg / body weight may be administered based on the values ​​above. Therefore, one or more doses of approximately 0.5 mg / kg, 2.0 mg / kg, 5.0 mg / kg, or 10 mg / kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives approximately 2 to approximately 20, or for example, approximately 6, doses of the IL2 agonist). An initial higher loading dose may be administered, followed by one or more lower doses. However, other dosing regimens may be useful. The progress of this therapy can be easily monitored by conventional techniques and assays.

[0267] The IL2 agonists of this disclosure are generally used in amounts effective to achieve the intended purpose. For use in treating or preventing a medical condition, the IL2 agonists or their pharmaceutically effective compositions of this disclosure are administered or applied in therapeutically effective doses. Determining the therapeutically effective dose is well within the capabilities of those skilled in the art, in light of the detailed disclosures provided herein.

[0268] For systemic administration, the therapeutically effective dose can first be estimated from in vitro assays such as cell culture assays. Next, the EC determined by cell culture... 50 To achieve a circulating concentration range that includes [specific concentration range], doses can be formulated in animal models. Such information can be used to more accurately determine useful doses in humans.

[0269] The initial dose can also be estimated using techniques well known in the art, for example, from in vivo data of animal models. Those skilled in the art can easily optimize the human dose based on the animal data.

[0270] Dosage and intervals can be individually adjusted to provide sufficient plasma levels of IL-2 agonist to maintain therapeutic effect. Typical patient doses for injectable administration range from approximately 0.1 to 50 mg / kg / day, typically from approximately 0.5 to 1 mg / kg / day. Therapeutably effective plasma levels can be achieved by administering multiple doses daily. Plasma levels can be measured, for example, by ELISA HPLC.

[0271] In the case of local administration or selective uptake, the effective local concentration of the IL2 agonist may not be related to the plasma concentration. Those skilled in the art will be able to optimize the therapeutically effective local dose without excessive experimentation.

[0272] The therapeutically effective doses of IL2 agonists described herein generally provide therapeutic benefits without causing substantial toxicity. The toxicity and therapeutic effects of IL2 agonists can be determined by standard pharmaceutical procedures in cell culture or experimental animals (see, for example, Examples 7 and 8). Using cell culture assays and animal experiments, LD 50 (A lethal dose in 50% of the population) and ED 50 (The therapeutically effective dose for 50% of the population) can be determined. The dose-to-toxicity ratio is the therapeutic index, or LD. 50 / ED 50 It can be expressed as a ratio. IL2 agonists exhibiting a large therapeutic index are preferred. In one embodiment, the IL2 agonist according to this disclosure exhibits a high therapeutic index. Data obtained from cell culture assays and animal experiments can be used to formulate a range of dosages suitable for human use. The dosages are ED with little or no toxicity. 50 It is preferable that the circulating concentration be within the range including [specific component]. The dosage may vary within this range depending on various factors, such as the dosage form used, the route of administration utilized, and the patient's condition. The exact formulation, route of administration, and dosage can be selected by the individual physician, taking into account the patient's condition. (See, for example, Fingl et al., 1975, In: The Pharmacological Basis of Therapeutics, Ch.1, p.1, which is incorporated herein by reference in its entirety).

[0273] The attending physician of a patient treated with the IL-2 agonist described herein will know how and when to discontinue, interrupt, or adjust the administration in the event of toxicity, organ dysfunction, etc. Conversely, the attending physician will also know how to adjust the treatment to a higher level if the clinical response is inadequate (to eliminate toxicity). The scale of the dose in managing the disorder of interest will vary depending on the severity of the condition being treated, the route of administration, etc. The severity of the condition may be assessed in part by, for example, standard prognostic assessment methods. Furthermore, the dose and possibly the dose frequency will also vary depending on the age, weight, and response of the individual patient.

[0274] Due to their lower toxicity, the IL2 agonists of this disclosure may have a higher maximum therapeutic dose than wild-type IL2; however, IL2 agonists containing a stabilized portion are typically administered at lower doses than wild-type IL2 due to their longer half-life.

[0275] 6.11. Combination Therapy The IL2 agonists according to this disclosure may be administered in combination with one or more other agents in therapy. For example, the IL2 agonists according to this disclosure may be administered concurrently with at least one additional therapeutic agent. The term “therapeutic agent” encompasses any agent administered to a subject in need of such treatment to treat a symptom or disease of interest. Such additional therapeutic agents may include any active ingredients suitable for the specific indication being treated, preferably those having complementary activities that do not adversely affect each other. In certain embodiments, the additional therapeutic agent is an immunomodulator, a cell proliferation inhibitor, a cell adhesion inhibitor, a cytotoxic agent, an activator of cell apoptosis, or an agent that increases the sensitivity of cells to apoptosis-inducing substances. In certain embodiments, the additional therapeutic agent is an anticancer agent, e.g., a microtubule disruptor, an antimetabolite, a topoisomerase inhibitor, a DNA insertor, an alkylating agent, a hormone therapy, a kinase inhibitor, a receptor antagonist, an activator of tumor cell apoptosis, or an anti-angiogenic agent.

[0276] Such other agents are preferably present in combination in amounts effective for the intended purpose. The effective amount of such other agents depends on the amount of IL2 agonist used, the type of disorder or treatment, and the other factors mentioned above. IL2 agonists are generally used in the same doses and routes of administration described herein, or in about 1–99% of the doses described herein, or in any dose and route that is determined to be empirically / clinically appropriate.

[0277] Such combination therapies described above include combination administration (where two or more therapeutic agents are contained in the same or separate compositions) and separate administrations, in which case the administration of the IL2 agonist of this disclosure may occur before, concurrently with, and / or after the administration of additional therapeutic agents and / or adjuvants. The IL2 agonist of this disclosure may also be used in combination with radiotherapy.

[0278] 6.11.1. Combination therapy using IL-2 agonist therapy and immunotherapy The IL2 agonists of this disclosure can be advantageously used in combination with chimeric antigen receptor ("CAR") expressing cells, such as CAR-expressing T ("CAR-T") cells, for example, CAR-T in the treatment of cancer or autoimmune diseases. In some embodiments, CAR-T cells are recognized by the targeting moiety of the IL2 agonist. The targeting moiety can recognize T cell receptors or other cell surface molecules on CAR-T cells. In some embodiments, the targeting moiety of the IL2 agonist can bind to the extracellular domain of the CAR, for example, the antigen-binding domain.

[0279] Pre-treatment or lymphocyte depletion therapy, such as regimens of cyclophosphamide and fludarabine, can also be administered to subjects receiving CAR and IL2 agonist therapy. Such therapies are typically administered a few days before the administration of CAR-expressing cells to the subject. For example, cyclophosphamide can be administered two days before infusion of CAR-expressing cells, e.g., on days -8 and -7 (infusion day is zero), and fludarabine can be administered to the subject for five consecutive days from day -6 to day -2. In one embodiment, 60 mg / kg of cyclophosphamide is administered to the subject. In another embodiment, 25 mg / m² 2 Fludarabine is administered to the subject. In one embodiment, there is a no-treatment day -1 day immediately preceding the injection of CAR-expressing cells into the subject.

[0280] CAR-expressing cells are 10 4 ~10 9 Cells / kg body weight, preferably 10 5~10 6 The T cell composition can be administered in amounts within the range of cells / kg body weight (including all integer values ​​within that range). Multiple doses of these T cell compositions may also be administered. In some embodiments, CAR-expressing cells are 1 × 10⁶ 6 ~1 × 10 11 Cell or 1 × 10 7 ~1 × 10 8 It is administered in cellular doses.

[0281] CAR-expressing cells can be activated with anti-CD3 and / or anti-CD28 antibodies in conjunction with IL2 expansion before administration to human subjects. CAR-expressing cells, such as T cells, are preferably self-referential to the target, but may also be of allogeneic origin.

[0282] In one embodiment, the IL2 agonist is administered to human subjects by bolus injection for four consecutive days starting from the day of administration of the population of CAR-expressing cells. In one embodiment, the IL2 agonist is administered to human subjects by bolus injection for at least 5 consecutive days starting from the day of administration of the population of CAR-expressing cells.

[0283] IL2 agonists can be administered for longer periods, such as one week, two weeks, one month, or longer. The frequency of administration can be reduced, for example, after the depletion of CAR-expressing cells. For example, IL2 agonists can be initially administered daily, and then the frequency of administration can be reduced to weekly.

[0284] IL2 therapy can be initiated on the same day as CAR-expressing cell administration, or 1, 2, 3, 4, 5, 6 days, or 1 week later. In one embodiment, the cell population includes T cells obtained from subjects engineered to recombinantly express CARs.

[0285] In one embodiment, IL2 agonist plasma levels are maintained for 1-2 weeks after administering a population of cells to the subject. In one embodiment, IL2 agonist plasma levels are maintained for one month after administering a population of cells to the subject.

[0286] 6.11.1.1. CAR components A typical CAR includes an antigen-binding domain, e.g., the antigen-binding domain of an antibody, and an extracellular domain linked to an intracellular signaling block (e.g., the CD3ζ signaling region of a T cell receptor) that includes a CD3 signaling domain that induces T cell activation after antigen binding. The antigen-binding domain may be in the form of an scFv, as described in Section 6.4.2.1.

[0287] The antigen-binding domain is typically linked to the signaling domain via a linker (e.g., a linker as described in Section 6.7), an optional spacer (e.g., as described in Section 6.11.1.1.1), an optional hinge (e.g., as described in Section 6.11.1.1.2), a transmembrane domain (e.g., as described in Section 6.11.1.1.3), and an intracellular signaling block (e.g., as described in Section 6.11.1.1.4).

[0288] 6.11.1.1.1. Spacer Domain In certain embodiments, the antigen-binding domain of the CAR (followed by an optional linker) is followed by one or more “spacer domains,” which refer to regions that detach the antigen-binding domain from the effector cell surface, enabling appropriate cell / cell contact, antigen binding, and activation (Patel et al., 1999, Gene Therapy 6:412-419). The spacer domains may originate from any source, whether natural, synthetic, semi-synthetic, or recombinant. In certain embodiments, the spacer domains include, but are not limited to, parts of the immunoglobulin, one or more heavy chain constant regions, e.g., CH2 and CH3. The spacer domains may include amino acid sequences of naturally occurring or modified immunoglobulin hinge regions.

[0289] In one embodiment, the spacer domain includes the CH2 and CH3 domains of IgG1 or IgG4. 6.11.1.1.2. Hinged Domain The antigen-binding domain of a CAR is generally followed by one or more "hinge domains" (optionally downstream of a linker and / or spacer), which play a role in positioning the antigen-binding domain away from the effector cell surface, enabling proper cell / cell contact, antigen binding, and activation. CARs generally contain one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domains may originate from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domains may contain amino acid sequences from naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge regions.

[0290] "Modified hinge region" means (a) a naturally occurring hinge region with up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region having a length of at least 10 amino acids (e.g., at least 12, 13, 14, or 15 amino acids) and having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), or (c) a portion of a naturally occurring hinge region containing a core hinge region (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length, or at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in length). In certain embodiments, one or more cysteine ​​residues in the naturally occurring immunoglobulin hinge region may be replaced by one or more other amino acid residues (e.g., one or more serine residues). The modified immunoglobulin hinge region may have, alternatively or additionally, proline residues in the wild-type immunoglobulin hinge region that have been replaced by other amino acid residues (e.g., serine residues).

[0291] Other exemplary hinge domains suitable for use in CARs described herein include hinge regions derived from the extracellular regions of type 1 membrane proteins such as CD8α, CD4, CD28, and CD7, which may or may not have their wild-type hinge regions modified. In another embodiment, the hinge domain includes the CD8α hinge region.

[0292] 6.11.1.1.3. Transmembrane (TM) domain The “transmembrane domain” is the portion of the CAR that fuses the extracellular binding portion with the intracellular signaling domain, thereby fixing the CAR to the plasma membrane of an immunoeffector cell. As used herein, the term “transmembrane domain” refers to any polypeptide structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane (e.g., a mammalian cell membrane).

[0293] The TM domain may originate from any of the following sources: natural, synthetic, semi-synthetic, or recombinant. The TM domain may originate from the T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, or the alpha, beta, or zeta chain of PD1 (e.g., including at least its transmembrane region). In certain embodiments, the TM domain is synthetic and mainly contains hydrophobic residues such as leucine and valine.

[0294] In certain embodiments, the CAR includes a CD3ζ transmembrane domain (e.g., a transmembrane domain containing the amino acid sequence LCYLLDGILFIYGVILTALFL (SEQ ID NO: 86) or LDPKLCYLLDGILFIYGVILTALFLRVK (SEQ ID NO: 87)), a CD28 transmembrane domain (e.g., a transmembrane domain containing the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 88)), or a CD8α transmembrane domain (e.g., a transmembrane domain containing the amino acid sequence KPTTTPAPRPPTPAPTIASQPLSLR PEACRPAAGGAVHTRGLDFA (SEQ ID NO: 89)).

[0295] The TM domain may be followed by a short linker, preferably of a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, which connects the TM domain to the intracellular signaling domain of the CAR. Glycine-serine-based linkers (e.g., linkers according to Section 6.7) provide particularly suitable linkers.

[0296] 6.11.1.1.4. Intracellular signal transduction domains CARs typically contain an intracellular signaling domain. The “intracellular signaling domain” refers to the portion of the CAR that is involved in transmitting effective antigen-binding messages into the interior of immune effector cells to induce effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity (including the release of cytotoxic factors to target cells bound to the CAR, or other cellular responses induced by antigen binding to the extracellular CAR domain).

[0297] The term "effector function" refers to the specific function of an immune effector cell. The effector function of a T cell may be, for example, cytolytic activity, or activity that assists or includes cytokine secretion. Therefore, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector function signals and directs the cell to perform a specific function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire domain. To the extent that a shortened portion of an intracellular signaling domain is used, such a shortened portion can be used in place of the entire domain, as long as it transmits effector function signals. The term "intracellular signaling domain" means that it includes any shortened portion of an intracellular signaling domain sufficient to transmit effector function signals.

[0298] It is known that signals generated solely through the TCR are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, it can be said that T cell activation is mediated by two distinct classes of intracellular signaling domains: a primary signaling domain (e.g., the TCR / CD3 complex) that initiates antigen-dependent primary activation via the TCR, and a co-stimulatory signaling domain that acts in an antigen-independent manner to provide secondary or co-stimulatory signals. In preferred embodiments, the CAR contemplated herein comprises an intracellular signaling domain comprising one or more “co-stimulatory signaling domains” and “primary signaling domains.”

[0299] Primary signaling domains modulate the primary activation of the TCR complex either stimulatingly or inhibitorily. Primary signaling domains that act stimulatingly may include immunoreceptor tyrosine-based activation motifs or signaling motifs known as ITAMs.

[0300] Exemplary examples of ITAMs containing primary signaling domains particularly useful in the methods of this disclosure include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In a particularly preferred embodiment, the CAR comprises a CD3ζ primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling and co-stimulatory signaling domains may be tandem-linked to the carboxyl terminus of the transmembrane domain in any order.

[0301] The CARs contemplated herein include one or more costimulatory signaling domains for enhancing the efficacy and expansion of T cells expressing CAR receptors. As used herein, the terms “costimulatory signaling domain” or “costimulatory domain” refer to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that, upon binding to an antigen, provides a second signal necessary for the efficient activation and function of T lymphocytes. Exemplary examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70.

[0302] In some embodiments, the CD3 signaling region is linked to a co-stimulatory endodomain of CD28, 4-1BB (also known as CD137), CD70, or OX40 (also known as CD134), or a combination thereof, or has two signaling domains of CD3ζ in tandem. These endodomains enable potent T cell activation during TCR recognition by antigen-presenting cells (APCs), improving cytokine production and proliferation of CAR-T cells.

[0303] In another embodiment, the CAR includes CD28 and CD137 co-stimulatory signaling domains and a CD3ζ primary signaling domain. In yet another embodiment, the CAR includes CD28 and CD134 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0304] In one embodiment, the CAR includes CD137 and CD134 co-stimulatory signaling domains and a CD3ζ primary signaling domain. An exemplary CD3ζ signaling region may include one of the following amino acid sequences: LDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELN LGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(Sequence ID 90). RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN PQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPR (Sequence ID 91) An exemplary CD28 signaling region may include one of the following amino acid sequences: KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGV LACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPP RDFAAYRS (Sequence ID 92).

[0305] RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP PRDFAAYRS (Sequence ID 93) KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP(Sequence ID 94) FWVLVVVGGVLACYSLLVTVAFIIFWV (Sequence ID 88) RSKRSRLLHSDY MNMTPRRPGPTRKHYQPYAPPRDFAAYRS (Sequence ID 93).

[0306] An exemplary CD137(41BB) signaling region may include the following amino acid sequence: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (Sequence ID 95).

[0307] 6.11.1.1.5. Tags In some embodiments, the CAR includes a tag used to identify the CAR, for example, the V5 epitope tag is derived from a small epitope (Pk) present on the P and V proteins of the paramyxovirus 5 (SV5). The V5 tag is typically used with all 14 amino acids (GKPIPNPLLGLDST) (SEQ ID NO: 96), but it is also used with a shorter 9-amino acid sequence (IPNPLLGLD) (SEQ ID NO: 97).

[0308] 6.11.1.1.6. Signal Peptides In some embodiments, the CAR includes a signal peptide. The signal peptide facilitates the expression of the CAR on the cell surface. Signal peptides, including naturally occurring protein signal peptides or synthetically produced unnatural signal peptides, that are suitable for use with the CARs described herein will be apparent to those skilled in the art. In some embodiments, the signal peptide is positioned at the N-terminus of the antigen-binding moiety of the CAR. During the expression and processing of the CAR in cells, such as T cells, the signal peptide is cleaved and therefore typically not present in the mature molecule.

[0309] 6.11.1.2. Preparation of CART cells To generate CART cells ex vivo, PBMCs, peripheral blood lymphocytes, or T cells enriched therefrom can be enlarged before and / or after introducing CAR-encoding nucleic acids into the cells, for example, by viral transduction.

[0310] T cells useful for CART cell generation can be isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation using a PERCOLL® gradient or countercurrent centrifugation. CD3+, CD28 +Specific subpopulations of T cells, such as CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3x28) conjugate beads, e.g., DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient to positively select the desired T cells. In one embodiment, the period is approximately 30 minutes. In further embodiments, the period is 30 minutes to 36 hours or longer, within the range of any integer value in between. In further embodiments, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours. In one preferred embodiment, the incubation period is 24 hours. When isolating T cells from patients with leukemia, using longer incubation times, such as 24 hours, can increase the cell yield. In any situation where T cells are scarce compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from an immunocompromised individual, longer incubation times can be used to isolate T cells. Furthermore, longer incubation times can increase the capture efficiency of CD8+ T cells. Thus, subpopulations of T cells can be preferentially selected at the start of culture or at any other point in the process, or against them, simply by shortening or lengthening the time that allows T cells to bind to CD3 / CD28 beads, and / or by increasing or decreasing the ratio of beads to T cells. In addition, subpopulations of T cells can be preferentially selected at the start of culture or at any other desired point in time, or against them, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces. Those skilled in the art will also recognize that multiple rounds of selection can be used in the context of this disclosure. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in the activation and expansion processes.Cells that are "not selected" can also undergo further rounds of selection.

[0311] Enrichment of T cell populations by negative selection can be achieved by a combination of antibodies directed to specific surface markers on negatively selected cells. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. T reg cells can also be depleted by anti-C25 conjugate beads or other similar selection methods.

[0312] In certain embodiments, for example, in applications involving the treatment of autoimmune diseases as described in Section 6.11.1.4, typically CD4+, CD25+, CD62L hi GITR + , and FoxP3 + It may be desirable to enrich or positively select regulatory T cells that express FoxP3. T reg can also be induced by recombinant expression of FoxP3.

[0313] Before or after gene modification of T cells to express a desired CAR, T cells can be activated and expanded using methods known in the art, such as those described in U.S. Patents No. 7,144,575, 7,067,318, 7,172,869, 7,232,566, or 7,175,843.

[0314] Generally, T cells useful in the methods of this disclosure are expanded by contacting a surface to which a drug that stimulates CD3 / TCR complex-related signaling and a ligand that stimulates co-stimulatory molecules on the surface of the T cells are attached. In particular, a T cell population may be stimulated by contact with an anti-CD3 antibody, or its antigen-binding fragment, or an anti-CD2 antibody immobilized on the surface, or by contact with a protein kinase C activator (e.g., bryostatin) in combination with a calcium ionophore. For co-stimulation of accessory molecules on the surface of T cells, ligands that bind to the accessory molecules are used. For example, a T cell population may be contacted with an anti-CD3 antibody and optionally an anti-CD28 antibody, for example, on anti-CD3 and anti-CD28 beads, under conditions suitable for stimulating T cell proliferation.

[0315] CAR-expressing cells can also be conditioned with IL12 before administration to human subjects (e.g., Emtage et al., 2003, J.Immunother. 16(2):97-106, incorporated herein by reference).

[0316] 6.11.1.3. Cancer Immunotherapy Accordingly, the present disclosure provides a method for doing so in a human subject requiring cancer treatment, comprising administering an effective amount of the present disclosure of an IL2 agonist to a subject and to CAR-expressing cells, e.g., CAR-expressing T cells (or "CART cells"). T cell subtypes particularly useful for cancer treatment are T cells with potent CAR-mediated cytotoxicity, e.g., CD3+CD8+ T cells, which can be prepared as described in 6.11.1.2 above.

[0317] For the treatment of cancer, the extracellular domain of CARs can target tumor-associated antigens, for example, as described in Section 6.4.1. In certain embodiments, tumor-associated antigens are CD20, EGFR, FITC, CD19, CD22, CD33, PSMA, GD2, EGFR variants, ROR1, c-Met, HER2, CEA, mesoserine, GM2, CD7, CD10, CD30, CD34, CD38, CD41, CD44, CD74, CD123, CD133, CD171, MUC16, MUC1, CS1 (CD319), IL-13Ra2, BCMA, Lewis Y, IgG kappa chain, folate receptor-alpha, PSCA, or EpCAM. In certain embodiments, CARs are designed to target CD22 to treat diffuse large B-cell lymphoma.

[0318] CARs are designed to target mesoserine to treat mesothelioma, pancreatic cancer, ovarian cancer, and other cancers. CARs are designed to target CD33 / IL3Ra to treat conditions such as acute myeloid leukemia.

[0319] CARs are designed to target c-Met to treat triple-negative breast cancer, non-small cell lung cancer, and other cancers. CARs are designed to target PSMA to treat conditions such as prostate cancer.

[0320] CARs are designed to target the glycolipid F77 to treat prostate cancer and other conditions. CARs are designed to target EGFRvIII to treat conditions such as glioblastoma.

[0321] CARs are designed to target GD-2 to treat neuroblastoma, melanoma, and other conditions. CARs are designed to target the NY-ESO-1 TCR to treat myeloma, sarcoma, melanoma, and other conditions.

[0322] CARs are designed to target the MAGE A3 TCR to treat myeloma, sarcoma, melanoma, and other conditions. Particularly useful in CAR-IL2 agonist combination therapy are IL2 agonists that contain a targeting moiety that recognizes cell surface antigens present on the surface of CAR-expressing lymphocytes.

[0323] When an IL2 agonist administered in combination with CAR therapy has a peptide-MHC targeting moiety, CAR-expressing cells preferably have a CD4 that recognizes the peptide-MHC complex in the TCR. + or CD8 + These are cells. For example, CAR-expressing cells are T lymphocytes, and their TCRs specifically bind to pMHC complexes present on IL2 agonists, leading to the functional activation and survival of CAR-expressing cells.

[0324] In some embodiments, the CAR itself includes an antigen-binding domain (e.g., an scFv domain) that targets a pMHC complex in an IL2 agonist, e.g., any pMHC fusion described in Section 6.4.3. An exemplary CAR that targets a pMHC complex having an HPV peptide (e.g., a peptide corresponding to amino acid residues 11-19 or 82-90 of the HPV16E7 polypeptide) is disclosed in U.S. Patent No. 10,806,780B2, which is incorporated herein by reference in its entirety. In some embodiments, CAR is used in the case of U.S. Patent No. 10,806,780B2, with Sequence IDs 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 202, 218 / 226, 234 / 242, 250 / 258, 266 / 274, 282 / 290, 298 / 306, 3 This comprises heavy chain variable domain (VH) and light chain variable domain (VL) amino acid sequence pairs selected from any one of 14 / 322, 330 / 338, 346 / 354, 362 / 370, 378 / 386, 394 / 402, 410 / 418, 426 / 434, 442 / 450, 458 / 466, 474 / 482, 490 / 498, 506 / 514, and 522 / 530, each of which is incorporated herein by reference. In certain embodiments, the VH-VL pair includes SEQ ID NOs. 2 and 10 of U.S. Patent No. 10,806,780B2, SEQ ID NOs. 34 and 42 of U.S. Patent No. 10,806,780B2, SEQ ID NOs. 82 and 90 of U.S. Patent No. 10,806,780B2, SEQ ID NOs. 194 and 202 of U.S. Patent No. 10,806,780B2, or SEQ ID NOs. 506 and 514 of U.S. Patent No. 10,806,780B2, each of which is incorporated herein by reference. CAR-IL2 agonist combination therapy with CARs targeting pMHCs having HPV peptides can be used to treat HPV-positive cancers, such as squamous cell carcinomas, such as cervical cancer, head and neck small cell carcinoma, anogenital cancer, and oropharyngeal cancer.

[0325] 6.11.1.4. Immunotherapy for Autoimmune Diseases Chimeric antigen receptor (CAR) T cells have become a powerful treatment option for hematological malignancies. Using the same idea of ​​modifying T cells to efficiently target disease requirements, scientists have efficiently engineered T cells with predetermined antigen specificity via transfection with viral vectors encoding chimeric antigen receptors (CARs). CAR-modified T cells engineered in a way that is not MHC-restricted have the advantage of having a wide range of applications, particularly in transplantation and autoimmunity.

[0326] CAR-expressing Treg cells can be prepared as described in 6.11.1.2 above. Particularly useful in CAR-IL2 agonist combination therapy are IL2 agonists that contain a targeting moiety that recognizes cell surface antigens present on the surface of pMHC cloned from CAR-expressing lymphocytes, such as autoimmune target cells.

[0327] For use in the treatment of autoimmune diseases, the extracellular domain of CARs is preferably specific to a target antigen or ligand associated with the autoimmune response. Such modification activates Tregs redirected at the site of inflammation, thereby suppressing the pro-inflammatory effector immune response. reg Examples of autoimmune diseases that can be targeted by this therapy include multiple sclerosis, inflammatory bowel disease (IBD), rheumatoid arthritis, systemic lupus erythematosus, Crohn's disease, psoriasis, type 1 diabetes, Sjögren's disease, myasthenia gravis (MG), Hashimoto's thyroiditis, Graves' disease, and uveitis.

[0328] In certain embodiments, Tregs are engineered to express CARs that target antigens or ligands specific to the following: Inflammatory bowel disease (IBD), the antigen or ligand is expressed in the affected colon or ileum. 〇 Rheumatoid arthritis, the antigen or ligand is an antigen present in collagen epitopes or joints. ○Type 1 diabetes mellitus or autoimmune islet inflammatory disease, where the antigen or ligand is a pancreatic β-cell antigen. In multiple sclerosis, the antigen or ligand is, for example, myelin basic protein (MBP) antigen or MOG-1 or MOG2-2, or a neuronal antigen. 〇 Autoimmune thyroiditis, where the antigen or ligand is a thyroid antigen. 〇 Autoimmune gastritis, where the antigen or ligand is a gastric antigen. 〇 Autoimmune uveitis or uveoretinitis, where the antigen or ligand is the S antigen or another uveal or retinal antigen. Autoimmune orchitis, where the antigen or ligand is a testicular antigen. 〇 Autoimmune folliculitis, where the antigen or ligand is an ovarian antigen. 〇 Psoriasis, where the antigen or ligand is a keratinocyte antigen or another antigen present in the dermis or epidermis. 〇 Vitiligo, where the antigen or ligand is a melanocyte antigen such as melanin or tyrosinase. 〇 Autoimmune prostatitis, where the antigen or ligand is a prostate antigen. ○ Any unwanted immune response, antigen, or ligand is an activating antigen, or another antigen expressed on T effector cells present at the site of the unwanted response. ○Tissue rejection, the antigen or ligand is an MHC specific to the transplanted tissue, and 〇Inflammation is a condition in which an antigen or ligand is expressed on non-lymphoid cells of the hematopoietic lineage involved in inflammation.

[0329] In one embodiment, T cells may be engineered to express chimeric autoantigen receptor (CAAR) T cells in order to specifically eliminate B cells that cause autoimmune diseases. Therefore, references to CAR-expressing T cells include references to CAAR expression unless the context otherwise indicates. [Examples]

[0330] 7.1. Materials and Methods 7.1.1. Production of IL2 and IL15 agonists Constructs encoding IL2 and IL15 mutaine (specified as IL2M_ or IL15M_ as needed), as well as Fc controls, were generated with or without the presence of the targeted moieties (specified as T) described in Tables 5A and 5B below (or containing the modules described therein), respectively. Constructs were expressed in Expi293F® cells by transient transfection (Thermo Fisher Scientific). Proteins from the Expi293F supernatant were purified using a ProteinMaker system (Protein BioSolutions, Gaithersburg, MD) equipped with a HiTrap Protein G HP column (GE Healthcare). After single-step elution, mutaine was neutralized, dialyzed to final buffer of phosphate-buffered saline (PBS) containing 5% glycerol, aliquoted, and stored at -80°C.

[0331] [Table 6-1]

[0332] [Table 6-2]

[0333] [Table 6-3]

[0334] [Table 6-4]

[0335] [Table 6-5]

[0336] [Table 6-6]

[0337] [Table 6-7]

[0338] [Table 6-8]

[0339] [Table 7-1]

[0340] [Table 7-2]

[0341] [Table 7-3]

[0342] [Table 7-4]

[0343] [Table 7-5]

[0344] 7.1.2. Intracellular staining of pSTAT5 in human PBMCs Frozen human PBMCs were thawed and incubated overnight in IL-2-free medium. The following day, the cells were treated with serial dilutions of different IL-2 variants and fixed with BD Cytofix® fixation buffer at 37°C for 12 minutes. The cells were then permeabilized with pre-cooled BD Phosflow® Perm Buffer III on ice for 20 minutes. The cells were then washed twice with FACS buffer (PBS + 2% FBS) and incubated at room temperature in the dark for 45 minutes with a staining cocktail containing Alexa Fluor® 647 conjugate anti-STAT5 pY694 (BD Biosciences). To identify different lymphocyte populations within PBMCs, the following antibody panels were also included in the staining cocktail: BUV496-anti-CD8, BUV395-anti-CD4, BV421-anti-NKp46, BV711-anti-CD56, BV786-anti-CD3, Alexa Fluor® 488-anti-FoxP3, and PE-anti-CD25 (BD Biosciences). Cells were washed twice with FACS buffer before data acquisition using a BD LSRFortessa® X-20 flow cytometer. Raw data were analyzed using FlowJo v10.

[0345] 7.1.3. Size exclusion chromatography and multi-angle light scattering The oligomeric states of different muteins were evaluated using a combination of size exclusion ultrahigh performance liquid chromatography (SEC) and multi-angle light scattering (MALS). SEC analysis was performed using a Waters Acquity UPLC H-Class system. 10 μg of each protein sample was injected into a two-column tandem setup consisting of Acquity BEH SEC columns (200 Å, 1.7 μm, 4.6 x 150 mm). The flow rate was set to 0.3 ml / min. The mobile phase buffer contained 10 mM sodium phosphate, 500 mM NaCl, and pH 7.0. Changes in UV absorbance, light scattering, and refractive index at 280 nm were monitored using Wyatt Optilab T-Rex and Wyatt-uDawn Treos LS detectors.

[0346] 7.1.4. FACS-binding assay To analyze the binding of antibody-IL-2 mutein to target antigens on the cell surface, HEK293 cells stably expressing the antigen target were collected and resuspended in FACS buffer (PBS + 2% FBS). For each binding assay, 50,000–100,000 cells were incubated with serial dilutions of IL-2 mutein in FACS buffer at 4°C for 30 minutes. The cells were washed twice with FACS buffer and incubated with a 1:500 dilution of APC-F(ab)'2 anti-mouse IgG Fcγ fragment (Jackson ImmunoResearch Laboratories) at 4°C for 30 minutes. At the end of incubation, the cells were washed twice with FACS buffer and analyzed using a BD FACSCelesta® flow cytometer.

[0347] 7.1.5. Inoculation, treatment, and measurement of tumors Total 3 x 10 5 MC38 cells were subcutaneously injected into the flank of 6-8 week old female C57BL / 6J mice (Jackson Laboratory). Tumor size was 80-100 mm. 3 Mice were randomized when they reached a certain stage and treated intraperitoneally with different IL-2 / IL-15 agonists. Treatment was repeated every other day or twice a week for a further four times. Tumors were measured twice a week using a digital caliper, and tumor size was measured as length x width. 2 Calculated as / 2. In tumor studies where survival was recorded, loss of survival was defined as death, or when the tumor was 20 mm in any dimension or 2250 mm in total volume. 3 It was defined as when it reached that point.

[0348] 7.1.6. FACS analysis of tumors, spleen, and blood Tumor cells were harvested and treated with a mild MACS® dissociative agent (Miltenyi Biotec.) to produce single-cell suspensions. Cells were counted and stained with a cocktail of fluorescently labeled antibodies diluted in BD Horizon Brilliant Buffer.

[0349] After treatment with IL2 mutaine, spleens and blood were collected from tumor- or non-tumor-bearing mice. The spleens were pulverized through a 70 μm Corning® cell strainer to produce single-cell suspensions. Next, the spleens and blood were treated with ACK lysis buffer (Lonza) to lyse red blood cells (RBCs). After RBC lysis, lymphocytes were counted and stained with an antibody cocktail diluted in BD Horizon Brilliant Buffer. All stained samples were analyzed using a BD LSRFortessa® X-20 flow cytometer. Raw data were processed using FlowJo v10.

[0350] 7.1.7. Antigen-specific T cells and chimeric antigen receptor (CAR) T cells Ovalbumin (257-264)-specific mouse OT-I T cells were isolated from the spleen of OT-I TCR transgenic mice (Jackson Laboratory). CMV pp65 (495-503)-specific human T cells were expanded from PBMCs of CMV+ donors and obtained from ASTARTE Biologics (catalog no. 1049). CAR T cells were generated by stimulating CD3+ T cells with CD3 / CD28 microbeads and 100 U / ml recombinant human IL2 before transduction with lentivirus at MOI=5. Transduced cells were then expanded for 17 days with CD3 / CD28 microbeads and 100 U / ml recombinant human IL2, after which the medium was replaced with IL2-deficient CTS-supplemented OpTmizer® medium (Gibco). After overnight incubation at 37°C and 5% CO2, the pSTAT5 assay was performed as described in Section 7.1.2.

[0351] 7.2. Example 1: In vitro activity of IL-2Rα-attenuated IL2 mutein To test the ability of IL2 M1 to induce IL2 receptor signaling in various immune cell populations, 4 × 10⁶ 5Human PBMCs were stimulated at 37°C for 20 minutes with increasing concentrations of IL2M0 or IL2M1. As an indicator of IL2 receptor-mediated signaling, intracellular STAT5 phosphorylation levels were measured by flow cytometry in immune cell subsets as described in Section 7.1.2. Figures 4A–4C show STAT5 activity in gated Tregs (Figure 4A), CD8+ T cells (Figure 4B), and NK cells (Figure 4C). Compared to IL2M0, IL2M1 maintains comparable activity against CD8+ T cells and NK cells lacking detectable IL-2Rα expression. However, its activity against IL-2Rα+ Tregs is several orders of magnitude lower than that of IL2-Fc. Thus, IL2M1 loses preferential activity against Tregs in vitro compared to other effector cell populations.

[0352] 7.3. Example 2: Activity of IL-2Rα-attenuated IL-2 mutein against immune cell populations in vivo C57BL / 6J mice received intraperitoneal injections of PBS, 15 μg of IL2M0, or IL2M1 for six consecutive days. One day after the last injection, the spleen was harvested for flow cytometry analysis. The number of Treg, NK cells, and CD8+ T cells in the spleen of the treated mice are shown in Figures 5A-5C, respectively. The relative frequencies of CD8+ and CD4+ T cells within TCRβ+ T cells were quantified (Figure 5D).

[0353] IL2M0 (IL2-Fc) preferentially expands Treg cells in vivo, but this Treg selectivity was lost with IL2M1 (CD122-biased human IL2-Fc fusion). In contrast, IL2M1 induces specific expansion of NK and CD8+ T cells while minimizing its impact on the Treg population. Therefore, IL2M1 can reorganize peripheral lymphocyte compartments by selectively expanding effector cell populations.

[0354] 7.4. Example 3: The antitumor activity of IL2 and IL-2Rα attenuated IL2 mutein as monotherapy and in combination with anti-PD1. C57BL / 6J mouse with 3x10 5 MC38 tumor cells were subcutaneously inoculated on day 0, and the average tumor size was 100 mm. 3 Mice were randomized on day 8 when they reached [a certain stage]. Next, mice were treated intraperitoneally with a total of five injections of isotype (10 mg / kg), IL2M0 (0.75 mg / kg) + isotype (10 mg / kg), IL2M1 (0.75 mg / kg) + isotype (10 mg / kg), anti-mPD1 (10 mg / kg), IL2M0 (0.75 mg / kg) + anti-mPD1 (10 mg / kg), or IL2M1 (0.75 mg / kg) + anti-mPD1 (10 mg / kg), either every other day (for IL2M0 and IL2M1) or twice a week (for isotype and anti-mPD1). Figure 6 shows the mean tumor volume (mm²) in each treatment group. 3 Figure 6A shows the SD (+SD) and Kaplan-Meier survival curves (Figure 6B). Loss of survival is defined as death, or when the tumor reaches 20 mm in any dimension or 2250 mm in total volume. 3 It was defined as when it reached that point.

[0355] As monotherapy, IL2M0 (IL2-Fc) significantly inhibits the progression of established tumors. When combined with anti-PD1, it further synergistically promotes tumor regression and sustained tumor-free survival. Surprisingly, despite its ability to specifically enlarge NK and CD8+ T cells, CD122-biased IL2M1 shows little antitumor effect, even in the presence of anti-PD1.

[0356] 7.5. Example 4: Study on the administration of IL2M0 C57BL / 6J mouse with 3x10 5 MC38 tumor cells were subcutaneously inoculated on day 0, and the average tumor size was 90 mm. 3 Randomization occurred on day 8 when the mice reached the specified dose. Next, the mice were treated intraperitoneally with IL2M0 at the indicated dose, administered by injection every other day for a total of five times. Figure 7 shows the mean tumor volume (mm²). 3Figure 7A shows the SD (+SD), Kaplan-Meier survival curve (Figure 7B), and individual tumor growth curves (7C.1-7C.5). Loss of survival is defined as death, or when the tumor reaches 20 mm in any dimension or 2250 mm in total volume. 3 It was defined as when it reached that point.

[0357] This study demonstrates that the antitumor effect of IL2M0 is highly dependent on the injected dose, with the highest dose being the most effective. Starting with 5 μg / dose and observing the effect, increasing the dose to 10 μg and 20 μg / dose resulted in better tumor control and survival benefits.

[0358] 7.6. Example 5: Antitumor activity and toxicity of IL2M1 compared to IL2M0 Two different studies used 3 × 10⁶ C57BL / 6J mice. 5 MC38 tumor cells were subcutaneously inoculated on day 0, and the average tumor size was 90 mm. 3 On day 8 (Study #1), the average tumor size reached 70 mm. 3 On day 7 (Study #2), mice were randomized. Next, mice were treated intraperitoneally with either IL2M0 or IL2 M1 at the indicated doses, administered by injection every other day for a total of 5 times. Mean tumor volume (mm) in each treatment group. 3 Figures 8A and 8B show the results for Study #1 and Study #2, respectively, and the percentage of weight change (Figure 8C, from Study #2). Arrows indicate the treatment day.

[0359] IL2M1 remained ineffective in controlling tumor progression up to 40 μg / dose, while IL2M0 at 10 μg / dose resulted in a significant delay in tumor growth (Figure 8A). Moderate effects were observed when IL2M1 was administered at 75 or 100 μg / dose (Figure 8B). However, at these doses, it caused severe toxicity, including significant weight loss (Figure 8C), decreased activity, and increased mortality. It has not yet been determined whether the effects observed in the high-dose IL2M1 group were caused by anti-tumor immunity or tumor malnutrition resulting from the host mouse disease.

[0360] Despite being less effective than 10 μg / dose IL2M0 in controlling tumor growth, 100 μg / dose IL2M1 induced a significant expansion of peripheral NK and CD8+ T cells, with less pronounced effects on Tregs than those induced by 10 μg / dose IL2M0. However, such a significant expansion of the peripheral effector cell population did not translate into a more effective antitumor response.

[0361] 7.7. Example 6: Antitumor activity and toxicity of IL15 mutein IL15 shares the same β / γ receptor subunit as IL2, but does not bind to IL2-Rα. IL15 is typically expressed on the surface of bone marrow cells as part of the IL15 / IL15Rα complex, and is subsequently presented to activate surrounding lymphocytes (Figure 1). IL15M1, containing the IL15-IL15Rα fusion, is thought to mimic the trans-presentation of IL15 by IL15Rα, specifically engaging with IL-2Rβ / γ, and thus triggering similar signaling to IL2-Rα-attenuated IL2 mutein (e.g., IL2M1).

[0362] The antitumor activity and toxicity of IL15M1 were tested. 3 × 10⁶ mice were used to administer the drug to C57BL / 6J mice. 5 MC38 tumor cells were subcutaneously inoculated on day 0, and the average tumor size was 70 mm. 3 Mice were randomized on day 7 when they reached [specific stage]. Next, mice were treated intraperitoneally with IL2M0 and IL15M1 at indicated doses, administered by injection every other day for a total of five times. Figure 9 shows the mean tumor volume (mm²) in each treatment group. 3 Figure 9A shows the +SD (spheric standard deviation) and the percentage change in body weight (Figure 9B). Arrows indicate the treatment day. On day 11, blood was collected from the indicated groups as described in Section 7.1.6 and analyzed by flow cytometry. The number of lymphocyte populations indicated was quantified (Figure 9C.1-C.4).

[0363] A moderate but dose-dependent antitumor effect was observed after IL15M1 treatment (Figure 9A). However, similar to IL2M1, IL15M1 treatment simultaneously induced dose-dependent toxicity in tumor-bearing mice (Figure 9B). IL15M1 also induced dramatic expansion of peripheral lymphocytes, mainly NK and CD8+ T cells, but did not induce Tregs in the blood of these tumor-bearing mice (Figures 9C.1 - C.4). Together with the example of IL2M1, these results indicate that selective stimulation of the IL-2Rβ / γ receptor by either the mutant IL2 or IL15 mutein with the loss of IL-2Rα binding results in a suboptimal therapeutic index and limited efficacy with regard to toxicity. They also suggest that the expanded peripheral NK and CD8+ T cells by IL2M1 and IL15M1 are inefficient in controlling tumor growth.

[0364] 7.8. Example 7: Activity of IL-2Rα-deleted IL2 mutein on intratumoral lymphocytes C57BL / 6J mice were subcutaneously inoculated with 3×10 5 MC38 tumor cells on day 0 and randomized on day 7 when the mean tumor size reached 100 mm 3 Next, the mice were intraperitoneally treated every other day with PBS, anti-mPD1 (10 mg / kg), IL2M0 (0.75 mg / kg) + anti-mPD1 (10 mg / kg), or IL2M1 (0.75 mg / kg) + anti-mPD1 (10 mg / kg). After a total of 3 administrations, the tumors were harvested on day 12 to isolate and analyze tumor infiltrating lymphocytes (TIL). Figures 10A.1 - 10A.5 show the density tSNE plots of total CD45 + TIL from each treatment group. The results of quantification of the percentages of clusters 1 - 4 within the total TIL highlighted in Figures 10A.1 - 10A.5 are shown in Figure 10B. The tSNE plots of total TIL with the expression of defining markers of different lymphocyte populations overlaid are shown in Figures 10C.1 - 10C.7. These results indicate that IL2M0 induces more prominent expansion of CD8+ T cells in the tumor than IL2M1.

[0365] TCRβ +T cells were further gated out from total TILs. Ki67 within CD8 + T cells within tumors after each treatment + IL-2Rα + Representative FACS plots and quantification of frequencies of cells are shown in FIGS. 10D and 10E, respectively. Ki67 in tumors + IL-2Rα + CD8 + Results of quantification of the density of T cells are shown in FIG. 10F.

[0366] FoxP3 within CD4 + T cells within tumors + IL-2Rα + Representative FACS plots and quantification of frequencies of Tregs are shown in FIGS. 10G and 10H. FoxP3 in tumors + IL-2Rα + CD4 + Results of quantification of the density of Tregs are shown in FIG. 10I. These results indicate that IL2M0 increases the proliferation of IL-2Rα+CD8+T cells against Tregs within tumors.

[0367] The expression of IL-2Rα and PD1 was evaluated in CD8+TILs. The expression of these markers was overlaid on the tSNE plot of total CD45+TILs, and the results are shown in FIGS. 10J - 10L. The expression profile shows a subpopulation of tumor-infiltrating CD8+T cells that co-express IL-2Rα and PD1. These results indicate that IL-2Rα and PD1 are co-expressed on some tumor-infiltrating CD8+T cells within tumors. Considering the dynamic expression of these two proteins on antigen-specific CD8+T cells after activation, their co-expression might have occurred in a broader population of CD8+T cells at a time point not captured here (Kalia et al., 2010, Immunity 32(1):91 - 103).

[0368] As described in Section 7.1.6, blood and spleen were collected from the same tumor-bearing mice for flow cytometry analysis. The results of quantifying the frequency of NK cells and Tregs in the spleen are shown in Figures 10M and 10N. The results of quantifying the frequency of NK cells and Tregs in the blood are shown in Figures 10P and 10Q. The results of quantifying the relative frequency of CD8+ and CD4+ T cells within TCRβ+ T cells in the spleen are shown in Figure 10O, and the results for the blood are shown in Figure 10R. These results indicate that IL2M1 is associated with NK cells and CD8+ T cells relative to Tregs. + While IL2M0 specifically enlarges T cells, it preferentially enlarges Treg cells in the spleen and blood.

[0369] The immune cell profiles induced by IL2M0 and IL2M1 differ significantly between the periphery and tumors. In the blood and spleen, IL2M0 preferentially enlarges Tregs, while IL2M1 selectively enlarges NK cells and CD8+ T cells. In tumors, IL2M0 enlarges CD8+ T cells but not Tregs, while IL2M1 only slightly increases NK cells. These observations may explain why the significant enlargement of peripheral NK and CD8+ T cells induced by IL-2Rα-attenuated IL2 mutein or IL15 variants does not translate into a favorable antitumor response, and therefore such enlargement does not extend to the tumor microenvironment. They also explain why IL2M0, which enlarges peripheral suppressive Tregs, can induce effective antitumor immunity. When activated by antigens within the tumor, tumor-specific T cells upmodulate IL-2Rα, making them more sensitive to IL2M0 treatment. In summary, these results suggest that maintaining the ability to participate in IL-2Rα is necessary for the antitumor effects of IL2-related molecules.

[0370] 7.9. Example 8: Tumor Tregs are less responsive to IL2 stimulation than splenic Tregs. As described in Section 7.1.6, tumors and spleens were collected from MC38 tumor-bearing C57BL / 6J mice to isolate lymphocytes. Digested tumor cells were further subjected to MACS® Cell Separation using CD45 MicroBeads to enrich TILs. 4 × 10 5 TIL or 2x10 6 Splenocytes were stimulated in vitro with increasing concentrations of recombinant human IL2 at 37°C for 20 minutes. Figure 11 shows the percentage of cells that underwent STAT5 phosphorylation in gated Treg (Figure 11A) and CD8+ T cells (Figure 11B), as determined by FACS according to the assay in Section 7.1.2.

[0371] Recombinant human IL-2 showed similar EC50s on tumor and spleen-derived Tregs. However, while IL-2 was able to induce STAT5 phosphorylation in almost all spleen Tregs, only a small fraction of tumor Tregs showed STAT5 phosphorylation after IL-2 stimulation. In contrast, tumor-derived CD8+ T cells responded to IL-2 treatment with lower EC50s than their spleen counterparts. These results suggest that tumor Tregs are less responsive to IL-2 stimulation than peripheral Tregs, while tumor-infiltrating CD8+ T cells may be more sensitive to IL-2 stimulation than their spleen counterparts. They also reveal potential heterogeneity within the tumor Treg compartment.

[0372] 7.10. Example 9: Activity of IL2M2 and IL2M3 on lymphocyte populations Human PBMCs were stimulated with increased concentrations of IL2M0, IL2M2, and IL2M3. Intracellular STAT5 phosphorylation levels were determined by FACS as described in Section 7.1.2, and the results for gated Tregs are shown in Figure 12A, CD8. + T cells are shown in Figure 12B, and NK cells are shown in Figure 12C.

[0373] IL2M2 and IL2M3 showed similar activity to each other in different lymphocyte populations. Compared to IL2M0, they showed decreased activity for all cell types tested. Overall, they are attenuated for all IL2 receptors while still maintaining selectivity for IL-2Rα+ cells.

[0374] C57BL / 6J mice were hydrodynamically injected with 5 μg of plasmid DNA encoding IL2M0, IL2M1, IL2M2, or IL2M3 on day 0. The proteins were continuously expressed and secreted by hepatocytes to maintain stable serum concentrations. The percentage change in body weight for each treatment group after plasmid injection is shown in Figure 12D. In contrast to IL2M0 and IL2M1, injection of IL2M2 and ILM3 did not result in any detectable weight loss, suggesting a significant reduction in toxicity.

[0375] 7.11. Example 10: Antitumor activity of IL2M2 monotherapy in a syngeneic mouse model Balbc / J mice were subcutaneously inoculated with Colon 26 or 4T1 tumor cells on day 0, and the average tumor size was 80 mm. 3 Randomization occurred on day 10 or 11, when the mice reached [specific stage]. Immediately after randomization, mice were hydrodynamically injected with the indicated dose of plasmid DNA encoding human Fc or IL2M2 under the human ubiquitin C promoter. The protein was continuously expressed and secreted by hepatocytes to maintain a stable serum concentration. The mean tumor volume was measured for each treatment group. Results for Colon 26 and 4T1 (mm) 3 The values ​​(as +SD) are shown in Figures 13A and 13B. The arrows indicate the hydrodynamic injection date.

[0376] MC38 tumor cells were subcutaneously inoculated into C57BL / 6J mice on day 0, and the average tumor size was 80 mm. 3 Randomization occurred on day 7, when the mice reached [a certain stage]. Next, mice were intraperitoneally treated with either PBS control, IL2M0 (15 μg), or IL2M2 (50 μg) via injection every 3 days for a total of 4 injections. The mean tumor volume was measured for each treatment group, and the results (mm²) were determined. 3Figure 13C shows the result (as +SD).

[0377] Treatment with IL2M2, either by hydrodynamic DNA delivery or protein injection, inhibited tumor progression in multiple syngeneic tumor models (Figure 13). Despite being approximately 100 times weaker than IL2M0 against multiple lymphocyte populations (Figures 12A-C), a dose of IL2M2 approximately three times higher than IL2M0 was able to achieve an antitumor effect comparable to that of IL2M0.

[0378] 7.12. Example 11: Activity of IL2M4 and IL2M5 on lymphocyte populations Human PBMCs were stimulated with increased concentrations of IL2M4 (Fc-IL2 fusion protein) or IL2M5. Intracellular STAT5 phosphorylation levels were measured by FACS as described in Section 7.1.2. Gated Treg (Figure 14A), CD8 + The results for T cells (Figure 14B) and NK cells (Figure 14C) are shown in Figure 14.

[0379] Compared to IL2M4, IL2M5 showed decreased activity against all cell types tested. However, the degree of attenuation differed among different lymphocyte populations, with IL-2Rα-NK and CD8+ T cells being more affected than IL-2Rα+ Tregs. Overall, IL2M5 was generally attenuated, but its selectivity for IL-2Rα+ cells increased.

[0380] 7.13. Example 12: Antitumor effect of IL2M5 monotherapy MC38 tumor cells were subcutaneously inoculated into C57BL / 6J mice on day 0, and the average tumor size was 80 mm. 3 Randomization occurred on day 7, when the mice reached a certain stage. Next, the mice were treated intraperitoneally with PBS, IL2M4 (15 μg), or IL2M5 (100 μg) via injection every 3 days for a total of 4 injections. Mean tumor volume was measured. Results for each treatment group (mm²) 3 Figure 15A shows the results for each tumor (with +SD), and Figures 15B-15D show the results for each individual tumor.

[0381] Given that IL2M5 is significantly attenuated compared to IL2M4, it was used at high doses in tumor studies. Despite being more than 100 times weaker than IL2M4 against multiple lymphocyte populations (Figure 14A-C), doses of IL2M5 approximately six times higher achieved more effective tumor control than IL2M4. Complete tumor regression and tumor-free survival were observed in three out of five mice treated with 100 μg / dose of IL2M5.

[0382] 7.14. Example 13: Oligomerization state of IL-2Rα-containing mutein The oligomeric states of IL2M2 and IL2M3 were evaluated using a combination of size exclusion ultrahigh performance liquid chromatography (SEC) and multi-angle light scattering (MALS). IL2M2 contains multiple high molecular weight species, with the dominant species (approximately 60%) exhibiting a molar mass consistent with the dimer (Figure 16A). In contrast, IL2M3 shows a less pronounced tendency towards oligomerization. It exists primarily as a dimer (approximately 77%), along with two trace amounts of high molecular weight species (Figure 16B). Arrows indicate the determined molecular weights and the relative percentages of each major population. The equilibrium between the different structures is illustrated in Figures 3A and 3B (for IL2M2 and IL2M3, respectively).

[0383] 7.15. Example 14: T1-IL2M3 retains binding to cell surface PD1. The binding of T1-IL2M3 containing the anti-PD1 targeting moiety was evaluated by FACS as described in Section 7.1.4. As shown in Figure 17, T1-IL2M3 and the parental anti-mPD1 antibody showed comparable binding to human HEK293 cells that stably express mouse PD1 on their surface, suggesting that the effect of targeted binding by fusing the antibody to IL2M3 is minimal.

[0384] 7.16. Example 15: T1-IL2M3 shows excellent antitumor effect against the combination of anti-PD1 and IL2M3. C57BL / 6J mouse with 3x10 5 MC38 tumor cells were subcutaneously inoculated on day 0, and the average tumor size was 100 mm. 3Mice were randomized on day 7 when they reached [a certain stage]. Next, the mice were intraperitoneally treated with a total of five injections twice a week, using one of the following: isotype (1 mg / kg), anti-mPD1 (1 mg / kg), isotype-IL2M3 (0.5 mg / kg) + anti-mPD1 (0.33 mg / kg), isotype-IL2M3 (1.5 mg / kg) + anti-mPD1 (1 mg / kg), T1-IL2M3 (0.5 mg / kg) + isotype (0.33 mg / kg), or T1-IL2M3 (1.5 mg / kg) + isotype (1 mg / kg).

[0385] The average tumor volume was measured. Results for each treatment group (mm 3 Figure 18A shows the results for each tumor (+SD), and Figures 18B.1 to 18B.4 show the results for each individual tumor. Isotype-IL2M3 + anti-PD1 failed to confer tumor control, while T1-IL2M3 + isotype showed a significant effect at both doses tested, with more mice experiencing complete tumor regression at higher doses. The amount of IL2M3 delivered by a 0.5 mg / kg dose of T1-IL2M3 was very small, equivalent to 5.7 μg / dose of IL2M3. This result highlights the superior antitumor effect of T1-IL2M3 compared to the anti-PD1 and IL2M3 combination. It also suggests that PD1 targeting significantly reduced the amount of IL2M3 required to achieve efficient tumor control.

[0386] Blood was collected from tumor-carrying mice on day 15 and analyzed by FACS. CD8 + CD44 in T cells high CD62L low Cells and PD1 + Cells, and CD4 + FoxP3 in T cells + IL-2Rα + The frequencies of Tregs are shown in Figures 18C.1-18D.2 and 18E.1-18E.2, respectively.

[0387] Compared to the untargeted isotype-IL2M3, T1-IL2M3 is CD44 hi CD62L lo and PD1+ This activated effector induces specific expansion of memory CD8+ T cells. At the same time, T1-IL2M3 causes less undesirable proliferation of Tregs than isotype-IL2M3. This result indicates that the T1-IL2M3 fusion can redirect IL2M3 to antigen-activated CD8+ T cells expressing PD1. Activated T cells upregulate PD1 and inhibit T cells. In addition to blocking PD1 signaling in these cells, T1-IL2M3 can specifically reactivate and expand these cells by stimulating IL2 signaling. A schematic diagram of the proposed mechanism of action of T1-IL2M3 is shown in Figure 19.

[0388] 7.17. Example 16: The anti-mPD1-IL2 mutein 3 fusion showed superior antitumor efficacy compared to the combination of isotype-IL2 mutein 3 and parental anti-PD1 antibody in several mouse syngeneic tumor models. The therapeutic effects of T1 IL2M3 (anti-mPD1-IL2 mutain 3) were evaluated in two different mouse strains (C57BL / 6J and BALB / cJ) in syngeneic mouse tumor models of lung cancer, skin cancer, breast cancer, and colon cancer (using LLC1, B16F10, 4T1, and colon-26 tumor cells, respectively).

[0389] C57BL / 6J mouse, (a) 2 × 10 5 LLC1 tumor cells were subcutaneously inoculated on day 0, with an average tumor size of 90 mm. 3 Randomization occurred on day 7 when (b) 3 × 10 5 B16F10 tumor cells were subcutaneously inoculated on day 0, with an average tumor size of 80 mm. 3 Mice were randomized on day 7 when they reached [a certain stage]. Next, the mice were intraperitoneally treated with isotype (1 mg / kg), isotype-IL2 mutein 3 (1.5 mg / kg) + anti-mPD1 (1 mg / kg), or anti-mPD1-IL2 mutein 3 (1.5 mg / kg) + isotype (1 mg / kg) twice a week for a total of four or five injections.

[0390] BALB / cJ mouse, (a) 5 x 10 5 4T1 tumor cells were subcutaneously inoculated on day 0, and the average tumor size was 60 mm. 3 (b) Randomized on day 8 when the number of patients reached (b) 1 × 10 6 Colon-26 tumor cells were subcutaneously inoculated on day 0, with an average tumor size of 100 mm. 3 Randomization occurred on day 12 when the mice reached a certain stage. Next, the mice were intraperitoneally treated with either isotype (1 mg / kg), isotype-IL2 mutein 3 (0.5 mg / kg) + anti-mPD1 (0.33 mg / kg), or anti-mPD1-IL2 mutein 3 (0.5 mg / kg) + isotype (0.33 mg / kg) via injection twice a week for a total of five times.

[0391] Average tumor volume (mm) of each treatment group 3 Figures 20A-20D show the SEM (+SEM) results. The arrows indicate the treatment date. In all models studied, anti-mPD1-IL2 mutein 3 showed superior antitumor activity compared to the combination of isotype-IL2 mutein 3 and anti-mPD1. Treatment with anti-mPD1-IL2 mutein 3 resulted in complete tumor regression in most treated mice in the Colon-26 model (Figure 20D). In other models that have shown resistance to conventional immunotherapy (LLC1, B16F10, 4T1) (Mosely et al., 2016, Cancer Immunol Res 5(1):29-41), anti-mPD1-IL2 mutein 3 was able to slow the progression of established tumors (Figures 20A-20C).

[0392] 7.18. Example 17: The anti-LAG3-IL2 mutain 3 fusion shows superior antitumor efficacy compared to the anti-LAG3 + isotype-IL2 mutain 3 combination. MC38 tumor cells were subcutaneously inoculated into C57BL / 6J mice on day 0, and the average tumor size was 50 mm. 3Mice were randomized on day 10 when they reached a certain stage. Next, the mice were treated intraperitoneally with a total of five injections twice a week with one of the following: isotype (1 mg / kg), anti-mLAG3 (1 mg / kg), isotype-IL2 mutein 3 (0.5 mg / kg) + anti-mLAG3 (0.33 mg / kg), isotype-IL2 mutein 3 (1.5 mg / kg) + anti-mLAG3 (1 mg / kg), T6-IL2M3 (anti-mLAG3-IL2 mutein 3) (0.5 mg / kg) + isotype (0.33 mg / kg), or T6-IL2M3 (anti-mLAG3-IL2 mutein 3) (1.5 mg / kg) + isotype (1 mg / kg).

[0393] Separately, C57BL / 6J mice were subcutaneously inoculated with MC38 tumor cells on day 0, and the average tumor size was 80 mm. 3 Mice were randomized on day 9 when they reached a certain stage. Next, the mice were treated intraperitoneally with a total of four injections twice a week, using one of the following: isotype (0.33 mg / kg), isotype-IL2 mutein 3 (0.5 mg / kg) + anti-mLAG3 (0.33 mg / kg) + anti-mPD1 (0.33 mg / kg), anti-mLAG3-IL2 mutein 3 (0.5 mg / kg) + isotype (0.33 mg / kg), anti-mLAG3-IL2 mutein 3 (0.5 mg / kg) + anti-mPD1 (0.33 mg / kg), or anti-mLAG3-IL2 mutein 3 (0.5 mg / kg) + anti-mPD1 (5 mg / kg).

[0394] Average tumor volume (mm) of each treatment group 3 Figures 21A-21B show the SEM (+SEM) results. The arrows indicate the treatment date. The molar amount of IL2M3 delivered by a 0.5 mg / kg dose of isotype-IL2mutein3 or anti-mLAG3-IL2mutein3 is equivalent to the molar amount of IL2M2 or IL2M3 delivered by a 5.7 μg / kg dose. While isotype-IL2mutein3 + anti-mLAG3 failed to confer tumor control at the studied doses, anti-mLAG3-IL2mutein3 + isotype demonstrated a dose-dependently enhanced antitumor effect (Figure 21A). This result provides another example of how tumor-responsive T cell targeting can enhance the antitumor effect of IL2M3 by reducing the amount of IL2M3 required to achieve efficient tumor control.

[0395] While anti-mLAG3-IL2M3 alone resulted in partial control of tumor growth, its combination with anti-mPD1 significantly enhanced the effect, leading to complete tumor regression in more mice (Figure 21B). This result suggests that combining IL2 mutein 3 targeted by a non-competitive tumor-reactive T cell-targeting antibody with PD1 blockade can provide more potent antitumor immunity.

[0396] 7.19. Example 18: Peptide MHC-IL2 mutain fusion enables selective stimulation of antigen-specific mouse CD8 T cells 1.5 × 10⁶ mice derived from OT-I TCR transgenic mice (obtained from Jackson Laboratory) or control C57BL / 6J mice. 6 Total splenocytes were stimulated at 37°C for 20 minutes with increasing concentrations of T4-IL2M6 or T5-IL2M6. Intracellular STAT5 phosphorylation in gated CD8+ T cells was assessed by flow cytometry as described in Section 7.1.2.

[0397] The results are shown in Figure 22B. Compared to T5-IL2M6, which targets the TCR for unrelated antigens, T4-IL2M6 shows two orders of magnitude higher efficacy in inducing STAT5 phosphorylation in T4-specific CD8+ OT-I T cells, but not in non-specific CD8+ T cells derived from control mice.

[0398] Gated conventional CD4 cells derived from the same mouse spleen + We measured the levels of intracellular STAT5 phosphorylation in T cells and Treg cells. The results are shown in Figures 23A-23B. Similar activity was observed in T4-IL2M6 and T5-IL2M6 of both cell types. CD8 + Unlike T cells, conventional CD4 cells derived from the same OT-I mouse + T cells and Tregs do not express T4-specific OT-I TCRs. Therefore, the selectivity of T4-IL2M6 was lost in these cells.

[0399] 7.20. Example 19: pMHC-IL2 mutain fusion is antigen-specific human CD8 + This enables selective stimulation of T cells. 1.5 × 10 4 CMV pp65-specific human CD8+ T cells were stimulated at 37°C for 20 minutes with increasing concentrations of T2-IL2M6 or T3-IL2M6. Intracellular STAT5 phosphorylation levels were assessed by flow cytometry as described in Section 7.1.2.

[0400] The results are shown in Figure 24. Compared to T3-IL2M6, which targets the TCR for the HPV 16E7 antigen, T2-IL2M6 shows several orders of magnitude higher efficacy in inducing STAT5 phosphorylation in these CMV pp65-specific T cells.

[0401] 7.21. Example 20: Selective CAR-T expansion by peptide-MHC targeted IL2 mutain 7.21.1. Structure of CAR structures T3 (HPV16 E7) 11-19 V recognizes the HLA-A2 peptide, the huCD8 transmembrane domain, the CD137(4-1BB) costimulatory domain, and the CD3ζ signaling domain. L -V H The chimeric antigen receptor containing scFv (shown in Figure 25A) is a single antibody, 17363N V L and V HThe CAR was constructed using the sequence. As a negative control for the CAR, non-transduced T cells from the same donor were used. This CAR was cloned into a pLVX lentiviral vector having an EF1a promoter and containing a P2A sequence upstream of the eGFP sequence to track the CAR-transduced cells. A VSV pseudotyped lentivirus was produced for subsequent transduction carried out using an infection multiplicity (MOI) of 5. Exemplary FACS profiles of cells transduced with the CAR construct are shown in Figures 25B–25C.

[0402] 7.21.2. Generation and Expansion of CAR T Cells After thawing CD3+ T cells derived from a donor homozygous for HLA-A2, they were treated with Dynabeads® Human T-Expander CD3 / CD28 microbeads and aldesleukin (100 IU / mL) for 1.5 × 10⁻¹⁴ cells. 6 Cells were stimulated at a density of 1 / mL for 24 hours. Subsequently, the CD3 / CD28 microbeads were removed by magnetic separation, and then the activated T cells were transduced with the CAR construct by spin-fection at 2440 rpm for 90 minutes. Non-transduced T cells from the same donor were used as a negative control for the CAR. Next, transduced and non-transduced cells were supplemented with Dynabeads® Human T-Expander CD3 / CD28 microbeads (1 bead per T cell) and expanded in CTS-supplemented OpTmizer® medium for 19 days. Cell density was increased to 1–1.5 × 10⁻⁶. 6 The culture was maintained at 100 IU / mL, and aldethleukin was added to the culture every 48 hours to maintain a concentration of 100 IU / mL. At the end of the expansion, the microbeads were removed and the cells were cryopreserved.

[0403] The activity of various peptide-MHC targeted IL2 muteins, illustrated in Figures 26A and 26B, containing attenuated IL2 (H16A, F42A) (also known as IL2(2m)), was tested: T7-IL2M7 (monovalent to IL2 and possessing an HPV peptide-MHC complex), T8-IL2M7 (monovalent to IL2 and possessing a CMV peptide-MHC complex), T3-IL2M6 (divalent to IL2 and possessing an HPV peptide-MHC complex), and T2-IL2M6 (monovalent to IL2 and possessing a CMV peptide-MHC complex). CAR-T cells were thawed and washed twice with complete RPMI medium. The thawed cells were then subjected to 2.8 × 10⁶ filtration in complete RPMI medium without aldethleukin. 6 The cells were allowed to stand overnight at / mL. After standing overnight, the levels of intracellular STAT5 phosphorylation in these gated CD4+ or CD8+ populations of CAR T cells were assessed by flow cytometry as described in Section 7.1.2.

[0404] 7.21.3.Results The results are shown in Figures 27A (CD4+ T cells) and 27B (CD8+ T cells). CAR targeting and HPV16 E7 11-19 By gated to CD4+ and CD8+ CAR-T cells engineered to express the peptide-HLA-A2 complex, IL2 mutain T7-IL2M7 and T3-IL2M6 (both HPV16 E7) can be obtained. 11-19 The peptide-HLA-A2 targeting portion is CMV pp65 495-503 It exhibits several orders of magnitude higher efficacy in inducing STAT5 phosphorylation in T8-IL2M7 and T2-IL2M6, which target specific T cells.

[0405] 7.22. Example 21: Selective CAR-T expansion by peptide-MHC targeted IL2 mutain 7.22.1. Generation and Expansion of CAR T Cells After thawing CD3+ T cells derived from a donor homozygous for HLA-A2, they were mixed with Dynabeads® Human T-Expander CD3 / CD28 microbeads and 3.3 × 10⁶ cells.-10 The cells were stimulated with recombinant aldethleukin M for 48 hours. Subsequently, the CD3 / CD28 microbeads were removed by magnetic separation, and then the activated T cells were transduced with the CAR construct shown in Figure 27A by spin-fection at 2440 rpm for 90 minutes. Non-transduced T cells from the same donor were used as a negative control for the CAR. Next, the transduced and non-transduced cells were added to CTS-supplemented OpTmizer® medium with Dynabeads® Human T-Expander CD3 / CD28 microbeads in a 1:1 ratio to the T cells and 3.3 × 10⁶ microbeads. -10 The cells were expanded with M aldethleukin for 4 days. Next, activated T cells were harvested, washed twice with OpTmizer® medium, and the CD3 / CD28 microbeads were removed by magnetic separation. The T cells were then allowed to stand overnight at 37°C in CTS-supplemented OpTmizer® medium lacking aldethleukin. The transduced and non-transduced T cells were resuspended and placed in CTS-supplemented OpTmizer® medium at a rate of 1 × 10⁶ in the presence of Dynabeads® human T-Expander CD3 / CD28 microbeads (1 bead per 1 T cell). 6 The cultures were then incubated at / mL. Next, each culture was treated with the respective biological agent at 3.3 × 10⁻⁶. -10 Stimulation was performed with a concentration of M. Each T cell enlargement culture was supplemented with the biologic every 48 hours, and the concentration increased from 1 to 1.5 × 10⁶. 6 The cells were maintained at a density of / mL for 17 days. On days 3, 9, and 17 of the expansion, T cell counts were taken, and aliquots of cells were collected to identify viable eGFP. Pos (CAR+) and eGFP Neg We quantified the change in the ratio of (non-CAR) T cells to non-transduced T cells. Using non-transduced T cells expanded with each biopharmaceutical, we defined the eGFP signaling for CAR-T expansion at each time point.

[0406] 7.22.2. Characterization of CAR-T cells At several point in time during CAR-T cell expansion, aliquots of cells were collected, and viable eGFP+(CAR+) cells were quantified by flow cytometry. To identify different T cell populations and IL2Rα expression, the following antibody combinations were used: BUV395 anti-CD4 (BD Biosciences), BUV737 anti-CD4 (BD Biosciences), PE-CY7 anti-CD8 (Biolegend), APC-Cy7 anti-CD8 (Biolegend), and BV605 anti-CD25 (BD Biosciences). To characterize subsets and phenotypes of memory T cells, BV421 anti-CCR7 (Biolegend), PE-CF594 anti-CD45RO (BD Biosciences), PerCP-Cy5.5 anti-CD45RO (BD Biosciences), BUV395 anti-PD1 (BD Biosciences), and PE-CF594 anti-CD57 (BD Biosciences) were used. Survival rates were assessed using 4',6-diamidino-2-phenylindole (DAPI) (ThermoFisher) or AQUA viability dye (ThermoFisher). All samples were acquired using a BDFortessa® X-20 or Biorad Ze5 flow cytometer. Raw data were processed using FlowJo v10.

[0407] 7.22.3. Results and Discussion To investigate the potential of attenuated IL-2 binding titers in conjunction with increased titers of a given targeting region that mediates selective CAR-T expansion, selective CAR-T expansion was examined in response to a series of engineered biologics. In the monovalent format, one copy of attenuated IL-2 is combined with one copy of the targeting region. In the bivalent format, two copies of attenuated IL-2 are combined with two copies of the targeting region. To control non-selective expansion, a targeting region not recognized by CAR-T cells (CMV pp65) was used. 495-503 The study utilized scHLA-A2) containing the peptide. The structures of the tested biologics are shown in Figures 26A and 26B.

[0408] The results are shown in Figure 28. As shown in Figures 28A.1 to 28A.16, viable CAR-T cells (eGFP) were developed before transduced T cells expanded in response to scMHC-peptide-targeted IL2 mutein (shown as day 0). Pos The frequency of ) cells was 38%. By day 3 of expansion, the frequency of viable CAR-T cells increased to approximately 43% in each culture, except for the bivalent T3-IL2M6 construct, where the frequency of CARs decreased to 36%. By day 9, the most selective CAR-T enrichment was observed in response to monovalent T7-IL2M7, with the percentage of viable CAR-T cells increasing to 75%. Notably, even without this bioagent binding to CAR scFv, a less significant increase in the percentage of CAR-T cells was also observed in response to monovalent T8-IL2M7. Due to the lack of antigen recall response (according to the supplier) using matched donor PBMCs, this expansion was not attributed to cytomegalovirus peptide (pp65). 495-503 While unlikely to be due to the existing T cell repertoire against ), this possibility cannot be completely ruled out. From days 9 to 17 of the expansion, the percentage of CARs responding to monovalent T7-IL2M7 and T8-IL2M7 remained the same. A slight increase in CAR-T frequency was observed in response to IL2 (aldesleukin) and divalent T3-IL2M6, but no further changes were observed in response to divalent T2-IL2M6.

[0409] To evaluate the potential of biologics that simultaneously bind to CAR scFv and deliver attenuated IL2 signaling to mediate selective expansion of CAR-T cells, we are using CAR-T cells (EGFP). Pos The total number of viable T cells was plotted against the ratio of eGFPNeg (non-CAR T cells) to non-CAR T cells. As shown in Figures 28B.1-B.4, eGFP Pos / eGFP NegAs the ratio increased from 0.73 to 3.12, selective enrichment of CAR-T cells occurred between days 3 and 9 in response to T7-IL2M7 (white circle). In addition, the number of T cells more than doubled. By day 17, the end of the culture, the maximum number of expanded total T cells in response to aldesleukin (black square), followed by monovalent T7-IL2M7 (white circle), was observed, but CAR Pos and CAR Neg The proportion of T cells was highest in cultures expanded with T7-IL2M7, indicating that selective and targeted expansion of CAR-T cells occurred. As shown in Figure 28C, the absolute number of viable CAR-T cells for each expansion condition is plotted as a function of time during culture. IL-2 (aldesleukin) resulted in the largest total number of T cells (Figure 28B.4), but selective CAR-T expansion resulted in monovalent T7-IL2M7 generating the largest number of viable CAR-T cells (Figure 28C). Minimal CAR-T expansion occurred in response to monovalent T8-IL2M7, highlighting the importance of the targeted region. Notably, CAR-T expansion was low in response to bivalent T3-IL2M6, potentially indicating hyperactivation of CAR-T cells leading to cell death. Alternatively, bivalent T3-IL2M6 may cross-link individual CAR-T cells, resulting in CAR-T fractorides. These studies demonstrate that monovalent format structures are excellent at mediating selective CAR-T expansion.

[0410] 7.23. Example 22: In vivo pharmacokinetic evaluation of peptide-MHC-targeted IL2 mutein 7.23.1. Method To evaluate the pharmacokinetics of IL2 mutein in plasma, both immunocompetent C57BL / 6J mice and immunodeficient NSG (NOD-scid IL2Rgnull) mice were intraperitoneally injected with a single dose of 12 μg of each protein. Blood samples were collected 2, 24, 48, and 72 hours post-administration, and plasma was isolated by centrifugation at 10,000 RPM for 10 minutes. 1 μl of each plasma sample was analyzed by SDS-PAGE, followed by immunoblotting with an antibody against human IgG Fc. 2 ng of each purified protein (spiked into 1 μl of naive plasma) was loaded onto the same gel to estimate the absolute level of each protein in the plasma sample.

[0411] 7.23.2.Results The results are shown in Figure 29. Compared to IL2M0 (referred to as IL2-Fc in Figure 29), the targeted IL2 muteins shown in Figures 26A and 26B showed improved PK in a binding-dependent manner. In addition, compared to IL2-Fc, both the bivalent and monovalent constructs showed delayed clearance in circulation, with the monovalent constructs (T7-IL2M7 and T8-IL2M7) lasting the longest. This result suggests that attenuation of IL2 significantly reduces its receptor-mediated clearance in a binding-dependent manner, and therefore may increase the likelihood that targeted attenuated IL2 muteins reach target cells. Despite their more durable presence in circulation, bivalent T2-IL2M6 was eliminated from the blood faster than the targeted portion alone (T2-Fc), suggesting that the clearance of T2-IL2M6 is primarily driven by IL2M6.

[0412] 8. Specific embodiments, references While various specific embodiments have been illustrated and described, it will be understood that various modifications can be made without departing from the spirit and scope of this disclosure. This disclosure is illustrated by the numbered embodiments described below.

[0413] In embodiments numbered below and preferred aspects of the following claims, the IL2 domain, IL2 receptor, Fc domain, MHC domain, β2M, and variants thereof preferably include the amino acid sequences of human IL2, human IL2 receptor, human Fc domain, human MHC domain, human β2M, and variants thereof, for example, variants having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with respect to such human sequences.

[0414] 1. It is an IL2 agonist, (a) The IL2 portion including the IL2 domain, (b) Optionally, the multimerized portion and (c) Optionally, the target portion and, (d) Optionally, an IL2 agonist including a stabilizing portion.

[0415] 2. (a) IL2M0, (b) IL2M1, (c)IL2M2, (d) IL2M3, (e) IL2M4, (f) IL2M5, (g) IL2M6, or (h) An IL2 agonist according to Embodiment 1, comprising IL2 mutaine having the configuration of IL2M7.

[0416] 3. An IL2 agonist according to Embodiment 1 or Embodiment 2, wherein the IL2 portion is IL2-Rα biased IL2 mutaine. 4. An IL2 agonist according to any one of Embodiments 1 to 3, wherein the IL2 moiety and / or IL2 agonist has 50 to 1,000 times reduced binding to human IL2-Rβ compared to wild-type IL2.

[0417] 5. An IL2 agonist according to any one of Embodiments 1 to 4, wherein the IL2 moiety and / or IL2 agonist is attenuated by up to 100-fold, up to 500-fold, up to 1,000-fold, or up to 5,000-fold compared to wild-type human IL2.

[0418] 6. An IL2 agonist according to any one of Embodiments 1 to 5, wherein the ratio of the binding affinity of the IL2 agonist to the high-affinity IL2 receptor to the binding affinity of the IL2 agonist to the intermediate-affinity IL2 receptor is equivalent to or greater than the corresponding ratio of wild-type IL2.

[0419] 7. An IL2 agonist according to any one of embodiments 1 to 6, wherein the IL2 moiety and / or IL2 agonist has an E50 of a high-affinity IL2 receptor that is 100 to 10,000 times lower than the E50 of an intermediate-affinity IL2 receptor.

[0420] 8. The IL2 agonist according to Embodiment 1, wherein the IL2 portion is IL2-Rβ biased IL2 mutaine. 9. An IL2 agonist according to Embodiment 1 or 8, wherein the IL2 moiety and / or IL2 agonist has (a) 50 to 1,000 times or (b) 50 to 5,000 times reduced binding to human IL2-Rα compared to wild-type IL2.

[0421] 10. An IL2 agonist according to any one of embodiments 1, 8, and 9, wherein the IL2 moiety and / or IL2 agonist has up to 50-fold attenuation of binding to human IL2-Rβ compared to wild-type human IL2.

[0422] 11. An IL2 agonist according to any one of Embodiments 1 and 8-10, wherein the ratio of the binding affinity of the IL2 moiety and / or IL2 agonist to the intermediate affinity IL2 receptor to the binding affinity of the IL2 moiety and / or IL2 agonist to the high affinity IL2 receptor is equal to or greater than the corresponding ratio of wild-type IL2.

[0423] 12. An IL2 agonist according to any one of Embodiments 1 and 7-11, wherein the IL2 moiety and / or IL2 agonist has an E50 of a high-affinity IL2 receptor that is 10 to 100 times lower than the E50 of an intermediate-affinity IL2 receptor.

[0424] 13. An IL2 agonist according to any one of embodiments 1 to 12, wherein the IL2 moiety and / or IL2 agonist has a reduced binding affinity to the high-affinity IL2 receptor compared to wild-type IL2.

[0425] 14. The binding affinity is reduced by up to 1,000 times or up to 5,000 times, or the binding affinity is (a) 10x to 1,000x, (b) 50x to 5,000x, (c) up to 10x; (d) up to 50x; (e) up to 100 times, or (f) The IL2 agonist according to Embodiment 13, which is attenuated by up to 200 times.

[0426] 15. An IL2 agonist according to any one of Embodiments 1 to 14, wherein the IL2 moiety and / or IL2 agonist has higher cytokine activity against tumor-reactive lymphocytes than against peripheral lymphocytes.

[0427] 16. The IL2 agonist according to Embodiment 15, wherein the IL2 moiety and / or IL2 agonist has at least 5-fold or at least 10-fold higher cytokine activity against tumor-reactive lymphocytes than against peripheral lymphocytes.

[0428] 17. An IL2 agonist according to any one of embodiments 1 to 16, having at least one therapeutic index. 18. An IL2 agonist according to Embodiment 17, having at least 2 therapeutic indices.

[0429] 19. An IL2 agonist according to Embodiment 17, having a therapeutic index of at least 5. 20. An IL2 agonist according to Embodiment 17, having at least 10 therapeutic indices. 21. An IL2 agonist according to Embodiment 17, having a therapeutic index of at least 50.

[0430] 22. An IL2 agonist according to any one of embodiments 17 to 21, having a maximum therapeutic index of 500. 23. An IL2 agonist according to any one of embodiments 17 to 21, having a maximum therapeutic index of 250.

[0431] 24. An IL2 agonist according to any one of embodiments 1 to 23, having a therapeutic index of approximately 2. 25. An IL2 agonist according to any one of embodiments 1 to 23, having a therapeutic index of approximately 10.

[0432] 26. An IL2 agonist according to any one of embodiments 1 to 23, having a therapeutic index of approximately 20. 27. An IL2 agonist according to any one of embodiments 1 to 23, having a therapeutic index of approximately 50.

[0433] 28. An IL2 agonist according to any one of embodiments 1 to 23, having a therapeutic index of approximately 100. 29. An IL2 agonist according to any one of embodiments 1 to 23, having a therapeutic index of approximately 200.

[0434] 30. An IL2 agonist according to any one of embodiments 1 to 29, wherein the IL2 portion and / or IL2 agonist are not pegged. 31. An IL2 agonist according to any one of Embodiments 1 to 30, wherein the IL2 portion and / or IL2 agonist does not contain cytokines other than IL2.

[0435] 32. An IL2 agonist according to any one of Embodiments 1 to 31, wherein the IL2 portion and / or IL2 agonist does not contain an anti-IL2 antibody or antibody fragment. 33. An IL2 agonist according to any one of Embodiments 1 to 32, wherein the IL2 portion and / or IL2 agonist does not contain an anti-DNA antibody or antibody fragment.

[0436] 34. An IL2 agonist according to any one of Embodiments 1 to 33, wherein the IL2 domain does not contain a substitution at position D20 compared to human IL2. 35. An IL2 agonist according to any one of Embodiments 1 to 34, wherein the IL2 domain does not contain a substitution at position Q126 compared to human IL2.

[0437] 36. An IL2 agonist according to any one of Embodiments 1 to 35, wherein the IL2 portion and / or IL2 agonist does not contain a non-binding variable domain. 37. An IL2 agonist according to any one of Embodiments 1 to 36, wherein the IL2 portion comprises an IL2 domain having an amino acid sequence having at least about 90% sequence identity with mature human IL2.

[0438] 38. An IL2 agonist according to any one of Embodiments 1 to 36, wherein the IL2 portion comprises an IL2 domain having an amino acid sequence having at least about 93% sequence identity with mature human IL2.

[0439] 39. An IL2 agonist according to any one of Embodiments 1 to 36, wherein the IL2 portion comprises an IL2 domain having an amino acid sequence having at least about 96% sequence identity with mature human IL2.

[0440] 40. An IL2 agonist according to any one of Embodiments 1 to 39, comprising an IL2 variant having an amino acid substitution C125S, C125A, or C125V in the IL2 portion.

[0441] 41. An IL2 agonist according to any one of Embodiments 1 to 40, wherein the IL2 domain comprises an IL2 sequence having one or more substitutions to reduce O-linked glycosylation. 42. The IL2 agonist according to Embodiment 41, wherein the IL2 domain has a substitution at a position corresponding to residue 3 of human IL2.

[0442] 43. The IL2 agonist according to Embodiment 42, wherein the amino acid substitution is T3A, T3G, T3Q, T3E, T3N, T3D, T3R, or T3K. 44. An IL2 agonist according to any one of Embodiments 1 to 43, wherein the IL2 domain has a neutral amino acid substitution at the position corresponding to methionine 104 of human IL2.

[0443] 45. The IL2 agonist according to Embodiment 44, wherein the neutral amino acid is alanine. 46. ​​An IL2 agonist according to any one of Embodiments 1 to 45, wherein the IL2 domain is a full-length human IL2 domain.

[0444] 47. An IL2 agonist according to any one of Embodiments 1 to 45, wherein the IL2 domain has an N-terminal alanine deletion compared to fully mature human IL2. 48. An IL2 agonist according to any one of claims 1 to 47, wherein the IL2 domain comprises an IL2 variant having an amino acid substitution at the N88 position.

[0445] 49. The IL2 agonist according to Embodiment 48, wherein the amino acid substitution is N88D. 50. An IL2 agonist according to any one of Embodiments 1 to 47, wherein the IL2 domain comprises an IL2 variant having amino acid substitutions at the H16 and F42 positions.

[0446] 51. The IL2 agonist according to Embodiment 50, wherein the amino acid substitutions are H16A and F42A. 52. An IL2 agonist according to any one of Embodiments 1 to 51, wherein the IL2 portion comprises an IL-2Rα domain containing an IL2 binding portion of IL-2Rα fused to the IL2 domain.

[0447] 53. The IL2 agonist according to Embodiment 52, wherein the IL2-Rα domain or the IL2 binding portion of IL-2Rα is the extracellular domain of IL-2Rα or its IL2 binding portion.

[0448] 54. The IL2-Rα domain or IL2 binding portion contains or comprises an amino acid sequence having at least approximately 90%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100% sequence identity with the IL2 binding portion of human IL-2Rα, and optionally, the binding portion is (a) at least 160 amino acids, at least 161 amino acids, at least 162 amino acids, at least 164 amino acids, or at least 165 amino acids of human IL2-Rα, and / or (b) An IL2 agonist according to Embodiment 52 or 53, having up to 251, up to 240, up to 230, up to 220, up to 210, up to 200, up to 190, up to 180, or up to 170 amino acids in the extracellular domain of human IL2-Rα.

[0449] 55. An IL2 agonist according to any one of embodiments 52 to 54, wherein the IL2-Rα domain or IL2 binding moiety has an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with amino acids 22 to 186, 22 to 240, and / or 22 to 272 of IL-2Rα.

[0450] 56. An IL2 agonist according to any one of embodiments 52 to 55, wherein the IL2-Rα domain or IL2 binding moiety contains or comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with amino acids 22 to 186, with or without up to 5, at least 10, at least 15, at least 20, at least 30, or at least 40 additional amino acids at the C-terminus of amino acid residue 186 of human IL2-Rα.

[0451] 57. An IL2 agonist according to any one of embodiments 52 to 56, wherein the IL-2Rα domain is the N-terminus of the IL2 domain. 58. An IL2 agonist according to any one of embodiments 52 to 56, wherein the IL-2Rα domain is the C-terminus of the IL2 domain.

[0452] 59. An IL2 agonist according to any one of embodiments 52 to 58, wherein the IL2 domain and the IL2-Rα domain are connected via a linker. 60. The IL2 agonist according to Embodiment 59, wherein the linker is a glycine-serine linker or comprises one.

[0453] 61. The IL2 agonist according to Embodiment 60, wherein the linker contains the amino acid sequence G4S (SEQ ID NO: 57). 62. An IL2 agonist according to Embodiment 61, wherein the linker is a multimer of amino acid sequence G4S (SEQ ID NO: 57) or comprises the same.

[0454] 63. The IL2 agonist according to Embodiment 62, wherein the multimer comprises 2, 3, 4, 5, or 6 repeats of the amino acid sequence G4S (SEQ ID NO: 57). 64. An IL2 agonist according to any one of embodiments 1 to 63, comprising a multimerized and / or stabilizing portion.

[0455] 65. The IL2 agonist according to Embodiment 64, wherein the multimerized portion and / or stabilizing portion is or contains an Fc domain. 66. The IL2 agonist according to Embodiment 65, wherein the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

[0456] 67. An IL2 agonist according to embodiment 65 or 66, wherein the Fc domain reduces the effector function. 68. An IL2 agonist according to Embodiment 67, wherein the Fc domain comprises, for example, the amino acid sequence or portion thereof of SEQ ID NO: 31 of WO2014 / 121087, as described in Table 4 and / or Section 6.5.1.1.

[0457] 69. An IL2 agonist according to any one of embodiments 1 to 68, comprising a stabilizing portion. 70. The IL2 agonist according to Embodiment 69, wherein the stabilizing portion is human serum albumin, a human serum albumin binder, XTEN, PAS, a carbohydrate, polysialic acid, a hydrophilic polymer, a fatty acid, or an Fc domain.

[0458] 71. The IL2 agonist according to Embodiment 70, wherein the stabilizing portion is a human serum albumin binder. 72. The IL2 agonist according to Embodiment 71, wherein the human serum albumin conjugate is adnectin PKE, AlbudAb, or an albumin-binding domain.

[0459] 73. The IL2 agonist according to embodiment 72, wherein the stabilizing portion is an Fc domain. 74. The IL2 agonist according to Embodiment 73, wherein the Fc domain is a monomer Fc domain.

[0460] 75. An IL2 agonist according to embodiment 73 or 74, wherein the Fc domain reduces the effector function. 76. The IL2 agonist according to Embodiment 70, wherein the stabilizing portion is a hydrophilic polymer.

[0461] 77. The IL2 agonist according to Embodiment 76, wherein the hydrophilic polymer is PEG. 78. An IL2 agonist according to Embodiment 77, wherein PEG has a molecular weight in the range of approximately 7.5 kDa to approximately 80 kDa.

[0462] 79. An IL2 agonist according to Embodiment 78, wherein PEG has a molecular weight in the range of approximately 30 kDa to approximately 60 kDa, and optionally, a molecular weight of approximately 50 kDa. 80. An IL2 agonist according to any one of embodiments 76 to 79, wherein a hydrophilic molecule is attached to the IL-2Rβ bond surface of IL2.

[0463] 81. An IL2 agonist according to any one of embodiments 1 to 80, which is a dimer. 82. The IL2 agonist described in Embodiment 81, which is a homodimer. 83. An IL2 agonist according to embodiment 81, which is a heterodimer.

[0464] 84. A monomer, which is an IL2 agonist according to any one of Embodiments 1 to 80. 85. An IL2 agonist according to any one of Embodiments 1 to 84, which is monovalent with respect to the IL2 portion.

[0465] 86. An IL2 agonist according to any one of Embodiments 1 to 85, which is divalent with respect to the IL2 portion. 87. An IL2 agonist according to any one of Embodiments 1 to 86, which is an IL2 agonist with orientation 1, or includes the same.

[0466] 88. (a) Part of IL2, (i) With or without substitution with C125 to reduce aggregation (e.g., C125S, C125A, or C125V), the IL2 or IL2 variant (e.g., IL2 N88D) domain, (ii) Linker (for example, as described in Section 6.7), for example, containing 10 or more amino acids and / or (G4S) n Linkers containing or consisting of (Sequence ID 57), and optionally n≧2, for example, N is 3, 4, 5 or more, and (iii) The IL2 portion of IL-2Rα, including the IL2 binding portion, (b) Linkers (e.g., as described in Section 6.7), e.g. (G4S) n A linker containing or consisting of (sequence number 57), where n ≥ 1, for example, N is 1, 2, 3, 4, 5, or more. (c) An IL2 agonist according to any one of Embodiments 1 to 87, comprising an Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections).

[0467] 89. An IL2 agonist according to any one of Embodiments 1 to 86, which is an IL2 agonist with orientation 2, or includes the same. 90.(a)Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections), (b) Linkers (e.g., as described in Section 6.7), e.g. (G4S) n A linker containing or consisting of (sequence number 57), where n ≥ 1, for example, N is 1, 2, 3, 4, 5, or more. (c) An IL2 agonist according to any one of Embodiments 1 to 89, comprising an IL2 or IL2 variant (e.g., IL2 N88D) domain, with or without substitution with C125 to reduce aggregation (e.g., C125S, C125A, or C125V).

[0468] 91.(a) Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections) (b) Linkers (e.g., as described in Section 6.7), e.g. (G4S) n A linker containing or consisting of (sequence number 57), where n ≥ 1, for example, N is 1, 2, 3, 4, 5, or more. (c) Part of IL2, (i) With or without substitution with C125 to reduce aggregation (e.g., C125S, C125A, or C125V), the IL2 or IL2 variant (e.g., IL2 N88D) domain, (ii) Linker (for example, as described in Section 6.7), for example, containing 10 or more amino acids and / or (G4S) n Linkers containing or consisting of (Sequence ID 57), and optionally n≧2, for example, N is 3, 4, 5 or more, and (iii) An IL2 agonist according to any one of embodiments 1 to 89, comprising an IL2 portion including the IL2 binding portion of IL2Rα.

[0469] 92. An IL2 agonist according to any one of Embodiments 1 to 86, which is or contains an IL2 agonist with orientation 3. 93. (a) a heavy chain variable region of scFv or Fab (associating with the corresponding light chain variable region on a separate polypeptide) (for example, as described in Section 6.4.2 and its subsections), (b) Linkers (e.g., as described in Section 6.7), e.g. (G4S) n A linker containing or consisting of (sequence number 57), where n ≥ 1, for example, N is 1, 2, 3, 4, 5, or more. (c) Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections) (d) Linkers (e.g., as described in Section 6.7), e.g. (G4S) n A linker containing or consisting of (sequence number 57), where n ≥ 1, for example, N is 1, 2, 3, 4, 5, or more. (e) Part of IL2, (i) With or without substitution with C125 to reduce aggregation (e.g., C125S, C125A, or C125V), the IL2 or IL2 variant (e.g., IL2 N88D) domain, (ii) Linker (for example, as described in Section 6.7), for example, containing 10 or more amino acids and / or (G4S) n Linkers containing or consisting of (Sequence ID 57), and optionally n≧2, for example, N is 3, 4, 5 or more, and (iii) An IL2 agonist according to any one of Embodiments 1 to 92, comprising an IL2 portion including an IL2 binding portion of IL-2Rα (for example, as described in Section 6.3).

[0470] 94.(a) A peptide-MHC complex (for example, as described in Section 6.4.3), (i) MHC peptide, (ii) Linkers (for example, as described in Section 6.7 or its subsections, for example, Section 6.7.1), (iii) Optionally, the β2-microglobulin (β2m) domain, (iv) Optionally, a linker (for example, as described in Section 6.7 or its subsection, for example, Section 6.7.1), and (v) A peptide-MHC complex containing MHC, (b) Optionally, a linker (e.g., as described in Section 6.7), e.g. (G4S) nA linker containing or consisting of (sequence number 57), where n ≥ 1, for example, N is 1, 2, 3, 4, 5, or more. (c) Fc domain (for example, IgG1 or IgG4, with or without substitutions that reduce glycosylation and / or effector function, as described in Section 6.5.1 and its subsections) (d) Linkers (e.g., as described in Section 6.7), e.g. (G4S) n A linker containing or consisting of (sequence number 57), where n ≥ 1, for example, N is 1, 2, 3, 4, 5, or more. (e) Part of IL2, (i) With or without substitution with C125 to reduce aggregation (e.g., C125S, C125A, or C125V), the IL2 or IL2 variant (e.g., IL2 N88D) domain, (ii) Linker (for example, as described in Section 6.7), for example, containing 10 or more amino acids and / or (G4S) n Linkers containing or consisting of (Sequence ID 57), and optionally n≧2, for example, N is 3, 4, 5 or more, and (iii) An IL2 agonist according to any one of Embodiments 1 to 92, comprising an IL2 portion including an IL2 binding portion of IL-2Rα (for example, as described in Section 6.3).

[0471] 95. An IL2 agonist according to any one of Embodiments 1 to 86, which is an IL2 agonist with orientation 4, or includes the same. 96.(a) The first polypeptide, (i) Targeting moieties, e.g., peptide-MHC complexes (e.g., as described in Section 6.4.3), Fab domains (e.g., as described in Section 6.4.2.2) (e.g., the heavy chain of Fab associating with a third polypeptide including the light chain of Fab), or scFv domains (e.g., as described in Section 6.4.2.1), (ii) optional linkers (for example, those described in Section 6.7), and (iii) A first polypeptide comprising a first Fc domain, (b) A second polypeptide, (i) With or without substitution with C125 to reduce aggregation (e.g., C125S, C125A, or C125V), IL2 portion containing IL2 or IL2 variant domains (e.g., IL2 domains with substitution H16A, F42A, also called IL2(2m) as described in Section 6.3), (ii) optional linkers (for example, those described in Section 6.7), and (iii) an IL2 agonist according to any one of embodiments 1 to 86 and 95, comprising a second polypeptide comprising a first Fc domain and a second Fc domain that can be dimerized (e.g., heterodimerized) (e.g., as described in section 6.5.1.2).

[0472] 97.(a) The first polypeptide, (i) A peptide-MHC complex (for example, as described in Section 6.4.3), (1) MHC peptide, (2) Linkers (for example, as described in Section 6.7 or its subsections, for example, Section 6.7.1), (3) Optionally, the β2-microglobulin (β2m) domain, (4) Optionally, linkers (for example, those described in Section 6.7 or its subsections, for example, Section 6.7.1), and (5) MHC-containing peptide-MHC complex, (ii) optionally, a linker (for example, as described in Section 6.7), and (iii) A first polypeptide comprising a first Fc domain, (b) A second polypeptide, (i) IL2 moiety containing IL2 or IL2 variant (e.g., IL2 H16A, F42A) domains, with or without substitution at C125 to reduce aggregation (e.g., C125S, C125A, or C125V), (ii) Optionally, a linker (for example, as described in Section 6.7), and (iii) an IL2 agonist according to any one of embodiments 1 to 86, 95, and 96, comprising a second polypeptide having a second Fc domain that is not identical to the first Fc domain but can be heterodimerized (for example, as described in Section 6.5.1.2).

[0473] 98. An IL2 agonist according to any one of Embodiments 1, 87, and 88, comprising (a) an amino acid sequence having the composition of IL2M0, or (b) an amino acid sequence of IL2M0.

[0474] 99. An IL2 agonist according to any one of Embodiments 1, 87, and 88, having (a) an amino acid sequence having the structure of IL2M2 (e.g., IL2 moiety - optional linker - IL2Rα - optional linker - Fc domain), or (b) an amino acid sequence of IL2M2.

[0475] 100. An IL2 agonist according to any one of Embodiments 1 and 91-94, having (a) an amino acid sequence having the structure of IL2M3 (e.g., IL2Rα-optional linker-IL2 moiety-optional linker-Fc domain), or (b) an amino acid sequence of IL2M3.

[0476] 101. An IL2 agonist according to any one of Embodiments 1, 89, and 90, having (a) an amino acid sequence having the structure of IL2M4 (e.g., Fc domain - optional linker - IL2 portion), or (b) an amino acid sequence of IL2M4.

[0477] 102. An IL2 agonist according to any one of Embodiments 1, 89, and 90, having (a) an amino acid sequence having the configuration of IL2M5 (e.g., Fc domain - optional linker - IL2 portion), or (b) an amino acid sequence comprising IL2M5.

[0478] 103. An IL2 agonist according to any one of Embodiments 1 and 95-97, having (a) an amino acid sequence having the configuration of IL2M6 (e.g., Fc domain - optional linker - IL2 portion), or (b) an amino acid sequence of IL2M6.

[0479] 104. (a) In each case, optionally, an amino acid sequence having the structure of IL2M7 (e.g., IL2 portion - optional linker - Fc domain) that associates with a polypeptide chain containing a targeting portion - optional linker - Fc domain, e.g., a polypeptide chain containing the sequence or structure of T7 or T8, or (b) an IL2 agonist according to any one of Embodiments 1 and 95-97, comprising the amino acid sequence of IL2M7.

[0480] 105. IL2 agonist, (a) The IL2 portion includes the IL2 domain and the IL2 binding portion of IL2-Rα, (b) An IL2 agonist containing an Fc domain.

[0481] 106. The IL2 agonist according to Embodiment 105, wherein the IL2 binding portion of IL2-Rα is the N-terminus of the IL2 domain. 107. The IL2 agonist according to Embodiment 105, wherein the IL2 binding portion of IL2-Rα is the C-terminus of the IL2 domain.

[0482] 108. An IL2 agonist according to any one of embodiments 105 to 107, wherein the Fc domain is the N-terminus of the IL2 portion. 109. An IL2 agonist according to any one of embodiments 105 to 107, wherein the Fc domain is the C-terminus of the IL2 portion.

[0483] 110. An IL2 agonist according to any one of embodiments 105 to 109, wherein the IL2 domain and the IL2 binding portion of IL2-Rα are linked via a linker, and optionally the linker comprises 10 or more amino acids, or 15 or more amino acids.

[0484] 111. An IL2 agonist according to Embodiment 110, wherein the linker is G4S (Sequence ID 57) or a multimer thereof, or includes the same. 112. The IL2 agonist according to Embodiment 111, wherein the linker includes a single G4S (Sequence ID 57).

[0485] 113. The IL2 agonist according to Embodiment 111, wherein the linker includes two, three, four, or five repeats of G4S (SEQ ID NO: 57). 114. An IL2 agonist according to any one of embodiments 105 to 113, wherein the IL2 moiety and the Fc domain are linked via a linker, and optionally the linker comprises five or more or ten or more amino acids.

[0486] 115. An IL2 agonist according to Embodiment 114, wherein the linker is G4S (Sequence ID 57) or a multimer thereof, or includes the same. 116. The IL2 agonist according to Embodiment 115, wherein the linker includes a single G4S (Sequence ID 57).

[0487] 117. The IL2 agonist according to Embodiment 115, wherein the linker includes two, three, four, or five repeats of G4S (SEQ ID NO: 57). 118. An IL2 agonist according to Embodiment 105, having (a) an amino acid sequence having the structure of IL2M2 (e.g., IL2 moiety - optional linker - IL2Rα - optional linker - Fc domain), or (b) an amino acid sequence of IL2M2.

[0488] 119. An IL2 agonist according to Embodiment 105, having (a) an amino acid sequence having the structure of IL2M3 (e.g., IL2α moiety - optional linker - IL2R - optional linker - Fc domain), or (b) an amino acid sequence of IL2M3.

[0489] 120. An IL2 agonist according to any one of embodiments 1 to 119, comprising a targeting portion. 121. The target area is (a) Binds to tumor-associated antigens, (b) Binds to tumor microenvironment antigens, (c) Binds to cell surface molecules of tumor-reactive lymphocytes, (d) Binds to checkpoint inhibitors, (e) Binds to the peptide-MHC complex, (f) It is a peptide-MHC complex, or (g) An IL2 agonist according to Embodiment 120 that binds to an antigen associated with or targeted by an autoimmune response.

[0490] 122. The IL2 agonist according to Embodiment 121, wherein the targeting portion binds to a tumor-associated antigen. 123. Tumor-associated antigens include fibroblast-activating protein (FAP), the A1 domain of tenascin-C (TNC A1), and the A2 domain of tenascin-C (TNC A2) Fibronectin extradomain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophyllin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) or immunogenic epitopes thereoPSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, MAGE-tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, M AGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-tumor antigen family (e.g., GAGE-1, GAGE-2, GAGE -3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, and γ-catenin, p120ctn, gp100Viral products such as Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2, and GD2 ganglioside, human papillomavirus proteins, Smad family of tumor antigens, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, cerebral glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (TCR-related (heteromultimer), CD22 (B cell receptor), CD23 (low binding affinity IgE receptor), CD30 (cytokine receptor), CD33 (bone marrow cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R- (IL-6 receptor), CD20, MCSP, PDGFβR (β-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA An IL2 agonist according to Embodiment 122, wherein the agonist is IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF-II, IGFI receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) or extra domain B (EDB) of fibronectin, or A1 domain (TnC A1) of tenascin-C.

[0491] 124. An IL2 agonist according to Embodiment 121 or 122, wherein the tumor-associated antigen is a viral antigen. 125. An IL2 agonist according to Embodiment 124, wherein the viral antigen is Epstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, or HIV gp120, HPV E6, HPV E7, CMV early membrane antigen (EMA), or CMV late membrane antigen (LMA).

[0492] 126. The IL2 agonist according to Embodiment 121, wherein the targeting portion binds to a tumor microenvironment antigen. 127. The IL2 agonist according to Embodiment 126, wherein the tumor microenvironment antigen is an extracellular matrix protein.

[0493] 128. An IL2 agonist according to Embodiment 127, wherein the extracellular matrix protein is syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch, tenascin, collagen, and matrixin.

[0494] 129. The IL2 agonist according to Embodiment 121, wherein the targeting portion binds to cell surface molecules of tumor lymphocytes. 130. An IL2 agonist according to Embodiment 129, wherein the cell surface molecule is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, LAG3, TIM3, or B7-H3.

[0495] 131. The IL2 agonist according to Embodiment 130, wherein the cell surface molecule is PD1. 132. The IL2 agonist according to Embodiment 131, wherein the targeting portion is an anti-PD1 antibody or its antigen-binding fragment.

[0496] 133. An IL2 agonist according to Embodiment 132, wherein an anti-PD1 antibody or its antigen-binding fragment inhibits PD1 signaling. 134. An IL2 agonist according to Embodiment 132, wherein the anti-PD1 antibody or its antigen-binding fragment does not inhibit PD1 signaling.

[0497] 135. The IL2 agonist according to Embodiment 130, wherein the cell surface molecule is LAG3. 136. The IL2 agonist according to Embodiment 121, wherein the targeting portion binds to a checkpoint inhibitor.

[0498] 137. An IL2 agonist according to Embodiment 136, wherein the checkpoint inhibitor is CTLA-4, PD1, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, VISTA, PSGL1, or CHK2.

[0499] 138. An IL2 agonist according to Embodiment 137, wherein the checkpoint inhibitor is PD1. 139. An IL2 agonist according to Embodiment 138, wherein the targeting portion is an anti-PD1 antibody or its antigen-binding fragment.

[0500] 140. An IL2 agonist according to Embodiment 139, wherein an anti-PD1 antibody or its antigen-binding fragment inhibits PD1 signaling. 141. An IL2 agonist according to Embodiment 139, wherein the anti-PD1 antibody or its antigen-binding fragment does not inhibit PD1 signaling.

[0501] 142. An IL2 agonist according to Embodiment 137, wherein the checkpoint inhibitor is LAG3. 143. The IL2 agonist according to Embodiment 138, wherein the targeting moiety binds to an MHC-peptide complex.

[0502] 144. The IL2 agonist according to Embodiment 143, wherein the peptide in the peptide-MHC complex contains a tumorigenic antigen. 145. Oncogenic antigens include LCMV-derived peptides gp33-41, APF (126-134), BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100 (209-217), HBV core (18-27), Her2 / neu (369-377; V2v9); HPV E7 (11-20), HPV E7 (11-19), HPV An IL2 agonist according to Embodiment 144, which is E7(82-90), KLK4(11-19), LMP1(125-133), MAG-A3(112-120), NYESO1(157-165, C165A), NYESO1(157-165, C165V), p54 WT(264-272), PAP-3(136-143), PSMA(4-12), PSMA(135-145), Survivin(96-014), Tyrosinase(369-377, 371D), or WT1(126-134).

[0503] 146. The targeted portion is (a) CDR-H1 having an amino acid sequence selected from any of SEQ ID NOs: 4, 20, 36, 52, 68, 84, 100, 116, 132, 148, 164, 180, 196, 212, 220, 236, 252, 268, 284, 300, 316, 332, 348, 364, 380, 396, 412, 428, 444, 460, 476, 492, 508, and 524 of International Patent Publication No. 2019 / 005897A1 (incorporated herein by reference), (b) CDR-H2 having an amino acid sequence selected from any of SEQ ID NOs: 6, 22, 38, 54, 70, 86, 102, 118, 134, 150, 166, 182, 198, 214, 222, 238, 254, 270, 286, 302, 318, 334, 350, 366, 382, ​​414, 430, 446, 462, 478, 494, 510, and 526 of International Patent Publication No. 2019 / 005897A1 (incorporated herein by reference), (c) CDR-H3 having an amino acid sequence selected from any of SEQ ID NOs: 8, 24, 40, 56, 72, 88, 104, 120, 136, 152, 168, 184, 200, 216, 224, 240, 256, 272, 288, 304, 320, 336, 352, 368, 384, 400, 416, 432, 448, 464, 480, 496, 512, and 528 of International Patent Publication No. 2019 / 005897A1 (incorporated herein by reference), (d) CDR-L1 having an amino acid sequence selected from any of SEQ ID NOs: 12, 28, 44, 60, 76, 92, 108, 124, 140, 156, 172, 188, 204, 204, 228, 244, 260, 276, 292, 308, 324, 340, 356, 372, 388, 404, 420, 436, 452, 468, 484, 500, 516, and 532 of International Patent Publication No. 2019 / 005897A1 (incorporated herein by reference), (e) CDR-L2 having an amino acid sequence selected from any of SEQ ID NOs: 14, 30, 46, 62, 78, 94, 110, 126, 142, 158, 174, 190, 206, 230, 246, 262, 278, 294, 310, 326, 342, 358, 374, 390, 406, 422, 438, 454, 470, 486, 502, 518, and 534 of International Patent Publication No. 2019 / 005897A1 (incorporated herein by reference), and (f) An IL2 agonist according to any one of Embodiments 143 to 145, comprising an antibody or antigen-binding fragment thereof having a complementation-determining region ("CDR") containing CDR-L3 having an amino acid sequence selected from any of SEQ ID NOs. 16, 32, 48, 64, 80, 96, 112, 128, 144, 160, 176, 192, 208, 232, 248, 264, 280, 296, 312, 328, 344, 360, 376, 392, 408, 424, 440, 456, 472, 488, 504, 520, and 536 of International Patent Publication No. 2019 / 005897A1 (incorporated herein by reference).

[0504] 147. An antibody or antigen-binding fragment is used in sequence numbers 2 / 10, 18 / 26, 34 / 42, 50 / 58, 66 / 74, 82 / 90, 98 / 106, 114 / 122, 130 / 138, 146 / 154, 162 / 170, 178 / 186, 194 / 202, 210 / 202, 218 / 226, 234 / 242, 250 / 258, 2 of International Patent Publication No. 2019 / 005897A1 (incorporated herein by reference). An IL2 agonist according to Embodiment 146, having a VH-VL amino acid sequence pair selected from any of 66 / 274, 282 / 290, 298 / 306, 314 / 322, 330 / 338, 346 / 354, 362 / 370, 378 / 386, 394 / 402, 410 / 418, 426 / 434, 442 / 450, 458 / 466, 474 / 482, 490 / 498, 506 / 514, and 522 / 530.

[0505] 148. An IL2 agonist according to Embodiment 147, wherein the antibody or antigen-binding fragment has a VH-VL amino acid sequence pair selected from any of SEQ ID NOs: 2 / 10, 34 / 42, 82 / 90, 194 / 202, 282 / 290, and 506 / 514 of International Patent Publication No. 2019 / 005897A1 (incorporated herein by reference).

[0506] 149. The IL2 agonist according to Embodiment 121, wherein the targeting portion binds to an antigen associated with or targeted by an autoimmune response. 150. An IL2 agonist according to Embodiment 149, wherein the peptide is derived from gliadin, GAD 65, IA-2, insulin B chain, glatiramer acetate (GA), akethylcholine receptor (AChR), p205, insulin, thyroid-stimulating hormone, tyrosinase, TRP I, or myelin antigen.

[0507] 151. An IL2 agonist according to Embodiment 150, wherein the peptide is derived from IL-4R, IL-6R, or DLL4. 152. An IL2 agonist according to any one of embodiments 121 to 151, wherein the targeting portion is an antibody or its antigen-binding fragment.

[0508] 153. The IL2 agonist according to embodiment 152, wherein the targeting portion is Fab. 154. The IL2 agonist according to embodiment 152, wherein the targeting portion is scFv. 155. The IL2 agonist according to Embodiment 121, wherein the targeting portion is a peptide-MHC complex.

[0509] 156. The IL2 agonist according to Embodiment 155, wherein the peptide-MHC complex binds to the T cell receptor of tumor lymphocytes. 157. An IL2 agonist according to Embodiment 155 or 156, wherein the peptide in the peptide-MHC complex contains a tumorigenic antigen.

[0510] 158. Oncogenic antigens include LCMV-derived peptides gp33-41, APF (126-134), BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100 (209-217), HBV core (18-27), Her2 / neu (369-377; V2v9); HPV E7 (11-20), HPV E7 (11-19), HPV An IL2 agonist according to Embodiment 157, which is E7(82-90), KLK4(11-19), LMP1(125-133), MAG-A3(112-120), NYESO1(157-165, C165A), NYESO1(157-165, C165V), p54 WT(264-272), PAP-3(136-143), PSMA(4-12), PSMA(135-145), Survivin(96-014), Tyrosinase(369-377, 371D), or WT1(126-134).

[0511] 159. The IL2 agonist according to Embodiment 155, wherein the peptide in the peptide-MHC complex contains a viral antigen. 160. An IL2 agonist according to Embodiment 159, wherein the viral antigen is CMVpp65 or HPV16E7.

[0512] 161. An IL2 agonist according to any one of embodiments 155 to 160, wherein the peptide-MHC complex further comprises β2 microglobulin or a fragment thereof. 162. The IL2 agonist according to Embodiment 161, wherein the peptide MHC complex contains a type I MHC domain.

[0513] 163. The IL2 agonist according to Embodiment 162, wherein the peptide-MHC complex comprises an MHC peptide, a linker, a β2-microglobulin domain, a linker, and a type I MHC domain, oriented from N to C-terminus.

[0514] 164. The IL2 agonist according to Embodiment 163, wherein the linker connecting the MHC peptide and the β2-microglobulin domain contains the amino acid sequence GCGGS (SEQ ID NO: 77).

[0515] 165. An IL2 agonist according to any one of embodiments 155 to 160, wherein the peptide-MHC complex does not contain β2 microglobulin or a fragment thereof. 166. The IL2 agonist according to Embodiment 165, wherein the peptide MHC complex contains a type II MHC domain.

[0516] 167. A nucleic acid or a group of nucleic acids encoding an IL2 agonist according to any one of embodiments 1 to 166. 168. A host cell manipulated to express an IL2 agonist according to any one of Embodiments 1 to 166 or a nucleic acid according to Embodiment 167.

[0517] 169. A method for producing an IL2 agonist according to any one of Embodiments 1 to 166, comprising culturing the host cells described in Embodiment 168 and recovering the IL2 agonist expressed thereby.

[0518] 170. A pharmaceutical composition comprising an IL2 agonist according to any one of Embodiments 1 to 166 and an excipient. 171. A method for treating cancer, comprising administering to a subject in need thereof an IL2 agonist according to any one of Embodiments 1 to 166 or a pharmaceutical composition according to Embodiment 170.

[0519] 172. A method of treating cancer, provided to those who need it. (a) Chimeric antigen receptor ("CAR") T cells ("CART cells"), and (b) A method comprising administering an IL2 agonist comprising a targeting moiety that binds to a T cell receptor on a CART cell or another cell surface molecule on a CART cell, wherein the targeting moiety can optionally bind to the extracellular domain of a CAR.

[0520] 173. The method according to Embodiment 172, wherein the IL2 agonist is monovalent with respect to the IL2 portion and / or the targeting portion. 174. The method according to Embodiment 172 or 173, wherein the IL2 agonist is an IL2 agonist described in any one of Embodiments 1 to 166, preferably an IL2 agonist described in any one of Embodiments 94 to 97, 121, and 155 to 166.

[0521] 175. The method according to any one of embodiments 172 to 174, wherein the CART cells are not engineered to express a variant IL2-Rβ receptor. 176. The method according to any one of embodiments 172 to 174, wherein the CART cells are not manipulated to express any variant IL2 receptor.

[0522] 177. The method according to any one of embodiments 172 to 176, wherein the IL2 agonist is administered to the subject within one week of administration of CART cells. 178. The method according to Embodiment 177, wherein the IL2 agonist is administered to the subject on the same day as the administration of CART cells.

[0523] 179. The method according to any one of embodiments 172 to 178, comprising administering an IL2 agonist to a subject for a period of at least two weeks. 180. The method according to Embodiment 179, wherein the IL2 agonist is administered by continuous infusion.

[0524] 181. The method according to Embodiment 179, wherein the IL2 agonist is administered by daily doses for at least a portion of a period of at least two weeks. 182. The IL2 agonist is administered according to a divided dose regimen, and the divided dose regimen is (a) Administer the IL2 agonist at the first dose frequency for at least the first part of a two-week period, (b) The method according to Embodiment 179, comprising administering an IL2 agonist at a second dose frequency for a portion of the period after at least two weeks.

[0525] 183. The method according to Embodiment 182, wherein the first administration frequency is daily. 184. The method according to Embodiment 182 or 183, wherein the second administration frequency is less frequent than the first administration frequency.

[0526] 185. The method according to Embodiment 184, wherein the second administration frequency is weekly. 186. The method according to any one of Embodiments 182 to 185, wherein the subject is transitioned from a first administration frequency to a second administration frequency simultaneously with or after the depletion of CART cells.

[0527] 187. The method according to any one of embodiments 171 to 186, further comprising administering an anti-PD1 antibody to the subject. 188. The method according to Embodiment 187, wherein the anti-PD1 antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108.

[0528] 189. The method according to any one of embodiments 172 to 188, wherein the CAR is designed to target one of the targets identified in Section 6.11.1.3. 190. The method according to any one of embodiments 172 to 189, wherein CAR is configured according to Section 6.11.1.1 and its subsections.

[0529] 191. The method according to any one of embodiments 172 to 190, wherein the targeting portion comprises a pMHC recognized by the antigen-binding domain of the CAR. 192. A method for treating autoimmune diseases, provided to those in need. (a) Chimeric antigen receptor ("CAR") T cells ("CART cells"), and (b) A method comprising administering an IL2 agonist comprising a targeting moiety that binds to a cell surface antigen present on CART cells, wherein the targeting moiety can optionally bind to the extracellular domain of CAR.

[0530] 193. The method of Embodiment 192, wherein the IL2 agonist is monovalent with respect to the IL2 portion and / or the targeting portion. 194. The method according to Embodiment 192 or 193, wherein the IL2 agonist is the IL2 agonist described in any one of Embodiments 1 to 166, preferably the IL2 agonist described in any one of Embodiments 94 to 97, 121, and 155 to 166.

[0531] 195. The method according to any one of Embodiments 192 to 194, comprising a pMHC cloned from an autoimmune target cell and / or from which the targeting portion is recognized by the antigen-binding domain of the CAR.

[0532] 196. The method according to any one of embodiments 192 to 195, wherein the CART cells are Treg cells. 197. The method according to any one of embodiments 192 to 196, wherein the CAR is designed to target one of the targets identified in Section 6.11.1.4.

[0533] 198. The method according to any one of embodiments 192 to 197, wherein CAR is configured according to Section 6.11.1.1 and its subsections. 199. IL2 agonist, (a) Fc domain and (b) The Fc domain includes an IL2 portion at its C-terminus, and the IL2 portion includes an IL2 domain and an IL2-Rα domain, An IL2 agonist wherein, optionally, the IL2 agonist has one or more characteristics of the IL2 agonists described in any one of Embodiments 1 to 36.

[0534] 200. An IL...

Claims

1. It is an IL2 agonist, (a) FC domain and (b) An IL2 agonist comprising an IL2 portion at the C-terminus of the Fc domain, wherein the IL2 portion comprises an IL2 domain and an IL2-Rα domain.

2. The IL2 agonist according to claim 1, wherein the IL2 domain is the N-terminus of the IL2-Rα domain.

3. The IL2 agonist according to claim 1, wherein the IL2 domain is the C-terminus of the IL2-Rα domain.

4. The aforementioned IL2 portion, (a) Sequence identity of at least about 90% or at least about 95% to mature human IL2, (b) N-terminal alanine deletion compared to mature human IL2, (c) An IL2 variant having an amino acid substitution at the N88 position compared to wild-type IL2 (optionally selected, the amino acid substitution is N88D), (d) Compared to wild-type IL2, amino acid substitutions C125S, C125A, or C125V, An IL2 agonist according to any one of claims 1 to 3, comprising an IL2 domain having an amino acid sequence having any combination of (e), (a), (b), (c), and / or (d).

5. The IL2-Rα domain contains or comprises an amino acid sequence having at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with respect to the IL2 binding portion of human IL-2Rα, and optionally the binding portion is (a) at least 160 amino acids, at least 161 amino acids, at least 162 amino acids, at least 164 amino acids, or at least 165 amino acids of human IL2-Rα, and / or (b) An IL2 agonist according to any one of claims 1 to 4, having a maximum of 251, 240, 230, 220, 210, 200, 190, 180, or 170 amino acids in the extracellular domain of human IL2-Rα.

6. The IL2 agonist according to any one of claims 1 to 5, wherein the IL2 domain and the IL2-Rα domain are connected via a linker ("IL2 partial linker").

7. The IL2 agonist according to claim 6, wherein the IL2 partial linker has a length of at least 10 or at least 15 amino acids.

8. The IL2 agonist according to claim 6 or 7, wherein the IL2 partial linker is a glycine-serine linker or comprises one.

9. The aforementioned IL2 partial linker has an amino acid sequence G ４ An IL2 agonist according to any one of claims 6 to 8, comprising S (Sequence ID 57).

10. The IL2 partial linker, the amino acid sequence G ４ The IL2 agonist according to claim 9, which is a multimer of S (Sequence ID 57) or comprises the same.

11. The multimer is the amino acid sequence G ４ The IL2 agonist according to claim 10, comprising two, three, four, five, six or more repeats of S (Sequence ID 57).

12. The IL2 agonist according to any one of claims 1 to 11, wherein the Fc domain and the IL2 portion are connected via a linker ("Fc-IL2 linker").

13. The IL2 agonist according to claim 12, wherein the Fc-IL2 linker has a length of at least 5 or at least 10 amino acids.

14. The IL2 agonist according to claim 12 or 13, wherein the Fc-IL2 linker is a glycine-serine linker or comprises the same.

15. The Fc-IL2 linker, the amino acid sequence G ４ An IL2 agonist according to any one of claims 12 to 14, comprising S (Sequence ID 57).

16. The Fc-IL2 linker, the amino acid sequence G ４ The IL2 agonist according to claim 9, which is a multimer of S (Sequence ID 57) or comprises the same.

17. The multimer is the amino acid sequence G ４ The IL2 agonist according to claim 10, comprising two, three, four, five, six or more repeats of S (Sequence ID 57).

18. The IL2 agonist according to any one of claims 1 to 17, wherein the Fc domain is an IgG1, IgG2, IgG3, or IgG4 Fc domain.

19. The IL2 agonist according to claim 18, wherein the Fc domain reduces the effector function.

20. An IL2 agonist according to any one of claims 1 to 19, which is a dimer.

21. The IL2 agonist according to claim 20, which is a homodimer.

22. The IL2 agonist according to claim 20, which is a heterodimer.

23. The IL2 agonist according to any one of claims 1 to 22, wherein the IL2 portion is divalent.

24. An IL2 agonist according to any one of claims 1 to 23, comprising a targeting portion.

25. The IL2 agonist according to claim 24, wherein the targeting portion is the N-terminus of the Fc domain.

26. The target portion, (a) Binds to tumor-associated antigens, (b) Binds to tumor microenvironment antigens, (c) Binds to cell surface molecules of tumor-reactive lymphocytes, (d) Binds to checkpoint inhibitors, (e) Binds to the peptide-MHC complex, (f) It is a peptide-MHC complex, or (g) The IL2 agonist according to claim 24 or 25, which binds to an antigen associated with or targeted by an autoimmune response.

27. The IL2 agonist according to claim 26, wherein the targeting portion binds to a tumor-associated antigen.

28. The tumor-associated antigens include fibroblast-activating protein (FAP), tenascin-C A1 domain (TNC A1), tenascin-C A2 domain (TNC A2), fibronectin extradomain B (EDB), melanoma-associated chondroitin sulfate proteoglycan (MCSP), MART-1 / Melan-A, gp100, dipeptidyl peptidase IV (DPPIV), adenosine deaminase-binding protein (ADAbp), cyclophyllin b, colorectal-associated antigen (CRC)-C017-1A / GA733, carcinoembryonic antigen (CEA), and its immunogen. Sexual epitopes CAP-1 and CAP-2, etv6, aml1, prostate-specific antigen (PSA) or immunogenic epitopes thereoPSA-1, PSA-2, and PSA-3, prostate-specific membrane antigen (PSMA), T cell receptor / CD3-zeta chain, MAGE-tumor antigen family (e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, M AGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5), GAGE-tumor antigen family (e.g., GAGE-1, GAGE-2, GAGE -3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9), BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, and γ-catenin, p120ctn, gp100Viral products such as Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposis protein (APC), fodrin, connexin 37, Ig-idiotype, p15, gp75, GM2, and GD2 gangliosides, human papillomavirus proteins, Smad family of tumor antigens, Imp-1, P1A, EBV-encoded nuclear antigen (EBNA)-1, cerebral glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40), SSX-1, SSX-4, SSX-5, SCP-1, and CT-7, c-erbB-2, Her2, EGFR, IGF-1R, CD2 (T cell surface antigen), CD3 (TCR-related (heteromultimer), CD22 (B cell receptor), CD23 (low binding affinity IgE receptor), CD30 (cytokine receptor), CD33 (bone marrow cell surface antigen), CD40 (tumor necrosis factor receptor), IL-6R- (IL-6 receptor), CD20, MCSP, PDGFβR (β-platelet-derived growth factor receptor), ErbB2 epithelial cell adhesion molecule (EpCAM), EGFR variant III (EGFRvIII), CD19, disialoganglioside GD2, ductal epithelial mucin, gp36, TAG-72, glioma-associated antigen, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, MN-CA An IL2 agonist according to claim 27, wherein the agonist is IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostase-specific antigen (PSA), PAP, LAGA-1a, p53, prostein, PSMA, survival and telomerase, prostate cancer tumor antigen-1 (PCTA-1), ELF2M, neutrophil elastase, ephrin B2, insulin growth factor (IGF1)-I, IGF-II, IGF-1 receptor, 5T4, ROR1, Nkp30, NKG2D, tumor stromal antigen, extra domain A (EDA) or extra domain B (EDB) of fibronectin, or A1 domain (TnC A1) of tenascin-C.

29. The IL2 agonist according to claim 26 or 27, wherein the tumor-associated antigen is a viral antigen.

30. The IL2 agonist according to claim 29, wherein the viral antigen is Epstein-Barr virus LMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, or HIV gp120, HPV E6, HPV E7, CMV early membrane antigen (EMA), or CMV late membrane antigen (LMA).

31. The IL2 agonist according to claim 26, wherein the targeting portion binds to tumor microenvironment antigens.

32. The IL2 agonist according to claim 31, wherein the tumor microenvironment antigen is an extracellular matrix protein.

33. The IL2 agonist according to claim 32, wherein the extracellular matrix protein is syndecan, heparanase, integrin, osteopontin, link, cadherin, laminin, laminin-type EGF, lectin, fibronectin, notch, tenascin, collagen, and matrixin.

34. The IL2 agonist according to claim 26, wherein the targeting portion binds to cell surface molecules of tumor lymphocytes.

35. The IL2 agonist according to claim 34, wherein the cell surface molecule is CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, LAG3, TIM3, or B7-H3.

36. The IL2 agonist according to claim 35, wherein the cell surface molecule is PD1.

37. The IL2 agonist according to claim 36, wherein the targeting portion is an anti-PD1 antibody or its antigen-binding fragment.

38. The IL2 agonist according to claim 35, wherein the cell surface molecule is LAG3.

39. The IL2 agonist according to claim 26, wherein the targeting portion binds to a checkpoint inhibitor.

40. The IL2 agonist according to claim 39, wherein the checkpoint inhibitor is CTLA-4, PD1, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, VISTA, PSGL1, or CHK2.

41. The IL2 agonist according to claim 40, wherein the checkpoint inhibitor is PD1.

42. The IL2 agonist according to claim 40, wherein the checkpoint inhibitor is LAG3.

43. The IL2 agonist according to claim 41, wherein the targeting portion is bound to an MHC-peptide complex.

44. The IL2 agonist according to claim 43, wherein the peptide in the peptide-MHC complex comprises a tumorigenic antigen.

45. The aforementioned tumor-generating antigens are LCMV-derived peptides gp33-41, APF (126-134), BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100 (209-217), HBV core (18-27), Her2 / neu (369-377; V2v9); HPV E7 (11-20), HPV E7 (11-19), HPV An IL2 agonist according to claim 44, which is E7 (82-90), KLK4 (11-19), LMP1 (125-133), MAG-A3 (112-120), NYESO1 (157-165, C165A), NYESO1 (157-165, C165V), p54 WT (264-272), PAP-3 (136-143), PSMA (4-12), PSMA (135-145), Survivin (96-014), Tyrosinase (369-377, 371D), or WT1 (126-134).

46. The IL2 agonist according to any one of claims 26 to 45, wherein the targeting portion is an antibody or its antigen-binding fragment.

47. The IL2 agonist according to claim 46, wherein the targeting portion is Fab.

48. The IL2 agonist according to claim 46, wherein the targeting portion is scFv.

49. The IL2 agonist according to claim 26, wherein the targeting portion is a peptide-MHC complex.

50. The IL2 agonist according to claim 49, wherein the peptide-MHC complex binds to the T cell receptor of tumor lymphocytes.

51. The IL2 agonist according to claim 49 or 50, wherein the peptide in the peptide-MHC complex comprises a tumorigenic antigen.

52. The aforementioned tumor-generating antigens are LCMV-derived peptides gp33-41, APF (126-134), BALF (276-284), CEA (571-579), CMV pp65 (495-503), FLU-M1 (58-66), gp100 (154-162), gp100 (209-217), HBV core (18-27), Her2 / neu (369-377; V2v9); HPV E7 (11-20), HPV E7 (11-19), HPV An IL2 agonist according to claim 51, which is E7 (82-90), KLK4 (11-19), LMP1 (125-133), MAG-A3 (112-120), NYESO1 (157-165, C165A), NYESO1 (157-165, C165V), p54 WT (264-272), PAP-3 (136-143), PSMA (4-12), PSMA (135-145), Survivin (96-014), Tyrosinase (369-377, 371D), or WT1 (126-134).

53. The IL2 agonist according to claim 49, wherein the peptide in the peptide-MHC complex contains a viral antigen.

54. The IL2 agonist according to claim 53, wherein the viral antigen is CMVpp65 or HPV16E7.

55. The IL2 agonist according to any one of claims 49 to 54, wherein the peptide-MHC complex further comprises β2-microglobulin or a fragment thereof.

56. The IL2 agonist according to claim 55, wherein the peptide MHC complex comprises a type I MHC domain.

57. The IL2 agonist according to claim 56, wherein the peptide-MHC complex comprises an MHC peptide, a linker, a β2-microglobulin domain, a linker, and a type I MHC domain, oriented from N to C-terminus.

58. The IL2 agonist according to claim 57, wherein the linker connecting the MHC peptide and the β2-microglobulin domain comprises the amino acid sequence GCGGS.

59. The IL2 agonist according to any one of claims 49 to 54, wherein the peptide-MHC complex does not contain β2 microglobulin or a fragment thereof.

60. The IL2 agonist according to claim 59, wherein the peptide MHC complex comprises a type II MHC domain.

61. A nucleic acid or a plurality of nucleic acids encoding an IL2 agonist according to any one of claims 1 to 60.

62. A host cell manipulated to express an IL2 agonist according to any one of claims 1 to 60 or a nucleic acid according to claim 61.

63. A method for producing an IL2 agonist according to any one of claims 1 to 60, comprising culturing the host cells according to claim 62 and recovering the IL2 agonist expressed thereby.

64. A pharmaceutical composition comprising an IL2 agonist according to any one of claims 1 to 60 and an excipient.

65. A method for treating cancer, comprising administering to a subject in need of such treatment an IL2 agonist according to any one of claims 1 to 60 or a pharmaceutical composition according to claim 64.

66. A method of treating cancer, for those who need it. (a) Chimeric antigen receptor ("CAR") T cells ("CART cells"), and (b) A method comprising administering an IL2 agonist according to any one of claims 1 to 26, comprising a targeting moiety that binds to a T cell receptor on the CART cell or another cell surface molecule on the CART cell, wherein the targeting moiety can optionally bind to the extracellular domain of the CAR.

67. The method according to claim 65 or 66, further comprising administering an anti-PD1 antibody to the subject.

68. The method according to claim 67, wherein the anti-PD1 antibody is MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, or BGB-108.

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